Sample records for total chromosomal dna
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Updating the maize karyotype by chromosome DNA sizing
2018-01-01
The karyotype is a basic concept regarding the genome, fundamentally described by the number and morphological features of all chromosomes. Chromosome class, centromeric index, intra- and interchromosomal asymmetry index, and constriction localization are important in clinical, systematic and evolutionary approaches. In spite of the advances in karyotype characterization made over the last years, new data about the chromosomes can be generated from quantitative methods, such as image cytometry. Therefore, using Zea mays L., this study aimed to update the species’ karyotype by supplementing information on chromosome DNA sizing. After adjustment of the procedures, chromosome morphometry and class as well as knob localization enabled describing the Z. mays karyotype. In addition, applying image cytometry, DNA sizing was unprecedentedly measured for the arms and satellite of all chromosomes. This way, unambiguous identification of the chromosome pairs, and hence the assembly of 51 karyograms, were only possible after the DNA sizing of each chromosome, their arms and satellite portions. These accurate, quantitative and reproducible data also enabled determining the distribution and variation of DNA content in each chromosome. From this, a correlation between DNA amount and total chromosome length evidenced that the mean DNA content of chromosome 9 was higher than that of chromosome 8. The chromosomal DNA sizing updated the Z. mays karyotype, providing insights into its dynamic genome with regards to the organization of the ten chromosomes and their respective portions. Considering the results and the relevance of cytogenetics in the current scenario of comparative sequencing and genomics, chromosomal DNA sizing should be incorporated as an additional parameter for karyotype definition. Based on this study, it can be affirmed that cytogenetic approaches go beyond the simple morphological description of chromosomes. PMID:29293613
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Chromosome-specific DNA Repeat Probes
Energy Technology Data Exchange (ETDEWEB)
Baumgartner, Adolf; Weier, Jingly Fung; Weier, Heinz-Ulrich G.
2006-03-16
In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with {alpha}-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.
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DNA-damage response during mitosis induces whole-chromosome missegregation.
Bakhoum, Samuel F; Kabeche, Lilian; Murnane, John P; Zaki, Bassem I; Compton, Duane A
2014-11-01
Many cancers display both structural (s-CIN) and numerical (w-CIN) chromosomal instabilities. Defective chromosome segregation during mitosis has been shown to cause DNA damage that induces structural rearrangements of chromosomes (s-CIN). In contrast, whether DNA damage can disrupt mitotic processes to generate whole chromosomal instability (w-CIN) is unknown. Here, we show that activation of the DNA-damage response (DDR) during mitosis selectively stabilizes kinetochore-microtubule (k-MT) attachments to chromosomes through Aurora-A and PLK1 kinases, thereby increasing the frequency of lagging chromosomes during anaphase. Inhibition of DDR proteins, ATM or CHK2, abolishes the effect of DNA damage on k-MTs and chromosome segregation, whereas activation of the DDR in the absence of DNA damage is sufficient to induce chromosome segregation errors. Finally, inhibiting the DDR during mitosis in cancer cells with persistent DNA damage suppresses inherent chromosome segregation defects. Thus, the DDR during mitosis inappropriately stabilizes k-MTs, creating a link between s-CIN and w-CIN. The genome-protective role of the DDR depends on its ability to delay cell division until damaged DNA can be fully repaired. Here, we show that when DNA damage is induced during mitosis, the DDR unexpectedly induces errors in the segregation of entire chromosomes, thus linking structural and numerical chromosomal instabilities. ©2014 American Association for Cancer Research.
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Delayed chromosomal instability induced by DNA damage
International Nuclear Information System (INIS)
Morgan, W.F.; Marder, B.A.; Day, J.P.
1994-01-01
Cellular exposure to DNA damaging agents rapidly results in a dose dependent increase in chromosomal breakage and gross structural chromosomal rearrangements. Over recent years, evidence has been accumulating indicating genomic instability can manifest multiple generations after cellular exposure to physical and chemical DNA damaging agents. Genomic instability manifests in the progeny of surviving cells, and has been implicated in mutation, gene application, cellular transformation, and cell killing. To investigate chromosome instability following DNA damage, we have used fluorescence in situ hybridization to detect chromosomal rearrangements in a human/hamster somatic hybrid cell line following exposure to ionizing radiation. Delayed chromosomal instability was detected when multiple populations of uniquely arranged metaphases were observed in clonal isolates raised from single cells surviving X-irradiation many generations after exposure. At higher radiation doses, chromosomal instability was observed in a relatively high frequency of surviving clones and, in general, those clones showed delayed chromosome instability also showed reduced survival as measured by colony forming ability
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Chromosomal instability drives metastasis through a cytosolic DNA response.
Bakhoum, Samuel F; Ngo, Bryan; Laughney, Ashley M; Cavallo, Julie-Ann; Murphy, Charles J; Ly, Peter; Shah, Pragya; Sriram, Roshan K; Watkins, Thomas B K; Taunk, Neil K; Duran, Mercedes; Pauli, Chantal; Shaw, Christine; Chadalavada, Kalyani; Rajasekhar, Vinagolu K; Genovese, Giulio; Venkatesan, Subramanian; Birkbak, Nicolai J; McGranahan, Nicholas; Lundquist, Mark; LaPlant, Quincey; Healey, John H; Elemento, Olivier; Chung, Christine H; Lee, Nancy Y; Imielenski, Marcin; Nanjangud, Gouri; Pe'er, Dana; Cleveland, Don W; Powell, Simon N; Lammerding, Jan; Swanton, Charles; Cantley, Lewis C
2018-01-25
Chromosomal instability is a hallmark of cancer that results from ongoing errors in chromosome segregation during mitosis. Although chromosomal instability is a major driver of tumour evolution, its role in metastasis has not been established. Here we show that chromosomal instability promotes metastasis by sustaining a tumour cell-autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signalling. Genetic suppression of chromosomal instability markedly delays metastasis even in highly aneuploid tumour models, whereas continuous chromosome segregation errors promote cellular invasion and metastasis in a STING-dependent manner. By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumour cells co-opt chronic activation of innate immune pathways to spread to distant organs.
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Human Chromosome 7: DNA Sequence and Biology
Scherer, Stephen W.; Cheung, Joseph; MacDonald, Jeffrey R.; Osborne, Lucy R.; Nakabayashi, Kazuhiko; Herbrick, Jo-Anne; Carson, Andrew R.; Parker-Katiraee, Layla; Skaug, Jennifer; Khaja, Razi; Zhang, Junjun; Hudek, Alexander K.; Li, Martin; Haddad, May; Duggan, Gavin E.
2003-01-01
DNA sequence and annotation of the entire human chromosome 7, encompassing nearly 158 million nucleotides of DNA and 1917 gene structures, are presented. To generate a higher order description, additional structural features such as imprinted genes, fragile sites, and segmental duplications were integrated at the level of the DNA sequence with medical genetic data, including 440 chromosome rearrangement breakpoints associated with disease. This approach enabled the discovery of candidate gene...
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Chromosomal DNA replication of Vicia faba cells
International Nuclear Information System (INIS)
Ikushima, Takaji
1976-01-01
The chromosomal DNA replication of higher plant cells has been investigated by DNA fiber autoradiography. The nuclear DNA fibers of Vicia root meristematic cells are organized into many tandem arrays of replication units or replicons which exist as clusters with respect to replication. DNA is replicated bidirectionally from the initiation points at the average rate of 0.15 μm/min at 20 0 C, and the average interinitiation interval is about 16 μm. The manner of chromosomal DNA replication in this higher plant is similar to that found in other eukaryotic cells at a subchromosomal level. (auth.)
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Function of Junk: Pericentromeric Satellite DNA in Chromosome Maintenance.
Jagannathan, Madhav; Yamashita, Yukiko M
2018-04-02
Satellite DNAs are simple tandem repeats that exist at centromeric and pericentromeric regions on eukaryotic chromosomes. Unlike the centromeric satellite DNA that comprises the vast majority of natural centromeres, function(s) for the much more abundant pericentromeric satellite repeats are poorly understood. In fact, the lack of coding potential allied with rapid divergence of repeat sequences across eukaryotes has led to their dismissal as "junk DNA" or "selfish parasites." Although implicated in various biological processes, a conserved function for pericentromeric satellite DNA remains unidentified. We have addressed the role of satellite DNA through studying chromocenters, a cytological aggregation of pericentromeric satellite DNA from multiple chromosomes into DNA-dense nuclear foci. We have shown that multivalent satellite DNA-binding proteins cross-link pericentromeric satellite DNA on chromosomes into chromocenters. Disruption of chromocenters results in the formation of micronuclei, which arise by budding off the nucleus during interphase. We propose a model that satellite DNAs are critical chromosome elements that are recognized by satellite DNA-binding proteins and incorporated into chromocenters. We suggest that chromocenters function to preserve the entire chromosomal complement in a single nucleus, a fundamental and unquestioned feature of eukaryotic genomes. We speculate that the rapid divergence of satellite DNA sequences between closely related species results in discordant chromocenter function and may underlie speciation and hybrid incompatibility. © 2017 Jagannathan and Yamashita; Published by Cold Spring Harbor Laboratory Press.
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DNA Repair Defects and Chromosomal Aberrations
Hada, Megumi; George, K. A.; Huff, J. L.; Pluth, J. M.; Cucinotta, F. A.
2009-01-01
Yields of chromosome aberrations were assessed in cells deficient in DNA doublestrand break (DSB) repair, after exposure to acute or to low-dose-rate (0.018 Gy/hr) gamma rays or acute high LET iron nuclei. We studied several cell lines including fibroblasts deficient in ATM (ataxia telangiectasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. Chromosomes were analyzed using the fluorescence in situ hybridization (FISH) chromosome painting method in cells at the first division post irradiation, and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma irradiation induced greater yields of both simple and complex exchanges in the DSB repair-defective cells than in the normal cells. The quadratic dose-response terms for both simple and complex chromosome exchanges were significantly higher for the ATM- and NBS-deficient lines than for normal fibroblasts. However, in the NBS cells the linear dose-response term was significantly higher only for simple exchanges. The large increases in the quadratic dose-response terms in these repair-defective cell lines points the importance of the functions of ATM and NBS in chromatin modifications to facilitate correct DSB repair and minimize the formation of aberrations. The differences found between ATM- and NBS-deficient cells at low doses suggest that important questions should with regard to applying observations of radiation sensitivity at high dose to low-dose exposures. For aberrations induced by iron nuclei, regression models preferred purely linear dose responses for simple exchanges and quadratic dose responses for complex exchanges. Relative biological effectiveness (RBE) factors of all of
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A comparison of the DNA and chromosome repair kinetics after #betta# irradiation
International Nuclear Information System (INIS)
Hittelman, W.N.; Pollard, M.
1982-01-01
The kinetics of repair at the chromosome and DNA levels were compared after #betta# irradiation of Chinese hamster ovary cells (CHO). Induction and repair of DNA damage were measured by the alkaline and neutral elution techniques, while chromosome damage and repair were determined by the technique of premature chromosome condensation. During and after #betta# irradiation, significant DNA repair occurred within 2 min. This fast repair could be inhibited by EDTA and pyrophosphate and probably reflected polynucleotide ligase activity. A slower component of DNA repair was detected between 15 and 60 min after irradiation, by which time most of the DNA had been repaired. In contrast, chromosome repair was not detectable until 45 min after irradiation, and nearly half of the chromatid breaks were repaired by 60 min. Cycloheximide, an inhibitor of protein synthesis, prevented chromosome break repair, yet had no effect on the immediate formation of chromatid exchanges or DNA repair. These results suggest the following: (1) the rapidly repairing DNA lesions are not important in the repair of chromosomes; (2) chromosome damage involves only a minority of the DNA lesions measured by alkaline and neutral DNA elution; and (3) chromosome repair may involve more than simply the repair of damaged DNA that can be detected by the alkaline and neutral elution assays
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Satellite DNA-based artificial chromosomes for use in gene therapy.
Hadlaczky, G
2001-04-01
Satellite DNA-based artificial chromosomes (SATACs) can be made by induced de novo chromosome formation in cells of different mammalian species. These artificially generated accessory chromosomes are composed of predictable DNA sequences and they contain defined genetic information. Prototype human SATACs have been successfully constructed in different cell types from 'neutral' endogenous DNA sequences from the short arm of the human chromosome 15. SATACs have already passed a number of hurdles crucial to their further development as gene therapy vectors, including: large-scale purification; transfer of purified artificial chromosomes into different cells and embryos; generation of transgenic animals and germline transmission with purified SATACs; and the tissue-specific expression of a therapeutic gene from an artificial chromosome in the milk of transgenic animals.
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Human sperm sex chromosome disomy and sperm DNA damage assessed by the neutral comet assay.
McAuliffe, M E; Williams, P L; Korrick, S A; Dadd, R; Marchetti, F; Martenies, S E; Perry, M J
2014-10-10
Is there an association between human sperm sex chromosome disomy and sperm DNA damage? An increase in human sperm XY disomy was associated with higher comet extent; however, there was no other consistent association of sex chromosome disomies with DNA damage. There is limited published research on the association between sex chromosome disomy and sperm DNA damage and the findings are not consistent across studies. We conducted a cross-sectional study of 190 men (25% ever smoker, 75% never smoker) from subfertile couples presenting at the Massachusetts General Hospital Fertility Clinic from January 2000 to May 2003. Multiprobe fluorescence in situ hybridization for chromosomes X, Y and 18 was used to determine XX, YY, XY and total sex chromosome disomy in sperm nuclei using an automated scoring method. The neutral comet assay was used to measure sperm DNA damage, as reflected by comet extent, percentage DNA in the comet tail, and tail distributed moment. Univariate and multiple linear regression models were constructed with sex chromosome disomy (separate models for each of the four disomic conditions) as the independent variable, and DNA damage parameters (separate models for each measure of DNA damage) as the dependent variable. Men with current or past smoking history had significantly greater comet extent (µm: regression coefficients with 95% CI) [XX18: 15.17 (1.98, 28.36); YY18: 14.68 (1.50, 27.86); XY18: 15.41 (2.37, 28.45); Total Sex Chromosome Disomy: 15.23 (2.09, 28.38)], and tail distributed moment [XX18: 3.01 (0.30, 5.72); YY18: 2.95 (0.24, 5.67); XY18: 3.04 (0.36, 5.72); Total Sex Chromosome Disomy: 3.10 (0.31, 5.71)] than men who had never smoked. In regression models adjusted for age and smoking, there was a positive association between XY disomy and comet extent. For an increase in XY disomy from 0.56 to 1.47% (representing the 25th to 75th percentile), there was a mean increase of 5.08 µm in comet extent. No other statistically significant
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Forensic use of Y-chromosome DNA: a general overview
M.H. Kayser (Manfred)
2017-01-01
textabstractThe male-specific part of the human Y chromosome is widely used in forensic DNA analysis, particularly in cases where standard autosomal DNA profiling is not informative. A Y-chromosomal gene fragment is applied for inferring the biological sex of a crime scene trace donor. Haplotypes
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The study of human Y chromosome variation through ancient DNA.
Kivisild, Toomas
2017-05-01
High throughput sequencing methods have completely transformed the study of human Y chromosome variation by offering a genome-scale view on genetic variation retrieved from ancient human remains in context of a growing number of high coverage whole Y chromosome sequence data from living populations from across the world. The ancient Y chromosome sequences are providing us the first exciting glimpses into the past variation of male-specific compartment of the genome and the opportunity to evaluate models based on previously made inferences from patterns of genetic variation in living populations. Analyses of the ancient Y chromosome sequences are challenging not only because of issues generally related to ancient DNA work, such as DNA damage-induced mutations and low content of endogenous DNA in most human remains, but also because of specific properties of the Y chromosome, such as its highly repetitive nature and high homology with the X chromosome. Shotgun sequencing of uniquely mapping regions of the Y chromosomes to sufficiently high coverage is still challenging and costly in poorly preserved samples. To increase the coverage of specific target SNPs capture-based methods have been developed and used in recent years to generate Y chromosome sequence data from hundreds of prehistoric skeletal remains. Besides the prospects of testing directly as how much genetic change in a given time period has accompanied changes in material culture the sequencing of ancient Y chromosomes allows us also to better understand the rate at which mutations accumulate and get fixed over time. This review considers genome-scale evidence on ancient Y chromosome diversity that has recently started to accumulate in geographic areas favourable to DNA preservation. More specifically the review focuses on examples of regional continuity and change of the Y chromosome haplogroups in North Eurasia and in the New World.
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Homologous subfamilies of human alphoid repetitive DNA on different nucleolus organizing chromosomes
International Nuclear Information System (INIS)
Joergensen, A.L.; Bostock, C.J.; Bak, A.L.
1987-01-01
The organization of alphoid repeated sequences on human nucleolus-organizing (NOR) chromosomes 13, 21, and 22 has been investigated. Analysis of hybridization of alphoid DNA probes to Southern transfers of restriction enzyme-digested DNA fragments from hybrid cells containing single human chromosomes shows that chromosomes 13 and 21 share one subfamily of alphoid repeats, whereas a different subfamily may be held in common by chromosomes 13 and 22. The sequences of cloned 680-base-pair EcoRI fragments of the alphoid DNA from chromosomes 13 and 21 show that the basic unit of this subfamily is indistinguishable on each chromosome. The sequence of cloned 1020-base-pair Xba I fragments from chromosome 22 is related to, but distinguishable from, that of the 680-base-pair EcoRI alphoid subfamily of chromosomes 13 and 21. These results suggest that, at some point after they originated and were homogenized, different subfamilies of alphoid sequences must have exchanged between chromosomes 13 and 21 and separately between chromosomes 13 and 22
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Defective double-strand DNA break repair and chromosomal translocations by MYC overexpression.
Karlsson, Asa; Deb-Basu, Debabrita; Cherry, Athena; Turner, Stephanie; Ford, James; Felsher, Dean W
2003-08-19
DNA repair mechanisms are essential for the maintenance of genomic integrity. Disruption of gene products responsible for DNA repair can result in chromosomal damage. Improperly repaired chromosomal damage can result in the loss of chromosomes or the generation of chromosomal deletions or translocations, which can lead to tumorigenesis. The MYC protooncogene is a transcription factor whose overexpression is frequently associated with human neoplasia. MYC has not been previously implicated in a role in DNA repair. Here we report that the overexpression of MYC disrupts the repair of double-strand DNA breaks, resulting in a several-magnitude increase in chromosomal breaks and translocations. We found that MYC inhibited the repair of gamma irradiation DNA breaks in normal human cells and blocked the repair of a single double-strand break engineered to occur in an immortal cell line. By spectral karyotypic analysis, we found that MYC even within one cell division cycle resulted in a several-magnitude increase in the frequency of chromosomal breaks and translocations in normal human cells. Hence, MYC overexpression may be a previously undescribed example of a dominant mutator that may fuel tumorigenesis by inducing chromosomal damage.
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Aten, J. A.; Buys, C. H.; van der Veen, A. Y.; Mesa, J. R.; Yu, L. C.; Gray, J. W.; Osinga, J.; Stap, J.
1987-01-01
A number of structurally unrelated DNA intercalators have been studied as stabilizers of mitotic chromosomes during isolation from rodent and human metaphase cells. Seven out of the nine intercalators tested were found to be useful as chromosome stabilizing agents. Chromosome suspensions prepared in
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Molecular fundamentals of chromosomal mutagenesis
International Nuclear Information System (INIS)
Ganassi, E.Eh.; Zaichkina, S.I.; Malakhova, L.V.
1987-01-01
Precise quantitative correlation between the yield of chromosome structure damages and the yield of DNA damages is shown when comparing data on molecular and cytogenetic investigations carried out in cultural Mammalia cells. As the chromosome structure damage is to be connected with the damage of its carcass structure, then it is natural that DNA damage in loop regions is not to affect considerably the structure, while DNA damage lying on the loop base and connected with the chromosome carcass is to play a determining role in chromosomal mutagenesis. This DNA constitutes 1-2% from the total quantity of nuclear DNA. If one accepts that damages of these regions of DNA are ''hot'' points of chromosomal mutagenesis, then it becomes clear why 1-2% of preparation damages in a cell are realized in chromosome structural damages
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Fragile sites, dysfunctional telomere and chromosome fusions: What is 5S rDNA role?
Barros, Alain Victor; Wolski, Michele Andressa Vier; Nogaroto, Viviane; Almeida, Mara Cristina; Moreira-Filho, Orlando; Vicari, Marcelo Ricardo
2017-04-15
Repetitive DNA regions are known as fragile chromosomal sites which present a high flexibility and low stability. Our focus was characterize fragile sites in 5S rDNA regions. The Ancistrus sp. species shows a diploid number of 50 and an indicative Robertsonian fusion at chromosomal pair 1. Two sequences of 5S rDNA were identified: 5S.1 rDNA and 5S.2 rDNA. The first sequence gathers the necessary structures to gene expression and shows a functional secondary structure prediction. Otherwise, the 5S.2 rDNA sequence does not contain the upstream sequences that are required to expression, furthermore its structure prediction reveals a nonfunctional ribosomal RNA. The chromosomal mapping revealed several 5S.1 and 5S.2 rDNA clusters. In addition, the 5S.2 rDNA clusters were found in acrocentric and metacentric chromosomes proximal regions. The pair 1 5S.2 rDNA cluster is co-located with interstitial telomeric sites (ITS). Our results indicate that its clusters are hotspots to chromosomal breaks. During the meiotic prophase bouquet arrangement, double strand breaks (DSBs) at proximal 5S.2 rDNA of acrocentric chromosomes could lead to homologous and non-homologous repair mechanisms as Robertsonian fusions. Still, ITS sites provides chromosomal instability, resulting in telomeric recombination via TRF2 shelterin protein and a series of breakage-fusion-bridge cycles. Our proposal is that 5S rDNA derived sequences, act as chromosomal fragile sites in association with some chromosomal rearrangements of Loricariidae. Copyright © 2017 Elsevier B.V. All rights reserved.
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Implications for x-chromosome regulation from studies of human x-chromosome DNA
International Nuclear Information System (INIS)
Wolf, S.F.; Migeon, B.R.
1983-01-01
It is clear that there must be multiple events involved in the regulation of the mammalian X chromosome. The initial event, occurring about the time of implantation results in inactivation of all but a single X chromosome in diploid cells. A popular working hypothesis is that DNA modification, such as methylation or sequence rearrangement, might be responsible for maintenance of the inactive state. Methylation is particularly attractive, since the preference for methylating half-methylated sites might result in perpetuation of the differentiated state. In this paper we discuss several facets of our studies of X inactivation; specifically, our general strategy, studies of X DNA methylation, and studies of loci that escape inactivation. 47 references, 8 figures, 2 tables
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International Nuclear Information System (INIS)
Matsunaga, S.; Kawano, S.; Michimoto, T.; Higashiyama, T.; Nakao, S.; Sakai, A.; Kuroiwa, T.
1999-01-01
Silene latifolia has heteromorphic sex chromosomes, the X and Y chromosomes. The Y chromosome, which is thought to carry the male determining gene, was isolated by UV laser microdissection and amplified by degenerate oligonucleotide-primed PCR. In situ chromosome suppression of the amplified Y chromosome DNA in the presence of female genomic DNA as a competitor showed that the microdissected Y chromosome DNA did not specifically hybridize to the Y chromosome, but-hybridized to all chromosomes. This result suggests that the Y chromosome does not contain Y chromosome-enriched repetitive sequences. A repetitive sequence in the microdissected Y chromosome, RMY1, was isolated while screening repetitive sequences in the amplified Y chromosome. Part of the nucleotide sequence shared a similarity to that of X-43.1, which was isolated from microdissected X chromosomes. Since fluorescence in situ hybridization analysis with RMY1 demonstrated that RMY1 was localized at the ends of the chromosome, RMY1 may be a subtelomeric repetitive sequence. Regarding the sex chromosomes, RMY1 was detected at both ends of the X chromosome and at one end near the pseudoautosomal region of the Y chromosome. The different localization of RMY1 on the sex chromosomes provides a clue to the problem of how the sex chromosomes arose from autosomes
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DNA Catenation Maintains Structure of Human Metaphase Chromosomes
DEFF Research Database (Denmark)
L. V. Bauer, David; Marie, Rodolphe; Rasmussen, Kristian Hagsted
2012-01-01
Mitotic chromosome structure is pivotal to cell division but difficult to observe in fine detail using conventional methods. DNA catenation has been implicated in both sister chromatid cohesion and chromosome condensation, but has never been observed directly. We have used a lab-on-a-chip microfl...
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Genetic maps of polymorphic DNA loci on rat chromosome 1
Energy Technology Data Exchange (ETDEWEB)
Ding, Yan-Ping; Remmers, E.F.; Longman, R.E. [National Institutes of Health, Bethesda, MD (United States)] [and others
1996-09-01
Genetic linkage maps of loci defined by polymorphic DNA markers on rat chromosome 1 were constructed by genotyping F2 progeny of F344/N x LEW/N, BN/SsN x LEW/N, and DA/Bkl x F344/Hsd inbred rat strains. In total, 43 markers were mapped, of which 3 were restriction fragment length polymorphisms and the others were simple sequence length polymorphisms. Nineteen of these markers were associated with genes. Six markers for five genes, {gamma}-aminobutyric acid receptor {beta}3 (Gabrb3), syntaxin 2 (Stx2), adrenergic receptor {beta}3 (Gabrb3), syntaxin 2 (Stx2), adrenergic receptor {beta}1 (Adrb1), carcinoembryonic antigen gene family member 1 (Cgm1), and lipogenic protein S14 (Lpgp), and 20 anonymous loci were not previously reported. Thirteen gene loci (Myl2, Aldoa, Tnt, Igf2, Prkcg, Cgm4, Calm3, Cgm3, Psbp1, Sa, Hbb, Ins1, and Tcp1) were previously mapped. Comparative mapping analysis indicated that the large portion of rat chromosome 1 is homologous to mouse chromosome 7, although the homologous to mouse chromosome 7, although the homologs of two rat genes are located on mouse chromosomes 17 and 19. Homologs of the rat chromosome 1 genes that we mapped are located on human chromosomes 6, 10, 11, 12, 15, 16, and 19. 38 refs., 1 fig., 3 tabs.
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International Nuclear Information System (INIS)
Iliakis, George; Pantelias, G.E.; Okayasu, Ryuichi; Seaner, Robert
1987-01-01
The effect of 125 I-decay on cell lethality, and induction of chromosome and DNA damage, was studied in synchronous non-cycling, G 1 -phase CHO-cells. Neutral filter elution was used to assay repair of DNA double-strand breaks (dsbs), and premature chromosome condensation was used to assay repair of chromosome fragments and induction of ring chromosomes. The results indicate very little repair at the cell survival level (repair of PLD). At the DNA level an efficient repair of DNA dsbs was observed, with kinetics similar to those observed after exposure to X-rays. At the chromosome level a fast repair of prematurely condensed chromosome fragments was observed, with a concomitant increase in the number of ring chromosomes induced. The repair kinetics of chromosome fragments and DNA dsbs were very similar, suggesting that DNA dsbs may underlie chromosome fragmentation. (author)
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Chromosomal Bands Affected by Acute Oil Exposure and DNA Repair Errors
Zock, Jan-Paul; Giraldo, Jesús; Pozo-Rodríguez, Francisco; Espinosa, Ana; Rodríguez-Trigo, Gema; Verea, Hector; Castaño-Vinyals, Gemma; Gómez, Federico P.; Antó, Josep M.; Coll, Maria Dolors; Barberà, Joan Albert; Fuster, Carme
2013-01-01
Background In a previous study, we showed that individuals who had participated in oil clean-up tasks after the wreckage of the Prestige presented an increase of structural chromosomal alterations two years after the acute exposure had occurred. Other studies have also reported the presence of DNA damage during acute oil exposure, but little is known about the long term persistence of chromosomal alterations, which can be considered as a marker of cancer risk. Objectives We analyzed whether the breakpoints involved in chromosomal damage can help to assess the risk of cancer as well as to investigate their possible association with DNA repair efficiency. Methods Cytogenetic analyses were carried out on the same individuals of our previous study and DNA repair errors were assessed in cultures with aphidicolin. Results Three chromosomal bands, 2q21, 3q27 and 5q31, were most affected by acute oil exposure. The dysfunction in DNA repair mechanisms, expressed as chromosomal damage, was significantly higher in exposed-oil participants than in those not exposed (p= 0.016). Conclusion The present study shows that breaks in 2q21, 3q27 and 5q31 chromosomal bands, which are commonly involved in hematological cancer, could be considered useful genotoxic oil biomarkers. Moreover, breakages in these bands could induce chromosomal instability, which can explain the increased risk of cancer (leukemia and lymphomas) reported in chronically benzene-exposed individuals. In addition, it has been determined that the individuals who participated in clean-up of the oil spill presented an alteration of their DNA repair mechanisms two years after exposure. PMID:24303039
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International Nuclear Information System (INIS)
Lee, C.S.
1975-01-01
It is postulated that certain repetitious DNA components play a role in the recombination processes during chromosome rearrangements. When the distribution of silver grain densities after the in situ hybridization of repetitious DNA and the distribution of chromosome breaks due to x-irradiation are compared, a strong correlation is found for the euchromatic portion of the D. melanogaster salivary X chromosome. These observations justify the postulate above that certain repetitious DNA provides homologous regions in the DNA of broken chromosome ends necessary for proper recombinatory joining. (U.S.)
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Correspondence: chromosomal localization of uv-induced unscheduled DNA synthesis
International Nuclear Information System (INIS)
Berliner, J.; Mello, R.S.; Norman, A.
1976-01-01
We have measured the grain density - the number of grains per unit length - over the centromere and noncentromere regions of metaphase chromosomes in autoradiographs of human lymphocytes. When the chromosomes were labeled in G 0 by uv-induced unscheduled DNA synthesis, the grain density was two to four times larger over the centromere than over the noncentromere regions. When the labeling was done by scheduled DNA synthesis in S or unscheduled synthesis in M, the grain densities were approximately equal over both regions
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Analysis of repetitive DNA in chromosomes by flow cytometry
Brind'Amour, Julie; Lansdorp, Peter M.
We developed a flow cytometry method, chromosome flow fluorescence in situ hybridization (FISH), called CFF, to analyze repetitive DNA in chromosomes using FISH with directly labeled peptide nucleic acid (PNA) probes. We used CFF to measure the abundance of interstitial telomeric sequences in
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The DNA sequence of the human X chromosome
Ross, Mark T.; Grafham, Darren V.; Coffey, Alison J.; Scherer, Steven; McLay, Kirsten; Muzny, Donna; Platzer, Matthias; Howell, Gareth R.; Burrows, Christine; Bird, Christine P.; Frankish, Adam; Lovell, Frances L.; Howe, Kevin L.; Ashurst, Jennifer L.; Fulton, Robert S.; Sudbrak, Ralf; Wen, Gaiping; Jones, Matthew C.; Hurles, Matthew E.; Andrews, T. Daniel; Scott, Carol E.; Searle, Stephen; Ramser, Juliane; Whittaker, Adam; Deadman, Rebecca; Carter, Nigel P.; Hunt, Sarah E.; Chen, Rui; Cree, Andrew; Gunaratne, Preethi; Havlak, Paul; Hodgson, Anne; Metzker, Michael L.; Richards, Stephen; Scott, Graham; Steffen, David; Sodergren, Erica; Wheeler, David A.; Worley, Kim C.; Ainscough, Rachael; Ambrose, Kerrie D.; Ansari-Lari, M. Ali; Aradhya, Swaroop; Ashwell, Robert I. S.; Babbage, Anne K.; Bagguley, Claire L.; Ballabio, Andrea; Banerjee, Ruby; Barker, Gary E.; Barlow, Karen F.; Barrett, Ian P.; Bates, Karen N.; Beare, David M.; Beasley, Helen; Beasley, Oliver; Beck, Alfred; Bethel, Graeme; Blechschmidt, Karin; Brady, Nicola; Bray-Allen, Sarah; Bridgeman, Anne M.; Brown, Andrew J.; Brown, Mary J.; Bonnin, David; Bruford, Elspeth A.; Buhay, Christian; Burch, Paula; Burford, Deborah; Burgess, Joanne; Burrill, Wayne; Burton, John; Bye, Jackie M.; Carder, Carol; Carrel, Laura; Chako, Joseph; Chapman, Joanne C.; Chavez, Dean; Chen, Ellson; Chen, Guan; Chen, Yuan; Chen, Zhijian; Chinault, Craig; Ciccodicola, Alfredo; Clark, Sue Y.; Clarke, Graham; Clee, Chris M.; Clegg, Sheila; Clerc-Blankenburg, Kerstin; Clifford, Karen; Cobley, Vicky; Cole, Charlotte G.; Conquer, Jen S.; Corby, Nicole; Connor, Richard E.; David, Robert; Davies, Joy; Davis, Clay; Davis, John; Delgado, Oliver; DeShazo, Denise; Dhami, Pawandeep; Ding, Yan; Dinh, Huyen; Dodsworth, Steve; Draper, Heather; Dugan-Rocha, Shannon; Dunham, Andrew; Dunn, Matthew; Durbin, K. James; Dutta, Ireena; Eades, Tamsin; Ellwood, Matthew; Emery-Cohen, Alexandra; Errington, Helen; Evans, Kathryn L.; Faulkner, Louisa; Francis, Fiona; Frankland, John; Fraser, Audrey E.; Galgoczy, Petra; Gilbert, James; Gill, Rachel; Glöckner, Gernot; Gregory, Simon G.; Gribble, Susan; Griffiths, Coline; Grocock, Russell; Gu, Yanghong; Gwilliam, Rhian; Hamilton, Cerissa; Hart, Elizabeth A.; Hawes, Alicia; Heath, Paul D.; Heitmann, Katja; Hennig, Steffen; Hernandez, Judith; Hinzmann, Bernd; Ho, Sarah; Hoffs, Michael; Howden, Phillip J.; Huckle, Elizabeth J.; Hume, Jennifer; Hunt, Paul J.; Hunt, Adrienne R.; Isherwood, Judith; Jacob, Leni; Johnson, David; Jones, Sally; de Jong, Pieter J.; Joseph, Shirin S.; Keenan, Stephen; Kelly, Susan; Kershaw, Joanne K.; Khan, Ziad; Kioschis, Petra; Klages, Sven; Knights, Andrew J.; Kosiura, Anna; Kovar-Smith, Christie; Laird, Gavin K.; Langford, Cordelia; Lawlor, Stephanie; Leversha, Margaret; Lewis, Lora; Liu, Wen; Lloyd, Christine; Lloyd, David M.; Loulseged, Hermela; Loveland, Jane E.; Lovell, Jamieson D.; Lozado, Ryan; Lu, Jing; Lyne, Rachael; Ma, Jie; Maheshwari, Manjula; Matthews, Lucy H.; McDowall, Jennifer; McLaren, Stuart; McMurray, Amanda; Meidl, Patrick; Meitinger, Thomas; Milne, Sarah; Miner, George; Mistry, Shailesh L.; Morgan, Margaret; Morris, Sidney; Müller, Ines; Mullikin, James C.; Nguyen, Ngoc; Nordsiek, Gabriele; Nyakatura, Gerald; O’Dell, Christopher N.; Okwuonu, Geoffery; Palmer, Sophie; Pandian, Richard; Parker, David; Parrish, Julia; Pasternak, Shiran; Patel, Dina; Pearce, Alex V.; Pearson, Danita M.; Pelan, Sarah E.; Perez, Lesette; Porter, Keith M.; Ramsey, Yvonne; Reichwald, Kathrin; Rhodes, Susan; Ridler, Kerry A.; Schlessinger, David; Schueler, Mary G.; Sehra, Harminder K.; Shaw-Smith, Charles; Shen, Hua; Sheridan, Elizabeth M.; Shownkeen, Ratna; Skuce, Carl D.; Smith, Michelle L.; Sotheran, Elizabeth C.; Steingruber, Helen E.; Steward, Charles A.; Storey, Roy; Swann, R. Mark; Swarbreck, David; Tabor, Paul E.; Taudien, Stefan; Taylor, Tineace; Teague, Brian; Thomas, Karen; Thorpe, Andrea; Timms, Kirsten; Tracey, Alan; Trevanion, Steve; Tromans, Anthony C.; d’Urso, Michele; Verduzco, Daniel; Villasana, Donna; Waldron, Lenee; Wall, Melanie; Wang, Qiaoyan; Warren, James; Warry, Georgina L.; Wei, Xuehong; West, Anthony; Whitehead, Siobhan L.; Whiteley, Mathew N.; Wilkinson, Jane E.; Willey, David L.; Williams, Gabrielle; Williams, Leanne; Williamson, Angela; Williamson, Helen; Wilming, Laurens; Woodmansey, Rebecca L.; Wray, Paul W.; Yen, Jennifer; Zhang, Jingkun; Zhou, Jianling; Zoghbi, Huda; Zorilla, Sara; Buck, David; Reinhardt, Richard; Poustka, Annemarie; Rosenthal, André; Lehrach, Hans; Meindl, Alfons; Minx, Patrick J.; Hillier, LaDeana W.; Willard, Huntington F.; Wilson, Richard K.; Waterston, Robert H.; Rice, Catherine M.; Vaudin, Mark; Coulson, Alan; Nelson, David L.; Weinstock, George; Sulston, John E.; Durbin, Richard; Hubbard, Tim; Gibbs, Richard A.; Beck, Stephan; Rogers, Jane; Bentley, David R.
2009-01-01
The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence. PMID:15772651
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A device for extraction, manipulation and stretching of DNA from single human chromosomes
DEFF Research Database (Denmark)
Rasmussen, Kristian Hagsted; Marie, Rodolphe; Moresco, Jacob Lange
2011-01-01
by time-lapse imaging; pressure-driven flow was then used to shunt the chromosomal DNA package into a nanoslit. A long linear DNA strand (>1.3 Mbp) was seen to stretch out from the DNA package and along the length of the nanoslit. Delivery of DNA in its native metaphase chromosome package as well...
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Directory of Open Access Journals (Sweden)
Duílio M Z de A Silva
Full Text Available Supernumerary (B chromosomes have been shown to contain a wide variety of repetitive sequences. For this reason, fluorescent in situ hybridisation (FISH is a useful tool for ascertaining the origin of these genomic elements, especially when combined with painting from microdissected B chromosomes. In order to investigate the origin of B chromosomes in the fish species Astyanax paranae, these two approaches were used along with PCR amplification of specific DNA sequences obtained from the B chromosomes and its comparison with those residing in the A chromosomes. Remarkably, chromosome painting with the one-arm metacentric B chromosome probe showed hybridization signals on entire B chromosome, while FISH mapping revealed the presence of H1 histone and 18S rDNA genes symmetrically placed in both arms of the B chromosome. These results support the hypothesis that the B chromosome of A. paranae is an isochromosome. Additionally, the chromosome pairs Nos. 2 or 23 are considered the possible B chromosome ancestors since both contain syntenic H1 and 18S rRNA sequences. The analysis of DNA sequence fragments of the histone and rRNA genes obtained from the microdissected B chromosomes showed high similarity with those obtained from 0B individuals, which supports the intraspecific origin of B chromosomes in A. paranae. Finally, the population hereby analysed showed a female-biased B chromosome presence suggesting that B chromosomes in this species could influence sex determinism.
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Mapping of rDNA on the chromosomes of Eleusine species by fluorescence in situ hybridization.
Bisht, M S; Mukai, Y
2000-12-01
Mapping of rDNA sites on the chromosomes of four diploid and two tetraploid species of Eleusine has provided valuable information on genome relationship between the species. Presence of 18S-5.8S-26S rDNA on the largest pair of the chromosomes, location of 5S rDNA at four sites on two pairs of chromosomes and presence of 18S-5.8S-26S and 5S rDNA at same location on one pair of chromosomes have clearly differentiated E. multiflora from rest of the species of Eleusine. The two tetraploid species, E. coracana and E. africana have the same number of 18S-5.8S-26S and 5S rDNA sites and located at similar position on the chromosomes. Diploid species, E. indica, E. floccifolia and E. tristachya have the same 18S-5.8S-26S sites and location on the chromosomes which also resembled with the two pairs of 18S-5.8S-26S rDNA locations in tetraploid species, E. coracana and E. africana. The 5S rDNA sites on chromosomes of E. indica and E. floccifolia were also comparable to the 5S rDNA sites of E. africana and E. coracana. The similarity of the rDNA sites and their location on chromosomes in the three diploid and two polyploid species also supports the view that genome donors to tetraploid species may be from these diploid species.
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Huh, Yang Hoon; Cohen, Justin; Sherley, James L
2013-10-15
Immortal strands are the targeted chromosomal DNA strands of nonrandom sister chromatid segregation, a mitotic chromosome segregation pattern unique to asymmetrically self-renewing distributed stem cells (DSCs). By nonrandom segregation, immortal DNA strands become the oldest DNA strands in asymmetrically self-renewing DSCs. Nonrandom segregation of immortal DNA strands may limit DSC mutagenesis, preserve DSC fate, and contribute to DSC aging. The mechanisms responsible for specification and maintenance of immortal DNA strands are unknown. To discover clues to these mechanisms, we investigated the 5-methylcytosine and 5-hydroxymethylcytosine (5hmC) content on chromosomes in mouse hair follicle DSCs during nonrandom segregation. Although 5-methylcytosine content did not differ significantly, the relative content of 5hmC was significantly higher in chromosomes containing immortal DNA strands than in opposed mitotic chromosomes containing younger mortal DNA strands. The difference in relative 5hmC content was caused by the loss of 5hmC from mortal chromosomes. These findings implicate higher 5hmC as a specific molecular determinant of immortal DNA strand chromosomes. Because 5hmC is an intermediate during DNA demethylation, we propose a ten-eleven translocase enzyme mechanism for both the specification and maintenance of nonrandomly segregated immortal DNA strands. The proposed mechanism reveals a means by which DSCs "know" the generational age of immortal DNA strands. The mechanism is supported by molecular expression data and accounts for the selection of newly replicated DNA strands when nonrandom segregation is initiated. These mechanistic insights also provide a possible basis for another characteristic property of immortal DNA strands, their guanine ribonucleotide dependency.
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Chromosomal location of the human gene for DNA polymerase β
International Nuclear Information System (INIS)
McBride, O.W.; Zmudzka, B.Z.; Wilson, S.H.
1987-01-01
Inhibition studies indicate that DNA polymerase β has a synthetic role in DNA repair after exposure of mammalian cells to some types of DNA-damaging agents. The primary structure of the enzyme is highly conserved in vertebrates, and nearly full-length cDNAs for the enzyme were recently cloned from mammalian cDNA libraries. Southern blot analysis of DNA from a panel of human-rodent somatic cell hybrids, using portions of the cDNA as probe, indicates that the gene for human DNA polymerase β is single copy and located on the short arm or proximal long arm of chromosome 8 (8pter-8q22). A restriction fragment length polymorphism (RFLP) was detected in normal individuals by using a probe from the 5' end of the cDNA, and this RFLP probably is due to an insertion or duplication of DNA in 20-25% of the population. This restriction site can be used as one marker for chromosome 8 genetic linkage studies and for family studies of traits potentially involving this DNA repair gene
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Hu, Yufei; Chen, Zhiyu; Zhuang, Chuxiong; Huang, Jilei
2017-06-01
Transferred DNA (T-DNA) from Agrobacterium tumefaciens can be integrated into the plant genome. The double-stranded break repair (DSBR) pathway is a major model for T-DNA integration. From this model, we expect that two ends of a T-DNA molecule would invade into a single DNA double-stranded break (DSB) or independent DSBs in the plant genome. We call the later phenomenon a heterogeneous T-DNA integration, which has never been observed. In this work, we demonstrated it in an Arabidopsis T-DNA insertion mutant seb19. To resolve the chromosomal structural changes caused by T-DNA integration at both the nucleotide and chromosome levels, we performed inverse PCR, genome resequencing, fluorescence in situ hybridization and linkage analysis. We found, in seb19, a single T-DNA connected two different chromosomal loci and caused complex chromosomal rearrangements. The specific break-junction pattern in seb19 is consistent with the result of heterogeneous T-DNA integration but not of recombination between two T-DNA insertions. We demonstrated that, in seb19, heterogeneous T-DNA integration evoked a cascade of incorrect repair of seven DSBs on chromosomes 4 and 5, and then produced translocation, inversion, duplication and deletion. Heterogeneous T-DNA integration supports the DSBR model and suggests that two ends of a T-DNA molecule could be integrated into the plant genome independently. Our results also show a new origin of chromosomal abnormalities. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.
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Chromosome painting in plants.
Schubert, I.; Fransz, P.F.; Fuchs, J.; Jong, de J.H.
2001-01-01
The current 'state-of-art' as to chromosome painting in plants is reviewed. We define different situations described as painting so far: i) Genomic in situ hybridisation (GISH) with total genomic DNA to distinguish alien chromosomes on the basis of divergent dispersed repeats, ii) 'Chromosomal in
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Image cytometry: nuclear and chromosomal DNA quantification.
Carvalho, Carlos Roberto; Clarindo, Wellington Ronildo; Abreu, Isabella Santiago
2011-01-01
Image cytometry (ICM) associates microscopy, digital image and software technologies, and has been particularly useful in spatial and densitometric cytological analyses, such as DNA ploidy and DNA content measurements. Basically, ICM integrates methodologies of optical microscopy calibration, standard density filters, digital CCD camera, and image analysis softwares for quantitative applications. Apart from all system calibration and setup, cytological protocols must provide good slide preparations for efficient and reliable ICM analysis. In this chapter, procedures for ICM applications employed in our laboratory are described. Protocols shown here for human DNA ploidy determination and quantification of nuclear and chromosomal DNA content in plants could be used as described, or adapted for other studies.
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Coupling amplified DNA from flow-sorted chromosomes to high-density SNP mapping in barley
Directory of Open Access Journals (Sweden)
Bartoš Jan
2008-06-01
Full Text Available Abstract Background Flow cytometry facilitates sorting of single chromosomes and chromosome arms which can be used for targeted genome analysis. However, the recovery of microgram amounts of DNA needed for some assays requires sorting of millions of chromosomes which is laborious and time consuming. Yet, many genomic applications such as development of genetic maps or physical mapping do not require large DNA fragments. In such cases time-consuming de novo sorting can be minimized by utilizing whole-genome amplification. Results Here we report a protocol optimized in barley including amplification of DNA from only ten thousand chromosomes, which can be isolated in less than one hour. Flow-sorted chromosomes were treated with proteinase K and amplified using Phi29 multiple displacement amplification (MDA. Overnight amplification in a 20-microlitre reaction produced 3.7 – 5.7 micrograms DNA with a majority of products between 5 and 30 kb. To determine the purity of sorted fractions and potential amplification bias we used quantitative PCR for specific genes on each chromosome. To extend the analysis to a whole genome level we performed an oligonucleotide pool assay (OPA for interrogation of 1524 loci, of which 1153 loci had known genetic map positions. Analysis of unamplified genomic DNA of barley cv. Akcent using this OPA resulted in 1426 markers with present calls. Comparison with three replicates of amplified genomic DNA revealed >99% concordance. DNA samples from amplified chromosome 1H and a fraction containing chromosomes 2H – 7H were examined. In addition to loci with known map positions, 349 loci with unknown map positions were included. Based on this analysis 40 new loci were mapped to 1H. Conclusion The results indicate a significant potential of using this approach for physical mapping. Moreover, the study showed that multiple displacement amplification of flow-sorted chromosomes is highly efficient and representative which
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Plantago lagopus B Chromosome Is Enriched in 5S rDNA-Derived Satellite DNA
Czech Academy of Sciences Publication Activity Database
Kumke, K.; Macas, Jiří; Fuchs, J.; Altschmied, L.; Kour, J.; Dhar, M.K.; Houben, A.
2016-01-01
Roč. 148, č. 1 (2016), s. 68-73 ISSN 1424-8581 R&D Projects: GA ČR GBP501/12/G090 Institutional support: RVO:60077344 Keywords : Polymorhpic A chromosome segment * Satellite repeat * Supernumerary chromosome * 5S rDNA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.354, year: 2016
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Bernardino, Andrezza C S; Cabral-de-Mello, Diogo C; Machado, Carolina B; Palacios-Gimenez, Octavio M; Santos, Neide; Loreto, Vilma
2017-01-01
B chromosomes, extra elements present in the karyotypes of some eukaryote species, have been described in the grasshopper Xyleus discoideus angulatus. Although some studies have proposed an autosomal origin of the B chromosome in X. d. angulatus, little is known about its repetitive DNA composition and evolutionary dynamics. The aim of the present work was to shed light on the B chromosome evolution in X. d. angulatus by cytogenetic analysis of 27 populations from Pernambuco and Ceará states (Brazil). The frequency of B chromosomes in the different populations was determined, and chromosome measurements and fluorescence in situ hybridization (FISH) with C0t-DNA and telomeric and B chromosome sequences were performed in cells from B-carrying individuals. The results revealed variations in B chromosome prevalence among the populations and showed that some B chromosomes were smaller in certain populations. FISH produced similar patterns for the C0t-DNA probe in all hybridized individuals, whereas telomeric and B chromosome probes, obtained by microdissection, exhibited variations in their distribution. These results indicate the presence of 3 morphotypes of B chromosomes in X. d. angulatus, with variation in repetitive DNA composition during their evolution. In this species, B chromosomes have an intraspecific origin and probably arose from the pericentromeric region of A chromosomes. © 2017 S. Karger AG, Basel.
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DistAMo: A web-based tool to characterize DNA-motif distribution on bacterial chromosomes
Directory of Open Access Journals (Sweden)
Patrick eSobetzko
2016-03-01
Full Text Available Short DNA motifs are involved in a multitude of functions such as for example chromosome segregation, DNA replication or mismatch repair. Distribution of such motifs is often not random and the specific chromosomal pattern relates to the respective motif function. Computational approaches which quantitatively assess such chromosomal motif patterns are necessary. Here we present a new computer tool DistAMo (Distribution Analysis of DNA Motifs. The algorithm uses codon redundancy to calculate the relative abundance of short DNA motifs from single genes to entire chromosomes. Comparative genomics analyses of the GATC-motif distribution in γ-proteobacterial genomes using DistAMo revealed that (i genes beside the replication origin are enriched in GATCs, (ii genome-wide GATC distribution follows a distinct pattern and (iii genes involved in DNA replication and repair are enriched in GATCs. These features are specific for bacterial chromosomes encoding a Dam methyltransferase. The new software is available as a stand-alone or as an easy-to-use web-based server version at http://www.computational.bio.uni-giessen.de/distamo.
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Huang, D; Wu, W; Zhou, Y; Hu, Z; Lu, L
2004-05-01
Construction of single chromosomal DNA libraries by means of chromosome microdissection and microcloning will be useful for genomic research, especially for those species that have not been extensively studied genetically. Application of the technology of microdissection and microcloning to woody fruit plants has not been reported hitherto, largely due to the generally small sizes of metaphase chromosomes and the difficulty of chromosome preparation. The present study was performed to establish a method for single chromosome microdissection and microcloning in woody fruit species using pomelo as a model. The standard karyotype of a pomelo cultivar ( Citrus grandis cv. Guanxi) was established based on 20 prometaphase photomicrographs. According to the standard karyotype, chromosome 1 was identified and isolated with fine glass microneedles controlled by a micromanipulator. DNA fragments ranging from 0.3 kb to 2 kb were acquired from the isolated single chromosome 1 via two rounds of PCR mediated by Sau3A linker adaptors and then cloned into T-easy vectors to generate a DNA library of chromosome 1. Approximately 30,000 recombinant clones were obtained. Evaluation based on 108 randomly selected clones showed that the sizes of the cloned inserts varied from 0.5 kb to 1.5 kb with an average of 860 bp. Our research suggests that microdissection and microcloning of single small chromosomes in woody plants is feasible.
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Directory of Open Access Journals (Sweden)
Mauro Nirchio
2007-01-01
Full Text Available The karyotype and chromosomal characteristics of the characid fish Triportheus venezuelensis were investigated using differential staining techniques (C-banding, Ag-NOR staining and fluorescent in situ hybridization (FISH with an 18S rDNA probe. The diploid chromosome number (2n = 52, karyotype composition and sex chromosome determination system of the ZZ/ZW type were the same as previously described in other species of the genus Triportheus. However, extensive variation regarding nucleolus organizer regions (NOR different from other species was observed. 18S rDNA sequences were distributed on nine chromosome pairs, but the number of chromosomes with Ag-NORs was usually lower, reaching a maximum of four chromosomes. When sequential staining experiments were performed, it was demonstrated that: 1. active NORs usually corresponded to segments with 18S rDNA genes identified in FISH experiments; 2. several 18S rDNA sequences were not silver-stained, suggesting that they do not correspond to active NORs; and 3. some chromosomes with silver-stained regions did not display any 18S rDNA signals. These findings characterize an extensive polymorphism associated with the NOR-bearing chromosomes of T. venezuelensis and emphasize the importance of combining traditional and molecular techniques in chromosome studies.
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Śliwińska-Jewsiewicka, A; Kuciński, M; Kirtiklis, L; Dobosz, S; Ocalewicz, K; Jankun, Malgorzata
2015-08-01
Brook trout Salvelinus fontinalis (Mitchill, 1814) chromosomes have been analyzed using conventional and molecular cytogenetic techniques enabling characteristics and chromosomal location of heterochromatin, nucleolus organizer regions (NORs), ribosomal RNA-encoding genes and telomeric DNA sequences. The C-banding and chromosome digestion with the restriction endonucleases demonstrated distribution and heterogeneity of the heterochromatin in the brook trout genome. DNA sequences of the ribosomal RNA genes, namely the nucleolus-forming 28S (major) and non-nucleolus-forming 5S (minor) rDNAs, were physically mapped using fluorescence in situ hybridization (FISH) and primed in situ labelling. The minor rDNA locus was located on the subtelo-acrocentric chromosome pair No. 9, whereas the major rDNA loci were dispersed on 14 chromosome pairs, showing a considerable inter-individual variation in the number and location. The major and minor rDNA loci were located at different chromosomes. Multichromosomal location (3-6 sites) of the NORs was demonstrated by silver nitrate (AgNO3) impregnation. All Ag-positive i.e. active NORs corresponded to the GC-rich blocks of heterochromatin. FISH with telomeric probe showed the presence of the interstitial telomeric site (ITS) adjacent to the NOR/28S rDNA site on the chromosome 11. This ITS was presumably remnant of the chromosome rearrangement(s) leading to the genomic redistribution of the rDNA sequences. Comparative analysis of the cytogenetic data among several related salmonid species confirmed huge variation in the number and the chromosomal location of rRNA gene clusters in the Salvelinus genome.
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Radiation induced chromosome aberrations and interphase DNA geometry
International Nuclear Information System (INIS)
Nasazzi, N.; Di Giorgio, M.; Otero, D.
1995-01-01
Ionizing radiation induces DNA double strand breaks (DSBs) and their interaction and illegitimate recombination produces chromosome aberrations. Stable chromosome aberrations comprise inter-chromosomal events (translocations) and intra-chromosomal events (inversions). Assuming DSBs induction and interaction is completely random and neglecting proximity effects, the expected ratio of translocations to inversions is F=86, based on chromosome arm lengths. We analyzed the number of translocations and inversions using G-banding, in 16 lymphocyte cultures from blood samples acutely irradiated with γ-rays (dose range: 0.5Gy-3Gy). Our results give F=13.5, significantly smaller than F=86. Literature data show similar small F values but strongly spread. The excess of inversions could be explained by a 'proximity effect', it means that more proximate DSBs have an extra probability of interaction. Therefore, it is possible to postulate a special chromosome arrangement during irradiation and the subsequent interval. We propose a model where individual chromosomes show spherical confinement with some degree of overlapping and DSBs induction proportional to cross section. We assume a DSBs interaction probability function with cut-off length = 1 μ. We propose that large spread in F data could be due to temporal variation in overlapping and spatial chromosome confinement. (author). 14 refs
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Dog Y chromosomal DNA sequence: identification, sequencing and SNP discovery
Directory of Open Access Journals (Sweden)
Kirkness Ewen
2006-10-01
Full Text Available Abstract Background Population genetic studies of dogs have so far mainly been based on analysis of mitochondrial DNA, describing only the history of female dogs. To get a picture of the male history, as well as a second independent marker, there is a need for studies of biallelic Y-chromosome polymorphisms. However, there are no biallelic polymorphisms reported, and only 3200 bp of non-repetitive dog Y-chromosome sequence deposited in GenBank, necessitating the identification of dog Y chromosome sequence and the search for polymorphisms therein. The genome has been only partially sequenced for one male dog, disallowing mapping of the sequence into specific chromosomes. However, by comparing the male genome sequence to the complete female dog genome sequence, candidate Y-chromosome sequence may be identified by exclusion. Results The male dog genome sequence was analysed by Blast search against the human genome to identify sequences with a best match to the human Y chromosome and to the female dog genome to identify those absent in the female genome. Candidate sequences were then tested for male specificity by PCR of five male and five female dogs. 32 sequences from the male genome, with a total length of 24 kbp, were identified as male specific, based on a match to the human Y chromosome, absence in the female dog genome and male specific PCR results. 14437 bp were then sequenced for 10 male dogs originating from Europe, Southwest Asia, Siberia, East Asia, Africa and America. Nine haplotypes were found, which were defined by 14 substitutions. The genetic distance between the haplotypes indicates that they originate from at least five wolf haplotypes. There was no obvious trend in the geographic distribution of the haplotypes. Conclusion We have identified 24159 bp of dog Y-chromosome sequence to be used for population genetic studies. We sequenced 14437 bp in a worldwide collection of dogs, identifying 14 SNPs for future SNP analyses, and
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Bueno, Danilo; Palacios-Gimenez, Octavio Manuel; Martí, Dardo Andrea; Mariguela, Tatiane Casagrande; Cabral-de-Mello, Diogo Cavalcanti
2016-08-01
The 5S ribosomal DNA (rDNA) sequences are subject of dynamic evolution at chromosomal and molecular levels, evolving through concerted and/or birth-and-death fashion. Among grasshoppers, the chromosomal location for this sequence was established for some species, but little molecular information was obtained to infer evolutionary patterns. Here, we integrated data from chromosomal and nucleotide sequence analysis for 5S rDNA in two Abracris species aiming to identify evolutionary dynamics. For both species, two arrays were identified, a larger sequence (named type-I) that consisted of the entire 5S rDNA gene plus NTS (non-transcribed spacer) and a smaller (named type-II) with truncated 5S rDNA gene plus short NTS that was considered a pseudogene. For type-I sequences, the gene corresponding region contained the internal control region and poly-T motif and the NTS presented partial transposable elements. Between the species, nucleotide differences for type-I were noticed, while type-II was identical, suggesting pseudogenization in a common ancestor. At chromosomal point to view, the type-II was placed in one bivalent, while type-I occurred in multiple copies in distinct chromosomes. In Abracris, the evolution of 5S rDNA was apparently influenced by the chromosomal distribution of clusters (single or multiple location), resulting in a mixed mechanism integrating concerted and birth-and-death evolution depending on the unit.
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Directory of Open Access Journals (Sweden)
Jaime Gosálvez
2013-02-01
Full Text Available We aimed to evaluate the association between the progressive stages of cervical neoplasia and DNA damage in 5-bp classical satellite DNA sequences from chromosome-1 in cervical epithelium and in peripheral blood lymphocytes using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH. A hospital-based unmatched case-control study was conducted in 2011 with a sample of 30 women grouped according to disease stage and selected according to histological diagnosis; 10 with low-grade squamous intraepithelial lesions (LG-SIL, 10 with high-grade SIL (HG-SIL, and 10 with no cervical lesions, from the Unidad Medica de Alta Especialidad of The Mexican Social Security Institute, IMSS, Mexico. Specific chromosome damage levels in 5-bp classical satellite DNA sequences from chromosome-1 were evaluated in cervical epithelium and peripheral blood lymphocytes using the DBD-FISH technique. Whole-genome DNA hybridization was used as a reference for the level of damage. Results of Kruskal-Wallis test showed a significant increase according to neoplastic development in both tissues. The instability of 5-bp classical satellite DNA sequences from chromosome-1 was evidenced using chromosome-orientation FISH. In conclusion, we suggest that the progression to malignant transformation involves an increase in the instability of 5-bp classical satellite DNA sequences from chromosome-1.
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Cortés-Gutiérrez, Elva I; Ortíz-Hernández, Brenda L; Dávila-Rodríguez, Martha I; Cerda-Flores, Ricardo M; Fernández, José Luis; López-Fernández, Carmen; Gosálvez, Jaime
2013-02-19
We aimed to evaluate the association between the progressive stages of cervical neoplasia and DNA damage in 5-bp classical satellite DNA sequences from chromosome-1 in cervical epithelium and in peripheral blood lymphocytes using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH). A hospital-based unmatched case-control study was conducted in 2011 with a sample of 30 women grouped according to disease stage and selected according to histological diagnosis; 10 with low-grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 with no cervical lesions, from the Unidad Medica de Alta Especialidad of The Mexican Social Security Institute, IMSS, Mexico. Specific chromosome damage levels in 5-bp classical satellite DNA sequences from chromosome-1 were evaluated in cervical epithelium and peripheral blood lymphocytes using the DBD-FISH technique. Whole-genome DNA hybridization was used as a reference for the level of damage. Results of Kruskal-Wallis test showed a significant increase according to neoplastic development in both tissues. The instability of 5-bp classical satellite DNA sequences from chromosome-1 was evidenced using chromosome-orientation FISH. In conclusion, we suggest that the progression to malignant transformation involves an increase in the instability of 5-bp classical satellite DNA sequences from chromosome-1.
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Directory of Open Access Journals (Sweden)
Stougaard Magnus
2007-11-01
represented by metaphase spreads. An optimized protocol for chromosome spreading in diagnostic well-slides was used for the detection of circularized padlock probes amplified by target primed rolling circle DNA synthesis from condensed metaphase chromosomes. We were able to detect a 40 nucleotide sequence in the male specific repetitive satellite I sequence and a 32 nucleotide sequence in the repetitive kringle IV domain in the apolipoprotein(a gene. Our overall conclusion is that whilst this type of reaction indeed can be brought to work on nuclear genomes, including metaphase chromosomes, the total efficiency of this multistep reaction is at present relatively low (1–10% of target sites picked up, meaning that it is best suited for the detection of targets that exist in multiple copies per cell. Changing this will require substantial efforts to systematically increase the efficiency in each step.
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Integration of hepatitis B virus DNA in chromosome-specific satellite sequences
International Nuclear Information System (INIS)
Shaul, Y.; Garcia, P.D.; Schonberg, S.; Rutter, W.J.
1986-01-01
The authors previously reported the cloning and detailed analysis of the integrated hepatitis B virus sequences in a human hepatoma cell line. They report here the integration of at least one of hepatitis B virus at human satellite DNA sequences. The majority of the cellular sequences identified by this satellite were organized as a multimeric composition of a 0.6-kilobase EcoRI fragment. This clone hybridized in situ almost exclusively to the centromeric heterochromatin of chromosomes 1 and 16 and to a lower extent to chromosome 2 and to the heterochromatic region of the Y chromosome. The immediate flanking host sequence appeared as a hierarchy of repeating units which were almost identical to a previously reported human satellite III DNA sequence
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Non-Random Distribution of 5S rDNA Sites and Its Association with 45S rDNA in Plant Chromosomes.
Roa, Fernando; Guerra, Marcelo
2015-01-01
5S and 45S rDNA sites are the best mapped chromosome regions in eukaryotic chromosomes. In this work, a database was built gathering information about the position and number of 5S rDNA sites in 784 plant species, aiming to identify patterns of distribution along the chromosomes and its correlation with the position of 45S rDNA sites. Data revealed that in most karyotypes (54.5%, including polyploids) two 5S rDNA sites (a single pair) are present, with 58.7% of all sites occurring in the short arm, mainly in the proximal region. In karyotypes of angiosperms with only 1 pair of sites (single sites) they are mostly found in the proximal region (52.0%), whereas in karyotypes with multiple sites the location varies according to the average chromosome size. Karyotypes with multiple sites and small chromosomes (6 µm) more commonly show terminal or interstitial sites. In species with holokinetic chromosomes, the modal value of sites per karyotype was also 2, but they were found mainly in a terminal position. Adjacent 5S and 45S rDNA sites were often found in the short arm, reflecting the preferential distribution of both sites in this arm. The high frequency of genera with at least 1 species with adjacent 5S and 45S sites reveals that this association appeared several times during angiosperm evolution, but it has been maintained only rarely as the dominant array in plant genera. © 2015 S. Karger AG, Basel.
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U1 snDNA clusters in grasshoppers: chromosomal dynamics and genomic organization
Anjos, A; Ruiz-Ruano, F J; Camacho, J P M; Loreto, V; Cabrero, J; de Souza, M J; Cabral-de-Mello, D C
2015-01-01
The spliceosome, constituted by a protein set associated with small nuclear RNA (snRNA), is responsible for mRNA maturation through intron removal. Among snRNA genes, U1 is generally a conserved repetitive sequence. To unveil the chromosomal/genomic dynamics of this multigene family in grasshoppers, we mapped U1 genes by fluorescence in situ hybridization in 70 species belonging to the families Proscopiidae, Pyrgomorphidae, Ommexechidae, Romaleidae and Acrididae. Evident clusters were observed in all species, indicating that, at least, some U1 repeats are tandemly arrayed. High conservation was observed in the first four families, with most species carrying a single U1 cluster, frequently located in the third or fourth longest autosome. By contrast, extensive variation was observed among Acrididae, from a single chromosome pair carrying U1 to all chromosome pairs carrying it, with occasional occurrence of two or more clusters in the same chromosome. DNA sequence analysis in Eyprepocnemis plorans (species carrying U1 clusters on seven different chromosome pairs) and Locusta migratoria (carrying U1 in a single chromosome pair) supported the coexistence of functional and pseudogenic lineages. One of these pseudogenic lineages was truncated in the same nucleotide position in both species, suggesting that it was present in a common ancestor to both species. At least in E. plorans, this U1 snDNA pseudogenic lineage was associated with 5S rDNA and short interspersed elements (SINE)-like mobile elements. Given that we conclude in grasshoppers that the U1 snDNA had evolved under the birth-and-death model and that its intragenomic spread might be related with mobile elements. PMID:25248465
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Atomic force microscopy on chromosomes, chromatin and DNA: a review.
Kalle, Wouter; Strappe, Padraig
2012-12-01
The purpose of this review is to discuss the achievements and progress that has been made in the use of atomic force microscopy in DNA related research in the last 25 years. For this review DNA related research is split up in chromosomal-, chromatin- and DNA focused research to achieve a logical flow from large- to smaller structures. The focus of this review is not only on the AFM as imaging tool but also on the AFM as measuring tool using force spectroscopy, as therein lays its greatest advantage and future. The amazing technological and experimental progress that has been made during the last 25 years is too extensive to fully cover in this review but some key developments and experiments have been described to give an overview of the evolution of AFM use from 'imaging tool' to 'measurement tool' on chromosomes, chromatin and DNA. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.
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Rearrangement of a common cellular DNA domain on chromosome 4 in human primary liver tumors
International Nuclear Information System (INIS)
Pasquinelli, C.; Garreau, F.; Bougueleret, L.; Cariani, E.; Thiers, V.; Croissant, O.; Hadchouel, M.; Tiollais, P.; Brechot, C.; Grzeschik, K.H.
1988-01-01
Hepatitis B virus (HBV) DNA integration has been shown to occur frequently in human hepatocellular carcinomas. The authors have investigated whether common cellular DNA domains might be rearranged, possibly by HBV integration, in human primary liver tumors. Unique cellular DNA sequences adjacent to an HBV integration site were isolated from a patient with hepatitis B surface antigen-positive hepatocellular carcinoma. These probes detected rearrangement of this cellular region of chromosomal DNA in 3 of 50 additional primary liver tumors studied. Of these three tumor samples, two contained HBV DNA, without an apparent link between the viral DNA and the rearranged allele; HBV DNA sequences were not detected in the third tumor sample. By use of a panel of somatic cell hybrids, these unique cellular DNA sequences were shown to be located on chromosome 4. Therefore, this region of chromosomal DNA might be implicated in the formation of different tumors at one step of liver cell transformation, possible related to HBV integration
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Bugno-Poniewierska, Monika; Solek, Przemysław; Wronski, Mariusz; Potocki, Leszek; Jezewska-Witkowska, Grażyna; Wnuk, Maciej
2014-12-01
The molecular structure of B chromosomes (Bs) is relatively well studied. Previous research demonstrates that Bs of various species usually contain two types of repetitive DNA sequences, satellite DNA and ribosomal DNA, but Bs also contain genes encoding histone proteins and many others. However, many questions remain regarding the origin and function of these chromosomes. Here, we focused on the comparative cytogenetic characteristics of the red fox and Chinese raccoon dog B chromosomes with particular attention to the distribution of repetitive DNA sequences and their methylation status. We confirmed that the small Bs of the red fox show a typical fluorescent telomeric distal signal, whereas medium-sized Bs of the Chinese raccoon dog were characterized by clusters of telomeric sequences along their length. We also found different DNA methylation patterns for the B chromosomes of both species. Therefore, we concluded that DNA methylation may maintain the transcriptional inactivation of DNA sequences localized to B chromosomes and may prevent genetic unbalancing and several negative phenotypic effects. © 2014 The Authors.
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Feng, Wenyi; Di Rienzi, Sara C; Raghuraman, M K; Brewer, Bonita J
2011-10-01
Chromosome breakage as a result of replication stress has been hypothesized to be the direct consequence of defective replication fork progression, or "collapsed" replication forks. However, direct and genome-wide evidence that collapsed replication forks give rise to chromosome breakage is still lacking. Previously we showed that a yeast replication checkpoint mutant mec1-1, after transient exposure to replication impediment imposed by hydroxyurea (HU), failed to complete DNA replication, accumulated single-stranded DNA (ssDNA) at the replication forks, and fragmented its chromosomes. In this study, by following replication fork progression genome-wide via ssDNA detection and by direct mapping of chromosome breakage after HU exposure, we have tested the hypothesis that the chromosome breakage in mec1 cells occurs at collapsed replication forks. We demonstrate that sites of chromosome breakage indeed correlate with replication fork locations. Moreover, ssDNA can be detected prior to chromosome breakage, suggesting that ssDNA accumulation is the common precursor to double strand breaks at collapsed replication forks.
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DNA amount of X and B chromosomes in the grasshoppers Eyprepocnemis plorans and Locusta migratoria.
Ruiz-Ruano, F J; Ruiz-Estévez, M; Rodríguez-Pérez, J; López-Pino, J L; Cabrero, J; Camacho, J P M
2011-01-01
We analyzed the DNA amount in X and B chromosomes of 2 XX/X0 grasshopper species (Eyprepocnemis plorans and Locusta migratoria), by means of Feulgen image analysis densitometry (FIAD), using previous estimates in L. migratoria as standard (5.89 pg). We first analyzed spermatids of 0B males and found a bimodal distribution of integrated optical densities (IODs), suggesting that one peak corresponded to +X and the other to -X spermatids. The difference between the 2 peaks corresponded to the X chromosome DNA amount, which was 1.28 pg in E. plorans and 0.80 pg in L. migratoria. In addition, the +X peak in E. plorans gave an estimate of the C-value in this species (10.39 pg). We next analyzed diplotene cells from 1B males in E. plorans and +B males in L. migratoria (a species where Bs are mitotically unstable and no integer B number can be defined for an individual) and measured B chromosome IOD relative to X chromosome IOD, within the same cell, taking advantage of the similar degree of condensation for both positively heteropycnotic chromosomes at this meiotic stage. From this proportion, we estimated the DNA amount for 3 different B chromosome variants found in individuals from 3 E. plorans Spanish populations (0.54 pg for B1 from Saladares, 0.51 pg for B2 from Salobreña and 0.64 for B24 from Torrox). Likewise, we estimated the DNA amount of the B chromosome in L. migratoria to be 0.15 pg. To automate measurements, we wrote a GPL3 licensed Python program (pyFIA). We discuss the utility of the present approach for estimating X and B chromosome DNA amount in a variety of situations, and the meaning of the DNA amount estimates for X and B chromosomes in these 2 species. Copyright © 2011 S. Karger AG, Basel.
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Directory of Open Access Journals (Sweden)
Gomes de Oliveira Sarah
2012-11-01
Full Text Available Abstract Background To contribute to the knowledge of coleopteran cytogenetics, especially with respect to the genomic content of B chromosomes, we analyzed the composition and organization of repetitive DNA sequences in the Coprophanaeus cyanescens karyotype. We used conventional staining and the application of fluorescence in situ hybridization (FISH mapping using as probes C0t-1 DNA fraction, the 18S and 5S rRNA genes, and the LOA-like non-LTR transposable element (TE. Results The conventional analysis detected 3 individuals (among 50 analyzed carrying one small metacentric and mitotically unstable B chromosome. The FISH analysis revealed a pericentromeric block of C0t-1 DNA in the B chromosome but no 18S or 5S rDNA clusters in this extra element. Using the LOA-like TE probe, the FISH analysis revealed large pericentromeric blocks in eight autosomal bivalents and in the B chromosome, and a pericentromeric block extending to the short arm in one autosomal pair. No positive hybridization signal was observed for the LOA-like element in the sex chromosomes. Conclusions The results indicate that the origin of the B chromosome is associated with the autosomal elements, as demonstrated by the hybridization with C0t-1 DNA and the LOA-like TE. The present study is the first report on the cytogenetic mapping of a TE in coleopteran chromosomes. These TEs could have been involved in the origin and evolution of the B chromosome in C. cyanescens.
