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Sample records for open gene dna

  1. DNA Copy Number Variants of Known Glaucoma Genes in Relation to Primary Open-Angle Glaucoma

    Science.gov (United States)

    Liu, Yutao; Garrett, Melanie E.; Yaspan, Brian L.; Bailey, Jessica Cooke; Loomis, Stephanie J.; Brilliant, Murray; Budenz, Donald L.; Christen, William G.; Fingert, John H.; Gaasterland, Douglas; Gaasterland, Terry; Kang, Jae H.; Lee, Richard K.; Lichter, Paul; Moroi, Sayoko E.; Realini, Anthony; Richards, Julia E.; Schuman, Joel S.; Scott, William K.; Singh, Kuldev; Sit, Arthur J.; Vollrath, Douglas; Weinreb, Robert; Wollstein, Gadi; Zack, Donald J.; Zhang, Kang; Pericak-Vance, Margaret A.; Haines, Jonathan L.; Pasquale, Louis R.; Wiggs, Janey L.; Allingham, R. Rand; Ashley-Koch, Allison E.; Hauser, Michael A.

    2014-01-01

    Purpose. We examined the role of DNA copy number variants (CNVs) of known glaucoma genes in relation to primary open angle glaucoma (POAG). Methods. Our study included DNA samples from two studies (NEIGHBOR and GLAUGEN). All the samples were genotyped with the Illumina Human660W_Quad_v1 BeadChip. After removing non–blood-derived and amplified DNA samples, we applied quality control steps based on the mean Log R Ratio and the mean B allele frequency. Subsequently, data from 3057 DNA samples (1599 cases and 1458 controls) were analyzed with PennCNV software. We defined CNVs as those ≥5 kilobases (kb) in size and interrogated by ≥5 consecutive probes. We further limited our investigation to CNVs in known POAG-related genes, including CDKN2B-AS1, TMCO1, SIX1/SIX6, CAV1/CAV2, the LRP12-ZFPM2 region, GAS7, ATOH7, FNDC3B, CYP1B1, MYOC, OPTN, WDR36, SRBD1, TBK1, and GALC. Results. Genomic duplications of CDKN2B-AS1 and TMCO1 were each found in a single case. Two cases carried duplications in the GAS7 region. Genomic deletions of SIX6 and ATOH7 were each identified in one case. One case carried a TBK1 deletion and another case carried a TBK1 duplication. No controls had duplications or deletions in these six genes. A single control had a duplication in the MYOC region. Deletions of GALC were observed in five cases and two controls. Conclusions. The CNV analysis of a large set of cases and controls revealed the presence of rare CNVs in known POAG susceptibility genes. Our data suggest that these rare CNVs may contribute to POAG pathogenesis and merit functional evaluation. PMID:25414181

  2. An 18.3 kb DNA fragment from yeast chromosome VII carries four unknown open reading frames, the gene for an Asn synthase, remnants of Ty and three tRNA genes.

    Science.gov (United States)

    Van Dyck, L; Tettelin, H; Purnelle, B; Goffeau, A

    1997-02-01

    An 18.3 kb DNA segment from yeast Saccharomyces cerevisiae VII encompasses the previously characterized MEP1, NUP57 and PPT1 genes as well as seven new open reading frames (ORFs) of at least 100 residues. G6358 is an ubiquitous glutamine-dependent asparagine synthase. G6362 is membrane protein highly homologous to a protein of unknown function in the yeast Schizosaccharomyces pombe. Three ORFs (G6324, G6335 and G6365) have no significant homology with previously reported proteins of characteristic motifs. G6321 and G6359, enclosed in longer ORFs, are not likely to be coding. The segment also contains tRNA genes for Asn, Arg and Ile as well as sigma element and two solo deltas. ORFs and genetic elements are named according to a preliminary working nomenclature.

  3. DNA fusion gene vaccines

    DEFF Research Database (Denmark)

    Holst, Peter Johannes; Bassi, Maria Rosaria; Thomsen, Allan Randrup

    2010-01-01

    DNA vaccines are versatile and safe, but limited immunogenicity has prevented their use in the clinical setting. Experimentally, immunogenicity may be enhanced by the use of new delivery technologies, by coadministration of cytokines and pathogen-associated molecular patterns, or by fusion...... of antigens into molecular domains that enhance antigen presentation. More specifically, the immunogenicity of DNA vaccines may benefit from increased protein synthesis, increased T-cell help and MHC class I presentation, and the addition of a range of specific cytokines and pathogen-associated molecular...... patterns that increase activation of the innate immune system. Importantly, viral-vectored vaccines that act through the induction of one or more of these factors also may benefit from cytokine coadministration and increased antigen presentation. In order to increase immunogenicity to the level achieved...

  4. Opening up the DNA methylome of dementia.

    Science.gov (United States)

    Delgado-Morales, R; Esteller, M

    2017-01-03

    Dementia is a complex clinical condition characterized by several cognitive impairments that interfere with patient independence in executing everyday tasks. Various neurodegenerative disorders have dementia in common among their clinical manifestations. In addition, these diseases, such as Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies and frontotemporal dementia, share molecular alterations at the neuropathological level. In recent years, the field of neuroepigenetics has expanded massively and it is now clear that epigenetic processes, such as DNA methylation, are mechanisms involved in both normal and pathological brain function. Despite the persistent methodological and conceptual caveats, it has been reported that several genes fundamental to the development of neurodegenerative disorders are deregulated by aberrant methylation patterns of their promoters, and even common epigenetic signatures for some dementia-associated pathologies have been identified. Therefore, understanding the epigenetic mechanisms that are altered in dementia, especially those associated with the initial phases, will allow us not only to understand the etiopathology of dementia and its progression but also to design effective therapies to reduce this global public health problem. This review provides an in-depth summary of our current knowledge about DNA methylation in dementia, focusing exclusively on the analyses performed in human brain.Molecular Psychiatry advance online publication, 3 January 2017; doi:10.1038/mp.2016.242.

  5. Sensing DNA Opening in Transcription Using Quenchable Förster Resonance Energy Transfer

    NARCIS (Netherlands)

    Cordes, Thorben; Santoso, Yusdi; Tomescu, Alexandra I.; Gryte, Kristofer; Hwang, Ling Chin; Camará, Beatriz; Wigneshweraraj, Sivaramesh; Kapanidis, Achillefs N.

    2010-01-01

    Many biological processes, such as gene transcription and replication, involve opening and closing of short regions of double-stranded DNA (dsDNA). Few techniques, however, can study these processes in real time or at the single-molecule level. Here, we present a Förster resonance energy transfer (F

  6. Discovery of metabolically stabilized electronegative polyacridine-PEG peptide DNA open polyplexes.

    Science.gov (United States)

    Fernandez, Christian A; Baumhover, Nicholas J; Anderson, Kevin; Rice, Kevin G

    2010-04-21

    Cationic condensing peptides and polymers bind electrostatically to DNA to form cationic polyplexes. While many cationic polyplexes are able to achieve in vitro transfection mediated through electrostatic interactions, few have been able to mediate gene transfer in vivo. The present study describes the development and testing of polyacridine PEG-peptides that bind to plasmid DNA by intercalation resulting in electronegative open polyplex DNA. Polyacridine PEG-peptides were prepared by chemically conjugating 6-(9-acridinylamino) hexanoic acid onto side chains of Lys in PEG-Cys-Trp-(Lys)(3, 4, or 5). The resulting PEG-Cys-Trp-(Lys-(Acr))(3, 4, or 5) peptides bound tightly to DNA by polyintercalation, rather than electrostatic binding. Unlike polycationic polyplexes, polyacridine PEG-peptide polyplexes were anionic and open coiled, as revealed by zeta potential and atomic force microscopy. PEG-Cys-Trp-(Lys-(Acr))(5) showed the highest DNA binding affinity and the greatest ability to protect DNA from metabolism by DNase. Polyacridine PEG-peptide DNA open polyplexes were dosed intramuscularly and electroporated in mice to demonstrate their functional activity in gene transfer. These results establish polyacridine PEG-peptide DNA open polyplexes as a novel gene delivery method for in vivo use.

  7. Filtering "genic" open reading frames from genomic DNA samples for advanced annotation

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    Sblattero Daniele

    2011-06-01

    Full Text Available Abstract Background In order to carry out experimental gene annotation, DNA encoding open reading frames (ORFs derived from real genes (termed "genic" in the correct frame is required. When genes are correctly assigned, isolation of genic DNA for functional annotation can be carried out by PCR. However, not all genes are correctly assigned, and even when correctly assigned, gene products are often incorrectly folded when expressed in heterologous hosts. This is a problem that can sometimes be overcome by the expression of protein fragments encoding domains, rather than full-length proteins. One possible method to isolate DNA encoding such domains would to "filter" complex DNA (cDNA libraries, genomic and metagenomic DNA for gene fragments that confer a selectable phenotype relying on correct folding, with all such domains present in a complex DNA sample, termed the “domainome”. Results In this paper we discuss the preparation of diverse genic ORF libraries from randomly fragmented genomic DNA using ß-lactamase to filter out the open reading frames. By cloning DNA fragments between leader sequences and the mature ß-lactamase gene, colonies can be selected for resistance to ampicillin, conferred by correct folding of the lactamase gene. Our experiments demonstrate that the majority of surviving colonies contain genic open reading frames, suggesting that ß-lactamase is acting as a selectable folding reporter. Furthermore, different leaders (Sec, TAT and SRP, normally translocating different protein classes, filter different genic fragment subsets, indicating that their use increases the fraction of the “domainone” that is accessible. Conclusions The availability of ORF libraries, obtained with the filtering method described here, combined with screening methods such as phage display and protein-protein interaction studies, or with protein structure determination projects, can lead to the identification and structural determination of

  8. Single gene retrieval from thermally degraded DNA

    Indian Academy of Sciences (India)

    Lianwen Zhang; Lianwen Zhang

    2005-12-01

    To simulate single gene retrieval from ancient DNA, several related factors have been investigated. By monitoring a 889 bp polymerase chain reaction (PCR) product and genomic DNA degradation, we find that heat and oxygen (especially heat) are both crucial factors influencing DNA degradation. The heat influence, mainly represented by temperature and heating time, affects the DNA degradation via DNA depurination followed by cleavage of nearby phosphodiesters. The heating time influence is temperature-dependent. By reactive oxygen species (ROS) scavenging and 1,3-diphenyl-isobenzofuran (DPBF) bleaching experiments the influence of oxygen on DNA thermal degradation was shown to occur via a singlet oxygen pathway. A comparative study of the thermal degradation of cellular DNA and isolated DNA showed that cellular lipids can aggravate DNA thermal degradation. These results confirm the possibility of gene amplification from thermally degraded DNA. They can be used to evaluate the feasibility of the retrieval of single gene from ancient remains.

  9. Primary open-angle glaucoma genes

    OpenAIRE

    Fingert, J.H.

    2011-01-01

    A substantial fraction of glaucoma has a genetic basis. About 5% of primary open angle glaucoma (POAG) is currently attributed to single-gene or Mendelian forms of glaucoma (ie glaucoma caused by mutations in myocilinor optineurin). Mutations in these genes have a high likelihood of leading to glaucoma and are rarely seen in normal subjects. Other cases of POAG have a more complex genetic basis and are caused by the combined effects of many genetic and environmental risk factors, each of whic...

  10. [Patentability of DNA sequences: the debate remains open].

    Science.gov (United States)

    Martín Uranga, Amelia

    2013-01-01

    The patentability of human genes was from the beginning of the discussion concerning the Directive on the legal protection of biotechnological inventions, an issue that provoked debates among politicians, scientists, lawyers and civil society itself. Although Directive 98/44 tried to settle the matter by stating that to support the patentability of human genes, it should know what role they fulfill, which protein they encode, all of this as an essential requirement to test its industrial application. However, following the judgment of 13 June 2013 (Supreme Court of the United States of America in the case of Association for Molecular Pathology et al. versus Myriad Genetics Inc.) the debate on this issue has been reopened. There are several issues to be considered, taking into account that the patents on DNA & Gene Sequences have played an important incentive to increase the interest in biotechnology applied to human health. On the other hand, this is a paradigm shift in the R & D of biopharmaceutical companies, and it has moved from an in house research model to a model of open innovation, a model of collaboration between large corporations with biotech SMEs and public and private research centers. This model of innovation, impacts on the issue of the industrial property, and therefore it will be necessary to clearly define what each party brings to the relationship and how they are expected to share the results. But all of this, with the ultimate goal that the patients have access to treatments and medications most innovative, safe and effective.

  11. Open source and DIY hardware for DNA nanotechnology labs.

    Science.gov (United States)

    Damase, Tulsi R; Stephens, Daniel; Spencer, Adam; Allen, Peter B

    A set of instruments and specialized equipment is necessary to equip a laboratory to work with DNA. Reducing the barrier to entry for DNA manipulation should enable and encourage new labs to enter the field. We present three examples of open source/DIY technology with significantly reduced costs relative to commercial equipment. This includes a gel scanner, a horizontal PAGE gel mold, and a homogenizer for generating DNA-coated particles. The overall cost savings obtained by using open source/DIY equipment was between 50 and 90%.

  12. Open source and DIY hardware for DNA nanotechnology labs

    Directory of Open Access Journals (Sweden)

    Tulsi R. Damase

    2015-08-01

    Full Text Available A set of instruments and specialized equipment is necessary to equip a laboratory to work with DNA. Reducing the barrier to entry for DNA manipulation should enable and encourage new labs to enter the field. We present three examples of open source/DIY technology with significantly reduced costs relative to commercial equipment. This includes a gel scanner, a horizontal PAGE gel mold, and a homogenizer for generating DNA-coated particles. The overall cost savings obtained by using open source/DIY equipment was between 50 and 90%.

  13. DNA vaccination of pigs with open reading frame 1-7 of PRRS virus

    DEFF Research Database (Denmark)

    Barfoed, Annette Malene; Blixenkrone-Møller, Merete; Jensen, Merethe Holm

    2004-01-01

    We cloned all open reading frames of a Danish isolate of porcine reproductive and respiratory syndrome (PRRS) virus in DNA vaccination vectors. Pigs were vaccinated using a gene gun with each single construct (ORF1, ORF2, ORF3, ORF4, ORF5, ORF6, or ORF7) or combinations thereof. Vaccination...

  14. The DNA sequence of a 7941 bp fragment of the left arm of chromosome VII of Saccharomyces cerevisiae contains four open reading frames including the multicopy suppressor gene of the pop2 mutation and a putative serine/threonine protein kinase gene.

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    Coglievina, M; Bertani, I; Klima, R; Zaccaria, P; Bruschi, C V

    1995-06-30

    We report the sequence of a 7941 bp DNA fragment from the left arm of chromosome VII of Saccharomyces cerevisiae which contains four open reading frames (ORFs) of greater than 100 amino acid residues. ORF biC834 shows 100% bp identity with the recently identified multicopy suppressor gene of the pop2 mutation (MPT5); its deduced protein product carries an eight-repeat domain region, homologous to that found in the hypothetical regulatory YGL023 protein of S. cerevisiae and the Pumilio protein of Drosophila. ORF biE560 protein exhibits patterns typical of serine/threonine protein kinases, with which it shares high degrees of homology.

  15. Sites and gene products involved in lambdoid phage DNA packaging.

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    Smith, M P; Feiss, M

    1993-04-01

    21 is a temperate lambdoid coliphage, and the genes that encode the head proteins of lambda and 21 are descended from a common ancestral bacteriophage. The sequencing of terminase genes 1 and 2 of 21 was completed, along with that of a segment at the right end of 21 DNA that includes the R4 sequence. The R4 sequence, a site that is likely involved in termination of DNA packaging, was found to be very similar to the R4 sequences of lambda and phi 80, suggesting that R4 is a recognition site that is not phage specific. DNA packaging by 21 is dependent on a host protein, integration host factor. A series of mutations in gene 1 (her mutations), which allow integration host factor-independent DNA packaging by 21, were found to be missense changes that affect predicted alpha-helixes in gp1. gp2, the large terminase subunit, is predicted to contain an ATP-binding domain and, perhaps, a second domain important for the cos-cutting activity of terminase. orf1, an open reading frame analogous in position to FI, a lambda gene involved in DNA packaging, shares some sequence identity with FI. orf1 was inactivated with nonsense and insertion mutations; these mutations were found not to affect phage growth. 21 was also not able to complement a lambda FI mutant.

  16. Coordinate expression of Escherichia coli dnaA and dnaN genes.

    Science.gov (United States)

    Sako, T; Sakakibara, Y

    1980-01-01

    The defects of temperature-sensitive dnaA and dnaN mutants of Escherichia coli are complemented by a recombinant lambda phage, which carries the bacterial DNA segment composed of two EcoRI segments of 1.0 and 3.3 kilobases. Derivatives of the phage, which have an insertion segment of Tn3 in the dnaA gene, are much less active in expressing the dnaN gene function than the parent phage. The dnaN gene activity was determined as the efficiency of superinfecting phage to suppress loss of the viability of lambda lysogenic dnaN59 cells at the non-permissive temperature. Deletions that include the end of the dnaA gene distal to the dnaN gene also reduce the expression of the dnaN gene function. Deletion and insertion in the dnaN gene do not affect the expression of the dnaA gene function. The expression of the dnaN gene function by the dnaA- dnaN+ phages remains weak upon simultaneous infection with dnaA+ dnaN- phages. Thus the insertion and deletion of the dnaA gene influence in cis the expresion of the dnaN gene. We propose that the dnaA and dnaN genes constitute an operon, where the former is upstream to the latter.

  17. NMR analysis of base-pair opening kinetics in DNA

    Science.gov (United States)

    Szulik, Marta W.; Voehler, Markus; Stone, Michael P.

    2014-01-01

    Base pairing in nucleic acids plays a crucial role in their structure and function. Differences in the base pair opening and closing kinetics of individual double stranded DNA sequences or between chemically modified base pairs provide insight into the recognition of these base pairs by DNA processing enzymes. This unit describes how to quantify the kinetics for localized base pairs by observing changes in the imino proton signals by nuclear magnetic resonance spectroscopy. The determination of all relevant parameters using state of the art techniques and NMR instrumentation, including cryoprobes, is discussed. PMID:25501592

  18. Identification of the dnaA and dnaN gene products of Escherichia coli.

    Science.gov (United States)

    Yuasa, S; Sakakibara, Y

    1980-01-01

    A specialized transducing lambda phage carrying the dnaN genes of Escherichia coli specifies two proteins of about 41 and 48 kilodaltons (kd). The temperature-sensitive mutations, dnaN59 and dnaA167, were found to result in altered isoelectric points of the 41 and 48 kd proteins, respectively. Thus the dnaN gene product was identified as a weakly acidic 41 and 48 kd protein. The synthesis of the dnaN gene product is greatly reduced by insertion of a transposon Tn3 in the dnaA gene and by deletion in the gene at the distal end to the dnaN gene. Temperature-sensitive dnaA mutations, on the dnaN gene product. These results indicate that the synthesis of the dnaN gene product is dependent on the structural integrity of the dnaA gene.

  19. DNA repair genes in the Megavirales pangenome.

    Science.gov (United States)

    Blanc-Mathieu, Romain; Ogata, Hiroyuki

    2016-06-01

    The order 'Megavirales' represents a group of eukaryotic viruses with a large genome encoding a few hundred up to two thousand five hundred genes. Several members of Megavirales possess genes involved in major DNA repair pathways. Some of these genes were likely inherited from an ancient virus world and some others were derived from the genomes of their hosts. Here we examine molecular phylogenies of key DNA repair enzymes in light of recent hypotheses on the origin of Megavirales, and propose that the last common ancestors of the individual families of the order Megavirales already possessed DNA repair functions to achieve and maintain a moderately large genome and that this repair capacity gradually increased, in a family-dependent manner, during their recent evolution.

  20. A superfamily of DNA transposons targeting multicopy small RNA genes.

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    Kenji K Kojima

    Full Text Available Target-specific integration of transposable elements for multicopy genes, such as ribosomal RNA and small nuclear RNA (snRNA genes, is of great interest because of the relatively harmless nature, stable inheritance and possible application for targeted gene delivery of target-specific transposable elements. To date, such strict target specificity has been observed only among non-LTR retrotransposons. We here report a new superfamily of sequence-specific DNA transposons, designated Dada. Dada encodes a DDE-type transposase that shows a distant similarity to transposases encoded by eukaryotic MuDR, hAT, P and Kolobok transposons, as well as the prokaryotic IS256 insertion element. Dada generates 6-7 bp target site duplications upon insertion. One family of Dada DNA transposons targets a specific site inside the U6 snRNA genes and are found in various fish species, water flea, oyster and polycheate worm. Other target sequences of the Dada transposons are U1 snRNA genes and different tRNA genes. The targets are well conserved in multicopy genes, indicating that copy number and sequence conservation are the primary constraints on the target choice of Dada transposons. Dada also opens a new frontier for target-specific gene delivery application.

  1. Organization and transcription of the dnaA and dnaN genes of Escherichia coli.

    Science.gov (United States)

    Sakakibara, Y; Tsukano, H; Sako, T

    1981-01-01

    The locations of the linked dnaA and dnaN genes of Escherichia coli in a specialized transducing lambda phage genome have been determined by electron microscopic heteroduplex analysis, using phages with deletions or insertions in the dnaA or dnaN gene. The transcription initiation sites for the dna genes were also localized by electron microscopic analysis of DNA-RBA heteroduplex molecules formed between the E. coli DNA fragment of the phage genome and the in vitro transcription products of the fragment. The dnaN gene was found to be transcribed in the same direction as the dnaA gene, and predominantly from the promoter of the dnaA gene.

  2. Differential expression of genes identified by suppression subtractive hybridization in petals of opening carnation flowers.

    Science.gov (United States)

    Harada, Taro; Torii, Yuka; Morita, Shigeto; Masumura, Takehiro; Satoh, Shigeru

    2010-05-01

    Flower opening is an event accompanied by morphological changes in petals which include elongation, expansion, and outward-curving. Petal cell growth is a fundamental process that underlies such phenomena, but its molecular mechanism remains largely unknown. Suppression subtractive hybridization was performed between petals during the early elongation period (stage 1) and during the opening period (stage 5) in carnation flowers and a pair of subtraction libraries abundant in differentially expressed genes was constructed at each stage. 393 cDNA clones picked up by differential screening out of 1728 clones were sequenced and 235 different cDNA fragments were identified, among which 211 did not match any known nucleotide sequence of carnation genes in the databases. BLASTX search of nucleotide sequences revealed that putative functions of the translational products can be classified into several categories including transcription, signalling, cell wall modification, lipid metabolism, and transport. Open reading frames of 15 selected genes were successfully determined by rapid amplification of cDNA ends (RACE). Time-course analysis of these genes by real-time RT-PCR showed that transcript levels of several genes correlatively fluctuate in petals of opening carnation flowers, suggesting an association with the morphological changes by elongation or curving. Based on the results, it is suggested that the growth of carnation petals is controlled by co-ordinated gene expression during the progress of flower opening. In addition, the possible roles of some key genes in the initiation of cell growth, the construction of the cell wall and cuticle, and transport across membranes were discussed.

  3. Structural analysis of the dnaA and dnaN genes of Escherichia coli.

    Science.gov (United States)

    Ohmori, H; Kimura, M; Nagata, T; Sakakibara, Y

    1984-05-01

    The nucleotide sequence of the entire region containing the Escherichia coli dnaA and dnaN genes was determined. Base substitutions by such mutations as dnaA46, dnaA167, dnaN59, and dnaN806 were also identified. Analyses of coding frames, the mutational base substitutions, and other data indicate that dnaN follows dnaA, both have the same orientation, and are separated by only 4 bp. The deduced amino acid sequence specifies Mrs and isoelectric points consistent with those of the previously identified gene products. The transcriptional initiation site of the dnaA gene was assigned by analysis of in vitro RNA products. Examination of the intercistronic sequence and analysis of in vitro transcription supported the notion that the dnaA and dnaN genes constitute a single operon.

  4. Discoordinate gene expression in the dnaA-dnaN operon of Escherichia coli.

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    Quiñones, A; Messer, W

    1988-07-01

    The dnaN gene of Escherichia coli encodes the beta-subunit of the DNA polymerase III holoenzyme. Previous work has established that dnaN lies immediately downstream of dnaA and that both genes may be cotranscribed from the dnaA promoters; no promoter for dnaN has been described. We investigated the in vivo regulation of transcription of the dnaN gene by transcriptional fusions to the galK gene, translational fusion to the lacZ gene and S1 mapping analysis. We found that there are at least three dnaN promoters residing entirely in the reading frame of the preceding dnaA gene, and that transcription from these promoters can occur independently of dnaA transcription which, however, extends at least up to dnaN. Furthermore, we found evidence for the inducibility of the dnaN promoters in a dam background under conditions of simultaneously reduced dnaA transcription. These results are consistent with the hypothesis that although dnaA and dnaN are organized in an operon considerable discoordinate transcription can occur, thus uncoupling dnaN and dnaA regulation, when needed.

  5. The first determination of DNA sequence of a specific gene.

    Science.gov (United States)

    Inouye, Masayori

    2016-05-10

    How and when the first DNA sequence of a gene was determined? In 1977, F. Sanger came up with an innovative technology to sequence DNA by using chain terminators, and determined the entire DNA sequence of the 5375-base genome of bacteriophage φX 174 (Sanger et al., 1977). While this Sanger's achievement has been recognized as the first DNA sequencing of genes, we had determined DNA sequence of a gene, albeit a partial sequence, 11 years before the Sanger's DNA sequence (Okada et al., 1966).

  6. Genome analysis of DNA repair genes in the alpha proteobacterium Caulobacter crescentus

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    Menck Carlos FM

    2007-03-01

    Full Text Available Abstract Background The integrity of DNA molecules is fundamental for maintaining life. The DNA repair proteins protect organisms against genetic damage, by removal of DNA lesions or helping to tolerate them. DNA repair genes are best known from the gamma-proteobacterium Escherichia coli, which is the most understood bacterial model. However, genome sequencing raises questions regarding uniformity and ubiquity of these DNA repair genes and pathways, reinforcing the need for identifying genes and proteins, which may respond to DNA damage in other bacteria. Results In this study, we employed a bioinformatic approach, to analyse and describe the open reading frames potentially related to DNA repair from the genome of the alpha-proteobacterium Caulobacter crescentus. This was performed by comparison with known DNA repair related genes found in public databases. As expected, although C. crescentus and E. coli bacteria belong to separate phylogenetic groups, many of their DNA repair genes are very similar. However, some important DNA repair genes are absent in the C. crescentus genome and other interesting functionally related gene duplications are present, which do not occur in E. coli. These include DNA ligases, exonuclease III (xthA, endonuclease III (nth, O6-methylguanine-DNA methyltransferase (ada gene, photolyase-like genes, and uracil-DNA-glycosylases. On the other hand, the genes imuA and imuB, which are involved in DNA damage induced mutagenesis, have recently been described in C. crescentus, but are absent in E. coli. Particularly interesting are the potential atypical phylogeny of one of the photolyase genes in alpha-proteobacteria, indicating an origin by horizontal transfer, and the duplication of the Ada orthologs, which have diverse structural configurations, including one that is still unique for C. crescentus. Conclusion The absence and the presence of certain genes are discussed and predictions are made considering the particular

  7. Brief Guide to Genomics: DNA, Genes and Genomes

    Science.gov (United States)

    ... Breve guía de genómica A Brief Guide to Genomics DNA, Genes and Genomes Deoxyribonucleic acid (DNA) is ... genetic basis for health and disease. Implications of Genomics for Medical Science Virtually every human ailment has ...

  8. Structure of an 'open' clamp type II topoisomerase-DNA complex provides a mechanism for DNA capture and transport.

    Science.gov (United States)

    Laponogov, Ivan; Veselkov, Dennis A; Crevel, Isabelle M-T; Pan, Xiao-Su; Fisher, L Mark; Sanderson, Mark R

    2013-11-01

    Type II topoisomerases regulate DNA supercoiling and chromosome segregation. They act as ATP-operated clamps that capture a DNA duplex and pass it through a transient DNA break in a second DNA segment via the sequential opening and closure of ATPase-, G-DNA- and C-gates. Here, we present the first 'open clamp' structures of a 3-gate topoisomerase II-DNA complex, the seminal complex engaged in DNA recognition and capture. A high-resolution structure was solved for a (full-length ParE-ParC55)2 dimer of Streptococcus pneumoniae topoisomerase IV bound to two DNA molecules: a closed DNA gate in a B-A-B form double-helical conformation and a second B-form duplex associated with closed C-gate helices at a novel site neighbouring the catalytically important β-pinwheel DNA-binding domain. The protein N gate is present in an 'arms-wide-open' state with the undimerized N-terminal ParE ATPase domains connected to TOPRIM domains via a flexible joint and folded back allowing ready access both for gate and transported DNA segments and cleavage-stabilizing antibacterial drugs. The structure shows the molecular conformations of all three gates at 3.7 Å, the highest resolution achieved for the full complex to date, and illuminates the mechanism of DNA capture and transport by a type II topoisomerase.