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Multiple DNA binding proteins contribute to timing of chromosome replication in E. coli
DEFF Research Database (Denmark)
Riber, Leise; Frimodt-Møller, Jakob; Charbon, Godefroid
2016-01-01
Chromosome replication in Escherichia coli is initiated from a single origin, oriC. Initiation involves a number of DNA binding proteins, but only DnaA is essential and specific for the initiation process. DnaA is an AAA+ protein that binds both ATP and ADP with similar high affinities. Dna...... replication is initiated, or the time window in which all origins present in a single cell are initiated, i.e. initiation synchrony, or both. Overall, these DNA binding proteins modulate the initiation frequency from oriC by: (i) binding directly to oriC to affect DnaA binding, (ii) altering the DNA topology...... in or around oriC, (iii) altering the nucleotide bound status of DnaA by interacting with non-coding chromosomal sequences, distant from oriC, that are important for DnaA activity. Thus, although DnaA is the key protein for initiation of replication, other DNA-binding proteins act not only on ori...
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The DNA sequence and biology of human chromosome 19
Energy Technology Data Exchange (ETDEWEB)
Grimwood, J; Gordon, L A; Olsen, A; Terry, A; Schmutz, J; Lamerdin, J; Hellsten, U; Goodstein, D; Couronne, O; Tran-Gyamfi, M
2004-04-06
Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high GC content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9% of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in Mendelian disorders, including familial hypercholesterolemia and insulin-resistant diabetes. Nearly one quarter of these genes belong to tandemly arranged families, encompassing more than 25% of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, and segments of coding and non-coding conservation with the distant fish species Takifugu.
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Guerra, Marcelo; García, Miguel A
2004-02-01
Cuscuta is a widely distributed genus of holoparasitic plants. Holocentric chromosomes have been reported only in species of one of its subgenera (Cuscuta subg. Cuscuta). In this work, a representative of this subgenus, Cuscuta approximata, was investigated looking for its mitotic and meiotic chromosome behaviour and the heterochromatin distribution. The mitotic chromosomes showed neither primary constriction nor Rabl orientation whereas the meiotic ones exhibited the typical quadripartite structure characteristic of holocentrics, supporting the assumption of holocentric chromosomes as a synapomorphy of Cuscuta subg. Cuscuta. Chromosomes and interphase nuclei displayed many heterochromatic blocks that stained deeply with hematoxylin, 4',6-diamidino-2-phenylindole (DAPI), or after C banding. The banded karyotype showed terminal or subterminal bands in all chromosomes and central bands in some of them. The single pair of 45S rDNA sites was observed at the end of the largest chromosome pair, close to a DAPI band and a 5S rDNA site. Two other 5S rDNA site pairs were found, both closely associated with DAPI bands. The noteworthy giant nuclei of glandular cells of petals and ovary wall exhibited large chromocentres typical of polytenic nuclei. The chromosomal location of heterochromatin and rDNA sites and the structure of the endoreplicated nuclei of C. approximata seemed to be similar to those known in monocentric nuclei, suggesting that centromeric organization has little or no effect on chromatin organization.
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Disruption of Maternal DNA Repair Increases Sperm-DerivedChromosomal Aberrations
Energy Technology Data Exchange (ETDEWEB)
Marchetti, Francesco; Essers, Jeroun; Kanaar, Roland; Wyrobek,Andrew J.
2007-02-07
The final weeks of male germ cell differentiation occur in aDNA repair-deficient environment and normal development depends on theability of the egg to repair DNA damage in the fertilizing sperm. Geneticdisruption of maternal DNA double-strand break repair pathways in micesignificantly increased the frequency of zygotes with chromosomalstructural aberrations after paternal exposure to ionizing radiation.These findings demonstrate that radiation-induced DNA sperm lesions arerepaired after fertilization by maternal factors and suggest that geneticvariation in maternal DNA repair can modulate the risk of early pregnancylosses and of children with chromosomal aberrations of paternalorigin.
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Role of DNA polymerase α in chromosomal aberration production by ionizing radiation
International Nuclear Information System (INIS)
Bender, M.A.
1983-01-01
Aphidicolin is a tetracyclic diterpinoid fungal antibiotic which inhibits DNA synthesis in eukaryotic cells by interfering specifically with DNA polymerase α, apparently by binding to and inactivating the DNA-polymerase α complex. We have shown that aphidicolin, like other inhibitors of DNA synthesis, both induces chromosomal aberrations in human peripheral lymphocytes, and, as a post-treatment, interacts synergistically with x rays to produce greatly enhanced aberration yields. The present experiments explore the effects of aphidicolin in human lymphocytes in the post-DNA-synthetic G 2 phase of the cell cycle. These experiments utilized labeling with tritiated thymidine to positively identify cells in the S phase at the time of treatment, and used serial colcemid collections and fixations to determine aberration yields over as much of the G 2 phase as feasible. Because DNA polymerase α is the only DNA synthetic or repair enzyme known to be affected by aphidicolin, we infer that this enzyme is directly involved in the repair of DNA lesions which can result in visible chromosomal aberrations. (DT)
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Chromosomal Replication Complexity: A Novel DNA Metrics and Genome Instability Factor.
Directory of Open Access Journals (Sweden)
Andrei Kuzminov
2016-10-01
Full Text Available As the ratio of the copy number of the most replicated to the unreplicated regions in the same chromosome, the definition of chromosomal replication complexity (CRC appears to leave little room for variation, being either two during S-phase or one otherwise. However, bacteria dividing faster than they replicate their chromosome spike CRC to four and even eight. A recent experimental inquiry about the limits of CRC in Escherichia coli revealed two major reasons to avoid elevating it further: (i increased chromosomal fragmentation and (ii complications with subsequent double-strand break repair. Remarkably, examples of stable elevated CRC in eukaryotic chromosomes are well known under various terms like "differential replication," "underreplication," "DNA puffs," "onion-skin replication," or "re-replication" and highlight the phenomenon of static replication fork (sRF. To accurately describe the resulting "amplification by overinitiation," I propose a new term: "replification" (subchromosomal overreplication. In both prokaryotes and eukaryotes, replification, via sRF processing, causes double-strand DNA breaks and, with their repair elevating chromosomal rearrangements, represents a novel genome instability factor. I suggest how static replication bubbles could be stabilized and speculate that some tandem duplications represent such persistent static bubbles. Moreover, I propose how static replication bubbles could be transformed into tandem duplications, double minutes, or inverted triplications. Possible experimental tests of these models are discussed.
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The chromosomal constitution of fish hybrid lineage revealed by 5S rDNA FISH.
Zhang, Chun; Ye, Lihai; Chen, Yiyi; Xiao, Jun; Wu, Yanhong; Tao, Min; Xiao, Yamei; Liu, Shaojun
2015-12-03
The establishment of the bisexual fertile fish hybrid lineage including the allodiploid and allotetraploid hybrids, from interspecific hybridization of red crucian carp (Carassius auratus red var. 2n = 100, 2n = AA) (♀) × common carp (Cyprinus carpio L. 2n = 100, 2n = BB) (♂), provided a good platform to investigate genetic relationship between the parents and their hybrid progenies. The chromosomal inheritance of diploid and allotetraploid hybrid progenies in successive generations, was studied by applying 5S rDNA fluorescence in situ hybridization. Signals of 5S rDNA distinguished the chromosomal constitution of common carp (B-genome) from red crucian carp (A-genome), in which two strong signals were observed on the first submetacentric chromosome, while no major signal was found in common carp. After fish hybridization, one strong signal of 5S rDNA was detected in the same locus on the chromosome of diploid hybrids. As expected, two strong signals were observed in 4nF3 tetraploid hybrids offspring and it is worth mentioning that two strong signals were detected in a separating bivalent of a primary spermatocyte in 4nF3. Furthermore, the mitosis of heterozygous chromosomes was shown normal and stable with blastular tissue histological studies. We revealed that 5S rDNA signal can be applied to discern A-genome from B-genome, and that 5S rDNA bearing chromosomes can be stably passed down in successive generations. Our work provided a significant method in fish breeding and this is important for studies in fish evolutionary biology.
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Rodrigues, Débora Silva; Rivera, Miryan; Lourenço, Luciana Bolsoni
2012-03-20
For anurans, knowledge of 5S rDNA is scarce. For Engystomops species, chromosomal homeologies are difficult to recognize due to the high level of inter- and intraspecific cytogenetic variation. In an attempt to better compare the karyotypes of the Amazonian species Engystomops freibergi and Engystomops petersi, and to extend the knowledge of 5S rDNA organization in anurans, the 5S rDNA sequences of Amazonian Engystomops species were isolated, characterized, and mapped. Two types of 5S rDNA, which were readily differentiated by their NTS (non-transcribed spacer) sizes and compositions, were isolated from specimens of E. freibergi from Brazil and E. petersi from two Ecuadorian localities (Puyo and Yasuní). In the E. freibergi karyotypes, the entire type I 5S rDNA repeating unit hybridized to the pericentromeric region of 3p, whereas the entire type II 5S rDNA repeating unit mapped to the distal region of 6q, suggesting a differential localization of these sequences. The type I NTS probe clearly detected the 3p pericentromeric region in the karyotypes of E. freibergi and E. petersi from Puyo and the 5p pericentromeric region in the karyotype of E. petersi from Yasuní, but no distal or interstitial signals were observed. Interestingly, this probe also detected many centromeric regions in the three karyotypes, suggesting the presence of a satellite DNA family derived from 5S rDNA. The type II NTS probe detected only distal 6q regions in the three karyotypes, corroborating the differential distribution of the two types of 5S rDNA. Because the 5S rDNA types found in Engystomops are related to those of Physalaemus with respect to their nucleotide sequences and chromosomal locations, their origin likely preceded the evolutionary divergence of these genera. In addition, our data indicated homeology between Chromosome 5 in E. petersi from Yasuní and Chromosomes 3 in E. freibergi and E. petersi from Puyo. In addition, the chromosomal location of the type II 5S rDNA
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Fine organization of genomic regions tagged to the 5S rDNA locus of the bread wheat 5B chromosome.
Sergeeva, Ekaterina M; Shcherban, Andrey B; Adonina, Irina G; Nesterov, Michail A; Beletsky, Alexey V; Rakitin, Andrey L; Mardanov, Andrey V; Ravin, Nikolai V; Salina, Elena A
2017-11-14
The multigene family encoding the 5S rRNA, one of the most important structurally-functional part of the large ribosomal subunit, is an obligate component of all eukaryotic genomes. 5S rDNA has long been a favored target for cytological and phylogenetic studies due to the inherent peculiarities of its structural organization, such as the tandem arrays of repetitive units and their high interspecific divergence. The complex polyploid nature of the genome of bread wheat, Triticum aestivum, and the technically difficult task of sequencing clusters of tandem repeats mean that the detailed organization of extended genomic regions containing 5S rRNA genes remains unclear. This is despite the recent progress made in wheat genomic sequencing. Using pyrosequencing of BAC clones, in this work we studied the organization of two distinct 5S rDNA-tagged regions of the 5BS chromosome of bread wheat. Three BAC-clones containing 5S rDNA were identified in the 5BS chromosome-specific BAC-library of Triticum aestivum. Using the results of pyrosequencing and assembling, we obtained six 5S rDNA- containing contigs with a total length of 140,417 bp, and two sets (pools) of individual 5S rDNA sequences belonging to separate, but closely located genomic regions on the 5BS chromosome. Both regions are characterized by the presence of approximately 70-80 copies of 5S rDNA, however, they are completely different in their structural organization. The first region contained highly diverged short-type 5S rDNA units that were disrupted by multiple insertions of transposable elements. The second region contained the more conserved long-type 5S rDNA, organized as a single tandem array. FISH using probes specific to both 5S rDNA unit types showed differences in the distribution and intensity of signals on the chromosomes of polyploid wheat species and their diploid progenitors. A detailed structural organization of two closely located 5S rDNA-tagged genomic regions on the 5BS chromosome of bread
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GEMC1 is a TopBP1 interacting protein required for chromosomal DNA replication
Balestrini, Alessia; Cosentino, Claudia; Errico, Alessia; Garner, Elizabeth; Costanzo, Vincenzo
2010-01-01
Many factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication in complex multicellular organisms is poorly understood. Here, we report the identification of GEMC1, a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus egg extract we show that Xenopus GEMC1 (xGEMC1) binds to checkpoint and replication factor TopBP1, which promotes xGEMC1 binding to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 directly interacts with replication factors such as Cdc45 and Cdk2-CyclinE by which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication whereas depletion of xGEMC1 prevents DNA replication onset due to impairment of Cdc45 loading onto chromatin. Likewise, inhibition of GEMC1 expression by morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in higher eukaryotes by mediating TopBP1 and Cdk2 dependent recruitment of Cdc45 onto replication origins. PMID:20383140
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Malagón, Talía; Burchell, Ann N; El-Zein, Mariam; Guénoun, Julie; Tellier, Pierre-Paul; Coutlée, François; Franco, Eduardo L
2017-12-05
Detection of human papillomavirus (HPV) DNA in genital samples may not always represent true infections but may be depositions from infected sexual partners. We examined whether sexual risk factors and a biomarker (Y chromosome DNA) were associated with genital HPV partner concordance and estimated the fraction of HPV detections potentially attributable to partner deposition. The HITCH study enrolled young women attending a university or college in Montréal, Canada, and their male partners, from 2005 to 2010. We tested baseline genital samples for Y chromosome DNA and HPV DNA using polymerase chain reaction. Type-specific HPV concordance was 42.4% in partnerships where at least one partner was HPV DNA positive. Y chromosome DNA predicted type-specific HPV concordance in univariate analyses, but in multivariable models the independent predictors of concordance were days since last vaginal sex (26.5% higher concordance 0-1 vs 8-14 days after last vaginal sex) and condom use (22.6% higher concordance in never vs always users). We estimated that 14.1% (95% confidence interval [CI], 6.3-21.9%) of HPV DNA detections in genital samples were attributable to vaginal sex in the past week. A substantial proportion of HPV DNA detections may be depositions due to recent unprotected vaginal sex. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
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Liu, Qiang; Cao, Jia; Li, Ke Qiu; Miao, Xu Hong; Li, Guang; Fan, Fei Yue; Zhao, Yong Cheng
2009-05-01
It has been known that the pollutants of electronic wastes (E-wastes) can lead to severe pollution to the environment. It has been reported that about 50% to 80% of E-wastes from developed countries are exported to Asia and Africa. It has become a major global environmental problem to deal with 'E-wastes'. E-waste recycling has remained primitive in Jinghai, China. This not only produces enormous environmental pollution but also can bring about toxic or genotoxic effects on the human body, threatening the health of both current residents and future generations living in the local environment. The concentration of lead in the blood of children in the E-waste polluted area in China is higher than that of the control area. But little is known about the cytogenetic effect to human beings caused by the pollution of E-wastes. In the present study, experiments have been performed to investigate the genetics of permanent residents of three villages with numerous E-waste disposal sites and to analyze the harmful effects of exposure to E-wastes. In total, 171 villagers (exposed group) were randomly selected from permanent residents of three villages located in Jinghai County of Tianjin, China, where there has been massive disposal of E-wastes. Thirty villagers were selected from the neighboring towns without E-waste disposal sites to serve as controls. Chromosomal aberrations and cytokinesis blocking micronucleus were performed to detect the cytogenetic effect, dic + r (dicentric and ring chromosome), monomer, fragments (acentric fragments, minute chromosomes, and acentric rings), translocation, satellite, quadriradial, total aberrations, and micronuclear rate were scored for each subject. DNA damage was detected using comet assay; the DNA percentage in the comet tail (TDNA%), tail moment (TM), and Olive tail moment (OTM) were recorded to describe DNA damage to lymphocytes. The total chromosome aberration rates (5.50%) and micronuclear rates (16.99%) of the exposure group
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Cloning and comparative mapping of a human chromosome 4-specific alpha satellite DNA sequence
Energy Technology Data Exchange (ETDEWEB)
D' Aiuto, L.; Marzella, R.; Archidiacono, N.; Rocchi, M. (Universita di Bari (Italy)); Antonacci, R. (Instituto Anatomia Umana Normale, Modena (Italy))
1993-11-01
The authors have isolated and characterized two human alphoid DNA clones: p4n1/4 and pZ4.1. Clone p4n1/4 identifies specifically the centromeric region of chromosome 4; pZ4.1 recognizes a subset of alphoid DNA shared by chromosomes 4 and 9. The specificity was determined using fluorescence in situ hybridization experiments on metaphase spreads and Southern blotting analysis of human-hamster somatic cell hybrids. The genomic organization of both subsets was also investigated. Comparative mapping on chimpanzee and gorilla chromosomes was performed. p4n1/4 hybridizes to chimpanzee chromosomes 11 and 13, homologs of human chromosomes 9 and 2q, respectively. On gorilla metaphase spreads, p4n1/4 hybridizes exclusively to the centromeric region of chromosome 19, partially homologous to human chromosome 17. No hybridization signal was detected on chromosome 3 of both chimpanzee and gorilla, in both species homolog of human chromosome 4. Identical comparative mapping results were obtained using pZ4.1 probe, although the latter recognizes an alphoid subset distinct from the one recognized by p4n1/4. The implications of these results in the evolution of centromeric regions of primate chromosomes are discussed. 33 refs., 4 figs.
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Stephanou, Nicolas C; Gao, Feng; Bongiorno, Paola; Ehrt, Sabine; Schnappinger, Dirk; Shuman, Stewart; Glickman, Michael S
2007-07-01
Bacterial nonhomologous end joining (NHEJ) is a recently described DNA repair pathway best characterized in mycobacteria. Bacterial NHEJ proteins LigD and Ku have been analyzed biochemically, and their roles in linear plasmid repair in vivo have been verified genetically; yet the contributions of NHEJ to repair of chromosomal DNA damage are unknown. Here we use an extensive set of NHEJ- and homologous recombination (HR)-deficient Mycobacterium smegmatis strains to probe the importance of HR and NHEJ in repairing diverse types of chromosomal DNA damage. An M. smegmatis Delta recA Delta ku double mutant has no apparent growth defect in vitro. Loss of the NHEJ components Ku and LigD had no effect on sensitivity to UV radiation, methyl methanesulfonate, or quinolone antibiotics. NHEJ deficiency had no effect on sensitivity to ionizing radiation in logarithmic- or early-stationary-phase cells but was required for ionizing radiation resistance in late stationary phase in 7H9 but not LB medium. In addition, NHEJ components were required for repair of I-SceI mediated chromosomal double-strand breaks (DSBs), and in the absence of HR, the NHEJ pathway rapidly mutates the chromosomal break site. The molecular outcomes of NHEJ-mediated chromosomal DSB repair involve predominantly single-nucleotide insertions at the break site, similar to previous findings using plasmid substrates. These findings demonstrate that prokaryotic NHEJ is specifically required for DSB repair in late stationary phase and can mediate mutagenic repair of homing endonuclease-generated chromosomal DSBs.
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Gómez-Herreros, Fernando; Zagnoli-Vieira, Guido; Ntai, Ioanna; Martínez-Macías, María Isabel; Anderson, Rhona M; Herrero-Ruíz, Andrés; Caldecott, Keith W
2017-08-10
DNA double-strand breaks (DSBs) induced by abortive topoisomerase II (TOP2) activity are a potential source of genome instability and chromosome translocation. TOP2-induced DNA double-strand breaks are rejoined in part by tyrosyl-DNA phosphodiesterase 2 (TDP2)-dependent non-homologous end-joining (NHEJ), but whether this process suppresses or promotes TOP2-induced translocations is unclear. Here, we show that TDP2 rejoins DSBs induced during transcription-dependent TOP2 activity in breast cancer cells and at the translocation 'hotspot', MLL. Moreover, we find that TDP2 suppresses chromosome rearrangements induced by TOP2 and reduces TOP2-induced chromosome translocations that arise during gene transcription. Interestingly, however, we implicate TDP2-dependent NHEJ in the formation of a rare subclass of translocations associated previously with therapy-related leukemia and characterized by junction sequences with 4-bp of perfect homology. Collectively, these data highlight the threat posed by TOP2-induced DSBs during transcription and demonstrate the importance of TDP2-dependent non-homologous end-joining in protecting both gene transcription and genome stability.DNA double-strand breaks (DSBs) induced by topoisomerase II (TOP2) are rejoined by TDP2-dependent non-homologous end-joining (NHEJ) but whether this promotes or suppresses translocations is not clear. Here the authors show that TDP2 suppresses chromosome translocations from DSBs introduced during gene transcription.
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GEMC1 is a TopBP1-interacting protein required for chromosomal DNA replication.
Balestrini, Alessia; Cosentino, Claudia; Errico, Alessia; Garner, Elizabeth; Costanzo, Vincenzo
2010-05-01
Many of the factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication is poorly understood in multicellular organisms. Here, we report the identification of GEMC1 (geminin coiled-coil containing protein 1), a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus laevis egg extract we show that Xenopus GEMC1 (xGEMC1) binds to the checkpoint and replication factor TopBP1, which promotes binding of xGEMC1 to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 interacts directly with replication factors such as Cdc45 and the kinase Cdk2-CyclinE, through which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication, whereas depletion of xGEMC1 prevents the onset of DNA replication owing to the impairment of Cdc45 loading onto chromatin. Similarly, inhibition of GEMC1 expression with morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in multicellular organisms by mediating TopBP1- and Cdk2-dependent recruitment of Cdc45 onto replication origins.
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De novo formed satellite DNA-based mammalian artificial chromosomes and their possible applications.
Katona, Robert L
2015-02-01
Mammalian artificial chromosomes (MACs) are non-integrating, autonomously replicating natural chromosome-based vectors that may carry a vast amount of genetic material, which in turn enable potentially prolonged, safe, and regulated therapeutic transgene expression and render MACs as attractive genetic vectors for "gene replacement" or for controlling differentiation pathways in target cells. Satellite-DNA-based artificial chromosomes (SATACs) can be made by induced de novo chromosome formation in cells of different mammalian and plant species. These artificially generated accessory chromosomes are composed of predictable DNA sequences, and they contain defined genetic information. SATACs have already passed a number of obstacles crucial to their further development as gene therapy vectors, including large-scale purification, transfer of purified artificial chromosomes into different cells and embryos, generation of transgenic animals and germline transmission with purified SATACs, and the tissue-specific expression of a therapeutic gene from an artificial chromosome in the milk of transgenic animals. SATACs could be used in cell therapy protocols. For these methods, the most versatile target cell would be one that was pluripotent and self-renewing to address multiple disease target cell types, thus making multilineage stem cells, such as adult derived early progenitor cells and embryonic stem cells, as attractive universal host cells.
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Chromosomal locations of four minor rDNA loci and a marker microsatellite sequence in barley
DEFF Research Database (Denmark)
Pedersen, C.; Linde-Laursen, I.
1994-01-01
is located about 54% out on the short arm of chromosome 4 and it has not previously been reported in barley. We have designated the new locus Nor-I6. rDNA loci on homoeologous group 4 chromosomes have not yet been reported in other Triticeae species. The origin of these 4 minor rDNA loci is discussed...
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DNA and chromosome breaks induced by 123I-estrogen in CHO cells
International Nuclear Information System (INIS)
Schwartz, J.L.
1997-01-01
The effects of the Auger electron-emitting isotope I-123, covalently bound to estrogen, on DNA single- and double-strand breakage and on chromosome breakage was determined in estrogen positive Chinese hamster ovary (CHO-ER) cells. Exposure to the 123 I-estrogen induced both single- and double-strand breaks with a ratio of single- to double-strand breaks of 2.2. The corresponding ratio with 60 Co gamma rays was 15.6. The dose-response was biphasic suggesting that either receptor sites are saturated at high does, or that there is a nonrandom distribution of breaks induced by the 123 I-estrogen. The 123 I-estrogen treatment induced chromosome aberrations with an efficiency of about 1 aberration for each 1,000 disintegrations per cell. This corresponds to the mean lethal dose of 123 I-estrogen for these cells suggesting that the lethal event induced by the Auger electron emitter bound to estrogen is a chromosome aberration. Most of the chromosome-type aberrations were dicentrics and rings, suggesting that 123 I-estrogen-induced chromosome breaks are rejoined. The F-ratio, the ratio of dicentrics to centric rings, was 5.8 ± 1.7, which is similar to that seen with high LET radiations. Their results suggest that I-123 bound to estrogen is an efficient clastogenic agent, that the cytotoxic damage produced by I-123 bound to estrogen is very like high LET-induced damage, and the I-123 in the estrogen-receptor-DNA complex is probably in close proximity to the sugar-phosphate backbone of the DNA
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Data Mining Empowers the Generation of a Novel Class of Chromosome-specific DNA Probes
Energy Technology Data Exchange (ETDEWEB)
Zeng, Hui; Weier, Heinz-Ulrich G.; Kwan, Johnson; Wang, Mei; O' Brien, Benjamin
2011-03-08
Probes that allow accurate delineation of chromosome-specific DNA sequences in interphase or metaphase cell nuclei have become important clinical tools that deliver life-saving information about the gender or chromosomal make-up of a product of conception or the probability of an embryo to implant, as well as the definition of tumor-specific genetic signatures. Often such highly specific DNA probes are proprietary in nature and have been the result of extensive probe selection and optimization procedures. We describe a novel approach that eliminates costly and time consuming probe selection and testing by applying data mining and common bioinformatics tools. Similar to a rational drug design process in which drug-protein interactions are modeled in the computer, the rational probe design described here uses a set of criteria and publicly available bioinformatics software to select the desired probe molecules from libraries comprised of hundreds of thousands of probe molecules. Examples describe the selection of DNA probes for the human X and Y chromosomes, both with unprecedented performance, but in a similar fashion, this approach can be applied to other chromosomes or species.
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Directory of Open Access Journals (Sweden)
Albert Victor A
2011-09-01
Full Text Available Abstract Background Paphiopedilum is a horticulturally and ecologically important genus of ca. 80 species of lady's slipper orchids native to Southeast Asia. These plants have long been of interest regarding their chromosomal evolution, which involves a progressive aneuploid series based on either fission or fusion of centromeres. Chromosome number is positively correlated with genome size, so rearrangement processes must include either insertion or deletion of DNA segments. We have conducted Fluorescence In Situ Hybridization (FISH studies using 5S and 25S ribosomal DNA (rDNA probes to survey for rearrangements, duplications, and phylogenetically-correlated variation within Paphiopedilum. We further studied sequence variation of the non-transcribed spacers of 5S rDNA (5S-NTS to examine their complex duplication history, including the possibility that concerted evolutionary forces may homogenize diversity. Results 5S and 25S rDNA loci among Paphiopedilum species, representing all key phylogenetic lineages, exhibit a considerable diversity that correlates well with recognized evolutionary groups. 25S rDNA signals range from 2 (representing 1 locus to 9, the latter representing hemizygosity. 5S loci display extensive structural variation, and show from 2 specific signals to many, both major and minor and highly dispersed. The dispersed signals mainly occur at centromeric and subtelomeric positions, which are hotspots for chromosomal breakpoints. Phylogenetic analysis of cloned 5S rDNA non-transcribed spacer (5S-NTS sequences showed evidence for both ancient and recent post-speciation duplication events, as well as interlocus and intralocus diversity. Conclusions Paphiopedilum species display many chromosomal rearrangements - for example, duplications, translocations, and inversions - but only weak concerted evolutionary forces among highly duplicated 5S arrays, which suggests that double-strand break repair processes are dynamic and ongoing. These
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Inagaki, Hidehito; Ohye, Tamae; Kogo, Hiroshi; Kato, Takema; Bolor, Hasbaira; Taniguchi, Mariko; Shaikh, Tamim H; Emanuel, Beverly S; Kurahashi, Hiroki
2009-02-01
Chromosomal aberrations have been thought to be random events. However, recent findings introduce a new paradigm in which certain DNA segments have the potential to adopt unusual conformations that lead to genomic instability and nonrandom chromosomal rearrangement. One of the best-studied examples is the palindromic AT-rich repeat (PATRR), which induces recurrent constitutional translocations in humans. Here, we established a plasmid-based model that promotes frequent intermolecular rearrangements between two PATRRs in HEK293 cells. In this model system, the proportion of PATRR plasmid that extrudes a cruciform structure correlates to the levels of rearrangement. Our data suggest that PATRR-mediated translocations are attributable to unusual DNA conformations that confer a common pathway for chromosomal rearrangements in humans.
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International Nuclear Information System (INIS)
Tsai-Pflugfelder, M.; Liu, L.F.; Liu, A.A.; Tewey, K.M.; Whang-Peng, J.; Knutsen, T.; Huebner, K.; Croce, C.M.; Wang, J.C.
1988-01-01
Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage λ was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a λgt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes
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International Nuclear Information System (INIS)
Barenfeld, L.S.; Pleskach, N.M.; Bildin, V.N.; Prokofjeva, V.V.; Mikhelson, V.M.
1986-01-01
The rate of DNA synthesis after γ-irradiation was studied either by analysis of the steady-state distribution of daughter [ 3 H]DNA in alkaline sucrose gradients or by direct assay of the amount of [ 3 H]thymidine incorporated into DNA of fibroblasts derived from a normal donor (LCH882) and from Down's syndrome (LCH944), Werner's syndrome (WS1LE) and xeroderma pigmentosum (XP2LE) patients with chromosomal sensitivity to ionizing radiation. Doses of γ-irradiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition of DNA synthesis in the cells from the affected individuals. The radioresistant DNA synthesis in Down's syndrome cells was mainly due to a much lower inhibition of replicon initiation than that in normal cells; these cells were also more resistant to damage that inhibited replicon elongation. Our data suggest that radioresistant DNA synthesis may be an intrinsic feature of all genetic disorders showing increased radiosensitivity in terms of chromosome aberrations. (orig.)
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Relationship of DNA lesions and their repair to chromosomal aberration production
International Nuclear Information System (INIS)
Bender, M.A.
1979-01-01
Recent work on the roles of specific kinds of DNA lesions and their enzymatic repair systems in the production of chromosomal aberrations seems consistent with a simple molecular model of chromosomal aberrations formation. Evidence from experiments with the human repair-deficient genetic diseases xeroderma pigmentosom, ataxia telangiectasia, and Fanconi's anemia is reviewed in the light of the contributions to aberration production of single and double polynucleotide strand breaks, base damage, polynucleotide strand crosslinks, and pyrimidine cyclobutane dimers
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Relationship of DNA lesions and their repair to chromosomal aberration production
Energy Technology Data Exchange (ETDEWEB)
Bender, M.A.
1979-01-01
Recent work on the roles of specific kinds of DNA lesions and their enzymatic repair systems in the production of chromosomal aberrations seems consistent with a simple molecular model of chromosomal aberrations formation. Evidence from experiments with the human repair-deficient genetic diseases xeroderma pigmentosom, ataxia telangiectasia, and Fanconi's anemia is reviewed in the light of the contributions to aberration production of single and double polynucleotide strand breaks, base damage, polynucleotide strand crosslinks, and pyrimidine cyclobutane dimers.
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Self-cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage.
Suspène, Rodolphe; Mussil, Bianka; Laude, Hélène; Caval, Vincent; Berry, Noémie; Bouzidi, Mohamed S; Thiers, Valérie; Wain-Hobson, Simon; Vartanian, Jean-Pierre
2017-04-07
Foreign and self-cytoplasmic DNA are recognized by numerous DNA sensor molecules leading to the production of type I interferons. Such DNA agonists should be degraded otherwise cells would be chronically stressed. Most human APOBEC3 cytidine deaminases can initiate catabolism of cytoplasmic mitochondrial DNA. Using the human myeloid cell line THP-1 with an interferon inducible APOBEC3A gene, we show that cytoplasmic DNA triggers interferon α and β production through the RNA polymerase III transcription/RIG-I pathway leading to massive upregulation of APOBEC3A. By catalyzing C→U editing in single stranded DNA fragments, the enzyme prevents them from re-annealing so attenuating the danger signal. The price to pay is chromosomal DNA damage in the form of CG→TA mutations and double stranded DNA breaks which, in the context of chronic inflammation, could drive cells down the path toward cancer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
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Directory of Open Access Journals (Sweden)
Asmahan Bekada
Full Text Available North Africa is considered a distinct geographic and ethnic entity within Africa. Although modern humans originated in this Continent, studies of mitochondrial DNA (mtDNA and Y-chromosome genealogical markers provide evidence that the North African gene pool has been shaped by the back-migration of several Eurasian lineages in Paleolithic and Neolithic times. More recent influences from sub-Saharan Africa and Mediterranean Europe are also evident. The presence of East-West and North-South haplogroup frequency gradients strongly reinforces the genetic complexity of this region. However, this genetic scenario is beset with a notable gap, which is the lack of consistent information for Algeria, the largest country in the Maghreb. To fill this gap, we analyzed a sample of 240 unrelated subjects from a northwest Algeria cosmopolitan population using mtDNA sequences and Y-chromosome biallelic polymorphisms, focusing on the fine dissection of haplogroups E and R, which are the most prevalent in North Africa and Europe respectively. The Eurasian component in Algeria reached 80% for mtDNA and 90% for Y-chromosome. However, within them, the North African genetic component for mtDNA (U6 and M1; 20% is significantly smaller than the paternal (E-M81 and E-V65; 70%. The unexpected presence of the European-derived Y-chromosome lineages R-M412, R-S116, R-U152 and R-M529 in Algeria and the rest of the Maghreb could be the counterparts of the mtDNA H1, H3 and V subgroups, pointing to direct maritime contacts between the European and North African sides of the western Mediterranean. Female influx of sub-Saharan Africans into Algeria (20% is also significantly greater than the male (10%. In spite of these sexual asymmetries, the Algerian uniparental profiles faithfully correlate between each other and with the geography.
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Directory of Open Access Journals (Sweden)
Sebastian Pita
2013-05-01
Full Text Available In this study, we used fluorescence in situ hybridisation to determine the chromosomal location of 45S rDNA clusters in 10 species of the tribe Rhodniini (Hemiptera: Reduviidae: Triatominae. The results showed striking inter and intraspecific variability, with the location of the rDNA clusters restricted to sex chromosomes with two patterns: either on one (X chromosome or both sex chromosomes (X and Y chromosomes. This variation occurs within a genus that has an unchanging diploid chromosome number (2n = 22, including 20 autosomes and 2 sex chromosomes and a similar chromosome size and genomic DNA content, reflecting a genome dynamic not revealed by these chromosome traits. The rDNA variation in closely related species and the intraspecific polymorphism in Rhodnius ecuadoriensis suggested that the chromosomal position of rDNA clusters might be a useful marker to identify recently diverged species or populations. We discuss the ancestral position of ribosomal genes in the tribe Rhodniini and the possible mechanisms involved in the variation of the rDNA clusters, including the loss of rDNA loci on the Y chromosome, transposition and ectopic pairing. The last two processes involve chromosomal exchanges between both sex chromosomes, in contrast to the widely accepted idea that the achiasmatic sex chromosomes of Heteroptera do not interchange sequences.
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DNA AND THE FINE STRUCTURE OF SYNAPTIC CHROMOSOMES IN THE DOMESTIC ROOSTER (GALLUS DOMESTICUS)
Coleman, James R.; Moses, Montrose J.
1964-01-01
The indium trichloride method of Watson and Aldridge (38) for staining nucleic acids for electron microscopy was employed to study the relationship of DNA to the structure of the synaptinemal complex in meiotic prophase chromosomes of the domestic rooster. The selectivity of the method was demonstrated in untreated and DNase-digested testis material by comparing the distribution of indium staining in the electron microscope to Feulgen staining and ultraviolet absorption in thicker sections seen with the light microscope. Following staining by indium, DNA was found mainly in the microfibril component of the synaptinemal complex. When DNA was known to have been removed from aldehyde-fixed material by digestion with DNase, indium stainability was also lost. However, staining of the digested material with non-selective heavy metal techniques demonstrated the presence of material other than DNA in the microfibrils and showed that little alteration in appearance of the chromosome resulted from DNA removal. The two dense lateral axial elements of the synaptinemal complex, but not the central one to any extent, also contained DNA, together with non-DNA material. PMID:14228519
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Mazzoleni, Sofia; Rovatsos, Michail; Schillaci, Odessa; Dumas, Francesca
2018-01-01
Abstract We explored the topology of 18S and 28S rDNA units by fluorescence in situ hybridization (FISH) in the karyotypes of thirteen species representatives from major groups of Primates and Tupaia minor (Günther, 1876) (Scandentia), in order to expand our knowledge of Primate genome reshuffling and to identify the possible dispersion mechanisms of rDNA sequences. We documented that rDNA probe signals were identified on one to six pairs of chromosomes, both acrocentric and metacentric ones. In addition, we examined the potential homology of chromosomes bearing rDNA genes across different species and in a wide phylogenetic perspective, based on the DAPI-inverted pattern and their synteny to human. Our analysis revealed an extensive variability in the topology of the rDNA signals across studied species. In some cases, closely related species show signals on homologous chromosomes, thus representing synapomorphies, while in other cases, signal was detected on distinct chromosomes, leading to species specific patterns. These results led us to support the hypothesis that different mechanisms are responsible for the distribution of the ribosomal DNA cluster in Primates. PMID:29416829
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Directory of Open Access Journals (Sweden)
Sofia Mazzoleni
2018-01-01
Full Text Available We explored the topology of 18S and 28S rDNA units by fluorescence in situ hybridization (FISH in the karyotypes of thirteen species representatives from major groups of Primates and Tupaia minor (Günther, 1876 (Scandentia, in order to expand our knowledge of Primate genome reshuffling and to identify the possible dispersion mechanisms of rDNA sequences. We documented that rDNA probe signals were identified on one to six pairs of chromosomes, both acrocentric and metacentric ones. In addition, we examined the potential homology of chromosomes bearing rDNA genes across different species and in a wide phylogenetic perspective, based on the DAPI-inverted pattern and their synteny to human. Our analysis revealed an extensive variability in the topology of the rDNA signals across studied species. In some cases, closely related species show signals on homologous chromosomes, thus representing synapomorphies, while in other cases, signal was detected on distinct chromosomes, leading to species specific patterns. These results led us to support the hypothesis that different mechanisms are responsible for the distribution of the ribosomal DNA cluster in Primates.
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International Nuclear Information System (INIS)
Stoilov, L.M.; Mullenders, L.H.F.; Natarajan, A.T.
1994-01-01
The effect of caffeine on radiation-induced chromosomal aberrations and DNA strand breaks in unstimulated human lymphocytes was investigated. When present prior to and during the radiation exposure, caffeine treatment was found to cause either potentiation or protection against induction of chromosomal aberrations depending on the concentration and temperature. When the nucleoid sedimentation technique was applied, enhancement or reduction of radiation-induced DNA strand breaks by caffeine was also found to be dependent on temperature and caffeine concentration. It is proposed that caffeine, in addition to its suspected ability to influence DNA repair, can also influence the induction of DNA damage, leading to alterations in the yield of chromosomal aberrations
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Energy Technology Data Exchange (ETDEWEB)
Stoilov, L.M. (Department of Molecular Genetics, Institute of Genetics, Sofia (Bulgaria)); Mullenders, L.H.F.; Natarajan, A.T. (J.A. Cohen Institute, Interuniversity Research Institute for Radiopathology and Radiation Protection, Leiden (Netherlands))
1994-12-01
The effect of caffeine on radiation-induced chromosomal aberrations and DNA strand breaks in unstimulated human lymphocytes was investigated. When present prior to and during the radiation exposure, caffeine treatment was found to cause either potentiation or protection against induction of chromosomal aberrations depending on the concentration and temperature. When the nucleoid sedimentation technique was applied, enhancement or reduction of radiation-induced DNA strand breaks by caffeine was also found to be dependent on temperature and caffeine concentration. It is proposed that caffeine, in addition to its suspected ability to influence DNA repair, can also influence the induction of DNA damage, leading to alterations in the yield of chromosomal aberrations.
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Directory of Open Access Journals (Sweden)
Marx Kenneth A
2006-03-01
Full Text Available Abstract Background The centromeres in yeast (S. cerevisiae are organized by short DNA sequences (125 bp on each chromosome consisting of 2 conserved elements: CDEI and CDEIII spaced by a CDEII region. CDEI and CDEIII are critical sequence specific protein binding sites necessary for correct centromere formation and following assembly with proteins, are positioned near each other on a specialized nucleosome. Hegemann et al. BioEssays 1993, 15: 451–460 reported single base DNA mutants within the critical CDEI and CDEIII binding sites on the centromere of chromosome 6 and quantitated centromere loss of function, which they measured as loss rates for the different chromosome 6 mutants during cell division. Olson et al. Proc Natl Acad Sci USA 1998, 95: 11163–11168 reported the use of protein-DNA crystallography data to produce a DNA dinucleotide protein deformability energetic scale (PD-scale that describes local DNA deformability by sequence specific binding proteins. We have used the PD-scale to investigate the DNA sequence dependence of the yeast chromosome 6 mutants' loss rate data. Each single base mutant changes 2 PD-scale values at that changed base position relative to the wild type. In this study, we have utilized these mutants to demonstrate a correlation between the change in DNA deformability of the CDEI and CDEIII core sites and the overall experimentally measured chromosome loss rates of the chromosome 6 mutants. Results In the CDE I and CDEIII core binding regions an increase in the magnitude of change in deformability of chromosome 6 single base mutants with respect to the wild type correlates to an increase in the measured chromosome loss rate. These correlations were found to be significant relative to 105 Monte Carlo randomizations of the dinucleotide PD-scale applied to the same calculation. A net loss of deformability also tends to increase the loss rate. Binding site position specific, 4 data-point correlations were also
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Directory of Open Access Journals (Sweden)
Repin Mikhail V
2009-06-01
Full Text Available Abstract Background The objective of this work is to obtain the correct relative DNA contents of chromosomes in the normal male and female human diploid genomes for the use at FISH analysis of radiation-induced chromosome aberrations. Results The relative DNA contents of chromosomes in the male and female human diploid genomes have been calculated from the publicly available international Human Genome Project data. New sequence-based data on the relative DNA contents of human chromosomes were compared with the data recommended by the International Atomic Energy Agency in 2001. The differences in the values of the relative DNA contents of chromosomes obtained by using different approaches for 15 human chromosomes, mainly for large chromosomes, were below 2%. For the chromosomes 13, 17, 20 and 22 the differences were above 5%. Conclusion New sequence-based data on the relative DNA contents of chromosomes in the normal male and female human diploid genomes were obtained. This approach, based on the genome sequence, can be recommended for the use in radiation molecular cytogenetics.
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Satellite DNA Sequences in Canidae and Their Chromosome Distribution in Dog and Red Fox.
Vozdova, Miluse; Kubickova, Svatava; Cernohorska, Halina; Fröhlich, Jan; Rubes, Jiri
2016-01-01
Satellite DNA is a characteristic component of mammalian centromeric heterochromatin, and a comparative analysis of its evolutionary dynamics can be used for phylogenetic studies. We analysed satellite and satellite-like DNA sequences available in NCBI for 4 species of the family Canidae (red fox, Vulpes vulpes, VVU; domestic dog, Canis familiaris, CFA; arctic fox, Vulpes lagopus, VLA; raccoon dog, Nyctereutes procyonoides procyonoides, NPR) by comparative sequence analysis, which revealed 86-90% intraspecies and 76-79% interspecies similarity. Comparative fluorescence in situ hybridisation in the red fox and dog showed signals of the red fox satellite probe in canine and vulpine autosomal centromeres, on VVUY, B chromosomes, and in the distal parts of VVU9q and VVU10p which were shown to contain nucleolus organiser regions. The CFA satellite probe stained autosomal centromeres only in the dog. The CFA satellite-like DNA did not show any significant sequence similarity with the satellite DNA of any species analysed and was localised to the centromeres of 9 canine chromosome pairs. No significant heterochromatin block was detected on the B chromosomes of the red fox. Our results show extensive heterogeneity of satellite sequences among Canidae and prove close evolutionary relationships between the red and arctic fox. © 2017 S. Karger AG, Basel.
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Eĭdel'man, Iu A; Slanina, S V; Sal'nikov, I V; Andreev, S G
2012-12-01
The knowledge of radiation-induced chromosomal aberration (CA) mechanisms is required in many fields of radiation genetics, radiation biology, biodosimetry, etc. However, these mechanisms are yet to be quantitatively characterised. One of the reasons is that the relationships between primary lesions of DNA/chromatin/chromosomes and dose-response curves for CA are unknown because the pathways of lesion interactions in an interphase nucleus are currently inaccessible for direct experimental observation. This article aims for the comparative analysis of two principally different scenarios of formation of simple and complex interchromosomal exchange aberrations: by lesion interactions at chromosome territories' surface vs. in the whole space of the nucleus. The analysis was based on quantitative mechanistic modelling of different levels of structures and processes involved in CA formation: chromosome structure in an interphase nucleus, induction, repair and interactions of DNA lesions. It was shown that the restricted diffusion of chromosomal loci, predicted by computational modelling of chromosome organization, results in lesion interactions in the whole space of the nucleus being impossible. At the same time, predicted features of subchromosomal dynamics agrees well with in vivo observations and does not contradict the mechanism of CA formation at the surface of chromosome territories. On the other hand, the "surface mechanism" of CA formation, despite having certain qualities, proved to be insufficient to explain high frequency of complex exchange aberrations observed by mFISH technique. The alternative mechanism, CA formation on nuclear centres is expected to be sufficient to explain frequent complex exchanges.
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Intrinsic bent DNA sites in the chromosomal replication origin of Xylella fastidiosa 9a5c
Directory of Open Access Journals (Sweden)
F. Gimenes
2008-04-01
Full Text Available The features of the nucleotide sequences in both replication and promoter regions have been investigated in many organisms. Intrinsically bent DNA sites associated with transcription have been described in several prokaryotic organisms. The aim of the present study was to investigate intrinsic bent DNA sites in the segment that holds the chromosomal replication origin, oriC, of Xylella fastidiosa 9a5c. Electrophoretic behavior analyses, as well as in silico analyses of both the 2-D projection and helical parameters, were performed. The chromosomal segment analyzed contains the initial sequence of the rpmH gene, an intergenic region, the dnaA gene, the oriC sequence, and the 5' partial sequence of the dnaN gene. The analysis revealed fragments with reduced electrophoretic mobility, which indicates the presence of curved DNA segments. The analysis of the helical parameter ENDS ratio revealed three bent DNA sites (b1, b2, and b3 located in the rpmH-dnaA intergenic region, the dnaA gene, and the oriC 5' end, respectively. The chromosomal segment of X. fastidiosa analyzed here is rich in phased AT tracts and in CAnT motifs. The 2-D projection indicated a segment whose structure was determined by the cumulative effect of all bent DNA sites. Further, the in silico analysis of the three different bacterial oriC sequences indicated similar negative roll and twist >34.00° values. The DnaA box sequences, and other motifs in them, may be associated with the intrinsic DNA curvature.
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Discrimination of chromosome by autoradiography
International Nuclear Information System (INIS)
Masubuchi, Masanori
1975-01-01
This paper describes discrimination of chromosome by autoradiography. In this method, the difference in DNA synthetic phase between each chromosome was used as a standard, and the used chromosome was in metaphase, as morphological characteristics were markedly in this phase. Cell cycle and autoradiography with 3 H-thymidine were also examined. In order to discriminate chromosome by autoradiography, it was effective to utilize the labelled pattern in late DNA synthetic phase, where asynchronous replication of chromosome appeared most obviously. DNA synthesis in chromosome was examined in each DNA synthetic phase by culturing the chromosome after the treatment with 3 H-thymidine and altering the time to prepare chromosome specimen. Discrimination of chromosome in plants and animals by autoradiography was also mentioned. It was noticed as a structural and functional discrimination of chromosome to observe amino acid uptake into chromosome protein and to utilize the difference in labelled pattern between the sites of chromosome. (K. Serizawa)
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Radiation hybrid mapping of human chromosome 18
International Nuclear Information System (INIS)
Francke, U.; Moon, A.J.; Chang, E.; Foellmer, B.; Strauss, B.; Haschke, A.; Chihlin Hsieh; Geigl, E.M.; Welch, S.
1990-01-01
The authors have generated a Chinese hamster V79/380-6 HPRT minus x human leukocyte hybrid cell line (18/V79) with chromosome 18 as the only human chromosome that is retained at high frequency without specific selection. Hybrid cells were selected in HAT medium, and 164 individual colonies were isolated. Of 110 colonies screened for human DNA by PCR amplification using a primer specific for human Alu repeats 67 (61%) were positive. These were expanded in culture for large-scale DNA preparations. Retesting expanded clones by PCR with Alu and LINE primers has revealed unique patterns of amplification products. In situ hybridization of biotin labelled total human DNA to metaphase spreads from various hybrids revealed the presence of one or more human DNA fragments integrated in hamster chromosomes. The authors have generated a resource that should allow the construction of a radiation map, to be compared with the YAC contig map also under construction in their laboratory
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Energy Technology Data Exchange (ETDEWEB)
Cheng, S.V.; Nadeau, J.H.; Tanzi, R.E.; Watkins, P.C.; Jagadesh, J.; Taylor, B.A.; Haines, J.L.; Sacchi, N.; Gusella, J.F. (Harvard Medical School, Boston, MA (USA))
1988-08-01
Mouse trisomy 16 has been proposed as an animal model of Down syndrome (DS), since this chromosome contains homologues of several loci from the q22 band of human chromosome 21. The recent mapping of the defect causing familial Alzheimer disease (FAD) and the locus encoding the Alzheimer amyloid {beta} precursor protein (APP) to human chromosome 21 has prompted a more detailed examination of the extent of conservation of this linkage group between the two species. Using anonymous DNA probes and cloned genes from human chromosome 21 in a combination of recombinant inbred and interspecific mouse backcross analyses, the authors have established that the linkage group shared by mouse chromosome 16 includes not only the critical DS region of human chromosome 21 but also the APP gene and FAD-linked markers. Extending from the anonymous DNA locus D21S52 to ETS2, the linkage map of six loci spans 39% recombination in man but only 6.4% recombination in the mouse. A break in synteny occurs distal to ETS2, with the homologue of the human marker D21S56 mapping to mouse chromosome 17. Conservation of the linkage relationships of markers in the FAD region suggests that the murine homologue of the FAD locus probably maps to chromosome 16 and that detailed comparison of the corresponding region in both species could facilitate identification of the primary defect in this disorder. The break in synteny between the terminal portion of human chromosome 21 and mouse chromosome 16 indicates, however, that mouse trisomy 16 may not represent a complete model of DS.
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International Nuclear Information System (INIS)
Cheng, S.V.; Nadeau, J.H.; Tanzi, R.E.; Watkins, P.C.; Jagadesh, J.; Taylor, B.A.; Haines, J.L.; Sacchi, N.; Gusella, J.F.
1988-01-01
Mouse trisomy 16 has been proposed as an animal model of Down syndrome (DS), since this chromosome contains homologues of several loci from the q22 band of human chromosome 21. The recent mapping of the defect causing familial Alzheimer disease (FAD) and the locus encoding the Alzheimer amyloid β precursor protein (APP) to human chromosome 21 has prompted a more detailed examination of the extent of conservation of this linkage group between the two species. Using anonymous DNA probes and cloned genes from human chromosome 21 in a combination of recombinant inbred and interspecific mouse backcross analyses, the authors have established that the linkage group shared by mouse chromosome 16 includes not only the critical DS region of human chromosome 21 but also the APP gene and FAD-linked markers. Extending from the anonymous DNA locus D21S52 to ETS2, the linkage map of six loci spans 39% recombination in man but only 6.4% recombination in the mouse. A break in synteny occurs distal to ETS2, with the homologue of the human marker D21S56 mapping to mouse chromosome 17. Conservation of the linkage relationships of markers in the FAD region suggests that the murine homologue of the FAD locus probably maps to chromosome 16 and that detailed comparison of the corresponding region in both species could facilitate identification of the primary defect in this disorder. The break in synteny between the terminal portion of human chromosome 21 and mouse chromosome 16 indicates, however, that mouse trisomy 16 may not represent a complete model of DS
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George, K. A.; Hada, M.; Patel, Z.; Huff, J.; Pluth, J. M.; Cucinotta, F. A.
2009-01-01
Chromosome aberration yields were assessed in DNA double-strand break repair (DSB) deficient cells after acute doses of gamma-rays or high-LET iron nuclei, or low dose-rate (0.018 Gy/hr) gamma-rays. We studied several cell lines including fibroblasts deficient in ATM (product of the gene that is mutated in ataxia telangiectasia patients) or NBS (product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase, DNA-PK activity. Chromosomes were analyzed using the fluorescence in-situ hybridization (FISH) chromosome painting method in cells at the first division post-irradiation and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma radiation induced higher yields of both simple and complex exchanges in the DSB repair defective cells than in the normal cells. The quadratic dose-response terms for both chromosome exchange types were significantly higher for the ATM and NBS defective lines than for normal fibroblasts. However, the linear dose-response term was significantly higher only for simple exchanges in the NBS cells. Large increases in the quadratic dose response terms indicate the important roles of ATM and NBS in chromatin modifications that facilitate correct DSB repair and minimize aberration formation. Differences in the response of AT and NBS deficient cells at lower doses suggests important questions about the applicability of observations of radiation sensitivity at high dose to low dose exposures. For all iron nuclei irradiated cells, regression models preferred purely linear and quadratic dose responses for simple and complex exchanges, respectively. All the DNA repair defective cell lines had lower Relative biological effectiveness (RBE) values than normal cells, the lowest being for the DNA-PK-deficient cells, which was near unity. To further
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International Nuclear Information System (INIS)
Fornace, A.J. Jr.; Nagasawa, H.; Little, J.B.
1980-01-01
By the alkaline elution technique, the repair of x-ray-induced DNA single strand breaks and DNA-protein cross-links was investigated in stationary phase, contact-inhibited mouse cells. During the first hour of repair, approximately 90% of x-ray induced single strand breaks were rejoined whereas most of the remaining breaks were rejoined more slowly during the next 5 h. The number of residual non-rejoined single strand breaks was approximately proportional to the x-ray dose at early repair times. DNA-protein cross-links were removed at a slower rate - T 1/2 approximately 10 to 12 h. Cells were subcultured at low density at various times after irradiation and scored for colony survival, and chromosome aberrations in the first mitosis after sub-culture. Both cell lethality and the frequency of chromosome aberrations decreased during the first several hours of repair, reaching a minimum level by 6 h; this decrease correlated temporally with the repair of the slowly rejoining DNA strand breaks. The possible relationship of DNA repair to changes in survival and chromosome aberrations is discussed
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DEFF Research Database (Denmark)
Duigou, Stephane; Knudsen, Kristine Groth; Skovgaard, Ole
2006-01-01
Although the two Vibrio cholerae chromosomes initiate replication in a coordinated fashion, we show here that each chromosome appears to have a specific replication initiator. DnaA overproduction promoted overinitiation of chromosome I and not chromosome II. In contrast, overproduction of RctB, a...
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Li, Ping; Jin, Hui; Yu, Hong-Guo
2014-01-01
During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. PMID:25103240
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Aly, Fayza M; Kotb, Ahmed M; Hammad, Seddik
2018-04-01
Todays, bioactive compounds extracted from Spirulina platensis have been intensively studied for their therapeutical values. Therefore, in the present study, we aimed to evaluate the effects of S. platensis extract on DNA damage and chromosomal aberrations induced by cadmium in rats. Four groups of male albino rats (n = 7 rats) were used. The first group served as a control group and received distilled water. The second group was exposed intraperitoneally to cadmium chloride (CdCl 2 ) (3.5 mg/kg body weight dissolved in 2 ml distilled water). The third group included the rats that were orally treated with S. platensis extract (1 g/kg dissolved in 5 ml distilled water, every other day for 30 days). The fourth group included the rats that were intraperitoneally and orally exposed to cadmium chloride and S. platensis, respectively. The experiment in all groups was extended for 60 days. The results of cadmium-mediated toxicity revealed significant genetic effects (DNA fragmentation, deletion or disappearance of some base pairs of DNA, and appearance of few base pairs according to ISSR-PCR analysis). Moreover, chromosomes showed structural aberrations such as reduction of chromosomal number, chromosomal ring, chromatid deletions, chromosomal fragmentations, and dicentric chromosomes. Surprisingly, S. platensis extract plus CdCl 2 -treated group showed less genetic effects compared with CdCl 2 alone. Further, S. platensis extract upon CdCl 2 toxicity was associated with less chromosomal aberration number and nearly normal appearance of DNA fragments as indicated by the bone marrow and ISSR-PCR analysis, respectively. In conclusion, the present novel study showed that co-treatment with S. platensis extract could reduce the genotoxic effects of CdCl 2 in rats.
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Directory of Open Access Journals (Sweden)
Ben Beheshti
2003-01-01
Full Text Available Conventional comparative genomic hybridization (CGH profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1 no amplifications evident, 2 a small MYCN amplicon as the only detectable imbalance, and 3 a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.
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Fragile DNA Motifs Trigger Mutagenesis at Distant Chromosomal Loci in Saccharomyces cerevisiae
Saini, Natalie; Zhang, Yu; Nishida, Yuri; Sheng, Ziwei; Choudhury, Shilpa; Mieczkowski, Piotr; Lobachev, Kirill S.
2013-01-01
DNA sequences capable of adopting non-canonical secondary structures have been associated with gross-chromosomal rearrangements in humans and model organisms. Previously, we have shown that long inverted repeats that form hairpin and cruciform structures and triplex-forming GAA/TTC repeats induce the formation of double-strand breaks which trigger genome instability in yeast. In this study, we demonstrate that breakage at both inverted repeats and GAA/TTC repeats is augmented by defects in DNA replication. Increased fragility is associated with increased mutation levels in the reporter genes located as far as 8 kb from both sides of the repeats. The increase in mutations was dependent on the presence of inverted or GAA/TTC repeats and activity of the translesion polymerase Polζ. Mutagenesis induced by inverted repeats also required Sae2 which opens hairpin-capped breaks and initiates end resection. The amount of breakage at the repeats is an important determinant of mutations as a perfect palindromic sequence with inherently increased fragility was also found to elevate mutation rates even in replication-proficient strains. We hypothesize that the underlying mechanism for mutagenesis induced by fragile motifs involves the formation of long single-stranded regions in the broken chromosome, invasion of the undamaged sister chromatid for repair, and faulty DNA synthesis employing Polζ. These data demonstrate that repeat-mediated breaks pose a dual threat to eukaryotic genome integrity by inducing chromosomal aberrations as well as mutations in flanking genes. PMID:23785298
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Pure chromosome-specific PCR libraries from single sorted chromosomes
VanDevanter, D. R.; Choongkittaworn, N. M.; Dyer, K. A.; Aten, J. A.; Otto, P.; Behler, C.; Bryant, E. M.; Rabinovitch, P. S.
1994-01-01
Chromosome-specific DNA libraries can be very useful in molecular and cytogenetic genome mapping studies. We have developed a rapid and simple method for the generation of chromosome-specific DNA sequences that relies on polymerase chain reaction (PCR) amplification of a single flow-sorted
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Directory of Open Access Journals (Sweden)
Anurag Kumar Sinha
2017-10-01
Full Text Available Marker frequency analysis of the Escherichia coli recB mutant chromosome has revealed a deficit of DNA in a specific zone of the terminus, centred on the dif/TerC region. Using fluorescence microscopy of a marked chromosomal site, we show that the dif region is lost after replication completion, at the time of cell division, in one daughter cell only, and that the phenomenon is transmitted to progeny. Analysis by marker frequency and microscopy shows that the position of DNA loss is not defined by the replication fork merging point since it still occurs in the dif/TerC region when the replication fork trap is displaced in strains harbouring ectopic Ter sites. Terminus DNA loss in the recB mutant is also independent of dimer resolution by XerCD at dif and of Topo IV action close to dif. It occurs in the terminus region, at the point of inversion of the GC skew, which is also the point of convergence of specific sequence motifs like KOPS and Chi sites, regardless of whether the convergence of GC skew is at dif (wild-type or a newly created sequence. In the absence of FtsK-driven DNA translocation, terminus DNA loss is less precisely targeted to the KOPS convergence sequence, but occurs at a similar frequency and follows the same pattern as in FtsK+ cells. Importantly, using ftsIts, ftsAts division mutants and cephalexin treated cells, we show that DNA loss of the dif region in the recB mutant is decreased by the inactivation of cell division. We propose that it results from septum-induced chromosome breakage, and largely contributes to the low viability of the recB mutant.
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Bourras, Salim; Meyer, Michel; Grandaubert, Jonathan; Lapalu, Nicolas; Fudal, Isabelle; Linglin, Juliette; Ollivier, Benedicte; Blaise, Françoise; Balesdent, Marie-Hélène; Rouxel, Thierry
2012-08-01
The ever-increasing generation of sequence data is accompanied by unsatisfactory functional annotation, and complex genomes, such as those of plants and filamentous fungi, show a large number of genes with no predicted or known function. For functional annotation of unknown or hypothetical genes, the production of collections of mutants using Agrobacterium tumefaciens-mediated transformation (ATMT) associated with genotyping and phenotyping has gained wide acceptance. ATMT is also widely used to identify pathogenicity determinants in pathogenic fungi. A systematic analysis of T-DNA borders was performed in an ATMT-mutagenized collection of the phytopathogenic fungus Leptosphaeria maculans to evaluate the features of T-DNA integration in its particular transposable element-rich compartmentalized genome. A total of 318 T-DNA tags were recovered and analyzed for biases in chromosome and genic compartments, existence of CG/AT skews at the insertion site, and occurrence of microhomologies between the T-DNA left border (LB) and the target sequence. Functional annotation of targeted genes was done using the Gene Ontology annotation. The T-DNA integration mainly targeted gene-rich, transcriptionally active regions, and it favored biological processes consistent with the physiological status of a germinating spore. T-DNA integration was strongly biased toward regulatory regions, and mainly promoters. Consistent with the T-DNA intranuclear-targeting model, the density of T-DNA insertion correlated with CG skew near the transcription initiation site. The existence of microhomologies between promoter sequences and the T-DNA LB flanking sequence was also consistent with T-DNA integration to host DNA mediated by homologous recombination based on the microhomology-mediated end-joining pathway.
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Directory of Open Access Journals (Sweden)
Sandra M Axiak-Bechtel
Full Text Available Fetal microchimerism has been suggested to play contradictory roles in women's health, with factors including age of the recipient, time elapsed since microchimerism occurred, and microchimeric cell type modulating disease. Both beneficial and harmful effects have been identified in wound healing and tissue regeneration, immune mediated disease, and cancer. This area of research is relatively new, and hindered by the time course from occurrence of fetal microchimerism to the multi-factorial development of disease. Dogs represent an excellent model for study of fetal microchimerism, as they share our environment, have a naturally condensed lifespan, and spontaneously develop immune-mediated diseases and cancers similar to their human counterparts. However, fetal microchimerism has not been described in dogs. These experiments sought preliminary evidence that dogs develop fetal microchimerism following pregnancy. We hypothesized that Y chromosomal DNA would be detected in the peripheral blood mononuclear cells of female dogs collected within two months of parturition. We further hypothesized that Y chromosomal DNA would be detected in banked whole blood DNA samples from parous female Golden Retrievers with at least one male puppy in a prior litter. Amplification of DNA extracted from five female Golden Retrievers that had whelped within the two months prior to collection revealed strong positive bands for the Y chromosome. Of banked, parous samples, 36% yielded positive bands for the Y chromosome. This is the first report of persistent Y chromosomal DNA in post-partum female dogs and these results suggest that fetal microchimerism occurs in the canine species. Evaluation of the contributions of fetal microchimeric cells to disease processes in dogs as a model for human disease is warranted.
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Axiak-Bechtel, Sandra M; Kumar, Senthil R; Hansen, Sarah A; Bryan, Jeffrey N
2013-01-01
Fetal microchimerism has been suggested to play contradictory roles in women's health, with factors including age of the recipient, time elapsed since microchimerism occurred, and microchimeric cell type modulating disease. Both beneficial and harmful effects have been identified in wound healing and tissue regeneration, immune mediated disease, and cancer. This area of research is relatively new, and hindered by the time course from occurrence of fetal microchimerism to the multi-factorial development of disease. Dogs represent an excellent model for study of fetal microchimerism, as they share our environment, have a naturally condensed lifespan, and spontaneously develop immune-mediated diseases and cancers similar to their human counterparts. However, fetal microchimerism has not been described in dogs. These experiments sought preliminary evidence that dogs develop fetal microchimerism following pregnancy. We hypothesized that Y chromosomal DNA would be detected in the peripheral blood mononuclear cells of female dogs collected within two months of parturition. We further hypothesized that Y chromosomal DNA would be detected in banked whole blood DNA samples from parous female Golden Retrievers with at least one male puppy in a prior litter. Amplification of DNA extracted from five female Golden Retrievers that had whelped within the two months prior to collection revealed strong positive bands for the Y chromosome. Of banked, parous samples, 36% yielded positive bands for the Y chromosome. This is the first report of persistent Y chromosomal DNA in post-partum female dogs and these results suggest that fetal microchimerism occurs in the canine species. Evaluation of the contributions of fetal microchimeric cells to disease processes in dogs as a model for human disease is warranted.
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Chechetkin, V R; Lobzin, V V
2017-08-07
Using state-of-the-art techniques combining imaging methods and high-throughput genomic mapping tools leaded to the significant progress in detailing chromosome architecture of various organisms. However, a gap still remains between the rapidly growing structural data on the chromosome folding and the large-scale genome organization. Could a part of information on the chromosome folding be obtained directly from underlying genomic DNA sequences abundantly stored in the databanks? To answer this question, we developed an original discrete double Fourier transform (DDFT). DDFT serves for the detection of large-scale genome regularities associated with domains/units at the different levels of hierarchical chromosome folding. The method is versatile and can be applied to both genomic DNA sequences and corresponding physico-chemical parameters such as base-pairing free energy. The latter characteristic is closely related to the replication and transcription and can also be used for the assessment of temperature or supercoiling effects on the chromosome folding. We tested the method on the genome of E. coli K-12 and found good correspondence with the annotated domains/units established experimentally. As a brief illustration of further abilities of DDFT, the study of large-scale genome organization for bacteriophage PHIX174 and bacterium Caulobacter crescentus was also added. The combined experimental, modeling, and bioinformatic DDFT analysis should yield more complete knowledge on the chromosome architecture and genome organization. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Directory of Open Access Journals (Sweden)
Roselaine Cristina Pereira
Full Text Available ABSTRACT: Chromosome doubling of Italian ryegrass genotypes ( Lolium multiflorum Lam. adapted to the brazilian edaphoclimatic conditions is an important strategy used by breeders and aims to obtain more vigorous genotypes with better forage quality and disease resistance. The effectiveness of chromosome doubling can be measured by genetic stability and fertility rates of plants over generations. However, a common problem in the polyploidization process is the regeneration of mixoploid plants that have impaired fertility and genetic stability. The objective of this study was to verify if progenies of recently tetraploidized plants remain stable regarding DNA content and chromosome number, over two generations. Progenies of L. multiflorum plants artificially tetraploidized with colchicine treatment were evaluated. Chromosome counting and estimates of the DNA content were used to evaluate the genetic stability. The percentage of tetraploid plants (4X increased over generations (18%, 34% and 91% in cycle 0, 1 and 2, respectively. All progenies identified as tetraploid by flow citometry showed variation in chromosome number (mixoploidy, but produced viable seeds. Results showed that stabilization in chromosome number and DNA content in tetraploidized plant progenies requires time and that the success of this procedure depends on a continuous and accurate screening and selection.
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DNA Probe Pooling for Rapid Delineation of Chromosomal Breakpoints
Energy Technology Data Exchange (ETDEWEB)
Lu, Chun-Mei; Kwan, Johnson; Baumgartner, Adolf; Weier, Jingly F.; Wang, Mei; Escudero, Tomas; Munne' , Santiago; Zitzelsberger, Horst F.; Weier, Heinz-Ulrich
2009-01-30
Structural chromosome aberrations are hallmarks of many human genetic diseases. The precise mapping of translocation breakpoints in tumors is important for identification of genes with altered levels of expression, prediction of tumor progression, therapy response, or length of disease-free survival as well as the preparation of probes for detection of tumor cells in peripheral blood. Similarly, in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) for carriers of balanced, reciprocal translocations benefit from accurate breakpoint maps in the preparation of patient-specific DNA probes followed by a selection of normal or balanced oocytes or embryos. We expedited the process of breakpoint mapping and preparation of case-specific probes by utilizing physically mapped bacterial artificial chromosome (BAC) clones. Historically, breakpoint mapping is based on the definition of the smallest interval between proximal and distal probes. Thus, many of the DNA probes prepared for multi-clone and multi-color mapping experiments do not generate additional information. Our pooling protocol described here with examples from thyroid cancer research and PGD accelerates the delineation of translocation breakpoints without sacrificing resolution. The turnaround time from clone selection to mapping results using tumor or IVF patient samples can be as short as three to four days.
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Delayed chromosomal instability caused by large deletion
International Nuclear Information System (INIS)
Ojima, M.; Suzuki, K.; Kodama, S.; Watanabe, M.
2003-01-01
Full text: There is accumulating evidence that genomic instability, manifested by the expression of delayed phenotypes, is induced by X-irradiation but not by ultraviolet (UV) light. It is well known that ionizing radiation, such as X-rays, induces DNA double strand breaks, but UV-light mainly causes base damage like pyrimidine dimers and (6-4) photoproducts. Although the mechanism of radiation-induced genomic instability has not been thoroughly explained, it is suggested that DNA double strand breaks contribute the induction of genomic instability. We examined here whether X-ray induced gene deletion at the hprt locus induces delayed instability in chromosome X. SV40-immortalized normal human fibroblasts, GM638, were irradiated with X-rays (3, 6 Gy), and the hprt mutants were isolated in the presence of 6-thioguanine (6-TG). A 2-fold and a 60-fold increase in mutation frequency were found by 3 Gy and 6 Gy irradiation, respectively. The molecular structure of the hprt mutations was determined by multiplex polymerase chain reaction of nine exons. Approximately 60% of 3 Gy mutants lost a part or the entire hprt gene, and the other mutants showed point mutations like spontaneous mutants. All 6 Gy mutants show total gene deletion. The chromosomes of the hprt mutants were analyzed by Whole Human Chromosome X Paint FISH or Xq telomere FISH. None of the point or partial gene deletion mutants showed aberrations of X-chromosome, however total gene deletion mutants induced translocations and dicentrics involving chromosome X. These results suggest that large deletion caused by DNA double strand breaks destabilizes chromosome structure, which may be involved in an induction of radiation-induced genomic instability
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Chitty, Lyn S; Hudgins, Louanne; Norton, Mary E
2018-02-01
Noninvasive prenatal testing (NIPT) using cell-free DNA (cfDNA) from maternal serum has been clinically available since 2011. This technology has revolutionized our ability to screen for the common aneuploidies trisomy 21 (Down syndrome), trisomy 18, and trisomy 13. More recently, clinical laboratories have offered screening for other chromosome abnormalities including sex chromosome abnormalities and copy number variants (CNV) without little published data on the sensitivity, specificity, and positive predictive value. In this debate, the pros and cons of performing prenatal screening via cfDNA for all chromosome abnormalities is discussed. At the time of the debate in 2017, the general consensus was that the literature does not yet support using this technology to screen for all chromosome abnormalities and that education is key for both providers and the patients so that the decision-making process is as informed as possible. © 2018 John Wiley & Sons, Ltd.
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DEFF Research Database (Denmark)
Lukas, Claudia; Savic, Velibor; Bekker-Jensen, Simon
2011-01-01
stress increases the frequency of chromosomal lesions that are transmitted to daughter cells. Throughout G1, these lesions are sequestered in nuclear compartments marked by p53-binding protein 1 (53BP1) and other chromatin-associated genome caretakers. We show that the number of such 53BP1 nuclear bodies...... increases after genetic ablation of BLM, a DNA helicase associated with dissolution of entangled DNA. Conversely, 53BP1 nuclear bodies are partially suppressed by knocking down SMC2, a condensin subunit required for mechanical stability of mitotic chromosomes. Finally, we provide evidence that 53BP1 nuclear...... bodies shield chromosomal fragile sites sequestered in these compartments against erosion. Together, these data indicate that restoration of DNA or chromatin integrity at loci prone to replication problems requires mitotic transmission to the next cell generations....
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Ponomarev, A. L.; Huff, J. L.; Cucinotta, F. A.
2011-01-01
Future long-tem space travel will face challenges from radiation concerns as the space environment poses health risk to humans in space from radiations with high biological efficiency and adverse post-flight long-term effects. Solar particles events may dramatically affect the crew performance, while Galactic Cosmic Rays will induce a chronic exposure to high-linear-energy-transfer (LET) particles. These types of radiation, not present on the ground level, can increase the probability of a fatal cancer later in astronaut life. No feasible shielding is possible from radiation in space, especially for the heavy ion component, as suggested solutions will require a dramatic increase in the mass of the mission. Our research group focuses on fundamental research and strategic analysis leading to better shielding design and to better understanding of the biological mechanisms of radiation damage. We present our recent effort to model DNA damage and tissue damage using computational models based on the physics of heavy ion radiation, DNA structure and DNA damage and repair in human cells. Our particular area of expertise include the clustered DNA damage from high-LET radiation, the visualization of DSBs (DNA double strand breaks) via DNA damage foci, image analysis and the statistics of the foci for different experimental situations, chromosomal aberration formation through DSB misrepair, the kinetics of DSB repair leading to a model-derived spectrum of chromosomal aberrations, and, finally, the simulation of human tissue and the pattern of apoptotic cell damage. This compendium of theoretical and experimental data sheds light on the complex nature of radiation interacting with human DNA, cells and tissues, which can lead to mutagenesis and carcinogenesis later in human life after the space mission.