  9. Genetic spell-checking: gene editing using single-stranded DNA oligonucleotides.

    Science.gov (United States)

    Rivera-Torres, Natalia; Kmiec, Eric B

    2016-02-01

    Single-stranded oligonucleotides (ssODNs) can be used to direct the exchange of a single nucleotide or the repair of a single base within the coding region of a gene in a process that is known, generically, as gene editing. These molecules are composed of either all DNA residues or a mixture of RNA and DNA bases and utilize inherent metabolic functions to execute the genetic alteration within the context of a chromosome. The mechanism of action of gene editing is now being elucidated as well as an understanding of its regulatory circuitry, work that has been particularly important in establishing a foundation for designing effective gene editing strategies in plants. Double-strand DNA breakage and the activation of the DNA damage response pathway play key roles in determining the frequency with which gene editing activity takes place. Cellular regulators respond to such damage and their action impacts the success or failure of a particular nucleotide exchange reaction. A consequence of such activation is the natural slowing of replication fork progression, which naturally creates a more open chromatin configuration, thereby increasing access of the oligonucleotide to the DNA template. Herein, how critical reaction parameters influence the effectiveness of gene editing is discussed. Functional interrelationships between DNA damage, the activation of DNA response pathways and the stalling of replication forks are presented in detail as potential targets for increasing the frequency of gene editing by ssODNs in plants and plant cells.

  10. Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by Oligomerized Pool ENgineering (OPEN.

    Directory of Open Access Journals (Sweden)

    Jonathan E Foley

    Full Text Available BACKGROUND: Customized zinc finger nucleases (ZFNs form the basis of a broadly applicable tool for highly efficient genome modification. ZFNs are artificial restriction endonucleases consisting of a non-specific nuclease domain fused to a zinc finger array which can be engineered to recognize specific DNA sequences of interest. Recent proof-of-principle experiments have shown that targeted knockout mutations can be efficiently generated in endogenous zebrafish genes via non-homologous end-joining-mediated repair of ZFN-induced DNA double-stranded breaks. The Zinc Finger Consortium, a group of academic laboratories committed to the development of engineered zinc finger technology, recently described the first rapid, highly effective, and publicly available method for engineering zinc finger arrays. The Consortium has previously used this new method (known as OPEN for Oligomerized Pool ENgineering to generate high quality ZFN pairs that function in human and plant cells. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that OPEN can also be used to generate ZFNs that function efficiently in zebrafish. Using OPEN, we successfully engineered ZFN pairs for five endogenous zebrafish genes: tfr2, dopamine transporter, telomerase, hif1aa, and gridlock. Each of these ZFN pairs induces targeted insertions and deletions with high efficiency at its endogenous gene target in somatic zebrafish cells. In addition, these mutations are transmitted through the germline with sufficiently high frequency such that only a small number of fish need to be screened to identify founders. Finally, in silico analysis demonstrates that one or more potential OPEN ZFN sites can be found within the first three coding exons of more than 25,000 different endogenous zebrafish gene transcripts. CONCLUSIONS AND SIGNIFICANCE: In summary, our study nearly triples the total number of endogenous zebrafish genes successfully modified using ZFNs (from three to eight and suggests that OPEN

  11. Sequence and transcription analysis of the human cytomegalovirus DNA polymerase gene

    Energy Technology Data Exchange (ETDEWEB)

    Kouzarides, T.; Bankier, A.T.; Satchwell, S.C.; Weston, K.; Tomlinson, P.; Barrell, B.G.

    1987-01-01

    DNA sequence analysis has revealed that the gene coding for the human cytomegalovirus (HCMV) DNA polymerase is present within the long unique region of the virus genome. Identification is based on extensive amino acid homology between the predicted HCMV open reading frame HFLF2 and the DNA polymerase of herpes simplex virus type 1. The authors present here a 5280 base-pair DNA sequence containing the HCMV pol gene, along with the analysis of transcripts encoded within this region. Since HCMV pol also shows homology to the predicted Epstein-Barr virus pol, they were able to analyze the extent of homology between the DNA polymerases of three distantly related herpes viruses, HCMV, Epstein-Barr virus, and herpes simplex virus. The comparison shows that these DNA polymerases exhibit considerable amino acid homology and highlights a number of highly conserved regions; two such regions show homology to sequences within the adenovirus type 2 DNA polymerase. The HCMV pol gene is flanked by open reading frames with homology to those of other herpes viruses; upstream, there is a reading frame homologous to the glycoprotein B gene of herpes simplex virus type I and Epstein-Barr virus, and downstream there is a reading frame homologous to BFLF2 of Epstein-Barr virus.

  12. Construction and Nonclinical Testing of a Puumala Virus Synthetic M Gene-Based DNA Vaccine

    Science.gov (United States)

    2012-12-12

    DNA vaccine constructsf Amino acid positiona PUUV M segment amino acid DNA vaccine amino acid DTK/ Ufa -97b K27c P360d Hallnas B1e PUU- M(x22) PUU- M...G 1097 S S S S L L S a M gene segment open reading frame amino acid position. b DTK/ Ufa -97, PUUV strain DTK/ Ufa (GenBank accession no. BAF49040). c

  13. DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing

    Science.gov (United States)

    Nishina, Kazutaka; Piao, Wenying; Yoshida-Tanaka, Kie; Sujino, Yumiko; Nishina, Tomoko; Yamamoto, Tsuyoshi; Nitta, Keiko; Yoshioka, Kotaro; Kuwahara, Hiroya; Yasuhara, Hidenori; Baba, Takeshi; Ono, Fumiko; Miyata, Kanjiro; Miyake, Koichi; Seth, Punit P.; Low, Audrey; Yoshida, Masayuki; Bennett, C. Frank; Kataoka, Kazunori; Mizusawa, Hidehiro; Obika, Satoshi; Yokota, Takanori

    2015-01-01

    Antisense oligonucleotides (ASOs) are recognized therapeutic agents for the modulation of specific genes at the post-transcriptional level. Similar to any medical drugs, there are opportunities to improve their efficacy and safety. Here we develop a short DNA/RNA heteroduplex oligonucleotide (HDO) with a structure different from double-stranded RNA used for short interfering RNA and single-stranded DNA used for ASO. A DNA/locked nucleotide acid gapmer duplex with an α-tocopherol-conjugated complementary RNA (Toc-HDO) is significantly more potent at reducing the expression of the targeted mRNA in liver compared with the parent single-stranded gapmer ASO. Toc-HDO also improves the phenotype in disease models more effectively. In addition, the high potency of Toc-HDO results in a reduction of liver dysfunction observed in the parent ASO at a similar silencing effect. HDO technology offers a novel concept of therapeutic oligonucleotides, and the development of this molecular design opens a new therapeutic field. PMID:26258894

  14. Functional cooperation of the dnaE and dnaN gene products in Escherichia coli.

    OpenAIRE

    Kuwabara, N; Uchida, H.

    1981-01-01

    A system was designed to isolate second-site intergenic suppressors of a thermosensitive mutation of the dnaE gene of Escherichia coli. The dnaE gene codes for the alpha subunit of DNA polymerase III [McHenry, C. S. & Crow, W. (1979) J. Biol. Chem. 254, 1748-1753]. One such suppressor, named sueA77, was finely mapped and found to be located at 82 min on the E. coli chromosome, between dnaA and recF, and within the dnaN gene [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553]....

  15. Induction of dnaN and dnaQ gene expression in Escherichia coli by alkylation damage to DNA.

    Science.gov (United States)

    Quiñones, A; Kaasch, J; Kaasch, M; Messer, W

    1989-02-01

    The dnaN and dnaQ genes encode the beta-subunit and the epsilon-subunit of the DNA polymerase III holoenzyme. By transcriptional fusions to the galK gene, translational fusions to lacZ and comparative S1 mapping analysis, we investigated the in-vivo regulation of dnaN and dnaQ. We found that DNA damage caused by the alkylating agent methyl methanesulphonate (MMS) leads to a significant induction in dnaN and dnaQ gene expression suggesting a requirement of increased amounts of at least some DNA polymerase III holoenzyme subunits for recovery from DNA damage caused by MMS. These results are first evidences that subunits of the DNA polymerase III holoenzyme are DNA damage inducible. This MMS induction of dnaN and dnaQ gene expression is unrelated to the adaptive response. It was not observed in lexA and recA mutants which abolish the induction of the SOS response.

  16. Expression of protein-coding genes embedded in ribosomal DNA

    DEFF Research Database (Denmark)

    Johansen, Steinar D; Haugen, Peik; Nielsen, Henrik

    2007-01-01

    Ribosomal DNA (rDNA) is a specialised chromosomal location that is dedicated to high-level transcription of ribosomal RNA genes. Interestingly, rDNAs are frequently interrupted by parasitic elements, some of which carry protein genes. These are non-LTR retrotransposons and group II introns...... that encode reverse transcriptase-like genes, and group I introns and archaeal introns that encode homing endonuclease genes (HEGs). Although rDNA-embedded protein genes are widespread in nuclei, organelles and bacteria, there is surprisingly little information available on how these genes are expressed....... Exceptions include a handful of HEGs from group I introns. Recent studies have revealed unusual and essential roles of group I and group I-like ribozymes in the endogenous expression of HEGs. Here we discuss general aspects of rDNA-embedded protein genes and focus on HEG expression from group I introns...

  17. Yeast DNA sequences initiating gene expression in Escherichia coli.

    Science.gov (United States)

    Lewin, Astrid; Tran, Thi Tuyen; Jacob, Daniela; Mayer, Martin; Freytag, Barbara; Appel, Bernd

    2004-01-01

    DNA transfer between pro- and eukaryotes occurs either during natural horizontal gene transfer or as a result of the employment of gene technology. We analysed the capacity of DNA sequences from a eukaryotic donor organism (Saccharomyces cerevisiae) to serve as promoter region in a prokaryotic recipient (Escherichia coli) by creating fusions between promoterless luxAB genes from Vibrio harveyi and random DNA sequences from S. cerevisiae and measuring the luminescence of transformed E. coli. Fifty-four out of 100 randomly analysed S. cerevisiae DNA sequences caused considerable gene expression in E. coli. Determination of transcription start sites within six selected yeast sequences in E. coli confirmed the existence of bacterial -10 and -35 consensus sequences at appropriate distances upstream from transcription initiation sites. Our results demonstrate that the probability of transcription of transferred eukaryotic DNA in bacteria is extremely high and does not require the insertion of the transferred DNA behind a promoter of the recipient genome.

  18. Epigenetic changes of DNA repair genes in cancer

    Institute of Scientific and Technical Information of China (English)

    Christoph Lahtz; Gerd P. Pfeifer

    2011-01-01

    'Every Hour Hurts, The Last One Kills'. That is an old saying about getting old. Every day, thousands of DNA damaging events take place in each cell of our body, but efficient DNA repair systems have evolved to prevent that. However, our DNA repair system and that of most other organisms are not as perfect as that of Deinococcus radiodurans, for example, which is able to repair massive amounts of DNA damage at one time. In many instances, accumulation of DNA damage has been linked to cancer, and genetic deficiencies in specific DNA repair genes are associated with tumor-prone phenotypes. In addition to mutations, which can be either inherited or somatically acquired, epigenetic silencing of DNA repair genes may promote tumorigenesis. This review will summarize current knowledge of the epigenetic inactivation of different DNA repair components in human cancer.

  19. Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons

    Directory of Open Access Journals (Sweden)

    De Paepe Anne

    2004-02-01

    Full Text Available Abstract Background Activation of proto-oncogenes by DNA amplification is an important mechanism in the development and maintenance of cancer cells. Until recently, identification of the targeted genes relied on labour intensive and time consuming positional cloning methods. In this study, we outline a straightforward and efficient strategy for fast and comprehensive cloning of amplified and overexpressed genes. Results As a proof of principle, we analyzed neuroblastoma cell line IMR-32, with at least two amplification sites along the short arm of chromosome 2. In a first step, overexpressed cDNA clones were isolated using a PCR based subtractive cloning method. Subsequent deposition of these clones on a custom microarray and hybridization with IMR-32 DNA, resulted in the identification of clones that were overexpressed due to gene amplification. Using this approach, amplification of all previously reported amplified genes in this cell line was detected. Furthermore, four additional clones were found to be amplified, including the TEM8 gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences. Conclusions The combinatorial strategy of subtractive cDNA cloning and array CGH analysis allows comprehensive amplicon dissection, which opens perspectives for improved identification of hitherto unknown targeted oncogenes in cancer cells.

  20. A putative helicase, the SUA5, PMR1, tRNALys1 genes and four open reading frames have been detected in the DNA sequence of an 8.8 kb fragment of the left arm of chromosome VII of Saccharomyces cerevisiae.

    Science.gov (United States)

    Klima, R; Coglievina, M; Zaccaria, P; Bertani, I; Bruschi, C V

    1996-09-01

    We report the sequence of an 8.8 kb segment of DNA from the left arm of chromosome VII of Saccharomyces cerevisiae. The sequence reveals seven open reading frames (ORFs) G1651, G1654, G1660, G1663, G1666, G1667 and G1669 greater than 100 amino acids in length and the tRNALys1 gene. ORF G1651 shows 100% identity with the ROK1 protein which is a putative RNA helicase of the 'DEAD box' protein family. ORF G1654 exhibits a motif highly conserved in ATP/GTP binding proteins generally referred to as 'P-loop'. From FastA analysis, G1660 and G1666 were found to be previously sequenced genes, respectively SUA5 and PMR1. The three other ORFs identified are partially (G1663) or completely (G1667 and G1669) overlapping with the PMR1 sequence on the complementary strand. This feature, together with their low codon adaptation indexes and the absence of significant homology with known proteins suggest that they do not correspond to real genes.

  1. DNA sequence, products, and transcriptional pattern of the genes involved in production of the DNA replication inhibitor microcin B17.

    Science.gov (United States)

    Genilloud, O; Moreno, F; Kolter, R

    1989-02-01

    The 3.8-kilobase segment of plasmid DNA that contains the genes required for production of the DNA replication inhibitor microcin B17 was sequenced. The sequence contains four open reading frames which were shown to be translated in vivo by the construction of fusions to lacZ. The location of these open reading frames fits well with the location of the four microcin B17 production genes, mcbABCD, identified previously through genetic complementation. The products of the four genes have been identified, and the observed molecular weights of the proteins agree with those predicted from the nucleotide sequence. The transcription of these genes was studied by using fusions to lacZ and physical mapping of mRNA start sites. Three promoters were identified in this region. The major promoter for all the genes is a growth phase-regulated OmpR-dependent promoter located upstream of mcbA. A second promoter is located within mcbC and is responsible for a low-level basal expression of mcbD. A third promoter, located within mcbD, promotes transcription in the reverse direction starting within mcbD and extending through mcbC. The resulting mRNA appears to be an untranslated antisense transcript that could play a regulatory role in the expression of these genes.