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Gan, Yimei; Liu, Fang; Chen, Dan; Wu, Qiong; Qin, Qin; Wang, Chunying; Li, Shaohui; Zhang, Xiangdi; Wang, Yuhong; Wang, Kunbo
2013-01-01
We investigated the locations of 5S and 45S rDNA in Gossypium diploid A, B, D, E, F, G genomes and tetraploid genome (AD) using multi-probe fluorescent in situ hybridization (FISH) for evolution analysis in Gossypium genus. The rDNA numbers and sizes, and synteny relationships between 5S and 45S were revealed using 5S and 45S as double-probe for all species, and the rDNA-bearing chromosomes were identified for A, D and AD genomes with one more probe that is single-chromosome-specific BAC clone from G. hirsutum (A1D1). Two to four 45S and one 5S loci were found in diploid-species except two 5S loci in G. incanum (E4), the same as that in tetraploid species. The 45S on the 7th and 9th chromosomes and the 5S on the 9th chromosomes seemed to be conserved in A, D and AD genomes. In the species of B, E, F and G genomes, the rDNA numbers, sizes, and synteny relationships were first reported in this paper. The rDNA pattern agrees with previously reported phylogenetic history with some disagreements. Combined with the whole-genome sequencing data from G. raimondii (D5) and the conserved cotton karyotype, it is suggested that the expansion, decrease and transposition of rDNA other than chromosome rearrangements might occur during the Gossypium evolution.
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Hannes, Femke; Van Houdt, Jeroen; Quarrell, Oliver W; Poot, Martin; Hochstenbach, Ron; Fryns, Jean-Pierre; Vermeesch, Joris R
2010-12-01
Constitutional developmental disorders are frequently caused by terminal chromosomal deletions. The mechanisms and/or architectural features that might underlie those chromosome breakages remain largely unexplored. Because telomeres are the vital DNA protein complexes stabilizing linear chromosomes against chromosome degradation, fusion, and incomplete replication, those terminal-deleted chromosomes acquired new telomeres either by telomere healing or by telomere capture. To unravel the mechanisms leading to chromosomal breakage and healing, we sequenced nine chromosome 4p terminal deletion boundaries. A computational analysis of the breakpoint flanking region, including 12 previously published pure terminal breakage sites, was performed in order to identify architectural features that might be involved in this process. All terminal 4p truncations were likely stabilized by telomerase-mediated telomere healing. In the majority of breakpoints multiple genetic elements have a potential to induce secondary structures and an enrichment in replication stalling site motifs were identified. These findings suggest DNA replication stalling-induced chromosome breakage during early development is the first mechanistic step leading toward terminal deletion syndromes. © 2010 Wiley-Liss, Inc.
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Schalbetter, Stephanie A; Mansoubi, Sahar; Chambers, Anna L; Downs, Jessica A; Baxter, Jonathan
2015-08-18
Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein-DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein-DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin.
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DEFF Research Database (Denmark)
Malmström, Helena; Vretemark, Maria; Tillmar, Andreas
2012-01-01
-chromosomal and autosomal SNPs and compared the results with haplogroup frequencies of modern Swedes to investigate paternal relations. Possible maternal kinship was investigated by deep FLX-sequencing of overlapping mtDNA amplicons. The authenticity of the sequences was examined using data from independent extractions......, massive clonal data, the c-statistics, and real-time quantitative data. We show that the males carry the same Y-chromosomal haplogroup and thus we cannot reject a father-son type of relation. Further, as shown by the mtDNA analyses, none of the individuals are maternally related. We conclude...
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Terzoudi, Georgia I; Karakosta, Maria; Pantelias, Antonio; Hatzi, Vasiliki I; Karachristou, Ioanna; Pantelias, Gabriel
2015-11-01
Combination of next-generation DNA sequencing, single nucleotide polymorphism array analyses and bioinformatics has revealed the striking phenomenon of chromothripsis, described as complex genomic rearrangements acquired in a single catastrophic event affecting one or a few chromosomes. Via an unproven mechanism, it is postulated that mechanical stress causes chromosome shattering into small lengths of DNA, which are then randomly reassembled by DNA repair machinery. Chromothripsis is currently examined as an alternative mechanism of oncogenesis, in contrast to the present paradigm that considers a stepwise development of cancer. While evidence for the mechanism(s) underlying chromosome shattering during cancer development remains elusive, a number of hypotheses have been proposed to explain chromothripsis, including ionizing radiation, DNA replication stress, breakage-fusion-bridge cycles, micronuclei formation and premature chromosome compaction. In the present work, we provide experimental evidence on the mechanistic basis of chromothripsis and on how chromosomes can get locally shattered in a single catastrophic event. Considering the dynamic nature of chromatin nucleoprotein complex, capable of rapid unfolding, disassembling, assembling and refolding, we first show that chromatin condensation at repairing or replicating DNA sites induces the mechanical stress needed for chromosome shattering to ensue. Premature chromosome condensation is then used to visualize the dynamic nature of interphase chromatin and demonstrate that such mechanical stress and chromosome shattering can also occur in chromosomes within micronuclei or asynchronous multinucleate cells when primary nuclei enter mitosis. Following an aberrant mitosis, chromosomes could find themselves in the wrong place at the wrong time so that they may undergo massive DNA breakage and rearrangement in a single catastrophic event. Specifically, our results support the hypothesis that premature chromosome
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International Nuclear Information System (INIS)
Kruminya, A.R.; Kroshkina, V.G.; Yurov, Yu.B.; Aleksandrov, I.A.; Mitkevich, S.P.; Gindilis, V.M.
1988-01-01
The chromosomal distribution of the cloned PHS05 fragment of human alphoid DNA was studied by in situ hybridization in 38 individuals. It was shown that this DNA fraction is primarily localized in the pericentric regions of practically all chromosomes of the set. Significant interchromosomal differences and a weakly expressed interindividual polymorphism were discovered in the copying ability of this class of repeating DNA sequences; associations were not found between the results of hybridization and the pattern of Q-polymorphism
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Inter-chromosomal heterogeneity in the formation of radiation induced chromosomal aberrations
International Nuclear Information System (INIS)
Natarajan, A.T.; Vermeulen, S.; Boei, J.J.W.A.
1997-01-01
It is generally assumed that radiation induced chromosomal lesions are distributed randomly and repaired randomly among the genome. Recent studies using fluorescent in situ hybridization (FISH) and chromosome specific DNA libraries indicate that some chromosomes are more sensitive for radiation induced aberration formation than others. Chromosome No. 4 in human and chromosome No. 8 in Chinese hamster have been found to involve more in exchange aberrations than others, when calculated on the basis of their DNA content. Painting with arm specific chromosome libraries indicate that the frequencies of radiation induced intra-chromosome exchanges (i.e., between the arms of a chromosome, such as centric rings and inversions) are far in excess than one would expect on the basis of the frequencies of observed inter-chromosomal exchanges. The possible factors leading to the observed heterogeneity will be discussed
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Demarre, Gaëlle; Chattoraj, Dhruba K
2010-05-06
DNA adenine methylation is widely used to control many DNA transactions, including replication. In Escherichia coli, methylation serves to silence newly synthesized (hemimethylated) sister origins. SeqA, a protein that binds to hemimethylated DNA, mediates the silencing, and this is necessary to restrict replication to once per cell cycle. The methylation, however, is not essential for replication initiation per se but appeared so when the origins (oriI and oriII) of the two Vibrio cholerae chromosomes were used to drive plasmid replication in E. coli. Here we show that, as in the case of E. coli, methylation is not essential for oriI when it drives chromosomal replication and is needed for once-per-cell-cycle replication in a SeqA-dependent fashion. We found that oriII also needs SeqA for once-per-cell-cycle replication and, additionally, full methylation for efficient initiator binding. The requirement for initiator binding might suffice to make methylation an essential function in V. cholerae. The structure of oriII suggests that it originated from a plasmid, but unlike plasmids, oriII makes use of methylation for once-per-cell-cycle replication, the norm for chromosomal but not plasmid replication.
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Niemi, Marianna; Bläuer, Auli; Iso-Touru, Terhi; Harjula, Janne; Nyström Edmark, Veronica; Rannamäe, Eve; Lõugas, Lembi; Sajantila, Antti; Lidén, Kerstin; Taavitsainen, Jussi-Pekka
2015-01-01
Background Ancient DNA analysis offers a way to detect changes in populations over time. To date, most studies of ancient cattle have focused on their domestication in prehistory, while only a limited number of studies have analysed later periods. Conversely, the genetic structure of modern cattle populations is well known given the undertaking of several molecular and population genetic studies. Results Bones and teeth from ancient cattle populations from the North-East Baltic Sea region dated to the Prehistoric (Late Bronze and Iron Age, 5 samples), Medieval (14), and Post-Medieval (26) periods were investigated by sequencing 667 base pairs (bp) from the mitochondrial DNA (mtDNA) and 155 bp of intron 19 in the Y-chromosomal UTY gene. Comparison of maternal (mtDNA haplotypes) genetic diversity in ancient cattle (45 samples) with modern cattle populations in Europe and Asia (2094 samples) revealed 30 ancient mtDNA haplotypes, 24 of which were shared with modern breeds, while 6 were unique to the ancient samples. Of seven Y-chromosomal sequences determined from ancient samples, six were Y2 and one Y1 haplotype. Combined data including Swedish samples from the same periods (64 samples) was compared with the occurrence of Y-chromosomal haplotypes in modern cattle (1614 samples). Conclusions The diversity of haplogroups was highest in the Prehistoric samples, where many haplotypes were unique. The Medieval and Post-Medieval samples also show a high diversity with new haplotypes. Some of these haplotypes have become frequent in modern breeds in the Nordic Countries and North-Western Russia while other haplotypes have remained in only a few local breeds or seem to have been lost. A temporal shift in Y-chromosomal haplotypes from Y2 to Y1 was detected that corresponds with the appearance of new mtDNA haplotypes in the Medieval and Post-Medieval period. This suggests a replacement of the Prehistoric mtDNA and Y chromosomal haplotypes by new types of cattle. PMID:25992976
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Directory of Open Access Journals (Sweden)
María Poggio
2011-05-01
Full Text Available In the present work, we analysed the male meiosis, the content and distribution of heterochromatin and the number and location of nucleolus organizing regions in Microtomus lunifer (Berg, 1900 by means of standard technique, C- and fluorescent bandings, and fluorescent in situ hybridization with an 18S rDNA probe. This species is the second one cytogenetically analysed within the Hammacerinae. Its male diploid chromosome number is 31 (2n=28+X1X2Y, including a minute pair of m-chromosomes. The diploid autosomal number and the presence of m-chromosomes are similar to those reported in M. conspicillaris (Drury, 1782 (2n=28+XY. However, M. lunifer has a multiple sex chromosome system X1X2Y (male that could have originated by fragmentation of the ancestral X chromosome. Taking into account that M. conspicillaris and M. lunifer are the only two species within Reduviidae that possess m-chromosomes, the presence of this pair could be a synapomorphy for the species of this genus. C- and fluorescent bandings showed that the amount of heterochromatin in M. lunifer was small, and only a small CMA3 bright band was observed in the largest autosomal pair at one terminal region. FISH with the 18S rDNA probe demonstrated that ribosomal genes were terminally placed on the largest autosomal pair. Our present results led us to propose that the location of rDNA genes could be associated with variants of the sex chromosome systems in relation with a kind of the sex chromosome systems within this family. Furthermore, the terminal location of NOR in the largest autosomal pair allowed us to use it as a chromosome marker and, thus, to infer that the kinetic activity of both ends is not a random process, and there is an inversion of this activity.
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International Nuclear Information System (INIS)
Shaham, M.; Adler, B.; Ganguly, S.; Chaganti, R.S.K.
1987-01-01
Cultured cells from individuals affected with Fanconi anemia (FA) exhibit spontaneous chromosome breakage and hypersensitivity to the cell killing and clastogenic effects of the difunctional alkylating agent diepoxybutane (DEB). The authors report here the correction of both of these DEB-hypersensitivity phenotypes of FA cells achieved by cotransfection of normal placental of Chinese hamster lung cell DNA and the plasmid pSV2-neo-SVgpt. Transfectants were selected for clonogenic survival after treatment with DEB at a dose of 5 μgml. At this dose of DEB, the clonogenicity of normal fibroblasts was reduced to 50% and that of FA fibroblasts was reduced to zero. DEB-resistant (DEB/sup r/) colonies selected in this system exhibited a normal response to DEB-induced chromosome breakage and resistance to repeated DEB treatment. The neo and gpt sequences were detected by Southern blot analysis of DNA from one of four DEB/sup r/ colonies independently derived from transfection of human DNA and one of three DEB/sup r/ colonies independently derived from transfection of Chinese hamster DNA. The results demonstrate that DNA sequences that complement the two hallmark cellular phenotypes (cellular and chromosomal hypersensitivity to alkylating agents) of FA are present in human as well as Chinese hamster DNA. The cloning of these genes using transfection strategies can be expected to enable molecular characterization of FA
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Mlinarec, Jelena; Chester, Mike; Siljak-Yakovlev, Sonja; Papes, Drazena; Leitch, Andrew R; Besendorfer, Visnja
2009-01-01
The structure, abundance and location of repetitive DNA sequences on chromosomes can characterize the nature of higher plant genomes. Here we report on three new repeat DNA families isolated from Anemone hortensis L.; (i) AhTR1, a family of satellite DNA (stDNA) composed of a 554-561 bp long EcoRV monomer; (ii) AhTR2, a stDNA family composed of a 743 bp long HindIII monomer and; (iii) AhDR, a repeat family composed of a 945 bp long HindIII fragment that exhibits some sequence similarity to Ty3/gypsy-like retroelements. Fluorescence in-situ hybridization (FISH) to metaphase chromosomes of A. hortensis (2n = 16) revealed that both AhTR1 and AhTR2 sequences co-localized with DAPI-positive AT-rich heterochromatic regions. AhTR1 sequences occur at intercalary DAPI bands while AhTR2 sequences occur at 8-10 terminally located heterochromatic blocks. In contrast AhDR sequences are dispersed over all chromosomes as expected of a Ty3/gypsy-like element. AhTR2 and AhTR1 repeat families include polyA- and polyT-tracks, AT/TA-motifs and a pentanucleotide sequence (CAAAA) that may have consequences for chromatin packing and sequence homogeneity. AhTR2 repeats also contain TTTAGGG motifs and degenerate variants. We suggest that they arose by interspersion of telomeric repeats with subtelomeric repeats, before hybrid unit(s) amplified through the heterochromatic domain. The three repetitive DNA families together occupy approximately 10% of the A. hortensis genome. Comparative analyses of eight Anemone species revealed that the divergence of the A. hortensis genome was accompanied by considerable modification and/or amplification of repeats.
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Directory of Open Access Journals (Sweden)
R. K. Chahota
2011-11-01
Full Text Available The fluorescent in situ hybridization (FISH technique has been applied to somatic chromosomes in the medicinally important species, Bunium persicum, to elucidate its karyotypes. The bicolour FISH technique involving 18S-5.8S-26S and 5S ribosomal RNA genes as probes was used to assign physical localization and measurement of rDNA sites on homologous pairs of chromosomes. The two 18S-5.8S-26S rRNA gene sites were at the terminal regions of the short arms of the chromosomes 1 and 2 involving NOR region of chromosome 1. The 5S rDNA sites were found on subtelomeric region of the long arm of the chromosome number 5 and at interstitial regions of the short arm of chromosome 7. Based on direct visual analysis of chromosome length, morphology and position of FISH signals, a pioneer attempt has been made to construct metaphase karyotype in B. persicum, an endangered medicinal plant of North Western Himalayas.
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Chromosomal localization of the human diazepam binding inhibitor gene
International Nuclear Information System (INIS)
DeBernardi, M.A.; Crowe, R.R.; Mocchetti, I.; Shows, T.B.; Eddy, R.L.; Costa, E.
1988-01-01
The authors have used in situ chromosome hybridization and human-mouse somatic cell hybrids to map the gene(s) for human diazepam binding inhibitor (DBI), an endogenous putative modulator of the γ-aminobutyric acid receptor acting at the allosteric regulatory center of this receptor that includes the benzodiazepine recognition site. In 784 chromosome spreads hybridized with human DBI cDNA, the distribution of 1,476 labeled sites revealed a significant clustering of autoradiographic grains (11.3% of total label) on the long arm of chromosome 2 (2q). Furthermore, 63.5% of the grains found on 2q were located on 2q12-21, suggesting regional mapping of DBI gene(s) to this segment. Secondary hybridization signals were frequently observed on other chromosomes and they were statistically significant mainly for chromosomes 5, 6, 11, and 14. In addition, DNA from 32 human-mouse cell hybrids was digested with BamHI and probed with human DBI cDNA. A 3.5-kilobase band, which probably represents the human DBI gene, was assigned to chromosome 2. Four higher molecular weight bands, also detected in BamHI digests, could not be unequivocally assigned. A chromosome 2 location was excluded for the 27-, 13-, and 10-kilobase bands. These results assign a human DBI gene to chromosome 2 (2q12-21) and indicate that three of the four homologous sequences detected by the human DBI probe are located on three other chromosomes
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Directory of Open Access Journals (Sweden)
Olesya G. Buleu
2017-09-01
Full Text Available The karyotypes of three species of Pyrgomorphidae grasshoppers were studied: Zonocerus elegans (Thunberg, 1815, Pyrgomorpha guentheri (Burr, 1899 and Atractomorpha lata (Mochulsky, 1866. Data on karyotypes of P. guentheri and Z. elegans are reported here for the first time. All species have karyotypes consisting of 19 acrocentric chromosomes in males and 20 acrocentric chromosomes in females (2n♂=19, NF=19; 2n♀=20, NF=20 and X0/XX sex determination system. A comparative analysis of the localization of C-heterochromatin, clusters of ribosomal DNA, and telomere repeats revealed inter-species diversity in these cytogenetic markers. These differences indicate that the karyotype divergence in the species studied is not associated with structural chromosome rearrangements, but with the evolution of repeated DNA sequences.
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International Nuclear Information System (INIS)
Chehab, F.F.; Kan, Y.W.; Law, M.L.; Hartz, J.; Kao, F.T.; Blostein, R.
1987-01-01
A 2.2-kilobase clone comprising a major portion of the coding sequence of the Na + , K + -ATPase α subunit was cloned from human placenta and its sequence was identical to that encoding the α subunit of human kidney and HeLa cells. Transfer blot analysis of the mRNA products of the Na + , K + -ATPase gene from various human tissues and cell lines revealed only one band (≅ 4.7 kilobases) under low and high stringency washing conditions. The levels of expression in the tissues were intestine > placenta > liver > pancreas, and in the cell lines the levels were human erythroleukemia > butyrate-induced colon > colon > brain > HeLa cells. mRNA was undetectable in reticulocytes, consistent with the authors failure to detect positive clones in a size-selected ( > 2 kilobases) λgt11 reticulocyte cDNA library. DNA analysis revealed by a polymorphic EcoRI band and chromosome localization by flow sorting and in situ hybridization showed that the α subunit is on the short is on the short arm (band p11-p13) of chromosome 1
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DEFF Research Database (Denmark)
Bagger Jørgensen, Rikke; Chen, B.Y.; Cheng, B.F.
1996-01-01
The alien C-genome chromosome in a Brassica campestris-alboglabra monosomic addition line was characterized by random amplified polymorphic DNA (RAPD) analysis. The alien chromosome carried three loci, E(c), W-c and Lap-1C, controlling synthesis of erucic acid, white flower colour and a fast...
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Directory of Open Access Journals (Sweden)
Gaëlle Demarre
2010-05-01
Full Text Available DNA adenine methylation is widely used to control many DNA transactions, including replication. In Escherichia coli, methylation serves to silence newly synthesized (hemimethylated sister origins. SeqA, a protein that binds to hemimethylated DNA, mediates the silencing, and this is necessary to restrict replication to once per cell cycle. The methylation, however, is not essential for replication initiation per se but appeared so when the origins (oriI and oriII of the two Vibrio cholerae chromosomes were used to drive plasmid replication in E. coli. Here we show that, as in the case of E. coli, methylation is not essential for oriI when it drives chromosomal replication and is needed for once-per-cell-cycle replication in a SeqA-dependent fashion. We found that oriII also needs SeqA for once-per-cell-cycle replication and, additionally, full methylation for efficient initiator binding. The requirement for initiator binding might suffice to make methylation an essential function in V. cholerae. The structure of oriII suggests that it originated from a plasmid, but unlike plasmids, oriII makes use of methylation for once-per-cell-cycle replication, the norm for chromosomal but not plasmid replication.
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Rybalko, S.; Larionov, S.; Poptsova, M.; Loskutov, A.
2011-10-01
Large-scale dynamical properties of complete chromosome DNA sequences of eukaryotes are considered. Using the proposed deterministic models with intermittency and symbolic dynamics we describe a wide spectrum of large-scale patterns inherent in these sequences, such as segmental duplications, tandem repeats, and other complex sequence structures. It is shown that the recently discovered gene number balance on the strands is not of a random nature, and certain subsystems of a complete chromosome DNA sequence exhibit the properties of deterministic chaos.
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DNA immunization with a herpes simplex virus 2 bacterial artificial chromosome
International Nuclear Information System (INIS)
Meseda, Clement A.; Schmeisser, Falko; Pedersen, Robin; Woerner, Amy; Weir, Jerry P.
2004-01-01
Construction of a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) is described. BAC vector sequences were inserted into the thymidine kinase gene of HSV-2 by homologous recombination. DNA from cells infected with the resulting recombinant virus was transformed into E. coli, and colonies containing the HSV-2 BAC (HSV2-BAC) were isolated and analyzed for the expected genotype. HSV2-BAC DNA was infectious when transfected back into mammalian cells and the resulting virus was thymidine kinase negative. When used to immunize mice, the HSV2-BAC DNA elicited a strong HSV-2 specific antibody response that was equal to or greater than live virus immunization. Further, HSV2-BAC immunization was protective when animals were challenged with a lethal dose of virus. The utility of the HSV2-BAC for construction of recombinant virus genomes was demonstrated by elimination of the HSV-2 glycoprotein D (gD) gene. A recombinant HSV-2 BAC with the gD gene deleted was isolated and shown to be incapable of producing infectious virus following transfection unless an HSV gD gene was expressed in a complementing cell line. Immunization of mice with the HSV2 gD-BAC also elicited an HSV-2 specific antibody response and was protective. The results demonstrate the feasibility of DNA immunization with HSV-2 bacterial artificial chromosomes for replicating and nonreplicating candidate HSV-2 vaccines, as well as the utility of BAC technology for construction and maintenance of novel HSV-2 vaccines. The results further suggest that such technology will be a powerful tool for dissecting the immune response to HSV-2
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Mapping replication origins in yeast chromosomes.
Brewer, B J; Fangman, W L
1991-07-01
The replicon hypothesis, first proposed in 1963 by Jacob and Brenner, states that DNA replication is controlled at sites called origins. Replication origins have been well studied in prokaryotes. However, the study of eukaryotic chromosomal origins has lagged behind, because until recently there has been no method for reliably determining the identity and location of origins from eukaryotic chromosomes. Here, we review a technique we developed with the yeast Saccharomyces cerevisiae that allows both the mapping of replication origins and an assessment of their activity. Two-dimensional agarose gel electrophoresis and Southern hybridization with total genomic DNA are used to determine whether a particular restriction fragment acquires the branched structure diagnostic of replication initiation. The technique has been used to localize origins in yeast chromosomes and assess their initiation efficiency. In some cases, origin activation is dependent upon the surrounding context. The technique is also being applied to a variety of eukaryotic organisms.
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HindIII RFLP on chromosome 8 detected with a calbindin 27 kDa cDNA probe, HBSC21
Energy Technology Data Exchange (ETDEWEB)
Parmentier, M; Vassart, G
1988-10-11
A 1.8 kb for EcoRI fragment of the human calbindin cDNA clone HBSC21 was subcloned into M13mp18 and used as a probe. HindIII identifies a 2 allele polymorphism with a band at 4.7 kb (A1) and a band at 4.3 kb (A2). A constant band is located at 5.3 kb. The calbindin 27 kDa gene was assigned to chromosome 8 using chinese hamster-human and mouse-human cell hybrids. Co-dominant segregation was demonstrated in 3 families (total of 20 individuals).
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Chromosomal instability in women with primary ovarian insufficiency.
Katari, Sunita; Aarabi, Mahmoud; Kintigh, Angela; Mann, Susan; Yatsenko, Svetlana A; Sanfilippo, Joseph S; Zeleznik, Anthony J; Rajkovic, Aleksandar
2018-02-07
What is the prevalence of somatic chromosomal instability among women with idiopathic primary ovarian insufficiency (POI)? A subset of women with idiopathic POI may have functional impairment in DNA repair leading to chromosomal instability in their soma. The formation and repair of DNA double-strand breaks during meiotic recombination are fundamental processes of gametogenesis. Oocytes with compromised DNA integrity are susceptible to apoptosis which could trigger premature ovarian aging and accelerated wastage of the human follicle reserve. Genomewide association studies, as well as whole exome sequencing, have implicated multiple genes involved in DNA damage repair. However, the prevalence of defective DNA damage repair in the soma of women with POI is unknown. In total, 46 women with POI and 15 family members were evaluated for excessive mitomycin-C (MMC)-induced chromosome breakage. Healthy fertile females (n = 20) and two lymphoblastoid cell lines served as negative and as positive controls, respectively. We performed a pilot functional study utilizing MMC to assess chromosomal instability in the peripheral blood of participants. A high-resolution array comparative genomic hybridization (aCGH) was performed on 16 POI patients to identify copy number variations (CNVs) for a set of 341 targeted genes implicated in DNA repair. Array CGH revealed three POI patients (3/16, 18.8%) with pathogenic CNVs. Excessive chromosomal breakage suggestive of a constitutional deficiency in DNA repair was detected in one POI patient with the 16p12.3 duplication. In two patients with negative chromosome breakage analysis, aCGH detected a Xq28 deletion comprising the Centrin EF-hand Protein 2 (CETN2) and HAUS Augmin Like Complex Subunit 7 (HAUS7) genes essential for meiotic DNA repair, and a duplication in the 3p22.2 region comprising a part of the ATPase domain of the MutL Homolog 1 (MLH1) gene. Peripheral lymphocytes, used as a surrogate tissue to quantify induced chromosome
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Badro, Danielle A; Douaihy, Bouchra; Haber, Marc; Youhanna, Sonia C; Salloum, Angélique; Ghassibe-Sabbagh, Michella; Johnsrud, Brian; Khazen, Georges; Matisoo-Smith, Elizabeth; Soria-Hernanz, David F; Wells, R Spencer; Tyler-Smith, Chris; Platt, Daniel E; Zalloua, Pierre A
2013-01-01
The Middle East was a funnel of human expansion out of Africa, a staging area for the Neolithic Agricultural Revolution, and the home to some of the earliest world empires. Post LGM expansions into the region and subsequent population movements created a striking genetic mosaic with distinct sex-based genetic differentiation. While prior studies have examined the mtDNA and Y-chromosome contrast in focal populations in the Middle East, none have undertaken a broad-spectrum survey including North and sub-Saharan Africa, Europe, and Middle Eastern populations. In this study 5,174 mtDNA and 4,658 Y-chromosome samples were investigated using PCA, MDS, mean-linkage clustering, AMOVA, and Fisher exact tests of F(ST)'s, R(ST)'s, and haplogroup frequencies. Geographic differentiation in affinities of Middle Eastern populations with Africa and Europe showed distinct contrasts between mtDNA and Y-chromosome data. Specifically, Lebanon's mtDNA shows a very strong association to Europe, while Yemen shows very strong affinity with Egypt and North and East Africa. Previous Y-chromosome results showed a Levantine coastal-inland contrast marked by J1 and J2, and a very strong North African component was evident throughout the Middle East. Neither of these patterns were observed in the mtDNA. While J2 has penetrated into Europe, the pattern of Y-chromosome diversity in Lebanon does not show the widespread affinities with Europe indicated by the mtDNA data. Lastly, while each population shows evidence of connections with expansions that now define the Middle East, Africa, and Europe, many of the populations in the Middle East show distinctive mtDNA and Y-haplogroup characteristics that indicate long standing settlement with relatively little impact from and movement into other populations.
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International Nuclear Information System (INIS)
Natarajan, A.T.; Darroudi, F.; Vermeulen, S.; Wiegant, J.
1992-01-01
DNA libraries of six human chromosomes were used to detect translocations in human lymphocytes induced by different doses of X-rays and fast neutrons. Results show that with X-rays, one can detect about 1.5 to 2.0 fold more translocations in comparison to dicentrics, whereas following fast neutron irradiation, the difference between these two classes of aberrations are significantly different at high doses. In addition, triple fluorescent in situ hybridization technique was used to study the frequencies of radiation-induced translocations involving a specific chromosome. Chromosome number 1 was found to be involved in translocations more frequently than chromosomes number 2, 3, 4, 8 and X. (author). 10 refs., 1 fig., 2 tabs
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Styl RFLP recognized by a human IRBP cDNA localized to chromosome 10
Energy Technology Data Exchange (ETDEWEB)
Chin, K S; Mathew, C G.P.; Fong, S L; Bridges, C D; Ponder, B A.J.
1988-02-25
A 2184 bp cDNA (H.4 IRBP) encoding human interstitial retinol-biding protein isolated from a human retina cDNA library in lambdagt10 by screening with a bovine IRBP cDNA probe. Styl identifies a 2-allele polymorphism with bands at 2.3 kb (Cl) and 1.95 kb (C2) and invariant bands at 1.1, 1.0 and 0.8kb. Codominant segregation was observed in two informative families. The RFLP was mapped to chromosome 10 using somatic cell hybrids. In situ hybridization suggests regional assignments near p11.2 -q11.2 with a secondary site of hybridization at q24-25.
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Electochemical detection of chromosome translocation
DEFF Research Database (Denmark)
Kwasny, Dorota; Dimaki, Maria; Silahtaroglu, Asli
2014-01-01
Cytogenetics is a study of the cell structure with a main focus on chromosomes content and their structure. Chromosome abnormalities, such as translocations may cause various genetic disorders and heametological malignancies. Chromosome translocations are structural rearrangements of two...... chromosomes that results in formation of derivative chromosomes with a mixed DNA sequence. The method currently used for their detection is Fluorescent In Situ Hybridization, which requires a use of expensive, fluorescently labeled probes that target the derivative chromosomes. We present here a double...... hybridization approach developed for label-free detection of the chromosome translocations. For specific translocation detection it is necessary to determine that the two DNA sequences forming a derivative chromosome are connected, which is achieved by two subsequent hybridization steps. The electrochemical...
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Chen, H.; Rossier, C.; Lalioti, M. D.; Lynn, A.; Chakravarti, A.; Perrin, G.; Antonarakis, S. E.
1996-01-01
In an effort to contribute to the transcript map of human chromosome 21 and the understanding of the pathophysiology of trisomy 21, we have used exon trapping to identify fragments of chromosome 21 genes. Two trapped exons, from pools of chromosome 21-specific cosmids, showed homology to the Drosophila white (w) gene. We subsequently cloned the corresponding cDNA for a human homologue of the Drosophila w gene (hW) from human retina and fetal brain cDNA libraries. The gene belongs to the ATP-b...
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International Nuclear Information System (INIS)
Hahn, P.J.; Giddings, L.; Lane, M.J.
1989-01-01
Restriction mapping of relatively large genomes (e.g. human) utilizing randomly generated DNA segments requires high mapping redundancy to successfully organize 'contigs' to represent the entire genome. The number of independent DNA segment maps required is dependent on the average size of a mapping segment; the larger the segment, the fewer required. The authors have developed a strategy for subcloning intact multimegabase subchromosomal fragments as double minute chromosomes. Such fragments could serve as primary mapping elements or as adjunct (linking) fragments to rapidly connect already existent contigs generated using yeast artificial chromosomes or cosmids. They present several lines of evidence supporting the viability of this approach. (1) X-ray treated EMT-6 mouse cells (7.5 Gr.) which are selected over several months with increasing levels of methotrexate (MTX) contain highly amplified circular DNA molecules (double minutes) which include the dihydrofolate reductase (DHFR) gene in a size range between 1,000 and 3,500 kilobases as determined by pulsed-field gel electrophoresis and these acentric chromosomal fragments have been stably maintained in culture for at least a year. (2) Preliminary data based on experiments involving fusion of X-irradiated Chinese Hamster Ovary (CH0 DG44) cells containing randomly inserted cotransfected Neomycin resistance and DHFR genes to mouse EMT-6 cells shows that the linked genes can be readily cotransferred as acentric subchromosomal fragment(s) suitable for gene amplification. (3) The studies of CHO cells with cell fusion transferred X-ray induced chromosomal fragments containing the natural CHO DHFR gene suggest that transferred chromosome fragments undergo gene amplification much more readily than nonfragmented endogenous DHFR genes
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Energy Technology Data Exchange (ETDEWEB)
Warburton, P.E.; Gosden, J.; Lawson, D. [Western General Hospital, Edinburgh (United Kingdom)] [and others
1996-04-15
Alpha satellite DNA is a tandemly repeated DNA family found at the centromeres of all primate chromosomes examined. The fundamental repeat units of alpha satellite DNA are diverged 169- to 172-bp monomers, often found to be organized in chromosome-specific higher-order repeat units. The chromosomes of human (Homo sapiens (HSA)), chimpanzee (Pan troglodytes (PTR) and Pan paniscus), and gorilla (Gorilla gorilla) share a remarkable similarity and synteny. It is of interest to ask if alpha satellite arrays at centromeres of homologous chromosomes between these species are closely related (evolving in an orthologous manner) or if the evolutionary processes that homogenize and spread these arrays within and between chromosomes result in nonorthologous evolution of arrays. By using PCR primers specific for human chromosome 17-specific alpha satellite DNA, we have amplified, cloned, and characterized a chromosome-specific subset from the PTR chimpanzee genome. Hybridization both on Southern blots and in situ as well as sequence analysis show that this subset is most closely related, as expected, to sequences on HSA 17. However, in situ hybridization reveals that this subset is not found on the homologous chromosome in chimpanzee (PTR 19), but instead on PTR 12, which is homologous to HSA 2p. 40 refs., 3 figs.
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Human tissue factor: cDNA sequence and chromosome localization of the gene
International Nuclear Information System (INIS)
Scarpati, E.M.; Wen, D.; Broze, G.J. Jr.; Miletich, J.P.; Flandermeyer, R.R.; Siegel, N.R.; Sadler, J.E.
1987-01-01
A human placenta cDNA library in λgt11 was screened for the expression of tissue factor antigens with rabbit polyclonal anti-human tissue factor immunoglobulin G. Among 4 million recombinant clones screened, one positive, λHTF8, expressed a protein that shared epitopes with authentic human brain tissue factor. The 1.1-kilobase cDNA insert of λHTF8 encoded a peptide that contained the amino-terminal protein sequence of human brain tissue factor. Northern blotting identified a major mRNA species of 2.2 kilobases and a minor species of ∼ 3.2 kilobases in poly(A) + RNA of placenta. Only 2.2-kilobase mRNA was detected in human brain and in the human monocytic U937 cell line. In U937 cells, the quantity of tissue factor mRNA was increased several fold by exposure of the cells to phorbol 12-myristate 13-acetate. Additional cDNA clones were selected by hybridization with the cDNA insert of λHTF8. These overlapping isolates span 2177 base pairs of the tissue factor cDNA sequence that includes a 5'-noncoding region of 75 base pairs, an open reading frame of 885 base pairs, a stop codon, a 3'-noncoding region of 1141 base pairs, and a poly(a) tail. The open reading frame encodes a 33-kilodalton protein of 295 amino acids. The predicted sequence includes a signal peptide of 32 or 34 amino acids, a probable extracellular factor VII binding domain of 217 or 219 amino acids, a transmembrane segment of 23 acids, and a cytoplasmic tail of 21 amino acids. There are three potential glycosylation sites with the sequence Asn-X-Thr/Ser. The 3'-noncoding region contains an inverted Alu family repetitive sequence. The tissue factor gene was localized to chromosome 1 by hybridization of the cDNA insert of λHTF8 to flow-sorted human chromosomes
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Radiation-induced genomic instability driven by de novo chromosomal rearrangement hot spots
International Nuclear Information System (INIS)
Grosovsky, A.J.; Allen, R.N.; Moore, S.R.
2003-01-01
Genomic instability has become generally recognized as a critical contributor to tumor progression by generating the necessary number of genetic alterations required for expression of a clinically significant malignancy. Our study of chromosomal instability investigates the hypothesis that chromosomal rearrangements can generate novel breakage-prone sites, resulting in instability acting predominantly in cis. Here we present an analysis of the karyotypic distribution of instability associated chromosomal rearrangements in TK6 and derivative human lymphoblasts. Karyotypic analysis performed on a total of 455 independent clones included 183 rearrangements distributed among 100 separate unstable clones. The results demonstrate that the breakpoints of chromosomal rearrangements in unstable clones are non-randomly distributed throughout the genome. This pattern is statistically significant, and incompatible with expectations for random breakage associated with loss or alteration of a trans-acting factor. Furthermore, specific chromosomal breakage hot spots associated with instability have been identified; these occur in several independent unstable clones and are often repeatedly broken and rejoined during the outgrowth of an individual clone. In complimentary studies, genomic instability was generated without any exposure to a DNA-damaging agent, but rather by transfection with alpha heterochromatin DNA. In a prospective analysis, human-hamster hybrid AL cells containing a single human chromosome 11 were transfected with heterochromatic alpha DNA repeats and clones were analyzed by chromosome 11 painting. Transfection with alpha DNA was associated with karyotypic heterogeneity in 40% of clones examined; control transfections with plasmid alone did not lead to karyotypic heterogeneity
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Czech Academy of Sciences Publication Activity Database
Puterová, J.; Razumova, O.; Martínek, T.; Alexandrov, O.; Divashuk, M.; Kubát, Z.; Hobza, Roman; Karlov, G.; Kejnovský, E.
2017-01-01
Roč. 9, č. 1 (2017), s. 197-212 ISSN 1759-6653 R&D Projects: GA ČR GBP501/12/G090 Institutional support: RVO:61389030 Keywords : sex-chromosomes * repetitive sequences * silene-latifolia * molecular cytogenetics * arabidopsis-thaliana * genome size * evolution * organization * alignment * database * sex chromosomes * genome composition * chromosomal localization * repetitive DNA Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Plant sciences, botany Impact factor: 3.979, year: 2016
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Milani, Diogo; Palacios-Gimenez, Octavio M; Cabral-de-Mello, Diogo C
2017-01-01
In this study, we describe a strategy to determine the presence of B chromosomes in the living grasshopper Abracris flavolineata by FISH using U2 snDNA as a probe in interphase hemolymph nuclei. In individuals without B chromosomes, (0B) 2 dot signals were noticed, corresponding to A complement U2 snDNA clusters. In +1B and +2B individuals, 4 or 8 additional signals were noticed, respectively. In all cases, the absence or presence of 1 or 2 B chromosomes correlated in hemolymph and in somatic or germline tissues, validating the efficiency of the marker. Our data suggest that the B chromosome of A. flavolineata is present in all somatic tissues. B-carrying individuals showed the same number of B chromosomes in germ and somatic cells, suggesting that the B is mitotically stable. The marker was used to compare B chromosome frequency in the analyzed population with a sample collected previously, in order to test for B frequency changes and differences of B chromosome prevalence among sexes, but no statistically significant differences were noticed. The identification of living animals harboring B chromosomes will be very useful in future studies of B chromosome transmission, as well as in functional studies involving RNA analysis, thus contributing to the understanding of evolutionary history and the possible role of the B chromosome in A. flavolineata. © 2017 S. Karger AG, Basel.
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Directory of Open Access Journals (Sweden)
Darija Strah
2015-12-01
Full Text Available Background: Chorionic villus sampling and amniocentesis as definitive diagnostic procedures represent a gold standard for prenatal diagnosis of chromosomal abnormalities. The methods are invasive and lead to a miscarriage and fetal loss in approximately 0.5–1 %. Non-invasive prenatal DNA testing (NIPT is based on the analysis of cell-free fetal DNA from maternal blood. It represents a highly accurate screening test for detecting the most common fetal chromosomal abnormalities. In our study we present the results of NIPT testing in the Diagnostic Center Strah, Slovenia, over the last 3 years.Methods: In our study, 123 pregnant women from 11th to 18th week of pregnancy were included. All of them had First trimester assessment of risk for trisomy 21, done before NIPT testing.Results: 5 of total 6 high-risk NIPT cases (including 3 cases of Down syndrome and 2 cases of Klinefelter’s syndrome were confirmed by fetal karyotyping. One case–Edwards syndrome was false positive. Patau syndrome, triple X syndrome or Turner syndrome were not observed in any of the cases. Furthermore, there were no false negative cases reported. In general, NIPT testing had 100 % sensitivity (95 % confidence interval: 46.29 %–100.00 % and 98.95 % specificity (95 % confidence interval: 93.44 %–99.95 %. In determining Down syndrome alone, specificity (95 % confidence interval: 95.25 %- 100.00 % and sensitivity (95 % confidence interval: 31.00 %–100.00 % turned out to be 100 %. In 2015, the average turnaround time for analysis was 8.3 days from the day when the sample was taken. Repeated blood sampling was required in 2 cases (redraw rate = 1.6 %.Conclusions: Our results confirm that NIPT represents a fast, safe and highly accurate advanced screening test for most common chromosomal abnormalities. In current clinical practice, NIPT would significantly decrease the number of unnecessary invasive procedures and the rate of fetal
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International Nuclear Information System (INIS)
Okayasu, Ryuichi; Okada, Maki; Noguchi, Mitsuho; Saito, Shiori; Okabe, Atsushi; Takakura, Kahoru
2005-01-01
We have studied cell survival and chromosome damage/repair in normal and non homologous end-joining (NHEJ) deficient human cells exposed to carbon ions (290 MeV/u, ∼70 keV/um), iron ions (500 MeV/u, ∼200 keV/um) and X-rays. In order to examine the effect of heavy ion on double strand break (DSB) repair machinery, the auto-phosphorylation of DNA-PKcs was also investigated. The important discoveries made during this period are: 200 keV/um iron irradiation induced additional molecular damage beyond that 70 keV/um carbon did. Iron irradiation not only caused an inefficient G1 chromosome repair, but also induced non-repairable DSB/chromosome damage. The auto-phosphorylation of DNA-PKcs was significantly affected by high linear energy transfer (LET) irradiation when compared to X-rays. These results indicate NHEJ machinery was markedly disturbed by high LET radiation when compared to low LET radiation. (author)
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Accumulation of chloroplast DNA sequences on the Y chromosome of Silene latifolia
Czech Academy of Sciences Publication Activity Database
Kejnovský, Eduard; Kubát, Zdeněk; Hobza, Roman; Lengerová, Martina; Sato, S.; Tabata, S.; Fukui, K.; Matsunaga, S.; Vyskot, Boris
2006-01-01
Roč. 128, 1-3 (2006), s. 167-175 ISSN 0016-6707 R&D Projects: GA ČR(CZ) GA204/05/2097; GA ČR(CZ) GD204/05/H505; GA AV ČR(CZ) 1QS500040507 Institutional research plan: CEZ:AV0Z50040507 Keywords : accumulation * chloroplast DNA * Y chromosome Subject RIV: BO - Biophysics Impact factor: 1.492, year: 2006
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Directory of Open Access Journals (Sweden)
Haishan Wang
Full Text Available We report on novel chromosomal characteristics of Haliotis discus hannai from a breeding population at Fujian, China. The karyotypes of H. discus hannai we obtained from an abalone farm include a common type 2n = 36 = 10M + 8SM (82% and two rare types 2n = 36 = 11M + 7SM (14% and 2n = 36 = 10M + 7SM + 1ST (4%. The results of silver staining showed that the NORs of H. discus hannai were usually located terminally on the long arms of chromosome pairs 14 and 17, NORs were also sometimes located terminally on the short arms of other chromosomes, either metacentric or submetacentric pairs. The number of Ag-nucleoli ranged from 2 to 8, and the mean number was 3.61 ± 0.93. Among the scored interphase cells, 41% had 3 detectable nucleoli and 37% had 4 nucleoli. The 18S rDNA FISH result is the first report of the location of 18S rDNA genes in H. discus hannai. The 18S rDNA locations were highly polymorphic in this species. Copies of the gene were observed in the terminal of long or/and short arms of submetacentric or/and metacentric chromosomes. Using FISH with probe for vertebrate-like telomeric sequences (CCCTAA3 displayed positive green FITC signals at telomere regions of all analyzed chromosome types. We found about 7% of chromosomes had breaks in prophase. A special form of nucleolus not previously described from H. discus hannai was observed in some interphase cells. It consists of many small silver-stained nucleoli gathered together to form a larger nucleolus and may correspond to prenucleolar bodies.
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International Nuclear Information System (INIS)
Waye, J.S.; Willard, H.F.
1989-01-01
The authors describe a class of human repetitive DNA, called β satellite, that, at a most fundamental level, exists as tandem arrays of diverged ∼68-base-pair monomer repeat units. The monomer units are organized as distinct subsets, each characterized by a multimeric higher-order repeat unit that is tandemly reiterated and represents a recent unit of amplification. They have cloned, characterized, and determined the sequence of two β satellite higher-order repeat units: one located on chromosome 9, the other on the acrocentric chromosomes (13, 14, 15, 21, and 22) and perhaps other sites in the genome. Analysis by pulsed-field gel electrophoresis reveals that these tandem arrays are localized in large domains that are marked by restriction fragment length polymorphisms. In total, β-satellite sequences comprise several million base pairs of DNA in the human genome. Analysis of this DNA family should permit insights into the nature of chromosome-specific and nonspecific modes of satellite DNA evolution and provide useful tools for probing the molecular organization and concerted evolution of the acrocentric chromosomes
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Rad54 and Mus81 cooperation promotes DNA damage repair and restrains chromosome missegregation
DEFF Research Database (Denmark)
Ghamrasni, S El; Cardoso, R; Li, L
2016-01-01
. The inefficient repair of DNA double-strand breaks (DSBs) in Rad54(-/-)Mus81(-/-) cells was accompanied by elevated levels of chromosome missegregation and cell death. Perhaps as a consequence, tumor incidence in Rad54(-/-)Mus81(-/-) mice remained comparable to that in Mus81(-/-) mice. Our study highlights...
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Binding of Multiple Rap1 Proteins Stimulates Chromosome Breakage Induction during DNA Replication.
Directory of Open Access Journals (Sweden)
Greicy H Goto
2015-08-01
Full Text Available Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB induction at both telomeric and non-telomeric regions. Consistent with the role of DSB induction, Rap1 stimulates nearby recombination events in a dosage-dependent manner. Rap1 recruits Rif1 and Rif2 to telomeres, but neither Rif1 nor Rif2 is required for DSB induction. Rap1-mediated DSB induction involves replication fork progression but inactivation of checkpoint kinase Mec1 does not affect DSB induction. Rap1 tethering shortens artificially elongated telomeres in parallel with telomerase inhibition, and this telomere shortening does not require homologous recombination. These results suggest that Rap1 contributes to telomere homeostasis by promoting chromosome breakage.
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Directory of Open Access Journals (Sweden)
E. Coluccia
2011-05-01
Full Text Available Muraenidae is a species-rich family, with relationships among genera and species and taxonomy that have not been completely clarified. Few cytogenetic studies have been conducted on this family, and all of them showed the same diploid chromosome number (2n=42 but with conspicuous karyotypic variation among species. The Mediterranean moray eel Gymnothorax unicolor was previously cytogenetically studied using classical techniques that allowed the characterization of its karyotype structure and the constitutive heterochromatin and argyrophilic nucleolar organizer regions (Ag-NORs distribution pattern. In the present study, we describe two new repetitive elements (called GuMboI and GuDdeI obtained from restricted genomic DNA of G. unicolor that were characterized by Southern blot and physically localized by in situ hybridization on metaphase chromosomes. As they are highly repetitive DNA sequences, they map in heterochromatic regions. However, while GuDdeI was localized in the centromeric regions, the GuMboI fraction was distributed on some centromeres and was co-localized with the nucleolus organizer region (NOR. Comparative analysis with other Mediterranean species such as Muraena helena pointed out that these DNA fractions are species-specific and could potentially be used for species discrimination. As a new contribution to the karyotype of this species, we found that the major ribosomal genes are localized on acrocentric chromosome 9 and that the telomeres of each chromosome are composed of a tandem repeat derived from a poly-TTAGGG DNA sequence, as it occurs in most vertebrate species. The results obtained add new information useful in comparative genomics at the chromosomal level and contribute to the cytogenetic knowledge regarding this fish family, which has not been extensively studied.
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Directory of Open Access Journals (Sweden)
Dimitrios Ioannou
Full Text Available The higher-order organization of chromatin is well-established, with chromosomes occupying distinct positions within the interphase nucleus. Chromatin is susceptible to, and constantly assaulted by both endogenous and exogenous threats. However, the effects of DNA damage on the spatial topology of chromosomes are hitherto, poorly understood. This study investigates the organization of all 24 human chromosomes in lymphocytes from six individuals prior to- and following in-vitro exposure to genotoxic agents: hydrogen peroxide and ultraviolet B. This study is the first to report reproducible distinct hierarchical radial organization of chromosomes with little inter-individual differences between subjects. Perturbed nuclear organization was observed following genotoxic exposure for both agents; however a greater effect was observed for hydrogen peroxide including: 1 More peripheral radial organization; 2 Alterations in the global distribution of chromosomes; and 3 More events of chromosome repositioning (18 events involving 10 chromosomes vs. 11 events involving 9 chromosomes for hydrogen peroxide and ultraviolet B respectively. Evidence is provided of chromosome repositioning and altered nuclear organization following in-vitro exposure to genotoxic agents, with notable differences observed between the two investigated agents. Repositioning of chromosomes following genotoxicity involved recurrent chromosomes and is most likely part of the genomes inherent response to DNA damage. The variances in nuclear organization observed between the two agents likely reflects differences in mobility and/or decondensation of chromatin as a result of differences in the type of DNA damage induced, chromatin regions targeted, and DNA repair mechanisms.
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Energy Technology Data Exchange (ETDEWEB)
Hwang, Gyoyeon [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biological Chemistry, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Deajeon (Korea, Republic of); Lee, Hansol [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Lee, Jiyeon, E-mail: jylee@kist.re.kr [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biological Chemistry, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Deajeon (Korea, Republic of)
2015-11-13
The telomere shortening in chromosomes implies the senescence, apoptosis, or oncogenic transformation of cells. Since detecting telomeres in aging and diseases like cancer, is important, the direct detection of telomeres has been a very useful biomarker. We propose a telomere detection method using a newly synthesized quantum dot (QD) based probe with oligonucleotide conjugation and direct fluorescence in situ hybridization (FISH). QD-oligonucleotides were prepared with metal coordination bonding based on platinum-guanine binding reported in our previous work. The QD-oligonucleotide conjugation method has an advantage where any sequence containing guanine at the end can be easily bound to the starting QD-Pt conjugate. A synthesized telomeric oligonucleotide was bound to the QD-Pt conjugate successfully and this probe hybridized specifically on the telomere of fabricated MV-4-11 and MOLT-4 chromosomes. Additionally, the QD-telomeric oligonucleotide probe successfully detected the telomeres on the CGH metaphase slide. Due to the excellent photostability and high quantum yield of QDs, the QD-oligonucleotide probe has high fluorescence intensity when compared to the organic dye-oligonucleotide probe. Our QD-oligonucleotide probe, conjugation method of this QD probe, and hybridization protocol with the chromosomes can be a useful tool for chromosome painting and FISH. - Highlights: • We prepared a probe linked between QD and telomeric oligonucleotide with platinum-guanine bonding. • Telomeres were detected by our new telomere probes successfully in three different human metaphase chromosomes. • QDPt-DNA probe has high fluorescence intensity in comparison with organic dye-DNA probe.
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Mannens, M.; Slater, R. M.; Heyting, C.; Geurts van Kessel, A.; Goedde-Salz, E.; Frants, R. R.; van Ommen, G. J.; Pearson, P. L.
1987-01-01
We are interested in the precise localization of various DNA probes on the short arm of chromosome 11 for our research on the aniridia-Wilms' tumor association (AWTA), assigned to region 11p13 (Knudson and Strong 1972; Riccardi et al. 1978). For this purpose we have screened lymphocyte DNA and
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CGG repeats associated with fragile X chromosome form left-handed Z-DNA structure
Czech Academy of Sciences Publication Activity Database
Renčiuk, Daniel; Kypr, Jaroslav; Vorlíčková, Michaela
2011-01-01
Roč. 95, č. 3 (2011), s. 174-181 ISSN 0006-3525 R&D Projects: GA ČR(CZ) GA202/07/0094; GA AV ČR(CZ) IAA100040701 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : fragile X chromosome syndrome * Z-DNA * trinucleotide repeats Subject RIV: BO - Biophysics Impact factor: 2.870, year: 2011
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Chromosome numbers and DNA content in some species of Mecardonia (Gratiolae, Plantaginaceae)
Sosa, María M.; Angulo, María B.; Greppi, Julián A.; Bugallo, Verónica
2016-01-01
Abstract Cytogenetic characterization and determination of DNA content by flow cytometry of five species of Mecardonia Ruiz et Pavon, 1798 (Gratiolae, Plantaginaceae) was performed. This is the first study of nuclear DNA content carried out in the genus. Mitotic analysis revealed a base chromosome number x = 11 for all entities and different ploidy levels, ranging from diploid (2n = 2x = 22) to hexaploid (2n = 6x = 66). The results include the first report of the chromosome numbers for Mecardonia flagellaris (Chamisso & Schlechtendal, 1827) (2n = 22), Mecardonia grandiflora (Bentham) Pennell, 1946 (2n = 22), Mecardonia kamogawae Greppi & Hagiwara, 2011 (2n = 66), and Mecardonia sp. (2n = 44). The three ploidy levels here reported suggest that polyploidy is common in Mecardonia and appear to be an important factor in the evolution of this genus. The 2C- and 1Cx-values were also estimated in all the species. The 2C-values ranged from 1.91 to 5.29 pg. The 1Cx-values ranged from 0.88 to 1.03 pg. The general tendency indicated a decrease in the 1Cx-value with increasing ploidy level. The significance of the results is discussed in relation to taxonomy of the genus. PMID:28123693
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Extensive duplication of the Wolbachia DNA in chromosome four of Drosophila ananassae.
Klasson, Lisa; Kumar, Nikhil; Bromley, Robin; Sieber, Karsten; Flowers, Melissa; Ott, Sandra H; Tallon, Luke J; Andersson, Siv G E; Dunning Hotopp, Julie C
2014-12-12
Lateral gene transfer (LGT) from bacterial Wolbachia endosymbionts has been detected in ~20% of arthropod and nematode genome sequencing projects. Many of these transfers are large and contain a substantial part of the Wolbachia genome. Here, we re-sequenced three D. ananassae genomes from Asia and the Pacific that contain large LGTs from Wolbachia. We find that multiple copies of the Wolbachia genome are transferred to the Drosophila nuclear genome in all three lines. In the D. ananassae line from Indonesia, the copies of Wolbachia DNA in the nuclear genome are nearly identical in size and sequence yielding an even coverage of mapped reads over the Wolbachia genome. In contrast, the D. ananassae lines from Hawaii and India show an uneven coverage of mapped reads over the Wolbachia genome suggesting that different parts of these LGTs are present in different copy numbers. In the Hawaii line, we find that this LGT is underrepresented in third instar larvae indicative of being heterochromatic. Fluorescence in situ hybridization of mitotic chromosomes confirms that the LGT in the Hawaii line is heterochromatic and represents ~20% of the sequence on chromosome 4 (dot chromosome, Muller element F). This collection of related lines contain large lateral gene transfers composed of multiple Wolbachia genomes that constitute >2% of the D. ananassae genome (~5 Mbp) and partially explain the abnormally large size of chromosome 4 in D. ananassae.
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Home collection of ejaculated semen would facilitate participation rates and geographic diversity in reproductive epidemiology studies. Our study addressed concerns that home collection and overnight mail return might induce chromosome/DNA damage. We collected semen from 10 hea...
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Filipino DNA variation at 12 X-chromosome short tandem repeat markers.
Salvador, Jazelyn M; Apaga, Dame Loveliness T; Delfin, Frederick C; Calacal, Gayvelline C; Dennis, Sheila Estacio; De Ungria, Maria Corazon A
2018-06-08
Demands for solving complex kinship scenarios where only distant relatives are available for testing have risen in the past years. In these instances, other genetic markers such as X-chromosome short tandem repeat (X-STR) markers are employed to supplement autosomal and Y-chromosomal STR DNA typing. However, prior to use, the degree of STR polymorphism in the population requires evaluation through generation of an allele or haplotype frequency population database. This population database is also used for statistical evaluation of DNA typing results. Here, we report X-STR data from 143 unrelated Filipino male individuals who were genotyped via conventional polymerase chain reaction-capillary electrophoresis (PCR-CE) using the 12 X-STR loci included in the Investigator ® Argus X-12 kit (Qiagen) and via massively parallel sequencing (MPS) of seven X-STR loci included in the ForenSeq ™ DNA Signature Prep kit of the MiSeq ® FGx ™ Forensic Genomics System (Illumina). Allele calls between PCR-CE and MPS systems were consistent (100% concordance) across seven overlapping X-STRs. Allele and haplotype frequencies and other parameters of forensic interest were calculated based on length (PCR-CE, 12 X-STRs) and sequence (MPS, seven X-STRs) variations observed in the population. Results of our study indicate that the 12 X-STRs in the PCR-CE system are highly informative for the Filipino population. MPS of seven X-STR loci identified 73 X-STR alleles compared with 55 X-STR alleles that were identified solely by length via PCR-CE. Of the 73 sequence-based alleles observed, six alleles have not been reported in the literature. The population data presented here may serve as a reference Philippine frequency database of X-STRs for forensic casework applications. Copyright © 2018 Elsevier B.V. All rights reserved.
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Wood, S; Starr, T V; Shukin, R J
1986-01-01
We have used a recombinant DNA library constructed from flow-sorted human chromosome 8 as a source of single-copy human probes. These probes have been screened for restriction fragment length polymorphism (RFLP) by hybridization to Southern transfers of genomic DNA from five unrelated individuals. We have detected six RFLPs distributed among four probes after screening 741 base pairs for restriction site variation. These RFLPs all behave as codominant Mendelian alleles. Two of the probes detect rare variants, while the other two detect RFLPs with PIC values of .36 and .16. Informative probes will be useful for the construction of a linkage map for chromosome 8 and for the localization of mutant alleles to this chromosome. Images Fig. 1 PMID:2879441
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Ohmine, Yuta; Satoh, Yukari; Kiyokawa, Kazuya; Yamamoto, Shinji; Moriguchi, Kazuki; Suzuki, Katsunori
2016-04-02
Plant pathogenic Agrobacterium strains can transfer T-DNA regions of their Ti plasmids to a broad range of eukaryotic hosts, including fungi, in vitro. In the recent decade, the yeast Saccharomyces cerevisiae is used as a model host to reveal important host proteins for the Agrobacterium-mediated transformation (AMT). Further investigation is required to understand the fundamental mechanism of AMT, including interaction at the cell surface, to expand the host range, and to develop new tools. In this study, we screened a yeast mutant library for low AMT mutant strains by advantage of a chromosome type T-DNA, which transfer is efficient and independent on integration into host chromosome. By the mutant screening, we identified four mutant strains (srs2Δ, rad52Δ, smi1Δ and erg28Δ), which showed considerably low AMT efficiency. Structural analysis of T-DNA product replicons in AMT colonies of mutants lacking each of the two DNA repair genes, SRS2 and RAD52, suggested that the genes act soon after T-DNA entry for modification of the chromosomal T-DNA to stably maintain them as linear replicons and to circularize certain T-DNA simultaneously. The cell wall synthesis regulator SMI1 might have a role in the cell surface interaction between the donor and recipient cells, but the smi1Δ mutant exhibited pleiotropic effect, i.e. low effector protein transport as well as low AMT for the chromosomal T-DNA, but relatively high AMT for integrative T-DNAs. The ergosterol synthesis regulator/enzyme-scaffold gene ERG28 probably contributes by sensing a congested environment, because growth of erg28Δ strain was unaffected by the presence of donor bacterial cells, while the growth of the wild-type and other mutant yeast strains was suppressed by their presence. RAD52 and the DNA helicase/anti-recombinase gene SRS2 are necessary to form and maintain artificial chromosomes through the AMT of chromosomal T-DNA. A sterol synthesis scaffold gene ERG28 is important in the high
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Gametocidal chromosomes enhancing chromosome aberration in common wheat induced by 5-azacytidine.
Su, W-Y; Cong, W-W; Shu, Y-J; Wang, D; Xu, G-H; Guo, C-H
2013-07-08
The gametocidal (Gc) chromosome from Aegilops spp induces chromosome mutation, which is introduced into common wheat as a tool of chromosome manipulation for genetic improvement. The Gc chromosome functions similar to a restriction-modification system in bacteria, in which DNA methylation is an important regulator. We treated root tips of wheat carrying Gc chromosomes with the hypomethylation agent 5-azacytidine; chromosome breakage and micronuclei were observed in these root tips. The frequency of aberrations differed in wheat containing different Gc chromosomes, suggesting different functions inducing chromosome breakage. Gc chromosome 3C caused the greatest degree of chromosome aberration, while Gc chromosome 3C(SAT) and 2C caused only slight chromosome aberration. Gc chromosome 3C induced different degrees of chromosome aberration in wheat varieties Triticum aestivum var. Chinese Spring and Norin 26, demonstrating an inhibition function in common wheat.
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Energy Technology Data Exchange (ETDEWEB)
Chehab, F.F.; Kan, Y.W.; Law, M.L.; Hartz, J.; Kao, F.T.; Blostein, R.
1987-11-01
A 2.2-kilobase clone comprising a major portion of the coding sequence of the Na/sup +/, K/sup +/-ATPase ..cap alpha.. subunit was cloned from human placenta and its sequence was identical to that encoding the ..cap alpha.. subunit of human kidney and HeLa cells. Transfer blot analysis of the mRNA products of the Na/sup +/, K/sup +/-ATPase gene from various human tissues and cell lines revealed only one band (approx. = 4.7 kilobases) under low and high stringency washing conditions. The levels of expression in the tissues were intestine > placenta > liver > pancreas, and in the cell lines the levels were human erythroleukemia > butyrate-induced colon > colon > brain > HeLa cells. mRNA was undetectable in reticulocytes, consistent with the authors failure to detect positive clones in a size-selected ( > 2 kilobases) lambdagt11 reticulocyte cDNA library. DNA analysis revealed by a polymorphic EcoRI band and chromosome localization by flow sorting and in situ hybridization showed that the ..cap alpha.. subunit is on the short is on the short arm (band p11-p13) of chromosome 1.
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Chromosomal instability induced by ionizing radiation
International Nuclear Information System (INIS)
Morgan, W.F.; Marder, B.A.; Day, J.P.
1995-01-01
There is accumulating evidence indicating genomic instability can manifest multiple generations after cellular exposure to DNA damaging agents. For instance, some cells surviving exposure to ionizing radiations show delayed reproductive cell death, delayed mutation and / or delayed chromosomal instability. Such instability, especially chromosome destabilization has been implicated in mutation, gene amplification, cellular transformation, and cell killing. To investigate chromosomal instability following DNA damage, we have used fluorescence in situ hybridization to detect chromosomal rearrangements in a human/hamster somatic hybrid cell line following exposure to ionizing radiation. Delayed chromosomal instability was detected when multiple populations of uniquely arranged metaphases were observed in clonal isolates raised from single cells. The relationship between delayed chromosomal destabilization and other endpoints of genomic instability, namely; delayed mutation and gene amplification will be discussed, as will the potential cytogenetic and molecular mechanisms contributing to delayed chromosomal instability
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Directory of Open Access Journals (Sweden)
Jyoti K. Jha
2017-04-01
Full Text Available Replication of Vibrio cholerae chromosome 2 (Chr2 depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations in rctB that reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in a dimerization domain which is folded similarly to the initiator of an iteron plasmid—the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2 initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding.
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Energy Technology Data Exchange (ETDEWEB)
1993-12-31
This volume contains the abstracts of oral and poster presentations made at the LVIII Cold Spring Harbor Symposium on Quantitative Biology entitles DNA & Chromosomes. The meeting was held June 2--June 9, 1993 at Cold Spring Harbor, New York.
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Different radiosensitization effects of the halogenated compounds on the human chromosome in vitro
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Kang, Y.S.
1976-01-01
Unscheduled DNA synthesis and chromosome aberrations were compared following X- or UV-irradiation or methyl methanesulfonate treatment in cultures of HeLa S 3 or KB cells or human and rabbit lymphocytes. The sensitization by incorporation of the halouridines BUdR and IUdR was also investigated. Unscheduled DNA synthesis occurred in two established cell lines after irradiation with 0 to 10 kR of X-rays. The rate of unscheduled synthesis was dose dependent and differed for the two cell lines. The unscheduled synthesis was not correlated with the modal chromosome number nor with the number of aberrations produced. UV-irradiated rabbit lymphocytes exhibited unscheduled DNA synthesis which saturated after a dose of 250 ergs/mm 2 . In contrast the incorporation of BUdR or IUdR eliminated this saturation and caused an increasing effect with increasing dose up to 1000 ergs/mm 2 . The degree of sensitization varied between the two halo-uridines, BUdR being more effective at high doses while IUdR was a more potent sensitizer at low doses. Chromosome aberrations were not directly related to unscheduled DNA synthesis but were sensitized by halo-uridine incorporation. In this case IUdR was more potent than BUdR at all doses studied. Methyl methanesulfonate was an effective producer of chromosome aberration in human lymphocytes of both the chromosome and chromatid type. Prior incorporation of BUdR or IUdR did not increase the total aberration produced but did increase the number of chromosome type aberration at the expense of the chromatid type
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Taiwan Y-chromosomal DNA variation and its relationship with Island Southeast Asia
2014-01-01
Background Much of the data resolution of the haploid non-recombining Y chromosome (NRY) haplogroup O in East Asia are still rudimentary and could be an explanatory factor for current debates on the settlement history of Island Southeast Asia (ISEA). Here, 81 slowly evolving markers (mostly SNPs) and 17 Y-chromosomal short tandem repeats were used to achieve higher level molecular resolution. Our aim is to investigate if the distribution of NRY DNA variation in Taiwan and ISEA is consistent with a single pre-Neolithic expansion scenario from Southeast China to all ISEA, or if it better fits an expansion model from Taiwan (the OOT model), or whether a more complex history of settlement and dispersals throughout ISEA should be envisioned. Results We examined DNA samples from 1658 individuals from Vietnam, Thailand, Fujian, Taiwan (Han, plain tribes and 14 indigenous groups), the Philippines and Indonesia. While haplogroups O1a*-M119, O1a1*-P203, O1a2-M50 and O3a2-P201 follow a decreasing cline from Taiwan towards Western Indonesia, O2a1-M95/M88, O3a*-M324, O3a1c-IMS-JST002611 and O3a2c1a-M133 decline northward from Western Indonesia towards Taiwan. Compared to the Taiwan plain tribe minority groups the Taiwanese Austronesian speaking groups show little genetic paternal contribution from Han. They are also characterized by low Y-chromosome diversity, thus testifying for fast drift in these populations. However, in contrast to data provided from other regions of the genome, Y-chromosome gene diversity in Taiwan mountain tribes significantly increases from North to South. Conclusion The geographic distribution and the diversity accumulated in the O1a*-M119, O1a1*-P203, O1a2-M50 and O3a2-P201 haplogroups on one hand, and in the O2a1-M95/M88, O3a*-M324, O3a1c-IMS-JST002611 and O3a2c1a-M133 haplogroups on the other, support a pincer model of dispersals and gene flow from the mainland to the islands which likely started during the late upper Paleolithic, 18,000 to 15
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Delineating Rearrangements in Single Yeast Artificial Chromosomes by Quantitative DNA Fiber Mapping
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Weier, Heinz-Ulrich G.; Greulich-Bode, Karin M.; Wu, Jenny; Duell, Thomas
2009-09-18
Cloning of large chunks of human genomic DNA in recombinant systems such as yeast or bacterial artificial chromosomes has greatly facilitated the construction of physical maps, the positional cloning of disease genes or the preparation of patient-specific DNA probes for diagnostic purposes. For this process to work efficiently, the DNA cloning process and subsequent clone propagation need to maintain stable inserts that are neither deleted nor otherwise rearranged. Some regions of the human genome; however, appear to have a higher propensity than others to rearrange in any host system. Thus, techniques to detect and accurately characterize such rearrangements need to be developed. We developed a technique termed 'Quantitative DNA Fiber Mapping (QDFM)' that allows accurate tagging of sequence elements of interest with near kilobase accuracy and optimized it for delineation of rearrangements in recombinant DNA clones. This paper demonstrates the power of this microscopic approach by investigating YAC rearrangements. In our examples, high-resolution physical maps for regions within the immunoglobulin lambda variant gene cluster were constructed for three different YAC clones carrying deletions of 95 kb and more. Rearrangements within YACs could be demonstrated unambiguously by pairwise mapping of cosmids along YAC DNA molecules. When coverage by YAC clones was not available, distances between cosmid clones were estimated by hybridization of cosmids onto DNA fibers prepared from human genomic DNA. In addition, the QDFM technology provides essential information about clone stability facilitating closure of the maps of the human genome as well as those of model organisms.
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Chromosomal aberrations in subjects exposed to ionizing radiation
International Nuclear Information System (INIS)
Jovicic, D.; Milacic, S.; Kovacevic, R.; Tanaskovic, I.
2006-01-01
Occupational exposure is particularly delicate because of chronic exposure to low doses of ionizing radiation and its cumulative effect, where it is important to consider the biological response of body to given conditions of exposure. The objective of this study was the observation of the recovery of the DNA damages in subjects working in the radiation area in two different intervals.Group I, consisting of 30 subjects, was exposed to ionizing radiation and unstable chromosomal aberrations were identified. Group II included the same, re-examined subjects (30) 9 months later. It was verified that 5 (16.67%) subjects still had unstable chromosomal aberrations, although they had been excluded from radiation area Controls groups (C) consisted of 64 subjects that were not exposed to mutagenic agents.The comparison of the control group with the two studied groups revealed the reduction of the unstable aberrations (p<0.05). The total effective doses, which increased with the years spent in radiation area, reflected the yield of chromosomal aberrations. The presence of chromosomal aberrations in some subjects, after the exclusion from the ionising radiation exposure, suggests that the time needed for the recovery of the DNA damages is different, which indicates the individual differences in radiosensitivity as well as different of the reparatory cellular response. (author)
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Sivaramakurup Sreekumar
2010-01-01
Full Text Available In silkworms, sex-limited strains are either obtained spontaneously or induced by X-rays or gamma rays. When a fragment of an autosome carrying a dominant allele of those genes responsible for certain characters is translocated onto a W chromosome, the female of the successive generations will express these phenotypic characters and sex discrimination can be facilitated. Gensei-kouken strains are sex-limited strains of silkworms developed by irradiating the pupae with gamma rays, by which a portion of the second chromosome is translocated onto the W chromosome. In these improved strains, the females are yellow-blooded and spin yellow cocoons. By using the EST-cDNA clones mapped on the Z chromosome, we identified the sex according to the polymorphic banding pattern or intensity of the signals. Furthermore, by using the clones on the second chromosome, the region of the second chromosome translocated onto the W chromosome was also defined. In both the A95 and A 96 strains selected for the present study, only the mid-portion of the second chromosome was translocated. The differences in length of the fragments translocated in these strains are discussed.
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International Nuclear Information System (INIS)
Beetstra, Sasja; Thomas, Philip; Salisbury, Carolyn; Turner, Julie; Fenech, Michael
2005-01-01
Folic acid deficiency can lead to uracil incorporation into DNA, hypomethylation of DNA, inefficient DNA repair and increase chromosome malsegregation and breakage. Because ionising radiation increases demand for efficient DNA repair and also causes chromosome breaks we hypothesised that folic acid deficiency may increase sensitivity to radiation-induced chromosome breakage. We tested this hypothesis by using the cytokinesis-block micronucleus assay in 10 day WIL2-NS cell cultures at four different folic acid concentrations (0.2, 2, 20, and 200 nM) that span the 'normal' physiological range in humans. The study showed a significant dose-dependent increase in frequency of binucleated cells with micronuclei and/or nucleoplasmic bridges with decreasing folic acid concentration (P < 0.0001, P = 0.028, respectively). These biomarkers of chromosomal instability were also increased in cells irradiated (1.5 Gy γ-rays) on day 9 relative to un-irradiated controls (P < 0.05). Folic acid deficiency and γ-irradiation were shown to have a significant interactive effect on frequency of cells containing micronuclei (two-way ANOVA, interaction P 0.0039) such that the frequency of radiation-induced micronucleated cells (i.e. after subtracting base-line frequency of un-irradiated controls) increased with decreasing folic acid concentration (P-trend < 0.0001). Aneuploidy of chromosome 21, apoptosis and necrosis were increased by folic acid deficiency but not by ionising radiation. The results of this study show that folate status has an important impact on chromosomal stability and is an important modifying factor of cellular sensitivity to radiation-induced genome damage
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You, Zhiying; De Falco, Mariarosaria; Kamada, Katsuhiko; Pisani, Francesca M.; Masai, Hisao
2013-01-01
The Mini-chromosome maintenance (Mcm) proteins are essential as central components for the DNA unwinding machinery during eukaryotic DNA replication. DNA primase activity is required at the DNA replication fork to synthesize short RNA primers for DNA chain elongation on the lagging strand. Although direct physical and functional interactions between helicase and primase have been known in many prokaryotic and viral systems, potential interactions between helicase and primase have not been explored in eukaryotes. Using purified Mcm and DNA primase complexes, a direct physical interaction is detected in pull-down assays between the Mcm2∼7 complex and the hetero-dimeric DNA primase composed of the p48 and p58 subunits. The Mcm4/6/7 complex co-sediments with the primase and the DNA polymerase α-primase complex in glycerol gradient centrifugation and forms a Mcm4/6/7-primase-DNA ternary complex in gel-shift assays. Both the Mcm4/6/7 and Mcm2∼7 complexes stimulate RNA primer synthesis by DNA primase in vitro. However, primase inhibits the Mcm4/6/7 helicase activity and this inhibition is abolished by the addition of competitor DNA. In contrast, the ATP hydrolysis activity of Mcm4/6/7 complex is not affected by primase. Mcm and primase proteins mutually stimulate their DNA-binding activities. Our findings indicate that a direct physical interaction between primase and Mcm proteins may facilitate priming reaction by the former protein, suggesting that efficient DNA synthesis through helicase-primase interactions may be conserved in eukaryotic chromosomes. PMID:23977294
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Zhiying You
Full Text Available The Mini-chromosome maintenance (Mcm proteins are essential as central components for the DNA unwinding machinery during eukaryotic DNA replication. DNA primase activity is required at the DNA replication fork to synthesize short RNA primers for DNA chain elongation on the lagging strand. Although direct physical and functional interactions between helicase and primase have been known in many prokaryotic and viral systems, potential interactions between helicase and primase have not been explored in eukaryotes. Using purified Mcm and DNA primase complexes, a direct physical interaction is detected in pull-down assays between the Mcm2~7 complex and the hetero-dimeric DNA primase composed of the p48 and p58 subunits. The Mcm4/6/7 complex co-sediments with the primase and the DNA polymerase α-primase complex in glycerol gradient centrifugation and forms a Mcm4/6/7-primase-DNA ternary complex in gel-shift assays. Both the Mcm4/6/7 and Mcm2~7 complexes stimulate RNA primer synthesis by DNA primase in vitro. However, primase inhibits the Mcm4/6/7 helicase activity and this inhibition is abolished by the addition of competitor DNA. In contrast, the ATP hydrolysis activity of Mcm4/6/7 complex is not affected by primase. Mcm and primase proteins mutually stimulate their DNA-binding activities. Our findings indicate that a direct physical interaction between primase and Mcm proteins may facilitate priming reaction by the former protein, suggesting that efficient DNA synthesis through helicase-primase interactions may be conserved in eukaryotic chromosomes.
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Jiang, Shu-Mei; Hu, Jun; Yin, Wei-Bo; Chen, Yu-Hong; Wang, Richard R-C; Hu, Zan-Min
2005-09-01
Homology-based gene/gene-analog cloning method has been extensively applied in isolation of RGAs (resistance gene analogs) in various plant species. However, serious interference of sequences on homoeologous chromosomes in polyploidy species usually occurred when cloning RGAs in a specific chromosome. In this research, the techniques of chromosome microdissection combined with homology-based cloning were used to clone RGAs from a specific chromosome of Wheat-Thinopyrum alien addition line TAi-27, which was derived from common wheat and Thinopyrum intermedium with a pair of chromosomes from Th. intermedium. The alien chromosomes carry genes for resistance to BYDV. The alien chromosome in TAi-27 was isolated by a glass needle and digested with proteinase K. The DNA of the alien chromosome was amplified by two rounds of Sau3A linker adaptor-mediated PCR. RGAs were amplified by PCR with the degenerated primers designed based on conserved domains of published resistance genes (R genes) by using the alien chromosome DNA, genomic DNA and cDNA of Th. intermedium, TAi-27 and 3B-2 (a parent of TAi-27) as templates. A total of seven RGAs were obtained and sequenced. Of which, a constitutively expressed single-copy NBS-LRR type RGA ACR 3 was amplified from the dissected alien chromosome of TAi-27, TcDR 2 and TcDR 3 were from cDNA of Th. intermedium, AcDR 3 was from cDNA of TAi-27, FcDR 2 was from cDNA of 3B-2, AR 2 was from genomic DNA of TAi-27 and TR 2 was from genomic DNA of Th. intermedium. Sequence homology analyses showed that the above RGAs were highly homologous with known resistance genes or resistance gene analogs and belonged to NBS-LRR type of R genes. ACR 3 was recovered by PCR from genomic DNA and cDNA of Th. intermedium and TAi-27, but not from 3B-2. Southern hybridization using the digested genomic DNA of Th. intermedium, TAi-27 and 3B-2 as the template and ACR 3 as the probe showed that there is only one copy of ACR 3 in the genome of Th. intermedium and TAi
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Christiansen, H; Sahin, K; Berthold, F; Hero, B; Terpe, H J; Lampert, F
1995-01-01
A comparison of the prognostic impact of five molecular variables in a large series was made, including tests of their nonrandom association and multivariate analysis. Molecular data were available for 377 patients and MYCN amplification, cytogenetic chromosome 1p deletion, loss of chromosome 1p heterozygosity, DNA ploidy and CD44 expression were investigated. Their interdependence and influence on event-free survival was tested uni- and multivariately using Pearson's chi 2-test, Kaplan-Meier estimates, log rank tests and the Cox's regression model. MYCN amplification was present in 18% (58/322) of cases and predicted poorer prognosis in localised (P < 0.001), metastatic (P = 0.002) and even 4S (P = 0.040) disease. CD44 expression was found in 86% (127/148) of cases, and was a marker for favourable outcome in patients with neuroblastoma stages 1-3 (P = 0.003) and 4 (P = 0.017). Chromosome 1p deletion was cytogenetically detected in 51% (28/55), and indicated reduced event-free survival in localised neuroblastoma (P = 0.020). DNA ploidy and loss of heterozygosity on chromosome 1p were of less prognostic value. Most factors of prognostic significance were associated with each other. By multivariate analysis, MYCN was selected as the only relevant factor. Risk estimation of high discriminating power is, therefore, possible for patients with localised and metastatic neuroblastoma using stage and MYCN.
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International Nuclear Information System (INIS)
McMahon, Laura W.; Zhang Pan; Sridharan, Deepa M.; Lefferts, Joel A.; Lambert, Muriel W.
2009-01-01
Nonerythroid α-spectrin (αIISp) is a structural protein involved in repair of DNA interstrand cross-links and is deficient in cells from patients with Fanconi anemia (FA), which are defective in ability to repair cross-links. In order to further demonstrate the importance of the role that αIISp plays in normal human cells and in the repair defect in FA, αIISp was knocked down in normal cells using siRNA. Depletion of αIISp in normal cells by siRNA resulted in chromosomal instability and cellular hypersensitivity to DNA interstrand cross-linking agents. An increased number of chromosomal aberrations were observed and, following treatment with a DNA interstrand cross-linking agent, mitomycin C, cells showed decreased cell growth and survival and decreased formation of damage-induced αIISp and XPF nuclear foci. Thus depletion of αIISp in normal cells leads to a number of defects observed in FA cells, such as chromosome instability and a deficiency in cross-link repair.
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International Nuclear Information System (INIS)
Fornace, A.J. Jr.; Nagasawa, H.; Little, J.B.
1980-01-01
The repair of X-ray-induced DNA single strand breaks and DNA-protein cross-links was investigated in stationary phase, contact-inhibited mouse cells by the alkaline-elution technique. Approx. 90% of X-ray-induced single strand breaks were rejoined during the first hour of repair, whereas most of the remaining breaks were rejoined more slowly during the next 5 h. At early repair times, the number of residual non-rejoined sungle strand breaks was approx. proportional to the X-ray dose. DNA-protein cross-links were removed at a slower rate (Tsub(1/2) approx. 10-12 h). Cells were held in stationary growth for various periods of time after irradiation before subculture at low density to score for colony survival (potentially lethal damage repair), chromosome aberrations in the first mitosis, and sister-chromatid exchanges in the second mitosis. Both cell killing and the frequency of chromosome aberrations decreased during the first several hours of recovery, reaching a minimum level by 6 h; this decrease correlated temporally with the repair of the slowly rejoining DNA-strand breaks. Relatively few sister-chromatid exchanges were observed when the cells were subcultured immediately after X-ray. The exchange frequency rose to maximum levels after a 4-h recovery interval, and returned to control levels after 12 h of recovery. The possible relationship of DNA repair to these changes in survival, chromosome aberrations, and sister-chromatid exchanges during liquid-holding recovery is discussed. (orig.)
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International Nuclear Information System (INIS)
Que Tran; Tien Hoang Hung
1997-01-01
Induced chromosome aberrations (ch. ab.) in exposed Human peripheral blood lymphocyte have been used to assay radio.bio.doses, because of their characters such as: the maintaining Go phase in cell cycle in body, the distribution of cell in blood system and the distribution of ch. ab. in exposed cells of body and among chromosomes of karyotype. The frequency of ch. ab. reflected the quantity of radiation dose, dose rate and radiation energy. The dependence between radiation dose and frequency of ch. ab. was illustrated by the mathematic equations. The distribution of induced ch. ab. among the cells exposed to uniform radiation fields was Poisson's, but the distribution of ch. ab. among chromosomes in karyotype depended on radiation field and mononucleotid sequence of DNA molecular of each chromosome. The minimum influence of mononucleotid sequence of DNA molecular in inform ch. ab. will be advantageous state for dose-assessments. The location of induced ch. ab. in exposed Human lymphocyte had been determined by karyotype analyses. The data of statistic analyse had improved that the number of ch. ab. depended on the size of chromosomes in karyotype. The equal distribution of ch. ab.among chromosomes in karyotype provided the objectiveness and the accuracy of using the chromosomal aberrant analysis technique on bio-dosimetry. (author)
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Leclerc Xavier
2009-04-01
Full Text Available Abstract Background Current strategies for gene therapy of inherited diseases consist in adding functional copies of the gene that is defective. An attractive alternative to these approaches would be to correct the endogenous mutated gene in the affected individual. This study presents a quantitative comparison of the repair efficiency using different forms of donor nucleic acids, including synthetic DNA oligonucleotides, double stranded DNA fragments with sizes ranging from 200 to 2200 bp and sequences carried by a recombinant adeno-associated virus (rAAV-1. Evaluation of each gene repair strategy was carried out using two different reporter systems, a mutated eGFP gene or a dual construct with a functional eGFP and an inactive luciferase gene, in several different cell systems. Gene targeting events were scored either following transient co-transfection of reporter plasmids and donor DNAs, or in a system where a reporter construct was stably integrated into the chromosome. Results In both episomal and chromosomal assays, DNA fragments were more efficient at gene repair than oligonucleotides or rAAV-1. Furthermore, the gene targeting frequency could be significantly increased by using DNA repair stimulating drugs such as doxorubicin and phleomycin. Conclusion Our results show that it is possible to obtain repair frequencies of 1% of the transfected cell population under optimized transfection protocols when cells were pretreated with phleomycin using rAAV-1 and dsDNA fragments.
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Czech Academy of Sciences Publication Activity Database
Rössner ml., Pavel; Rössnerová, Andrea; Beskid, Olena; Tabashidze, Nana; Líbalová, Helena; Uhlířová, Kateřina; Topinka, Jan; Šrám, Radim
763-764, MAY-JUN 2014 (2014), s. 28-38 ISSN 0027-5107 R&D Projects: GA ČR GAP503/11/0084 Institutional support: RVO:68378041 Keywords : benzo[a]pyrene * chromosome aberrations * double-strand DNA breaks Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 3.680, year: 2014
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International Nuclear Information System (INIS)
Terzoudi, Georgia I.; Hatzi, Vasiliki I.; Donta-Bakoyianni, Catherine; Pantelias, Gabriel E.
2011-01-01
The formation of diverse chromosomal aberrations following irradiation and the variability in radiosensitivity at different cell-cycle stages remain a long standing controversy, probably because most of the studies have focused on elucidating the enzymatic mechanisms involved using simple DNA substrates. Yet, recognition, processing and repair of DNA damage occur within the nucleoprotein complex of chromatin which is dynamic in nature, capable of rapid unfolding, disassembling, assembling and refolding. The present work reviews experimental work designed to investigate the impact of chromatin dynamics and chromosome conformation changes during cell-cycle in the formation of chromosomal aberrations. Using conventional cytogenetics and premature chromosome condensation to visualize interphase chromatin, the data presented support the hypothesis that chromatin dynamic changes during cell-cycle are important determinants in the conversion of sub-microscopic DNA lesions into chromatid breaks. Consequently, the type and yield of radiation-induced chromosomal aberrations at a given cell-cycle-stage depends on the combined effect of DNA repair processes and chromatin dynamics, which is cell-cycle-regulated and subject to up- or down-regulation following radiation exposure or genetic alterations. This new hypothesis is used to explain the variability in radiosensitivity observed at various cell-cycle-stages, among mutant cells and cells of different origin, or among different individuals, and to revisit unresolved issues and unanswered questions. In addition, it is used to better understand hypersensitivity of AT cells and to provide an improved predictive G2-assay for evaluating radiosensitivity at individual level. Finally, experimental data at single cell level obtained using hybrid cells suggest that the proposed hypothesis applies only to the irradiated component of the hybrid.
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Malagón, Talía; Burchell, Ann; El-Zein, Mariam; Guénoun, Julie; Tellier, Pierre-Paul; Coutlée, François; Franco, Eduardo L
2018-01-01
Y chromosome DNA from male epithelial and sperm cells was detected in vaginal samples after unprotected sex in experimental studies. We assessed the strength of this association in an observational setting to examine the utility of Y chromosome DNA as a biomarker of recent sexual behaviors in epidemiological studies. The HPV (human papillomavirus) Infection and Transmission Among Couples Through Heterosexual Activity cohort study enrolled 502 women attending a university or college in Montréal, Canada, and their male partners from 2005 to 2010. Participants completed self-administered questionnaires. We used real-time polymerase chain reaction to test women's baseline vaginal samples for Y chromosome DNA and assessed which sexual behaviors were independent predictors of Y chromosome DNA positivity and quantity with logistic and negative binomial regression. Y chromosome DNA positivity decreased from 77% in women in partnerships reporting vaginal sex 0 to 1 day ago to 13% in women in partnerships reporting last vaginal sex of 15 or more days ago (adjusted odds ratio, 0.09; 95% confidence interval, 0.02-0.36). The mean proportion of exfoliated vaginal sample cells with Y chromosome DNA was much lower for women who reported always using condoms (0.01%) than for women who reported never using condoms (2.07%) (adjusted ratio, 26.8; 95% confidence interval, 8.9-80.5). No association was found with reported oral/digital sex frequency or concurrency of partnerships. Y chromosome DNA quantity is strongly associated with days since last vaginal sex and lack of condom use in observational settings. Y chromosome DNA quantity may prove useful as a correlate of recent vaginal sex in observational studies lacking data on sexual behavior, such as surveillance studies of human papillomavirus infection prevalence.
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Micro-Raman spectroscopy of chromosomes
de Mul, F.F.M.; van Welle, A.G.M.; Otto, Cornelis; Greve, Jan
1984-01-01
Raman spectra of intact chromosomes (Chinese hamster), recorded with a microspectrometer, are reported. The spectra could be assigned to protein and DNA contributions. Protein and DNA conformations and the ratio of base pairs in DNA were determined.
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Waye, J S; Willard, H F
1986-09-01
The centromeric regions of all human chromosomes are characterized by distinct subsets of a diverse tandemly repeated DNA family, alpha satellite. On human chromosome 17, the predominant form of alpha satellite is a 2.7-kilobase-pair higher-order repeat unit consisting of 16 alphoid monomers. We present the complete nucleotide sequence of the 16-monomer repeat, which is present in 500 to 1,000 copies per chromosome 17, as well as that of a less abundant 15-monomer repeat, also from chromosome 17. These repeat units were approximately 98% identical in sequence, differing by the exclusion of precisely 1 monomer from the 15-monomer repeat. Homologous unequal crossing-over is suggested as a probable mechanism by which the different repeat lengths on chromosome 17 were generated, and the putative site of such a recombination event is identified. The monomer organization of the chromosome 17 higher-order repeat unit is based, in part, on tandemly repeated pentamers. A similar pentameric suborganization has been previously demonstrated for alpha satellite of the human X chromosome. Despite the organizational similarities, substantial sequence divergence distinguishes these subsets. Hybridization experiments indicate that the chromosome 17 and X subsets are more similar to each other than to the subsets found on several other human chromosomes. We suggest that the chromosome 17 and X alpha satellite subsets may be related components of a larger alphoid subfamily which have evolved from a common ancestral repeat into the contemporary chromosome-specific subsets.
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Choo, K H; Vissel, B; Brown, R; Filby, R G; Earle, E
1988-02-25
The authors report a new subfamily of alpha satellite DNA (pTRA-2) which is found on all the human acrocentric chromosomes. The alphoid nature of the cloned DNA was established by partial sequencing. Southern analysis of restriction enzyme-digested DNA fragments from mouse/human hybrid cells containing only human chromosome 21 showed that the predominant higher-order repeating unit for pTRA-2 is a 3.9 kb structure. Analysis of a consensus in situ hybridization profile derived from 13 normal individuals revealed the localization of 73% of all centromeric autoradiographic grains over the five acrocentric chromosomes, with the following distribution: 20.4%, 21.5%, 17.1%, 7.3% and 6.5% on chromosomes 13, 14, 21, 15 and 22 respectively. An average of 1.4% of grains was found on the centromere of each of the remaining 19 nonacrocentric chromosomes. These results indicate the presence of a common subfamily of alpha satellite DNA on the five acrocentric chromosomes and suggest an evolutionary process consistent with recombination exchange of sequences between the nonhomologues. The results further suggests that such exchanges are more selective for chromosomes 13, 14 and 21 than for chromosomes 15 and 22. The possible role of centromeric alpha satellite DNA in the aetiology of 13q14q and 14q21q Robertsonian translocation involving the common and nonrandom association of chromosomes 13 and 14, and 14 and 21 is discussed.
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Formation of pyrimidine dimers in Simian virus 40 chromosomes and DNA in vitro: effects of salt
International Nuclear Information System (INIS)
Edenberg, H.J.
1984-01-01
Simian virus 40 chromosomes were used to determine whether packaging of DNA into chromatin affected the yield of cylcobutane pyrimidine dimers introduced by ultraviolet light (254 nm). SV40 chromatin and purified SV40 DNA (radioactively labeled with different isotopes) were mixed and irradiated in vitro. The proteins were extracted and pyrimidine dimers detected as sites sensitive to the UV-endonuclease encoded by bacteriophage T4. When irradiation was carried out in the presence of at least 0.05 M NaCl the same number of dimers were formed in chromatin as in free DNA. Irradiation in the absence of NaCl, however, reduced the relative yield of dimers in chromatin to 89% of that in free DNA. Different methods of chromatin preparation did not influence these results. (author)
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High chromosomal instability in workers occupationally exposed to solvents and paint removers.
Villalba-Campos, Mónica; Chuaire-Noack, Lilian; Sánchez-Corredor, Magda Carolina; Rondón-Lagos, Milena
2016-01-01
Painters are exposed to an extensive variety of harmful substances like aromatic hydrocarbons used as solvents and paint removers, some of which have shown clastogenic activity. These substances constitute a complex mixture of chemicals which contain well-known genotoxicants, such as Benzene, Toluene and Xylene. Thus, chronic occupational exposure to such substances may be considered to possess genotoxic risk. In Colombia the information available around the genotoxic damage (Chromosomal and DNA damage) in car paint shop workers is limited and the knowledge of this damage could contribute not only to a better understanding of the carcinogenic effect of this kind of substances but also could be used as biomarkers of occupational exposure to genotoxic agents. In this study, the genotoxic effect of aromatic hydrocarbons was assessed in peripheral blood lymphocytes of 24 workers occupationally exposed and 24 unexposed donors, by using Cytogenetic analysis and comet assay. A high frequency of Chromosomal alterations was found in the exposed group in comparison with those observed in the unexposed group. Among the total of CAs observed in the exposed group, fragilities were most frequently found (100 %), followed by chromosomal breaks (58 %), structural (41.2 %) and numerical chromosomal alterations (21 %). Numerical chromosomal alterations, fragilities and chromosomal breaks showed significant differences between exposed and unexposed groups. Among the fragilities, fra(9)(q12) was the most frequently observed. DNA damage index was also significantly higher in the exposed group compared to the unexposed group (p car paint shops workers and are also indicative of high chromosomal instability. The high frequency of both Chromosomal Alterations and DNA Damage Index observed in this study indicates an urgent need of intervention not only to prevent the increased risk of developing cancer but also to the application of strict health control and motivation to the use of
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Coutelle Charles
2006-03-01
Full Text Available Abstract Background Minicircle DNA is the non-replicating product of intramolecular site-specific recombination within a bacterial minicircle producer plasmid. Minicircle DNA can be engineered to contain predominantly human sequences which have a low content of CpG dinucleotides and thus reduced immunotoxicity for humans, whilst the immunogenic bacterial origin and antibiotic resistance marker gene sequences are entirely removed by site-specific recombination. This property makes minicircle DNA an excellent vector for non-viral gene therapy. Large-scale production of minicircle DNA requires a bacterial strain expressing tightly controlled site-specific recombinase, such as Cre recombinase. As recombinant plasmids tend to be more stable in RecA-deficient strains, we aimed to construct a recA- bacterial strain for generation of minicircle vector DNA with less chance of unwanted deletions. Results We describe here the construction of the RecA-deficient minicircle DNA producer Escherichia coli HB101Cre with a chromosomally located Cre recombinase gene under the tight control of the araC regulon. The Cre gene expression cassette was inserted into the chromosomal lacZ gene by creating transient homologous recombination proficiency in the recA- strain HB101 using plasmid-born recET genes and homology-mediated chromosomal "pop-in, pop-out" of the plasmid pBAD75Cre containing the Cre gene and a temperature sensitive replication origin. Favourably for the Cre gene placement, at the "pop-out" step, the observed frequency of RecET-led recombination between the proximal regions of homology was 10 times higher than between the distal regions. Using the minicircle producing plasmid pFIXluc containing mutant loxP66 and loxP71 sites, we isolated pure minicircle DNA from the obtained recA- producer strain HB101Cre. The minicircle DNA preparation consisted of monomeric and, unexpectedly, also multimeric minicircle DNA forms, all containing the hybrid loxP66
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G.J.A. Arkesteijn (Ger); C.A.J. Erpelinck (Claudia); A.C.M. Martens (Anton); A. Hagenbeek (Anton)
1995-01-01
textabstractFlow cytometry was used to measure the fluorescence intensity of nuclei that were subjected to fluorescent in situ hybridization in suspension with chromosome specific DNA probes. Paraformaldehyde-fixed nuclei were protein digested with trypsin and hybridized simultaneously with a
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Hanifah, Y A; Hiramatsu, K
1994-12-01
Methicillin-resistant Staphylococcus aureus (MRSA) infection has been endemic in the University Hospital, Kuala Lumpur since the late 1970s. Fifty isolates of MRSA obtained from clinical specimens of patients with nosocomial infections associated with this organism have been studied by pulsed-field gel electrophoresis (PFGE) of its chromosomal DNA fragments to discrimate between strains and to identify the predominant strain. Twenty-one chromosomal patterns were observed which could be further grouped into nine types. The predominant strain was Type 9-b (40% of isolates) found mainly in the Orthopaedic and Surgical Units. Outbreak strains found in the Special Care Nursery were of Type 1, entirely different from those of the surgical ward S2, which were of Type 9-b. Type 8 strains were found mainly at one end of the hospital building where the maternity, paediatric and orthopaedic units were situated. Genomic DNA fingerprinting by PFGE is recommended as a useful and effective tool for the purpose of epidemiological studies of MSRA infections, particularly for nosocomial infections.
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Directory of Open Access Journals (Sweden)
Beleza Sandra
2009-04-01
Full Text Available Abstract Background Current information about the expansion of Bantu-speaking peoples is hampered by the scarcity of genetic data from well identified populations from southern Africa. Here, we fill an important gap in the analysis of the western edge of the Bantu migrations by studying for the first time the patterns of Y-chromosome, mtDNA and lactase persistence genetic variation in four representative groups living around the Namib Desert in southwestern Angola (Ovimbundu, Ganguela, Nyaneka-Nkumbi and Kuvale. We assessed the differentiation between these populations and their levels of admixture with Khoe-San groups, and examined their relationship with other sub-Saharan populations. We further combined our dataset with previously published data on Y-chromosome and mtDNA variation to explore a general isolation with migration model and infer the demographic parameters underlying current genetic diversity in Bantu populations. Results Correspondence analysis, lineage sharing patterns and admixture estimates indicate that the gene pool from southwestern Angola is predominantly derived from West-Central Africa. The pastoralist Herero-speaking Kuvale people were additionally characterized by relatively high frequencies of Y-chromosome (12% and mtDNA (22% Khoe-San lineages, as well as by the presence of the -14010C lactase persistence mutation (6%, which likely originated in non-Bantu pastoralists from East Africa. Inferred demographic parameters show that both male and female populations underwent significant size growth after the split between the western and eastern branches of Bantu expansions occurring 4000 years ago. However, males had lower population sizes and migration rates than females throughout the Bantu dispersals. Conclusion Genetic variation in southwestern Angola essentially results from the encounter of an offshoot of West-Central Africa with autochthonous Khoisan-speaking peoples from the south. Interactions between the Bantus
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Pulsed-field gel electrophoresis of bacterial chromosomes.
Mawer, Julia S P; Leach, David R F
2013-01-01
The separation of fragments of DNA by agarose gel electrophoresis is integral to laboratory life. Nevertheless, standard agarose gel electrophoresis cannot resolve fragments bigger than 50 kb. Pulsed-field gel electrophoresis is a technique that has been developed to overcome the limitations of standard agarose gel electrophoresis. Entire linear eukaryotic chromosomes, or large fragments of a chromosome that have been generated by the action of rare-cutting restriction endonucleases, can be separated using this technique. As a result, pulsed-field gel electrophoresis has many applications, from karyotype analysis of microbial genomes, to the analysis of chromosomal strand breaks and their repair intermediates, to the study of DNA replication and the identification of origins of replication. This chapter presents a detailed protocol for the preparation of Escherichia coli chromosomal DNA that has been embedded in agarose plugs, digested with the rare-cutting endonuclease NotI, and separated by contour-clamped homogeneous field electrophoresis. The principles in this protocol can be applied to the separation of all fragments of DNA whose size range is between 40 kb and 1 Mb.
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Directory of Open Access Journals (Sweden)
Eliane Mariza Dortas Maffei
2004-01-01
Full Text Available The aim of this study was to describe mitotic and meiotic chromosomes of Cycloneda sanguinea using C-banding, fluorescent in situ hybridization (FISH rDNA probes, and sequential FISH/Ag-NOR staining. The chromosome number was 2n = 18 + XX for females and 2n = 18 + Xy for males. The X chromosome was metacentric and the Y chromosome was very small. During meiosis, the karyotypic meioformula was n = 9 + Xy p, and sex chromosomes configured a parachute at metaphase I. At the beginning of pachytene, bivalents were still individualized, and sex chromosomes were associated end-to-end through the heteropycnotic region of the X chromosome. Later in pachytene, further condensation led to the formation of a pseudo-ring by the sex bivalent. All chromosomes showed pericentromeric heterochromatin. FISH and sequential FISH/Ag-NOR staining evidenced the location of the nucleolar organizer region in one pair of autosomes (at spermatogonial metaphase. During meiosis, these genes were mapped to a region outside the sex vesicle by FISH, although Xy p was deeply stained with silver at metaphase I. These results suggest that these argyrophilic substances are of a nucleolar protein nature, and seem to be synthesized by a pair of autosomes and imported during meiosis (prophase I to the sex pair, during the association of the sex chromosomes.
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Preparation and bivariate analysis of suspensions of human chromosomes
Energy Technology Data Exchange (ETDEWEB)
van den Engh, G.J.; Trask, B.J.; Gray, J.W.; Langlois, R.G.; Yu, L.C.
1985-01-01
Chromosomes were isolated from a variety of human cell types using a HEPES-buffered hypotonic solution (pH 8.0) containing KCl, MgSO/sub 4/ dithioerythritol, and RNase. The chromosomes isolated by this procedure could be stained with a variety of fluorescent stains including propidium iodide, chromomycin A3, and Hoeschst 33258. Addition of sodium citrate to the stained chromosomes was found to improve the total fluorescence resolution. High-quality bivariate Hoeschst vs. chromomycin fluorescence distributions were obtained for chromosomes isolated from a human fibroblast cell strain, a human colon carcinoma cell line, and human peripheral blood lymphocyte cultures. Good flow karyotypes were also obtained from primary amniotic cell cultures. The Hoeschst vs. chromomycin flow karyotypes of a given cell line, made at different times and at dye concentrations varying over fourfold ranges, show little variation in the relative peak positions of the chromosomes. The size of the DNA in chromosomes isolated using this procedure ranges from 20 to 50 kilobases. The described isolation procedure is simple, it yields high-quality flow karyotypes, and it can be used to prepare chromosomes from clinical samples. 22 references, 7 figures, 1 table.
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A CTAB Procedure Of Total Genomic DNA Extraction For Medicinal Mushrooms
International Nuclear Information System (INIS)
Azhar Mohamad; Muhammad Hussaini Mohd Mustafa; Muhammad Hanif Azhari Noor; Rosnani Abdul Rashid; Hasan Hamdani Hasan Mutaat; Meswan Meskom; Mat Rasol Awang
2014-01-01
Medicinal mushroom is defined as mushrooms used in medicine or medical research. Isolation of intact, high-molecular-mass genomic DNA is essential for many molecular biology applications including Polymerase Chain Reaction (PCR), endonuclease restriction digestion, Southern blot analysis, and genomic library construction. The most important and prerequisite towards reliable molecular biology work is the total genomic DNA of a sample must be in good quality. Five freshly samples of medicinal mushroom were used in this work known as Auriculariapolytricha, Lentinus edode, Pleurotus sayorcaju, Sczhizopyllum commune and Ganodermalucidum. 5 mg of each sample were used to extraction the DNA, prepared in 3 replications and repeated twice. PCR based technique by using ISSR markers were used in checking the amplification ability of the total genomic extraction. A standard Doyle and Doyle protocol for genomic DNA extraction was modified in optimizing the total genomic DNA from the medicinal mushroom.The modification parameters were percentage of CTAB, incubation period and temperature. The results reveal that each sample required a certain combinations of time and period of incubation. Besides, percentage of CTAB in the buffer was found significant in giving a high yielding of extracted total genomic DNA. The extracted total genomic DNA from the medicinal mushroom yielded from 39.7 ng/ μl to 919.1 ng/ μl. The different yield among the samples found to be corresponded to polysaccharide content in the medicinal mushrooms. The objective of this works is to optimize total genomic DNA extraction of medicinal mushrooms towards a high quality intact genomic DNA for molecular activities. (author)
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Marín, J C; Romero, K; Rivera, R; Johnson, W E; González, B A
2017-10-01
Investigations of genetic diversity and domestication in South American camelids (SAC) have relied on autosomal microsatellite and maternally-inherited mitochondrial data. We present the first integrated analysis of domestic and wild SAC combining male and female sex-specific markers (male specific Y-chromosome and female-specific mtDNA sequence variation) to assess: (i) hypotheses about the origin of domestic camelids, (ii) directionality of introgression among domestic and/or wild taxa as evidence of hybridization and (iii) currently recognized subspecies patterns. Three male-specific Y-chromosome markers and control region sequences of mitochondrial DNA are studied here. Although no sequence variation was found in SRY and ZFY, there were seven variable sites in DBY generating five haplotypes on the Y-chromosome. The haplotype network showed clear separation between haplogroups of guanaco-llama and vicuña-alpaca, indicating two genetically distinct patrilineages with near absence of shared haplotypes between guanacos and vicuñas. Although we document some examples of directional hybridization, the patterns strongly support the hypothesis that llama (Lama glama) is derived from guanaco (Lama guanicoe) and the alpaca (Vicugna pacos) from vicuña (Vicugna vicugna). Within male guanacos we identified a haplogroup formed by three haplotypes with different geographical distributions, the northernmost of which (Peru and northern Chile) was also observed in llamas, supporting the commonly held hypothesis that llamas were domesticated from the northernmost populations of guanacos (L. g. cacilensis). Southern guanacos shared the other two haplotypes. A second haplogroup, consisting of two haplotypes, was mostly present in vicuñas and alpacas. However, Y-chromosome variation did not distinguish the two subspecies of vicuñas. © 2017 Stichting International Foundation for Animal Genetics.
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Involvement of DNA repair in telomere maintenance and chromosomal instability in human cells
International Nuclear Information System (INIS)
Ayouaz, Ali
2008-01-01
Telomeres are a major actor of cell immortalization, precursor of a carcinogenesis process. Thus, it appears that the maintenance of telomeres is crucial in the implementation of carcinogenesis process. Due to their structures and under some conditions, telomeres can be assimilated in some respects to chromosomal breakages. Within this perspective, this research thesis aims at determining under which circumstances telomeres can be taken as targets by DNA repair mechanisms. More precisely, the author addressed the respective contributions of two repair mechanisms (the Non-Homologous End-Joining or NHEJ, and Homologous Recombination or HR) in the maintenance of telomere integrity. The author first discusses knowledge related to the interaction between chromosomal extremities and repair mechanisms. Then, he defines the behaviour of these mechanisms with respect to telomeres. He shows that, in absence of recombination mechanisms, the integrity of telomeres is not affected. Finally, he reports the attempt to determine their respective contributions in telomeric homeostasis [fr
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Directory of Open Access Journals (Sweden)
Hamiyet Donmez-Altuntas
2017-01-01
Full Text Available Thyroid nodules are a common clinical problem worldwide. Although thyroid cancer accounts for a small percentage of thyroid nodules, the majority are benign. 8-Hydroxy-2′-deoxyguanosine (8-OHdG levels are a marker of oxidative stress and play a key role in the initiation and development of a range of diseases and cancer types. This study evaluates cytokinesis-block micronucleus cytome (CBMN-cyt assay parameters and plasma 8-OHdG levels and their association with thyroid nodule size and thyroid hormones in patients with multinodular goiter. The study included 32 patients with multinodular goiter and 18 age- and sex-matched healthy controls. CBMN-cyt assay parameters in peripheral blood lymphocytes of patients with multinodular goiter and controls were evaluated, and plasma 8-OHdG levels were measured. The micronucleus (MN frequency (chromosomal DNA damage, apoptotic and necrotic cells (cytotoxicity, and plasma 8-OHdG levels (oxidative DNA damage were significantly higher among patients with multinodular goiter. Our study is the first report of increased chromosomal and oxidative DNA damage in patients with multinodular goiter, which may predict an increased risk of thyroid cancer in these patients. MN frequency and plasma 8-OHdG levels may be markers of the carcinogenic potential of multinodular goiters and could be used for early detection of different cancer types, including thyroid cancer.
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Energy Technology Data Exchange (ETDEWEB)
Jha, Jyoti K.; Li, Mi; Ghirlando, Rodolfo; Miller Jenkins, Lisa M.; Wlodawer, Alexander; Chattoraj, Dhruba; Dunny, Gary M.
2017-04-18
Replication of Vibrio cholerae chromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations in
rctB that reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in a dimerization domain which is folded similarly to the initiator of an iteron plasmid—the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition) when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2 initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding. IMPORTANCE The capacity of proteins to undergo remodeling provides opportunities to control their function. However, remodeling remains a poorly understood aspect of the structure-function paradigm due to its dynamic nature. Here we have studied remodeling of the initiator of replication ofVibrio cholerae Chr2 by the molecular chaperone, DnaK. We show that DnaK binds to a site on the Chr2 initiator (RctB) that -
Wong, H-P; Mozdarani, H; Finnegan, C; McIlrath, J; Bryant, P E; Slijepcevic, P
2004-01-01
Interstitial telomeric sites (ITSs) in chromosomes from DNA repair-proficient mammalian cells are sensitive to both spontaneous and radiation-induced chromosome breakage. Exact mechanisms of this chromosome breakage sensitivity are not known. To investigate factors that predispose ITSs to chromosome breakage we used murine scid cells. These cells lack functional DNA-PKcs, an enzyme involved in the repair of DNA double-strand breaks. Interestingly, our results revealed lack of both spontaneous and radiation-induced chromosome breakage at ITSs found in scid chromosomes. Therefore, it is possible that increased sensitivity of ITSs to chromosome breakage is associated with the functional DNA double-strand break repair machinery. To investigate if this is the case we used scid cells in which DNA-PKcs deficiency was corrected. Our results revealed complete disappearance of ITSs in scid cells with functional DNA-PKcs, presumably through chromosome breakage at ITSs, but their unchanged frequency in positive and negative control cells. Therefore, our results indicate that the functional DNA double-strand break machinery is required for elevated sensitivity of ITSs to chromosome breakage. Interestingly, we observed significant differences in mitotic chromosome condensation between scid cells and their counterparts with restored DNA-PKcs activity suggesting that lack of functional DNA-PKcs may cause a defect in chromatin organization. Increased condensation of mitotic chromosomes in the scid background was also confirmed in vivo. Therefore, our results indicate a previously unanticipated role of DNA-PKcs in chromatin organisation, which could contribute to the lack of ITS sensitivity to chromosome breakage in murine scid cells. Copyright 2003 S. Karger AG, Basel
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Anhidrotic ectodermal dysplasia gene region cloned in yeast artificial chromosomes
Energy Technology Data Exchange (ETDEWEB)
Kere, J. [Washington Univ. School of Medicine, St. Louis, MO (United States)]|[Univ. of Helsinki (Finland); Grzeschik, K.H. [Univ. of Marburg (Germany); Limon, J. [Medical Academy, Gdansk (Poland); Gremaud, M.; Schlessinger, D. [Washington Univ. School of Medicine, St. Louis, MO (United States); De La Chapelle, A. [Univ. of Helsinki (Finland)
1993-05-01
Anhidrotic ectodermal dysplasia (EDA), an X-chromosomal recessive disorder, is expressed in a few females with chromosomal translocations involving bands Xq12-q13. Using available DNA markers from the region and somatic cell hybrids the authors mapped the X-chromosomal breakpoints in two such translocations. The breakpoints were further mapped within a yeast artificial chromosome contig constructed by chromosome walking techniques. Genomic DNA markers that map between the two translocation breakpoints were recovered representing putative portions of the EDA gene. 32 refs., 3 figs., 1 tab.
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International Nuclear Information System (INIS)
Tagliasacchi, A.M.; Forino, L.M.C.; Cionini, P.G.; Cavallini, A.; Durante, M.; Cremonini, R.; Avanzi, S.
1984-01-01
Different regions of polytene chromosomes pair VI have been characterized by: 1. morphological observations, 2. incorporation of 3 H-thymidine and 3 H-uridine, 3. cytophotometry of DNA and associated proteins, 4. hybridization with satellite DNA and highly repeated DNA sequences. The collected data indicate that DNA and RNA puffs are organized by heterochromatic segments. DNA puffs show often a clustered pattern of labeling by 3 H-thymidine and RNA puffs are always labeled by 3 H-urindine. Each heterochromatic segment is characterized by a definite ratio between DNA and associated fastgreen stainable proteins. Satellite DNA binds mostly to heterochromatic blocks at centromers, highly repeated DNA sequences bind, with approximately the same frequency, to centromeric heterochromatin and to the main intercalary heterochromatic band. The telomeric portions of euchromatin seem to be also enriched in highly repeated DNA sequences. The results indicate that heterochromatic chromosome segments might be sites of intense localized DNA replication. The same chromosome regions are also engaged in an active transcription process. The response to hybridization suggests that heterochromatic blocks of chromosome pair VI are heterogeneous in nucleotide sequences. The present studies also indicate that DNA and RNA puffs organized by different chromosome sites are specific of particular steps of embryo differentiation. The observed metabolic aspects of the suspensior's polytene chromosomes are discussed in relation to the synthesis of growth regulators which is known to occur in the suspensor. (Author)
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Directory of Open Access Journals (Sweden)
Audrey Costes
2010-12-01
Full Text Available We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSB(Cter as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSB(Cter interactome using GFP fusions and by Tap-tag and biochemical analysis. It includes at least 12 proteins. The interactome was previously shown to include PriA, RecG, and RecQ and extended in this study by addition of DnaE, SbcC, RarA, RecJ, RecO, XseA, Ung, YpbB, and YrrC. Targeting of YpbB to active forks appears to depend on RecS, a RecQ paralogue, with which it forms a stable complex. Most of these SSB partners are conserved in bacteria, while others, such as the essential DNA polymerase DnaE, YrrC, and the YpbB/RecS complex, appear to be specific to B. subtilis. SSB(Cter deletion has a moderate impact on B. subtilis cell growth. However, it markedly affects the efficiency of repair of damaged genomic DNA and arrested replication forks. ssbΔCter mutant cells appear deficient in RecA loading on ssDNA, explaining their inefficiency in triggering the SOS response upon exposure to genotoxic agents. Together, our findings show that the bacterial SSB(Cter acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome.
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DNA damage and polyploidization.
Chow, Jeremy; Poon, Randy Y C
2010-01-01
A growing body of evidence indicates that polyploidization triggers chromosomal instability and contributes to tumorigenesis. DNA damage is increasingly being recognized for its roles in promoting polyploidization. Although elegant mechanisms known as the DNA damage checkpoints are responsible for halting the cell cycle after DNA damage, agents that uncouple the checkpoints can induce unscheduled entry into mitosis. Likewise, defects of the checkpoints in several disorders permit mitotic entry even in the presence of DNA damage. Forcing cells with damaged DNA into mitosis causes severe chromosome segregation defects, including lagging chromosomes, chromosomal fragments and chromosomal bridges. The presence of these lesions in the cleavage plane is believed to abort cytokinesis. It is postulated that if cytokinesis failure is coupled with defects of the p53-dependent postmitotic checkpoint pathway, cells can enter S phase and become polyploids. Progress in the past several years has unraveled some of the underlying principles of these pathways and underscored the important role of DNA damage in polyploidization. Furthermore, polyploidization per se may also be an important determinant of sensitivity to DNA damage, thereby may offer an opportunity for novel therapies.
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[Chromosome as a chronicler: Genetic dating, historical events, and DNA-genealogic temptation].
Balanovsky, O P; Zaporozhchenko, V V
2016-07-01
Nonrecombinant portions of the genome, Y chromosome and mitochondrial DNA, are widely used for research on human population gene pools and reconstruction of their history. These systems allow the genetic dating of clusters of emerging haplotypes. The main method for age estimations is ρ statistics, which is an average number of mutations from founder haplotype to all modern-day haplotypes. A researcher can estimate the age of the cluster by multiplying this number by the mutation rate. The second method of estimation, ASD, is used for STR haplotypes of the Y chromosome and is based on the squared difference in the number of repeats. In addition to the methods of calculation, methods of Bayesian modeling assume a new significance. They have greater computational cost and complexity, but they allow obtaining an a posteriori distribution of the value of interest that is the most consistent with experimental data. The mutation rate must be known for both calculation methods and modeling methods. It can be determined either during the analysis of lineages or by providing calibration points based on populations with known formation time. These two approaches resulted in rate estimations for Y-chromosomal STR haplotypes with threefold difference. This contradiction was only recently refuted through the use of sequence data for the complete Y chromosome; “whole-genomic” rates of single nucleotide mutations obtained by both methods are mutually consistent and mark the area of application for different rates of STR markers. An issue even more crucial than that of the rates is correlation of the reconstructed history of the haplogroup (a cluster of haplotypes) and the history of the population. Although the need for distinguishing “lineage history” and “population history” arose in the earliest days of phylogeographic research, reconstructing the population history using genetic dating requires a number of methods and conditions. It is known that population
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DNA hybridization sensing for cytogenetic analysis
DEFF Research Database (Denmark)
Kwasny, Dorota; Dapra, Johannes; Brøgger, Anna Line
2013-01-01
are rearrangements between two chromosome arms that results in two derivative chromosomes having a mixed DNA sequence. The current detection method is a Fluorescent In situ Hybridization, which requires a use of expensive, fluorescently labeled probes that target the DNA sequences of two chromosomes involved...... in the translocation (Kwasny et al., 2012). We have developed a new double hybridization assay that allows for sorting of the DNA chromosomal fragments into separate compartment, moreover allowing for detection of the translocation. To detect the translocation it is necessary to determine that the two DNA sequences...... forming a derivative chromosome are connected, which is achieved by two subsequent hybridization steps. The first example of the translocation detection was presented on lab-on-a-disc using fluorescently labeled DNA fragments, representing the derivative chromosome (Brøgger et al., 2012). To allow...
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Zhuo, L; Reed, K M; Phillips, R B
1995-06-01
Variation in the intergenic spacer (IGS) of the ribosomal DNA (rDNA) of lake trout (Salvelinus namaycush) was examined. Digestion of genomic DNA with restriction enzymes showed that almost every individual had a unique combination of length variants with most of this variation occurring within rather than between populations. Sequence analysis of a 2.3 kilobase (kb) EcoRI-DraI fragment spanning the 3' end of the 28S coding region and approximately 1.8 kb of the IGS revealed two blocks of repetitive DNA. Putative transcriptional termination sites were found approximately 220 bases (b) downstream from the end of the 28S coding region. Comparison of the 2.3-kb fragments with two longer (3.1 kb) fragments showed that the major difference in length resulted from variation in the number of short (89 b) repeats located 3' to the putative terminator. Repeat units within a single nucleolus organizer region (NOR) appeared relatively homogeneous and genetic analysis found variants to be stably inherited. A comparison of the number of spacer-length variants with the number of NORs found that the number of length variants per individual was always less than the number of NORs. Examination of spacer variants in five populations showed that populations with more NORs had more spacer variants, indicating that variants are present at different rDNA sites on nonhomologous chromosomes.
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International Nuclear Information System (INIS)
Rozenberg-Arska, M.; van Strijp, J.A.; Hoekstra, W.P.; Verhoef, J.
1984-01-01
Phagocytosis and killing by polymorphonuclear and mononuclear leukocytes are important host resistance factors against invading microorganisms. Evidence showing that killing is rapidly followed by degradation of bacterial components is limited. Therefore, we studied the fate of Escherichia coli DNA following phagocytosis of E. coli by polymorphonuclear and mononuclear leukocytes. [ 3 H]Thymidine-labeled, unencapsulated E. coli PC2166 and E. coli 048K1 were incubated in serum, washed, and added to leukocytes. Uptake and killing of the bacteria and degradation of DNA were measured. Although phagocytosis and killing by mononuclear leukocytes was less efficient than that by polymorphonuclear leukocytes, only mononuclear leukocytes were able to degrade E. coli PC2166 DNA. Within 2 h, 60% of the radioactivity added to mononuclear leukocytes was released into the supernate, of which 40% was acid soluble. DNA of E. coli 048K1 was not degraded. To further analyze the capacity of mononuclear leukocytes to degrade E. coli DNA, chromosomal and plasmid DNA was isolated from ingested bacteria and subjected to agarose gel-electrophoresis. Only chromosomal DNA was degraded after phagocytosis. Plasmid DNA of E. coli carrying a gene coding for ampicillin resistance remained intact for a 2-h period after ingestion, and was still able to transform recipient E. coli cells after this period. Although we observed no DNA degradation during phagocytosis by polymorphonuclear leukocytes, lysates of both polymorphonuclear and mononuclear leukocytes contained acid-DNase activity with a pH optimum of 4.9. However, the DNase activity of mononuclear leukocytes was 20 times higher than that of polymorphonuclear leukocytes. No difference was observed between DNase activity from polymorphonuclear and mononuclear leukocytes from a chronic granulomatous disease patient with DNase activity from control polymorphonuclear and mononuclear leukocytes
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The mapping of novel genes to human chromosome 19
Energy Technology Data Exchange (ETDEWEB)
Buenaventura, J.M. [Sarah Lawrence College, Bronxville, NY (United States)
1994-12-01
The principle goal of our laboratory is the discovery of new genes on human chromosome 19. One of the strategies to achieve this goal is through the use of cDNA clones known as {open_quotes}expressed sequence tags{close_quotes} (ESTs). ESTs, short segments of sequence from a cDNA clone that correspond to the mRNA, occur as unique regions in the genome and, therefore, can be used as markers for specific positions. In collaboration with researchers from Genethon in France, fifteen cDNA clones from a normalized human infant brain cDNA library were tested and determined to map to chromosome 19. A verification procedure is then followed to confirm assignment to chromosome 19. First, primers for each cDNA clone are developed and then amplified by polymerase chain reaction from genomic DNA. Next, a {sup 32}P-radiolabeled probe is made by polymerase chain reaction for each clone and then hybridized against filters containing an LLNL chromosome 19-specific cosmid library to find putative locations on the chromosome. The location is then verified by running a polymerase chain reactions from the positive cosmids. With the Browser database at LLNL, additional information about the positive cosmids can be found. Through use of the BLAST database at the National Library of Medicine, homologous sequences to the clones can be found. Among the fifteen cDNA clones received from Genethon, all have been amplified by polymerase chain reaction. Three have turned out as repetitive elements in the genome. Ten have been mapped to specific locations on chromosome 19. Putative locations have been found for the remaining two clones and thus verification testing will proceed.
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Someswara Rao, Chinta; Viswanadha Raju, S.
2016-01-01
In this paper, we consider correlation coefficient, rank correlation coefficient and cosine similarity measures for evaluating similarity between Homo sapiens and monkeys. We used DNA chromosomes of genome wide genes to determine the correlation between the chromosomal content and evolutionary relationship. The similarity among the H. sapiens and monkeys is measured for a total of 210 chromosomes related to 10 species. The similarity measures of these different species show the relationship b...
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Geant4.10 simulation of geometric model for metaphase chromosome
Energy Technology Data Exchange (ETDEWEB)
Rafat-Motavalli, L., E-mail: rafat@um.ac.ir; Miri-Hakimabad, H.; Bakhtiyari, E.
2016-04-01
In this paper, a geometric model of metaphase chromosome is explained. The model is constructed according to the packing ratio and dimension of the structure from nucleosome up to chromosome. A B-DNA base pair is used to construct 200 base pairs of nucleosomes. Each chromatin fiber loop, which is the unit of repeat, has 49,200 bp. This geometry is entered in Geant4.10 Monte Carlo simulation toolkit and can be extended to the whole metaphase chromosomes and any application in which a DNA geometrical model is needed. The chromosome base pairs, chromosome length, and relative length of chromosomes are calculated. The calculated relative length is compared to the relative length of human chromosomes.
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Geant4.10 simulation of geometric model for metaphase chromosome
International Nuclear Information System (INIS)
Rafat-Motavalli, L.; Miri-Hakimabad, H.; Bakhtiyari, E.
2016-01-01
In this paper, a geometric model of metaphase chromosome is explained. The model is constructed according to the packing ratio and dimension of the structure from nucleosome up to chromosome. A B-DNA base pair is used to construct 200 base pairs of nucleosomes. Each chromatin fiber loop, which is the unit of repeat, has 49,200 bp. This geometry is entered in Geant4.10 Monte Carlo simulation toolkit and can be extended to the whole metaphase chromosomes and any application in which a DNA geometrical model is needed. The chromosome base pairs, chromosome length, and relative length of chromosomes are calculated. The calculated relative length is compared to the relative length of human chromosomes.
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CHROMOSOMAL MULTIPLICITY IN BURKHOLDERIA CEPACIA
We have used CHEF gel electrophoresis to screen preparations of large DNA from different Burkholderia cepacia isolates for the presence of DNA species corresponding to the linearized forms of the three chromosomes of 3.4,2.5, and 0.9 Mb identified in B. cepacia strain 17616. DNA ...
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Palacios-Gimenez, Octavio M; Milani, Diogo; Lemos, Bernardo; Castillo, Elio R; Martí, Dardo A; Ramos, Erica; Martins, Cesar; Cabral-de-Mello, Diogo C
2018-01-08
Neo-sex chromosome systems arose independently multiple times in evolution, presenting the remarkable characteristic of repetitive DNAs accumulation. Among grasshoppers, occurrence of neo-XY was repeatedly noticed in Melanoplinae. Here we analyzed the most abundant tandem repeats of R. bergii (2n = 22, neo-XY♂) using deep Illumina sequencing and graph-based clustering in order to address the neo-sex chromosomes evolution. The analyses revealed ten families of satDNAs comprising about ~1% of the male genome, which occupied mainly C-positive regions of autosomes. Regarding the sex chromosomes, satDNAs were recorded within centromeric or interstitial regions of the neo-X chromosome and four satDNAs occurred in the neo-Y, two of them being exclusive (Rber248 and Rber299). Using a combination of probes we uncovered five well-defined cytological variants for neo-Y, originated by multiple paracentric inversions and satDNA amplification, besides fragmented neo-Y. These neo-Y variants were distinct in frequency between embryos and adult males. The genomic data together with cytogenetic mapping enabled us to better understand the neo-sex chromosome dynamics in grasshoppers, reinforcing differentiation of neo-X and neo-Y and revealing the occurrence of multiple additional rearrangements involved in the neo-Y evolution of R. bergii. We discussed the possible causes that led to differences in frequency for the neo-Y variants between embryos and adults. Finally we hypothesize about the role of DNA satellites in R. bergii as well as putative historical events involved in the evolution of the R. bergii neo-XY.
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Human Chromosome 21: Mapping of the chromosomes and cloning of cDNAs
Energy Technology Data Exchange (ETDEWEB)
Antonarakis, S.E.
1991-09-01
The objective of the research funded by DOE grant DE-FG02-89ER60857 from 6/15/89 to 8/31/91 was to contribute to the physical mapping of human chromosome 21 (HC21) by cloning large fragments of DNA into Yeast Artificial Chromosomes (YACs) and identify YACs that map on HC21. A total of 54 sequence tagged sites (STS) have been developed and mapped in our laboratory to HC21 and can be used as initial reference points for YAC identification and construction of overlapping clones. A small YAC library was constructed which is HC21 specific. DNA from somatic cell hybrid WAV17 or from flow-sorted HC21 was partially digested with EcoRI, ligated into vectors PJS97, PJS98, and YACs have been obtained with average size insert of more than 300 kb. This library has been deposited in D. Patterson's lab for the Joint YAC screening effort. Additional YAC libraries from ICI Pharmaceuticals or from Los Alamos National Laboratories have been screened with several STS and positive YACs have been identified. Work in progress includes screening of YAC libraries in order to construct overlapping clones, characterization of the cloning ends of YACs, characterization of additional STS and cloning of HC21 specific cDNAs. 15 refs., 2 figs., 5 tabs.
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Analysis of the Ceratitis capitata y chromosome using in situ hybridization to mitotic chromosomes
International Nuclear Information System (INIS)
Willhoeft, U.; Franz, G.
1998-01-01
In Ceratitis capitata the Y chromosome is responsible for sex-determination. We used fluorescence in situ hybridization (FISH) for cytogenetic analysis of mitotic chromosomes. FISH with the wild-type strain EgyptII and two repetitive DNA probes enabled us to differentiate between the short and the long arm of the Y chromosome and gives a much better resolution than C-banding of mitotic chromosomes. We identified the Y-chromosomal breakpoints in Y-autosome translocations using FISH. Even more complex rearrangements i.e. deletions and insertions in some translocation strains were detected by this method. A strategy for mapping the primary sex determination factor in Ceratitis capitata by FISH is presented. (author)
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Directory of Open Access Journals (Sweden)
Wang Yanjie
2010-09-01
Full Text Available Abstract Background Distant hybridization can result genome duplication and allopolyploid formation which may play a significant role in the origin and evolution of many plant species. It is unclear how the two or more divergent genomes coordinate in one nucleus with a single parental cytoplasm within allopolyploids. We used cytological and molecular methods to investigate the genetic and epigenetic instabilities associated with the process of distant hybridization and allopolyploid formation, measuring changes in chromosome number and DNA methylation across multiple generations. Results F1 plants from intergeneric hybridization between Raphanus sativus L. (2n = 18, RR and Brassica alboglabra Bailey (2n = 18, CC were obtained by hand crosses and subsequent embryo rescue. Random amplification of polymorphic DNA (RAPD markers were used to identify the F1 hybrid plants. The RAPD data indicated that the hybrids produced specific bands similar to those of parents and new bands that were not present in either parent. Chromosome number variation of somatic cells from allotetraploids in the F4 to F10 generations showed that intensive genetic changes occurred in the early generations of distant hybridization, leading to the formation of mixopolyploids with different chromosome numbers. DNA methylation variation was revealed using MSAP (methylation-sensitive amplification polymorphism, which showed that cytosine methylation patterns changed markedly in the process of hybridization and amphidiploid formation. Differences in cytosine methylation levels demonstrated an epigenetic instability of the allopolyploid of Raphanobrassica between the genetically stable and unstable generations. Conclusions Our results showed that chromosome instability occurred in the early generations of allopolyploidy and then the plants were reverted to largely euploidy in later generations. During this process, DNA methylation changed markedly. These results suggest that
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Directory of Open Access Journals (Sweden)
Gosalvez Jaime
2009-04-01
Full Text Available Abstract Background Fluoroquinolones are extensively used antibiotics that induce DNA double-strand breaks (DSBs by trapping DNA gyrase and topoisomerase IV on DNA. This effect is usually evaluated using biochemical or molecular procedures, but these are not effective at the single-cell level. We assessed ciprofloxacin (CIP-induced chromosomal DNA breakage in single-cell Escherichia coli by direct visualization of the DNA fragments that diffused from the nucleoid obtained after bacterial lysis in an agarose microgel on a slide. Results Exposing the E. coli strain TG1 to CIP starting at a minimum inhibitory concentration (MIC of 0.012 μg/ml and at increasing doses for 40 min increased the DNA fragmentation progressively. DNA damage started to be detectable at the MIC dose. At a dose of 1 μg/ml of CIP, DNA damage was visualized clearly immediately after processing, and the DNA fragmentation increased progressively with the antibiotic incubation time. The level of DNA damage was much higher when the bacteria were taken from liquid LB broth than from solid LB agar. CIP treatment produced a progressively slower rate of DNA damage in bacteria in the stationary phase than in the exponentially growing phase. Removing the antibiotic after the 40 min incubation resulted in progressive DSB repair activity with time. The magnitude of DNA repair was inversely related to CIP dose and was noticeable after incubation with CIP at 0.1 μg/ml but scarce after 10 μg/ml. The repair activity was not strictly related to viability. Four E. coli strains with identified mechanisms of reduced sensitivity to CIP were assessed using this procedure and produced DNA fragmentation levels that were inversely related to MIC dose, except those with very high MIC dose. Conclusion This procedure for determining DNA fragmentation is a simple and rapid test for studying and evaluating the effect of quinolones.
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International Nuclear Information System (INIS)
Bredberg, A.; Lambert, B.; Lindblad, A.; Swanbeck, G.; Wennersten, G.
1983-01-01
Exposure of human lymphocytes and skin fibroblasts in vitro to a single, clinically used dose of PUVA, i.e., 0.1 micrograms/ml of 8-methoxypsoralen (8-MOP) plus 0.9-4 J/cm2 of longwave ultraviolet radiation (UVA), lead to the formation of DNA damage as determined by alkaline elution, and to chromosome aberrations and sister chromatid exchanges (SCE). When lymphocyte-enriched plasma was obtained from psoriasis patients 2 h after oral intake of 8-MOP and then UVA irradiated (1.8-3.6 J/cm2) in vitro, an increased frequency of chromosome aberrations and SCE was observed. Normal levels of chromosome aberrations and SCE were found in lymphocytes of psoriasis patients after 3-30 weeks of PUVA treatment in vivo. A small but statistically significant increase in the SCE frequency was observed in the lymphocytes of psoriasis patients treated for 1-6 years with PUVA (mean 18.0 SCE/cell) as compared with before PUVA (mean 15.8, p less than 0.05). Skin fibroblasts of psoriasis patients analyzed 5 years after the start of PUVA treatment showed a normal number of SCE but a high fraction of filter-retained DNA in the alkaline elution assay, suggesting the presence of cross-linked DNA
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Huang, D; Wu, W; Lu, L
2004-05-01
Amplification of resistance gene analogs (RGAs) is both a useful method for acquiring DNA markers closely linked to disease resistance (R) genes and a potential approach for the rapid cloning of R genes in plants. However, the screening of target sequences from among the numerous amplified RGAs can be very laborious. The amplification of RGAs from specific chromosomes could greatly reduce the number of RGAs to be screened and, consequently, speed up the identification of target RGAs. We have developed two methods for amplifying RGAs from single chromosomes. Method 1 uses products of Sau3A linker adaptor-mediated PCR (LAM-PCR) from a single chromosome as the templates for RGA amplification, while Method 2 directly uses a single chromosomal DNA molecule as the template. Using a pair of degenerate primers designed on the basis of the conserved nucleotide-binding-site motifs in many R genes, RGAs were successfully amplified from single chromosomes of pomelo using both these methods. Sequencing and cluster analysis of RGA clones obtained from single chromosomes revealed the number, type and organization of R-gene clusters on the chromosomes. We suggest that Method 1 is suitable for analyzing chromosomes that are unidentifiable under a microscope, while Method 2 is more appropriate when chromosomes can be clearly identified.
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Directory of Open Access Journals (Sweden)
Tatyana Yu Vatolina
Full Text Available Salivary gland polytene chromosomes demonstrate banding pattern, genetic meaning of which is an enigma for decades. Till now it is not known how to mark the band/interband borders on physical map of DNA and structures of polytene chromosomes are not characterized in molecular and genetic terms. It is not known either similar banding pattern exists in chromosomes of regular diploid mitotically dividing nonpolytene cells. Using the newly developed approach permitting to identify the interband material and localization data of interband-specific proteins from modENCODE and other genome-wide projects, we identify physical limits of bands and interbands in small cytological region 9F13-10B3 of the X chromosome in D. melanogaster, as well as characterize their general molecular features. Our results suggests that the polytene and interphase cell line chromosomes have practically the same patterns of bands and interbands reflecting, probably, the basic principle of interphase chromosome organization. Two types of bands have been described in chromosomes, early and late-replicating, which differ in many aspects of their protein and genetic content. As appeared, origin recognition complexes are located almost totally in the interbands of chromosomes.
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Directory of Open Access Journals (Sweden)
Emanuele eLibertini
2015-12-01
Full Text Available BAH domain-containing protein 1 (BAHD1 is involved in heterochromatin formation and gene repression in human cells. BAHD1 also localizes to the inactive X chromosome (Xi, but the functional significance of this targeting is unknown. So far, research on this protein has been hampered by its low endogenous abundance and its role in epigenetic regulation remains poorly explored. In this work, we used whole-genome bisulfite sequencing (BS-seq to compare the DNA methylation profile of HEK293 cells expressing low levels of BAHD1 (HEK-CT to that of isogenic cells stably overexpressing BAHD1 (HEK-BAHD1. We show that increasing BAHD1 levels induces de novo DNA methylation on autosomes and a marked hypomethylation on the X chromosome (chrX. We identified 91,358 regions that have different methylation patterns in HEK-BAHD1 compared to HEK-CT cells (termed BAHD1-DMRs, of which 83,850 mapped on autosomes and 7,508 on the X chromosome (chrX. Autosomal BAHD1-DMRs were predominantly hypermethylated and located to satellites, interspersed repeats and intergenic regions. In contrast, BAHD1-DMRs on chrX were mainly hypomethylated and located to gene bodies and enhancers. We further found that BAHD1-DMRs display a higher-order organization by being clustered within large chromosomal domains. Half of these BAHD1-Associated differentially methylated Domains (BADs overlapped with lamina-associated domains (LADs. Based on these results, we propose that BAHD1-mediated heterochromatin formation is linked to DNA methylation and may play a role in the spatial architecture of the genome.
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Human hereditary diseases associated with elevated frequency of chromosome aberrations
International Nuclear Information System (INIS)
Ejima, Yosuke
1988-01-01
Human recessive diseases collectively known as chromosome breakage syndromes include Fanconi's anemia, Bloom's syndrome and ataxia telangiectasia. Cells from these patients show chromosome instabilities both spontaneously and following treatments with radiations or certain chemicals, where defects in DNA metabolisms are supposed to be involved. Cells from patients with ataxia telangiectasia are hypersensitive to ionizing radiations, though DNA replication is less affected than in normal cells. Chromatid-type as well as chromosom-type aberrations are induced in cells irradiated in G 0 or G 1 phases. These unusual responses to radiations may provide clues for understanding the link between DNA replicative response and cellular radiosensitivity. Alterations in cellular radiosensitivity or spontaneous chromosome instabilities are observed in some patients with congenital chromosome anomalies or dominant diseases, where underlying defects may be different from those in recessive diseases. (author)
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Human hereditary diseases associated with elevated frequency of chromosome aberrations
Energy Technology Data Exchange (ETDEWEB)
Ejima, Yosuke; Ikushima, Takaji (ed.)
1988-07-01
Human recessive diseases collectively known as chromosome breakage syndromes include Fanconi's anemia, Bloom's syndrome and ataxia telangiectasia. Cells from these patients show chromosome instabilities both spontaneously and following treatments with radiations or certain chemicals, where defects in DNA metabolisms are supposed to be involved. Cells from patients with ataxia telangiectasia are hypersensitive to ionizing radiations, though DNA replication is less affected than in normal cells. Chromatid-type as well as chromosom-type aberrations are induced in cells irradiated in G/sub 0/ or G/sub 1/ phases. These unusual responses to radiations may provide clues for understanding the link between DNA replicative response and cellular radiosensitivity. Alterations in cellular radiosensitivity or spontaneous chromosome instabilities are observed in some patients with congenital chromosome anomalies or dominant diseases, where underlying defects may be different from those in recessive diseases.
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Evidence of activity-specific, radial organization of mitotic chromosomes in Drosophila.
Directory of Open Access Journals (Sweden)
Yuri G Strukov
2011-01-01
Full Text Available The organization and the mechanisms of condensation of mitotic chromosomes remain unsolved despite many decades of efforts. The lack of resolution, tight compaction, and the absence of function-specific chromatin labels have been the key technical obstacles. The correlation between DNA sequence composition and its contribution to the chromosome-scale structure has been suggested before; it is unclear though if all DNA sequences equally participate in intra- or inter-chromatin or DNA-protein interactions that lead to formation of mitotic chromosomes and if their mitotic positions are reproduced radially. Using high-resolution fluorescence microscopy of live or minimally perturbed, fixed chromosomes in Drosophila embryonic cultures or tissues expressing MSL3-GFP fusion protein, we studied positioning of specific MSL3-binding sites. Actively transcribed, dosage compensated Drosophila genes are distributed along the euchromatic arm of the male X chromosome. Several novel features of mitotic chromosomes have been observed. MSL3-GFP is always found at the periphery of mitotic chromosomes, suggesting that active, dosage compensated genes are also found at the periphery of mitotic chromosomes. Furthermore, radial distribution of chromatin loci on mitotic chromosomes was found to be correlated with their functional activity as judged by core histone modifications. Histone modifications specific to active chromatin were found peripheral with respect to silent chromatin. MSL3-GFP-labeled chromatin loci become peripheral starting in late prophase. In early prophase, dosage compensated chromatin regions traverse the entire width of chromosomes. These findings suggest large-scale internal rearrangements within chromosomes during the prophase condensation step, arguing against consecutive coiling models. Our results suggest that the organization of mitotic chromosomes is reproducible not only longitudinally, as demonstrated by chromosome-specific banding
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Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression.
Aze, Antoine; Sannino, Vincenzo; Soffientini, Paolo; Bachi, Angela; Costanzo, Vincenzo
2016-06-01
Half of the human genome is made up of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using bacterial artificial chromosomes in Xenopus laevis egg extract. Using this approach we characterized the chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication-dependent enrichment of a network of DNA repair factors including the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR-dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to the inability of the single-stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of topoisomerase I-dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications for our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions.
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International Nuclear Information System (INIS)
Liu Qingjie; Feng Jiangbing; Lu Xue; Chen Deqing; Lv Huimin; Su Xu; Liu Yufei; Jia Kejun
2008-01-01
Objective: To explore if the occupational exposure to low dose thorium could induce adaptive response in peripheral lymphocytes. Methods: 40 individuals, who exposed to thorium and rare earth mixed dust (exposure group) or control in Baotou Steel Plant, were selected, and chromosome aberrations were analyzed. Then the peripheral blood samples were irradiated in vitro with 2 Gy 60 Co γ-rays, and unstable chromosome aberration or DNA stand breakage analysis using single cell gel electrophoresis was performed. Results: The dicentrics before 2 Gy exposure in exposure group was higher than that in control (P>0.05). But the dicentrics after 2 Gy exposure in exposure group was lower than that in control, but not significantly (P >0.05). The tricentrics in exposure group was significantly lower than that in control (U=3.1622, 0.001< P<0.002). The DNA strand breakage in control group was significantly higher than that in exposure group (t=25, P<0.001). Conclusions: Occupational exposure to low dose thorium could induce the adaptive response on chromosome aberration and DNA strand breakage in peripheral lymphocytes. (authors)
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Evolutionary dynamics of rDNA clusters on chromosomes of moths and butterflies (Lepidoptera)
Czech Academy of Sciences Publication Activity Database
Nguyen, Petr; Sahara, K.; Yoshido, A.; Marec, František
2010-01-01
Roč. 138, č. 3 (2010), s. 343-354 ISSN 0016-6707 R&D Projects: GA ČR GA206/06/1860; GA AV ČR IAA600960925 Grant - others:Student Grant Agency of the Faculty of Science, University of South Bohemia(CZ) SGA2006/01; Japan Society for the Promotion of Science(JP) 18380037; Japan Society for the Promotion of Science(JP) 191114; GA ČR(CZ) 521/08/H042 Institutional research plan: CEZ:AV0Z50070508 Keywords : ribosomal DNA * nucleolar organizer region * chromosome fusion Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.358, year: 2010
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Mitotic chromosome condensation in vertebrates
International Nuclear Information System (INIS)
Vagnarelli, Paola
2012-01-01
Work from several laboratories over the past 10–15 years has revealed that, within the interphase nucleus, chromosomes are organized into spatially distinct territories [T. Cremer, C. Cremer, Chromosome territories, nuclear architecture and gene regulation in mammalian cells, Nat. Rev. Genet. 2 (2001) 292–301 and T. Cremer, M. Cremer, S. Dietzel, S. Muller, I. Solovei, S. Fakan, Chromosome territories—a functional nuclear landscape, Curr. Opin. Cell Biol. 18 (2006) 307–316]. The overall compaction level and intranuclear location varies as a function of gene density for both entire chromosomes [J.A. Croft, J.M. Bridger, S. Boyle, P. Perry, P. Teague,W.A. Bickmore, Differences in the localization and morphology of chromosomes in the human nucleus, J. Cell Biol. 145 (1999) 1119–1131] and specific chromosomal regions [N.L. Mahy, P.E. Perry, S. Gilchrist, R.A. Baldock, W.A. Bickmore, Spatial organization of active and inactive genes and noncoding DNA within chromosome territories, J. Cell Biol. 157 (2002) 579–589] (Fig. 1A, A'). In prophase, when cyclin B activity reaches a high threshold, chromosome condensation occurs followed by Nuclear Envelope Breakdown (NEB) [1]. At this point vertebrate chromosomes appear as compact structures harboring an attachment point for the spindle microtubules physically recognizable as a primary constriction where the two sister chromatids are held together. The transition from an unshaped interphase chromosome to the highly structured mitotic chromosome (compare Figs. 1A and B) has fascinated researchers for several decades now; however a definite picture of how this process is achieved and regulated is not yet in our hands and it will require more investigation to comprehend the complete process. From a biochemical point of view a vertebrate mitotic chromosomes is composed of DNA, histone proteins (60%) and non-histone proteins (40%) [6]. I will discuss below what is known to date on the contribution of these two different
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Mitotic chromosome condensation in vertebrates
Energy Technology Data Exchange (ETDEWEB)
Vagnarelli, Paola, E-mail: P.Vagnarelli@ed.ac.uk
2012-07-15
Work from several laboratories over the past 10-15 years has revealed that, within the interphase nucleus, chromosomes are organized into spatially distinct territories [T. Cremer, C. Cremer, Chromosome territories, nuclear architecture and gene regulation in mammalian cells, Nat. Rev. Genet. 2 (2001) 292-301 and T. Cremer, M. Cremer, S. Dietzel, S. Muller, I. Solovei, S. Fakan, Chromosome territories-a functional nuclear landscape, Curr. Opin. Cell Biol. 18 (2006) 307-316]. The overall compaction level and intranuclear location varies as a function of gene density for both entire chromosomes [J.A. Croft, J.M. Bridger, S. Boyle, P. Perry, P. Teague,W.A. Bickmore, Differences in the localization and morphology of chromosomes in the human nucleus, J. Cell Biol. 145 (1999) 1119-1131] and specific chromosomal regions [N.L. Mahy, P.E. Perry, S. Gilchrist, R.A. Baldock, W.A. Bickmore, Spatial organization of active and inactive genes and noncoding DNA within chromosome territories, J. Cell Biol. 157 (2002) 579-589] (Fig. 1A, A'). In prophase, when cyclin B activity reaches a high threshold, chromosome condensation occurs followed by Nuclear Envelope Breakdown (NEB) [1]. At this point vertebrate chromosomes appear as compact structures harboring an attachment point for the spindle microtubules physically recognizable as a primary constriction where the two sister chromatids are held together. The transition from an unshaped interphase chromosome to the highly structured mitotic chromosome (compare Figs. 1A and B) has fascinated researchers for several decades now; however a definite picture of how this process is achieved and regulated is not yet in our hands and it will require more investigation to comprehend the complete process. From a biochemical point of view a vertebrate mitotic chromosomes is composed of DNA, histone proteins (60%) and non-histone proteins (40%) [6]. I will discuss below what is known to date on the contribution of these two different classes
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Abundance and Characterization of Perfect Microsatellites on the Cattle Y Chromosome.