  2. DNA-mediated gene transfer in plant protoplasts

    Energy Technology Data Exchange (ETDEWEB)

    U, Zang Kual; Riu, Key Zung; So, In Sup; Hong, Kyung Ae [Cheju National University, Cheju (Korea, Republic of)

    1994-12-31

    The neomycin phosphotransferase II gene(NPT-II) was introduced into geranium (Pelargonium zonale hybrids) protoplasts by using PEG or electroporation method. The presence of the introduced DNA in the protoplasts and the expressions of the gene in the transformed cells were examined. The presence of the NPT-II DNA in the protoplasts were detected by polymerase chain reaction. The expressions of NPT-II gene in the transformed cells were confirmed by the NPT-II assay. (author)

  3. 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

  4. DNA Ministrings: Highly Safe and Effective Gene Delivery Vectors

    Directory of Open Access Journals (Sweden)

    Nafiseh Nafissi

    2014-01-01

    Full Text Available Conventional plasmid DNA vectors play a significant role in gene therapy, but they also have considerable limitations: they can elicit adverse immune responses because of bacterial sequences they contain for maintenance and amplification in prokaryotes, their bioavailability is compromised because of their large molecular size, and they may be genotoxic. We constructed an in vivo platform to produce ministring DNA—mini linear covalently closed DNA vectors—that are devoid of unwanted bacterial sequences and encode only the gene(s of interest and necessary eukaryotic expression elements. Transfection of rapidly and slowly dividing human cells with ministring DNA coding for enhanced green fluorescent protein resulted in significantly improved transfection, bioavailability, and cytoplasmic kinetics compared with parental plasmid precursors and isogenic circular covalently closed DNA counterparts. Ministring DNA that integrated into the genome of human cells caused chromosomal disruption and apoptotic death of possibly oncogenic vector integrants; thus, they may be safer than plasmid and circular DNA vectors.

  5. A “GC-rich” method for mammalian gene expression:A dominant role of non-coding DNA GC content in the regulation of mammalian gene expression

    Institute of Scientific and Technical Information of China (English)

    Gilbert; Rishton; Matthew; (Mizhou); HUI

    2010-01-01

    High mammalian gene expression was obtained for more than twenty different proteins in different cell types by just a few laboratory scale stable gene transfections for each protein.The stable expression vectors were constructed by inserting a naturally-occurring 1.006 kb or a synthetic 0.733 kb DNA fragment(including intron) of extremely GC-rich at the 5’ or/and 3’ flanking regions of these protein genes or their gene promoters.This experiment is the first experimental evidence showing that a non-coding extremely GC-rich DNA fragment is a super "chromatin opening element" and plays an important role in mammalian gene expression.This experiment has further indicated that chromatin-based regulation of mammalian gene expression is at least partially embedded in DNA primary structure,namely DNA GC-content.

  6. Functional cooperation of the dnaE and dnaN gene products in Escherichia coli.

    Science.gov (United States)

    Kuwabara, N; Uchida, H

    1981-09-01

    A system was designed to isolate second-site intergenic suppressors of a thermosensitive mutation of the dnaE gene of Escherichia coli. The dnaE gene codes for the alpha subunit of DNA polymerase III [McHenry, C. S. & Crow, W. (1979) J. Biol. Chem. 254, 1748-1753]. One such suppressor, named sueA77, was finely mapped and found to be located at 82 min on the E. coli chromosome, between dnaA and recF, and within the dnaN gene [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553]. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme [Burgers, P. M. J., Kornberg, A. & Sakakibara, Y. (1981) Proc. Natl. Acad. Sci. USA 78, 5391-5395]. The sueA77 mutation was trans-dominant over its wild-type allele, and it suppressed different thermosensitive mutations of dnaE with different maximal permissive temperature. These properties were interpreted as providing genetic evidence for interaction of the dnaE and dnaN gene products in E. coli.

  7. Dynamic regulation of cerebral DNA repair genes by psychological stress

    DEFF Research Database (Denmark)

    Forsberg, Kristin; Aalling, Nadia; Wörtwein, Gitta

    2015-01-01

    for maintaining genomic integrity. The aim of the present study was to characterize the pattern of cerebral DNA repair enzyme regulation after stress through the quantification of a targeted range of gene products involved in different types of DNA repair. 72 male Sprague-Dawley rats were subjected to either...... was seen in HC, but with overall smaller effects and without the induction after acute stress. Nuclear DNA damage from oxidation as measured by the comet assay was unaffected by stress in both regions. We conclude that psychological stress have a dynamic influence on brain DNA repair gene expression...

  8. DNA purification and gene typing:Based on multifunctional nanobeads

    Institute of Scientific and Technical Information of China (English)

    XIE Xin; ZHANG Xu; GAO Huafang; ZHANG Huan; CHEN Depu; CHENG Jing; FEI Weiyang

    2004-01-01

    In this report, a universal protocol for extracting genomic DNA from whole blood, saliva, and bacterial culture by using magnetic nanobeads as solid-phase absorbents was presented. The enrichment of target cells and adsorption of DNA have been functionally integrated onto the surfaces of the carboxyl-modified magnetic nano-beads, and the DNA segments bound on the surface of the beads can be directly used as PCR templates to amplify a target gene. The PCR products were applied to an oligonucleotide array to perform gene typing. The protocol proves to be simple, rapid, biologically and chemically nonhazardous, and promising for the microfabrication of DNA preparation chip.

  9. Association between CYP1A1m1 gene polymorphism and primary open-angle glaucoma.

    Science.gov (United States)

    Costa, N B; Silva, C T X; Frare, A B; Silva, R E; Moura, K K V O

    2014-12-04

    The CYP1A1 gene is related to the generation of secondary metabolites that are capable of inducing DNA damage. The CYP1A1m1 polymorphism has been examined in many studies, and is located in a region near loci that have been linked to glaucoma, including the locus GLC1I. As a result, this polymorphism has been related to several diseases that are influenced by exposure to xenobiotic as well as primary open-angle glaucoma. We compared the prevalence of the CYP1A1m1 polymorphism in 152 Brazilian patients, 100 patients with primary open-angle glaucoma, and 52 normal controls using restriction fragment length polymorphism analysis. The frequency of the homozygous wild-type (w1/w1) CYP1A1 gene among patients with primary open-angle glaucoma (N = 100) was 16%, for genotype w1/m1, the frequency was 77%, and for m1/m1 it was 7%. Among the control group (N = 52), the frequency of the homozygous wild-type (w1/w1) CYP1A1 gene was 54%, the frequency of w1/m1 was 46%, and the frequency of m1/m1 was 0%. The presence of the CYP1A1m1 polymorphism may interfere with xenobiotic metabolism and exacerbate direct or indirect damage to the optic nerve. These CYP1A1m1 polymorphisms may be risk factors for primary open-angle glaucoma.

  10. A Central Swivel Point in the RFC Clamp Loader Controls PCNA Opening and Loading on DNA

    OpenAIRE

    Sakato, Miho; O’Donnell, Mike; Hingorani, Manju M.

    2011-01-01

    Replication Factor C (RFC) is a 5-subunit complex that loads PCNA clamps onto primer-template DNA (ptDNA) during replication. RFC subunits belong to the AAA+ superfamily, and their ATPase activity drives interactions between the clamp loader, clamp, and ptDNA, leading to topologically linked PCNA•ptDNA. We report the kinetics of transient events in S. cerevisiae RFC catalyzed PCNA loading, including ATP-induced RFC activation, PCNA opening, ptDNA binding, ATP hydrolysis, PCNA closing and PCNA...

  11. Opening of DNA double strands by helicases. Active versus passive opening

    CERN Document Server

    Betterton, M D

    2002-01-01

    Helicase opening of double-stranded nucleic acids may be "active" (the helicase directly destabilizes the dsNA to promote opening) or "passive" (the helicase binds ssNA available due to a thermal fluctuation which opens part of the dsNA). We describe helicase opening of dsNA, based on helicases which bind single NA strands and move towards the double-stranded region, using a discrete ``hopping'' model. The interaction between the helicase and the junction where the double strand opens is characterized by an interaction potential. The form of the potential determines whether the opening is active or passive. We calculate the rate of passive opening for the helicase PcrA, and show that the rate increases when the opening is active. Finally, we examine how to choose the interaction potential to optimize the rate of strand separation. One important result is our finding that active opening can increase the unwinding rate by 7 fold compared to passive opening.

  12. Regulation of mammalian horizontal gene transfer by apoptotic DNA fragmentation

    Science.gov (United States)

    Yan, B; Wang, H; Li, F; Li, C-Y

    2006-01-01

    Previously it was shown that horizontal DNA transfer between mammalian cells can occur through the uptake of apoptotic bodies, where genes from the apoptotic cells were transferred to neighbouring cells phagocytosing the apoptotic bodies. The regulation of this process is poorly understood. It was shown that the ability of cells as recipient of horizontally transferred DNA was enhanced by deficiency of p53 or p21. However, little is known with regard to the regulation of DNA from donor apoptotic cells. Here we report that the DNA fragmentation factor/caspase-activated DNase (DFF/CAD), which is the endonuclease responsible for DNA fragmentation during apoptosis, plays a significant role in regulation of horizontal DNA transfer. Cells with inhibited DFF/CAD function are poor donors for horizontal gene transfer (HGT) while their ability of being recipients of HGT is not affected. PMID:17146478

  13. Genes with stable DNA methylation levels show higher evolutionary conservation than genes with fluctuant DNA methylation levels.

    Science.gov (United States)

    Zhang, Ruijie; Lv, Wenhua; Luan, Meiwei; Zheng, Jiajia; Shi, Miao; Zhu, Hongjie; Li, Jin; Lv, Hongchao; Zhang, Mingming; Shang, Zhenwei; Duan, Lian; Jiang, Yongshuai

    2015-11-24

    Different human genes often exhibit different degrees of stability in their DNA methylation levels between tissues, samples or cell types. This may be related to the evolution of human genome. Thus, we compared the evolutionary conservation between two types of genes: genes with stable DNA methylation levels (SM genes) and genes with fluctuant DNA methylation levels (FM genes). For long-term evolutionary characteristics between species, we compared the percentage of the orthologous genes, evolutionary rate dn/ds and protein sequence identity. We found that the SM genes had greater percentages of the orthologous genes, lower dn/ds, and higher protein sequence identities in all the 21 species. These results indicated that the SM genes were more evolutionarily conserved than the FM genes. For short-term evolutionary characteristics among human populations, we compared the single nucleotide polymorphism (SNP) density, and the linkage disequilibrium (LD) degree in HapMap populations and 1000 genomes project populations. We observed that the SM genes had lower SNP densities, and higher degrees of LD in all the 11 HapMap populations and 13 1000 genomes project populations. These results mean that the SM genes had more stable chromosome genetic structures, and were more conserved than the FM genes.

  14. Visually Relating Gene Expression and in vivo DNA Binding Data

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Min-Yu; Mackey, Lester; Ker?,; nen, Soile V. E.; Weber, Gunther H.; Jordan, Michael I.; Knowles, David W.; Biggin, Mark D.; Hamann, Bernd

    2011-09-20

    Gene expression and in vivo DNA binding data provide important information for understanding gene regulatory networks: in vivo DNA binding data indicate genomic regions where transcription factors are bound, and expression data show the output resulting from this binding. Thus, there must be functional relationships between these two types of data. While visualization and data analysis tools exist for each data type alone, there is a lack of tools that can easily explore the relationship between them. We propose an approach that uses the average expression driven by multiple of ciscontrol regions to visually relate gene expression and in vivo DNA binding data. We demonstrate the utility of this tool with examples from the network controlling early Drosophila development. The results obtained support the idea that the level of occupancy of a transcription factor on DNA strongly determines the degree to which the factor regulates a target gene, and in some cases also controls whether the regulation is positive or negative.

  15. Global and gene specific DNA methylation changes during zebrafish development

    Science.gov (United States)

    DNA methylation is dynamic through the life of an organism. In this study, we measured the global and gene specific DNA methylation changes in zebrafish at different developmental stages. We found that the methylation percentage of cytosines was 11.75 ± 0.96% in 3.3 hour post fertilization (hpf) zeb...

  16. Structure and function of the DNA ligases encoded by the mammalian LIG3 gene

    OpenAIRE

    Tomkinson, Alan E.; Sallmyr, Annahita

    2013-01-01

    Among the mammalian genes encoding DNA ligases (LIG), the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different cellular functions. Notably, this nuclear gene encodes the only mitochondrial DNA ligase and so is essential for this organelle. In the nucleus, there is significant functional redundancy between DNA ligase IIIα and DNA ligase I in excision repair. In addition, DNA ligase IIIα is essential for DNA replication in the absence of the replicative DNA lig...

  17. Plasmid DNA gene therapy by electroporation: principles and recent advances.

    Science.gov (United States)

    Murakami, Tatsufumi; Sunada, Yoshihide

    2011-12-01

    Simple plasmid DNA injection is a safe and feasible gene transfer method, but it confers low transfection efficiency and transgene expression. This non-viral gene transfer method is enhanced by physical delivery methods, such as electroporation and the use of a gene gun. In vivo electroporation has been rapidly developed over the last two decades to deliver DNA to various tissues or organs. It is generally considered that membrane permeabilization and DNA electrophoresis play important roles in electro-gene transfer. Skeletal muscle is a well characterized target tissue for electroporation, because it is accessible and allows for long-lasting gene expression ( > one year). Skin is also a target tissue because of its accessibility and immunogenicity. Numerous studies have been performed using in vivo electroporation in animal models of disease. Clinical trials of DNA vaccines and immunotherapy for cancer treatment using in vivo electroporation have been initiated in patients with melanoma and prostate cancer. Furthermore, electroporation has been applied to DNA vaccines for infectious diseases to enhance immunogenicity, and the relevant clinical trials have been initiated. The gene gun approach is also being applied for the delivery of DNA vaccines against infectious diseases to the skin. Here, we review recent advances in the mechanism of in vivo electroporation, and summarize the findings of recent preclinical and clinical studies using this technology.