Ma, Zhi-Jie
2017-07-03
Microsatellites or simple sequence repeats (SSRs) are found in most organisms and play an important role in genomic organization and function. To characterize the abundance of SSRs (1-6 base-pairs [bp]) on the cattle Y chromsome, the relative frequency and density of perfect or uninterrupted SSRs based on the published Y chromosome sequence were examined. A total of 17,273 perfect SSRs were found, with total length of 324.78 kb, indicating that approximately 0.75% of the cattle Y chromosome sequence (43.30 Mb) comprises perfect SSRs, with an average length of 18.80 bp. The relative frequency and density were 398.92 loci/Mb and 7500.62 bp/Mb, respectively. The proportions of the six classes of perfect SSRs were highly variable on the cattle Y chromosome. Mononucleotide repeats had a total number of 8073 (46.74%) and an average length of 15.45 bp, and were the most abundant SSRs class, while the percentages of di-, tetra-, tri-, penta-, and hexa-nucleotide repeats were 22.86%, 11.98%, 11.58%, 6.65%, and 0.19%, respectively. Different classes of SSRs varied in their repeat number, with the highest being 42 for dinucleotides. Results reveal that repeat categories A, AC, AT, AAC, AGC, GTTT, CTTT, ATTT, and AACTG predominate on the Y chromosome. This study provides insight into the organization of cattle Y chromosome repetitive DNA, as well as information useful for developing more polymorphic cattle Y-chromosome-specific SSRs.
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Characterization of muntjac DNA
International Nuclear Information System (INIS)
Davis, R.C.
1981-01-01
Sister chromatid exchange (SCE) in muntjac chromosomes is generally proportional to the chromosomal DNA content, but the SCE frequency is reduced in the heterochromatic neck region of the X chromosome. The physical properties of muntjac DNA and the kinetics of repair of UV damage in muntjac heterochromatin and euchromatin were examined and compared with the distribution of sister chromatid exchange
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Characterization of muntjac DNA
Energy Technology Data Exchange (ETDEWEB)
Davis, R.C.
1981-05-27
Sister chromatid exchange (SCE) in muntjac chromosomes is generally proportional to the chromosomal DNA content, but the SCE frequency is reduced in the heterochromatic neck region of the X chromosome. The physical properties of muntjac DNA and the kinetics of repair of UV damage in muntjac heterochromatin and euchromatin were examined and compared with the distribution of sister chromatid exchange.
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Construction of BAC Libraries from Flow-Sorted Chromosomes.
Šafář, Jan; Šimková, Hana; Doležel, Jaroslav
2016-01-01
Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by constructing a library derived from a smaller part of the genome. Here we describe construction of BAC libraries from mitotic chromosomes purified by flow cytometric sorting. Chromosome-specific BAC libraries facilitate positional gene cloning, physical mapping, and sequencing in complex plant genomes.
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An ultra-high discrimination Y chromosome short tandem repeat multiplex DNA typing system.
Directory of Open Access Journals (Sweden)
Erin K Hanson
Full Text Available In forensic casework, Y chromosome short tandem repeat markers (Y-STRs are often used to identify a male donor DNA profile in the presence of excess quantities of female DNA, such as is found in many sexual assault investigations. Commercially available Y-STR multiplexes incorporating 12-17 loci are currently used in forensic casework (Promega's PowerPlex Y and Applied Biosystems' AmpFlSTR Yfiler. Despite the robustness of these commercial multiplex Y-STR systems and the ability to discriminate two male individuals in most cases, the coincidence match probabilities between unrelated males are modest compared with the standard set of autosomal STR markers. Hence there is still a need to develop new multiplex systems to supplement these for those cases where additional discriminatory power is desired or where there is a coincidental Y-STR match between potential male participants. Over 400 Y-STR loci have been identified on the Y chromosome. While these have the potential to increase the discrimination potential afforded by the commercially available kits, many have not been well characterized. In the present work, 91 loci were tested for their relative ability to increase the discrimination potential of the commonly used 'core' Y-STR loci. The result of this extensive evaluation was the development of an ultra high discrimination (UHD multiplex DNA typing system that allows for the robust co-amplification of 14 non-core Y-STR loci. Population studies with a mixed African American and American Caucasian sample set (n = 572 indicated that the overall discriminatory potential of the UHD multiplex was superior to all commercial kits tested. The combined use of the UHD multiplex and the Applied Biosystems' AmpFlSTR Yfiler kit resulted in 100% discrimination of all individuals within the sample set, which presages its potential to maximally augment currently available forensic casework markers. It could also find applications in human evolutionary
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Applications of pooled DNA samples to the assessment of population affinities: short tandem repeats.
Crawford, M H; Banerjee, P; Demarchi, D A; Zlojutro, M; McComb, J; Livshits, G; Henneberg, M; Mosher, M J; Schanfield, M S; Knowles, J A
2005-12-01
Pooled DNA samples have been used in association studies of Mendelian disease genes. This method involves combining equal quantities of DNA from patients and control subjects into separate pools and comparing the pools for distributions of genetic markers. In this study identical quantities of DNA from 300 individuals representing 6 populations were pooled and amplified for 296 loci using the touchdown polymerase chain reaction (PCR) method. The purpose of this study is to test the efficacy of pooled DNA markers in the reconstruction of the genetic structure of human populations. The populations sampled included Chuvash, Buryats, Kizhi, Native Americans, South Africans, and New York City whites. To test the accuracy of the allele-frequency distributions, we genotyped the Buryats and New York samples individually for six microsatellite markers and compared their frequencies to the allele frequencies derived from the electropherogram peak heights for the pooled DNA, producing a correlation of 0.9811 with a variance of less than 0.04. Two-dimensional scaling of genetic distances among the six populations produced clusters that reflected known historical relationships. A distance matrix was created using all 296 loci, and matrices based on individual chromosomes were correlated against the total matrix. As expected, the largest chromosomes had the highest correlations with the total matrix, whereas one of the smallest chromosomes, chromosome 22, had the lowest correlation and differed most from the combined STR distance matrix.
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International Nuclear Information System (INIS)
Chung, Hai Won; Kim, Su Young; Kim, Byung Mo; Kim, Sun Jin; Ha, Sung Whan; Kim, Tae Hwan; Cho, Chul Koo
2000-01-01
Comparative study was performed for the assessment of DNA damage and Chromosomal aberration in human lymphocyte exposed to low dose radiation using Fluorescence In Situ Hybridization(FISH) and Single Cell Gel Electrophoresis(SCGE). Chromosomal aberrations in human lymphocyte exposed to radiation at doses of 5, 10, 30 and 50cGy were analysed with whole chromosome-specific probes by human chromosome 1, 2 and 4 according to PAINT system. FISH with chromosome-specific probe has been used to be a valid and rapid method for detection of chromosome rearrangements induced by low dose radiation. The frequencies of stable translocation per cell equivalents were 0.0116, 0.0375, 0.0407, 0.0727 and 0.0814 for 0, 5, 10, 30 and 50cGy, respectively, and those of dicentric were 0.00, 0.0125, 0.174, 0.0291 and 0.0407 respectively. Radiation induced DNA damage in human lymphocyte in a dose-dependent manner at low doses from 5cGy to 50cGy, which were analysed by single Cell Gel Electrophoresis(SCGE). From above results, FISH seemed to be useful for radiation biodosimetry by which the frequencies of stable aberrations in human lymphocyte can be observed more easily than by conventional method and SCGE also seemed to be sensitive method for detecting DNA damage by low dose radiation exposure, so that those methods will improve our technique to perform meaningful biodosimetry for radiation at low doses
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Directory of Open Access Journals (Sweden)
Laura S Burrack
2016-09-01
Full Text Available Assembly of kinetochore complexes, involving greater than one hundred proteins, is essential for chromosome segregation and genome stability. Neocentromeres, or new centromeres, occur when kinetochores assemble de novo, at DNA loci not previously associated with kinetochore proteins, and they restore chromosome segregation to chromosomes lacking a functional centromere. Neocentromeres have been observed in a number of diseases and may play an evolutionary role in adaptation or speciation. However, the consequences of neocentromere formation on chromosome missegregation rates, gene expression, and three-dimensional (3D nuclear structure are not well understood. Here, we used Candida albicans, an organism with small, epigenetically-inherited centromeres, as a model system to study the functions of twenty different neocentromere loci along a single chromosome, chromosome 5. Comparison of neocentromere properties relative to native centromere functions revealed that all twenty neocentromeres mediated chromosome segregation, albeit to different degrees. Some neocentromeres also caused reduced levels of transcription from genes found within the neocentromere region. Furthermore, like native centromeres, neocentromeres clustered in 3D with active/functional centromeres, indicating that formation of a new centromere mediates the reorganization of 3D nuclear architecture. This demonstrates that centromere clustering depends on epigenetically defined function and not on the primary DNA sequence, and that neocentromere function is independent of its distance from the native centromere position. Together, the results show that a neocentromere can form at many loci along a chromosome and can support the assembly of a functional kinetochore that exhibits native centromere functions including chromosome segregation accuracy and centromere clustering within the nucleus.
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Pita, Sebastián; Mora, Pablo; Vela, Jesús; Palomeque, Teresa; Sánchez, Antonio; Panzera, Francisco; Lorite, Pedro
2018-04-24
Chagas disease or American trypanosomiasis affects six to seven million people worldwide, mostly in Latin America. This disease is transmitted by hematophagous insects known as "kissing bugs" (Hemiptera, Triatominae), with Triatoma infestans and Rhodnius prolixus being the two most important vector species. Despite the fact that both species present the same diploid chromosome number (2 n = 22), they have remarkable differences in their total DNA content, chromosome structure and genome organization. Variations in the DNA genome size are expected to be due to differences in the amount of repetitive DNA sequences. The T. infestans genome-wide analysis revealed the existence of 42 satellite DNA families. BLAST searches of these sequences against the R. prolixus genome assembly revealed that only four of these satellite DNA families are shared between both species, suggesting a great differentiation between the Triatoma and Rhodnius genomes. Fluorescence in situ hybridization (FISH) location of these repetitive DNAs in both species showed that they are dispersed on the euchromatic regions of all autosomes and the X chromosome. Regarding the Y chromosome, these common satellite DNAs are absent in T. infestans but they are present in the R. prolixus Y chromosome. These results support a different origin and/or evolution in the Y chromosome of both species.
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Y chromosome STR typing in crime casework.
Roewer, Lutz
2009-01-01
Since the beginning of the nineties the field of forensic Y chromosome analysis has been successfully developed to become commonplace in laboratories working in crime casework all over the world. The ability to identify male-specific DNA renders highly variable Y-chromosomal polymorphisms, the STR sequences, an invaluable addition to the standard panel of autosomal loci used in forensic genetics. The male-specificity makes the Y chromosome especially useful in cases of male/female cell admixture, namely in sexual assault cases. On the other hand, the haploidy and patrilineal inheritance complicates the interpretation of a Y-STR match, because male relatives share for several generations an identical Y-STR profile. Since paternal relatives tend to live in the geographic and cultural territory of their ancestors, the Y chromosome analysis has a potential to make inferences on the population of origin of a given DNA profile. This review addresses the fields of application of Y chromosome haplotyping, the interpretation of results, databasing efforts and population genetics aspects.
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International Nuclear Information System (INIS)
Yanishevsky, R.M.
1975-01-01
Human diploid cell cultures, strain WI-38, have a finite proliferative capacity and have been proposed as a model of biological aging. To identify the cell cycle phase of the nondividing cells, cultures of various ages were exposed to 3 Hdt for 48 hours to label dividing cells, then the cycle phase was identified for individual cells by one of two methods, and finally, the proliferative status of the same cells was scored by autoradiographic evidence of 3 HdT uptake. The methods to identify the cycle phase were: determination of DNA strain content by Feulgen scanning cytophotometry, and determination of chromosome constitution by the technique of premature chromosome condensation (PCC). Preliminary experiments showed the effect of continuous exposure to various levels of 3 HdT on cell growth. High levels of 3 HdT inhibited cell cycle traverse: the cell number and labeling index curves reached a plateau; the cell volume increased; the cells accumulated with 4C DNA contents and it appeared that they blocked in G 2 phase. This pattern is consistent with a radiation effect. (U.S.)
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Accuracy of preimplantation genetic diagnosis (PGD) of single gene and chromosomal disorders
Energy Technology Data Exchange (ETDEWEB)
Verlinsky, Y.; Strom, C.; Rechitsky, S. [Reproductive Genetics Institute, Chicage, IL (United States)] [and others
1994-09-01
We have developed a polar body inferred approach for preconception diagnosis of single gene and chromosomal disorders. Preconception PCR or FISH analysis was performed in a total of 310 first polar bodies for the following genetic conditions: cystic fibrosis, hemophilia A, alpha-1-antitrypsin deficiency, Tay Sachs disease, retinitis pigmentosa and common chromosomal trisomies. An important advantage of this approach is the avoidance of sperm (DNA) contamination, which is the major problem of PGD. We are currently applying FISH analysis of biopsied blastomeres, in combination with PCR or separately, and have demonstrated a significant improvement of the accuracy of PGD of X-linked disorders at this stage. Our data have also demonstrated feasibility of the application of FISH technique for PGD of chromosomal disorders. It was possible to detect chromosomal non-disjunctions and chromatid malsegregations in the first meiotic division, as well as to evaluate chromosomal mutations originating from the second meiotic nondisjunction.
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Wang, Dan; Zhao, Jieyu; Bai, Yan; Ao, You; Guo, Changhong
2017-08-10
Gametocidal (Gc) chromosomes can ensure their preferential transmission by killing the gametes without themselves through causing chromosome breakage and therefore have been exploited as an effective tool for genetic breeding. However, to date very little is known about the molecular mechanism of Gc action. In this study, we used methylation-sensitive amplified polymorphism (MSAP) technique to assess the extent and pattern of cytosine methylation alterations at the whole genome level between two lines of wheat Gc addition line and their common wheat parent. The results indicated that the overall levels of cytosine methylation of two studied Gc addition lines (CS-3C and CS-3C3C, 48.68% and 48.65%, respectively) were significantly increased when compared to common wheat CS (41.31%) and no matter fully methylated or hemimethylated rates enhanced in Gc addition lines. A set of 30 isolated fragments that showed different DNA methylation or demethylation patterns between the three lines were sequenced and the results indicated that 8 fragments showed significant homology to known sequences, of which three were homologous to MITE transposon (Miniature inverted-repeat transposable elements), LTR-retrotransposon WIS-1p and retrotransposon Gypsy , respectively. Overall, our results showed that DNA methylation could play a role in the Gc action.
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Directory of Open Access Journals (Sweden)
Dan Wang
2017-08-01
Full Text Available Gametocidal (Gc chromosomes can ensure their preferential transmission by killing the gametes without themselves through causing chromosome breakage and therefore have been exploited as an effective tool for genetic breeding. However, to date very little is known about the molecular mechanism of Gc action. In this study, we used methylation-sensitive amplified polymorphism (MSAP technique to assess the extent and pattern of cytosine methylation alterations at the whole genome level between two lines of wheat Gc addition line and their common wheat parent. The results indicated that the overall levels of cytosine methylation of two studied Gc addition lines (CS–3C and CS–3C3C, 48.68% and 48.65%, respectively were significantly increased when compared to common wheat CS (41.31% and no matter fully methylated or hemimethylated rates enhanced in Gc addition lines. A set of 30 isolated fragments that showed different DNA methylation or demethylation patterns between the three lines were sequenced and the results indicated that 8 fragments showed significant homology to known sequences, of which three were homologous to MITE transposon (Miniature inverted–repeat transposable elements, LTR-retrotransposon WIS-1p and retrotransposon Gypsy, respectively. Overall, our results showed that DNA methylation could play a role in the Gc action.
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Cytogenetic analysis of quinoa chromosomes using nanoscale imaging and spectroscopy techniques
Yangquanwei, Zhong; Neethirajan, Suresh; Karunakaran, Chithra
2013-11-01
Here we present a high-resolution chromosomal spectral map derived from synchrotron-based soft X-ray spectromicroscopy applied to quinoa species. The label-free characterization of quinoa metaphase chromosomes shows that it consists of organized substructures of DNA-protein complex. The analysis of spectra of chromosomes using the scanning transmission X-ray microscope (STXM) and its superposition of the pattern with the atomic force microscopy (AFM) and scanning electron microscopy (SEM) images proves that it is possible to precisely locate the gene loci and the DNA packaging inside the chromosomes. STXM has been successfully used to distinguish and quantify the DNA and protein components inside the quinoa chromosomes by visualizing the interphase at up to 30-nm spatial resolution. Our study represents the successful attempt of non-intrusive interrogation and integrating imaging techniques of chromosomes using synchrotron STXM and AFM techniques. The methodology developed for 3-D imaging of chromosomes with chemical specificity and temporal resolution will allow the nanoscale imaging tools to emerge from scientific research and development into broad practical applications such as gene loci tools and biomarker libraries.
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Directory of Open Access Journals (Sweden)
Evert van den Broek
2017-07-01
Full Text Available Development of cancer is driven by somatic alterations, including numerical and structural chromosomal aberrations. Currently, several computational methods are available and are widely applied to detect numerical copy number aberrations (CNAs of chromosomal segments in tumor genomes. However, there is lack of computational methods that systematically detect structural chromosomal aberrations by virtue of the genomic location of CNA-associated chromosomal breaks and identify genes that appear non-randomly affected by chromosomal breakpoints across (large series of tumor samples. ‘GeneBreak’ is developed to systematically identify genes recurrently affected by the genomic location of chromosomal CNA-associated breaks by a genome-wide approach, which can be applied to DNA copy number data obtained by array-Comparative Genomic Hybridization (CGH or by (low-pass whole genome sequencing (WGS. First, ‘GeneBreak’ collects the genomic locations of chromosomal CNA-associated breaks that were previously pinpointed by the segmentation algorithm that was applied to obtain CNA profiles. Next, a tailored annotation approach for breakpoint-to-gene mapping is implemented. Finally, dedicated cohort-based statistics is incorporated with correction for covariates that influence the probability to be a breakpoint gene. In addition, multiple testing correction is integrated to reveal recurrent breakpoint events. This easy-to-use algorithm, ‘GeneBreak’, is implemented in R (www.cran.r-project.org and is available from Bioconductor (www.bioconductor.org/packages/release/bioc/html/GeneBreak.html.
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Using 3-color chromosome painting to decide between chromosome aberration models
International Nuclear Information System (INIS)
Lucas, J.N.; Sachs, R.K.
1993-01-01
Ionizing radiation produces chromosome aberrations when DNA double strand breaks (DSB) interact pairwise. For more than 30 years there have been two main, competing theories of such binary DSB interactions. The classical theory asserts that an unrepaired DSB makes two ends which separate, with each end subsequently able to join any similar (non-telomeric) end. The exchange theory asserts that the two DSB ends remain associated until repair or a reciprocal chromosome exchange involving a second DSB occurs. The authors conducted an experiment to test these models, using 3-color chromosome painting. After in vitro irradiation of resting human lymphocytes, they observed cells with three-color triplets at first metaphase: three derivative chromosomes having permuted colors, as if three broken chromosomes had played musical chairs. On the exchange model in its standard form such 3-color triplets cannot occur. On the classical model the expected frequency can be calculated. They report data and computer calculations which exclude the exchange model and favor the classical model
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Directory of Open Access Journals (Sweden)
Eugenia E Montiel
Full Text Available R2 non-LTR retrotransposons exclusively insert into the 28S rRNA genes of their host, and are expressed by co-transcription with the rDNA unit. The grasshopper Eyprepocnemis plorans contains transcribed rDNA clusters on most of its A chromosomes, as well as non-transcribed rDNA clusters on the parasitic B chromosomes found in many populations. Here the structure of the E. plorans R2 element, its abundance relative to the number of rDNA units and its retrotransposition activity were determined. Animals screened from five populations contained on average over 12,000 rDNA units on their A chromosomes, but surprisingly only about 100 R2 elements. Monitoring the patterns of R2 insertions in individuals from these populations revealed only low levels of retrotransposition. The low rates of R2 insertion observed in E. plorans differ from the high levels of R2 insertion previously observed in insect species that have many fewer rDNA units. It is proposed that high levels of R2 are strongly selected against in E. plorans, because the rDNA transcription machinery in this species is unable to differentiate between R2-inserted and uninserted units. The B chromosomes of E. plorans contain an additional 7,000 to 15,000 rDNA units, but in contrast to the A chromosomes, from 150 to over 1,500 R2 elements. The higher concentration of R2 in the inactive B chromosomes rDNA clusters suggests these chromosomes can act as a sink for R2 insertions thus further reducing the level of insertions on the A chromosomes. These studies suggest an interesting evolutionary relationship between the parasitic B chromosomes and R2 elements.
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International Nuclear Information System (INIS)
Finocchiaro, G.; Taroni, F.; Martin, A.L.; Colombo, I.; Tarelli, G.T.; DiDonato, S.; Rocchi, M.
1991-01-01
The authors have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA encoding such a peptide. A 60-base-pair (bp) oligonucleotide synthesized on the basis of the sequence from this fragment was used for the screening of a cDNA library from human liver and hybridized to a cDNA insert of 2255 bp. This cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH 2 -terminal leader peptide. The assignment of this open reading frame to human liver CPTase is confirmed by matches to seven different amino acid sequences of tryptic peptides derived from pure human CPTase and by the 82.2% homology with the amino acid sequence of rat CPTase. The NH 2 -terminal region of CPTase contains a leucine-proline motif that is shared by carnitine acetyl- and octanoyltransferases and by choline acetyltransferase. The gene encoding CPTase was assigned to human chromosome 1, region 1q12-1pter, by hybridization of CPTase cDNA with a DNA panel of 19 human-hanster somatic cell hybrids
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Katre, Amar N; Damle, Sg
2013-09-01
It is now understood and accepted that there is a direct transmission of mutans streptococci (MS) from the mother to the child. There is also a direct correlation between the levels of MS in the mother and the caries status of the child. Advanced technologies in molecular biology like chromosomal DNA fngerprinting have established beyond doubt that the mother and the child bear similar strains of MS. A study was designed with the aim of comparing the MS strains between the father, mother and the child in Indian families. A group of 20 Indian families comprising of the father, mother and child were selected and divided into caries free and caries active groups. Mixed salivary samples were collected from the individuals and were cultured for the growth of Mutans streptococci. The colonies were counted on a colony counter and a comparison was made between the mutans streptococcal counts of the mother and the caries status of the child. Further, the genotypes of the father, mother and the child were isolated and compared using the technique of chromosomal DNA fngerprinting. Following electrophoresis, the band pattern obtained was compared for similarities or differences. The results of the same were tabulated and evaluated statistically. When the colony counts of the mother (in CFU/ml) were compared with the 'dft' status of the child, a positive correlation was seen in group II. Intergroup comparison using the unpaired T test was statistically signifcant. Electrophoretic analysis of the chromosomal DNA on the agarose gels revealed identical band patterns in 13 mother-child pairs, which was statistically signifcant. Three of the father-child pairs showed identical band patterns, which was statistically signifcant. Intergroup comparison using Chi-square test was not statistically signifcant. One may conclude that irrespective of the caries status of the child, majority of the mother child pairs share identical strains of MS and hence the mother is the primary source of
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Nanoneedle insertion into the cell nucleus does not induce double-strand breaks in chromosomal DNA.
Ryu, Seunghwan; Kawamura, Ryuzo; Naka, Ryohei; Silberberg, Yaron R; Nakamura, Noriyuki; Nakamura, Chikashi
2013-09-01
An atomic force microscope probe can be formed into an ultra-sharp cylindrical shape (a nanoneedle) using micro-fabrication techniques such as focused ion beam etching. This nanoneedle can be effectively inserted through the plasma membrane of a living cell to not only access the cytosol, but also to penetrate through the nuclear membrane. This technique shows great potential as a tool for performing intranuclear measurements and manipulations. Repeated insertions of a nanoneedle into a live cell were previously shown not to affect cell viability. However, the effect of nanoneedle insertion on the nucleus and nuclear components is still unknown. DNA is the most crucial component of the nucleus for proper cell function and may be physically damaged by a nanoneedle. To investigate the integrity of DNA following nanoneedle insertion, the occurrence of DNA double-strand breaks (DSBs) was assessed. The results showed that there was no chromosomal DNA damage due to nanoneedle insertion into the nucleus, as indicated by the expression level of γ-H2AX, a molecular marker of DSBs. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
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Molecular genetic approach to human meningioma: loss of genes on chromosome 22
International Nuclear Information System (INIS)
Seizinger, B.R.; De La Monte, S.; Atkins, L.; Gusella, J.F.; Martuza, R.L.
1987-01-01
A molecular genetic approach employing polymorphic DNA markers has been used to investigate the role of chromosomal aberrations in meningioma, one of the most common tumors of the human nervous system. Comparison of the alleles detected by DNA markers in tumor DNA versus DNA from normal tissue revealed chromosomal alterations present in primary surgical specimens. In agreement with cytogenetic studies of cultured meningiomas, the most frequent alteration detected was loss of heterozygosity on chromosome 22. Forty of 51 patients were constitutionally heterozygous for at least one chromosome 22 DNA marker. Seventeen of the 40 constitutionally heterozygotic patients (43%) displayed hemizygosity for the corresponding marker in their meningioma tumor tissues. Loss of heterozygosity was also detected at a significantly lower frequency for markers on several other autosomes. In view of the striking association between acoustic neuroma and meningioma in bilateral acoustic neurofibromatosis and the discovery that acoustic neuromas display specific loss of genes on chromosome 22, the authors propose that a common mechanism involving chromosome 22 is operative in the development of both tumor types. Fine-structure mapping to reveal partial deletions in meningiomas may provide the means to clone and characterize a gene (or genes) of importance for tumorigenesis in this and possibly other clinically associated tumors of the human nervous system
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International Nuclear Information System (INIS)
Cleaver, J.E.; Rose, R.; Mitchell, D.L.
1990-01-01
Episomal plasmids and viruses in mammalian cells present small targets for X-ray-induced DNA damage. At doses up to 100 Gy, DNA strand breaks or endonuclease III-sensitive sites were not discernible in 10.3-kb Epstein-Barr virus-based plasmid DNA or in 4.9-kb defective simian virus 40 DNA. DNA replication in these small molecules, however, was inhibited strongly by X-ray doses of greater than or equal to 20 Gy, decreasing to only 20 to 40% of control values. Inhibition was relieved slightly by growth in caffeine but was increased by growth in 3-aminobenzamide. Inhibition of DNA replication in episomal DNA molecules that are too small to sustain significant damage directly to their DNA may be due to either (a) a trans-acting diffusible factor that transfers the consequences of DNA breakage to episomes and to other replicating molecules, (b) a cis-acting mechanism in which episomes are structurally linked to genomic chromatin, and replication of both episomal and chromosomal replicons is under common control, or (c) radiation damage on other cellular structures unrelated to DNA. The resolution of these cellular mechanisms may shed light on the X-ray-resistant replication in ataxia-telangiectasia and may suggest strategies for molecular characterization of potential trans- or cis-acting factors
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Effect of aspirin on chromosome aberration and DNA damage induced by X-rays in mice
Niikawa, M.; Chuuriki, K.; Shibuya, K.; Seo, M.; Nagase, H.
In order to reveal the anticlastogenic potency of aspirin, we evaluated the suppressive ability of aspirin on chromosome aberrations induced by X-ray. Aspirin at doses of 0.5, 5 and 50 mg/kg was administrated intraperitoneally or orally at 0.5 h after or before the X-ray irradiation. The anticlastogenic activity of aspirin on chromosome aberrations induced by X-ray was determined in the mouse micronucleus test and alkaline single cell gel electrophoresis (SCG) assay in vivo. The frequency by polychromatic erythrocytes with micronuclei (MNPCEs) was decreased by about 19-61% at 0.5 h after and about 23-62% at 0.5 h before the X-ray irradiation. DNA damage by X-ray was significantly decreased by oral administration of aspirin at 0.5 h after or before the X-ray irradiation for the SCG assay. We consider aspirin can be used as preventive agents against exposure of X-ray.
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Cohesin in determining chromosome architecture
Energy Technology Data Exchange (ETDEWEB)
Haering, Christian H., E-mail: christian.haering@embl.de [Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg (Germany); Jessberger, Rolf, E-mail: rolf.jessberger@tu-dresden.de [Institute of Physiological Chemistry, Dresden University of Technology, Dresden (Germany)
2012-07-15
Cells use ring-like structured protein complexes for various tasks in DNA dynamics. The tripartite cohesin ring is particularly suited to determine chromosome architecture, for it is large and dynamic, may acquire different forms, and is involved in several distinct nuclear processes. This review focuses on cohesin's role in structuring chromosomes during mitotic and meiotic cell divisions and during interphase.
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International Nuclear Information System (INIS)
Helbig, R.; Speit, G.; Zdzienicka, M.Z.
1995-01-01
The radiosensitive Chinese hamster cell line XR-V15B was used to study the effect of decreased rejoining of DNA double-strand breaks (DSBs) on gene mutations and chromosome aberrations. XR-V15B cells are hypersensitive to the cytotoxic effects of neocarzinostatin (NCS) and methyl methanesulfonate (MMS). Both mutagens induced more chromosome aberrations in XR-V15B cells than in the parental cell strain. The clastogenic action of NCS was characterized by the induction of predominantly chromosome-type aberrations in cells of both strains, whereas MMS induced mainly chromatid aberrations. The frequency of induced gene mutations at the hprt locus was not increased compared to the parental V79 cells when considering the same survival level. Molecular analysis by multiplex polymerase chain reaction (PCR) of mutants induced by NCS revealed a high frequency of deletions in cells of both cell lines. Methyl methane-sulfonate induced mainly mutations without visible change in the PCR pattern, which probably represent point mutations. Our findings suggest a link between a defect in DNA DSB repair and increased cytotoxic and clastogenic effects. However, a decreased ability to rejoin DNA DSBs does not seem to influence the incidence and types of gene mutations at the hprt locus induced by NCS and MMS. 28 refs., 4 figs., 3 tabs
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Ohmido, Nobuko; Iwata, Aiko; Kato, Seiji; Wako, Toshiyuki; Fukui, Kiichi
2018-01-01
A quantitative pachytene chromosome map of rice (Oryza sativa L.) was developed using imaging methods. The map depicts not only distribution patterns of chromomeres specific to pachytene chromosomes, but also the higher order information of chromosomal structures, such as heterochromatin (condensed regions), euchromatin (decondensed regions), the primary constrictions (centromeres), and the secondary constriction (nucleolar organizing regions, NOR). These features were image analyzed and quantitatively mapped onto the map by Chromosome Image Analyzing System ver. 4.0 (CHIAS IV). Correlation between H3K9me2, an epigenetic marker and formation and/or maintenance of heterochromatin, thus was, clearly visualized. Then the pachytene chromosome map was unified with the existing somatic chromosome and linkage maps by physically mapping common DNA markers among them, such as a rice A genome specific tandem repeat sequence (TrsA), 5S and 45S ribosomal RNA genes, five bacterial artificial chromosome (BAC) clones, four P1 bacteriophage artificial chromosome (PAC) clones using multicolor fluorescence in situ hybridization (FISH). Detailed comparison between the locations of the DNA probes on the pachytene chromosomes using multicolor FISH, and the linkage map enabled determination of the chromosome number and short/long arms of individual pachytene chromosomes using the chromosome number and arm assignment designated for the linkage map. As a result, the quantitative pachytene chromosome map was unified with two other major rice chromosome maps representing somatic prometaphase chromosomes and genetic linkages. In conclusion, the unification of the three rice maps serves as an indispensable basic information, not only for an in-depth comparison between genetic and chromosomal data, but also for practical breeding programs.
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The Detection and Analysis of Chromosome Fragile Sites
DEFF Research Database (Denmark)
Bjerregaard, Victoria A; Özer, Özgün; Hickson, Ian D
2018-01-01
A fragile site is a chromosomal locus that is prone to form a gap or constriction visible within a condensed metaphase chromosome, particularly following exposure of cells to DNA replication stress. Based on their frequency, fragile sites are classified as either common (CFSs; present in all...... for detection and analysis of chromosome fragile sites....
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International Nuclear Information System (INIS)
Cremer, C.; Cremer, T.
1986-01-01
Cells of synchronized and of asynchronously growing cultures of a V79 Chinese hamster line were microirradiated with a low power laser-UV-microbeam of wavelength 257 nm. Ultraviolet light was either focused onto a small part of the nucleus (mode I) or distributed over the whole nucleus (mode II). Following microirradiation, the cells were incubated for 7-20 h with caffeine (1-2 mM) until chromosome preparation was performed. After both modes of microirradiation, shattering of the entire chromosome complement (generalized chromosome shattering, GCS) was observed. It is suggested that the probability by which GCS is induced depends on the total number of DNA lesions rather than on their distribution in the chromatin. The results are consistent with the prediction of a 'factor depletion model' which assumes that in a given cell, GCS takes place both in irradiated and non-irradiated chromosomes if the total number of daughter strand-repair sites surpasses a threshold value. (Auth.)
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Ullerich, Fritz-Helmut; Schöttke, Michael
2006-06-01
The karyotypes and C-banding patterns of Chrysomya species C. marginalis, C. phaonis, C. pinguis, C. saffranea, C. megacephala (New Guinean strain), Lucilia sericata, and Protophormia terraenovae are described. All species are amphogenic and have similar chromosome complements (2n = 12), including an XY-XX sex-chromosome pair varying in size and morphology between species. Additionally, the C-banding pattern of the monogenic species Chrysomya albiceps is presented. The DNA contents of these and of further species Chrysomya rufifacies, Chrysomya varipes, and Chrysomya putoria were assessed on mitotic metaphases by Feulgen cytophotometry. The average 2C DNA value of the male genomes ranged from 1.04 pg in C. varipes to 2.31 pg in C. pinguis. The DNA content of metaphase X chromosomes varied from 0.013 pg (= 1.23% of the total genome) in C. varipes to 0.277 pg (12.20%) in L. sericata; that of Y chromosomes ranged from 0.003 pg (0.27%) in C. varipes to 0.104 pg (5.59%) in L. sericata. In most species, the corresponding 5 large chromosome pairs showed similar relative DNA contents. The data suggest that the interspecific DNA differences in most species are mainly due to quantitative variation of (repetitive) sequences lying outside the centromeric heterochromatin blocks of the large chromosomes. The results are also discussed with regard to phylogenetic relationships of some species.
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Someswara Rao, Chinta; Viswanadha Raju, S
2016-03-01
In this paper, we consider correlation coefficient, rank correlation coefficient and cosine similarity measures for evaluating similarity between Homo sapiens and monkeys. We used DNA chromosomes of genome wide genes to determine the correlation between the chromosomal content and evolutionary relationship. The similarity among the H. sapiens and monkeys is measured for a total of 210 chromosomes related to 10 species. The similarity measures of these different species show the relationship between the H. sapiens and monkey. This similarity will be helpful at theft identification, maternity identification, disease identification, etc.
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The biology and polymer physics underlying large-scale chromosome organization.
Sazer, Shelley; Schiessel, Helmut
2018-02-01
Chromosome large-scale organization is a beautiful example of the interplay between physics and biology. DNA molecules are polymers and thus belong to the class of molecules for which physicists have developed models and formulated testable hypotheses to understand their arrangement and dynamic properties in solution, based on the principles of polymer physics. Biologists documented and discovered the biochemical basis for the structure, function and dynamic spatial organization of chromosomes in cells. The underlying principles of chromosome organization have recently been revealed in unprecedented detail using high-resolution chromosome capture technology that can simultaneously detect chromosome contact sites throughout the genome. These independent lines of investigation have now converged on a model in which DNA loops, generated by the loop extrusion mechanism, are the basic organizational and functional units of the chromosome. © 2017 The Authors. Traffic published by John Wiley & Sons Ltd.
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International Nuclear Information System (INIS)
Jordan, R.; Schwartz, J.L.
1994-01-01
Fluorescence in situ hybridization procedures were used to examine the influence of chromosome locus on the frequency and type of chromosome aberrations induced by 60 Co γ rays in the human lymphoblastoid cell line TK6. Aberrations involving the X chromosome were compared to those involving the similarly sized autosome chromosome 7. When corrected for DNA content, acentric fragments were induced with equal frequency in the X and 7 chromosomes. Dose-dependent increases in chromosomal interchanges involving chromosome 7 were noted, and the frequencies of balanced translocations and dicentrics produced were approximately equal. Chromosome interchanges involving the X chromosome were rare and showed no apparent dose dependence. Thus, while chromosomes 7 and X are equally sensitive to the induction of chromosome breaks, the X chromosome is much less likely to interact with autosomes than chromosome 7. The noninvolvement of the X chromosome in translocations with autosomes may reflect a more peripheral and separate location for the X chromosome in the mammalian nucleus. 20 refs., 2 figs., 1 tab
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Kolano, B; Gardunia, B W; Michalska, M; Bonifacio, A; Fairbanks, D; Maughan, P J; Coleman, C E; Stevens, M R; Jellen, E N; Maluszynska, J
2011-09-01
The chromosomal organization of two novel repetitive DNA sequences isolated from the Chenopodium quinoa Willd. genome was analyzed across the genomes of selected Chenopodium species. Fluorescence in situ hybridization (FISH) analysis with the repetitive DNA clone 18-24J in the closely related allotetraploids C. quinoa and Chenopodium berlandieri Moq. (2n = 4x = 36) evidenced hybridization signals that were mainly present on 18 chromosomes; however, in the allohexaploid Chenopodium album L. (2n = 6x = 54), cross-hybridization was observed on all of the chromosomes. In situ hybridization with rRNA gene probes indicated that during the evolution of polyploidy, the chenopods lost some of their rDNA loci. Reprobing with rDNA indicated that in the subgenome labeled with 18-24J, one 35S rRNA locus and at least half of the 5S rDNA loci were present. A second analyzed sequence, 12-13P, localized exclusively in pericentromeric regions of each chromosome of C. quinoa and related species. The intensity of the FISH signals differed considerably among chromosomes. The pattern observed on C. quinoa chromosomes after FISH with 12-13P was very similar to GISH results, suggesting that the 12-13P sequence constitutes a major part of the repetitive DNA of C. quinoa.
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Chromosome heteromorphisms in the Japanese, 3
International Nuclear Information System (INIS)
Sofuni, Toshio; Awa, A.A.
1982-12-01
The type and frequency of chromosome variants detected by the C-staining method were ascertained in 1,857 individuals residing in Hiroshima. The most frequent heteromorphic variant was the total inversion of the C-band in chromosome 9 found in 27 individuals (1.45%). The total inversion of the C-band in chromosome 1 was not seen in this sample, but the partial inversion of the C-band in chromosome 1 was found in 18 persons (0.97%). Partial inversion was also detected in the C-band in chromosome 9 in 22 individuals (1.18%). In chromosome 16, neither total nor partial inversion of the C-band was observed in the present study. The frequencies of chromosomes 1, 9, and 16 with a very large C-band were 0.70%, 0.22%, and 0.54%, respectively. Aside from these (1, 9, and 16) a very large C-band was found occasionally in chromosomes 4, 5, 6, 11, 12, 14, and 15, and an unusual insertion of the Y chromosome was observed. A total of 128 C-band variants (6.89%) was found in the 1,857 Hiroshima residents. (author)
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Strong Accumulation of Chloroplast DNA in the Y Chromosomes of Rumex acetosa and Silene latifolia
Czech Academy of Sciences Publication Activity Database
Šteflová, Pavlína; Hobza, Roman; Vyskot, Boris; Kejnovský, Eduard
2014-01-01
Roč. 142, č. 1 (2014), s. 59-65 ISSN 1424-8581 R&D Projects: GA ČR(CZ) GAP305/10/0930; GA ČR(CZ) GAP501/10/0102; GA ČR(CZ) GBP501/12/G090; GA ČR GAP501/12/2220; GA ČR(CZ) GA522/09/0083; GA MŠk(CZ) LO1204 Institutional research plan: CEZ:AV0Z50040702 Institutional support: RVO:68081707 Keywords : Chloroplast DNA * Rumex acetosa * Sex chromosomes Subject RIV: BO - Biophysics Impact factor: 1.561, year: 2014
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Serfaty, Dayane Martins Barbosa; Carvalho, Natália Dayane Moura; Gross, Maria Claudia; Gordo, Marcelo; Schneider, Carlos Henrique
2017-10-01
Saguinus is the largest and most complex genus of the subfamily Callitrichinae, with 23 species distributed from the south of Central America to the north of South America with Saguinus midas having the largest geographical distribution while Saguinus bicolor has a very restricted one, affected by the population expansion in the state of Amazonas. Considering the phylogenetic proximity of the two species along with evidence on the existence of hybrids between them, as well as cytogenetic studies on Saguinus describing a conserved karyotypic macrostructure, we carried out a physical mapping of DNA repeated sequences in the mitotic chromosome of both species, since these sequences are less susceptible to evolutionary pressure and possibly perform an important function in speciation. Both species presented 2n = 46 chromosomes; in S. midas, chromosome Y is the smallest. Multiple ribosomal sites occur in both species, but chromosome pairs three and four may be regarded as markers that differ the species when subjected to G banding and distribution of retroelement LINE 1, suggesting that it may be cytogenetic marker in which it can contribute to identification of first generation hybrids in contact zone. Saguinus bicolor also presented differences in the LINE 1 distribution pattern for sexual chromosome X in individuals from different urban fragments, probably due to geographical isolation. In this context, cytogenetic analyses reveal a differential genomic organization pattern between species S. midas and S. bicolor, in addition to indicating that individuals from different urban fragments have been accumulating differences because of the isolation between them.
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An XmnI RFLP detected with a cDNA probe for the CYP2C gene locus on chromosome 10
Energy Technology Data Exchange (ETDEWEB)
Gough, A C; Spurr, N K [Clare Hall Laboratories, Herts (England); Meehan, R R; Miles, J S; Wolf, C R [Univ. Department of Biochemistry, Edinburgh (England)
1989-06-12
A 700 bp fragment of the cDNA clone for the human cytochrome P450 gene cloned in pUC9 at the PstI restriction site. XmnI detects a two allele polymorphism with bands at 10.00 kb (A1), 4.8 kb (A2) and constants bands at 13.0, 8.3, 4.6, 3.1, 2.8, 2.5, 2.3, 2.2, 1.8 and 1.5 kb. A total of 16 unrelated individuals of Caucasian origin were screened for A1 (.625) and A2 (.375). The probe was assigned to chromosome 10q24.1-q24.3 using a panel of human-rodent somatic cell hybrids and in situ hybridization. Co-dominant inheritance was observed in 3 families from CEPH, K1329 2 K1331 and K1333.
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Bueno, Danilo; Palacios-Gimenez, Octavio Manuel; Cabral-de-Mello, Diogo Cavalcanti
2013-01-01
Supernumerary chromosomes (B chromosomes) occur in approximately 15% of eukaryote species. Although these chromosomes have been extensively studied, knowledge concerning their specific molecular composition is lacking in most cases. The accumulation of repetitive DNAs is one remarkable characteristic of B chromosomes, and the occurrence of distinct types of multigene families, satellite DNAs and some transposable elements have been reported. Here, we describe the organization of repetitive DNAs in the A complement and B chromosome system in the grasshopper species Abracris flavolineata using classical cytogenetic techniques and FISH analysis using probes for five multigene families, telomeric repeats and repetitive C0t-1 DNA fractions. The 18S rRNA and H3 histone multigene families are highly variable and well distributed in A. flavolineata chromosomes, which contrasts with the conservation of U snRNA genes and less variable distribution of 5S rDNA sequences. The H3 histone gene was an extensively distributed with clusters occurring in all chromosomes. Repetitive DNAs were concentrated in C-positive regions, including the pericentromeric region and small chromosomal arms, with some occurrence in C-negative regions, but abundance was low in the B chromosome. Finally, the first demonstration of the U2 snRNA gene in B chromosomes in A. flavolineata may shed light on its possible origin. These results provide new information regarding chromosomal variability for repetitive DNAs in grasshoppers and the specific molecular composition of B chromosomes. PMID:23826099
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Hopkins, Jessica; Hwang, Grace; Jacob, Justin; Sapp, Nicklas; Bedigian, Rick; Oka, Kazuhiro; Overbeek, Paul; Murray, Steve; Jordan, Philip W
2014-07-01
Cohesins are important for chromosome structure and chromosome segregation during mitosis and meiosis. Cohesins are composed of two structural maintenance of chromosomes (SMC1-SMC3) proteins that form a V-shaped heterodimer structure, which is bridged by a α-kleisin protein and a stromal antigen (STAG) protein. Previous studies in mouse have shown that there is one SMC1 protein (SMC1β), two α-kleisins (RAD21L and REC8) and one STAG protein (STAG3) that are meiosis-specific. During meiosis, homologous chromosomes must recombine with one another in the context of a tripartite structure known as the synaptonemal complex (SC). From interaction studies, it has been shown that there are at least four meiosis-specific forms of cohesin, which together with the mitotic cohesin complex, are lateral components of the SC. STAG3 is the only meiosis-specific subunit that is represented within all four meiosis-specific cohesin complexes. In Stag3 mutant germ cells, the protein level of other meiosis-specific cohesin subunits (SMC1β, RAD21L and REC8) is reduced, and their localization to chromosome axes is disrupted. In contrast, the mitotic cohesin complex remains intact and localizes robustly to the meiotic chromosome axes. The instability of meiosis-specific cohesins observed in Stag3 mutants results in aberrant DNA repair processes, and disruption of synapsis between homologous chromosomes. Furthermore, mutation of Stag3 results in perturbation of pericentromeric heterochromatin clustering, and disruption of centromere cohesion between sister chromatids during meiotic prophase. These defects result in early prophase I arrest and apoptosis in both male and female germ cells. The meiotic defects observed in Stag3 mutants are more severe when compared to single mutants for Smc1β, Rec8 and Rad21l, however they are not as severe as the Rec8, Rad21l double mutants. Taken together, our study demonstrates that STAG3 is required for the stability of all meiosis-specific cohesin
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Directory of Open Access Journals (Sweden)
Jessica Hopkins
2014-07-01
Full Text Available Cohesins are important for chromosome structure and chromosome segregation during mitosis and meiosis. Cohesins are composed of two structural maintenance of chromosomes (SMC1-SMC3 proteins that form a V-shaped heterodimer structure, which is bridged by a α-kleisin protein and a stromal antigen (STAG protein. Previous studies in mouse have shown that there is one SMC1 protein (SMC1β, two α-kleisins (RAD21L and REC8 and one STAG protein (STAG3 that are meiosis-specific. During meiosis, homologous chromosomes must recombine with one another in the context of a tripartite structure known as the synaptonemal complex (SC. From interaction studies, it has been shown that there are at least four meiosis-specific forms of cohesin, which together with the mitotic cohesin complex, are lateral components of the SC. STAG3 is the only meiosis-specific subunit that is represented within all four meiosis-specific cohesin complexes. In Stag3 mutant germ cells, the protein level of other meiosis-specific cohesin subunits (SMC1β, RAD21L and REC8 is reduced, and their localization to chromosome axes is disrupted. In contrast, the mitotic cohesin complex remains intact and localizes robustly to the meiotic chromosome axes. The instability of meiosis-specific cohesins observed in Stag3 mutants results in aberrant DNA repair processes, and disruption of synapsis between homologous chromosomes. Furthermore, mutation of Stag3 results in perturbation of pericentromeric heterochromatin clustering, and disruption of centromere cohesion between sister chromatids during meiotic prophase. These defects result in early prophase I arrest and apoptosis in both male and female germ cells. The meiotic defects observed in Stag3 mutants are more severe when compared to single mutants for Smc1β, Rec8 and Rad21l, however they are not as severe as the Rec8, Rad21l double mutants. Taken together, our study demonstrates that STAG3 is required for the stability of all meiosis
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Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells.
Sato, Hiroshi; Kato, Hiroki; Yamaza, Haruyoshi; Masuda, Keiji; Nguyen, Huong Thi Nguyen; Pham, Thanh Thi Mai; Han, Xu; Hirofuji, Yuta; Nonaka, Kazuaki
2017-01-01
Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk) gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV). Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.
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Afonso, Luis O B; Smith, Jack L; Ikonomou, Michael G; Devlin, Robert H
2002-01-01
Chinook salmon alevins were exposed during their labile period for sex differentiation to different concentrations of bleached kraft mill effluent (BKME), primary sewage effluent, secondary sewage effluent (SE), 17ss-estradiol, testosterone, and nonylphenol. After exposure for 29 days post hatching (DPH), fish were allowed to grow until 103 and 179 DPH, at which time their genetic sex was determined using Y-chromosomal DNA markers and their gonadal sex was determined by histology. Independent...
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Rudd, M Katharine; Mays, Robert W; Schwartz, Stuart; Willard, Huntington F
2003-11-01
Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes.
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Integrated and Total HIV-1 DNA Predict Ex Vivo Viral Outgrowth.
Directory of Open Access Journals (Sweden)
Maja Kiselinova
2016-03-01
Full Text Available The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4-2.6 between time point 1 and 2; and median of 31 days (IQR: 28-36 between time point 2 and 3. Patients were median of 6 years (IQR: 3-12 on ART, and plasma viral load (<50 copies/ml was suppressed for median of 4 years (IQR: 2-8. Total HIV-1 DNA, unspliced (us and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowth assay (VOA was performed, and integrated HIV-1 DNA and relative mRNA expression levels of HIV-1 restriction factors were quantified. No significant change was found for long- and short-term dynamics of all HIV-1 markers tested in peripheral blood. Integrated HIV-1 DNA was associated with total HIV-1 DNA (p<0.001, R² = 0.85, us HIV-1 RNA (p = 0.029, R² = 0.40, and VOA (p = 0.041, R2 = 0.44. Replication-competent virus was detected in 80% of patients by the VOA and it correlated with total HIV-1 DNA (p = 0.039, R² = 0.54. The mean quantification difference between Alu-PCR and VOA was 2.88 log10, and 2.23 log10 between total HIV-1 DNA and VOA. The levels of usHIV-1 RNA were inversely correlated with mRNA levels of several HIV-1 restriction factors (TRIM5α, SAMHD1, MX2, SLFN11, pSIP1. Our study reveals important correlations between the viral outgrowth and total and integrated HIV-1 DNA measures, suggesting that the total pool of HIV-1 DNA may predict the size of the
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DEFF Research Database (Denmark)
Rasmussen, Thomas Bruun; Risager, Peter Christian; Fahnøe, Ulrik
2013-01-01
Background Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described....... This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. Results A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable...
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... DNA What is mitochondrial DNA? What is mitochondrial DNA? Although most DNA is packaged in chromosomes within ... proteins. For more information about mitochondria and mitochondrial DNA: Molecular Expressions, a web site from the Florida ...
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Genome organization influences partner selection for chromosomal rearrangements
Wijchers, P.J.; de Laat, W.
2010-01-01
Chromosomal rearrangements occur as a consequence of the erroneous repair of DNA double-stranded breaks, and often underlie disease. The recurrent detection of specific tumorigenic rearrangements suggests that there is a mechanism behind chromosomal partner selection involving the shape of the
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Chromosomes of Protists: The crucible of evolution.
Soyer-Gobillard, Marie-Odile; Dolan, Michael F
2015-12-01
As early as 1925, the great protozoologist Edouard Chatton classified microorganisms into two categories, the prokaryotic and the eukaryotic microbes, based on light microscopical observation of their nuclear organization. Now, by means of transmission electron microscopy, we know that prokaryotic microbes are characterized by the absence of nuclear envelope surrounding the bacterial chromosome, which is more or less condensed and whose chromatin is deprived of histone proteins but presents specific basic proteins. Eukaryotic microbes, the protists, have nuclei surrounded by a nuclear envelope and have chromosomes more or less condensed, with chromatin-containing histone proteins organized into nucleosomes. The extraordinary diversity of mitotic systems presented by the 36 phyla of protists (according to Margulis et al., Handbook of Protoctista, 1990) is in contrast to the relative homogeneity of their chromosome structure and chromatin components. Dinoflagellates are the exception to this pattern. The phylum is composed of around 2000 species, and characterized by unique features including their nucleus (dinokaryon), dinomitosis, chromosome organization and chromatin composition. Although their DNA synthesis is typically eukaryotic, dinoflagellates are the only eukaryotes in which the chromatin, organized into quasi-permanently condensed chromosomes, is in some species devoid of histones and nucleosomes. In these cases, their chromatin contains specific DNA-binding basic proteins. The permanent compaction of their chromosomes throughout the cell cycle raises the question of the modalities of their division and their transcription. Successful in vitro reconstitution of nucleosomes using dinoflagellate DNA and heterologous corn histones raises questions about dinoflagellate evolution and phylogeny. [Int Microbiol 18(4):209-216 (2015)]. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.
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Sabri, Suriana; Steen, Jennifer A; Bongers, Mareike; Nielsen, Lars K; Vickers, Claudia E
2013-06-24
Metabolic engineering projects often require integration of multiple genes in order to control the desired phenotype. However, this often requires iterative rounds of engineering because many current insertion approaches are limited by the size of the DNA that can be transferred onto the chromosome. Consequently, construction of highly engineered strains is very time-consuming. A lack of well-characterised insertion loci is also problematic. A series of knock-in/knock-out (KIKO) vectors was constructed for integration of large DNA sequences onto the E. coli chromosome at well-defined loci. The KIKO plasmids target three nonessential genes/operons as insertion sites: arsB (an arsenite transporter); lacZ (β-galactosidase); and rbsA-rbsR (a ribose metabolism operon). Two homologous 'arms' target each insertion locus; insertion is mediated by λ Red recombinase through these arms. Between the arms is a multiple cloning site for the introduction of exogenous sequences and an antibiotic resistance marker (either chloramphenicol or kanamycin) for selection of positive recombinants. The resistance marker can subsequently be removed by flippase-mediated recombination. The insertion cassette is flanked by hairpin loops to isolate it from the effects of external transcription at the integration locus. To characterize each target locus, a xylanase reporter gene (xynA) was integrated onto the chromosomes of E. coli strains W and K-12 using the KIKO vectors. Expression levels varied between loci, with the arsB locus consistently showing the highest level of expression. To demonstrate the simultaneous use of all three loci in one strain, xynA, green fluorescent protein (gfp) and a sucrose catabolic operon (cscAKB) were introduced into lacZ, arsB and rbsAR respectively, and shown to be functional. The KIKO plasmids are a useful tool for efficient integration of large DNA fragments (including multiple genes and pathways) into E. coli. Chromosomal insertion provides stable
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Karyotype with 210 chromosomes in guaraná (Paullinia cupana 'Sorbilis').
de Freitas, Danival Vieira; Carvalho, Carlos Roberto; Filho, Firmino José do Nascimento; Astolfi-Filho, Spartaco
2007-05-01
The genus Paullinia includes the economically important P. cupana, known as guaraná in Brazil and more recently in the world market. Native Americans of the Maué and Andirá tribes cultivated P. cupana 'Sorbilis' in central Amazon, and the Barés cultivated the 'Typica' variety in the upper Negro River (Brazil). Cytological studies in the Sapindaceae family have concentrated on the diversity in number (from 2n = 14 to 96) and size of the chromosomes. In Paullinia, seven species have been karyotyped and all show 2n = 24. Meristem maceration, cellular dissociation and air-drying techniques were used for cytogenetic preparations and DNA content was determined by flow cytometry. Chromosome characterization and DNA content of Paullinia cupana Kunth 'Sorbilis' (Mart.) Ducke (Sapindaceae) were studied. The high chromosome number (2n = 210) fall into two cytomorphological groups: (a) a metacentric and submetacentric group showing 25 sets of three pairs of chromosomes (2-76); (b) a group containing only acrocentric showing 12 sets of two pairs of chromosomes (82-105), a homologous submetacentric pair (1) and an acrocentric pair (81). Mean nuclear DNA content of guaraná was 2C = 22.8 pg. A karyogram was set up showing a high chromosome number complement.
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Initiation of chromosomal replication in predatory bacterium Bdellovibrio bacteriovorus
Directory of Open Access Journals (Sweden)
Lukasz Makowski
2016-11-01
Full Text Available Bdellovibrio bacteriovorus is a small Gram-negative predatory bacterium that attacks other Gram-negative bacteria, including many animal, human, and plant pathogens. This bacterium exhibits a peculiar biphasic life cycle during which two different types of cells are produced: non-replicating highly motile cells (the free-living phase and replicating cells (the intracellular-growth phase. The process of chromosomal replication in B. bacteriovorus must therefore be temporally and spatially regulated to ensure that it is coordinated with cell differentiation and cell cycle progression. Recently, B. bacteriovorus has received considerable research interest due to its intriguing life cycle and great potential as a prospective antimicrobial agent. Although we know that chromosomal replication in bacteria is mainly regulated at the initiation step, no data exists about this process in B. bacteriovorus. We report the first characterization of key elements of initiation of chromosomal replication – DnaA protein and oriC region from the predatory bacterium, B. bacteriovorus. In vitro studies using different approaches demonstrate that the B. bacteriovorus oriC (BdoriC is specifically bound and unwound by the DnaA protein. Sequence comparison of the DnaA-binding sites enabled us to propose a consensus sequence for the B. bacteriovorus DnaA box (5’-NN(A/TTCCACA-3’. Surprisingly, in vitro analysis revealed that BdoriC is also bound and unwound by the host DnaA proteins (relatively distantly related from B. bacteriovorus. We compared the architecture of the DnaA–oriC complexes (orisomes in homologous (oriC and DnaA from B. bacteriovorus and heterologous (BdoriC and DnaA from prey, E. coli or P. aeruginosa systems. This work provides important new entry points toward improving our understanding of the initiation of chromosomal replication in this predatory bacterium.
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Chromosome aberration analysis for biological dosimetry: a review
International Nuclear Information System (INIS)
Paul, S.F.D.; Venkatachalam, P.; Jeevanram, R.K.
1996-01-01
Among various biological dosimetry techniques, dicentric chromosome aberration method appears to be the method of choice in analysing accidental radiation exposure in most of the laboratories. The major advantage of this method is its sensitivity as the number of dicentric chromosomes present in control population is too small and more importantly radiation induces mainly dicentric chromosome aberration among unstable aberration. This report brings out the historical development of various cytogenetic methods, the basic structure of DNA, chromosomes and different forms of chromosome aberrations. It also highlights the construction of dose-response curve for dicentric chromosome and its use in the estimation of radiation dose. (author)
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Cytometric analysis of irradiation damaged chromosomes
International Nuclear Information System (INIS)
Wilder, M.E.; Raju, M.R.
1982-01-01
Irradiation of cells in interphase results in dose-dependent damage to DNA which is discernable by flow-cytometric analysis of chromosomes. The quantity (and possibly the quality) of chromosomal changes is different in survival-matched doses of x and α irradiation. It may, therefore, be possible to use these methods for analysis of dose and type of exposure in unknown cases
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Identification of Y-Chromosome Sequences in Turner Syndrome.
Silva-Grecco, Roseane Lopes da; Trovó-Marqui, Alessandra Bernadete; Sousa, Tiago Alves de; Croce, Lilian Da; Balarin, Marly Aparecida Spadotto
2016-05-01
To investigate the presence of Y-chromosome sequences and determine their frequency in patients with Turner syndrome. The study included 23 patients with Turner syndrome from Brazil, who gave written informed consent for participating in the study. Cytogenetic analyses were performed in peripheral blood lymphocytes, with 100 metaphases per patient. Genomic DNA was also extracted from peripheral blood lymphocytes, and gene sequences DYZ1, DYZ3, ZFY and SRY were amplified by Polymerase Chain Reaction. The cytogenetic analysis showed a 45,X karyotype in 9 patients (39.2 %) and a mosaic pattern in 14 (60.8 %). In 8.7 % (2 out of 23) of the patients, Y-chromosome sequences were found. This prevalence is very similar to those reported previously. The initial karyotype analysis of these patients did not reveal Y-chromosome material, but they were found positive for Y-specific sequences in the lymphocyte DNA analysis. The PCR technique showed that 2 (8.7 %) of the patients with Turner syndrome had Y-chromosome sequences, both presenting marker chromosomes on cytogenetic analysis.
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Energy Technology Data Exchange (ETDEWEB)
Tan, K.L.; Baker, R.T.; Board, P.G. [Australian National Univ., Canberra (Australia)] [and others
1995-01-20
Until recently the Theta-class glutathione S-transferases (GSTs) were largely overlooked due to their low activity with the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) and their failure to bind to immobilized glutathione affinity matrices. Little is known about the number of genes in this class. Recently, Pemble et al. reported the cDNA cloning of a human Theta-class GST, termed GSTT1. In this study, we describe the molecular cloning of a cDNA encoding a second human Theta-class GST (GSTT2) from a {lambda}gt11 human liver 5{prime}-stretch cDNA library. The encoded protein contains 244 amino acids and has 78.3% sequence identity with the rat subunit 12 and only 55.0% identity with human GSTT1. GSTT2 has been mapped to chromosome 22 by somatic cell hybrid analysis. The precise position of the gene was localized to subband 22q11.2 by in situ hybridization. The absence of other regions of hybridization suggests that there are no closely related sequences (e.g., reverse transcribed pseudogenes) scattered throughout the genome and that if there are closely related genes, they must be clustered near GSTT2. Southern blot analysis of human DNA digested with BamHI shows that the size of the GSTT2 gene is relatively small, as the coding sequence falls within a 3.6-kb BamHI fragment. 35 refs., 6 figs.
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Chromosome mapping of repetitive sequences in four Serrasalmidae species (Characiformes
Directory of Open Access Journals (Sweden)
Leila Braga Ribeiro
2014-01-01
Full Text Available The Serrasalmidae family is composed of a number of commercially interesting species, mainly in the Amazon region where most of these fishes occur. In the present study, we investigated the genomic organization of the 18S and 5S rDNA and telomeric sequences in mitotic chromosomes of four species from the basal clade of the Serrasalmidae family: Colossoma macropomum, Mylossoma aureum, M. duriventre, and Piaractus mesopotamicus, in order to understand the chromosomal evolution in the family. All the species studied had diploid numbers 2n = 54 and exclusively biarmed chromosomes, but variations of the karyotypic formulas were observed. C-banding resulted in similar patterns among the analyzed species, with heterochromatic blocks mainly present in centromeric regions. The 18S rDNA mapping of C. macropomum and P. mesopotamicus revealed multiple sites of this gene; 5S rDNA sites were detected in two chromosome pairs in all species, although not all of them were homeologs. Hybridization with a telomeric probe revealed signals in the terminal portions of chromosomes in all the species and an interstitial signal was observed in one pair of C. macropomum.
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Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells
Directory of Open Access Journals (Sweden)
Hiroshi Sato
2017-01-01
Full Text Available Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV. Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.
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Genome Organization Drives Chromosome Fragility.
Canela, Andres; Maman, Yaakov; Jung, Seolkyoung; Wong, Nancy; Callen, Elsa; Day, Amanda; Kieffer-Kwon, Kyong-Rim; Pekowska, Aleksandra; Zhang, Hongliang; Rao, Suhas S P; Huang, Su-Chen; Mckinnon, Peter J; Aplan, Peter D; Pommier, Yves; Aiden, Erez Lieberman; Casellas, Rafael; Nussenzweig, André
2017-07-27
In this study, we show that evolutionarily conserved chromosome loop anchors bound by CCCTC-binding factor (CTCF) and cohesin are vulnerable to DNA double strand breaks (DSBs) mediated by topoisomerase 2B (TOP2B). Polymorphisms in the genome that redistribute CTCF/cohesin occupancy rewire DNA cleavage sites to novel loop anchors. While transcription- and replication-coupled genomic rearrangements have been well documented, we demonstrate that DSBs formed at loop anchors are largely transcription-, replication-, and cell-type-independent. DSBs are continuously formed throughout interphase, are enriched on both sides of strong topological domain borders, and frequently occur at breakpoint clusters commonly translocated in cancer. Thus, loop anchors serve as fragile sites that generate DSBs and chromosomal rearrangements. VIDEO ABSTRACT. Published by Elsevier Inc.
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Energy Technology Data Exchange (ETDEWEB)
Romac, S; Leong, P; Sockett, H; Hutchinson, F [Yale Univ., New Haven, CT (USA). Dept. of Molecular Biophysics and Biochemistry
1989-09-20
The DNA base sequence changes induced by mutagenesis with ultraviolet light have been determined in a gene on a chromosome of cultured Chinese hamster ovary (CHO) cells. The gene was the Excherichia coli gpt gene, of which a single copy was stably incorporated and expressed in the CHO cell genome. The cells were irradiated with ultraviolet light and gpt{sup -} colonies were selected by resistance to 6-thioguanine. The gpt gene was amplified from chromosomal DNA by use of the polymerase chain reaction (PCR) and the amplified DNA sequenced directly by the dideoxy method. Of the 58 sequenced mutants of independent origin 53 were base change mutations. Forty-one base substitutions were single base changes, ten had two adjacent (or tandem) base changes, and one had two base changes separated by a single base-pair. Only one mutant had a multiple base change mutation with two or more well separated base changes. In contrast much higher levels of such mutations were reported in ultraviolet mutagenesis of genes on a shuttle vector in primate cells. Two deletions of a single base-pair were observed and three deletions ranging from 6 to 37 base-pairs. The mutation spectrum in the gpt gene had similarities to the ultraviolet mutation spectra for several genes in prokaryotes, which suggests similarities in mutational mechanisms in prokaryotes and eukaryotes. (author).
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Pichler, Irene; Mueller, Jakob C; Stefanov, Stefan A; De Grandi, Alessandro; Volpato, Claudia Beu; Pinggera, Gerd K; Mayr, Agnes; Ogriseg, Martin; Ploner, Franz; Meitinger, Thomas; Pramstaller, Peter P
2006-08-01
Most of the inhabitants of South Tyrol in the eastern Italian Alps can be considered isolated populations because of their physical separation by mountain barriers and their sociocultural heritage. We analyzed the genetic structure of South Tyrolean populations using three types of genetic markers: Y-chromosome, mitochondrial DNA (mtDNA), and autosomal Alu markers. Using random samples taken from the populations of Val Venosta, Val Pusteria, Val Isarco, Val Badia, and Val Gardena, we calculated genetic diversity within and among the populations. Microsatellite diversity and unique event polymorphism diversity (on the Y chromosome) were substantially lower in the Ladin-speaking population of Val Badia compared to the neighboring German-speaking populations. In contrast, the genetic diversity of mtDNA haplotypes was lowest for the upper Val Venosta and Val Pusteria. These data suggest a low effective population size, or little admixture, for the gene pool of the Ladin-speaking population from Val Badia. Interestingly, this is more pronounced for Ladin males than for Ladin females. For the pattern of genetic Alu variation, both Ladin samples (Val Gardena and Val Badia) are among the samples with the lowest diversity. An admixture analysis of one German-speaking valley (Val Venosta) indicates a relatively high genetic contribution of Ladin origin. The reduced genetic diversity and a high genetic differentiation in the Rhaetoroman- and German-speaking South Tyrolean populations may constitute an important basis for future medical genetic research and gene mapping studies in South Tyrol.
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Sex chromosome diversity in Armenian toad grasshoppers (Orthoptera, Acridoidea, Pamphagidae)
Bugrov, Alexander G.; Jetybayev, Ilyas E.; Karagyan, Gayane H.; Rubtsov, Nicolay B.
2016-01-01
Abstract Although previous cytogenetic analysis of Pamphagidae grasshoppers pointed to considerable karyotype uniformity among most of the species in the family, our study of species from Armenia has discovered other, previously unknown karyotypes, differing from the standard for Pamphagidae mainly in having unusual sets of sex chromosomes. Asiotmethis turritus (Fischer von Waldheim, 1833), Paranocaracris rubripes (Fischer von Waldheim, 1846), and Nocaracris cyanipes (Fischer von Waldheim, 1846) were found to have the karyotype 2n♂=16+neo-XY and 2n♀=16+neo-XX, the neo-X chromosome being the result of centromeric fusion of an ancient acrocentric X chromosome and a large acrocentric autosome. The karyotype of Paranothrotes opacus (Brunner von Wattenwyl, 1882) was found to be 2n♂=14+X1X2Y and 2n♀=14+X1X1X2X2., the result of an additional chromosome rearrangement involving translocation of the neo-Y and another large autosome. Furthermore, evolution of the sex chromosomes in these species has involved different variants of heterochromatinization and miniaturization of the neo-Y. The karyotype of Eremopeza festiva (Saussure, 1884), in turn, appeared to have the standard sex determination system described earlier for Pamphagidae grasshoppers, 2n♂=18+X0 and 2n♀=18+XX, but all the chromosomes of this species were found to have small second C-positive arms. Using fluorescent in situ hybridization (FISH) with 18S rDNA and telomeric (TTAGG)n DNA repeats to yield new data on the structural organization of chromosomes in the species studied, we found that for most of them, clusters of repeats homologous to 18S rDNA localize on two, three or four pairs of autosomes and on the X. In Eremopeza festiva, however, FISH with labelled 18S rDNA painted C-positive regions of all autosomes and the X chromosome; clusters of telomeric repeats localized primarily on the ends of the chromosome arms. Overall, we conclude that the different stages of neo-Y degradation revealed in
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Destabilized SMC5/6 complex leads to chromosome breakage syndrome with severe lung disease
van der Crabben, Saskia N.; Hennus, Marije P.; McGregor, Grant A.; Ritter, Deborah I.; Nagamani, Sandesh C.S.; Wells, Owen S.; Harakalova, Magdalena; Chinn, Ivan K.; Alt, Aaron; Vondrova, Lucie; Hochstenbach, Ron; van Montfrans, Joris M.; Terheggen-Lagro, Suzanne W.; van Lieshout, Stef; van Roosmalen, Markus J.; Renkens, Ivo; Duran, Karen; Nijman, Isaac J.; Kloosterman, Wigard P.; Hennekam, Eric; van Hasselt, Peter M.; Wheeler, David A.; Palecek, Jan J.; Lehmann, Alan R.; Oliver, Antony W.; Pearl, Laurence H.; Plon, Sharon E.; Murray, Johanne M.
2016-01-01
The structural maintenance of chromosomes (SMC) family of proteins supports mitotic proliferation, meiosis, and DNA repair to control genomic stability. Impairments in chromosome maintenance are linked to rare chromosome breakage disorders. Here, we have identified a chromosome breakage syndrome associated with severe lung disease in early childhood. Four children from two unrelated kindreds died of severe pulmonary disease during infancy following viral pneumonia with evidence of combined T and B cell immunodeficiency. Whole exome sequencing revealed biallelic missense mutations in the NSMCE3 (also known as NDNL2) gene, which encodes a subunit of the SMC5/6 complex that is essential for DNA damage response and chromosome segregation. The NSMCE3 mutations disrupted interactions within the SMC5/6 complex, leading to destabilization of the complex. Patient cells showed chromosome rearrangements, micronuclei, sensitivity to replication stress and DNA damage, and defective homologous recombination. This work associates missense mutations in NSMCE3 with an autosomal recessive chromosome breakage syndrome that leads to defective T and B cell function and acute respiratory distress syndrome in early childhood. PMID:27427983
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DEFF Research Database (Denmark)
Skovgaard, Ole; Bak, Mads; Løbner-Olesen, Anders
2011-01-01
a combination of WGS and genome copy number analysis, for the identification of mutations that suppress the growth deficiency imposed by excessive initiations from the Escherichia coli origin of replication, oriC. The E. coli chromosome, like the majority of bacterial chromosomes, is circular, and DNA...... replication is initiated by assembling two replication complexes at the origin, oriC. These complexes then replicate the chromosome bidirectionally toward the terminus, ter. In a population of growing cells, this results in a copy number gradient, so that origin-proximal sequences are more frequent than...... origin-distal sequences. Major rearrangements in the chromosome are, therefore, readily identified by changes in copy number, i.e., certain sequences become over- or under-represented. Of the eight mutations analyzed in detail here, six were found to affect a single gene only, one was a large chromosomal...
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Directory of Open Access Journals (Sweden)
M Rajičić
Full Text Available B chromosomes (Bs are known for more than hundred years but their origin, structure and pattern of evolution are not well understood. In the past few years new methodological approaches, involving isolation of Bs followed by whole DNA amplification, DNA probe generation, and fluorescent in situ hybridization (FISH or the B chromosome DNA sequencing, has allowed detailed analysis of their origin and molecular structure in different species. In this study we explored the origin of Bs in the yellow-necked wood mouse, Apodemus flavicollis, using generation of microdissected DNA probes followed by FISH on metaphase chromosomes. Bs of A. flavicollis were successfully isolated and DNA was used as the template for B-specific probes for the first time. We revealed homology of DNA derived from the analyzed B chromosomes to the pericentromeric region (PR of sex chromosomes and subtelomeric region of two pairs of small autosomes, but lower homology to the rest of the Y chromosome. Moreover, all analysed Bs had the same structure regardless of their number per individual or the great geographic distance between examined populations from the Balkan Peninsula (Serbia and Eastern Europe (south region of Russia and central Belarus. Therefore, it was suggested that B chromosomes in A. flavicollis have a unique common origin from the PR of sex chromosomes, and/or similar evolutionary pattern.
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International Nuclear Information System (INIS)
Allen, R.N.; Ritter, L.; Moore, S.R.; Grosovsky, A.J.
2003-01-01
Full text: Studies in our lab have led to the hypothesis that chromosomal rearrangements can generate novel breakage-prone sites, resulting in chromosomal instability acting predominantly in cis. For example, specific breakage of large blocks of centromeric region heterochromatin on chromosome 16q by treatment with 2,6-diaminopurine (DAP) is associated with repeated rearrangement of chromosome 16q during outgrowth of DAP-treated clones, thereby establishing a link between the initial site of damage and the occurrence of persistent chromosomal instability. Similarly, karyotypic analysis of gamma ray induced instability demonstrated that chromosomal rearrangements in sub-clones were significantly clustered near the site of previously identified chromosomal rearrangement junctions in unstable parental clones. This study investigates the hypothesis that integration of transfected sequences into host chromosomes could create breakage-prone junction regions and persistent genomic instability without exposure to DNA-damage agents. These junctions may mimic the unstable chromosomal rearrangements induced by DAP or radiation, and thus provide a test of the broader hypothesis that instability can to some extent be attributed to the formation of novel chromosomal breakage hot spots. These experiments were performed using human-hamster hybrid AL cells containing a single human chromosome 11, which was used to monitor instability in a chromosomal painting assay. AL cells were transfected with a 2.5 Kb fragment containing multiple copies of the 180 bp human alpha heterochromatic repeat, which resulted in chromosomal instability in 41% of the transfected clones. Parallel exposure to gamma-radiation resulted in a similar level of chromosomal instability, although control transfections with plasmid alone did not lead to karyotypic instability. Chromosomal instability induced by integration of alpha heterochromatic repeats was also frequently associated with delayed reproductive
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Fang, Yuhui; Yuan, Jingya; Wang, Zhangjun; Wang, Haiyan; Xiao, Jin; Yang, Zhixi; Zhang, Ruiqi; Qi, Zengjun; Xu, Weigang; Hu, Lin; Wang, Xiu-E
2014-08-20
Hordeum californicum (2n = 2x = 14, HH) is resistant to several wheat diseases and tolerant to lower nitrogen. In this study, a molecular karyotype of H. californicum chromosomes in the Triticum aestivum L. cv. Chinese Spring (CS)-H. californicum amphidiploid (2n = 6x = 56, AABBDDHH) was established. By genomic in situ hybridization (GISH) and multicolor fluorescent in situ hybridization (FISH) using repetitive DNA clones (pTa71, pTa794 and pSc119.2) as probes, the H. californicum chromosomes could be differentiated from each other and from the wheat chromosomes unequivocally. Based on molecular karyotype and marker analyses, 12 wheat-alien chromosome lines, including four disomic addition lines (DAH1, DAH3, DAH5 and DAH6), five telosomic addition lines (MtH7L, MtH1S, MtH1L, DtH6S and DtH6L), one multiple addition line involving H. californicum chromosome H2, one disomic substitution line (DSH4) and one translocation line (TH7S/1BL), were identified from the progenies derived from the crosses of CS-H. californicum amphidiploid with common wheat varieties. A total of 482 EST (expressed sequence tag) or SSR (simple sequence repeat) markers specific for individual H. californicum chromosomes were identified, and 47, 50, 45, 49, 21, 51 and 40 markers were assigned to chromosomes H1, H2, H3, H4, H5, H6 and H7, respectively. According to the chromosome allocation of these markers, chromosomes H2, H3, H4, H5, and H7 of H. californicum have relationship with wheat homoeologous groups 5, 2, 6, 3, and 1, and hence could be designated as 5H(c), 2H(c), 6H(c), 3H(c) and 1H(c), respectively. The chromosomes H1 and H6 were designated as 7H(c) and 4H(c), respectively, by referring to SSR markers located on rye chromosomes. Copyright © 2014 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.
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Chromosomal organization of repetitive DNAs in Hordeum bogdanii and H. brevisubulatum (Poaceae
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Quanwen Dou
2016-10-01
Full Text Available Molecular karyotypes of H. bogdanii Wilensky, 1918 (2n = 14, and H. brevisubulatum Link, 1844 ssp. brevisubulatum (2n = 28, were characterized by physical mapping of several repetitive sequences. A total of 18 repeats, including all possible di- or trinucleotide SSR (simple sequence repeat motifs and satellite DNAs, such as pAs1, 5S rDNA, 45S rDNA, and pSc119.2, were used as probes for fluorescence in situ hybridization on root-tip metaphase chromosomes. Except for the SSR motifs AG, AT and GC, all the repeats we examined produced detectable hybridization signals on chromosomes of both species. A detailed molecular karyotype of the I genome of H. bogdanii is described for the first time, and each repetitive sequence is physically mapped. A high degree of chromosome variation, including aneuploidy and structural changes, was observed in H. brevisubulatum. Although the distribution of repeats in the chromosomes of H. brevisubulatum is different from that of H. bogdanii, similar patterns between the two species imply that the autopolyploid origin of H. brevisubulatum is from a Hordeum species with an I genome. A comparison of the I genome and the other Hordeum genomes, H, Xa and Xu, shows that colocalization of motifs AAC, ACT and CAT and colocalization of motifs AAG and AGG are characteristic of the I genome. In addition, we discuss the evolutionary significance of repeats in the genome during genome differentiation.
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International Nuclear Information System (INIS)
Cremer, C.; Gray, J.W.
1982-01-01
Asynchronously growing Chinese hamster cells (M3-1) were UV-irradiated (lambda = 254 nm) and then incubated with/without caffeine (2 mM) for 20 h. Microscopic evaluation of metaphase spreads revealed that after UV-irradiation alone (5.0 J/m 2 ) and caffeine treatment alone, the percentage of cells with condensed chromatin appearing fragmented and/or pulverized ('GCS-like' cells; GCS, Generalized Chromosome Shattering) was very low while it was high following the combined treatment. Cytogenetic and flow cytometric analysis of cells obtained by mechanical shaking cultures treated with UV and caffeine indicated that 'GCS-like' cells have the same DNA content as untreated cells in G2 phase and mitosis. (orig.)
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Directory of Open Access Journals (Sweden)
Karina de Cassia Faria
2002-01-01
Full Text Available The purpose of this work was to verify the ability of the enzyme Alu I to cleave and/or remove satellite DNA sequences from heterochromatic regions in chromosomes of bats, by identifying the occurrence of modifications in the pattern of fluorescence in situ hybridization with telomeric DNA. The localization and fluorescence intensity of the telomeric DNA sites of the Alu-digested and undigested chromosomes of species Eumops glaucinus, Carollia perspicillata, and Platyrrhinus lineatus were analyzed. Telomeric sequences were detected at the termini of chromosomes of all three species, although, in C. perspicillata, the signals were very faint or absent in most chromosomes. This finding was interpreted as being due to a reduced number of copies of the telomeric repeat, resulting from extensive telomeric association and/or rearrangements undergone by the chromosomes of Carollia. Fluorescent signals were also observed in centromeric and pericentromeric regions in several two-arm chromosomes of E. glaucinus and C. perspicillata. In E. glaucinus and P. lineatus, some interstitial and terminal telomeric sites were observed to be in association with regions of constitutive heterochromatin and ribosomal DNA (NORs. After digestion, these telomeric sites showed a significant decrease in signal intensity, indicating that enzyme Alu I cleaves and/or removes part of the satellite DNA present in these regions. These results suggest that the telomeric sequence is a component of the heterochromatin, and that the C-band- positive regions of bat chromosomes have a different DNA composition.
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Structure and chromosomal localization of the human lymphotoxin gene
International Nuclear Information System (INIS)
Nedwin, G.E.; Jarrett-Nedwin, J.; Smith, D.H.; Naylor, S.L.; Sakaguchi, A.Y.; Goeddel, D.V.; Gray, P.W.
1987-01-01
The authors have isolated, sequenced, and determined the chromosomal localization of the gene encoding human lymphotoxin (LT). The single copy gene was isolated from a human genomic library using a /sup 32/P-labeled 116 bp synthetic DNA fragment whose sequence was based on the NH/sub 2/-terminal amino acid sequence of LT. The gene spans 3 kb of DNA and is interrupted by three intervening sequences. The LT gene is located on human chromosome 6, as determined by Southern blot analysis of human-murine hybrid DNA. Putative transcriptional control regions and areas of homology with the promoters of interferon and other genes are identified
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Cloning an expressed gene shared by the human sex chromosomes
International Nuclear Information System (INIS)
Darling, S.M.; Banting, G.S.; Pym, B.; Wolfe, J.; Goodfellow, P.N.
1986-01-01
The existence of genes shared by mammalian sex chromosomes has been predicted on both evolutionary and functional grounds. However, the only experimental evidence for such genes in humans is the cell-surface antigen encoded by loci on the X and Y chromosomes (MIC2X and MIC2Y, respectively), which is recognized by the monoclonal antibody 12E7. Using the bacteriophage λgt11 expression system in Escherichia coli and immunoscreening techniques, the authors have isolated a cDNA clone whose primary product is recognized by 12E7. Southern blot analysis using somatic cell hybrids containing only the human X or Y chromosomes shows that the sequences reacting with the cDNA clone are localized to the sex chromosomes. In addition, the clone hybridizes to DNAs isolated from mouse cells that have been transfected with human DNA and selected for 12E7 expression on the fluorescence-activated cell sorter. The authors conclude that the cDNA clone encodes the 12E7 antigen, which is the primary product of the MIC2 loci. The clone was used to explore sequence homology between MIC2X and MIC2Y; these loci are closely related, if not identical
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Principles of Chromosome Architecture Revealed by Hi-C.
Eagen, Kyle P
2018-06-01
Chromosomes are folded and compacted in interphase nuclei, but the molecular basis of this folding is poorly understood. Chromosome conformation capture methods, such as Hi-C, combine chemical crosslinking of chromatin with fragmentation, DNA ligation, and high-throughput DNA sequencing to detect neighboring loci genome-wide. Hi-C has revealed the segregation of chromatin into active and inactive compartments and the folding of DNA into self-associating domains and loops. Depletion of CTCF, cohesin, or cohesin-associated proteins was recently shown to affect the majority of domains and loops in a manner that is consistent with a model of DNA folding through extrusion of chromatin loops. Compartmentation was not dependent on CTCF or cohesin. Hi-C contact maps represent the superimposition of CTCF/cohesin-dependent and -independent folding states. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Yasui, Hideshi; Nonomura, Ken-ichi; Iwata, Nobuo; 安井, 秀; 野々村, 賢一; 岩田, 伸夫
1997-01-01
Genomic in situ hybridization (GIS H) using total Oryza punctata Kotschy genomic DNA as a probe was applied to detect alien chromosomes transferred from O. punctata (W1514: 2n=2x=24: BB) to O. sativa Japonica cultivar, Nipponbare (2n=2x=24: AA). Only 12 chromosomes in the interspecific hybrids (2n=3x=36: AAB) between autotetraploid of O. sativa cultivar Nipponbare and a diploid strain of O. punctata (W1514) showed intense staining by FITC in mitotic metaphase spreads. Only one homologous pair...
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Genes on B chromosomes: old questions revisited with new tools.
Banaei-Moghaddam, Ali M; Martis, Mihaela M; Macas, Jiří; Gundlach, Heidrun; Himmelbach, Axel; Altschmied, Lothar; Mayer, Klaus F X; Houben, Andreas
2015-01-01
B chromosomes are supernumerary dispensable parts of the karyotype which appear in some individuals of some populations in some species. Often, they have been considered as 'junk DNA' or genomic parasites without functional genes. Due to recent advances in sequencing technologies, it became possible to investigate their DNA composition, transcriptional activity and effects on the host transcriptome profile in detail. Here, we review the most recent findings regarding the gene content of B chromosomes and their transcriptional activities and discuss these findings in the context of comparable biological phenomena, like sex chromosomes, aneuploidy and pseudogenes. Recent data suggest that B chromosomes carry transcriptionally active genic sequences which could affect the transcriptome profile of their host genome. These findings are gradually changing our view that B chromosomes are solely genetically inert selfish elements without any functional genes. This at one side could partly explain the deleterious effects which are associated with their presence. On the other hand it makes B chromosome a nice model for studying regulatory mechanisms of duplicated genes and their evolutionary consequences. Copyright © 2014 Elsevier B.V. All rights reserved.
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El Kafsi, Hela; Loux, Valentin; Mariadassou, Mahendra; Blin, Camille; Chiapello, Hélène; Abraham, Anne-Laure; Maguin, Emmanuelle; van de Guchte, Maarten
2017-01-01
The first Lactobacillus delbrueckii ssp. bulgaricus genome sequence revealed the presence of a very large inverted repeat (IR), a DNA sequence arrangement which thus far seemed inconceivable in a non-manipulated circular bacterial chromosome, at the replication terminus. This intriguing observation prompted us to investigate if similar IRs could be found in other bacteria. IRs with sizes varying from 38 to 76 kbp were found at the replication terminus of all 5 L. delbrueckii ssp. bulgaricus chromosomes analysed, but in none of 1373 other chromosomes. They represent the first naturally occurring very large IRs detected in circular bacterial genomes. A comparison of the L. bulgaricus replication terminus regions and the corresponding regions without IR in 5 L. delbrueckii ssp. lactis genomes leads us to propose a model for the formation and evolution of the IRs. The DNA sequence data are consistent with a novel model of chromosome rescue after premature replication termination or irreversible chromosome damage near the replication terminus, involving mechanisms analogous to those proposed in the formation of very large IRs in human cancer cells. We postulate that the L. delbrueckii ssp. bulgaricus-specific IRs in different strains derive from a single ancestral IR of at least 93 kbp. PMID:28281695
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Destabilized SMC5/6 complex leads to chromosome breakage syndrome with severe lung disease
van der Crabben, Saskia N; Hennus, Marije P; McGregor, Grant A; Ritter, Deborah I; Nagamani, Sandesh C S; Wells, Owen S; Harakalova, Magdalena; Chinn, Ivan K; Alt, Aaron; Vondrova, Lucie; Hochstenbach, Ron; van Montfrans, Joris M; Terheggen-Lagro, Suzanne W; van Lieshout, Stef; van Roosmalen, Markus J; Renkens, Ivo; Duran, Karen; Nijman, Isaäc J.; Kloosterman, Wigard P; Hennekam, Eric; Orange, Jordan S; van Hasselt, Peter M; Wheeler, David A; Palecek, Jan J; Lehmann, Alan R; Oliver, Antony W; Pearl, Laurence H; Plon, Sharon E; Murray, Johanne M; van Haaften, Gijs
The structural maintenance of chromosomes (SMC) family of proteins supports mitotic proliferation, meiosis, and DNA repair to control genomic stability. Impairments in chromosome maintenance are linked to rare chromosome breakage disorders. Here, we have identified a chromosome breakage syndrome
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Histone H2AFX Links Meiotic Chromosome Asynapsis to Prophase I Oocyte Loss in Mammals.
Directory of Open Access Journals (Sweden)
Jeffrey M Cloutier
2015-10-01
Full Text Available Chromosome abnormalities are common in the human population, causing germ cell loss at meiotic prophase I and infertility. The mechanisms driving this loss are unknown, but persistent meiotic DNA damage and asynapsis may be triggers. Here we investigate the contribution of these lesions to oocyte elimination in mice with chromosome abnormalities, e.g. Turner syndrome (XO and translocations. We show that asynapsed chromosomes trigger oocyte elimination at diplonema, which is linked to the presence of phosphorylated H2AFX (γH2AFX. We find that DNA double-strand break (DSB foci disappear on asynapsed chromosomes during pachynema, excluding persistent DNA damage as a likely cause, and demonstrating the existence in mammalian oocytes of a repair pathway for asynapsis-associated DNA DSBs. Importantly, deletion or point mutation of H2afx restores oocyte numbers in XO females to wild type (XX levels. Unexpectedly, we find that asynapsed supernumerary chromosomes do not elicit prophase I loss, despite being enriched for γH2AFX and other checkpoint proteins. These results suggest that oocyte loss cannot be explained simply by asynapsis checkpoint models, but is related to the gene content of asynapsed chromosomes. A similar mechanistic basis for oocyte loss may operate in humans with chromosome abnormalities.
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Histone H2AFX Links Meiotic Chromosome Asynapsis to Prophase I Oocyte Loss in Mammals
Cloutier, Jeffrey M.; Mahadevaiah, Shantha K.; ElInati, Elias; Nussenzweig, André; Tóth, Attila; Turner, James M. A.
2015-01-01
Chromosome abnormalities are common in the human population, causing germ cell loss at meiotic prophase I and infertility. The mechanisms driving this loss are unknown, but persistent meiotic DNA damage and asynapsis may be triggers. Here we investigate the contribution of these lesions to oocyte elimination in mice with chromosome abnormalities, e.g. Turner syndrome (XO) and translocations. We show that asynapsed chromosomes trigger oocyte elimination at diplonema, which is linked to the presence of phosphorylated H2AFX (γH2AFX). We find that DNA double-strand break (DSB) foci disappear on asynapsed chromosomes during pachynema, excluding persistent DNA damage as a likely cause, and demonstrating the existence in mammalian oocytes of a repair pathway for asynapsis-associated DNA DSBs. Importantly, deletion or point mutation of H2afx restores oocyte numbers in XO females to wild type (XX) levels. Unexpectedly, we find that asynapsed supernumerary chromosomes do not elicit prophase I loss, despite being enriched for γH2AFX and other checkpoint proteins. These results suggest that oocyte loss cannot be explained simply by asynapsis checkpoint models, but is related to the gene content of asynapsed chromosomes. A similar mechanistic basis for oocyte loss may operate in humans with chromosome abnormalities. PMID:26509888
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ParABS system in chromosome partitioning in the bacterium Myxococcus xanthus.
Directory of Open Access Journals (Sweden)
Antonio A Iniesta
Full Text Available Chromosome segregation is an essential cellular function in eukaryotic and prokaryotic cells. The ParABS system is a fundamental player for a mitosis-like process in chromosome partitioning in many bacterial species. This work shows that the social bacterium Myxococcus xanthus also uses the ParABS system for chromosome segregation. Its large prokaryotic genome of 9.1 Mb contains 22 parS sequences near the origin of replication, and it is shown here that M. xanthus ParB binds preferentially to a consensus parS sequence in vitro. ParB and ParA are essential for cell viability in M. xanthus as in Caulobacter crescentus, but unlike in many other bacteria. Absence of ParB results in anucleate cells, chromosome segregation defects and loss of viability. Analysis of ParA subcellular localization shows that it clusters at the poles in all cells, and in some, in the DNA-free cell division plane between two chromosomal DNA masses. This ParA localization pattern depends on ParB but not on FtsZ. ParB inhibits the nonspecific interaction of ParA with DNA, and ParA colocalizes with chromosomal DNA only when ParB is depleted. The subcellular localization of ParB suggests a single ParB-parS complex localized at the edge of the nucleoid, next to a polar ParA cluster, with a second ParB-parS complex migrating after the replication of parS takes place to the opposite nucleoid edge, next to the other polar ParA cluster.
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Chromosome reduction in Eleocharis maculosa (Cyperaceae).
da Silva, C R M; González-Elizondo, M S; Laforga Vanzela, A L
2008-01-01
Chromosome numbers in Cyperaceae lower than the typical basic number x = 5 have been described for only three species: Rhynchospora tenuis (n = 2), Fimbristylis umbellaris (n = 3) and Eleocharis subarticulata (n = 3). Eleocharis maculosa is recorded here as the fourth species of Cyperaceae that has a chromosome number lower than 2n = 10, with 2n = 8, 7 and 6. The karyotype differentiation in E. maculosa was studied using conventional staining (mitosis and meiosis), FISH with 45S and 5S rDNA and telomere probes. The results allow us to determine which chromosomes of the chromosome race with 2n = 10 fused to form the remaining reduced numbers, as well as to understand how the symploidy and translocation mechanisms were important in karyotype differentiation and the formation of chromosome races in Eleocharis. Copyright 2008 S. Karger AG, Basel.
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Chromosomal characterization of the bonytongue Arapaima gigas (Osteoglossiformes: Arapaimidae
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Debora Karla Marques
Full Text Available The mitotic chromosomes of the pirarucu Arapaima gigas inhabiting the middle Araguaia River and collected in the municipality of Araguaiana (MT, Brazil were studied. The chromosomes were analyzed through Giemsa staining, C-banding, Ag-NOR staining and in situ hybridization using an 18S rRNA gene probe. The karyotype had 2n=56 comprising 14 biarmed and 14 uniarmed chromosome pairs in both sexes. No cytologically distinguishable sex chromosome was identified. A single NOR-bearing chromosome pair was detected by Ag-NOR staining and confirmed by 18S rDNA- FISH. Faint constitutive heterochromatin was C-banded in the centromeric region of some chromosomes.
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Induction of chromosomal aberrations in human lymphocytes by fission neutrons
International Nuclear Information System (INIS)
Silva, Marcia Augusta da; Coelho, Paulo Rogerio Pinto; Bartolini, Paolo; Okazaki, Kayo
2009-01-01
Chromosome aberrations induced by sparsely ionizing radiation (low-LET) are well known and cytogenetic analyses of irradiated human lymphocytes have been widely applied to biological dosimetry. However, much less is known about chromosome aberrations induced by densely ionizing radiation (high LET), such as that of alpha particles or neutrons. Such particles induce DNA strand breaks, as well as chromosome breakage and rearrangements of high complexity. This damage is more localized and less efficiently repaired than after X- or γ-ray irradiation. This preferential production of complex aberrations by densely ionizing radiation is related to the unique energy deposition patterns, which produces highly localized multiple DNA damage at the chromosomal level. A better knowledge of the interactions between different types of radiation and cellular DNA is of importance, not only from the radiobiological viewpoint but also for dosimetric and therapeutic purposes. The objective of the present study was to analyse the cytogenetic effects of fission neutrons on peripheral blood lymphocytes in order to evaluate structural and numerical aberrations and number of cells in the different mitotic cycles. So, blood samples from five healthy donors, 22-25 years old, of both sexes, were irradiated in the Research Reactor IEA-R1 of our Institute (IPEN/CNEN-SP) with thermal and fast neutrons at doses of 0.2; 0.3; 0.5 and 1.0 Gy. The γ contribution to the total absorbed dose was about 30%. These doses were monitored by thermoluminescent dosemeters: LiF-600 (for neutrons) and LiF-700 (for γ-rays). The data concerning structural aberrations were evaluated with regard to three parameters: percentage of cells with aberrations, number of aberrations/cell and number of dicentric/cell. The cytogenetic results showed an increase in the three parameters after irradiation with neutrons, as a function of radiation dose. Apparently, there was no influence of neutrons on the kinetics of cellular
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Fetal sex determination in the first trimester of pregnancy using a Y chromosome-specific DNA probe
Energy Technology Data Exchange (ETDEWEB)
Zeng, Y.; Huang, S.; Chen, M.; Huang, Y.; Zhang, M.; Dong, J.; Ku, A.; Xu, S.
1987-05-01
Prenatal determination of fetal sex is important for the prevention of X-linked disorders such as hemophilia, Lesch-Nyhan syndrome and Duchenne muscular dystrophy. The complex procedures of prenatal diagnosis for X-linked disorders are unnecessary if the fetus is female, because usually no clinical symptoms ever appear in female. pY 3.4 probe used in this work for sex determination is a 3.4 kilobase human repeat sequence. The probe is specific for the Y chromosome of males and can be used for sex determination. The other prove pBLUR used in this paper as control is a widely dispersed, highly repeated human Alu family DNA sequence, represented equally in male and female DNA. On the basis of the relative densities of the autoradiographic spots produced by hybridization of fetal DNA with pY3.4 and pBLUR, the sex of fetus can be clearly identified. Further the authors can determine the radioactive intensity (cpm) of the hybridized DNA spots and the ratio of hybridization with Y3.4 to pBLUR (Y3.4/pBLUR x 10). Results show that the hybridization ratio of DNA from chorionic villi of male (1.03 +/- 0.24) is significantly higher than that of female (0.16 +/- 0.09). Therefore, sex determination of the fetus can be made, based on the ratio of pY3.4/pBLUR x 10. If necessary they can also use Southern hybridization with pY 3.4 probe of DNA isolated from chorionic villi to confirm the result of dot hybridization.
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Telomere dysfunction and chromosome instability
Energy Technology Data Exchange (ETDEWEB)
Murnane, John P., E-mail: jmurnane@radonc.ucsf.edu [Department of Radiation Oncology, University of California San Francisco, 2340 Sutter Street, San Francisco, CA 94143-1331 (United States)
2012-02-01
The ends of chromosomes are composed of a short repeat sequence and associated proteins that together form a cap, called a telomere, that keeps the ends from appearing as double-strand breaks (DSBs) and prevents chromosome fusion. The loss of telomeric repeat sequences or deficiencies in telomeric proteins can result in chromosome fusion and lead to chromosome instability. The similarity between chromosome rearrangements resulting from telomere loss and those found in cancer cells implicates telomere loss as an important mechanism for the chromosome instability contributing to human cancer. Telomere loss in cancer cells can occur through gradual shortening due to insufficient telomerase, the protein that maintains telomeres. However, cancer cells often have a high rate of spontaneous telomere loss despite the expression of telomerase, which has been proposed to result from a combination of oncogene-mediated replication stress and a deficiency in DSB repair in telomeric regions. Chromosome fusion in mammalian cells primarily involves nonhomologous end joining (NHEJ), which is the major form of DSB repair. Chromosome fusion initiates chromosome instability involving breakage-fusion-bridge (B/F/B) cycles, in which dicentric chromosomes form bridges and break as the cell attempts to divide, repeating the process in subsequent cell cycles. Fusion between sister chromatids results in large inverted repeats on the end of the chromosome, which amplify further following additional B/F/B cycles. B/F/B cycles continue until the chromosome acquires a new telomere, most often by translocation of the end of another chromosome. The instability is not confined to a chromosome that loses its telomere, because the instability is transferred to the chromosome donating a translocation. Moreover, the amplified regions are unstable and form extrachromosomal DNA that can reintegrate at new locations. Knowledge concerning the factors promoting telomere loss and its consequences is
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International Nuclear Information System (INIS)
Latt, S.A.; Lalande, M.; Donlon, T.
1986-01-01
This paper describes a few of the many possible examples in which application of a molecular cytogenetic approach can ultimately lead to a new, important understanding about the statics and dynamics of human chromosome structure. In the case of retinoblastoma, cytological observations of deletions and linkage analysis have positioned the retinoblastoma locus to bank 13q14. This locus is grossly deleted in some spontaneous tumors. It is still necessary to locate more precisely and characterize the nature of the retinoblastoma locus, as well as the basis for the heterogeneity in deletions removing one copy of this locus. One is left with the possibility that those deletions that may be observed cytologically reflect but the tip of the iceberg of deletions; detection of others may require molecular probes. A related question is the nature of the DNA sequences at the deletion boundaries and the role they play in promoting these deletions
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The alpha-spectrin gene is on chromosome 1 in mouse and man.
Huebner, K; Palumbo, A P; Isobe, M; Kozak, C A; Monaco, S; Rovera, G; Croce, C M; Curtis, P J
1985-06-01
By using alpha-spectrin cDNA clones of murine and human origin and somatic cell hybrids segregating either mouse or human chromosomes, the gene for alpha-spectrin has been mapped to chromosome 1 in both species. This assignment of the mouse alpha-spectrin gene to mouse chromosome 1 by DNA hybridization strengthens the previous identification of the alpha-spectrin locus in mouse with the sph locus, which previously was mapped by linkage analysis to mouse chromosome 1, distal to the Pep-3 locus. By in situ hybridization to human metaphase chromosomes, the human alpha-spectrin gene has been localized to 1q22-1q25; interestingly, the locus for a non-Rh-linked form of elliptocytosis has been provisionally mapped to band 1q2 by family linkage studies.
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Deng, Chuanliang; Bai, Lili; Fu, Shulan; Yin, Weibo; Zhang, Yingxin; Chen, Yuhong; Wang, Richard R-C; Zhang, Xiangqi; Han, Fangpu; Hu, Zanmin
2013-01-01
In this study, chromosome painting was developed and used to identify alien chromosomes in TAi-27, a wheat--Thinopyrum intermedium addition line, and the chromosomes of the three different genomes of Th. Intermedium. The smallest alien chromosome of TAi-27 was microdissected and its DNA amplified by DOP-PCR was used as a probe to hybridize with metaphase chromosomes of TAi-27 and Th. intermedium. Results showed that hybridization signals were observed in all regions of a pair of the smallest alien chromosomes and the pericentromeric area of another pair of alien chromosomes in TAi-27, indicating that the probe from microdissected chromosome is species specific. In Th. intermedium, 14 chromosomes had wide and strong hybridization signals distributed mainly on the pericentromere area and 9 chromosomes with narrow and weak signals on the pericentromere area. The remaining chromosomes displayed a very weak or no signal. Sequential FISH/GISH on Th. intermedium chromosomes using the DNAs of microdissected chromosome, Pseudoroegneria spicata (St genome) and pDbH12 (a J(s) genome specific probe) as the probes indicated that the microdissected chromosome belonged to the St genome, three genomes (J(s) , J and St) in Th. intermedium could be distinguished, in which there is no hybridization signal on J genome that is similar to the genome of Th. bessarabicum. Our results showed that the smallest alien chromosomes may represent a truncated chromosome and the repetitive sequence distribution might be similar in different chromosomes within the St genome. However, the repetitive sequence distributions are different within the J(s) genome, within a single chromosome, and among different genomes in Th. intermedium. Our results suggested that chromosome painting could be feasible in some plants and useful in detecting chromosome variation and repetitive sequence distribution in different genomes of polyploidy plants, which is helpful for understanding the evolution of different
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Yin, Weibo; Zhang, Yingxin; Chen, Yuhong; Wang, Richard R.-C.; Zhang, Xiangqi; Han, Fangpu; Hu, Zanmin
2013-01-01
In this study, chromosome painting was developed and used to identify alien chromosomes in TAi-27, a wheat - Thinopyrum intermedium addition line, and the chromosomes of the three different genomes of Th. Intermedium. The smallest alien chromosome of TAi-27 was microdissected and its DNA amplified by DOP-PCR was used as a probe to hybridize with metaphase chromosomes of TAi-27 and Th . intermedium . Results showed that hybridization signals were observed in all regions of a pair of the smallest alien chromosomes and the pericentromeric area of another pair of alien chromosomes in TAi-27, indicating that the probe from microdissected chromosome is species specific. In Th . intermedium , 14 chromosomes had wide and strong hybridization signals distributed mainly on the pericentromere area and 9 chromosomes with narrow and weak signals on the pericentromere area. The remaining chromosomes displayed a very weak or no signal. Sequential FISH/GISH on Th . intermedium chromosomes using the DNAs of microdissected chromosome, Pseudoroegneria spicata (St genome) and pDbH12 (a Js genome specific probe) as the probes indicated that the microdissected chromosome belonged to the St genome, three genomes (Js, J and St) in Th . intermedium could be distinguished, in which there is no hybridization signal on J genome that is similar to the genome of Th . bessarabicum . Our results showed that the smallest alien chromosomes may represent a truncated chromosome and the repetitive sequence distribution might be similar in different chromosomes within the St genome. However, the repetitive sequence distributions are different within the Js genome, within a single chromosome, and among different genomes in Th . intermedium . Our results suggested that chromosome painting could be feasible in some plants and useful in detecting chromosome variation and repetitive sequence distribution in different genomes of polyploidy plants, which is helpful for understanding the evolution of different
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Guevara, Evelyn K; Palo, Jukka U; Guillén, Sonia; Sajantila, Antti
2016-11-01
The ancient Chachapoya were an aggregate of several ethnic groups that shared a common language, religion, and material culture. They inhabited a territory at the juncture of the Andes and the Amazon basin. Their position between those ecozones most likely influenced their genetic composition. We attempted to better understand their population history by assessing the contemporary genetic diversity in the Chachapoya and three of their immediate neighbors (Huancas, Jivaro, and Cajamarca). We inferred signatures of demographic history and genetic affinities, and contrasted the findings with data from other populations on local and continental scales. We studied mitochondrial DNA (mtDNA; hypervariable segment [HVSI and HVSII]) and Y chromosome (23 short tandem repeats (STRs)) marker data in 382 modern individuals. We used Sanger sequencing for mtDNA and a commercially available kit for Y-chromosomal STR typing. The Chachapoya had affinities with various populations of Andean and Amazonian origin. When examining the Native American component, the Chachapoya displayed high levels of genetic diversity. Together with other parameters, for example, large Tajima's D and Fu's Fs, the data indicated no drastic reduction of the population size in the past. The high level of diversity in the Chachapoya, the lack of evidence of drift in the past, and genetic affinities with a broad range of populations in the Americas reflects an intricate population history in the region. The new genetic data from the Chachapoya indeed seems to point to a genetic complexity that is not yet resolved but beginning to be elucidated. Am. J. Hum. Biol. 28:857-867, 2016. © 2016Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
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Construction of a map of chromosome 16 by using radiation hybrids
International Nuclear Information System (INIS)
Ceccherini, I.; Romeo, G.; Lawrence, S.; Morton, N.E.; Breuning, M.H.; Harris, P.C.; Himmelbauer, H.; Frischauf, A.M.; Sutherland, G.R.; Germino, G.G.; Reeders, S.T.
1992-01-01
A human-hamster cell hybrid carrying a single copy of chromosome 16 as the only human genetic material was irradiated with a single dose of γ-rays and then fused with a thymidine kinase-deficient hamster cell line (RJKM) to generate radiation hybrids retaining unselected fragments of this human chromosome. In two experiments, 223 hybrids were isolated in hypoxanthine/aminopterine/thymidine (HAT) medium and screened with 38 DNA probes, corresponding to anonymous DNA or gene sequences localized on chromosome 16. The most likely order and location of the 38 DNA sequences were established by multiple pairwise analysis and scaled to estimate physical distance in megabases. The order and the distances thus obtained are mostly consistent with available data on genetic and physical mapping of these markers, illustrating the usefulness of radiation hybrids for mapping
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Directory of Open Access Journals (Sweden)
Elżbieta Warchałowska-Śliwa
2017-05-01
Full Text Available The present study focused on the evolution of the karyotype in four genera of the tribe Pholidopterini: Eupholidoptera Mařan, 1953, Parapholidoptera Mařan, 1953, Pholidoptera Wesmaël, 1838, Uvarovistia Mařan, 1953. Chromosomes were analyzed using fluorescence in situ hybridization (FISH with 18S rDNA and (TTAGGn telomeric probes, and classical techniques, such as C-banding, silver impregnation and fluorochrome DAPI/CMA3 staining. Most species retained the ancestral diploid chromosome number 2n = 31 (male or 32 (female, while some of the taxa, especially a group of species within genus Pholidoptera, evolved a reduced chromosome number 2n = 29. All species show the same sex determination system X0/XX. In some taxa, a pericentric inversion has changed the morphology of the ancestral acrocentric X chromosome to the biarmed X. The rDNA loci coincided with active NORs and C-band/CG-rich segments. A comparison of the location of the single rDNA/NOR in the genus Pholidoptera suggests that reduced chromosome number results from Robertsonian translocation between two pairs of autosomes, one carrying the rDNA/NOR. The results constitute a step towards better understanding of the chromosomal reorganization and evolution within the tribe Phaneropterini and the whole subfamily Tettigoniinae.
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Analysis of DNA polymerase activity in Petunia protoplasts treated with clastogenic agents
International Nuclear Information System (INIS)
Benediktsson, I.; Spampinato, C.P.; Andreo, C.S.; Schieder, O.
1994-01-01
Clastogenic agents, i.e. agents that can induce chromosome or DNA breakage, have been shown to enhance the role of direct gene transfer to protoplasts. The effect was analysed at the enzymatic level using protoplast homogenates as well as intact protoplasts. For that purpose existing procedures were modified to enable measurement of DNA polymerase in vivo. In the system used, external DNA was able to enter the cells without the addition of membrane-permeabilizing compounds. When comparing total DNA polymerase activity of protoplasts irradiated with X-rays or UV-light with that of untreated cells we did not observe significant differences. Incubation of protoplasts with high doses of bleomycin affected total DNA polymerase activity negatively. but dideoxythymidine triphosphate-sensitive activity was not influenced. We conclude that the DNA strand-breaks induced by low doses of X-rays. UV-light or bleomycin do not increase the total or the repair-DNA polymerase activity and. therefore. that the increase in the transformation rates after DNA strand-breaking is not preceded by enhanced DNA polymerase activity. (author)
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International Nuclear Information System (INIS)
Nasazzi, N.; Otero, D.; Di Giorgio, M.
1996-01-01
Ionizing radiation induces DNA double-strand breaks (DSBs) and their interaction and illegitimate recombination produces chromosomal aberrations. Stable chromosomal aberrations comprise inter-chromosomal events (translocations) and intra-chromosomal events (inversions). When DSBs induction and interaction is done at random, and the proximity effects are neglected, the expected relation between translocations and inversions is F=86, based on chromosome arm length. The number of translocations and inversions is analyzed by using G-banding in 16 lymphocytes cultures from blood samples acutely irradiated with γ-rays (dose range: 0,5 Gy - 3 Gy). The result obtained was: F=13,5, significantly smaller than F=86. Literature data show similar small F values, but strongly spread. The excess of inversions could be explained by a 'proximity effect', it means that more proximate DSBs have more interaction probability. Therefore, it is possible to postulate a special chromosome arrangement during irradiation and the subsequent interval. We propose a model where individual chromosomes show spherical confinement with some degree of overlapping and DSBs induction proportional to cross section. A DSBs interaction probability function with cut-off length= 1μ is assumed. According to our results, the confinement volume is ≅ 6.4% of the nuclear volume. Nevertheless, we presume that large spread in F data could be due to temporal variation in overlapping and spatial chromosomal confinement. (authors). 14 refs
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Chromosomal replicons of higher plants
International Nuclear Information System (INIS)
Van't Hof, J.
1987-01-01
This brief discussion of replicons of higher plants offers a glimpse into the properties of chromosomal DNA replication. It gives evidence that the S phase of unrelated plant species is comprised of temporally ordered replicon families that increase in number with genome size. This orderly process, which assures a normal inheritance of genetic material to recipient daughter cells, is maintained at the level of replicon clusters by two mutually exclusive mechanisms, one involving the rate at which single replicons replicate their allotment of DNA, and another by means of the tempo-pause. The same two mechanisms are used by cells to alter the pattern of chromosomal DNA replication just prior to and during normal development. Both mechanisms are genetically determined and produce genetic effects when disturbed of disrupted by additional non-conforming DNAs. Further insight into how these two mechanisms operate requires more molecular information about the nature of replicons and the factors that govern when a replicon family replicates. Plant material is a rich and ideal source for this information just awaiting exploitation. 63 refs
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Heritable alteration of DNA methylation induced by whole-chromosome aneuploidy in wheat.
Gao, Lihong; Diarso, Moussa; Zhang, Ai; Zhang, Huakun; Dong, Yuzhu; Liu, Lixia; Lv, Zhenling; Liu, Bao
2016-01-01
Aneuploidy causes changes in gene expression and phenotypes in all organisms studied. A previous study in the model plant Arabidopsis thaliana showed that aneuploidy-generated phenotypic changes can be inherited to euploid progenies and implicated an epigenetic underpinning of the heritable variations. Based on an analysis by amplified fragment length polymorphism and methylation-sensitive amplified fragment length polymorphism markers, we found that although genetic changes at the nucleotide sequence level were negligible, extensive changes in cytosine DNA methylation patterns occurred in all studied homeologous group 1 whole-chromosome aneuploid lines of common wheat (Triticum aestivum), with monosomic 1A showing the greatest amount of methylation changes. The changed methylation patterns were inherited by euploid progenies derived from the aneuploid parents. The aneuploidy-induced DNA methylation alterations and their heritability were verified at selected loci by bisulfite sequencing. Our data have provided empirical evidence supporting earlier suggestions that heritability of aneuploidy-generated, but aneuploidy-independent, phenotypic variations may have an epigenetic basis. That at least one type of aneuploidy - monosomic 1A - was able to cause significant epigenetic divergence of the aneuploid plants and their euploid progenies also lends support to recent suggestions that aneuploidy may have played an important and protracted role in polyploid genome evolution. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.
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International Nuclear Information System (INIS)
Game, J.C.; Sitney, K.C.; Cook, V.E.; Mortimer, R.K.
1989-01-01
The authors describe a system that uses pulsed-field gels for the physical detection of recombinant DNA molecules, double-strand DNA breaks (DSB) and sister-chromatid exchange in the yeast Saccharomyces cerevisiae. The system makes use of a circular variant of chromosome II (Chr. III). Meiotic recombination between this ring chromosome and a linear homolog produces new molecules of sizes distinguishable on gels from either parental molecule. They demonstrate that these recombinant molecules are not present either in strains with two linear Chr. III molecules or in rad50 mutants, which are defective in meiotic recombination. In conjunction with the molecular endpoints. They present data on the timing of commitment to meiotic recombination scored genetically. They have used x-rays to linearize circular Chr. III, both to develop a sensitive method for measuring frequency of DSB and as a means of detecting double-size circles originating in part from sister-chromatid exchange, which they find to be frequent during meiosis
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Johnston, Calum; Mortier-Barrière, Isabelle; Granadel, Chantal; Polard, Patrice; Martin, Bernard; Claverys, Jean-Pierre
2015-01-01
Homologous recombination (HR) is required for both genome maintenance and generation of diversity in eukaryotes and prokaryotes. This process initiates from single-stranded (ss) DNA and is driven by a universal recombinase, which promotes strand exchange between homologous sequences. The bacterial recombinase, RecA, is loaded onto ssDNA by recombinase loaders, RecBCD and RecFOR for genome maintenance. DprA was recently proposed as a third loader dedicated to genetic transformation. Here we assessed the role of RecFOR in transformation of the human pathogen Streptococcus pneumoniae. We firstly established that RecFOR proteins are not required for plasmid transformation, strongly suggesting that DprA ensures annealing of plasmid single-strands internalized in the process. We then observed no reduction in chromosomal transformation using a PCR fragment as donor, contrasting with the 10,000-fold drop in dprA - cells and demonstrating that RecFOR play no role in transformation. However, a ∼1.45-fold drop in transformation was observed with total chromosomal DNA in recFOR mutants. To account for this limited deficit, we hypothesized that transformation with chromosomal DNA stimulated unexpectedly high frequency (>30% of cells) formation of chromosome dimers as an intermediate in the generation of tandem duplications, and that RecFOR were crucial for dimer resolution. We validated this hypothesis, showing that the site-specific recombinase XerS was also crucial for dimer resolution. An even higher frequency of dimer formation (>80% of cells) was promoted by interspecies transformation with Streptococcus mitis chromosomal DNA, which contains numerous inversions compared to pneumococcal chromosome, each potentially promoting dimerization. In the absence of RecFOR and XerS, dimers persist, as confirmed by DAPI staining, and can limit the efficiency of transformation, since resulting in loss of transformant chromosome. These findings strengthen the view that different HR
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Directory of Open Access Journals (Sweden)
Calum Johnston
2015-01-01
Full Text Available Homologous recombination (HR is required for both genome maintenance and generation of diversity in eukaryotes and prokaryotes. This process initiates from single-stranded (ss DNA and is driven by a universal recombinase, which promotes strand exchange between homologous sequences. The bacterial recombinase, RecA, is loaded onto ssDNA by recombinase loaders, RecBCD and RecFOR for genome maintenance. DprA was recently proposed as a third loader dedicated to genetic transformation. Here we assessed the role of RecFOR in transformation of the human pathogen Streptococcus pneumoniae. We firstly established that RecFOR proteins are not required for plasmid transformation, strongly suggesting that DprA ensures annealing of plasmid single-strands internalized in the process. We then observed no reduction in chromosomal transformation using a PCR fragment as donor, contrasting with the 10,000-fold drop in dprA- cells and demonstrating that RecFOR play no role in transformation. However, a ∼1.45-fold drop in transformation was observed with total chromosomal DNA in recFOR mutants. To account for this limited deficit, we hypothesized that transformation with chromosomal DNA stimulated unexpectedly high frequency (>30% of cells formation of chromosome dimers as an intermediate in the generation of tandem duplications, and that RecFOR were crucial for dimer resolution. We validated this hypothesis, showing that the site-specific recombinase XerS was also crucial for dimer resolution. An even higher frequency of dimer formation (>80% of cells was promoted by interspecies transformation with Streptococcus mitis chromosomal DNA, which contains numerous inversions compared to pneumococcal chromosome, each potentially promoting dimerization. In the absence of RecFOR and XerS, dimers persist, as confirmed by DAPI staining, and can limit the efficiency of transformation, since resulting in loss of transformant chromosome. These findings strengthen the view that
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International Nuclear Information System (INIS)
Kelley-Clarke, Brenna; Ballestas, Mary E.; Komatsu, Takashi; Kaye, Kenneth M.
2007-01-01
The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) tethers KSHV terminal repeat (TR) DNA to mitotic chromosomes to efficiently segregate episomes to progeny nuclei. LANA contains N- and C-terminal chromosome binding regions. We now show that C-terminal LANA preferentially concentrates to paired dots at pericentromeric and peri-telomeric regions of a subset of mitotic chromosomes through residues 996-1139. Deletions within C-terminal LANA abolished both self-association and chromosome binding, consistent with a requirement for self-association to bind chromosomes. A deletion abolishing TR DNA binding did not affect chromosome targeting, indicating LANA's localization is not due to binding its recognition sequence in chromosomal DNA. LANA distributed similarly on human and non-human mitotic chromosomes. These results are consistent with C-terminal LANA interacting with a cell factor that concentrates at pericentromeric and peri-telomeric regions of mitotic chromosomes
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Hanson, Erin K; Ballantyne, Jack
2016-01-01
In some cases of sexual assault the victim may not report the assault for several days after the incident due to various factors. The ability to obtain an autosomal STR profile of the semen donor from a living victim rapidly diminishes as the post-coital interval is extended due to the presence of only a small amount of male DNA amidst an overwhelming amount of female DNA. Previously, we have utilized various technological tools to overcome the limitations of male DNA profiling in extended interval post-coital samples including the use of Y-chromosome STR profiling, cervical sample, and post-PCR purification permitting the recovery of Y-STR profiles of the male DNA from samples collected 5-6 days after intercourse. Despite this success, the reproductive biology literature reports the presence of spermatozoa in the human cervix up to 7-10 days post-coitus. Therefore, novel and improved methods for recovery of male profiles in extended interval post-coital samples were required. Here, we describe enhanced strategies, including Y-chromosome-targeted pre-amplification and next generation Y-STR amplification kits, that have resulted in the ability to obtain probative male profiles from samples collected 6-9 days after intercourse.
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Vibrio chromosome-specific families
DEFF Research Database (Denmark)
Lukjancenko, Oksana; Ussery, David
2014-01-01
We have compared chromosome-specific genes in a set of 18 finished Vibrio genomes, and, in addition, also calculated the pan- and core-genomes from a data set of more than 250 draft Vibrio genome sequences. These genomes come from 9 known species and 2 unknown species. Within the finished...... chromosomes, we find a core set of 1269 encoded protein families for chromosome 1, and a core of 252 encoded protein families for chromosome 2. Many of these core proteins are also found in the draft genomes (although which chromosome they are located on is unknown.) Of the chromosome specific core protein...... families, 1169 and 153 are uniquely found in chromosomes 1 and 2, respectively. Gene ontology (GO) terms for each of the protein families were determined, and the different sets for each chromosome were compared. A total of 363 different "Molecular Function" GO categories were found for chromosome 1...
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Increased Y-chromosome detection by SRY duplexing
DEFF Research Database (Denmark)
Hansen, Morten Høgh; Clausen, Frederik Banch; Dziegiel, Morten Hanefeld
2012-01-01
Determining fetal sex noninvasively is dependent of a robust assay. We designed a novel SRY assay and combined it with a SRY assay from literature forming a duplex assay with the same fluorescent dye to increase detection of Y-chromosome at low cell-free fetal DNA or chimeric DNA concentrations....
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Flow cytometry measurements of human chromosome kinetochore labeling
International Nuclear Information System (INIS)
Fantes, J.A.; Green, D.K.; Malloy, P.; Sumner, A.T.
1989-01-01
A method for the preparation and measurement of immunofluorescent human chromosome centromeres in suspension is described using CREST antibodies, which bind to the centromeric region of chromosomes. Fluorescein isothiocyanate (FITC)-conjugated antihuman antibodies provide the fluorescent label. Labeled chromosomes are examined on microscope slides and by flow cytometry. In both cases a dye which binds to DNA is added to provide identification of the chromosome groups. Sera from different CREST patients vary in their ability to bind to chromosome arms in addition to the centromeric region. Flow cytometry and microfluorimetry measurements have shown that with a given CREST serum the differences in kinetochore fluorescence between chromosomes are only minor. Flow cytometry experiments to relate the number of dicentric chromosomes, induced by in vitro radiation of peripheral blood cells to the slightly increased number of chromosomes with above-average kinetochore fluorescence did not produce decisive radiation dosimetry results
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Energy Technology Data Exchange (ETDEWEB)
Nigro, M.; Bernardeschi, M. [Department of Clinical and Experimental Medicine, Pisa University, Pisa (Italy); Costagliola, D. [Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Caserta (Italy); Della Torre, C. [Department of Physical, Earth and Environmental Sciences, University of Siena, Siena (Italy); Frenzilli, G., E-mail: giada@biomed.unipi.it [Department of Clinical and Experimental Medicine, Pisa University, Pisa (Italy); Guidi, P.; Lucchesi, P. [Department of Clinical and Experimental Medicine, Pisa University, Pisa (Italy); Mottola, F.; Santonastaso, M. [Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Caserta (Italy); Scarcelli, V. [Department of Clinical and Experimental Medicine, Pisa University, Pisa (Italy); Monaci, F.; Corsi, I. [Department of Physical, Earth and Environmental Sciences, University of Siena, Siena (Italy); Stingo, V.; Rocco, L. [Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Caserta (Italy)
2015-11-15
Highlights: • European sea bass was exposed to CdCl{sub 2} and n-TiO{sub 2} alone and in combination. • Genotoxicity was evaluated by RAPD-assay, comet assay and cytome assay. • CdCl{sub 2} induced DNA primary damage but not chromosomal damage. • n-TiO{sub 2} induced chromosomal damage but not DNA primary damage. • Co-exposure effects depend on the biomarker used. - Abstract: Due to the large production and growing use of titanium dioxide nanoparticles (n-TiO{sub 2}), their release in the marine environment and their potential interaction with existing toxic contaminants represent a growing concern for biota. Different end-points of genotoxicity were investigated in the European sea bass Dicentrarchus labrax exposed to n-TiO{sub 2} (1 mg L{sup −1}) either alone and combined with CdCl{sub 2} (0.1 mg L{sup −1}) for 7 days. DNA primary damage (comet assay), apoptotic cells (diffusion assay), occurrence of micronuclei and nuclear abnormalities (cytome assay) were assessed in peripheral erythrocytes and genomic stability (random amplified polymorphism DNA-PCR, RAPD assay) in muscle tissue. Results showed that genome template stability was reduced after CdCl{sub 2} and n-TiO{sub 2} exposure. Exposure to n-TiO{sub 2} alone was responsible for chromosomal alteration but ineffective in terms of DNA damage; while the opposite was observed in CdCl{sub 2} exposed specimens. Co-exposure apparently prevents the chromosomal damage and leads to a partial recovery of the genome template stability.
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Marker chromosome 21 identified by microdissection and FISH
Energy Technology Data Exchange (ETDEWEB)
Sun, Y.; Palmer, C.G. [Indiana Univ. School of Medicine, Indianapolis, IN (United States); Rubinstein, J. [Univ. Affiliated Cincinnati Center for Developmental Disorders, OH (United States)] [and others
1995-03-27
A child without Down`s syndrome but with developmental delay, short stature, and autistic behavior was found to be mosaic 46,XX/47,XX,+mar(21) de novo. The marker was a small ring or dot-like chromosome. Microdissection of the marker was performed. The dissected fragments were biotinylated with sequence-independent PCR as a probe pool for fluorescence in situ hybridization (FISH). FISH results suggested an acrocentric origin of the marker. Subsequent FISH with {alpha}-satellite DNA probes for acrocentric chromosomes and chromosome-specific 21 and 22 painting probes confirmed its origin from chromosome 21. 14 refs., 3 figs.
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Ramos, Laia; Daina, Gemma; Del Rey, Javier; Ribas-Maynou, Jordi; Fernández-Encinas, Alba; Martinez-Passarell, Olga; Boada, Montserrat; Benet, Jordi; Navarro, Joaquima
2015-09-01
To assess whether preimplantation genetic screening can successfully identify cytogenetically normal embryos in couples carrying balanced chromosome rearrangements in addition to increased sperm DNA fragmentation. Comprehensive preimplantation genetic screening was performed on three couples carrying chromosome rearrangements. Sperm DNA fragmentation was assessed for each patient. Academic center. One couple with the male partner carrying a chromosome 2 pericentric inversion and two couples with the male partners carrying a Robertsonian translocation (13:14 and 14:21, respectively). A single blastomere from each of the 18 cleavage-stage embryos obtained was analysed by metaphase comparative genomic hybridization. Single- and double-strand sperm DNA fragmentation was determined by the alkaline and neutral Comet assays. Single- and double-strand sperm DNA fragmentation values and incidence of chromosome imbalances in the blastomeres were analyzed. The obtained values of single-strand sperm DNA fragmentation were between 47% and 59%, and the double-strand sperm DNA fragmentation values were between 43% and 54%. No euploid embryos were observed in the couple showing the highest single-strand sperm DNA fragmentation. However, euploid embryos were observed in the other two couples: embryo transfer was performed, and pregnancy was achieved by the couple showing the lowest sperm DNA fragmentation values. Preimplantation genetic screening enables the detection of euploid embryos in couples affected by balanced chromosome rearrangements and increased sperm DNA fragmentation. Even though sperm DNA fragmentation may potentially have clinical consequences on fertility, comprehensive preimplantation genetic screening allows for the identification and transfer of euploid embryos. Copyright © 2015. Published by Elsevier Inc.
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DNA repair: keeping it together
DEFF Research Database (Denmark)
Lisby, Michael; Rothstein, Rodney
2004-01-01
A protein scaffold has been identified that holds a chromosome together in the event of a DNA double-strand break. This scaffold is dependent on Rad52 and the Rad50-Mre11-Xrs2 complex and withstands the pulling forces of the mitotic spindle during DNA damage checkpoint arrest.......A protein scaffold has been identified that holds a chromosome together in the event of a DNA double-strand break. This scaffold is dependent on Rad52 and the Rad50-Mre11-Xrs2 complex and withstands the pulling forces of the mitotic spindle during DNA damage checkpoint arrest....
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Scassa, María E; Marazita, Mariela C; Ceruti, Julieta M; Carcagno, Abel L; Sirkin, Pablo F; González-Cid, Marcela; Pignataro, Omar P; Cánepa, Eduardo T
2007-05-01
Genome integrity and cell proliferation and survival are regulated by an intricate network of pathways that includes cell cycle checkpoints, DNA repair and recombination, and programmed cell death. It makes sense that there should be a coordinated regulation of these different processes, but the components of such mechanisms remain unknown. In this report, we demonstrate that p19INK4d expression enhances cell survival under genotoxic conditions. By using p19INK4d-overexpressing clones, we demonstrated that p19INK4d expression correlates with the cellular resistance to UV treatment with increased DNA repair activity against UV-induced lesions. On the contrary, cells transfected with p19INK4d antisense cDNA show reduced ability to repair DNA damage and increased sensitivity to genotoxic insult when compared with their p19INK4d-overexpressing counterparts. Consistent with these findings, our studies also show that p19INK4d-overexpressing cells present not only a minor accumulation of UV-induced chromosomal aberrations but a lower frequency of spontaneous chromosome abnormalities than p19INK4d-antisense cells. Lastly, we suggest that p19INK4d effects are dissociated from its role as CDK4/6 inhibitor. The results presented herein support a crucial role for p19INK4d in regulating genomic stability and overall cell viability under conditions of genotoxic stress. We propose that p19INK4d would belong to a protein network that would integrate DNA repair, apoptotic and checkpoint mechanisms in order to maintain the genomic integrity.
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Chromosome duplication in Lolium multiflorum Lam.
Directory of Open Access Journals (Sweden)
Roselaine Cristina Pereira
2014-11-01
Full Text Available Artificial chromosome duplication of diploid genotypes of Lolium multiflorum (2n=2x=14 is worthy to breeding, and aims to increase the expression of traits with agronomic interest. The purpose of this study was to obtain polyploid plants of L. multiflorum from local diploid populations in order to exploit adaptation and future verification of the effects of polyploidy in agronomic traits. Seedlings were immersed in different colchicine solutions for an exposure time of 3h and 24h. Ploidy determination was made by the DNA content and certified by chromosomes counts. The plants confirmed as tetraploids were placed in a greenhouse, and, at flowering, pollen viability was evaluated, and seeds were harvested to assess the stability of the progenies. The percentage of polyploids obtained was 20%. Pollen viability of the tetraploids generated ranged from 58% to 69%. The tetraploid plants obtained in the experiment generated 164 progenies, of which 109 presented DNA content compatible with the tetraploid level, showing stability of chromosome duplication in the filial generation.
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Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange*
Borgogno, María V.; Monti, Mariela R.; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E.; Pezza, Roberto J.
2016-01-01
Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3′ end of the initiating DNA strand have a small effect, whereas most mismatches near the 5′ end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. PMID:26709229
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Isolation of anonymous, polymorphic DNA fragments from human chromosome 22q12-qter
J.P. Dumanski (Jan); A.H.M. Geurts van Kessel (Ad); M. Ruttledge (Martin); A. Wladis (Andreas); N. Sugawa (Noriaki); V.P. Collins (Peter); M. Nordenskjöld
1990-01-01
textabstractA series of 195 random chromosome 22-specific probes, equivalent to approximately 1% of the size of this chromosome, have been isolated from a chromosome 22-specific bacteriophage lambda genomic library. These probes were mapped to four different regions of chromosome 22 on a panel of
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Naturally occurring minichromosome platforms in chromosome engineering: an overview.
Raimondi, Elena
2011-01-01
Artificially modified chromosome vectors are non-integrating gene delivery platforms that can shuttle very large DNA fragments in various recipient cells: theoretically, no size limit exists for the chromosome segments that an engineered minichromosome can accommodate. Therefore, genetically manipulated chromosomes might be potentially ideal vector systems, especially when the complexity of higher eukaryotic genes is concerned. This review focuses on those chromosome vectors generated using spontaneously occurring small markers as starting material. The definition and manipulation of the centromere domain is one of the main obstacles in chromosome engineering: naturally occurring minichromosomes, due to their inherent small size, were helpful in defining some aspects of centromere function. In addition, several distinctive features of small marker chromosomes, like their appearance as supernumerary elements in otherwise normal karyotypes, have been successfully exploited to use them as gene delivery vectors. The key technologies employed for minichromosome engineering are: size reduction, gene targeting, and vector delivery in various recipient cells. In spite of the significant advances that have been recently achieved in all these fields, several unsolved problems limit the potential of artificially modified chromosomes. Still, these vector systems have been exploited in a number of applications where the investigation of the controlled expression of large DNA segments is needed. A typical example is the analysis of genes whose expression strictly depends on the chromosomal environment in which they are positioned, where engineered chromosomes can be envisaged as epigenetically regulated expression systems. A novel and exciting advance concerns the use of engineered minichromosomes to study the organization and dynamics of local chromatin structures.
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Structural variations of chromosome 1 R from rye cultivar Jingzhouheimai induced by irradiation
International Nuclear Information System (INIS)
Wang Conglei; Zhuang Lifang; Qi Zengjun
2012-01-01
Irradiated with 60 Co γ-rays (12 Gy), the pollen of wheat landrace Huixianhong-Secale cereal cv. Jingzhouheimai DA1R was pollinated to the emasculated spikes of Huixianhong. Analyzed with genomic in situ hybridization GISH using gDNA of rye cv. Jingzhouheimai as a probe, four plants with reciprocal translocation, four plants with large segmental translocation and one plant with distal segmental translocation, one plant with one telocentric chromosome were identified from 33 M 1 seeds. The results showed that the translocation frequency was 30.30% and of the total 11 breakage-fusion events, 1 involved centric regions and 10 involved interstitial regions. The experiment showed that pollen irradiation was an effective method to induce wheat alien chromosomal structural variations which could effectively by used in deletion mapping, chromosomal location of important agronomic genes and development of small segmental translocations with target genes. (authors)
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Recombinant DNA probe (PheA12) from the hc-(ERBA) gene on chromosome 3 detects a high frequency RFLP
Energy Technology Data Exchange (ETDEWEB)
Bale, A E; Usala, S; Weinberger, C; Weintraub, B D; McBride, O W [National Institutes of Health, Bethesda, MD (USA)
1988-08-11
A cDNA probe (phe A12), a 1.5 kb fragment, was obtained by screening a gt10 library with the v-erb-A gene, subcloned into pUC 8. BamHI identifies constant bands at 23 kb, 21 kb, 13 kb, and 7.0 kb and a simple two-allele polymorphism with a band at either 5.3 kb (A1) or 2.8 kb (A2). EcoRV identifies constant bands at 15 kb and 8.9 kb and two separate two-allele polymorphisms--13.5 kb (B1) vs. 10.5 kb (B2) and 3.3 kb (C1) vs. 1.6 kb (C2). It was mapped to chromosome 3 using laser sorted chromosomes. Co-dominant inheritance and cosegregation of BamHI and EcoRV polymorphism was demonstrated in 20 individuals in one large kindred.
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Mixing of maize and wheat genomic DNA by somatic hybridization in regenerated sterile maize plants.
Szarka, B.; Göntér, I.; Molnár-Láng, M.; Mórocz, S.; Dudits, D.
2002-07-01
Intergeneric somatic hybridization was performed between albino maize ( Zea mays L.) protoplasts and mesophyll protoplasts of wheat ( Triticum aestivum L.) by polyethylene glycol (PEG) treatments. None of the parental protoplasts were able to produce green plants without fusion. The maize cells regenerated only rudimentary albino plantlets of limited viability, and the wheat mesophyll protoplasts were unable to divide. PEG-mediated fusion treatments resulted in hybrid cells with mixed cytoplasm. Six months after fusion green embryogenic calli were selected as putative hybrids. The first-regenerates were discovered as aborted embryos. Regeneration of intact, green, maize-like plants needed 6 months of further subcultures on hormone-free medium. These plants were sterile, although had both male and female flowers. The cytological analysis of cells from callus tissues and root tips revealed 56 chromosomes, but intact wheat chromosomes were not observed. Using total DNA from hybrid plants, three RAPD primer combinations produced bands resembling the wheat profile. Genomic in situ hybridization (GISH) using total wheat DNA as a probe revealed the presence of wheat DNA islands in the maize chromosomal background. The increased viability and the restored green color were the most-significant new traits as compared to the original maize parent. Other intermediate morphological traits of plants with hybrid origin were not found.
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Directory of Open Access Journals (Sweden)
Kaitlin M Stimpson
2010-08-01
Full Text Available Genome rearrangement often produces chromosomes with two centromeres (dicentrics that are inherently unstable because of bridge formation and breakage during cell division. However, mammalian dicentrics, and particularly those in humans, can be quite stable, usually because one centromere is functionally silenced. Molecular mechanisms of centromere inactivation are poorly understood since there are few systems to experimentally create dicentric human chromosomes. Here, we describe a human cell culture model that enriches for de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. The induced dicentrics vary in structure near fusion breakpoints and like naturally-occurring dicentrics, exhibit various inter-centromeric distances. Many functional dicentrics persist for months after formation. Even those with distantly spaced centromeres remain functionally dicentric for 20 cell generations. Other dicentrics within the population reflect centromere inactivation. In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the alpha-satellite DNA array associated with CENP-A is reduced compared to the same array before dicentric formation. Extra-chromosomal fragments that contained CENP-A often appear in the same cells as dicentrics. Some of these fragments are derived from the same alpha-satellite DNA array as inactivated centromeres. Our results indicate that dicentric human chromosomes undergo alternative fates after formation. Many retain two active centromeres and are stable through multiple cell divisions. Others undergo centromere inactivation. This event occurs within a broad temporal window and can involve deletion of chromatin that marks the locus as a site for CENP-A maintenance/replenishment.
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Dynamic organization of genetic recombination proteins and chromosomes
International Nuclear Information System (INIS)
Essers, J.; Van Cappellen, G.; Van Drunen, E.; Theil, A.; Jaspers, N.N.G.J.; Houtsmuller, A.B.; Vermeulen, W.; Kanaar, R.
2003-01-01
Homologous recombination requires the co-ordinated action of the RAD52 group proteins, including Rad51, Rad52 and Rad54. Upon treatment of mammalian cells with ionizing radiation, these proteins accumulate into foci at sites of DSB induction. We probed the nature of the DNA damage-induced foci in living cells with the use of photobleaching techniques. These foci are not static assemblies of DNA repair proteins. Instead, they are dynamic structures of which Rad51 is a stable core component, while Rad52 and Rad54 reversibly interact with the structure. Furthermore, even though the RAD52 group proteins colocalize in the DNA damage-induced foci, the majority of the proteins are not part of the same multi-protein complex in the absence of DNA damage. Executing DNA transactions through dynamic multi-protein complexes, rather than stable holo-complexes, allows greater flexibility during the transaction. In case of DNA repair, for example, it allows cross talk between different DNA repair pathways and coupling to other DNA transactions, such as replication. In addition to the behavior of proteins in living cells, we have tracked chromosomes during cell division. Our results suggest that the relative position of chromosomes in the mother cell is conserved in its daughter cells
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Structure and evolution of the Y-chromosomal and mitochondrial DNA of cattle
Verkaar, Edward Louis Christian
2003-01-01
The research described in this thesis is focused on the structure and evolution of the bovine Y-chromosome and the use of paternal markers in molecular diagnostics. The Y-chromosome has emerged together with the X-chromosome early during the evolution of the mammals by differentiation of a pair of
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Histone Modification Associated with Initiation of DNA Replication | Center for Cancer Research
Before cells are able to divide, they must first duplicate their chromosomes accurately. DNA replication and packaging of DNA into chromosomes by histone proteins need to be coordinated by the cell to ensure proper transmission of genetic and epigenetic information to the next generation. Mammalian DNA replication begins at specific chromosomal sites, called replication
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Somatic DNA recombination yielding circular DNA and deletion of a genomic region in embryonic brain
International Nuclear Information System (INIS)
Maeda, Toyoki; Chijiiwa, Yoshiharu; Tsuji, Hideo; Sakoda, Saburo; Tani, Kenzaburo; Suzuki, Tomokazu
2004-01-01
In this study, a mouse genomic region is identified that undergoes DNA rearrangement and yields circular DNA in brain during embryogenesis. External region-directed inverse polymerase chain reaction on circular DNA extracted from late embryonic brain tissue repeatedly detected DNA of this region containing recombination joints. Wide-range genomic PCR and digestion-circularization PCR analysis showed this region underwent recombination accompanied with deletion of intervening sequences, including the circularized regions. This region was mapped by fluorescence in situ hybridization to C1 on mouse chromosome 16, where no gene and no physiological DNA rearrangement had been identified. DNA sequence in the region has segmental homology to an orthologous region on human chromosome 3q.13. These observations demonstrated somatic DNA recombination yielding genomic deletions in brain during embryogenesis
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Identification of Pneumocystis carinii chromosomes and mapping of five genes
DEFF Research Database (Denmark)
Lundgren, B; Cotton, R; Lundgren, J D
1990-01-01
Pulsed field gel electrophoresis was used to identify the chromosome-size DNA of Pneumocystis carinii, a major pathogen of immunocompromised patients. Thirteen chromosomes of rodent Pneumocystis carinii, ranging in size from 300 to 700 kilobases (kb), were identified. The minimum genome size for ...
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Panzera, Francisco; Ferreiro, María J; Pita, Sebastián; Calleros, Lucía; Pérez, Ruben; Basmadjián, Yester; Guevara, Yenny; Brenière, Simone Frédérique; Panzera, Yanina
2014-10-01
Chagas disease, one of the most important vector-borne diseases in the Americas, is caused by Trypanosoma cruzi and transmitted to humans by insects of the subfamily Triatominae. An effective control of this disease depends on elimination of vectors through spraying with insecticides. Genetic research can help insect control programs by identifying and characterizing vector populations. In southern Latin America, Triatoma infestans is the main vector and presents two distinct lineages, known as Andean and non-Andean chromosomal groups, that are highly differentiated by the amount of heterochromatin and genome size. Analyses with nuclear and mitochondrial sequences are not conclusive about resolving the origin and spread of T. infestans. The present paper includes the analyses of karyotypes, heterochromatin distribution and chromosomal mapping of the major ribosomal cluster (45S rDNA) to specimens throughout the distribution range of this species, including pyrethroid-resistant populations. A total of 417 specimens from seven different countries were analyzed. We show an unusual wide rDNA variability related to number and chromosomal position of the ribosomal genes, never before reported in species with holocentric chromosomes. Considering the chromosomal groups previously described, the ribosomal patterns are associated with a particular geographic distribution. Our results reveal that the differentiation process between both T. infestans chromosomal groups has involved significant genomic reorganization of essential coding sequences, besides the changes in heterochromatin and genomic size previously reported. The chromosomal markers also allowed us to detect the existence of a hybrid zone occupied by individuals derived from crosses between both chromosomal groups. Our genetic studies support the hypothesis of an Andean origin for T. infestans, and suggest that pyrethroid-resistant populations from the Argentinean-Bolivian border are most likely the result of
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Energy Technology Data Exchange (ETDEWEB)
Mullinger, A.M.; Johnson, R.T. (Cambridge Univ. (UK). Dept. of Zoology)
1985-02-01
DNA repair occurs in metaphase-arrested cells in response to ultraviolet irradiation. In the presence of the repair synthesis inhibitors, hydroxyurea and 1-..beta..-D-arabinofuranosylcytosine, the chromosomes of such cells are decondensed. The dose response of chromosome decondensation varies between different cell types. In human cells defective in excision repair there is much less chromosome decondensation in response to the same ultraviolet dose and time of repair inhibition. However, a simian virus 40-transformed muntjac cell displays pronounced chromosome decondensation but has limited incision ability. Both chromosome decondensation and single-strand break accumulation in the presence of inhibitors are reversed when DNA precursors are provided, but reversal after higher ultraviolet doses and longer period of incubation leads to recondensed chromosomes that are fragmented. Although the chromosomes of repair-inhibited metaphase cells are decondensed in fixed preparations, their morphology appears normal in intact cells. The cells also retain a capacity to induce prematurely condensed chromosomes (PCC) when fused with interphase cells: compared with control mitotic cells, the speed of induction is sometimes reduced but the final amount of PCC produced is similar.
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Singh, L; Jones, K W
1982-02-01
Satellite DNA (Bkm) from the W sex-determining chromosome of snakes, which is related to sequences on the mouse Y chromosome, has been used to analyze the DNA and chromosomes of sex-reversed (Sxr) XXSxr male mice. Such mice exhibit a male-specific Southern blot Bkm hybridization pattern, consistent with the presence of Y-chromosome DNA. In situ hybridization of Bkm to chromosomes of XXSxr mice shows an aberrant concentration of related sequences on the distal terminus of a large mouse chromosome. The XYSxr carrier male, however, shows a pair of small chromosomes, which are presumed to be aberrant Y derivatives. Meiosis in the XYSxr mouse involves transfer of chromatin rich in Bkm-related DNA from the Y-Y1 complex to the X distal terminus. We suggest that this event is responsible for the transmission of the Sxr trait.
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International Nuclear Information System (INIS)
Mestres, M.; Schmid, E.; Stephan, G.; Barrios, L.; Caballin, M. R.; Barquinero, J. F.
2004-01-01
The aim of the present study has been the evaluation of the incompleteness of α-particle induced chromosome aberrations by the simultaneous detection of all centromeres and telomeres present in human lymphocytes. For this purpose attached lymphocytes were irradiated at doses of 0.2, 0.5,0.7 and 1 Gy in a ''241Am source. FISH techniques were applied using pan-centromeric and pan-telomeric probes. All abnormal cells were digitalised and analysed using a Cytovision FISH workstation. A total of 378 incomplete chromosomes plus incomplete acentrics was found. Cases with more than 92 telomeres were not detected. The ratio between total incomplete elements and multicentrics was 1.00. The total number of acentric fragments was 822; 57% of then were complete fragments ace (+.+), 26% incomplete fragments ace (+,-), and 17% interstitial fragment ace (-.-). The percentage of incomplete aberrations is higher after high-LET than described for low-LET exposure. The results seem to indicate that compared to low-LET. after α-particle exposure it is more likely to repair the centromere-containing elements. (Author) 30 refs
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Shelterin Protects Chromosome Ends by Compacting Telomeric Chromatin
Bandaria, Jigar N.; Qin, Peiwu; Berk, Veysel; Chu, Steven; Yildiz, Ahmet
2016-01-01
SUMMARY Telomeres, repetitive DNA sequences at chromosome ends, are shielded against the DNA damage response (DDR) by the shelterin complex. To understand how shelterin protects telomere ends, we investigated the structural organization of telomeric chromatin in human cells using super-resolution microscopy. We found that telomeres form compact globular structures through a complex network of interactions between shelterin subunits and telomeric DNA, and not by DNA methylation, histone deacetylation or histone trimethylation at telomeres and subtelomeric regions. Mutations that abrogate shelterin assembly or removal of individual subunits from telomeres cause up to a 10-fold increase in telomere volume. Decompacted telomeres become more accessible to telomere-associated proteins and accumulate DDR signals. Recompaction of telomeric chromatin using an orthogonal method displaces DDR signals from telomeres. These results reveal the chromatin remodeling activity of shelterin and demonstrate that shelterin-mediated compaction of telomeric chromatin provides robust protection of chromosome ends against the DDR machinery. PMID:26871633
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Directory of Open Access Journals (Sweden)
Aneta Spoz
2015-11-01
Chromosomal location of ribosomal DNA sequences is useful for comparative cytogenetic fish studies due to their relatively fast rate of evolution. The results of species from the family Barbinae comparatively presented here for the first time, and they may support further taxonomical studies of the Barbus species.