  18. Sequence dependency of canonical base pair opening in the DNA double helix.

    Science.gov (United States)

    Lindahl, Viveca; Villa, Alessandra; Hess, Berk

    2017-04-01

    The flipping-out of a DNA base from the double helical structure is a key step of many cellular processes, such as DNA replication, modification and repair. Base pair opening is the first step of base flipping and the exact mechanism is still not well understood. We investigate sequence effects on base pair opening using extensive classical molecular dynamics simulations targeting the opening of 11 different canonical base pairs in two DNA sequences. Two popular biomolecular force fields are applied. To enhance sampling and calculate free energies, we bias the simulation along a simple distance coordinate using a newly developed adaptive sampling algorithm. The simulation is guided back and forth along the coordinate, allowing for multiple opening pathways. We compare the calculated free energies with those from an NMR study and check assumptions of the model used for interpreting the NMR data. Our results further show that the neighboring sequence is an important factor for the opening free energy, but also indicates that other sequence effects may play a role. All base pairs are observed to have a propensity for opening toward the major groove. The preferred opening base is cytosine for GC base pairs, while for AT there is sequence dependent competition between the two bases. For AT opening, we identify two non-canonical base pair interactions contributing to a local minimum in the free energy profile. For both AT and CG we observe long-lived interactions with water and with sodium ions at specific sites on the open base pair.

  19. Quantitative DNA methylation analysis of candidate genes in cervical cancer.

    Directory of Open Access Journals (Sweden)

    Erin M Siegel

    Full Text Available Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2. A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97-1.00, p-value = 0.003. Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated.

  20. Quantitative DNA methylation analysis of candidate genes in cervical cancer.

    Science.gov (United States)

    Siegel, Erin M; Riggs, Bridget M; Delmas, Amber L; Koch, Abby; Hakam, Ardeshir; Brown, Kevin D

    2015-01-01

    Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97-1.00, p-value = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated.

  1. Biased distribution of DNA uptake sequences towards genome maintenance genes

    DEFF Research Database (Denmark)

    Davidsen, T.; Rodland, E.A.; Lagesen, K.

    2004-01-01

    coding regions are the DNA uptake sequences (DUS) required for natural genetic transformation. More importantly, we found a significantly higher density of DUS within genes involved in DNA repair, recombination, restriction-modification and replication than in any other annotated gene group......Repeated sequence signatures are characteristic features of all genomic DNA. We have made a rigorous search for repeat genomic sequences in the human pathogens Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae and found that by far the most frequent 9-10mers residing within...

  2. DNA methylation of retrotransposons, DNA transposons and genes in sugar beet (Beta vulgaris L.).

    Science.gov (United States)

    Zakrzewski, Falk; Schmidt, Martin; Van Lijsebettens, Mieke; Schmidt, Thomas

    2017-03-03

    The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses activity of transposable elements (TEs), affects gene expression, and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of the worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%) in particular, satellite DNA, retrotransposons, and DNA transposons. Genome-wide cytosine methylation in the sugar beet genome was studied in leaves and leaf-derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H=A, C, and T), and CHH sites, whereas the TE pattern differed, depending on the classes 1 (retrotransposons) and 2 (DNA transposons), respectively. Along genes and TEs, the CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing toward a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome-wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared to leaves. This article is protected by copyright. All rights reserved.

  3. DNA-water interactions distinguish messenger RNA genes from transfer RNA genes.

    Science.gov (United States)

    Khandelwal, Garima; Jayaram, B

    2012-05-30

    Physicochemical properties of DNA sequences as a guide to developing insights into genome organization has received little attention. Here, we utilize the energetics of DNA to further advance the knowledge on its language at a molecular level. Specifically, we ask the question whether physicochemical properties of different functional units on genomes differ. We extract intramolecular and solvation energies of different DNA base pair steps from a comprehensive set of molecular dynamics simulations. We then investigate the solvation behavior of DNA sequences coding for mRNAs and tRNAs. Distinguishing mRNA genes from tRNA genes is a tricky problem in genome annotation without assumptions on length of DNA and secondary structure of the product of transcription. We find that solvation energetics of DNA behaves as an extremely efficient property in discriminating 2,063,537 genes coding for mRNAs from 56,251 genes coding for tRNAs in all (~1500) completely sequenced prokaryotic genomes.

  4. Polyamine-DNA interactions and development of gene delivery vehicles.

    Science.gov (United States)

    Thomas, T J; Tajmir-Riahi, H A; Thomas, Thresia

    2016-10-01

    Polyamines are positively charged organic cations under physiologic ionic and pH conditions and hence they interact with negatively charged macromolecules such as DNA and RNA. Although electrostatic interaction is the predominant mode of polyamine-nucleic acid interactions, site- and structure-specific binding has also been recognized. A major consequence of polyamine-DNA interaction is the collapse of DNA to nanoparticles of approximately 100 nm diameter. Electron and atomic force microscopic studies have shown that these nanoparticles are spheroids, toroids and rods. DNA transport to cells for gene therapy applications requires the condensation of DNA to nanoparticles and hence the study of polyamines and related compounds with nucleic acids has received technological importance. In addition to natural and synthetic polyamines, several amine-terminated or polyamine-substituted agents are under intense investigation for non-viral gene delivery vehicles.

  5. cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Finocchiaro, G.; Taroni, F.; Martin, A.L.; Colombo, I.; Tarelli, G.T.; DiDonato, S. (Istituto Nazionale Neurologico C. Besta, Milan (Italy)); Rocchi, M. (Istituto G. Gaslini, Genoa (Italy))

    1991-01-15

    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{sub 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{sub 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.

  6. Screening for candidate genes related to breast cancer with cDNA microarray analysis

    Institute of Scientific and Technical Information of China (English)

    Yu-Juan Xiang; Zhi-Gang Yu; Ming-Ming Guo; Qin-Ye Fu; Zhong-Bing Ma; De-Zong Gao; Qiang Zhang; Yu-Yang Li; Liang Li; Lu Liu; Chun-Miao Ye

    2015-01-01

    Objective: The aim of this study was to reveal the exact changes during the occurrence of breast cancer to explore significant new and promising genes or factors related to this disease. Methods: We compared the gene expression profiles of breast cancer tissues with its uninvolved normal breast tissues as controls using the cDNA microarray analysis in seven breast cancer patients. Further, one representative gene, named IFI30, was quanti-tatively analyzed by real-time PCR to confirm the result of the cDNA microarray analysis. Results: A total of 427 genes were identified with significantly differential expression, 221 genes were up-regulated and 206 genes were down-regulated. And the result of cDNA microarray analysis was validated by detection of IFI30 mRNA level changes by real-time PCR. Genes for cell proliferation, cell cycle, cell division, mitosis, apoptosis, and immune response were enriched in the up-regulated genes, while genes for cell adhesion, proteolysis, and transport were significantly enriched in the down-regulated genes in breast cancer tissues compared with normal breast tissues by a gene ontology analysis. Conclusion: Our present study revealed a range of differentially expressed genes between breast cancer tissues and normal breast tissues, and provide candidate genes for further study focusing on the pathogenesis and new biomarkers for breast cancer. Copyright © 2015, Chinese Medical Association Production. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

  7. Bioreducible cross-linked nanoshell enhances gene transfection of polycation/DNA polyplex in vivo.

    Science.gov (United States)

    Piao, Ji-Gang; Ding, Sheng-Gang; Yang, Lu; Hong, Chun-Yan; You, Ye-Zi

    2014-08-11

    In this study, we have prepared a self-cross-linking PEG-based branched polymer, which easily forms a bioreducible nanoshell around polyplexes of cationic polymer and DNA, simply via heating the polyplex dispersions in the presence of this self-cross-linking branched polymer. This nanoshell can prevent the polyplex from dissociation and aggregation in physiological fluids without inhibiting the electrostatic interactions between the polymer and DNA. Furthermore, glutathione (GSH) can act as a stimulus to open the nanoshell after it has entered the cell. The polyplexes coated with the bioreducible nanoshell show an obvious enhancement in gene transfection in vivo compared with bare polyplexes.

  8. Identification of a poxvirus gene encoding a uracil DNA glycosylase.

    OpenAIRE

    Upton, C; Stuart, D T; McFadden, G

    1993-01-01

    An open reading frame, BamHI D6R, from the central highly conserved region of the Shope fibroma virus (SFV) genome was sequenced and found to have significant homology to that of uracil DNA glycosylases from a number of organisms. Uracil DNA glycosylase catalyzes the initial step in the repair pathway that removes potentially mutagenic uracil from duplex DNA. The D6R polypeptide was expressed in reticulocyte lysates programmed with RNA transcribed from an expression vector containing the T7 R...

  9. Cloning and DNA sequence analysis of a Lactococcus bacteriophage lysin gene.

    Science.gov (United States)

    Shearman, C; Underwood, H; Jury, K; Gasson, M

    1989-08-01

    A gene for the lysin of Lactococcus lactis bacteriphage phi vML3 was cloned using an Escherichia coli/bacteriophage lambda host-vector system. The gene was detected by its expression of antimicrobial activity against L. lactis cells in a bioassay. The cloned fragment was analysed by sub-cloning on to E. coli plasmid vectors and by restriction endonuclease and deletion mapping. Its entire DNA sequence was determined and an open reading frame for the lysin structural gene was identified. The sequenced lysin gene would express a protein of 187 amino acids with a molecular weight of 21,090, which is in good agreement with that of a protein detected after in vitro transcription and translation of DNA encoding the gene. Expression of the lysin gene in E. coli and B. subtilis from an adjacent bacteriophage promoter was readily detected but in L. lactis expression of lysin was found to be lethal. The bacteriophage phi vML3 lysin had sequence homology with protein 15 of B. subtilis bacteriophage PZA. This protein is involved in DNA packaging during bacteriophage maturation rather than in host cell lysis. The cloning and analysis of the phi vML3 lysin gene is of importance in further understanding lactic streptococcal bacteriophages, for the development of positive selection vectors and for biotechnological applications of relevance to the dairy industry.

  10. DNA-energetics-based analyses suggest additional genes in prokaryotes

    Indian Academy of Sciences (India)

    Garima Khandelwal; Jalaj Gupta; B Jayaram

    2012-07-01

    We present here a novel methodology for predicting new genes in prokaryotic genomes on the basis of inherent energetics of DNA. Regions of higher thermodynamic stability were identified, which were filtered based on already known annotations to yield a set of potentially new genes. These were then processed for their compatibility with the stereo-chemical properties of proteins and tripeptide frequencies of proteins in Swissprot data, which results in a reliable set of new genes in a genome. Quite surprisingly, the methodology identifies new genes even in well-annotated genomes. Also, the methodology can handle genomes of any GC-content, size and number of annotated genes.

  11. The detection of HBV DNA with gold nanoparticle gene probes

    Institute of Scientific and Technical Information of China (English)

    Dong Xi; Xiaoping Luo; Qin Ning; Qianghua Lu; Kailun Yao; Zuli Liu

    2007-01-01

    Objective:Gold nanoparticle Hepatitis B virus (HBV) DNA probes were prepared, and their application for HBV DNA measurement was studied. Methods:Alkanethiol modified oligonucleotide was bound with self-made Au nanoparticles to form nanoparticle HBV DNA gene probes, through covalent binding of Au-S. By using a fluorescence-based method, the number of thiol-derivatized, single-stranded oligonucleotides and their hybridization efficiency with complementary oligonucleotides in solution was determined. With the aid of Au nanoparticle-supported mercapto-modified oligonucleotides serving as detection probes, and oligonucleotides immobilized on a nylon membrane surface acting as capturing probes,HBV DNA was detected visually by sandwich hybridization based on highly sensitive aggregation and silver staining. The modified nanoparticle HBV DNA gene probes were also used to detect the HBV DNA extracted from serum in patients with hepatitis B. Results:Compared with bare Au nanoparticles, oligonucleotide modified nanoparticles had a higher stability in NaCl solution or under high temperature environment and the absorbance peak of modified Au nanoparticles shifted from 520nm to 524nm. For Au nanoparticles, the maximal oligonucleotide surface coverage of hexaethiol 30-mer oligonucleotide was (132 ± 10) oligonucleotides per nanoparticle, and the percentage of hybridization strands on nanoparticles was (22 ± 3% ). Based on a two-probe sandwich hybridization/nanoparticle amplification/silver staining enhancement method, Au nanoparticle gene probes could detect as low as 10-11 mol/L composite HBV DNA molecules on a nylon membrane and the PCR products of HBV DNA visually. As made evident by transmission electron microscopy, the nanoparticles assembled into large network aggregates when nanoparticle HBV DNA gene probes were applied to detect HBV DNA molecules in liquid. Conclusion:Our results showed that successfully prepared Au nanoparticle HBV DNA gene probes could be used to

  12. Gene Expression Analysis Using Agilent DNA Microarrays

    DEFF Research Database (Denmark)

    Stangegaard, Michael

    2009-01-01

    of labeled cDNA added to each slide reduces dye-bias and slide to slide variation. Efficient mixing of the hybridization solution throughout the hybridization reaction increases signals several fold. The amount of near perfect target-probe hybrids may be reduced by efficient stringency washes...

  13. Hormonal induction of transfected genes depends on DNA topology.

    Science.gov (United States)

    Piña, B; Haché, R J; Arnemann, J; Chalepakis, G; Slater, E P; Beato, M

    1990-02-01

    Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thymidine kinase promoter of herpes simplex virus and the reporter gene chloramphenicol acetyltransferase of Escherichia coli respond to glucocorticoids and progestins when transfected into appropriate cells. In the human mammary tumor cell line T47D, the response to progestins, but not to glucocorticoids, is highly dependent on the topology of the transfected DNA. Although negatively supercoiled plasmids respond optimally to the synthetic progestin R5020, their linearized counterparts exhibit markedly reduced progestin inducibility. This is not due to changes in the efficiency of DNA transfection, since the amount of DNA incorporated into the cell nucleus is not significantly dependent on the initial topology of the plasmids. In contrast, cotransfection experiments with glucocorticoid receptor cDNA in the same cell line show no significant influence of DNA topology on induction by dexamethasone. A similar result was obtained with fibroblasts that contain endogenous glucocorticoid receptors. When the distance between receptor-binding sites or between the binding sites and the promoter was increased, the dependence of progestin induction on DNA topology was more pronounced. In contrast to the original plasmid, these constructs also revealed a similar topological dependence for induction by glucocorticoids. The differential influence of DNA topology is not due to differences in the affinity of the two hormone receptors for DNA of various topologies, but probably reflects an influence of DNA topology on the interaction between different DNA-bound receptor molecules and between receptors and other transcription factors.

  14. Plant DNA barcoding: from gene to genome.

    Science.gov (United States)

    Li, Xiwen; Yang, Yang; Henry, Robert J; Rossetto, Maurizio; Wang, Yitao; Chen, Shilin

    2015-02-01

    DNA barcoding is currently a widely used and effective tool that enables rapid and accurate identification of plant species; however, none of the available loci work across all species. Because single-locus DNA barcodes lack adequate variations in closely related taxa, recent barcoding studies have placed high emphasis on the use of whole-chloroplast genome sequences which are now more readily available as a consequence of improving sequencing technologies. While chloroplast genome sequencing can already deliver a reliable barcode for accurate plant identification it is not yet resource-effective and does not yet offer the speed of analysis provided by single-locus barcodes to unspecialized laboratory facilities. Here, we review the development of candidate barcodes and discuss the feasibility of using the chloroplast genome as a super-barcode. We advocate a new approach for DNA barcoding that, for selected groups of taxa, combines the best use of single-locus barcodes and super-barcodes for efficient plant identification. Specific barcodes might enhance our ability to distinguish closely related plants at the species and population levels.

  15. A central swivel point in the RFC clamp loader controls PCNA opening and loading on DNA.

    Science.gov (United States)

    Sakato, Miho; O'Donnell, Mike; Hingorani, Manju M

    2012-02-17

    Replication factor C (RFC) is a five-subunit complex that loads proliferating cell nuclear antigen (PCNA) clamps onto primer-template DNA (ptDNA) during replication. RFC subunits belong to the AAA(+) superfamily, and their ATPase activity drives interactions between the clamp loader, the clamp, and the ptDNA, leading to topologically linked PCNA·ptDNA. We report the kinetics of transient events in Saccharomyces cerevisiae RFC-catalyzed PCNA loading, including ATP-induced RFC activation, PCNA opening, ptDNA binding, ATP hydrolysis, PCNA closing, and PCNA·ptDNA release. This detailed perspective enables assessment of individual RFC-A, RFC-B, RFC-C, RFC-D, and RFC-E subunit functions in the reaction mechanism. Functions have been ascribed to RFC subunits previously based on a steady-state analysis of 'arginine-finger' ATPase mutants; however, pre-steady-state analysis provides a different view. The central subunit RFC-C serves as a critical swivel point in the clamp loader. ATP binding to this subunit initiates RFC activation, and the clamp loader adopts a spiral conformation that stabilizes PCNA in a corresponding open spiral. The importance of RFC subunit response to ATP binding decreases as RFC-C>RFC-D>RFC-B, with RFC-A being unnecessary. RFC-C-dependent activation of RFC also enables ptDNA binding, leading to the formation of the RFC·ATP·PCNA(open)·ptDNA complex. Subsequent ATP hydrolysis leads to complex dissociation, with RFC-D activity contributing the most to rapid ptDNA release. The pivotal role of the RFC-B/C/D subunit ATPase core in clamp loading is consistent with the similar central location of all three ATPase active subunits of the Escherichia coli clamp loader.

  16. DNA Polymorphisms in River Buffalo Leptin Gene

    Directory of Open Access Journals (Sweden)

    B. Moioli

    2010-02-01

    Full Text Available Leptin is a protein involved in the regulation of feed intake, fat metabolism, whole body energy balance, reproduction and hematopoiesis. In cattle Leptin gene has been considered a potential QTL influencing several production traits like meat production, milk performance and reproduction. Several studies on bovine leptin gene have found association between polymorphisms and traits like milk yield, feed intake, fat content, carcass and meat quality. With the aim to assess the presence of sequences polymorphisms in the Buffalo leptin gene, we sequenced the entire coding region and part of the introns on a panel of Italian River Buffalos. In this study we identified a new set of SNP (Single Nucleotide Polymorphism useful for association studies.

  17. Expression of the dnaN and dnaQ genes of Escherichia coli is inducible by mitomycin C.

    Science.gov (United States)

    Kaasch, M; Kaasch, J; Quiñones, A

    1989-10-01

    The dnaN and dnaQ genes encode the beta subunit and the epsilon subunit of the DNA polymerase III holoenzyme. Using translational fusions to lacZ we found that DNA damage caused by mitomycin C induces expression of the dnaA and dnaQ genes. This induction was not observed in lexA and recA mutants which block the induction of the SOS response, suggesting a relationship between the mechanism(s) of genetic control of DNA polymerase III holoenzyme and the SOS regulatory network. Nevertheless, there is evidence that the mitomycin C induction of dnaN and dnaQ is not a simple lexA-regulated process, because nalidixic acid (an excellent SOS inducer) does not increase dnaN and dnaQ gene expression, and the time course of induction is abnormally slow.

  18. Modulation of DNA binding by gene-specific transcription factors.

    Science.gov (United States)

    Schleif, Robert F

    2013-10-01

    The transcription of many genes, particularly in prokaryotes, is controlled by transcription factors whose activity can be modulated by controlling their DNA binding affinity. Understanding the molecular mechanisms by which DNA binding affinity is regulated is important, but because forming definitive conclusions usually requires detailed structural information in combination with data from extensive biophysical, biochemical, and sometimes genetic experiments, little is truly understood about this topic. This review describes the biological requirements placed upon DNA binding transcription factors and their consequent properties, particularly the ways that DNA binding affinity can be modulated and methods for its study. What is known and not known about the mechanisms modulating the DNA binding affinity of a number of prokaryotic transcription factors, including CAP and lac repressor, is provided.

  19. Depletion of mtDNA: syndromes and genes.

    Science.gov (United States)

    Alberio, Simona; Mineri, Rossana; Tiranti, Valeria; Zeviani, Massimo

    2007-01-01

    Maintenance of mitochondrial DNA (mtDNA) requires the concerted activity of several nuclear-encoded factors that participate in its replication, being part of the mitochondrial replisome or ensuring the balanced supply of dNTPs to mitochondria. In the past decade, a growing number of syndromes associated with dysfunction due to tissue-specific depletion of mtDNA (MDS) have been reported. This article reviews the current knowledge of the genes responsible for these disorders, the impact of different mutations in the epidemiology of MDS and their role in the pathogenic mechanisms underlying the different clinical presentations.

  20. Open chromatin encoded in DNA sequence is the signature of 'master' replication origins in human cells.

    Science.gov (United States)

    Audit, Benjamin; Zaghloul, Lamia; Vaillant, Cédric; Chevereau, Guillaume; d'Aubenton-Carafa, Yves; Thermes, Claude; Arneodo, Alain

    2009-10-01

    For years, progress in elucidating the mechanisms underlying replication initiation and its coupling to transcriptional activities and to local chromatin structure has been hampered by the small number (approximately 30) of well-established origins in the human genome and more generally in mammalian genomes. Recent in silico studies of compositional strand asymmetries revealed a high level of organization of human genes around 1000 putative replication origins. Here, by comparing with recently experimentally identified replication origins, we provide further support that these putative origins are active in vivo. We show that regions approximately 300-kb wide surrounding most of these putative replication origins that replicate early in the S phase are hypersensitive to DNase I cleavage, hypomethylated and present a significant enrichment in genomic energy barriers that impair nucleosome formation (nucleosome-free regions). This suggests that these putative replication origins are specified by an open chromatin structure favored by the DNA sequence. We discuss how this distinctive attribute makes these origins, further qualified as 'master' replication origins, priviledged loci for future research to decipher the human spatio-temporal replication program. Finally, we argue that these 'master' origins are likely to play a key role in genome dynamics during evolution and in pathological situations.

  1. Controlling gene expression by DNA mechanics: emerging insights and challenges.

    Science.gov (United States)

    Levens, David; Baranello, Laura; Kouzine, Fedor

    2016-11-01

    Transcription initiation is a major control point for the precise regulation of gene expression. Our knowledge of this process has been mainly derived from protein-centric studies wherein cis-regulatory DNA sequences play a passive role, mainly in arranging the protein machinery to coalesce at the transcription start sites of genes in a spatial and temporal-specific manner. However, this is a highly dynamic process in which molecular motors such as RNA polymerase II (RNAPII), helicases, and other transcription factors, alter the level of mechanical force in DNA, rather than simply a set of static DNA-protein interactions. The double helix is a fiber that responds to flexural and torsional stress, which if accumulated, can affect promoter output as well as change DNA and chromatin structure. The relationship between DNA mechanics and the control of early transcription initiation events has been under-investigated. Genomic techniques to display topological stress and conformational variation in DNA across the mammalian genome provide an exciting new insight on the role of DNA mechanics in the early stages of the transcription cycle. Without understanding how torsional and flexural stresses are generated, transmitted, and dissipated, no model of transcription will be complete and accurate.

  2. Zirconium(IV)-Catalyzed Ring Opening of on-DNA Epoxides in Water.

    Science.gov (United States)

    Fan, Lijun; Davie, Christopher P

    2017-05-04

    DNA-encoded library technology (ELT) has spurred wide interest in the pharmaceutical industry as a powerful tool for hit and lead generation. In recent years a number of "DNA-compatible" chemical modifications have been published and used to synthesize vastly diverse screening libraries. Herein we report a newly developed, zirconium tetrakis(dodecyl sulfate) [Zr(DS)4 ] catalyzed ring-opening of on-DNA epoxides in water with amines, including anilines. Subsequent cyclization of the resulting on-DNA β-amino alcohols leads to a variety of biologically interesting, nonaromatic heterocycles. Under these conditions, a library of 137 million on-DNA β-amino alcohols and their cyclization products was assembled. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Novel roles for selected genes in meiotic DNA processing.

    Directory of Open Access Journals (Sweden)

    Philip W Jordan

    2007-12-01

    Full Text Available High-throughput studies of the 6,200 genes of Saccharomyces cerevisiae have provided valuable data resources. However, these resources require a return to experimental analysis to test predictions. An in-silico screen, mining existing interaction, expression, localization, and phenotype datasets was developed with the aim of selecting minimally characterized genes involved in meiotic DNA processing. Based on our selection procedure, 81 deletion mutants were constructed and tested for phenotypic abnormalities. Eleven (13.6% genes were identified to have novel roles in meiotic DNA processes including DNA replication, recombination, and chromosome segregation. In particular, this analysis showed that Def1, a protein that facilitates ubiquitination of RNA polymerase II as a response to DNA damage, is required for efficient synapsis between homologues and normal levels of crossover recombination during meiosis. These characteristics are shared by a group of proteins required for Zip1 loading (ZMM proteins. Additionally, Soh1/Med31, a subunit of the RNA pol II mediator complex, Bre5, a ubiquitin protease cofactor and an uncharacterized protein, Rmr1/Ygl250w, are required for normal levels of gene conversion events during meiosis. We show how existing datasets may be used to define gene sets enriched for specific roles and how these can be evaluated by experimental analysis.

  4. Sequence dependency of canonical base pair opening in the DNA double helix.

    Directory of Open Access Journals (Sweden)

    Viveca Lindahl

    2017-04-01

    Full Text Available The flipping-out of a DNA base from the double helical structure is a key step of many cellular processes, such as DNA replication, modification and repair. Base pair opening is the first step of base flipping and the exact mechanism is still not well understood. We investigate sequence effects on base pair opening using extensive classical molecular dynamics simulations targeting the opening of 11 different canonical base pairs in two DNA sequences. Two popular biomolecular force fields are applied. To enhance sampling and calculate free energies, we bias the simulation along a simple distance coordinate using a newly developed adaptive sampling algorithm. The simulation is guided back and forth along the coordinate, allowing for multiple opening pathways. We compare the calculated free energies with those from an NMR study and check assumptions of the model used for interpreting the NMR data. Our results further show that the neighboring sequence is an important factor for the opening free energy, but also indicates that other sequence effects may play a role. All base pairs are observed to have a propensity for opening toward the major groove. The preferred opening base is cytosine for GC base pairs, while for AT there is sequence dependent competition between the two bases. For AT opening, we identify two non-canonical base pair interactions contributing to a local minimum in the free energy profile. For both AT and CG we observe long-lived interactions with water and with sodium ions at specific sites on the open base pair.

  5. The nucleotide sequence of the dnaA gene and the first part of the dnaN gene of Escherichia coli K-12.

    Science.gov (United States)

    Hansen, E B; Hansen, F G; von Meyenburg, K

    1982-11-25

    The nucleotide sequence of the dnaA gene and the first 10% of the dnaN gene was determined. From the nucleotide sequence the amino acid sequence of the dnaA gene product was derived. It is a basic protein of 467 amino acid residues with a molecular weight of 52.5 kD. The expression of the dnaA gene is in the counterclockwise direction like the one of the dnaN gene, for which potential startsites were found.

  6. Whole genome amplification of DNA for genotyping pharmacogenetics candidate genes.

    Directory of Open Access Journals (Sweden)

    Santosh ePhilips

    2012-03-01

    Full Text Available Whole genome amplification (WGA technologies can be used to amplify genomic DNA when only small amounts of DNA are available. The Multiple Displacement Amplification Phi polymerase based amplification has been shown to accurately amplify DNA for a variety of genotyping assays; however, it has not been tested for genotyping many of the clinically relevant genes important for pharmacogenetic studies, such as the cytochrome P450 genes, that are typically difficult to genotype due to multiple pseudogenes, copy number variations, and high similarity to other related genes. We evaluated whole genome amplified samples for Taqman™ genotyping of SNPs in a variety of pharmacogenetic genes. In 24 DNA samples from the Coriell human diversity panel, the call rates and concordance between amplified (~200-fold amplification and unamplified samples was 100% for two SNPs in CYP2D6 and one in ESR1. In samples from a breast cancer clinical trial (Trial 1, we compared the genotyping results in samples before and after WGA for four SNPs in CYP2D6, one SNP in CYP2C19, one SNP in CYP19A1, two SNPs in ESR1, and two SNPs in ESR2. The concordance rates were all >97%. Finally, we compared the allele frequencies of 143 SNPs determined in Trial 1 (whole genome amplified DNA to the allele frequencies determined in unamplified DNA samples from a separate trial (Trial 2 that enrolled a similar population. The call rates and allele frequencies between the two trials were 98% and 99.7%, respectively. We conclude that the whole genome amplified DNA is suitable for Taqman™ genotyping for a wide variety of pharmacogenetically relevant SNPs.

  7. Sequence-Dependence of the Energetics of Opening of AT Basepairs in DNA

    OpenAIRE

    Chen, Congju; Russu, Irina M.

    2004-01-01

    Proton exchange and nuclear magnetic resonance spectroscopy are being used to characterize the energetics of opening of AT/TA basepairs in the DNA dodecamer 5′-d(GCTATAAAAGGG)-3′/5′-d(CCCTTTTATAGC)-3′. The dodecamer contains the TATA box of the adenovirus major late promoter. The equilibrium constants for opening of each basepair are measured from the dependence of the exchange rates of imino protons on ammonia concentration. The enthalpy, entropy, and free energy changes in the opening react...