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Origin of amphibian and avian chromosomes by fission, fusion, and retention of ancestral chromosomes
Voss, Stephen R.; Kump, D. Kevin; Putta, Srikrishna; Pauly, Nathan; Reynolds, Anna; Henry, Rema J.; Basa, Saritha; Walker, John A.; Smith, Jeramiah J.
2011-01-01
Amphibian genomes differ greatly in DNA content and chromosome size, morphology, and number. Investigations of this diversity are needed to identify mechanisms that have shaped the evolution of vertebrate genomes. We used comparative mapping to investigate the organization of genes in the Mexican axolotl (Ambystoma mexicanum), a species that presents relatively few chromosomes (n = 14) and a gigantic genome (>20 pg/N). We show extensive conservation of synteny between Ambystoma, chicken, and human, and a positive correlation between the length of conserved segments and genome size. Ambystoma segments are estimated to be four to 51 times longer than homologous human and chicken segments. Strikingly, genes demarking the structures of 28 chicken chromosomes are ordered among linkage groups defining the Ambystoma genome, and we show that these same chromosomal segments are also conserved in a distantly related anuran amphibian (Xenopus tropicalis). Using linkage relationships from the amphibian maps, we predict that three chicken chromosomes originated by fusion, nine to 14 originated by fission, and 12–17 evolved directly from ancestral tetrapod chromosomes. We further show that some ancestral segments were fused prior to the divergence of salamanders and anurans, while others fused independently and randomly as chromosome numbers were reduced in lineages leading to Ambystoma and Xenopus. The maintenance of gene order relationships between chromosomal segments that have greatly expanded and contracted in salamander and chicken genomes, respectively, suggests selection to maintain synteny relationships and/or extremely low rates of chromosomal rearrangement. Overall, the results demonstrate the value of data from diverse, amphibian genomes in studies of vertebrate genome evolution. PMID:21482624
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Chromosome sizes of phytoplasmas composing major phylogenetic groups and subgroups.
Marcone, C; Neimark, H; Ragozzino, A; Lauer, U; Seemüller, E
1999-09-01
ABSTRACT Chromosome sizes of 71 phytoplasmas belonging to 12 major phylogenetic groups including several of the aster yellows subgroups were estimated from electrophoretic mobilities of full-length chromosomes in pulsed-field gels. Considerable variation in genome size, from 660 to 1,130 kilobases (kb), was observed among aster yellows phytoplasmas. Chromosome size heterogeneity was also observed in the stolbur phytoplasma group (range 860 to 1,350 kb); in this group, isolate STOLF contains the largest chromosome found in a phytoplasma to date. A wide range of chromosome sizes, from 670 to 1,075 kb, was also identified in the X-disease group. The other phytoplasmas examined, which included members of the apple proliferation, Italian alfalfa witches' broom, faba bean phyllody, pigeon pea witches' broom, sugarcane white leaf, Bermuda grass white leaf, ash yellows, clover proliferation, and elm yellows groups, all have chromosomes smaller than 1 megabase, and the size ranges within each of these groups is narrower than in the aster yellows, stolbur, and X-disease groups. The smallest chromosome, approximately 530 kb, was found in two Bermuda grass white leaf phytoplasma isolates. This not only is the smallest mollicute chromosome found to date, but also is the smallest chromosome known for any cell. More than one large DNA band was observed in several phytoplasma preparations. Possible explanations for the occurrence of more than one band may be infection of the host plant by different phytoplasmas, the presence of more than one chromosome in the same organism, or the presence of large extrachromosomal DNA elements.
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Chromosome Banding in Amphibia. XXXII. The Genus Xenopus (Anura, Pipidae).
Schmid, Michael; Steinlein, Claus
2015-01-01
Mitotic chromosomes of 16 species of the frog genus Xenopus were prepared from kidney and lung cell cultures. In the chromosomes of 7 species, high-resolution replication banding patterns could be induced by treating the cultures with 5-bromodeoxyuridine (BrdU) and deoxythymidine (dT) in succession, and in 6 of these species the BrdU/dT-banded chromosomes could be arranged into karyotypes. In the 3 species of the clade with 2n = 20 and 4n = 40 chromosomes (X. tropicalis, X. epitropicalis, X. new tetraploid 1), as well as in the 3 species with 4n = 36 chromosomes (X. laevis, X. borealis, X. muelleri), the BrdU/dT-banded karyotypes show a high degree of homoeology, though differences were detected between these groups. Translocations, inversions, insertions or sex-specific replication bands were not observed. Minor replication asynchronies found between chromosomes probably involve heterochromatic regions. BrdU/dT replication banding of Xenopus chromosomes provides the landmarks necessary for the exact physical mapping of genes and repetitive sequences. FISH with an X. laevis 5S rDNA probe detected multiple hybridization sites at or near the long-arm telomeric regions in most chromosomes of X. laevis and X. borealis, whereas in X. muelleri, the 5S rDNA sequences are located exclusively at the long-arm telomeres of a single chromosome pair. Staining with the AT base pair-specific fluorochrome quinacrine mustard revealed brightly fluorescing heterochromatic regions in the majority of X. borealis chromosomes which are absent in other Xenopus species.
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Satellite DNA: An Evolving Topic.
Garrido-Ramos, Manuel A
2017-09-18
Satellite DNA represents one of the most fascinating parts of the repetitive fraction of the eukaryotic genome. Since the discovery of highly repetitive tandem DNA in the 1960s, a lot of literature has extensively covered various topics related to the structure, organization, function, and evolution of such sequences. Today, with the advent of genomic tools, the study of satellite DNA has regained a great interest. Thus, Next-Generation Sequencing (NGS), together with high-throughput in silico analysis of the information contained in NGS reads, has revolutionized the analysis of the repetitive fraction of the eukaryotic genomes. The whole of the historical and current approaches to the topic gives us a broad view of the function and evolution of satellite DNA and its role in chromosomal evolution. Currently, we have extensive information on the molecular, chromosomal, biological, and population factors that affect the evolutionary fate of satellite DNA, knowledge that gives rise to a series of hypotheses that get on well with each other about the origin, spreading, and evolution of satellite DNA. In this paper, I review these hypotheses from a methodological, conceptual, and historical perspective and frame them in the context of chromosomal organization and evolution.
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Tracking alien chromosome in sativa background by genomic in situ hybridization
International Nuclear Information System (INIS)
Abbasi, F.M.; Iqbal, M.; Salim, M.
2004-01-01
Genomic in situ hybridization (GISH) was used to look into the genomic constitution of monosomic alien -addition line derived from O. sativa x O. brachyantha. Biotin label genomic DNA from O. brachyantha was used as probe. The probe hybridized to the brachyantha chromosome. No detectable hybridization signal was observed on sativa chromosomes. This differential painting of chromosome enables us to unequivocally discriminate brachyantha chromosome from those of sativa. Results showed the usefulness of GISH in the identification of a single alien chromosome in the sativa background. (author)
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Chromosome microdissection and cloning in human genome and genetic disease analysis
International Nuclear Information System (INIS)
Kao, Faten; Yu, Jingwei
1991-01-01
A procedure has been described for microdissection and microcloning of human chromosomal DNA sequences in which universal amplification of the dissected fragments by Mbo I linker adaptor and polymerase chain reaction is used. A very large library comprising 700,000 recombinant plasmid microclones from 30 dissected chromosomes of human chromosome 21 was constructed. Colony hybridization showed that 42% of the clones contained repetitive sequences and 58% contained single or low-copy sequences. The insert sizes generated by complete Mbo I cleavage ranged from 50 to 1,100 base pairs with a mean of 416 base pairs. Southern blot analysis of microclones from the library confirmed their human origin and chromosome 21 specificity. Some of these clones have also been regionally mapped to specific sites of chromosome 21 by using a regional mapping panel of cell hybrids. This chromosome microtechnology can generate large numbers of microclones with unique sequences from defined chromosomal regions and can be used for processes such as (i) isolating corresponding yeast artificial chromosome clones with large inserts, (ii) screening various cDNA libraries for isolating expressed sequences, and (iii) constructing region-specific libraries of the entire human genome. The studies described here demonstrate the power of this technology for high-resolution genome analysis and explicate their use in an efficient search for disease-associated genes localized to specific chromosomal regions
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Transposable elements in fish chromosomes: a study in the marine cobia species.
Costa, G W W F; Cioffi, M B; Bertollo, L A C; Molina, W F
2013-01-01
Rachycentron canadum, a unique representative of the Rachycentridae family, has been the subject of considerable biotechnological interest due to its potential use in marine fish farming. This species has undergone extensive research concerning the location of genes and multigene families on its chromosomes. Although most of the genome of some organisms is composed of repeated DNA sequences, aspects of the origin and dispersion of these elements are still largely unknown. The physical mapping of repetitive sequences on the chromosomes of R. canadum proved to be relevant for evolutionary and applied purposes. Therefore, here, we present the mapping by fluorescence in situ hybridization of the transposable element (TE) Tol2, the non-LTR retrotransposons Rex1 and Rex3, together with the 18S and 5S rRNA genes in the chromosome of this species. The Tol2 TE, belonging to the family of hAT transposons, is homogeneously distributed in the euchromatic regions of the chromosomes but with huge colocalization with the 18S rDNA sites. The hybridization signals for Rex1 and Rex3 revealed a semi-arbitrary distribution pattern, presenting differentiated dispersion in euchromatic and heterochromatic regions. Rex1 elements are associated preferentially in heterochromatic regions, while Rex3 shows a scarce distribution in the euchromatic regions of the chromosomes. The colocalization of TEs with 18S and 5S rDNA revealed complex chromosomal regions of repetitive sequences. In addition, the nonpreferential distribution of Rex1 and Rex3 in all heterochromatic regions, as well as the preferential distribution of the Tol2 transposon associated with 18S rDNA sequences, reveals a distinct pattern of organization of TEs in the genome of this species. A heterogeneous chromosomal colonization of TEs may confer different evolutionary rates to the heterochromatic regions of this species.
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Directory of Open Access Journals (Sweden)
Chuanliang Deng
Full Text Available In this study, chromosome painting was developed and used to identify alien chromosomes in TAi-27, a wheat--Thinopyrum intermedium addition line, and the chromosomes of the three different genomes of Th. Intermedium. The smallest alien chromosome of TAi-27 was microdissected and its DNA amplified by DOP-PCR was used as a probe to hybridize with metaphase chromosomes of TAi-27 and Th. intermedium. Results showed that hybridization signals were observed in all regions of a pair of the smallest alien chromosomes and the pericentromeric area of another pair of alien chromosomes in TAi-27, indicating that the probe from microdissected chromosome is species specific. In Th. intermedium, 14 chromosomes had wide and strong hybridization signals distributed mainly on the pericentromere area and 9 chromosomes with narrow and weak signals on the pericentromere area. The remaining chromosomes displayed a very weak or no signal. Sequential FISH/GISH on Th. intermedium chromosomes using the DNAs of microdissected chromosome, Pseudoroegneria spicata (St genome and pDbH12 (a J(s genome specific probe as the probes indicated that the microdissected chromosome belonged to the St genome, three genomes (J(s , J and St in Th. intermedium could be distinguished, in which there is no hybridization signal on J genome that is similar to the genome of Th. bessarabicum. Our results showed that the smallest alien chromosomes may represent a truncated chromosome and the repetitive sequence distribution might be similar in different chromosomes within the St genome. However, the repetitive sequence distributions are different within the J(s genome, within a single chromosome, and among different genomes in Th. intermedium. Our results suggested that chromosome painting could be feasible in some plants and useful in detecting chromosome variation and repetitive sequence distribution in different genomes of polyploidy plants, which is helpful for understanding the evolution of
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Castañeda, María; Odriozola, Adrián; Gómez, Javier; Zarrabeitia, María T
2013-07-01
We report the development of an effective system for analyzing X chromosome-linked mini short tandem repeat loci with reduced-size amplicons (less than 220 bp), useful for analyzing highly degraded DNA samples. To generate smaller amplicons, we redesigned primers for eight X-linked microsatellites (DXS7132, DXS10079, DXS10074, DXS10075, DXS6801, DXS6809, DXS6789, and DXS6799) and established efficient conditions for a multiplex PCR system (miniX). The validation tests confirmed that it has good sensitivity, requiring as little as 20 pg of DNA, and performs well with DNA from paraffin-embedded tissues, thus showing potential for improved analysis and identification of highly degraded and/or very limited DNA samples. Consequently, this system may help to solve complex forensic cases, particularly when autosomal markers convey insufficient information.
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Xiang, Yuqian; Zhang, Junyu; Li, Qiaoli; Zhou, Xinyao; Wang, Teng; Xu, Mingqing; Xia, Shihui; Xing, Qinghe; Wang, Lei; He, Lin; Zhao, Xinzhi
2014-09-01
Utilizing epigenetic (DNA methylation) differences to differentiate between maternal peripheral blood (PBL) and fetal (placental) DNA has been a promising strategy for non-invasive prenatal testing (NIPT). However, the differentially methylated regions (DMRs) have yet to be fully ascertained. In the present study, we performed genome-wide comparative methylome analysis between maternal PBL and placental DNA from pregnancies of first trimester by methylated DNA immunoprecipitation-sequencing (MeDIP-Seq) and Infinium HumanMethylation450 BeadChip assays. A total of 36 931 DMRs and 45 804 differentially methylated sites (DMSs) covering the whole genome, exclusive of the Y chromosome, were identified via MeDIP-Seq and Infinium 450k array, respectively, of which 3759 sites in 2188 regions were confirmed by both methods. Not only did we find the previously reported potential fetal DNA markers in our identified DMRs/DMSs but also we verified fully the identified DMRs/DMSs in the validation round by MassARRAY EpiTYPER. The screened potential fetal DNA markers may be used for NIPT on aneuploidies and other chromosomal diseases, such as cri du chat syndrome and velo-cardio-facial syndrome. In addition, these potential markers may have application in the early diagnosis of placental dysfunction, such as pre-eclampsia. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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International Nuclear Information System (INIS)
Rosen, H.; Orman, J.; Rakita, R.M.; Michel, B.R.; VanDevanter, D.R.
1990-01-01
Neutrophils and monocytes employ a diverse array of antimicrobial effector systems to support their host defense functions. The mechanisms of action of most of these systems are incompletely understood. The present report indicates that microbicidal activity by a neutrophil-derived antimicrobial system, consisting of myeloperoxidase, enzymatically generated hydrogen peroxide, and chloride ion, is accompanied by prompt cessation of DNA synthesis in Escherichia coli, as determined by markedly reduced incorporation of [ 3 H]thymidine into trichloracetic acid-precipitable material. Simultaneously, the myeloperoxidase system mediates a decline in the ability of E. coli membranes to bind hemimethylated DNA sequences containing the E. coli chromosomal origin of replication (oriC). Binding of oriC to the E. coli membrane is an essential element of orderly chromosomal DNA replication. Comparable early changes in DNA synthesis and DNA-membrane interactions were not observed with alternative oxidant or antibiotic-mediated microbicidal systems. It is proposed that oxidants generated by the myeloperoxidase system modify the E. coli membrane in such a fashion that oriC binding is markedly impaired. As a consequence chromosomal DNA replication is impaired and organisms can no longer replicate
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Yong, Lee C; Petersen, Martin R
2011-02-01
Experimental studies suggest that B vitamins such as niacin, folate, riboflavin, vitamin B6 and vitamin B12 may protect against DNA damage induced by ionising radiation (IR). However, to date, data from IR-exposed human populations are not available. We examined the intakes of these B vitamins and their food sources in relation to the frequency of chromosome translocations as a biomarker of cumulative DNA damage, in eighty-two male airline pilots. Dietary intakes were estimated by using a self-administered semi-quantitative FFQ. Translocations in peripheral blood lymphocytes were scored by using fluorescence in situ hybridisation whole-chromosome painting. Negative binomial regression was used to estimate rate ratios and 95 % CI, adjusted for age and occupational and lifestyle factors. We observed a significant inverse association between translocation frequency and dietary intake of niacin (P = 0·02): adjusted rate ratio for subjects in the highest tertile compared with the lowest tertile was 0·58 (95 % CI 0·40, 0·83). Translocation frequency was not associated with total niacin intake from food and supplements as well as dietary or total intake of folate, riboflavin or vitamin B6 or B12. However, the adjusted rate ratios were significant for subjects with ≥ median compared with food or a diet high in whole grains but low in red and processed meat may protect against cumulative DNA damage in IR-exposed persons.
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Comparative physical mapping between wheat chromosome arm 2BL and rice chromosome 4.
Lee, Tong Geon; Lee, Yong Jin; Kim, Dae Yeon; Seo, Yong Weon
2010-12-01
Physical maps of chromosomes provide a framework for organizing and integrating diverse genetic information. DNA microarrays are a valuable technique for physical mapping and can also be used to facilitate the discovery of single feature polymorphisms (SFPs). Wheat chromosome arm 2BL was physically mapped using a Wheat Genome Array onto near-isogenic lines (NILs) with the aid of wheat-rice synteny and mapped wheat EST information. Using high variance probe set (HVP) analysis, 314 HVPs constituting genes present on 2BL were identified. The 314 HVPs were grouped into 3 categories: HVPs that match only rice chromosome 4 (298 HVPs), those that match only wheat ESTs mapped on 2BL (1), and those that match both rice chromosome 4 and wheat ESTs mapped on 2BL (15). All HVPs were converted into gene sets, which represented either unique rice gene models or mapped wheat ESTs that matched identified HVPs. Comparative physical maps were constructed for 16 wheat gene sets and 271 rice gene sets. Of the 271 rice gene sets, 257 were mapped to the 18-35 Mb regions on rice chromosome 4. Based on HVP analysis and sequence similarity between the gene models in the rice chromosomes and mapped wheat ESTs, the outermost rice gene model that limits the translocation breakpoint to orthologous regions was identified.
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Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange.
Borgogno, María V; Monti, Mariela R; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E; Pezza, Roberto J
2016-03-04
Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3' end of the initiating DNA strand have a small effect, whereas most mismatches near the 5' end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy.
Directory of Open Access Journals (Sweden)
Debora Giorgi
Full Text Available The large size and complex polyploid nature of many genomes has often hampered genomics development, as is the case for several plants of high agronomic value. Isolating single chromosomes or chromosome arms via flow sorting offers a clue to resolve such complexity by focusing sequencing to a discrete and self-consistent part of the whole genome. The occurrence of sufficient differences in the size and or base-pair composition of the individual chromosomes, which is uncommon in plants, is critical for the success of flow sorting. We overcome this limitation by developing a robust method for labeling isolated chromosomes, named Fluorescent In situ Hybridization In suspension (FISHIS. FISHIS employs fluorescently labeled synthetic repetitive DNA probes, which are hybridized, in a wash-less procedure, to chromosomes in suspension following DNA alkaline denaturation. All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L. and bread (T. aestivum L. wheat, were flow-sorted, after FISHIS, at high purity. For the first time in eukaryotes, each individual chromosome of a diploid organism, Dasypyrum villosum (L. Candargy, was flow-sorted regardless of its size or base-pair related content. FISHIS-based chromosome sorting is a powerful and innovative flow cytogenetic tool which can develop new genomic resources from each plant species, where microsatellite DNA probes are available and high quality chromosome suspensions could be produced. The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement. It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations
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FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy.
Giorgi, Debora; Farina, Anna; Grosso, Valentina; Gennaro, Andrea; Ceoloni, Carla; Lucretti, Sergio
2013-01-01
The large size and complex polyploid nature of many genomes has often hampered genomics development, as is the case for several plants of high agronomic value. Isolating single chromosomes or chromosome arms via flow sorting offers a clue to resolve such complexity by focusing sequencing to a discrete and self-consistent part of the whole genome. The occurrence of sufficient differences in the size and or base-pair composition of the individual chromosomes, which is uncommon in plants, is critical for the success of flow sorting. We overcome this limitation by developing a robust method for labeling isolated chromosomes, named Fluorescent In situ Hybridization In suspension (FISHIS). FISHIS employs fluorescently labeled synthetic repetitive DNA probes, which are hybridized, in a wash-less procedure, to chromosomes in suspension following DNA alkaline denaturation. All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L.) and bread (T. aestivum L.) wheat, were flow-sorted, after FISHIS, at high purity. For the first time in eukaryotes, each individual chromosome of a diploid organism, Dasypyrum villosum (L.) Candargy, was flow-sorted regardless of its size or base-pair related content. FISHIS-based chromosome sorting is a powerful and innovative flow cytogenetic tool which can develop new genomic resources from each plant species, where microsatellite DNA probes are available and high quality chromosome suspensions could be produced. The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement. It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations or cytogenetic
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Birth and death of genes linked to chromosomal inversion
Furuta, Yoshikazu; Kawai, Mikihiko; Yahara, Koji; Takahashi, Noriko; Handa, Naofumi; Tsuru, Takeshi; Oshima, Kenshiro; Yoshida, Masaru; Azuma, Takeshi; Hattori, Masahira; Uchiyama, Ikuo; Kobayashi, Ichizo
2011-01-01
The birth and death of genes is central to adaptive evolution, yet the underlying genome dynamics remain elusive. The availability of closely related complete genome sequences helps to follow changes in gene contents and clarify their relationship to overall genome organization. Helicobacter pylori, bacteria in our stomach, are known for their extreme genome plasticity through mutation and recombination and will make a good target for such an analysis. In comparing their complete genome sequences, we found that gain and loss of genes (loci) for outer membrane proteins, which mediate host interaction, occurred at breakpoints of chromosomal inversions. Sequence comparison there revealed a unique mechanism of DNA duplication: DNA duplication associated with inversion. In this process, a DNA segment at one chromosomal locus is copied and inserted, in an inverted orientation, into a distant locus on the same chromosome, while the entire region between these two loci is also inverted. Recognition of this and three more inversion modes, which occur through reciprocal recombination between long or short sequence similarity or adjacent to a mobile element, allowed reconstruction of synteny evolution through inversion events in this species. These results will guide the interpretation of extensive DNA sequencing results for understanding long- and short-term genome evolution in various organisms and in cancer cells. PMID:21212362
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A mitosis-specific and R loop-driven ATR pathway promotes faithful chromosome segregation.
Kabeche, Lilian; Nguyen, Hai Dang; Buisson, Rémi; Zou, Lee
2018-01-05
The ataxia telangiectasia mutated and Rad3-related (ATR) kinase is crucial for DNA damage and replication stress responses. Here, we describe an unexpected role of ATR in mitosis. Acute inhibition or degradation of ATR in mitosis induces whole-chromosome missegregation. The effect of ATR ablation is not due to altered cyclin-dependent kinase 1 (CDK1) activity, DNA damage responses, or unscheduled DNA synthesis but to loss of an ATR function at centromeres. In mitosis, ATR localizes to centromeres through Aurora A-regulated association with centromere protein F (CENP-F), allowing ATR to engage replication protein A (RPA)-coated centromeric R loops. As ATR is activated at centromeres, it stimulates Aurora B through Chk1, preventing formation of lagging chromosomes. Thus, a mitosis-specific and R loop-driven ATR pathway acts at centromeres to promote faithful chromosome segregation, revealing functions of R loops and ATR in suppressing chromosome instability. Copyright © 2018, American Association for the Advancement of Science.
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Human oocyte chromosome analyses need a standardized ...
Indian Academy of Sciences (India)
Studies of DNA polymorphisms in human trisomic abor- tions and liveborn have ... Keywords. human oocyte chromosomes; cytogenetic analysis; aneuploidy; nondisjunction; predivision. Journal of .... oocytes and giant embryos. Hum. Reprod.
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Integration sites of Epstein-Barr virus genome on chromosomes of human lymphoblastoid cell lines
Energy Technology Data Exchange (ETDEWEB)
Wuu, K.D.; Chen, Y.J.; Wang-Wuu, S. [Institute of Genetics, Taipei (Taiwan, Province of China)
1994-09-01
Epstein-Barr virus (EBV) is the pathogen of infectious mononucleosis. The viral genome is present in more than 95% of the African cases of Burkitt lymphoma and it is usually maintained in episomal form in the tumor cells. Viral integration has been described only for Nanalwa which is a Burkitt lymphoma cell line lacking episomes. In order to examine the role of EBV in the immortalization of human Blymphocytes, we investigated whether the EBV integration into the human genome is essential. If the integration does occur, we would like to know whether the integration is randomly distributed or whether the viral DNA integrates preferentially at certain sites. Fourteen in vitro immortalized human lymphoblastoid cell lines (LCLs) were examined by fluorescence in situ hybridization (FISH) with a biotinylated EBV BamHI w DNA fragment as probe. The episomal form of EBV DNA was found in all cells of these cell lines, while only about 65% of the cells have the integrated viral DNA. This might suggest that integration is not a pre-requisite for cell immortalization. Although all chromosomes, except Y, have been found with integrated viral genome, chromsomes 1 and 5 are the most frequent EBV DNA carrier (p<0.05). Nine chromosome bands, namely, 1p31, 1q31, 2q32, 3q13, 3q26, 5q14, 6q24, 7q31 and 12q21, are preferential targets for EBV integration (p<0.001). Eighty percent of the total 938 EBV hybridization signals were found to be at G-band-positive area. This suggests that the mechanism of EBV integration might be different from that of the retroviruses, which specifically integrate to G-band-negative areas. Thus, we conclude that the integration of EBV to host genome is non-random and it may have something to do with the structure of chromosome and DNA sequences.
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International Nuclear Information System (INIS)
Geard, Charles R.; Jenkins, Gloria
1995-01-01
Purpose: To quantitatively assess all gamma-ray induced chromosomal changes confined to one human chromosome using fluorescence microscopy and in situ hybridization with a fluorescently labeled human chromosome specific nucleic acid probe. Methods and Materials: Synchronized human-hamster hybrid cells containing human chromosome 11 were obtained by a modified mitotic shake-off procedure. G1 phase cells (> 95%) were irradiated with 137 Cs gamma rays (0, 0.5, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0, and 10.0 Gy) at a dose rate of 1.1 Gy/min and mitotic cells collected 16-20 h later; chromosomal spreads were prepared, denatured, and hybridized with a fluorescein-tagged nucleic acid probe against total human DNA. Chromosomes were examined by fluorescence microscopy and all categories of change involving the human chromosome 11 as target, recorded. Results: Overall, of the 3104 human-hamster hybrid cells examined, 82.1% were euploid, of which 88.6% contained one copy of human chromosome 11, 6.2% contained two copies, and 5.2% contained 0 copies. This is compatible with mitotic nondisjunction in a small fraction of cells. Of the remaining 17.9% of cells, 85.2% were tetraploid cells with two copies of human chromosome 11. For all aberrations involving human chromosome 11 there was a linear relationship between yield and absorbed dose of 0.1 aberrations per chromosome per Gy. The yield of dicentrics, translocations, and terminal deletions that involve one lesion on the human chromosome was linear, while the yield of interstitial deletions that arise from two interacting lesions on the human chromosome was curvilinear. The frequencies of dicentrics and translocations were about equal, while there was a high (40-60%) incidence of incomplete exchanges between human and hamster chromosomes. Conclusions: Fluorescent in situ hybridization (FISH) procedures allow for the efficient detection of a broad range of induced changes in target chromosomes. Symmetrical exchanges induced in G1
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Makarov, M S; Chentsov, Iu S
2010-01-01
Giant nuclei from salivary glands of Chironomus plumosus were treated in situ with detergent, 2 M NaCl and nucleases in order to reveal residual nuclear matrix proteins (NMP). It was shown, that preceding stabilization of non-histone proteins with 2 mM CuCl2 allowed to visualize the structure of polythene chromosomes at every stage of the extraction of histones and DNA. Stabilized NPM of polythene chromosomes maintains their morphology and banding patterns, which is observed by light and electron microscopy, whereas internal fibril net or residual nucleoli are not found. In stabilized NPM of polythene chromosomes, topoisomerase IIalpha and SMC1 retain their localization that is typical of untreated chromosomes. NPM of polythene chromosomes also includes sites of DNA replication, visualized with BrDU incubation, and some RNA-components. So, we can conclude that structure of NPM from giant nuclei is equal to NPM from normal interphase nuclei, and that morphological features of polythene chromosomes depend on the presence of NMP.
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The colocalization transition of homologous chromosomes at meiosis
Nicodemi, Mario; Panning, Barbara; Prisco, Antonella
2008-06-01
Meiosis is the specialized cell division required in sexual reproduction. During its early stages, in the mother cell nucleus, homologous chromosomes recognize each other and colocalize in a crucial step that remains one of the most mysterious of meiosis. Starting from recent discoveries on the system molecular components and interactions, we discuss a statistical mechanics model of chromosome early pairing. Binding molecules mediate long-distance interaction of special DNA recognition sequences and, if their concentration exceeds a critical threshold, they induce a spontaneous colocalization transition of chromosomes, otherwise independently diffusing.
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Directory of Open Access Journals (Sweden)
Redkar Rajendra
2004-02-01
Full Text Available Abstract Background Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are closely related members of the B. cereus-group of bacilli. Suppressive subtractive hybridization (SSH was used to identify specific chromosomal sequences unique to B. anthracis. Results Two SSH libraries were generated. Genomic DNA from plasmid-cured B. anthracis was used as the tester DNA in both libraries, while genomic DNA from either B. cereus or B. thuringiensis served as the driver DNA. Progressive screening of the libraries by colony filter and Southern blot analyses identified 29 different clones that were specific for the B. anthracis chromosome relative not only to the respective driver DNAs, but also to seven other different strains of B. cereus and B. thuringiensis included in the process. The nucleotide sequences of the clones were compared with those found in genomic databases, revealing that over half of the clones were located into 2 regions on the B. anthracis chromosome. Conclusions Genes encoding potential cell wall synthesis proteins dominated one region, while bacteriophage-related sequences dominated the other region. The latter supports the hypothesis that acquisition of these bacteriophage sequences occurred during or after speciation of B. anthracis relative to B. cereus and B. thuringiensis. This study provides insight into the chromosomal differences between B. anthracis and its closest phylogenetic relatives.
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Loss of DNA topoisomerase I activity alters many cellular functions in Salmonella typhimurium
International Nuclear Information System (INIS)
Overbye, K.M.; Basu, S.K.; Margolin, P.
1983-01-01
In this paper is reported the absence of DNA topoisomerase I in S. typhimurium results in an increased level of the recBC DNase (exonuclease V) enzyme, an almost total abolition of both direct and indirect mutagenesis by alkylating agents, and altered characteristics in the formation of chromosomal tandem duplications. We also present evidence that modifications in DNA superhelicity may strongly affect the pattern of DNA degrafation initiated by treatment of recA mutant cells with bleomycin and mitomycin C. 43 references, 3 figures, 3 tables
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Chromosome painting analysis of X-ray-induced aberrations in human lymphocytes in vitro
International Nuclear Information System (INIS)
Matsuoka, A.; Hayashi, M.; Yamazaki, N.; Sofuni, T.
1994-01-01
Chromosomal rearrangements in human lymphocytes induced by X-rays (0, 0.5, 1.0 and 2.0 Gray) were analyzed using chromosome painting. DNA probes for human chromosomes 1, 3 or 4 alone, and a combination of 1 and 4, were used for analysis. The frequency of cells with rearrangements, i.e. reciprocal translocations, dicentrics, insertions, tricentrics and fragments, involving chromosome 4 increased with dose in both 48 and 72 h cultures. The number of translocations per cell also increased with dose at 48 and 72 h. Dicentrics increased with dose in 48 h but not in 72 h cultures. The estimated genomic frequency of aberrations per cell was comparable with results in banded cells. No difference was shown on the detection efficiency of chromosome rearrangements among the various DNA probes used. Since this technique does not necessarily require well-spread metaphases for analysis, it is possible to increase the number of analyzable metaphases compared with the banding technique. Chromosome painting is a simpler, more objective and practical method for detecting chromosome rearrangements than conventional banding analyses. (Author)
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Transfer of stem cells carrying engineered chromosomes with XY clone laser system.
Sinko, Ildiko; Katona, Robert L
2011-01-01
Current transgenic technologies for gene transfer into the germline of mammals cause a random integration of exogenous naked DNA into the host genome that can generate undesirable position effects as well as insertional mutations. The vectors used to generate transgenic animals are limited by the amount of foreign DNA they can carry. Mammalian artificial chromosomes have large DNA-carrying capacity and ability to replicate in parallel with, but without integration into, the host genome. Hence they are attractive vectors for transgenesis, cellular protein production, and gene therapy applications as well. ES cells mediated chromosome transfer by conventional blastocyst injection has a limitation in unpredictable germline transmission. The demonstrated protocol of laser-assisted microinjection of artificial chromosome containing ES cells into eight-cell mouse embryos protocol described here can solve the problem for faster production of germline transchromosomic mice.
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Splitting the chromosome: cutting the ties that bind sister chromatids.
Nasmyth, K; Peters, J M; Uhlmann, F
2001-01-01
In eukaryotic cells, replicated DNA molecules remain physically connected from their synthesis in S phase until they are separated during anaphase. This phenomenon, called sister chromatid cohesion, is essential for the temporal separation of DNA replication and mitosis and for the equal separation of the duplicated genome. Recent work has identified a number of chromosomal proteins required for cohesion. In this review we discuss how these proteins may connect sister chromatids and how they are removed from chromosomes to allow sister chromatid separation at the onset of anaphase.
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Directory of Open Access Journals (Sweden)
Pedram Kashiani
2012-01-01
Full Text Available A study of genetic variation among 10 pairs of chromosomes extracted from 13 tropical sweet corn inbred lines, using 99 microsatellite markers, revealed a wide range of genetic diversity. Allelic richness and the number of effective alleles per chromosome ranged from 2.78 to 4.33 and 1.96 to 3.47, respectively, with respective mean values of 3.62 and 2.73. According to the Shannon's information index (I and Nei's gene diversity coefficient (Nei, Chromosome 10 was the most informative chromosome (I = 1.311 and Nei = 0.703, while Chromosome 2 possessed the least (I = 0.762 and Nei = 0.456. Based on linkage disequilibrium (LD measurements for loci less than 50 cM apart on the same chromosome, all loci on Chromosomes 1, 6 and 7 were in equilibrium. Even so, there was a high proportion of genetic variation in Chromosomes 4, 5, 8, 9 and 10, thereby revealing their appropriateness for use in the genetic diversity investigations among tropical sweet corn lines. Chromosome 4, with the highest number of loci in linkage disequilibrium, was considered the best for marker-phenotype association and QTL mapping, followed by Chromosomes 5, 8, 9 and 10.
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Chen, Chao; Zhao, Xinqing; Jin, Yingyu; Zhao, Zongbao Kent; Suh, Joo-Won
2014-11-01
Bacterial artificial chromosomal (BAC) vectors are increasingly being used in cloning large DNA fragments containing complex biosynthetic pathways to facilitate heterologous production of microbial metabolites for drug development. To express inserted genes using Streptomyces species as the production hosts, an integration expression cassette is required to be inserted into the BAC vector, which includes genetic elements encoding a phage-specific attachment site, an integrase, an origin of transfer, a selection marker and a promoter. Due to the large sizes of DNA inserted into the BAC vectors, it is normally inefficient and time-consuming to assemble these fragments by routine PCR amplifications and restriction-ligations. Here we present a rapid method to insert fragments to construct BAC-based expression vectors. A DNA fragment of about 130 bp was designed, which contains upstream and downstream homologous sequences of both BAC vector and pIB139 plasmid carrying the whole integration expression cassette. In-Fusion cloning was performed using the designer DNA fragment to modify pIB139, followed by λ-RED-mediated recombination to obtain the BAC-based expression vector. We demonstrated the effectiveness of this method by rapid construction of a BAC-based expression vector with an insert of about 120 kb that contains the entire gene cluster for biosynthesis of immunosuppressant FK506. The empty BAC-based expression vector constructed in this study can be conveniently used for construction of BAC libraries using either microbial pure culture or environmental DNA, and the selected BAC clones can be directly used for heterologous expression. Alternatively, if a BAC library has already been constructed using a commercial BAC vector, the selected BAC vectors can be manipulated using the method described here to get the BAC-based expression vectors with desired gene clusters for heterologous expression. The rapid construction of a BAC-based expression vector facilitates
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DNA replication stress restricts ribosomal DNA copy number.
Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L
2017-09-01
Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.
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DNA replication stress restricts ribosomal DNA copy number
Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin
2017-01-01
Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237
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DNA replication stress restricts ribosomal DNA copy number.
Directory of Open Access Journals (Sweden)
Devika Salim
2017-09-01
Full Text Available Ribosomal RNAs (rRNAs in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.
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Positioning of NORs and NOR-bearing chromosomes in relation to nucleoli.
Kalmárová, Markéta; Smirnov, Evgeny; Masata, Martin; Koberna, Karel; Ligasová, Anna; Popov, Alexey; Raska, Ivan
2007-10-01
It is widely accepted that chromosomes occupy more or less fixed positions in mammalian interphase nucleus. However, relation between large-scale order of chromosome positioning and gene activity remains unclear. We used the model of the human ribosomal genes to address specific aspects of this problem. Ribosomal genes are organized at particular chromosomal sites in clusters termed nucleolus organizer regions (NORs). Only some NORs, called competent are generally accepted to be transcriptionally active during interphase. Importantly in this respect, the regularities in distribution of competent, and non-competent NORs among the specific chromosomes were already established in two human-derived cell lines: transformed HeLa and primary LEP cells. In the present study, using FISH and immunocytochemistry, we found that in HeLa and LEP cells the large-scale positioning of the NOR-bearing chromosomes with regard to nucleoli is linked to the transcription activity of rDNA. Namely, the tendency of rDNA-bearing chromosomes to associate with nucleoli correlates with the number of transcriptionally competent NORs in the respective chromosome homologs. Regarding the position of NORs, we found that not only competent but also most of the non-competent NORs are included in the nucleoli. Some intranucleolar NORs (supposedly non-competent) are situated on elongated chromatin protrusions connecting nucleoli with respective chromosome territories spatially distanced from nucleoli.
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Persistence of Breakage in Specific Chromosome Bands 6 Years after Acute Exposure to Oil.
Directory of Open Access Journals (Sweden)
Alexandra Francés
Full Text Available The identification of breakpoints involved in chromosomal damage could help to detect genes involved in genetic disorders, most notably cancer. Until now, only one published study, carried out by our group, has identified chromosome bands affected by exposure to oil from an oil spill. In that study, which was performed two years after the initial oil exposure in individuals who had participated in clean-up tasks following the wreck of the Prestige, three chromosomal bands (2q21, 3q27, 5q31 were found to be especially prone to breakage. A recent follow-up study, performed on the same individuals, revealed that the genotoxic damage had persisted six years after oil exposure.To determine whether there exist chromosome bands which are especially prone to breakages and to know if there is some correlation with those detected in the previous study. In addition, to investigate if the DNA repair problems detected previously persist in the present study.Follow-up study performed six years after the Prestige oil spill.Fishermen cooperatives in coastal villages.Fishermen highly exposed to oil spill who participated in previous genotoxic study six years after the oil.Chromosome damage in peripheral lymphocytes. For accurate identification of the breakpoints involved in chromosome damage of circulating lymphocytes, a sequential stain/G-banding technique was employed. To determine the most break-prone chromosome bands, two statistical methods, the Fragile Site Multinomial and the chi-square tests (where the bands were corrected by their length were used. To compare the chromosome lesions, structural chromosome alterations and gaps/breaks between two groups of individuals we used the GEE test which takes into account a possible within-individual correlation. Dysfunctions in DNA repair mechanisms, expressed as chromosome damage, were assessed in cultures with aphidicolin by the GEE test.Cytogenetic analyses were performed in 47 exposed individuals. A total of
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International Nuclear Information System (INIS)
Huang, Shuohao; Kawabe, Yoshinori; Ito, Akira; Kamihira, Masamichi
2012-01-01
Highlights: ► Adeno-associated virus (AAV) is capable of targeted integration in human cells. ► Integrase-defective retroviral vector (IDRV) enables a circular DNA delivery. ► A targeted integration system of IDRV DNA using the AAV integration mechanism. ► Targeted IDRV integration ameliorates the safety concerns for retroviral vectors. -- Abstract: Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.
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Carofiglio, Fabrizia; Sleddens-Linkels, Esther; Wassenaar, Evelyne; Inagaki, Akiko; van Cappellen, Wiggert A; Grootegoed, J Anton; Toth, Attila; Baarends, Willy M
2018-03-01
Repair of SPO11-dependent DNA double-strand breaks (DSBs) via homologous recombination (HR) is essential for stable homologous chromosome pairing and synapsis during meiotic prophase. Here, we induced radiation-induced DSBs to study meiotic recombination and homologous chromosome pairing in mouse meiocytes in the absence of SPO11 activity (Spo11 YF/YF model), and in the absence of both SPO11 and HORMAD1 (Spo11/Hormad1 dko). Within 30 min after 5 Gy irradiation of Spo11 YF/YF mice, 140-160 DSB repair foci were detected, which specifically localized to the synaptonemal complex axes. Repair of radiation-induced DSBs was incomplete in Spo11 YF/YF compared to Spo11 +/YF meiocytes. Still, repair of exogenous DSBs promoted partial recovery of chromosome pairing and synapsis in Spo11 YF/YF meiocytes. This indicates that at least part of the exogenous DSBs can be processed in an interhomolog recombination repair pathway. Interestingly, in a seperate experiment, using 3 Gy of irradiation, we observed that Spo11/Hormad1 dko spermatocytes contained fewer remaining DSB repair foci at 48 h after irradiation compared to irradiated Spo11 knockout spermatocytes. Together, these results show that recruitment of exogenous DSBs to the synaptonemal complex, in conjunction with repair of exogenous DSBs via the homologous chromosome, contributes to homology recognition. In addition, the data suggest a role for HORMAD1 in DNA repair pathway choice in mouse meiocytes. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.
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Bacterial Artificial Chromosome Mutagenesis Using Recombineering
Directory of Open Access Journals (Sweden)
Kumaran Narayanan
2011-01-01
Full Text Available Gene expression from bacterial artificial chromosome (BAC clones has been demonstrated to facilitate physiologically relevant levels compared to viral and nonviral cDNA vectors. BACs are large enough to transfer intact genes in their native chromosomal setting together with flanking regulatory elements to provide all the signals for correct spatiotemporal gene expression. Until recently, the use of BACs for functional studies has been limited because their large size has inherently presented a major obstacle for introducing modifications using conventional genetic engineering strategies. The development of in vivo homologous recombination strategies based on recombineering in E. coli has helped resolve this problem by enabling facile engineering of high molecular weight BAC DNA without dependence on suitably placed restriction enzymes or cloning steps. These techniques have considerably expanded the possibilities for studying functional genetics using BACs in vitro and in vivo.
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Exceptional Complex Chromosomal Rearrangements in Three Generations
Directory of Open Access Journals (Sweden)
Hannie Kartapradja
2015-01-01
Full Text Available We report an exceptional complex chromosomal rearrangement (CCR found in three individuals in a family that involves 4 chromosomes with 5 breakpoints. The CCR was ascertained in a phenotypically abnormal newborn with additional chromosomal material on the short arm of chromosome 4. Maternal karyotyping indicated that the mother carried an apparently balanced CCR involving chromosomes 4, 6, 11, and 18. Maternal transmission of the derivative chromosome 4 resulted in partial trisomy for chromosomes 6q and 18q and a partial monosomy of chromosome 4p in the proband. Further family studies found that the maternal grandmother carried the same apparently balanced CCR as the proband’s mother, which was confirmed using the whole chromosome painting (WCP FISH. High resolution whole genome microarray analysis of DNA from the proband’s mother found no evidence for copy number imbalance in the vicinity of the CCR translocation breakpoints, or elsewhere in the genome, providing evidence that the mother’s and grandmother’s CCRs were balanced at a molecular level. This structural rearrangement can be categorized as an exceptional CCR due to its complexity and is a rare example of an exceptional CCR being transmitted in balanced and/or unbalanced form across three generations.
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Chung, Kyong-Sook; Weber, Jaime A; Hipp, Andrew L
2011-01-01
High intraspecific cytogenetic variation in the sedge genus Carex (Cyperaceae) is hypothesized to be due to the "diffuse" or non-localized centromeres, which facilitate chromosome fission and fusion. If chromosome number changes are dominated by fission and fusion, then chromosome evolution will result primarily in changes in the potential for recombination among populations. Chromosome duplications, on the other hand, entail consequent opportunities for divergent evolution of paralogs. In this study, we evaluate whether genome size and chromosome number covary within species. We used flow cytometry to estimate genome sizes in Carex scoparia var. scoparia, sampling 99 plants (23 populations) in the Chicago region, and we used meiotic chromosome observations to document chromosome numbers and chromosome pairing relations. Chromosome numbers range from 2n = 62 to 2n = 68, and nuclear DNA 1C content from 0.342 to 0.361 pg DNA. Regressions of DNA content on chromosome number are nonsignificant for data analyzed by individual or population, and a regression model that excludes slope is favored over a model in which chromosome number predicts genome size. Chromosome rearrangements within cytogenetically variable Carex species are more likely a consequence of fission and fusion than of duplication and deletion. Moreover, neither genome size nor chromosome number is spatially autocorrelated, which suggests the potential for rapid chromosome evolution by fission and fusion at a relatively fine geographic scale (<350 km). These findings have important implications for ecological restoration and speciation within the largest angiosperm genus of the temperate zone.
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Chromosome mapping by FISH to metaphase and interphase nuclei. Final report
Energy Technology Data Exchange (ETDEWEB)
Trask, B.
1997-08-01
The overall specific aims of this project were: (1) to determine the large-scale structure of interphase and metaphase chromosomes, in order to establish new capabilities for genome mapping by fluorescence in situ hybridization (FISH); (2) to detect chromosome abnormalities associated with genetic disease and map DNA sequences relative to them in order to facilitate the identification of new genes with disease-causing mutations; (3) to establish medium resolution physical maps of selected chromosomal regions using a combined metaphase and interphase mapping strategy and to corroborate physical and genetic maps and integrate these maps with the cytogenetic map; (4) to analyze the polymorphism and sequence evolution of subtelomeric regions of human chromosomes; (5) to establish a state-of-the-art FISH and image processing facility in the Department of Molecular Biotechnology, University of Washington, in order to map DNA sequences rapidly and accurately to benefit the Human Genome Project.
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Chromosomal instability determines taxane response
DEFF Research Database (Denmark)
Swanton, C.; Nicke, B.; Schuett, M.
2009-01-01
chromosomal instability (CIN). Silencing 22/50 of these genes, many of which are involved in DNA repair, caused cancer cell death, suggesting that these genes are involved in the survival of aneuploid cells. Overexpression of these "CIN-survival'' genes is associated with poor outcome in estrogen receptor......-positive breast cancer and occurs frequently in basal-like and Her2-positive cases. In diploid cells, but not in chromosomally unstable cells, paclitaxel causes repression of CIN-survival genes, followed by cell death. In the OV01 ovarian cancer clinical trial, a high level of CIN was associated with taxane...
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Brown and polar bear Y chromosomes reveal extensive male-biased gene flow within brother lineages.
Bidon, Tobias; Janke, Axel; Fain, Steven R; Eiken, Hans Geir; Hagen, Snorre B; Saarma, Urmas; Hallström, Björn M; Lecomte, Nicolas; Hailer, Frank
2014-06-01
Brown and polar bears have become prominent examples in phylogeography, but previous phylogeographic studies relied largely on maternally inherited mitochondrial DNA (mtDNA) or were geographically restricted. The male-specific Y chromosome, a natural counterpart to mtDNA, has remained underexplored. Although this paternally inherited chromosome is indispensable for comprehensive analyses of phylogeographic patterns, technical difficulties and low variability have hampered its application in most mammals. We developed 13 novel Y-chromosomal sequence and microsatellite markers from the polar bear genome and screened these in a broad geographic sample of 130 brown and polar bears. We also analyzed a 390-kb-long Y-chromosomal scaffold using sequencing data from published male ursine genomes. Y chromosome evidence support the emerging understanding that brown and polar bears started to diverge no later than the Middle Pleistocene. Contrary to mtDNA patterns, we found 1) brown and polar bears to be reciprocally monophyletic sister (or rather brother) lineages, without signals of introgression, 2) male-biased gene flow across continents and on phylogeographic time scales, and 3) male dispersal that links the Alaskan ABC islands population to mainland brown bears. Due to female philopatry, mtDNA provides a highly structured estimate of population differentiation, while male-biased gene flow is a homogenizing force for nuclear genetic variation. Our findings highlight the importance of analyzing both maternally and paternally inherited loci for a comprehensive view of phylogeographic history, and that mtDNA-based phylogeographic studies of many mammals should be reevaluated. Recent advances in sequencing technology render the analysis of Y-chromosomal variation feasible, even in nonmodel organisms. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e
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Directory of Open Access Journals (Sweden)
Joseph C Sanchez
2017-10-01
Full Text Available A form of dwarfism known as Meier-Gorlin syndrome (MGS is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5. These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45. The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C. We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways-DNA replication and ribosome biogenesis.
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Sanchez, Joseph C; Kwan, Elizabeth X; Pohl, Thomas J; Amemiya, Haley M; Raghuraman, M K; Brewer, Bonita J
2017-10-01
A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C). We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA) locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways-DNA replication and ribosome biogenesis.
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Energy Technology Data Exchange (ETDEWEB)
Natarajan, A.T.; van Zeeland, A.A.; Zwanenburg, T.S.
1983-01-01
The influence of inhibitors of poly(ADP-ribose) polymerase such as 3-aminobenzamide (3AB) and benzamide (B) on the spontaneously occurring as well as mutagen induced chromosomal aberrations, sister chromatid exchanges (SCEs) and point mutations has been studied. In addition, the influence of 3AB on DNA repair was measured following treatment with physical and chemical mutagens. Post treatment of X-irradiated mammalian cells with 3AB increases the frequencies of induced chromosomal aberrations by a factor of 2 to 3. 3AB, when present in the medium containing bromodeoxyuridine(BrdUrd) during two cell cycles, increases the frequencies of SCEs in Chinese hamster ovary cells (CHO) in a concentration dependent manner leading to about a 10-fold increase at 10 mM concentration. The extent of increase in the frequencies of SCEs due to 1 mM 3AB in several human cell lines has been studied, including those derived from patients suffering from genetic diseases such as ataxia telangiectasia (A-T), Fanconi's anemia (FA), and Huntington's chorea. None of these syndromes showed any increased response when compared to normal cells. 3AB, however, increased the frequencies of spontaneously occurring chromosomal aberrations in A-T and FA cells. 3AB does not influence the frequencies of SCEs induced by UV or mitomycin C (MMC) in CHO cells. However, it increases the frequencies of SCEs induced by ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS). Under the conditions in which 3AB increases the frequencies of spontaneously occurring as well as induced SCEs, it does not increase the frequencies of point mutations in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus. 3AB does not influence the amount of repair replication following dimethylsulphate (DMS) treatment of human fibroblasts, or UV irradiated human lymphocytes.
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Plasmid and chromosome segregation in prokaryotes
DEFF Research Database (Denmark)
Møller-Jensen, Jakob; Bugge Jensen, Rasmus; Gerdes, Kenn
2000-01-01
Recent major advances in the understanding of prokaryotic DNA segregation have been achieved by using fluorescence microscopy to visualize the localization of cellular components. Plasmids and bacterial chromosomes are partitioned in a highly dynamic fashion, suggesting the presence of a mitotic...
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Energy Technology Data Exchange (ETDEWEB)
Sztrolovics, R.; Grover, J.; Roughley, P.J. [McGill Univ., Montreal (Canada)] [and others
1994-10-01
This report describes the cloning of the 3{prime}-untranslated region of the human fibromodulin cDNA and its use to map the gene. For somatic cell hybrids, the generation of the PCR product was concordant with the presence of chromosome 1 and discordant with the presence of all other chromosomes, confirming that the fibromodulin gene is located within region q32 of chromosome 1. The physical mapping of genes is a critical step in the process of identifying which genes may be responsible for various inherited disorders. Specifically, the mapping of the fibromodulin gene now provides the information necessary to evaluate its potential role in genetic disorders of connective tissues. The analysis of previously reported diseases mapped to chromosome 1 reveals two genes located in the proximity of the fibromodulin locus. These are Usher syndrome type II, a recessive disorder characterized by hearing loss and retinitis pigmentosa, and Van der Woude syndrome, a dominant condition associated with abnormalities such as cleft lip and palate and hyperdontia. The genes for both of these disorders have been projected to be localized to 1q32 of a physical map that integrates available genetic linkage and physical data. However, it seems improbable that either of these disorders, exhibiting restricted tissue involvement, could be linked to the fibromodulin gene, given the wide tissue distribution of the encoded proteoglycan, although it remains possible that the relative importance of the quantity and function of the proteoglycan may avry between tissues. 11 refs., 1 fig.
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An enzyme-catalyzed multistep DNA refolding mechanism in hairpin telomere formation.
Directory of Open Access Journals (Sweden)
Ke Shi
Full Text Available Hairpin telomeres of bacterial linear chromosomes are generated by a DNA cutting-rejoining enzyme protelomerase. Protelomerase resolves a concatenated dimer of chromosomes as the last step of chromosome replication, converting a palindromic DNA sequence at the junctions between chromosomes into covalently closed hairpins. The mechanism by which protelomerase transforms a duplex DNA substrate into the hairpin telomeres remains largely unknown. We report here a series of crystal structures of the protelomerase TelA bound to DNA that represent distinct stages along the reaction pathway. The structures suggest that TelA converts a linear duplex substrate into hairpin turns via a transient strand-refolding intermediate that involves DNA-base flipping and wobble base-pairs. The extremely compact di-nucleotide hairpin structure of the product is fully stabilized by TelA prior to strand ligation, which drives the reaction to completion. The enzyme-catalyzed, multistep strand refolding is a novel mechanism in DNA rearrangement reactions.
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Progress towards construction of a total restriction fragment map of a human chromosome.
H. Vissing; F.G. Grosveld (Frank); E. Solomon; G. Moore; N. Lench; N. Shennan; R. Williamson
1987-01-01
textabstractWe present an approach to the construction of an overlapping restriction fragment map of a single human chromosome. A genomic cosmid library genome was constructed from a mouse-human hybrid cell line containing chromosome 17 as its only human genetic component. Cosmids containing human
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BRCA1-mediated repression of select X chromosome genes
Directory of Open Access Journals (Sweden)
Ropers H Hilger
2004-09-01
Full Text Available Abstract Recently BRCA1 has been implicated in the regulation of gene expression from the X chromosome. In this study the influence of BRCA1 on expression of X chromosome genes was investigated. Complementary DNA microarrays were used to compare the expression levels of X chromosome genes in 18 BRCA1-associated ovarian cancers to those of the 13 "BRCA1-like" and 14 "BRCA2-like" sporadic tumors (as defined by previously reported expression profiling. Significance was determined using parametric statistics with P
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Directory of Open Access Journals (Sweden)
De Marzo Angelo M
2011-06-01
Full Text Available Abstract Background DNA methylation has been linked to genome regulation and dysregulation in health and disease respectively, and methods for characterizing genomic DNA methylation patterns are rapidly emerging. We have developed/refined methods for enrichment of methylated genomic fragments using the methyl-binding domain of the human MBD2 protein (MBD2-MBD followed by analysis with high-density tiling microarrays. This MBD-chip approach was used to characterize DNA methylation patterns across all non-repetitive sequences of human chromosomes 21 and 22 at high-resolution in normal and malignant prostate cells. Results Examining this data using computational methods that were designed specifically for DNA methylation tiling array data revealed widespread methylation of both gene promoter and non-promoter regions in cancer and normal cells. In addition to identifying several novel cancer hypermethylated 5' gene upstream regions that mediated epigenetic gene silencing, we also found several hypermethylated 3' gene downstream, intragenic and intergenic regions. The hypermethylated intragenic regions were highly enriched for overlap with intron-exon boundaries, suggesting a possible role in regulation of alternative transcriptional start sites, exon usage and/or splicing. The hypermethylated intergenic regions showed significant enrichment for conservation across vertebrate species. A sampling of these newly identified promoter (ADAMTS1 and SCARF2 genes and non-promoter (downstream or within DSCR9, C21orf57 and HLCS genes hypermethylated regions were effective in distinguishing malignant from normal prostate tissues and/or cell lines. Conclusions Comparison of chromosome-wide DNA methylation patterns in normal and malignant prostate cells revealed significant methylation of gene-proximal and conserved intergenic sequences. Such analyses can be easily extended for genome-wide methylation analysis in health and disease.
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Metaphase chromosome and nucleoid differences between CHO-K1 and its radiosensitive derivative xrs-5
International Nuclear Information System (INIS)
Schwartz, J.L.; Stephens, J.; Vaughan, A.T.M.
1992-05-01
The Chinese hamster ovary (CHO) cell line xrs-5 is a radiation-sensitive mutant isolated from CHO-K1 cells. The radiation sensitivity is associated with a defect in DNA double-strand break rejoining. Chromatin structure also appears altered in xrs-5 cells compared to the parental CHO-K1 cells. Metaphase chromosomes from xrs-5 are more condensed in appearance than CHO-K1 chromosomes. The overcondensed look is not the result of colcemid sensitivity. Electron microscopy studies suggest that xrs-5 metaphase chromosomes have larger loops of chromatin extending out from the chromosome core. There are also differences between CHO-K1 and xrs-5 cells in the size and fluorescence pattern of ethidium bromide-stained nucleoid preparations. These results suggest that there is a fundamental difference between CHO-K1 and xrs-5 in either the organization of the supercoiled loops of DNA attached to the nuclear matrix or in the nature of the proteins that attach the DNA to the matrix. These alterations in chromosome structure may underlie, in part, the radiation sensitivity of xrs-5 cells
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Metaphase chromosome and nucleoid differences between CHO-K1 and its radiosensitive derivative xrs-5
International Nuclear Information System (INIS)
Schwartz, J.L.; Stephens, J.; Vaughan, A.T.M.
1993-01-01
The Chinese hamster ovary (CHO) cell line xrs-5 is a radiation-sensitive mutant isolated from CHO-K1 cells. The radiosensitivity is associated with a defect in DNA double-strand break rejoining. Chromatin structure also appears altered in xrs-5 cells compared with the parental CHO-K1 cells. Metaphase chromosomes form xrs-5 are more condensed in appearance than CHO-K1 chromosomes. The overcondensed look is not the result of colcemid sensitivity. Electron microscopy studies suggest that xrs-5 metaphase chromosomes have larger loops of chromatin extending out from the chromosome core. There are also differences between CHO-K1 and xrs-5 cells in the size and fluorescence pattern of ethidium bromide-stained nucleoid preparations. These results suggest that there is a fundamental difference between CHO-K1 and xrs-5 in either the organization of the supercoiled loops of DNA attached to the nuclear matrix or in the nature of the proteins that attach the DNA to the matrix. These alterations in chromosome structure may underlie, in part the radiation sensitivity of xrs-5 cells. (Author)
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Hakumai, Yuichi; Shimomoto, Kouko; Ashiuchi, Makoto
2015-05-15
Extra-chromosomal DNA maintenance (EDM) as an important process in the propagation and genetic engineering of microbes. Bacillus subtilis EdmS (formerly PgsE), a protein comprising 55 amino acids, is a mediator of the EDM process. In this study, the effect of mutation of global regulators on B. subtilis EDM was examined. Mutation of the swrA gene abolished EdmS-mediated EDM. It is known that swrA predominantly regulates expression of the fla/che operon in B. subtilis. We therefore performed EDM analysis using fla/che-deletion mutants and identified an EDM-mediated EDM cooperator in the flgB-fliL region. Further genetic investigation identified the flagellation factor FliF is a crucial EDM cooperator. To our knowledge, this is the first observation of the moonlighting function of FliF in DNA maintenance. Copyright © 2015 Elsevier Inc. All rights reserved.
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Directory of Open Access Journals (Sweden)
Octavio M Palacios-Gimenez
Full Text Available A large percentage of eukaryotic genomes consist of repetitive DNA that plays an important role in the organization, size and evolution. In the case of crickets, chromosomal variability has been found using classical cytogenetics, but almost no information concerning the organization of their repetitive DNAs is available. To better understand the chromosomal organization and diversification of repetitive DNAs in crickets, we studied the chromosomes of two Gryllidae species with highly divergent karyotypes, i.e., 2n(♂ = 29,X0 (Gryllus assimilis and 2n = 9, neo-X1X2Y (Eneoptera surinamensis. The analyses were performed using classical cytogenetic techniques, repetitive DNA mapping and genome-size estimation. Conserved characteristics were observed, such as the occurrence of a small number of clusters of rDNAs and U snDNAs, in contrast to the multiple clusters/dispersal of the H3 histone genes. The positions of U2 snDNA and 18S rDNA are also conserved, being intermingled within the largest autosome. The distribution and base-pair composition of the heterochromatin and repetitive DNA pools of these organisms differed, suggesting reorganization. Although the microsatellite arrays had a similar distribution pattern, being dispersed along entire chromosomes, as has been observed in some grasshopper species, a band-like pattern was also observed in the E. surinamensis chromosomes, putatively due to their amplification and clustering. In addition to these differences, the genome of E. surinamensis is approximately 2.5 times larger than that of G. assimilis, which we hypothesize is due to the amplification of repetitive DNAs. Finally, we discuss the possible involvement of repetitive DNAs in the differentiation of the neo-sex chromosomes of E. surinamensis, as has been reported in other eukaryotic groups. This study provided an opportunity to explore the evolutionary dynamics of repetitive DNAs in two non-model species and will contribute to the
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Grudinina, N A; Sasina, L K; Noniashvili, E M; Neronova, E G; Pavlinova, L I; Suchkova, I O; Sofronov, G A; Patkin, E L
2015-01-01
Qualitative and quantitate analysis of DNA methylation in situ at the level of cells, chromosomes and chromosomal domains is extremely important for the diagnosis and treatment of various diseases, the study of ageing and the consequences of environmental impacts. An important question arises, whether the revealed in situ methylation pattern reflects DNA methylation per se and (or) availability of the DNA for antibodies, which in turn depends on the peculiarities of chromatin structure and chromosome condensation. These events can lead to an incorrect evaluation of the actual pattern of DNA methylation. To avoid this shortcoming as far as possible, we have modified the most widely used method of revealing 5-methylcytosine in situ with monoclonal antibodies. Here we have shown that the detection of DNA methylation staining of chromosomes including C-heterochromatin, chromosomal arms and sister chromatids is drastically dependent on pretreatment of chromosomal preparations for immunocytochemical study using fluorescent antibodies. Using undifferentiated stem cells of mouse embryonal carcinoma line F9, it has been found that change in preparations storage results in a sharp fluorescence decrease up to complete disappearance of the signal in centromeric heterochromatin. With the help of the method described in the work, we have first revealed the asymmetry of sister chromatids methylation in metaphase chromosomes of F9 cell and lymphocytes of human periphery blood. This may lead to asymmetry of transcriptional signature of daughter cells after division. The proposed here modification of 5-methylcytosine detection in situ provides a more complete characterization of methylation of chromosomes and chromosomal domains, compared to previously published methods.
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Yong, Lee C; Petersen, Martin R; Sigurdson, Alice J; Sampson, Laura A; Ward, Elizabeth M
2009-11-01
Dietary antioxidants may protect against DNA damage induced by endogenous and exogenous sources, including ionizing radiation (IR), but data from IR-exposed human populations are limited. The objective was to examine the association between the frequency of chromosome translocations, as a biomarker of cumulative DNA damage, and intakes of vitamins C and E and carotenoids in 82 male airline pilots. Dietary intakes were estimated by using a self-administered semiquantitative food-frequency questionnaire. Translocations were scored by using fluorescence in situ hybridization with whole chromosome paints. Negative binomial regression was used to estimate rate ratios and 95% CIs, adjusted for potential confounders. Significant and inverse associations were observed between translocation frequency and intakes of vitamin C, beta-carotene, beta-cryptoxanthin, and lutein-zeaxanthin from food (P food; total vitamin C or E from food and supplements; or vitamin C or E or multivitamin supplements. The adjusted rate ratios (95% CI) for > or =median compared with or =median compared with food: 0.27 (0.14, 0.55). High combined intakes of vitamins C and E, beta-carotene, beta-cryptoxanthin, and lutein-zeaxanthin from food, or a diet high in their food sources, may protect against cumulative DNA damage in IR-exposed persons.
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Trapping and breaking of in vivo nicked DNA during pulsed-field gel electrophoresis
Khan, Sharik R.; Kuzminov, Andrei
2013-01-01
Pulsed field gel electrophoresis (PFGE) offers a high-resolution approach to quantify chromosomal fragmentation in bacteria, measured as percent of chromosomal DNA entering the gel. The degree of separation in PFG depends upon the size of DNA, as well as various conditions of electrophoresis, such as electric field strength (FS), time of electrophoresis, switch time and buffer composition. Here we describe a new parameter, the structural integrity of the sample DNA itself, that influences its migration through PFGs. We show that sub-chromosomal fragments containing both spontaneous and DNA damage-induced nicks are prone to breakage during PFGE. Such breakage at single strand interruptions results in artefactual decrease in molecular weight of linear DNA making accurate determination of the number of double strand breaks difficult. While breakage of nicked sub-chromosomal fragments is FS-independent, some high molecular weight sub-chromosomal fragments are also trapped within wells under the standard PFGE conditions. This trapping can be minimized by lowering the field strength and increasing the time of electrophoresis. We discuss how breakage of nicked DNA may be mechanistically linked to trapping. Our results suggest how to optimize conditions for PFGE when quantifying chromosomal fragmentation induced by DNA damage. PMID:23770235
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An Estimate of the Total DNA in the Biosphere.
Landenmark, Hanna K E; Forgan, Duncan H; Cockell, Charles S
2015-06-01
Modern whole-organism genome analysis, in combination with biomass estimates, allows us to estimate a lower bound on the total information content in the biosphere: 5.3 × 1031 (±3.6 × 1031) megabases (Mb) of DNA. Given conservative estimates regarding DNA transcription rates, this information content suggests biosphere processing speeds exceeding yottaNOPS values (1024 Nucleotide Operations Per Second). Although prokaryotes evolved at least 3 billion years before plants and animals, we find that the information content of prokaryotes is similar to plants and animals at the present day. This information-based approach offers a new way to quantify anthropogenic and natural processes in the biosphere and its information diversity over time.
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Preparation and Fluorescent Analysis of Plant Metaphase Chromosomes.
Schwarzacher, Trude
2016-01-01
Good preparations are essential for informative analysis of both somatic and meiotic chromosomes, cytogenetics, and cell divisions. Fluorescent chromosome staining allows even small chromosomes to be visualized and counted, showing their morphology. Aneuploidies and polyploidies can be established for species, populations, or individuals while changes occurring in breeding lines during hybridization or tissue culture and transformation protocols can be assessed. The process of division can be followed during mitosis and meiosis including pairing and chiasma distribution, as well as DNA organization and structure during the evolution of chromosomes can be studied. This chapter presents protocols for pretreatment and fixation of material, including tips of how to grow plants to get good and healthy meristem with many divisions. The chromosome preparation technique is described using proteolytic enzymes, but acids can be used instead. Chromosome slide preparations are suitable for fluorochrome staining for fast screening (described in the chapter) or fluorescent in situ hybridization (see Schwarzacher and Heslop-Harrison, In situ hybridization. BIOS Scientific Publishers, Oxford, 2000).
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Paliwal, Anupam; Temkin, Alexis M; Kerkel, Kristi; Yale, Alexander; Yotova, Iveta; Drost, Natalia; Lax, Simon; Nhan-Chang, Chia-Ling; Powell, Charles; Borczuk, Alain; Aviv, Abraham; Wapner, Ronald; Chen, Xiaowei; Nagy, Peter L; Schork, Nicholas; Do, Catherine; Torkamani, Ali; Tycko, Benjamin
2013-08-01
Allele-specific DNA methylation (ASM) is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons), one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated) while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq) in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs), each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS) peaks near CTCF binding sites with ASM.
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Directory of Open Access Journals (Sweden)
Anupam Paliwal
2013-08-01
Full Text Available Allele-specific DNA methylation (ASM is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons, one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs, each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS peaks near CTCF binding sites with ASM.
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Chromosomal Arrangement of Phosphorelay Genes Couples Sporulation and DNA Replication.
Narula, Jatin; Kuchina, Anna; Lee, Dong-Yeon D; Fujita, Masaya; Süel, Gürol M; Igoshin, Oleg A
2015-07-16
Genes encoding proteins in a common regulatory network are frequently located close to one another on the chromosome to facilitate co-regulation or couple gene expression to growth rate. Contrasting with these observations, here, we demonstrate a functional role for the arrangement of Bacillus subtilis sporulation network genes on opposite sides of the chromosome. We show that the arrangement of two sporulation network genes, one located close to the origin and the other close to the terminus, leads to a transient gene dosage imbalance during chromosome replication. This imbalance is detected by the sporulation network to produce cell-cycle coordinated pulses of the sporulation master regulator Spo0A∼P. This pulsed response allows cells to decide between sporulation and continued vegetative growth during each cell cycle spent in starvation. The simplicity of this coordination mechanism suggests that it may be widely applicable in a variety of gene regulatory and stress-response settings. VIDEO ABSTRACT. Copyright © 2015 Elsevier Inc. All rights reserved.
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DNA damage and chromosome aberration induced by heavy-ion beams
International Nuclear Information System (INIS)
Takakura, Kahoru; Funada, Aya; Aoki, Mizuho; Furusawa, Yoshiya
2003-01-01
The aim of this study is to clarify the relation between cell death and chromosomal aberration in cultured human cells (human salivary gland (HSG) tumor cells and GM05389 human normal fibroblasts) irradiated with heavy ion beams on the basis of linear energy transfer (LET) values. The LET dependences of cell death were observed for the both cells by the method of colony assay. The LET dependences of the chromosomal aberrations, breaks and gaps, isochromatid breaks and exchanges were also observed for the both cells using the premature chromosome condensation (PCC) method. From these results it is suggested that exchange formation is essential for the cell death caused by heavy ion beam irradiation. It is suspected that the densely ionizing track structure of hight LET heavy ions inhibits the effective repair in the chromatid breaks and isochromatid breaks and finally induce much exchange in the cells, which should be essential cause of cell death. (author)
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Replication stress activates DNA repair synthesis in mitosis
DEFF Research Database (Denmark)
Minocherhomji, Sheroy; Ying, Songmin; Bjerregaard, Victoria A
2015-01-01
Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis. Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs). CFSs are difficult-to-replicate loci that manifest as gaps...... into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early...... mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN(+)) and replicative stress, we suggest...
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Genome landscape and evolutionary plasticity of chromosomes in malaria mosquitoes.
Directory of Open Access Journals (Sweden)
Ai Xia
2010-05-01
Full Text Available Nonrandom distribution of rearrangements is a common feature of eukaryotic chromosomes that is not well understood in terms of genome organization and evolution. In the major African malaria vector Anopheles gambiae, polymorphic inversions are highly nonuniformly distributed among five chromosomal arms and are associated with epidemiologically important adaptations. However, it is not clear whether the genomic content of the chromosomal arms is associated with inversion polymorphism and fixation rates.To better understand the evolutionary dynamics of chromosomal inversions, we created a physical map for an Asian malaria mosquito, Anopheles stephensi, and compared it with the genome of An. gambiae. We also developed and deployed novel Bayesian statistical models to analyze genome landscapes in individual chromosomal arms An. gambiae. Here, we demonstrate that, despite the paucity of inversion polymorphisms on the X chromosome, this chromosome has the fastest rate of inversion fixation and the highest density of transposable elements, simple DNA repeats, and GC content. The highly polymorphic and rapidly evolving autosomal 2R arm had overrepresentation of genes involved in cellular response to stress supporting the role of natural selection in maintaining adaptive polymorphic inversions. In addition, the 2R arm had the highest density of regions involved in segmental duplications that clustered in the breakpoint-rich zone of the arm. In contrast, the slower evolving 2L, 3R, and 3L, arms were enriched with matrix-attachment regions that potentially contribute to chromosome stability in the cell nucleus.These results highlight fundamental differences in evolutionary dynamics of the sex chromosome and autosomes and revealed the strong association between characteristics of the genome landscape and rates of chromosomal evolution. We conclude that a unique combination of various classes of genes and repetitive DNA in each arm, rather than a single type
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TRF2 Protein Interacts with Core Histones to Stabilize Chromosome Ends.