  8. DNA Methylation Occurred around Lowly Expressed Genes of Plastid DNA during Tomato Fruit Development.

    Science.gov (United States)

    Ngernprasirtsiri, J; Kobayashi, H; Akazawa, T

    1988-09-01

    We have analyzed DNA methylation of plastid DNA from fully ripened red fruits, green mature fruits, and green leaves of tomato (Lycopersicon esculentum var. Firstmore). Essentially identical restriction profiles were obtained between chromoplast and chloroplast DNAs by EcoRI digestion. BstNI/EcoRII and HpaII/MspI are pairs of isoschizomers that can discriminate between methylated and unmethylated DNAs. These endonucleases produced different restriction patterns of plastid DNAs from tomato fruits compared to tomato leaves. Moreover, we have found from Southern blots that methylation was not detected in DNA fragments containing certain genes that are actively expressed in chromoplasts, whereas DNA fragments bearing genes that are barely transcribed in chromoplasts are methylated.

  9. XRCC1 and XPD DNA repair gene polymorphisms: a potential risk factor for glaucoma in the Pakistani population

    NARCIS (Netherlands)

    Yousaf, S.; Khan, M.I.; Micheal, S.; Akhtar, F.; Ali, S.H.; Riaz, M.; Ali, M.; Lall, P.; Waheed, N.K.; Hollander, A.I. den; Ahmed, A.; Qamar, R.

    2011-01-01

    PURPOSE: The present study was designed to determine the association of polymorphisms of the DNA repair genes X-ray cross-complementing group 1 (XRCC1) (c.1316G>A [rs25487]) and xeroderma pigmentosum complementation group D (XPD) (c.2298A>C [rs13181]) with primary open-angle glaucoma (POAG) an

  10. Controlling gene expression by DNA mechanics: emerging insights and challenges

    OpenAIRE

    Levens, David; Baranello, Laura; Kouzine, Fedor

    2016-01-01

    Transcription initiation is a major control point for the precise regulation of gene expression. Our knowledge of this process has been mainly derived from protein-centric studies wherein cis-regulatory DNA sequences play a passive role, mainly in arranging the protein machinery to coalesce at the transcription start sites of genes in a spatial and temporal-specific manner. However, this is a highly dynamic process in which molecular motors such as RNA polymerase II (RNAPII), helicases, and o...

  11. A microfluidic DNA computing processor for gene expression analysis and gene drug synthesis.

    Science.gov (United States)

    Zhang, Yu; Yu, Hao; Qin, Jianhua; Lin, Bingcheng

    2009-11-06

    Boolean logic performs a logical operation on one or more logic input and produces a single logic output. Here, we describe a microfluidic DNA computing processor performing Boolean logic operations for gene expression analysis and gene drug synthesis. Multiple cancer-related genes were used as input molecules. Their expression levels were identified by interacting with the computing related DNA strands, which were designed according to the sequences of cancer-related genes and the suicide gene. When all the expressions of the cancer-related genes fit in with the diagnostic criteria, positive diagnosis would be confirmed and then a complete suicide gene (gene drug) could be synthesized as an output molecule. Microfluidic chip was employed as an effective platform to realize the computing process by integrating multistep biochemical reactions involving hybridization, displacement, denaturalization, and ligation. By combining the specific design of the computing related molecules and the integrated functions of the microfluidics, the microfluidic DNA computing processor is able to analyze the multiple gene expressions simultaneously and realize the corresponding gene drug synthesis with simplicity and fast speed, which demonstrates the potential of this platform for DNA computing in biomedical applications.

  12. Proteasome inhibition enhances resistance to DNA damage via upregulation of Rpn4-dependent DNA repair genes.

    Science.gov (United States)

    Karpov, Dmitry S; Spasskaya, Daria S; Tutyaeva, Vera V; Mironov, Alexander S; Karpov, Vadim L

    2013-09-17

    The 26S proteasome is an ATP-dependent multi-subunit protease complex and the major regulator of intracellular protein turnover and quality control. However, its role in the DNA damage response is controversial. We addressed this question in yeast by disrupting the transcriptional regulation of the PRE1 proteasomal gene. The mutant strain has decreased proteasome activity and is hyper-resistant to various DNA-damaging agents. We found that Rpn4-target genes MAG1, RAD23, and RAD52 are overexpressed in this strain due to Rpn4 stabilisation. These genes represent three different pathways of base excision, nucleotide excision and double strand break repair by homologous recombination (DSB-HR). Consistently, the proteasome mutant displays increased DSB-HR activity. Our data imply that the proteasome may have a negative role in DNA damage response.

  13. Dietary Berries and Ellagic Acid Prevent Oxidative DNA Damage and Modulate Expression of DNA Repair Genes

    Directory of Open Access Journals (Sweden)

    Ramesh C. Gupta

    2008-03-01

    Full Text Available DNA damage is a pre-requisite for the initiation of cancer and agents that reduce this damage are useful in cancer prevention. In this study, we evaluated the ability of whole berries and berry phytochemical, ellagic acid to reduce endogenous oxidative DNA damage. Ellagic acid was selected based on > 95% inhibition of 8-oxodeoxyguosine (8-oxodG and other unidentified oxidative DNA adducts induced by 4-hydroxy-17B;-estradiol and CuCl2 in vitro. Inhibition of the latter occurred at lower concentrations (10 u(microM than that for 8-oxodG (100 u(microM. In the in vivo study, female CD-1 mice (n=6 were fed either a control diet or diet supplemented with ellagic acid (400 ppm and dehydrated berries (5% w/w with varying ellagic acid contents -- blueberry (low, strawberry (medium and red raspberry (high, for 3 weeks. Blueberry and strawberry diets showed moderate reductions in endogenous DNA adducts (25%. However, both red raspberry and ellagic acid diets showed a significant reduction of 59% (p < 0.001 and 48% (p < 0.01, respectively. Both diets also resulted in a 3-8 fold over-expression of genes involved in DNA repair such as xeroderma pigmentosum group A complementing protein (XPA, DNA excision repair protein (ERCC5 and DNA ligase III (DNL3. These results suggest that red raspberry and ellagic acid reduce endogenous oxidative DNA damage by mechanisms which may involve increase in DNA repair.

  14. The gene expressions of DNA methylation/demethylation enzymes ...

    African Journals Online (AJOL)

    user

    2011-01-31

    Jan 31, 2011 ... methylation might be a potential regulation mechanism for transcription ... of Cox4 gene provide possible DNA methylation sites. Up till now .... Effects of hypothyroidism on expressions of Cox4, Dnmt1 and. Dnmt3a in the ...

  15. DNA polymorphism of HLA class II genes in alopecia areata

    DEFF Research Database (Denmark)

    Morling, N; Frentz, G; Fugger, L

    1992-01-01

    We investigated the DNA restriction polymorphism (RFLP) of the Major Histocompatibility Complex (MHC) class II genes: HLA-DQA, -DQB, -DPA, and -DPB in 20 Danish patients with alopecia areata (AA) and in healthy Danes. The frequency in AA of the DQB1*0301 and DQw7 associated DQB Bgl/II 4.2 kb...

  16. Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors.

    Science.gov (United States)

    Falkner, F G; Moss, B

    1988-06-01

    Mycophenolic acid, an inhibitor of purine metabolism, was shown to block the replication of vaccinia virus in normal cell lines. This observation led to the development of a dominant one-step plaque selection system, based on expression of the Escherichia coli gpt gene, for the isolation of recombinant vaccinia viruses. Synthesis of xanthine-guanine phosphoribosyltransferase enabled only the recombinant viruses to form large plaques in a selective medium containing mycophenolic acid, xanthine, and hypoxanthine. To utilize the selection system efficiently, we constructed a series of plasmids that contain the E. coli gpt gene and allow insertion of foreign genes into multiple unique restriction endonuclease sites in all three reading frames between the translation initiation codon of a strong late promoter and synthetic translation termination sequences. The selection-expression cassette is flanked by vaccinia virus DNA that directs homologous recombination into the virus genome. The new vectors allow high-level expression of complete or partial open reading frames and rapid construction of recombinant viruses by facilitating the cloning steps and by simplifying their isolation. The system was tested by cloning the E. coli beta-galactosidase gene; in 24 h, this enzyme accounted for approximately 3.5% of the total infected-cell protein.

  17. DNA repair and gene therapy: implications for translational uses.

    Science.gov (United States)

    Limp-Foster, M; Kelley, M R

    2000-01-01

    Gene therapy has been proposed to have implications in the treatment of cancer. By genetically manipulating the hematopoietic stem cell compartment with genes that confer resistance to chemotherapeutic agents, the dose escalation that is necessary to effectively treat the cancers could potentially be achieved. DNA repair genes are some of the potential candidates to confer increased resistance to chemotherapeutic agents. Although initial focus in this area has been on the direct reversal protein (MGMT), its protective ability is limited to those agents that produce O(6)-methylGuanine cross-links-agents that are not extensively used clinically (e.g., nitrosoureas). Furthermore, most alkylating agents attack more sites in DNA other than O(6)-methylGuanine, such that the protections afforded by MGMT may prevent the initial cytotoxicity, but at a price of increased mutational burden and potential secondary leukemias. Therefore, some of the genes that are being tested as candidates for gene transfer are base excision repair (BER) genes. We and others have found that overexpression of selective BER genes confers resistance to chemotherapeutic agents such as thiotepa, ionizing radiation, bleomycin, and other agents. As these "proof of concept" analyses mature, many more clinically relevant chemotherapeutic agents can be tested for BER protective ability.

  18. HLA class II genes: typing by DNA analysis.

    Science.gov (United States)

    Bidwell, J L; Bidwell, E A; Bradley, B A

    1990-04-01

    A detailed understanding of the structure and function of the human major histocompatibility complex (MHC) has ensued from studies by molecular biologist during the last decade. Virtually all of the HLA genes have now been cloned, and the nucleotide sequences of their different allelic forms have been determined. Typing for these HLA alleles is a fundamental prerequisite for tissue matching in allogeneic organ transplantation. Until very recently, typing procedures have been dominated by serological and cellular methods. The availability of cloned DNA from HLA genes has now permitted the technique of restriction fragment length polymorphism (RFLP) analysis to be applied, with remarkable success and advantage, to phenotyping of both HLA Class I and Class II determinants. For the HLA Class II genes DR and DQ, a simple two-stage RFLP analysis permits the accurate identification of all specificities defined by serology, and of many which are defined by cellular typing. At the present time, however, RFLP typing of HLA Class I genes is not as practicable or as informative as that for HLA Class II genes. The present clinical applications of HLA-DR and DQ RFLP typing are predominantly in phenotyping of living donors, including selection of HLA-matched volunteer bone marrow donors, in allograft survival studies, and in studies of HLA Class II-associated diseases. However, the time taken to perform RFLP analysis precludes its use for the typing of cadaveric kidney donors. Nucleotide sequence data for the alleles of HLA Class II genes have now permitted the development of allele-specific oligonucleotide (ASO) typing, a second category of DNA analysis. This has been greatly facilitated by the ability to amplify specific HLA Class II DNA 'target' sequences using the polymerase chain reaction (PCR) technique. The accuracy of DNA typing techniques should ensure that this methodology will eventually replace conventional HLA phenotyping.

  19. Aberrant DNA methylation in 5'regions of DNA methyltransferase genes in aborted bovine clones

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning.It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation.DNA methylation is established and maintained by DNA methyltransferases(DNMTs),therefore,it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs.Since DNA methylation can strongly inhibit gene expression,aberrant DNA methylation of DNMT genes may disturb gene expression.But presently,it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos.In our study,we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a,Dnmt3b,Dnmtl and Dnmt2 in four aborted bovine clones.Using bisulfite sequencing method,we found that 3 out of 4 aborted bovine clones(AF1,AF2 and AF3)showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b.indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed.However,the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF)fetuses.Besides,we found that tle 5'regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults.IVF fetuses,sperm and aborted clones.Together,our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.

  20. Integrated analysis of DNA copy number and gene expression microarray data using gene sets

    NARCIS (Netherlands)

    R.X. de Menezes (Renee); M. Boetzer (Marten); M. Sieswerda (Melle); G.J.B. van Ommen; J.M. Boer (Judith)

    2009-01-01

    textabstractBackground: Genes that play an important role in tumorigenesis are expected to show association between DNA copy number and RNA expression. Optimal power to find such associations can only be achieved if analysing copy number and gene expression jointly. Furthermore, some copy number

  1. Divergence of gene body DNA methylation and evolution of plant duplicate genes.

    Directory of Open Access Journals (Sweden)

    Jun Wang

    Full Text Available It has been shown that gene body DNA methylation is associated with gene expression. However, whether and how deviation of gene body DNA methylation between duplicate genes can influence their divergence remains largely unexplored. Here, we aim to elucidate the potential role of gene body DNA methylation in the fate of duplicate genes. We identified paralogous gene pairs from Arabidopsis and rice (Oryza sativa ssp. japonica genomes and reprocessed their single-base resolution methylome data. We show that methylation in paralogous genes nonlinearly correlates with several gene properties including exon number/gene length, expression level and mutation rate. Further, we demonstrated that divergence of methylation level and pattern in paralogs indeed positively correlate with their sequence and expression divergences. This result held even after controlling for other confounding factors known to influence the divergence of paralogs. We observed that methylation level divergence might be more relevant to the expression divergence of paralogs than methylation pattern divergence. Finally, we explored the mechanisms that might give rise to the divergence of gene body methylation in paralogs. We found that exonic methylation divergence more closely correlates with expression divergence than intronic methylation divergence. We show that genomic environments (e.g., flanked by transposable elements and repetitive sequences of paralogs generated by various duplication mechanisms are associated with the methylation divergence of paralogs. Overall, our results suggest that the changes in gene body DNA methylation could provide another avenue for duplicate genes to develop differential expression patterns and undergo different evolutionary fates in plant genomes.