Konishi, Akimitsu; Izumi, Takashi; Shimizu, Shigeomi
2016-09-23
Mammalian chromosome ends are protected by a specialized nucleoprotein complex called telomeres. Both shelterin, a telomere-specific multi-protein complex, and higher order telomeric chromatin structures combine to stabilize the chromosome ends. Here, we showed that TRF2, a component of shelterin, binds to core histones to protect chromosome ends from inappropriate DNA damage response and loss of telomeric DNA. The N-terminal Gly/Arg-rich domain (GAR domain) of TRF2 directly binds to the globular domain of core histones. The conserved arginine residues in the GAR domain of TRF2 are required for this interaction. A TRF2 mutant with these arginine residues substituted by alanine lost the ability to protect telomeres and induced rapid telomere shortening caused by the cleavage of a loop structure of the telomeric chromatin. These findings showed a previously unnoticed interaction between the shelterin complex and nucleosomal histones to stabilize the chromosome ends. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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TRF2 Protein Interacts with Core Histones to Stabilize Chromosome Ends*
Izumi, Takashi; Shimizu, Shigeomi
2016-01-01
Mammalian chromosome ends are protected by a specialized nucleoprotein complex called telomeres. Both shelterin, a telomere-specific multi-protein complex, and higher order telomeric chromatin structures combine to stabilize the chromosome ends. Here, we showed that TRF2, a component of shelterin, binds to core histones to protect chromosome ends from inappropriate DNA damage response and loss of telomeric DNA. The N-terminal Gly/Arg-rich domain (GAR domain) of TRF2 directly binds to the globular domain of core histones. The conserved arginine residues in the GAR domain of TRF2 are required for this interaction. A TRF2 mutant with these arginine residues substituted by alanine lost the ability to protect telomeres and induced rapid telomere shortening caused by the cleavage of a loop structure of the telomeric chromatin. These findings showed a previously unnoticed interaction between the shelterin complex and nucleosomal histones to stabilize the chromosome ends. PMID:27514743
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International Nuclear Information System (INIS)
Lopez Hidalgo, Juana Ines
2000-01-01
The characteristic of ionizing radiation suggests that induced chromosomal damage in the form of translocations would appear to be randomly distributed. However, the outcome of tests performed in vitro and in vivo (irradiated individuals) are contradictories. The most translocation-related chromosomes, as far as some studies reveal on one hand, appear to be less involved in accordance with others. These data, together with those related to molecular mechanisms involved in translocations production suggest that in G 0 -irradiated cells, the frequency and distribution of this kind of chromosomal rearrangement, does not take place at random. They seem to be affected by in-nucleus chromosome distribution, by each chromosome's DNA length and functional features, by the efficiency of DNA repair mechanisms, and by inter individual differences. The objective of this study was to establish the frequency pattern of each human chromosome involved in radio-induced translocations, as well as to analyze the importance the chromosome length, the activity of DNA polymerase- dependant repair mechanisms, and inter individual differences within the scope of such distribution. To achieve the goals, peripheral blood lymphocytes from healthy donors were irradiated in presence and absence of 2'-3' dideoxithimidine (ddThd), a Β - DNA polymerase inhibitor, which takes part in the base repair mechanism (B E R). The results showed that: The presence of ddThd during the irradiation increase the basal frequency of radioinduced translocations in 60 %. This result suggests that ddThd repair synthesis inhibition can be in itself a valid methodology for radiation-induced bases damage assessment, damage which if not BER-repaired may result in translocation-leading double strand breaks. A statistically significant correlation between translocation frequency and chromosome length, in terms of percentage of genome, has been noticed both in (basal) irradiation and in irradiation with ddThd inhibitor
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DEFF Research Database (Denmark)
Knudsen, Lisbeth E.; Norppa, H; Gamborg, M O
1999-01-01
We have studied the influence of individual susceptibility factors on the genotoxic effects of urban air pollution in 106 nonsmoking bus drivers and 101 postal workers in the Copenhagen metropolitan area. We used the frequency of chromosomal aberrations in peripheral blood lymphocytes as a biomar......We have studied the influence of individual susceptibility factors on the genotoxic effects of urban air pollution in 106 nonsmoking bus drivers and 101 postal workers in the Copenhagen metropolitan area. We used the frequency of chromosomal aberrations in peripheral blood lymphocytes...... that long-term exposure to urban air pollution (with traffic as the main contributor) induces chromosome damage in human somatic cells. Low DNA repair capacity and GSTM1 and NAT2 variants associated with reduced detoxification ability increase susceptibility to such damage. The effect of the GSTM1 genotype......, which was observed only in the bus drivers, appears to be associated with air pollution, whereas the NAT2 genotype effect, which affected all subjects, may influence the individual response to some other common exposure or the baseline level of chromosomal aberrations....
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Directory of Open Access Journals (Sweden)
Juliano S. Cabral
2006-01-01
Full Text Available We investigated four orchids of the genus Maxillaria (M. discolor, M. acicularis, M. notylioglossa and M. desvauxiana in regard to the position of heterochromatin blocks as revealed using chromomycin A3 (CMA and 4'-6-diamidino-2-phenylindole (DAPI fluorochrome staining and 5S and 45S rDNA sites using fluorescence in situ hybridization (FISH. The species showed differences in chromosome number and a diversified pattern of CMA+ and DAPI+ bands, including heteromorphism for CMA+ bands. The 5S and 45S rDNA sites also varied in number and most of them were co-localized with CMA+ bands. The relationship between 5S rDNA sites and CMA+ bands was more evident in M. notylioglossa, in which the brighter CMA+ bands were associated with large 5S rDNA sites. However, not all 5S and 45S rDNA sites were co-localized with CMA+ bands, probably due to technical constraints. We compare these results to banding data from other species and suggest that not all blocks of tandemly repetitive sequences, such as 5S rDNA sites, can be observed as heterochromatin blocks.
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Diniz, Débora; Moreira-Filho, Orlando; Bertollo, Luiz Antonio Carlos
2008-05-01
Chromosomes of Triportheus nematurus, a fish species from family Characidae, were analyzed in order to establish the conventional karyotype, location of C-band positive heterochromatin, Ag-NORs, GC- and AT-rich sites, and mapping of 18S and 5S rDNA with fluorescence in situ hybridization (FISH). The diploid number found was 2n = 52 chromosomes in both males and females. However, the females presented a pair of differentiated heteromorphic chromosomes, characterizing a ZZ/ZW sex chromosome system. The Z chromosome was metacentric and the largest one in the karyotype, bearing C-positive heterochromatin at pericentromeric and telomeric regions. The W chromosome was middle-sized submetacentric, appearing mostly heterochromatic after C-banding and presenting heterogeneous heterochromatin composed of GC- and AT-rich regions revealed by fluorochrome staining. Ag-NORs were also GC-rich and surrounded by heterochromatic regions, being located at the secondary constriction on the short arms of the second chromosome pair, in agreement with 18S rDNA sites detected with FISH. The 18S and 5S rDNA were aligned in tandem, representing an uncommon situation in fishes. The results obtained reinforce the basal condition of the ZZ/ZW sex system in the genus Triportheus, probably arisen prior to speciation in the group.
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Identification of Pneumocystis carinii chromosomes and mapping of five genes
DEFF Research Database (Denmark)
Lundgren, B; Cotton, R; Lundgren, J D
1990-01-01
Pulsed field gel electrophoresis was used to identify the chromosome-size DNA of Pneumocystis carinii, a major pathogen of immunocompromised patients. Thirteen chromosomes of rodent Pneumocystis carinii, ranging in size from 300 to 700 kilobases (kb), were identified. The minimum genome size for P....... carinii, estimated on the basis of the sizes of chromosomes, is 7,000 kb. Genetic heterogeneity among different P. carinii isolates was documented by demonstration of chromosomal size variability. By hybridization studies, the genes for topoisomerase I, dihydrofolate reductase, rRNA, actin......, and thymidylate synthase were mapped to single chromosomes of approximately 650, 590, 550, 460, and 350 kb, respectively. Hybridization studies further confirmed the genetic heterogeneity of P. carinii....
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The significance of chromosome deletions in atomic-bomb survivors
International Nuclear Information System (INIS)
Tanaka, Kimio; Shigeta, Chiharu; Oguma, Nobuo; Kamada, Nanao; Deng, Z.; Niimi, Masanobu; Aisaka, Tadaichi.
1986-01-01
In 39 A-bomb survivors 40 years after exposure at ≤ 1,000 m from ground zero, the frequency and features of chromosome deletions in peripheral lymphocytes were examined using a differential staining technique. Simultaneously, in vitro irradiation experiment with Cf-252 was made to infer chromosome aberrations occuring immediately after exposure. Californium-252 with 100 rad induced dicentric and ring chromosomes in 40 % of the cells and acentric fragments in 44 %. Among the A-bomb survivors, chromosome aberrations were observed in 651 (21 %) of the total 3,136 cells. There were 146 cells with deletions (22 % of abnormal cells; 5 % of the total cells), and 10 cells with acentric fragment (0.3 % of the total cells). The figure for deletions was far higher than that reported in the literature. A large number of deletions were seen in chromosomes no.4, no.21, and no.22, and a few deletions in chromosomes no.7 and no.20. Significance of chromosome deletions is discussed. (Namekawa, K.)
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Typing of Y chromosome SNPs with multiplex PCR methods
DEFF Research Database (Denmark)
Sanchez Sanchez, Juan Jose; Børsting, Claus; Morling, Niels
2005-01-01
We describe a method for the simultaneous typing of Y-chromosome single nucleotide polymorphism (SNP) markers by means of multiplex polymerase chain reaction (PCR) strategies that allow the detection of 35 Y chromosome SNPs on 25 amplicons from 100 to 200 pg of chromosomal deoxyribonucleic acid...... factors for the creation of larger SNP typing PCR multiplexes include careful selection of primers for the primary amplification and the SBE reaction, use of DNA primers with homogenous composition, and balancing the primer concentrations for both the amplification and the SBE reactions....
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Sun, Jiying; Shi, Lin; Kinomura, Aiko; Fukuto, Atsuhiko; Horikoshi, Yasunori; Oma, Yukako; Harata, Masahiko; Ikura, Masae; Ikura, Tsuyoshi; Kanaar, Roland; Tashiro, Satoshi
2018-05-08
Chromosomal translocations are hallmarks of various types of cancers and leukemias. However, the molecular mechanisms of chromosome translocations remain largely unknown. The ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, facilitates DNA repair to prevent chromosome abnormalities. Previously, we showed that ATM deficiency led to the 11q23 chromosome translocation, the most frequent chromosome abnormalities in secondary leukemia. Here, we show that ARP8, a subunit of the INO80 chromatin remodeling complex, is phosphorylated after etoposide treatment. The etoposide-induced phosphorylation of ARP8 is regulated by ATM and ATR, and attenuates its interaction with INO80. The ATM-regulated phosphorylation of ARP8 reduces the excessive loading of INO80 and RAD51 onto the breakpoint cluster region. These findings suggest that the phosphorylation of ARP8, regulated by ATM, plays an important role in maintaining the fidelity of DNA repair to prevent the etoposide-induced 11q23 abnormalities. © 2018, Sun et al.
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Rudd, M. Katharine; Mays, Robert W.; Schwartz, Stuart; Willard, Huntington F.
2003-01-01
Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fu...
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Suga, Koushirou; Mark Welch, David B; Tanaka, Yukari; Sakakura, Yoshitaka; Hagiwara, Atsushi
2008-06-01
The monogonont rotifer Brachionus plicatilis is an emerging model system for a diverse array of questions in limnological ecosystem dynamics, the evolution of sexual recombination, cryptic speciation, and the phylogeny of basal metazoans. We sequenced the complete mitochondrial genome of B. plicatilis sensu strictu NH1L and found that it is composed of 2 circular chromosomes, designated mtDNA-I (11,153 bp) and mtDNA-II (12,672 bp). Hybridization to DNA isolated from mitochondria demonstrated that mtDNA-I is present at 4 times the copy number of mtDNA-II. The only nucleotide similarity between the 2 chromosomes is a 4.9-kbp region of 99.5% identity including a transfer RNA (tRNA) gene and an extensive noncoding region that contains putative D-loop and control sequence. The mtDNA-I chromosome encodes 4 proteins (ATP6, COB, NAD1, and NAD2), 13 tRNAs, and the large and small subunit ribosomal RNAs; mtDNA-II encodes 8 proteins (COX1-3, NAD3-6, and NAD4L) and 9 tRNAs. Gene order is not conserved between B. plicatilis and its closest relative with a sequenced mitochondrial genome, the acanthocephalan Leptorhynchoides thecatus, or other sequenced mitochondrial genomes. Polymerase chain reaction assays and Southern hybridization to DNA from 18 strains of Brachionus suggest that the 2-chromosome structure has been stable for millions of years. The novel organization of the B. plicatilis mitochondrial genome into 2 nearly equal chromosomes of 4-fold different copy number may provide insight into the evolution of metazoan mitochondria and the phylogenetics of rotifers and other basal animal phyla.
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Identification of the facultative heterochromatic X chromosome in females of 25 rodent species.
Kanda, N; Yosida, T H
1979-01-01
Treatment of the chromosomes of 25 rodent species with a 50 degrees C hypotonic solution and Giemsa staining permitted identification of the heterochromatic X chromosome in 24 species. With this technique, the facultative of the heterochromatic X chromosome or the facultative portion of large, composite-type X chromosoms is stained darker than the other chromosomes, allowing it to be distinguished from the homologous euchromatic X chromosome in female metaphase cells. Intense staining of the single X chromosome was not observed in male metaphase cells. It is suggested that this differential staining of one of the two X chromosomes might be due to qualitative differences in chromosomal proteins rather than to differences in the degree of chromosomal condensation or in DNA base sequence.
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Directory of Open Access Journals (Sweden)
Bryan Utter
Full Text Available In Staphylococcus aureus, the disease impact of chromosomally integrated prophages on virulence is well described. However, the existence of extra-chromosomal prophages, both plasmidial and episomal, remains obscure. Despite the recent explosion in bacterial and bacteriophage genomic sequencing, studies have failed to specifically focus on extra-chromosomal elements. We selectively enriched and sequenced extra-chromosomal DNA from S. aureus isolates using Roche-454 technology and uncovered evidence for the widespread distribution of multiple extra-chromosomal prophages (ExPΦs throughout both antibiotic-sensitive and -resistant strains. We completely sequenced one such element comprised of a 43.8 kbp, circular ExPΦ (designated ФBU01 from a vancomycin-intermediate S. aureus (VISA strain. Assembly and annotation of ФBU01 revealed a number of putative virulence determinants encoded within a bacteriophage immune evasion cluster (IEC. Our identification of several potential ExPΦs and mobile genetic elements (MGEs also revealed numerous putative virulence factors and antibiotic resistance genes. We describe here a previously unidentified level of genetic diversity of stealth extra-chromosomal elements in S. aureus, including phages with a larger presence outside the chromosome that likely play a prominent role in pathogenesis and strain diversity driven by horizontal gene transfer (HGT.
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Boschman, G. A.; Rens, W.; Manders, E.; van Oven, C.; Barendsen, G. W.; Aten, J. A.
1990-01-01
Using slit-scan flow cytometry, the shape of human metaphase chromosomes, as expressed in their centromeric index (CI), and the DNA content of the chromosomes have been used as parameters in bivariate flow karyotyping. The resolution of the DNA vs CI flow karyogram of the larger chromosomes up to
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DNA Repair Decline During Mouse Spermiogenesis Results in the Accumulation of Heritable DNA Damage
Energy Technology Data Exchange (ETDEWEB)
Marchetti, Francesco; Marchetti, Francesco; Wyrobek, Andrew J.
2007-12-01
The post-meiotic phase of mouse spermatogenesis (spermiogenesis) is very sensitive to the genomic effects of environmental mutagens because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. We hypothesized that repeated exposures to mutagens during this repair-deficient phase result in the accumulation of heritable genomic damage in mouse sperm that leads to chromosomal aberrations in zygotes after fertilization. We used a combination of single or fractionated exposures to diepoxybutane (DEB), a component of tobacco smoke, to investigate how differential DNA repair efficiencies during the three weeks of spermiogenesis affected the accumulation of DEB-induced heritable damage in early spermatids (21-15 days before fertilization, dbf), late spermatids (14-8 dbf) and sperm (7-1 dbf). Analysis of chromosomal aberrations in zygotic metaphases using PAINT/DAPI showed that late spermatids and sperm are unable to repair DEB-induced DNA damage as demonstrated by significant increases (P<0.001) in the frequencies of zygotes with chromosomal aberrations. Comparisons between single and fractionated exposures suggested that the DNA repair-deficient window during late spermiogenesis may be less than two weeks in the mouse and that during this repair-deficient window there is accumulation of DNA damage in sperm. Finally, the dose-response study in sperm indicated a linear response for both single and repeated exposures. These findings show that the differential DNA repair capacity of post-meioitic male germ cells has a major impact on the risk of paternally transmitted heritable damage and suggest that chronic exposures that may occur in the weeks prior to fertilization because of occupational or lifestyle factors (i.e, smoking) can lead to an accumulation of genetic damage in sperm and result in heritable chromosomal aberrations of paternal origin.
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Chiatante, Giorgia; Giannuzzi, Giuliana; Calabrese, Francesco Maria; Eichler, Evan E; Ventura, Mario
2017-07-01
Dicentric chromosomes are products of genomic rearrangements that place two centromeres on the same chromosome. Due to the presence of two primary constrictions, they are inherently unstable and overcome their instability by epigenetically inactivating and/or deleting one of the two centromeres, thus resulting in functionally monocentric chromosomes that segregate normally during cell division. Our understanding to date of dicentric chromosome formation, behavior and fate has been largely inferred from observational studies in plants and humans as well as artificially produced de novo dicentrics in yeast and in human cells. We investigate the most recent product of a chromosome fusion event fixed in the human lineage, human chromosome 2, whose stability was acquired by the suppression of one centromere, resulting in a unique difference in chromosome number between humans (46 chromosomes) and our most closely related ape relatives (48 chromosomes). Using molecular cytogenetics, sequencing, and comparative sequence data, we deeply characterize the relicts of the chromosome 2q ancestral centromere and its flanking regions, gaining insight into the ancestral organization that can be easily broadened to all acrocentric chromosome centromeres. Moreover, our analyses offered the opportunity to trace the evolutionary history of rDNA and satellite III sequences among great apes, thus suggesting a new hypothesis for the preferential inactivation of some human centromeres, including IIq. Our results suggest two possible centromere inactivation models to explain the evolutionarily stabilization of human chromosome 2 over the last 5-6 million years. Our results strongly favor centromere excision through a one-step process. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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A mechanism of gene amplification driven by small DNA fragments.
Directory of Open Access Journals (Sweden)
Kuntal Mukherjee
Full Text Available DNA amplification is a molecular process that increases the copy number of a chromosomal tract and often causes elevated expression of the amplified gene(s. Although gene amplification is frequently observed in cancer and other degenerative disorders, the molecular mechanisms involved in the process of DNA copy number increase remain largely unknown. We hypothesized that small DNA fragments could be the trigger of DNA amplification events. Following our findings that small fragments of DNA in the form of DNA oligonucleotides can be highly recombinogenic, we have developed a system in the yeast Saccharomyces cerevisiae to capture events of chromosomal DNA amplification initiated by small DNA fragments. Here we demonstrate that small DNAs can amplify a chromosomal region, generating either tandem duplications or acentric extrachromosomal DNA circles. Small fragment-driven DNA amplification (SFDA occurs with a frequency that increases with the length of homology between the small DNAs and the target chromosomal regions. SFDA events are triggered even by small single-stranded molecules with as little as 20-nt homology with the genomic target. A double-strand break (DSB external to the chromosomal amplicon region stimulates the amplification event up to a factor of 20 and favors formation of extrachromosomal circles. SFDA is dependent on Rad52 and Rad59, partially dependent on Rad1, Rad10, and Pol32, and independent of Rad51, suggesting a single-strand annealing mechanism. Our results reveal a novel molecular model for gene amplification, in which small DNA fragments drive DNA amplification and define the boundaries of the amplicon region. As DNA fragments are frequently found both inside cells and in the extracellular environment, such as the serum of patients with cancer or other degenerative disorders, we propose that SFDA may be a common mechanism for DNA amplification in cancer cells, as well as a more general cause of DNA copy number variation
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Molecular mechanism in the formation of a human ring chromosome 21
International Nuclear Information System (INIS)
Wong, C.; Kazazian, H.H. Jr.; Stetten, G.; Earnshaw, W.C.; Antonarakis, S.E.; Van Keuren, M.L.
1989-01-01
The authors have characterized the structural rearrangements of a chromosome 21 that led to the de novo formation of a human ring chromosome 21 [r(21)]. Molecular cloning and chromosomal localization of the DNA regions flanking the ring junction provide evidence for a long arm to long arm fusion in formation of the r(21). In addition, the centromere and proximal long arm region of a maternal chromosome 21 are duplicated in the r(21). Therefore, the mechanism in formation of the r(21) was complex involving two sequential chromosomal rearrangements. (i) Duplication of the centromere and long arm of one maternal chromosome 21 occurred forming a rearranged intermediate. (ii) Chromosomal breaks in both the proximal and telomeric long arm regions on opposite arms of this rearranged chromosome occurred with subsequent reunion producing the r(21)
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Epigenetic telomere protection by Drosophila DNA damage response pathways.
Oikemus, Sarah R; Queiroz-Machado, Joana; Lai, KuanJu; McGinnis, Nadine; Sunkel, Claudio; Brodsky, Michael H
2006-05-01
Analysis of terminal deletion chromosomes indicates that a sequence-independent mechanism regulates protection of Drosophila telomeres. Mutations in Drosophila DNA damage response genes such as atm/tefu, mre11, or rad50 disrupt telomere protection and localization of the telomere-associated proteins HP1 and HOAP, suggesting that recognition of chromosome ends contributes to telomere protection. However, the partial telomere protection phenotype of these mutations limits the ability to test if they act in the epigenetic telomere protection mechanism. We examined the roles of the Drosophila atm and atr-atrip DNA damage response pathways and the nbs homolog in DNA damage responses and telomere protection. As in other organisms, the atm and atr-atrip pathways act in parallel to promote telomere protection. Cells lacking both pathways exhibit severe defects in telomere protection and fail to localize the protection protein HOAP to telomeres. Drosophila nbs is required for both atm- and atr-dependent DNA damage responses and acts in these pathways during DNA repair. The telomere fusion phenotype of nbs is consistent with defects in each of these activities. Cells defective in both the atm and atr pathways were used to examine if DNA damage response pathways regulate telomere protection without affecting telomere specific sequences. In these cells, chromosome fusion sites retain telomere-specific sequences, demonstrating that loss of these sequences is not responsible for loss of protection. Furthermore, terminally deleted chromosomes also fuse in these cells, directly implicating DNA damage response pathways in the epigenetic protection of telomeres. We propose that recognition of chromosome ends and recruitment of HP1 and HOAP by DNA damage response proteins is essential for the epigenetic protection of Drosophila telomeres. Given the conserved roles of DNA damage response proteins in telomere function, related mechanisms may act at the telomeres of other organisms.
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DEFF Research Database (Denmark)
Wollen Steen, Kristian; Doseth, Berit; westbye, Marianne
2012-01-01
Single-strand DNA binding proteins protect DNA from nucleolytic damage, prevent formation of secondary structures and prevent premature reannealing of DNA in DNA metabolic transactions. In eukaryotes, the nuclear single-strand DNA binding protein RPA is essential for chromosomal DNA replication...
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Taranta, Andrzej; Tien Sy, Bui; Zacher, Behrend Johan; Rogalska-Taranta, Magdalena; Manns, Michael Peter; Bock, Claus Thomas; Wursthorn, Karsten
2014-08-01
Hepatitis B virus (HBV) replicates via reverse transcription converting its partially double stranded genome into the covalently closed circular DNA (cccDNA). The long-lasting cccDNA serves as a replication intermediate in the nuclei of hepatocytes. It is an excellent, though evasive, parameter for monitoring the course of liver disease and treatment efficiency. To develop and test a new approach for HBV DNA quantification in serum and small-size liver samples. The p3io plasmid contains an HBV fragment and human β-actin gene (hACTB) as a standard. Respective TaqMan probes were labeled with different fluorescent dyes. A triplex real-time PCR for simultaneous quantification of total HBV DNA, cccDNA and hACTB could be established. Three-in-one method allows simultaneous analysis of 3 targets with a lower limit of quantification of 48 copies per 20 μl PCR reaction and a wide range of linearity (R(2)>0.99, pDNA samples from HBV infected patients. Total HBV DNA and cccDNA could be quantified in 32 and 22 of 33 FFPE preserved liver specimens, respectively. Total HBV DNA concentrations quantified by the 3io method remained comparable with Cobas TaqMan HBV Test v2.0. The three-in-one protocol allows the single step quantification of viral DNA in samples from different sources. Therefore lower sample input, faster data acquisition, a lowered error and significantly lower costs are the advantages of the method. Copyright © 2014 Elsevier B.V. All rights reserved.
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Petersen, Martin R; Sigurdson, Alice J; Sampson, Laura A; Ward, Elizabeth M
2009-01-01
Background: Dietary antioxidants may protect against DNA damage induced by endogenous and exogenous sources, including ionizing radiation (IR), but data from IR-exposed human populations are limited. Objective: The objective was to examine the association between the frequency of chromosome translocations, as a biomarker of cumulative DNA damage, and intakes of vitamins C and E and carotenoids in 82 male airline pilots. Design: Dietary intakes were estimated by using a self-administered semiquantitative food-frequency questionnaire. Translocations were scored by using fluorescence in situ hybridization with whole chromosome paints. Negative binomial regression was used to estimate rate ratios and 95% CIs, adjusted for potential confounders. Results: Significant and inverse associations were observed between translocation frequency and intakes of vitamin C, β-carotene, β-cryptoxanthin, and lutein-zeaxanthin from food (P food; total vitamin C or E from food and supplements; or vitamin C or E or multivitamin supplements. The adjusted rate ratios (95% CI) for ≥median compared with food: 0.27 (0.14, 0.55). Conclusion: High combined intakes of vitamins C and E, β-carotene, β-cryptoxanthin, and lutein-zeaxanthin from food, or a diet high in their food sources, may protect against cumulative DNA damage in IR-exposed persons. PMID:19793852
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Mansuroglu, Z; Josse, T; Gilleron, J; Billecocq, A; Leger, P; Bouloy, M; Bonnefoy, E
2010-01-01
Rift Valley fever virus (RVFV) is an emerging, highly pathogenic virus; RVFV infection can lead to encephalitis, retinitis, or fatal hepatitis associated with hemorrhagic fever in humans, as well as death, abortions, and fetal deformities in animals. RVFV nonstructural NSs protein, a major factor of the virulence, forms filamentous structures in the nuclei of infected cells. In order to further understand RVFV pathology, we investigated, by chromatin immunoprecipitation, immunofluorescence, fluorescence in situ hybridization, and confocal microscopy, the capacity of NSs to interact with the host genome. Our results demonstrate that even though cellular DNA is predominantly excluded from NSs filaments, NSs interacts with some specific DNA regions of the host genome such as clusters of pericentromeric gamma-satellite sequence. Targeting of these sequences by NSs was correlated with the induction of chromosome cohesion and segregation defects in RVFV-infected murine, as well as sheep cells. Using recombinant nonpathogenic virus rZHDeltaNSs210-230, expressing a NSs protein deleted of its region of interaction with cellular factor SAP30, we showed that the NSs-SAP30 interaction was essential for NSs to target pericentromeric sequences, as well as for induction of chromosome segregation defects. The effect of RVFV upon the inheritance of genetic information is discussed with respect to the pathology associated with fetal deformities and abortions, highlighting the main role played by cellular cofactor SAP30 on the establishment of NSs interactions with host DNA sequences and RVFV pathogenesis.
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International Nuclear Information System (INIS)
Friedland, W.; Kundrat, P.
2015-01-01
The module that simulates the kinetics and yields of radiation-induced chromosome aberrations within the biophysical code PARTRAC is described. Radiation track structures simulated by Monte Carlo methods are overlapped with multi-scale models of DNA and chromatin to assess the resulting DNA damage. Spatial mobility of individual DNA ends from double-strand breaks is modelled simultaneously with their processing by the non-homologous end-joining enzymes. To score diverse types of chromosome aberrations, the joined ends are classified regarding their original chromosomal location, orientation and the involvement of centromeres. A comparison with experimental data on dicentrics induced by gamma and alpha particles shows that their relative dose dependence is predicted correctly, although the absolute yields are overestimated. The critical model assumptions on chromatin mobility and on the initial damage recognition and chromatin remodelling steps and their future refinements to solve this issue are discussed. (authors)
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Yu, Shoukai; Lemos, Bernardo
2016-12-31
Ribosomal RNAs (rRNAs) account for >60% of all RNAs in eukaryotic cells and are encoded in the ribosomal DNA (rDNA) arrays. The rRNAs are produced from two sets of loci: the 5S rDNA array resides exclusively on human chromosome 1, whereas the 45S rDNA array resides on the short arm of five human acrocentric chromosomes. The 45S rDNA gives origin to the nucleolus, the nuclear organelle that is the site of ribosome biogenesis. Intriguingly, 5S and 45S rDNA arrays exhibit correlated copy number variation in lymphoblastoid cells (LCLs). Here we examined the genomic architecture and repeat content of the 5S and 45S rDNA arrays in multiple human genome assemblies (including PacBio MHAP assembly) and ascertained contacts between the rDNA arrays and the rest of the genome using Hi-C datasets from two human cell lines (erythroleukemia K562 and lymphoblastoid cells). Our analyses revealed that 5S and 45S arrays each have thousands of contacts in the folded genome, with rDNA-associated regions and genes dispersed across all chromosomes. The rDNA contact map displayed conserved and disparate features between two cell lines, and pointed to specific chromosomes, genomic regions, and genes with evidence of spatial proximity to the rDNA arrays; the data also showed a lack of direct physical interaction between the 5S and 45S rDNA arrays. Finally, the analysis identified an intriguing organization in the 5S array with Alu and 5S elements adjacent to one another and organized in opposite orientation along the array. Portraits of genome folding centered on the ribosomal DNA array could help understand the emergence of concerted variation, the control of 5S and 45S expression, as well as provide insights into an organelle that contributes to the spatial localization of human chromosomes during interphase. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
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Energy Technology Data Exchange (ETDEWEB)
Verbakel, Werner, E-mail: werner.verbakel@chem.kuleuven.be [Laboratory of Biomolecular Dynamics, Katholieke Universiteit Leuven, Celestijnenlaan 200G, Bus 2403, 3001 Heverlee (Belgium); Carmeliet, Geert, E-mail: geert.carmeliet@med.kuleuven.be [Laboratory of Experimental Medicine and Endocrinology, Katholieke Universiteit Leuven, Herestraat 49, Bus 902, 3000 Leuven (Belgium); Engelborghs, Yves, E-mail: yves.engelborghs@fys.kuleuven.be [Laboratory of Biomolecular Dynamics, Katholieke Universiteit Leuven, Celestijnenlaan 200G, Bus 2403, 3001 Heverlee (Belgium)
2011-08-12
Highlights: {yields} The SAP-like domain in NuSAP is a functional DNA-binding domain with preference for dsDNA. {yields} This SAP-like domain is essential for chromosome loading during early mitosis. {yields} NuSAP is highly dynamic on mitotic chromatin, as evident from photobleaching experiments. {yields} The SAP-like domain also mediates NuSAP-chromatin interaction in interphase nucleoplasm. -- Abstract: Nucleolar spindle associated protein (NuSAP) is a microtubule-stabilizing protein that localizes to chromosome arms and chromosome-proximal microtubules during mitosis and to the nucleus, with enrichment in the nucleoli, during interphase. The critical function of NuSAP is underscored by the finding that its depletion in HeLa cells results in various mitotic defects. Moreover, NuSAP is found overexpressed in multiple cancers and its expression levels often correlate with the aggressiveness of cancer. Due to its localization on chromosome arms and combination of microtubule-stabilizing and DNA-binding properties, NuSAP takes a special place within the extensive group of spindle assembly factors. In this study, we identify a SAP-like domain that shows DNA binding in vitro with a preference for dsDNA. Deletion of the SAP-like domain abolishes chromosome arm binding of NuSAP during mitosis, but is not sufficient to abrogate its chromosome-proximal localization after anaphase onset. Fluorescence recovery after photobleaching experiments revealed the highly dynamic nature of this NuSAP-chromatin interaction during mitosis. In interphase cells, NuSAP also interacts with chromatin through its SAP-like domain, as evident from its enrichment on dense chromatin regions and intranuclear mobility, measured by fluorescence correlation spectroscopy. The obtained results are in agreement with a model where NuSAP dynamically stabilizes newly formed microtubules on mitotic chromosomes to enhance chromosome positioning without immobilizing these microtubules. Interphase Nu
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International Nuclear Information System (INIS)
Verbakel, Werner; Carmeliet, Geert; Engelborghs, Yves
2011-01-01
Highlights: → The SAP-like domain in NuSAP is a functional DNA-binding domain with preference for dsDNA. → This SAP-like domain is essential for chromosome loading during early mitosis. → NuSAP is highly dynamic on mitotic chromatin, as evident from photobleaching experiments. → The SAP-like domain also mediates NuSAP-chromatin interaction in interphase nucleoplasm. -- Abstract: Nucleolar spindle associated protein (NuSAP) is a microtubule-stabilizing protein that localizes to chromosome arms and chromosome-proximal microtubules during mitosis and to the nucleus, with enrichment in the nucleoli, during interphase. The critical function of NuSAP is underscored by the finding that its depletion in HeLa cells results in various mitotic defects. Moreover, NuSAP is found overexpressed in multiple cancers and its expression levels often correlate with the aggressiveness of cancer. Due to its localization on chromosome arms and combination of microtubule-stabilizing and DNA-binding properties, NuSAP takes a special place within the extensive group of spindle assembly factors. In this study, we identify a SAP-like domain that shows DNA binding in vitro with a preference for dsDNA. Deletion of the SAP-like domain abolishes chromosome arm binding of NuSAP during mitosis, but is not sufficient to abrogate its chromosome-proximal localization after anaphase onset. Fluorescence recovery after photobleaching experiments revealed the highly dynamic nature of this NuSAP-chromatin interaction during mitosis. In interphase cells, NuSAP also interacts with chromatin through its SAP-like domain, as evident from its enrichment on dense chromatin regions and intranuclear mobility, measured by fluorescence correlation spectroscopy. The obtained results are in agreement with a model where NuSAP dynamically stabilizes newly formed microtubules on mitotic chromosomes to enhance chromosome positioning without immobilizing these microtubules. Interphase NuSAP-chromatin interaction
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International Nuclear Information System (INIS)
Krawczyk, P. M.; Stap, C.; Van Oven, C.; Hoebe, R.; Aten, J. A.
2006-01-01
For efficient repair of DNA double strand breaks (DSBs) cells rely on a process that involves the Mre11/Rad50/Nbs1 complex, which may help to protect non-repaired DNA ends from separating until they can be rejoined by DNA repair proteins. It has been observed that as a secondary effect, this process can lead to unintended clustering of multiple, initially separate, DSB-containing chromosome domains. This work demonstrates that neither inactivation of the major repair proteins XRCC3 and the DNA-dependent protein kinase (DNA-PK) nor inhibition of DNA-PK by vanillin influences the aggregation of DSB-containing chromosome domains. (authors)
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DEFF Research Database (Denmark)
Li, Zhongyi; Huang, Bingyan; Rampling, Lynette
2004-01-01
Rice bacterial artificial chromosome (BAC) clones have been identified that contain sequences orthologous to each EST localized to wheat chromosome 7AS deletion stocks by Southern blot hybridization. This information has been used to relate the DNA sequence included in each wheat deletion stock t...
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Transcription-based model for the induction of chromosomal exchange events by ionising radiation
International Nuclear Information System (INIS)
Radford, I.A.
2003-01-01
The mechanistic basis for chromosomal aberration formation, following exposure of mammalian cells to ionising radiation, has long been debated. Although chromosomal aberrations are probably initiated by DNA double-strand breaks (DSB), little is understood about the mechanisms that generate and modulate DNA rearrangement. Based on results from our laboratory and data from the literature, a novel model of chromosomal aberration formation has been suggested (Radford 2002). The basic postulates of this model are that: (1) DSB, primarily those involving multiple individual damage sites (i.e. complex DSB), are the critical initiating lesion; (2) only those DSB occurring in transcription units that are associated with transcription 'factories' (complexes containing multiple transcription units) induce chromosomal exchange events; (3) such DSB are brought into contact with a DNA topoisomerase I molecule through RNA polymerase II catalysed transcription and give rise to trapped DNA-topo I cleavage complexes; and (4) trapped complexes interact with another topo I molecule on a temporarily inactive transcription unit at the same transcription factory leading to DNA cleavage and subsequent strand exchange between the cleavage complexes. We have developed a method using inverse PCR that allows the detection and sequencing of putative ionising radiation-induced DNA rearrangements involving different regions of the human genome (Forrester and Radford 1998). The sequences detected by inverse PCR can provide a test of the prediction of the transcription-based model that ionising radiation-induced DNA rearrangements occur between sequences in active transcription units. Accordingly, reverse transcriptase PCR was used to determine if sequences involved in rearrangements were transcribed in the test cells. Consistent with the transcription-based model, nearly all of the sequences examined gave a positive result to reverse transcriptase PCR (Forrester and Radford unpublished)
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Ge, Jun; Lou, Zheng; Cui, Hong; Shang, Lei; Harshey, Rasika M
2011-09-01
Of all known transposable elements, phage Mu exhibits the highest transposition efficiency and the lowest target specificity. In vitro, MuB protein is responsible for target choice. In this work, we provide a comprehensive assessment of the genome-wide distribution of MuB and its relationship to Mu target selection using high-resolution Escherichia coli tiling DNA arrays. We have also assessed how MuB binding and Mu transposition are influenced by chromosome-organizing elements such as AT-rich DNA signatures, or the binding of the nucleoid-associated protein Fis, or processes such as transcription. The results confirm and extend previous biochemical and lower resolution in vivo data. Despite the generally random nature of Mu transposition and MuB binding, there were hot and cold insertion sites and MuB binding sites in the genome, and differences between the hottest and coldest sites were large. The new data also suggest that MuB distribution and subsequent Mu integration is responsive to DNA sequences that contribute to the structural organization of the chromosome.
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Detection of obesity QTLs on mouse chromosomes 1 and 7 by selective DNA pooling
Energy Technology Data Exchange (ETDEWEB)
Taylor, B.A.; Phillips, S.J. [Jackson Lab., Bar Harbor, ME (United States)
1996-06-15
The inheritance of obesity has been analyzed in an intercross between the lean 129/Sv mouse strain and the obesity-prone EL/Suz mouse strain. The weights of three major fat pads were determined on 4-month-old mice, and the sum of these weights, divided by body weight, was used as an adiposity index. The strategy of selective DNA pooling was used as a primary screen to identify putative quantitative trait loci (QTLs) affecting adiposity index. DNA pools representing the leanest 15% and fattest 15% of the F2 progeny were compared for differential allelic enrichment using widely dispersed microsatellite variants. To evaluate putative QTLs, individual genotyping and interval mapping were employed to estimate QTL effects and assess statistical significance. One QTL affecting adiposity index, which accounted for 12.3% of phenotypic variance in gender-merged data, was mapped to the central region of Chromosome (Chr) 7. The QTL allele inherited from EL conferred increased adiposity. A second QTL that accounts for 6.3% of phenotypic variance was identified on Chr 1 near D1Mitt211. At both QTLs, the data are consistent with dominant inheritance of the allele contributing to obesity. The possible relationships between these QTLs and previously described obesity QYLs, major obesity mutations, and candidate genes are discussed. 42 refs., 3 figs., 3 tabs.
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The relationships among lemons, limes and citron: a chromosomal comparison.
Carvalho, R; Soares Filho, W S; Brasileiro-Vidal, A C; Guerra, M
2005-01-01
Lemons, limes and citron constitute a group of closely related Citrus species, whose species delimitations and taxonomic relationships are unclear. In order to identify karyotypic similarities and species relationships within this group, the CMA+/DAPI- banding pattern and the distribution of the 5S and 45S rDNA sites of 10 accessions of lime, lemon, and citron were investigated. The four cultivars of C. limon analyzed showed the same pattern of CMA+ bands and rDNA sites, suggesting that they originated from a single germplasm, later differentiated by distinct somatic mutations. The lemons C. jambhiri, C. limonia and C. volkameriana displayed karyotypes very similar to each other, but they differed from C. limon by the absence of a single chromosome with one band in each telomere. The limes, C. aurantifolia and C. limettioides, seemed less related to each other and exhibited different heteromorphic chromosome pairs. In C. aurantifolia, the presence of a chromosome type unknown in all other Citrus species cytologically known so far supports the assumption that this accession may be derived from a hybrid with a species from the subgenus Papeda or from another genus. Citrus medica was the only homozygous accession of this group and all of its chromosome types were clearly represented in limes and lemons, some of them forming heteromorphic pairs. The analysis of the distribution of rDNA sites allowed a further refinement of the comparison among accessions. The lemons and limes were heterozygous for all rDNA sites, whereas C. medica was entirely homozygous. These data support the hypothesis that C. medica is a true species while the other nine accessions are hybrids. Copyright 2005 S. Karger AG, Basel.
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Divashuk, Mikhail G; Alexandrov, Oleg S; Razumova, Olga V; Kirov, Ilya V; Karlov, Gennady I
2014-01-01
Hemp (Cannabis sativa L.) was karyotyped using by DAPI/C-banding staining to provide chromosome measurements, and by fluorescence in situ hybridization with probes for 45 rDNA (pTa71), 5S rDNA (pCT4.2), a subtelomeric repeat (CS-1) and the Arabidopsis telomere probes. The karyotype has 18 autosomes plus a sex chromosome pair (XX in female and XY in male plants). The autosomes are difficult to distinguish morphologically, but three pairs could be distinguished using the probes. The Y chromosome is larger than the autosomes, and carries a fully heterochromatic DAPI positive arm and CS-1 repeats only on the less intensely DAPI-stained, euchromatic arm. The X is the largest chromosome of all, and carries CS-1 subtelomeric repeats on both arms. The meiotic configuration of the sex bivalent locates a pseudoautosomal region of the Y chromosome at the end of the euchromatic CS-1-carrying arm. Our molecular cytogenetic study of the C. sativa sex chromosomes is a starting point for helping to make C. sativa a promising model to study sex chromosome evolution.
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Directory of Open Access Journals (Sweden)
Olivieri Anna
2011-10-01
Full Text Available Abstract Background For millennia, the southern part of the Mesopotamia has been a wetland region generated by the Tigris and Euphrates rivers before flowing into the Gulf. This area has been occupied by human communities since ancient times and the present-day inhabitants, the Marsh Arabs, are considered the population with the strongest link to ancient Sumerians. Popular tradition, however, considers the Marsh Arabs as a foreign group, of unknown origin, which arrived in the marshlands when the rearing of water buffalo was introduced to the region. Results To shed some light on the paternal and maternal origin of this population, Y chromosome and mitochondrial DNA (mtDNA variation was surveyed in 143 Marsh Arabs and in a large sample of Iraqi controls. Analyses of the haplogroups and sub-haplogroups observed in the Marsh Arabs revealed a prevalent autochthonous Middle Eastern component for both male and female gene pools, with weak South-West Asian and African contributions, more evident in mtDNA. A higher male than female homogeneity is characteristic of the Marsh Arab gene pool, likely due to a strong male genetic drift determined by socio-cultural factors (patrilocality, polygamy, unequal male and female migration rates. Conclusions Evidence of genetic stratification ascribable to the Sumerian development was provided by the Y-chromosome data where the J1-Page08 branch reveals a local expansion, almost contemporary with the Sumerian City State period that characterized Southern Mesopotamia. On the other hand, a more ancient background shared with Northern Mesopotamia is revealed by the less represented Y-chromosome lineage J1-M267*. Overall our results indicate that the introduction of water buffalo breeding and rice farming, most likely from the Indian sub-continent, only marginally affected the gene pool of autochthonous people of the region. Furthermore, a prevalent Middle Eastern ancestry of the modern population of the marshes of
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Dynamics of DNA replication during premeiosis and early meiosis in wheat.
Rey, María-Dolores; Prieto, Pilar
2014-01-01
Meiosis is a specialised cell division that involves chromosome replication, two rounds of chromosome segregation and results in the formation of the gametes. Meiotic DNA replication generally precedes chromosome pairing, recombination and synapsis in sexually developing eukaryotes. In this work, replication has been studied during premeiosis and early meiosis in wheat using flow cytometry, which has allowed the quantification of the amount of DNA in wheat anther in each phase of the cell cycle during premeiosis and each stage of early meiosis. Flow cytometry has been revealed as a suitable and user-friendly tool to detect and quantify DNA replication during early meiosis in wheat. Chromosome replication was detected in wheat during premeiosis and early meiosis until the stage of pachytene, when chromosomes are associated in pairs to further recombine and correctly segregate in the gametes. In addition, the effect of the Ph1 locus, which controls chromosome pairing and affects replication in wheat, was also studied by flow cytometry. Here we showed that the Ph1 locus plays an important role on the length of meiotic DNA replication in wheat, particularly affecting the rate of replication during early meiosis in wheat.
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Smith, Owen K.; Aladjem, Mirit I.
2014-01-01
The DNA replication program is, in part, determined by the epigenetic landscape that governs local chromosome architecture and directs chromosome duplication. Replication must coordinate with other biochemical processes occurring concomitantly on chromatin, such as transcription and remodeling, to insure accurate duplication of both genetic and epigenetic features and to preserve genomic stability. The importance of genome architecture and chromatin looping in coordinating cellular processes on chromatin is illustrated by two recent sets of discoveries. First, chromatin-associated proteins that are not part of the core replication machinery were shown to affect the timing of DNA replication. These chromatin-associated proteins could be working in concert, or perhaps in competition, with the transcriptional machinery and with chromatin modifiers to determine the spatial and temporal organization of replication initiation events. Second, epigenetic interactions are mediated by DNA sequences that determine chromosomal replication. In this review we summarize recent findings and current models linking spatial and temporal regulation of the replication program with epigenetic signaling. We discuss these issues in the context of the genome’s three-dimensional structure with an emphasis on events occurring during the initiation of DNA replication. PMID:24905010
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Wilson, Paul J; Rutledge, Linda Y; Wheeldon, Tyler J; Patterson, Brent R; White, Bradley N
2012-09-01
There has been considerable discussion on the origin of the red wolf and eastern wolf and their evolution independent of the gray wolf. We analyzed mitochondrial DNA (mtDNA) and a Y-chromosome intron sequence in combination with Y-chromosome microsatellites from wolves and coyotes within the range of extensive wolf-coyote hybridization, that is, eastern North America. The detection of divergent Y-chromosome haplotypes in the historic range of the eastern wolf is concordant with earlier mtDNA findings, and the absence of these haplotypes in western coyotes supports the existence of the North American evolved eastern wolf (Canis lycaon). Having haplotypes observed exclusively in eastern North America as a result of insufficient sampling in the historic range of the coyote or that these lineages subsequently went extinct in western geographies is unlikely given that eastern-specific mtDNA and Y-chromosome haplotypes represent lineages divergent from those observed in extant western coyotes. By combining Y-chromosome and mtDNA distributional patterns, we identified hybrid genomes of eastern wolf, coyote, gray wolf, and potentially dog origin in Canis populations of central and eastern North America. The natural contemporary eastern Canis populations represent an important example of widespread introgression resulting in hybrid genomes across the original C. lycaon range that appears to be facilitated by the eastern wolf acting as a conduit for hybridization. Applying conventional taxonomic nomenclature and species-based conservation initiatives, particularly in human-modified landscapes, may be counterproductive to the effective management of these hybrids and fails to consider their evolutionary potential.
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Mode of ATM-dependent suppression of chromosome translocation
Energy Technology Data Exchange (ETDEWEB)
Yamauchi, Motohiro, E-mail: motoyama@nagasaki-u.ac.jp [Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan); Suzuki, Keiji; Oka, Yasuyoshi; Suzuki, Masatoshi; Kondo, Hisayoshi; Yamashita, Shunichi [Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan)
2011-12-09
Highlights: Black-Right-Pointing-Pointer We addressed how ATM suppresses frequency of chromosome translocation. Black-Right-Pointing-Pointer We found ATM/p53-dependent G1 checkpoint suppresses translocation frequency. Black-Right-Pointing-Pointer We found ATM and DNA-PKcs function in a common pathway to suppress translocation. -- Abstract: It is well documented that deficiency in ataxia telangiectasia mutated (ATM) protein leads to elevated frequency of chromosome translocation, however, it remains poorly understood how ATM suppresses translocation frequency. In the present study, we addressed the mechanism of ATM-dependent suppression of translocation frequency. To know frequency of translocation events in a whole genome at once, we performed centromere/telomere FISH and scored dicentric chromosomes, because dicentric and translocation occur with equal frequency and by identical mechanism. By centromere/telomere FISH analysis, we confirmed that chemical inhibition or RNAi-mediated knockdown of ATM causes 2 to 2.5-fold increase in dicentric frequency at first mitosis after 2 Gy of gamma-irradiation in G0/G1. The FISH analysis revealed that ATM/p53-dependent G1 checkpoint suppresses dicentric frequency, since RNAi-mediated knockdown of p53 elevated dicentric frequency by 1.5-fold. We found ATM also suppresses dicentric occurrence independently of its checkpoint role, as ATM inhibitor showed additional effect on dicentric frequency in the context of p53 depletion and Chk1/2 inactivation. Epistasis analysis using chemical inhibitors revealed that ATM kinase functions in the same pathway that requires kinase activity of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to suppress dicentric frequency. From the results in the present study, we conclude that ATM minimizes translocation frequency through its commitment to G1 checkpoint and DNA double-strand break repair pathway that requires kinase activity of DNA-PKcs.
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Chromosome sizes and phylogenetic relationships between serotypes of Actinobacillus pleuropneumoniae
Chevallier, Bruno; Dugourd, Dominique; Tarasiuk, Kazimirez; Harel, Josée; Gottschalk, Marcelo; Kobisch, Marylène; Frey, Joachim
2017-01-01
The genome size of Actinobacillus pleuropneumoniae was determined by pulsed field gel electrophoresis of AscI and ApaI digested chromosomal DNA. The genome size of the type strain 4074T (serotype 1) was determined to be 2404±40 kb. The chromosome sizes for the reference strains of the other serotypes range between 2.3 and 2.4 Mb. The restriction pattern profiles of AscI, ApaI and NheI digested chromosomes showed a high degree of polymorphism among the different serotype reference strains and ...
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International Nuclear Information System (INIS)
Sugiyama, M.; Tsuzuki, K.; Matsumoto, K.; Ogura, R.
1992-01-01
The effect of pretreatment with vitamin E on cytotoxicity, DNA single strand breaks, and chromosomal aberrations as well as on mutation induced by ultraviolet-B light (UV-B) was investigated in Chinese hamster V-79 cells. Cellular pretreatment with non-toxic levels of 25 μM α-tocopherol succinate (vitamin E) for 24h prior to exposure resulted in a 10-fold increase in cellular levels of α-tocopherol. Using a colony-forming assay, this pretreatment decreased the cytotoxicity of UV-B light. However, alkaline elution assays demonstrated that pretreatment with vitamin E did not affect the number of DNA single strand breaks caused by UV-B light. UV-B exposure produced a dose-dependent induction of chromosomal aberrations and mutations at the HGPRT locus, and neither of these actions of UV-B was influenced by pretreatment with the vitamin. These results suggest that vitamin E protects cells from UV-B-induced cytotoxicity, possibly through its ability to scavenge free radicals. The genotoxicity induced by UV-B light may not correlate directly with the cytotoxic action of this wavelength region in sunlight. (author)
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Chromosome End Repair and Genome Stability in Plasmodium falciparum
Directory of Open Access Journals (Sweden)
Susannah F. Calhoun
2017-08-01
Full Text Available The human malaria parasite Plasmodium falciparum replicates within circulating red blood cells, where it is subjected to conditions that frequently cause DNA damage. The repair of DNA double-stranded breaks (DSBs is thought to rely almost exclusively on homologous recombination (HR, due to a lack of efficient nonhomologous end joining. However, given that the parasite is haploid during this stage of its life cycle, the mechanisms involved in maintaining genome stability are poorly understood. Of particular interest are the subtelomeric regions of the chromosomes, which contain the majority of the multicopy variant antigen-encoding genes responsible for virulence and disease severity. Here, we show that parasites utilize a competitive balance between de novo telomere addition, also called “telomere healing,” and HR to stabilize chromosome ends. Products of both repair pathways were observed in response to DSBs that occurred spontaneously during routine in vitro culture or resulted from experimentally induced DSBs, demonstrating that both pathways are active in repairing DSBs within subtelomeric regions and that the pathway utilized was determined by the DNA sequences immediately surrounding the break. In combination, these two repair pathways enable parasites to efficiently maintain chromosome stability while also contributing to the generation of genetic diversity.
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Efficient identification of Y chromosome sequences in the human and Drosophila genomes
Carvalho, Antonio Bernardo; Clark, Andrew G.
2013-01-01
Notwithstanding their biological importance, Y chromosomes remain poorly known in most species. A major obstacle to their study is the identification of Y chromosome sequences; due to its high content of repetitive DNA, in most genome projects, the Y chromosome sequence is fragmented into a large number of small, unmapped scaffolds. Identification of Y-linked genes among these fragments has yielded important insights about the origin and evolution of Y chromosomes, but the process is labor intensive, restricting studies to a small number of species. Apart from these fragmentary assemblies, in a few mammalian species, the euchromatic sequence of the Y is essentially complete, owing to painstaking BAC mapping and sequencing. Here we use female short-read sequencing and k-mer comparison to identify Y-linked sequences in two very different genomes, Drosophila virilis and human. Using this method, essentially all D. virilis scaffolds were unambiguously classified as Y-linked or not Y-linked. We found 800 new scaffolds (totaling 8.5 Mbp), and four new genes in the Y chromosome of D. virilis, including JYalpha, a gene involved in hybrid male sterility. Our results also strongly support the preponderance of gene gains over gene losses in the evolution of the Drosophila Y. In the intensively studied human genome, used here as a positive control, we recovered all previously known genes or gene families, plus a small amount (283 kb) of new, unfinished sequence. Hence, this method works in large and complex genomes and can be applied to any species with sex chromosomes. PMID:23921660
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Efficient identification of Y chromosome sequences in the human and Drosophila genomes.
Carvalho, Antonio Bernardo; Clark, Andrew G
2013-11-01
Notwithstanding their biological importance, Y chromosomes remain poorly known in most species. A major obstacle to their study is the identification of Y chromosome sequences; due to its high content of repetitive DNA, in most genome projects, the Y chromosome sequence is fragmented into a large number of small, unmapped scaffolds. Identification of Y-linked genes among these fragments has yielded important insights about the origin and evolution of Y chromosomes, but the process is labor intensive, restricting studies to a small number of species. Apart from these fragmentary assemblies, in a few mammalian species, the euchromatic sequence of the Y is essentially complete, owing to painstaking BAC mapping and sequencing. Here we use female short-read sequencing and k-mer comparison to identify Y-linked sequences in two very different genomes, Drosophila virilis and human. Using this method, essentially all D. virilis scaffolds were unambiguously classified as Y-linked or not Y-linked. We found 800 new scaffolds (totaling 8.5 Mbp), and four new genes in the Y chromosome of D. virilis, including JYalpha, a gene involved in hybrid male sterility. Our results also strongly support the preponderance of gene gains over gene losses in the evolution of the Drosophila Y. In the intensively studied human genome, used here as a positive control, we recovered all previously known genes or gene families, plus a small amount (283 kb) of new, unfinished sequence. Hence, this method works in large and complex genomes and can be applied to any species with sex chromosomes.
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Froelich, Katrin; Mickler, Johannes; Steusloff, Gudrun; Technau, Antje; Ramos Tirado, Mario; Scherzed, Agmal; Hackenberg, Stephan; Radeloff, Andreas; Hagen, Rudolf; Kleinsasser, Norbert
2013-07-01
Adipose-derived stem cells (ASCs) are a promising mesenchymal cell source for tissue engineering approaches. To obtain an adequate cell amount, in vitro expansion of the cells may be required in some cases. To monitor potential contraindications for therapeutic applications in humans, DNA strand breaks and chromosomal aberrations in ASCs during in vitro expansion were examined. After isolation of ASC from human lipoaspirates of seven patients, in vitro expansion over 10 passages was performed. Cells from passages 1, 2, 3, 5 and 10 were used for the alkaline single-cell microgel electrophoresis (comet) assay to detect DNA single-strand breaks and alkali labile as well as incomplete excision repair sites. Chromosomal changes were examined by means of the chromosomal aberration test. During in vitro expansion, ASC showed no DNA single-strand breaks in the comet assay. With the chromosomal aberration test, however, a significant increase in chromosomal aberrations were detected. The study showed that although no DNA fragmentation could be determined, the safety of ASC cannot be ensured with respect to chromosome stability during in vitro expansion. Thus, reliable analyses for detecting ASC populations, which accumulate chromosomal aberrations or even undergo malignant transformation during extensive in vitro expansion, must be implemented as part of the safety evaluation of these cells for stem cell-based therapy. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
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The Emerging Role of the Cytoskeleton in Chromosome Dynamics
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Maya Spichal
2017-05-01
Full Text Available Chromosomes underlie a dynamic organization that fulfills functional roles in processes like transcription, DNA repair, nuclear envelope stability, and cell division. Chromosome dynamics depend on chromosome structure and cannot freely diffuse. Furthermore, chromosomes interact closely with their surrounding nuclear environment, which further constrains chromosome dynamics. Recently, several studies enlighten that cytoskeletal proteins regulate dynamic chromosome organization. Cytoskeletal polymers that include actin filaments, microtubules and intermediate filaments can connect to the nuclear envelope via Linker of the Nucleoskeleton and Cytoskeleton (LINC complexes and transfer forces onto chromosomes inside the nucleus. Monomers of these cytoplasmic polymers and related proteins can also enter the nucleus and play different roles in the interior of the nucleus than they do in the cytoplasm. Nuclear cytoskeletal proteins can act as chromatin remodelers alone or in complexes with other nuclear proteins. They can also act as transcription factors. Many of these mechanisms have been conserved during evolution, indicating that the cytoskeletal regulation of chromosome dynamics is an essential process. In this review, we discuss the different influences of cytoskeletal proteins on chromosome dynamics by focusing on the well-studied model organism budding yeast.
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Nassan, Feiby L; Chavarro, Jorge E; Mínguez-Alarcón, Lidia; Williams, Paige L; Tanrikut, Cigdem; Ford, Jennifer B; Dadd, Ramace; Perry, Melissa J; Hauser, Russ; Gaskins, Audrey J
2018-06-01
We examined associations of residential distance to major roadways, as a proxy for traffic-related air pollution exposures, with sperm characteristics and male reproductive hormones. The cohort included 797 men recruited from Massachusetts General Hospital Fertility Center between 2000 and 2015 to participate in fertility research studies. Men reported their residential addresses at enrollment and provided 1-6 semen samples and a blood sample during follow-up. We estimated the Euclidean distance to major roadways (e.g. interstates and highways: limited access highways, multi-lane highways (not limited access), other numbered routes, and major roads) using information from the Massachusetts Department of Geographic Information Systems. Semen parameters (1238 semen samples), sperm DNA integrity (389 semen samples), chromosomal disomy (101 semen samples), and serum reproductive hormones (405 serum samples) were assessed following standard procedures. Men in this cohort were primarily Caucasian (86%), not current smokers (92%), with a college or higher education (88%), and had an average age of 36 years and BMI of 27.7 kg/m 2 . The median (interquartile range) residential distance to a major roadway was 111 (37, 248) meters. Residential proximity to major roadways was not associated with semen parameters, sperm DNA integrity, chromosomal disomy, or serum reproductive hormone concentrations. The adjusted percent change (95% CI) in semen quality parameters associated with a 500 m increase in residential distance to a major roadway was -1.0% (-6.3, 4.5) for semen volume, 4.3% (-5.8, 15.7) for sperm concentration, 3.1% (-7.2, 14.5) for sperm count, 1.1% (-1.2, 3.4) for % total motile sperm, and 0.1% (-0.3, 0.5) for % morphologically normal sperm. Results were consistent when we modeled the semen parameters dichotomized according to WHO 2010 reference values. Residential distance to major roadways, as a proxy for traffic-related air pollution exposure, was not related
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Roles of Cohesin and Condensin in Chromosome Dynamics During Mammalian Meiosis
LEE, Jibak
2013-01-01
Meiosis is a key step for sexual reproduction in which chromosome number is halved by two successive meiotic divisions after a single round of DNA replication. In the first meiotic division (meiosis I), homologous chromosomes pair, synapse, and recombine with their partners in prophase I. As a result, homologous chromosomes are physically connected until metaphase I and then segregated from each other at the onset of anaphase I. In the subsequent second meiotic division (meiosis II), sister c...
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Trofimova, Irina; Krasikova, Alla
2016-12-01
Tandemly organized highly repetitive DNA sequences are crucial structural and functional elements of eukaryotic genomes. Despite extensive evidence, satellite DNA remains an enigmatic part of the eukaryotic genome, with biological role and significance of tandem repeat transcripts remaining rather obscure. Data on tandem repeats transcription in amphibian and avian model organisms is fragmentary despite their genomes being thoroughly characterized. Review systematically covers historical and modern data on transcription of amphibian and avian satellite DNA in somatic cells and during meiosis when chromosomes acquire special lampbrush form. We highlight how transcription of tandemly repetitive DNA sequences is organized in interphase nucleus and on lampbrush chromosomes. We offer LTR-activation hypotheses of widespread satellite DNA transcription initiation during oogenesis. Recent explanations are provided for the significance of high-yield production of non-coding RNA derived from tandemly organized highly repetitive DNA. In many cases the data on the transcription of satellite DNA can be extrapolated from lampbrush chromosomes to interphase chromosomes. Lampbrush chromosomes with applied novel technical approaches such as superresolution imaging, chromosome microdissection followed by high-throughput sequencing, dynamic observation in life-like conditions provide amazing opportunities for investigation mechanisms of the satellite DNA transcription.
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Modelling the induction of cell death and chromosome damage by therapeutic protons
Carante, M P
2015-01-01
A two-parameter biophysical model cal led BIANCA (BIophysical ANalysis of Cell death and chromosome Aberrations), which assumes a pivotal role for DNA cluster damage and for “lethal” chromosome aberrations, was applied to calculate cell death and chromosome aberrations for normal and radio-resistant cells along a 62-MeV eye melanoma proton beam. The yield of DNA “Cluster Lesions” and the probability for a chromosome fragment of not being rejoined with any partne r were adjustable parameters. In line with other works, the beam effectiveness at inducing both biological endpoints was found to increase with increasing depth, and high levels of damage were found also beyond the dose fall-off, due to the higher biological effectiveness of low-energy protons. This implies that assuming a constant RBE along the whole SOBP, as is currently done in clinical practice, may be sub-optimal, also implying a possible underestimation of normal tissue damage. Furthermore, the calculations suggested that fo...
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Davis, Ronald L.; Davidson, Norman
1984-01-01
Using the method of chromosomal walking, we have isolated a contiguous region of the Drosophila melanogaster X chromosome which corresponds to salivary gland chromosome bands 3C12 to 3D4. This five-band region contains approximately 100 kilobases of DNA, including those sequences comprising dunce, a gene which functions in memory and cyclic nucleotide metabolism. Genome blots of DNA from flies carrying several different chromosomal aberrations with breakpoints in the region have been probed w...
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Measurements of DNA Damage and Repair in Bacillus anthracis Sterne Spores by UV Radiation
2014-09-18
tightly into chromosomal structure, which serves to protect the DNA. The DNA is coiled several times and wrapped tightly around proteins. The wrapping...helps the DNA keep its chromatin structure. When DNA is to be replicated, enzymes unwind the chromosomal DNA. Therefore, DNA is not specifically used...2 See Appendix for Plasmid Information sheet. 30 the ice crystals disappeared . The tube was gently mixed by hand
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International Nuclear Information System (INIS)
Hoshi, Osamu; Ushiki, Tatsuo
2011-01-01
A novel technique using the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into replicating DNA is described for the analysis of replicating banding patterns of human metaphase chromosomes. Human lymphocytes were synchronized with excess thymidine and treated with EdU during the late S phase of the cell cycle. The incorporated EdU was then detected in metaphase chromosomes using Alexa Fluor® 488 azides, through the 1,3-dipolar cycloaddition reaction of organic azides with the terminal acetylene group of EdU. Chromosomes with incorporated EdU showed a banding pattern similar to G-banding of normal human chromosomes. Imaging by atomic force microscopy (AFM) in liquid conditions showed that the structure of the chromosomes was well preserved even after EdU treatment. Comparison between fluorescence microscopy and AFM images of the same chromosome 1 indicated the presence of ridges and grooves in the chromatid arm, features that have been previously reported in relation to G-banding. These results suggest an intimate relationship between EdU-induced replication bands and G- or R-bands in human chromosomes. This technique is thus useful for analyzing the structure of chromosomes in relation to their banding patterns following DNA replication in the S phase
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Gal, A; Wieringa, B; Smeets, D F; Bleeker-Wagemakers, L; Ropers, H H
1986-01-01
Norrie disease (ND), an X-linked recessive disorder, is characterized by congenital blindness followed by bulbar atrophy. We have examined a three-generation family in which ND is part of a complex X-linked syndrome with severe mental retardation, hypogonadism, growth disturbances, and increased susceptibility to infections as additional features. This syndrome is apparently due to an interstitial deletion, as evidenced by the failure of the L1.28 DNA probe (DXS7 locus, Xp11.3) to detect complementary DNA sequences on the defective X chromosome of an affected male and of several obligatory heterozygotes. Attempts to further define this deletion with other DNA probes from the proximal short arm of the X chromosome or by prometaphase chromosome analysis were unsuccessful.
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Moralli, Daniela; Monaco, Zoia L
2015-02-01
De novo artificial chromosomes expressing genes have been generated in human embryonic stem cells (hESc) and are maintained following differentiation into other cell types. Human artificial chromosomes (HAC) are small, functional, extrachromosomal elements, which behave as normal chromosomes in human cells. De novo HAC are generated following delivery of alpha satellite DNA into target cells. HAC are characterized by high levels of mitotic stability and are used as models to study centromere formation and chromosome organisation. They are successful and effective as gene expression vectors since they remain autonomous and can accommodate larger genes and regulatory regions for long-term expression studies in cells unlike other viral gene delivery vectors currently used. Transferring the essential DNA sequences for HAC formation intact across the cell membrane has been challenging for a number of years. A highly efficient delivery system based on HSV-1 amplicons has been used to target DNA directly to the ES cell nucleus and HAC stably generated in human embryonic stem cells (hESc) at high frequency. HAC were detected using an improved protocol for hESc chromosome harvesting, which consistently produced high-quality metaphase spreads that could routinely detect HAC in hESc. In tumour cells, the input DNA often integrated in the host chromosomes, but in the host ES genome, it remained intact. The hESc containing the HAC formed embryoid bodies, generated teratoma in mice, and differentiated into neuronal cells where the HAC were maintained. The HAC structure and chromatin composition was similar to the endogenous hESc chromosomes. This review will discuss the technological advances in HAC vector delivery using HSV-1 amplicons and the improvements in the identification of de novo HAC in hESc.
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Directory of Open Access Journals (Sweden)
Harpending Henry C
2008-07-01
Full Text Available Abstract Background Near the junction of three major continents, the Caucasus region has been an important thoroughfare for human migration. While the Caucasus Mountains have diverted human traffic to the few lowland regions that provide a gateway from north to south between the Caspian and Black Seas, highland populations have been isolated by their remote geographic location and their practice of patrilocal endogamy. We investigate how these cultural and historical differences between highland and lowland populations have affected patterns of genetic diversity. We test 1 whether the highland practice of patrilocal endogamy has generated sex-specific population relationships, and 2 whether the history of migration and military conquest associated with the lowland populations has left Central Asian genes in the Caucasus, by comparing genetic diversity and pairwise population relationships between Daghestani populations and reference populations throughout Europe and Asia for autosomal, mitochondrial, and Y-chromosomal markers. Results We found that the highland Daghestani populations had contrasting histories for the mitochondrial DNA and Y-chromosome data sets. Y-chromosomal haplogroup diversity was reduced among highland Daghestani populations when compared to other populations and to highland Daghestani mitochondrial DNA haplogroup diversity. Lowland Daghestani populations showed Turkish and Central Asian affinities for both mitochondrial and Y-chromosomal data sets. Autosomal population histories are strongly correlated to the pattern observed for the mitochondrial DNA data set, while the correlation between the mitochondrial DNA and Y-chromosome distance matrices was weak and not significant. Conclusion The reduced Y-chromosomal diversity exhibited by highland Daghestani populations is consistent with genetic drift caused by patrilocal endogamy. Mitochondrial and Y-chromosomal phylogeographic comparisons indicate a common Near Eastern
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Camara, Johanna Eltz; Skarstad, Kirsten; Crooke, Elliott
2003-05-01
Regulatory inactivation of DnaA helps ensure that the Escherichia coli chromosome is replicated only once per cell cycle, through accelerated hydrolysis of active replication initiator ATP-DnaA to inactive ADP-DnaA. Analysis of deltahda strains revealed that the regulatory inactivation of DnaA component Hda is necessary for maintaining controlled initiation but not for cell growth or viability.
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A geometrical model for DNA organization in bacteria.
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Mathias Buenemann
Full Text Available Recent experimental studies have revealed that bacteria, such as C. crescentus, show a remarkable spatial ordering of their chromosome. A strong linear correlation has been found between the position of genes on the chromosomal map and their spatial position in the cellular volume. We show that this correlation can be explained by a purely geometrical model. Namely, self-avoidance of DNA, specific positioning of one or few DNA loci (such as origin or terminus together with the action of DNA compaction proteins (that organize the chromosome into topological domains are sufficient to get a linear arrangement of the chromosome along the cell axis. We develop a Monte-Carlo method that allows us to test our model numerically and to analyze the dependence of the spatial ordering on various physiologically relevant parameters. We show that the proposed geometrical ordering mechanism is robust and universal (i.e. does not depend on specific bacterial details. The geometrical mechanism should work in all bacteria that have compacted chromosomes with spatially fixed regions. We use our model to make specific and experimentally testable predictions about the spatial arrangement of the chromosome in mutants of C. crescentus and the growth-stage dependent ordering in E. coli.
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van Koningsbruggen, Silvana; Gierlinski, Marek; Schofield, Pietá; Martin, David; Barton, Geoffey J; Ariyurek, Yavuz; den Dunnen, Johan T; Lamond, Angus I
2010-11-01
The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope.
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High-resolution YAC-cosmid-STS map of human chromosome 13.
Cayanis, E; Russo, J J; Kalachikov, S; Ye, X; Park, S H; Sunjevaric, I; Bonaldo, M F; Lawton, L; Venkatraj, V S; Schon, E; Soares, M B; Rothstein, R; Warburton, D; Edelman, I S; Zhang, P; Efstratiadis, A; Fischer, S G
1998-01-01
We have assembled a high-resolution physical map of human chromosome 13 DNA (approximately 114 Mb) from hybridization, PCR, and FISH mapping data using a specifically designed set of computer programs. Although the mapping of 13p is limited, 13q (approximately 98 Mb) is covered by an almost continuous contig of 736 YACs aligned to 597 contigs of cosmids. Of a total of 10,789 cosmids initially selected from a chromosome 13-specific cosmid library (16,896 colonies) using inter-Alu PCR probes from the YACs and probes for markers mapped to chromosome 13, 511 were assembled in contigs that were established from cross-hybridization relationships between the cosmids. The 13q YAC-cosmid map was annotated with 655 sequence tagged sites (STSs) with an average spacing of 1 STS per 150 kb. This set of STSs, each identified by a D number and cytogenetic location, includes database markers (198), expressed sequence tags (93), and STSs generated by sequencing of the ends of cosmid inserts (364). Additional annotation has been provided by positioning 197 cosmids mapped by FISH on 13q. The final (comprehensive) map, a list of STS primers, and raw data used in map assembly are available at our Web site (genome1.ccc.columbia.edu/ approximately genome/) and can serve as a resource to facilitate accurate localization of additional markers, provide substrates for sequencing, and assist in the discovery of chromosome 13 genes associated with hereditary diseases.
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X chromosome control of meiotic chromosome synapsis in mouse inter-subspecific hybrids.
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Tanmoy Bhattacharyya
2014-02-01
Full Text Available Hybrid sterility (HS belongs to reproductive isolation barriers that safeguard the integrity of species in statu nascendi. Although hybrid sterility occurs almost universally among animal and plant species, most of our current knowledge comes from the classical genetic studies on Drosophila interspecific crosses or introgressions. With the house mouse subspecies Mus m. musculus and Mus m. domesticus as a model, new research tools have become available for studies of the molecular mechanisms and genetic networks underlying HS. Here we used QTL analysis and intersubspecific chromosome substitution strains to identify a 4.7 Mb critical region on Chromosome X (Chr X harboring the Hstx2 HS locus, which causes asymmetrical spermatogenic arrest in reciprocal intersubspecific F1 hybrids. Subsequently, we mapped autosomal loci on Chrs 3, 9 and 13 that can abolish this asymmetry. Combination of immunofluorescent visualization of the proteins of synaptonemal complexes with whole-chromosome DNA FISH on pachytene spreads revealed that heterosubspecific, unlike consubspecific, homologous chromosomes are predisposed to asynapsis in F1 hybrid male and female meiosis. The asynapsis is under the trans- control of Hstx2 and Hst1/Prdm9 hybrid sterility genes in pachynemas of male but not female hybrids. The finding concurred with the fertility of intersubpecific F1 hybrid females homozygous for the Hstx2(Mmm allele and resolved the apparent conflict with the dominance theory of Haldane's rule. We propose that meiotic asynapsis in intersubspecific hybrids is a consequence of cis-acting mismatch between homologous chromosomes modulated by the trans-acting Hstx2 and Prdm9 hybrid male sterility genes.