  2. DNA-transporting nanoparticles : design and in vitro evaluation of DNA and formulation for non-viral gene delivery

    NARCIS (Netherlands)

    van Gaal, E.V.B.|info:eu-repo/dai/nl/30483629X

    2010-01-01

    The aim of gene therapy is to treat, cure or prevent a disease by replacing defective genes, introducing new genes or changing the expression of a person’s genes. Success of gene therapy is dependent on successful delivery of DNA from the site of administration into cell nuclei. Naturally occurring

  3. Cationic Polyene Phospholipids as DNA Carriers for Ocular Gene Therapy

    Directory of Open Access Journals (Sweden)

    Susana Machado

    2014-01-01

    Full Text Available Recent success in the treatment of congenital blindness demonstrates the potential of ocular gene therapy as a therapeutic approach. The eye is a good target due to its small size, minimal diffusion of therapeutic agent to the systemic circulation, and low immune and inflammatory responses. Currently, most approaches are based on viral vectors, but efforts continue towards the synthesis and evaluation of new nonviral carriers to improve nucleic acid delivery. Our objective is to evaluate the efficiency of novel cationic retinoic and carotenoic glycol phospholipids, designated C20-18, C20-20, and C30-20, to deliver DNA to human retinal pigmented epithelium (RPE cells. Liposomes were produced by solvent evaporation of ethanolic mixtures of the polyene compounds and coformulated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE or cholesterol (Chol. Addition of DNA to the liposomes formed lipoplexes, which were characterized for binding, size, biocompatibility, and transgene efficiency. Lipoplex formulations of suitable size and biocompatibility were assayed for DNA delivery, both qualitatively and quantitatively, using RPE cells and a GFP-encoding plasmid. The retinoic lipoplex formulation with DOPE revealed a transfection efficiency comparable to the known lipid references 3β-[N-(N′,N′-dimethylaminoethane-carbamoyl]-cholesterol (DC-Chol and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC and GeneJuice. The results demonstrate that cationic polyene phospholipids have potential as DNA carriers for ocular gene therapy.

  4. Analysis of Gene Expression Profile in Lung Adenosquamous Carcinoma Using cDNA Microarray

    Institute of Scientific and Technical Information of China (English)

    YANG Fei; YANG Jiong; JIANG Man; YE Bo; ZHANG Yu-xia; CHEN Hong-lei; XIA Dong; LIU Ming-qiu

    2004-01-01

    Gene expression profile of the lung adenosquamous carcinoma was characterized by using cDNA microarray chip containing 4 096 human genes. Among target genes, 508 differentially expressed genes were identified in adenosquamous carcinoma of the lung, 232 genes were overexpressed and 276 genes were underexpressed. Among them, 92 genes are cell signals transduction genes, 34 genes are proto-oncogenes and tumor suppressor genes or cell cycle related genes or cell apoptosis related genes, 29 genes are cell skeleton genes, 28 genes are DNA synthesis, repair and recombination genes, 12 genes are DNA binding and transcription genes. These genes may be associated with the occurence and development of adenosquamous carinome of the lung.

  5. Gene Expression Commons: an open platform for absolute gene expression profiling.

    Directory of Open Access Journals (Sweden)

    Jun Seita

    Full Text Available Gene expression profiling using microarrays has been limited to comparisons of gene expression between small numbers of samples within individual experiments. However, the unknown and variable sensitivities of each probeset have rendered the absolute expression of any given gene nearly impossible to estimate. We have overcome this limitation by using a very large number (>10,000 of varied microarray data as a common reference, so that statistical attributes of each probeset, such as the dynamic range and threshold between low and high expression, can be reliably discovered through meta-analysis. This strategy is implemented in a web-based platform named "Gene Expression Commons" (https://gexc.stanford.edu/ which contains data of 39 distinct highly purified mouse hematopoietic stem/progenitor/differentiated cell populations covering almost the entire hematopoietic system. Since the Gene Expression Commons is designed as an open platform, investigators can explore the expression level of any gene, search by expression patterns of interest, submit their own microarray data, and design their own working models representing biological relationship among samples.

  6. Gene Expression Commons: an open platform for absolute gene expression profiling.

    Science.gov (United States)

    Seita, Jun; Sahoo, Debashis; Rossi, Derrick J; Bhattacharya, Deepta; Serwold, Thomas; Inlay, Matthew A; Ehrlich, Lauren I R; Fathman, John W; Dill, David L; Weissman, Irving L

    2012-01-01

    Gene expression profiling using microarrays has been limited to comparisons of gene expression between small numbers of samples within individual experiments. However, the unknown and variable sensitivities of each probeset have rendered the absolute expression of any given gene nearly impossible to estimate. We have overcome this limitation by using a very large number (>10,000) of varied microarray data as a common reference, so that statistical attributes of each probeset, such as the dynamic range and threshold between low and high expression, can be reliably discovered through meta-analysis. This strategy is implemented in a web-based platform named "Gene Expression Commons" (https://gexc.stanford.edu/) which contains data of 39 distinct highly purified mouse hematopoietic stem/progenitor/differentiated cell populations covering almost the entire hematopoietic system. Since the Gene Expression Commons is designed as an open platform, investigators can explore the expression level of any gene, search by expression patterns of interest, submit their own microarray data, and design their own working models representing biological relationship among samples.

  7. Molecular characterization of the human excision repair gene ERCC-1: cDNA cloning and aminoacid homology with the yeast DNA repair gene RAD10.

    NARCIS (Netherlands)

    M. van Duin (Mark); J. de Wit (Jan); H. Odijk (Hanny); A. Westerveld (Andries); A. Yasui (Akira); M.H.M. Koken (Marcel); J.H.J. Hoeijmakers (Jan); D. Bootsma (Dirk)

    1986-01-01

    textabstractThe human excision repair gene ERCC-7 was cloned after DNA mediated gene transfer to the CHO mutant 43-38, which is sensitive to ultraviolet light and mitomycin-C. We describe the cloning and sequence analysis of the ERCC-7 cDNA and partial characterization of the gene. ERCC.1 has a size

  8. Independent component analysis of Alzheimer's DNA microarray gene expression data

    Directory of Open Access Journals (Sweden)

    Vanderburg Charles R

    2009-01-01

    Full Text Available Abstract Background Gene microarray technology is an effective tool to investigate the simultaneous activity of multiple cellular pathways from hundreds to thousands of genes. However, because data in the colossal amounts generated by DNA microarray technology are usually complex, noisy, high-dimensional, and often hindered by low statistical power, their exploitation is difficult. To overcome these problems, two kinds of unsupervised analysis methods for microarray data: principal component analysis (PCA and independent component analysis (ICA have been developed to accomplish the task. PCA projects the data into a new space spanned by the principal components that are mutually orthonormal to each other. The constraint of mutual orthogonality and second-order statistics technique within PCA algorithms, however, may not be applied to the biological systems studied. Extracting and characterizing the most informative features of the biological signals, however, require higher-order statistics. Results ICA is one of the unsupervised algorithms that can extract higher-order statistical structures from data and has been applied to DNA microarray gene expression data analysis. We performed FastICA method on DNA microarray gene expression data from Alzheimer's disease (AD hippocampal tissue samples and consequential gene clustering. Experimental results showed that the ICA method can improve the clustering results of AD samples and identify significant genes. More than 50 significant genes with high expression levels in severe AD were extracted, representing immunity-related protein, metal-related protein, membrane protein, lipoprotein, neuropeptide, cytoskeleton protein, cellular binding protein, and ribosomal protein. Within the aforementioned categories, our method also found 37 significant genes with low expression levels. Moreover, it is worth noting that some oncogenes and phosphorylation-related proteins are expressed in low levels. In

  9. MOLECULAR CLONING OF OVINE cDNA LEPTIN GENE

    Directory of Open Access Journals (Sweden)

    CLAUDIA TEREZIA SOCOL

    2013-12-01

    Full Text Available An efficient bacterial transformation system suitable for cloning the coding sequence of the ovine leptin gene in E. coli DH5α host cells using the pGEMT easy vector it is described in this paper. The necessity of producing leptin is based on the fact that the role of this molecule in the animal and human organism is still unknown, leptin not existing as commercial product on the Romanian market. The results obtained in the bacterial transformation, cloning, recombinant clones selection, control of the insertion experiments and DNA computational analysis represent the first steps in further genetic engineering experiments such as production of DNA libraries, DNA sequencing, protein expression, etc., for a further contribution in elucidating the role of leptin in the animal and human organism.

  10. cDNA-SRAP and Its Application in Differential Gene Expression Analysis: A Case Study in Erianthus arundinaceum

    Directory of Open Access Journals (Sweden)

    Youxiong Que

    2012-01-01

    Full Text Available Erianthus arundinaceum is a wild relative species of sugarcane. The aim of this research was to demonstrate the feasibility of cDNA-SRAP for differential gene expression and to explore the molecular mechanism of drought resistance in E. arundinaceum. cDNA-SRAP technique, for the first time, was applied in the analysis of differential gene expression in E. arundinaceum under drought stress. In total, eight differentially expressed genes with length of 185–427 bp were successfully isolated (GenBank Accession numbers: EU071770, EU071772, EU071774, EU071776, EU071777, EU071779, EU071780, and EU071781. Based on their homologies with genes in GenBank, these genes were assumed to encode ribonuclease III, vacuolar protein, ethylene insensitive protein, aerobactin biosynthesis protein, photosystem II protein, glucose transporter, leucine-rich repeat protein, and ammonia monooxygenase. Real-time PCR analysis on the expression profiling of gene (EU071774 encoding ethylene-insensitive protein and gene (EU071781 encoding ammonia monooxygenase revealed that the expression of these two genes was upregulated both by PEG and ABA treatments, suggesting that they may involve in the drought resistance of E. arundinaceum. This study constitutes the first report of genes activated in E. arundinaceum by drought stress and opens up the application of cDNA-SRAP in differential gene expression analysis in E. arundinaceum under certain stress conditions.

  11. Advances in Non-Viral DNA Vectors for Gene Therapy

    Directory of Open Access Journals (Sweden)

    Cinnamon L. Hardee

    2017-02-01

    Full Text Available Uses of viral vectors have thus far eclipsed uses of non-viral vectors for gene therapy delivery in the clinic. Viral vectors, however, have certain issues involving genome integration, the inability to be delivered repeatedly, and possible host rejection. Fortunately, development of non-viral DNA vectors has progressed steadily, especially in plasmid vector length reduction, now allowing these tools to fill in specifically where viral or other non-viral vectors may not be the best options. In this review, we examine the improvements made to non-viral DNA gene therapy vectors, highlight opportunities for their further development, address therapeutic needs for which their use is the logical choice, and discuss their future expansion into the clinic

  12. Advances in Non-Viral DNA Vectors for Gene Therapy

    Science.gov (United States)

    Hardee, Cinnamon L.; Arévalo-Soliz, Lirio Milenka; Hornstein, Benjamin D.; Zechiedrich, Lynn

    2017-01-01

    Uses of viral vectors have thus far eclipsed uses of non-viral vectors for gene therapy delivery in the clinic. Viral vectors, however, have certain issues involving genome integration, the inability to be delivered repeatedly, and possible host rejection. Fortunately, development of non-viral DNA vectors has progressed steadily, especially in plasmid vector length reduction, now allowing these tools to fill in specifically where viral or other non-viral vectors may not be the best options. In this review, we examine the improvements made to non-viral DNA gene therapy vectors, highlight opportunities for their further development, address therapeutic needs for which their use is the logical choice, and discuss their future expansion into the clinic. PMID:28208635

  13. Gene order computation using Alzheimer's DNA microarray gene expression data and the Ant Colony Optimisation algorithm.

    Science.gov (United States)

    Pang, Chaoyang; Jiang, Gang; Wang, Shipeng; Hu, Benqiong; Liu, Qingzhong; Deng, Youping; Huang, Xudong

    2012-01-01

    As Alzheimer's Disease (AD) is the most common form of dementia, the study of AD-related genes via biocomputation is an important research topic. One method of studying AD-related gene is to cluster similar genes together into a gene order. Gene order is a good clustering method as the results can be optimal globally while other clustering methods are only optimal locally. Herein we use the Ant Colony Optimisation (ACO)-based algorithm to calculate the gene order from an Alzheimer's DNA microarray dataset. We test it with four distance measurements: Pearson distance, Spearmen distance, Euclidean distance, and squared Euclidean distance. Our computing results indicate: a different distance formula generated a different quality of gene order, the squared Euclidean distance approach produced the optimal AD-related gene order.

  14. Evolving DNA motifs to predict GeneChip probe performance

    Directory of Open Access Journals (Sweden)

    Harrison AP

    2009-03-01

    Full Text Available Abstract Background Affymetrix High Density Oligonuclotide Arrays (HDONA simultaneously measure expression of thousands of genes using millions of probes. We use correlations between measurements for the same gene across 6685 human tissue samples from NCBI's GEO database to indicated the quality of individual HG-U133A probes. Low correlation indicates a poor probe. Results Regular expressions can be automatically created from a Backus-Naur form (BNF context-free grammar using strongly typed genetic programming. Conclusion The automatically produced motif is better at predicting poor DNA sequences than an existing human generated RE, suggesting runs of Cytosine and Guanine and mixtures should all be avoided.

  15. GenePublisher: automated analysis of DNA microarray data

    DEFF Research Database (Denmark)

    Knudsen, Steen; Workman, Christopher; Sicheritz-Ponten, T.

    2003-01-01

    GenePublisher, a system for automatic analysis of data from DNA microarray experiments, has been implemented with a web interface at http://www.cbs.dtu.dk/services/GenePublisher. Raw data are uploaded to the server together with aspecification of the data. The server performs normalization......, statistical analysis and visualization of the data. The results are run against databases of signal transduction pathways, metabolic pathways and promoter sequences in order to extract more information. The results of the entire analysis are summarized in report form and returned to the user....

  16. Adenovirus with DNA Packaging Gene Mutations Increased Virus Release

    Science.gov (United States)

    Wechman, Stephen L.; Rao, Xiao-Mei; McMasters, Kelly M.; Zhou, Heshan Sam

    2016-01-01

    Adenoviruses (Ads) have been extensively manipulated for the development of cancer selective replication, leading to cancer cell death or oncolysis. Clinical studies using E1-modified oncolytic Ads have shown that this therapeutic platform was safe, but with limited efficacy, indicating the necessity of targeting other viral genes for manipulation. To improve the therapeutic efficacy of oncolytic Ads, we treated the entire Ad genome repeatedly with UV-light and have isolated AdUV which efficiently lyses cancer cells as reported previously (Wechman, S. L. et al. Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection. Viruses 2016, 8, 6). In this report, we show that no mutations were observed in the early genes (E1 or E4) of AdUV while several mutations were observed within the Ad late genes which have structural or viral DNA packaging functions. This study also reported the increased release of AdUV from cancer cells. In this study, we found that AdUV inhibits tumor growth following intratumoral injection. These results indicate the potentially significant role of the viral late genes, in particular the DNA packaging genes, to enhance Ad oncolysis. PMID:27999391

  17. DnaC inactivation in Escherichia coli K-12 induces the SOS response and expression of nucleotide biosynthesis genes

    DEFF Research Database (Denmark)

    Løbner-Olesen, Anders; Slominska-Wojewodzka, Monika; Hansen, Flemming G.;

    2008-01-01

    Background: Initiation of chromosome replication in E. coli requires the DnaA and DnaC proteins and conditionally-lethal dnaA and dnaC mutants are often used to synchronize cell populations. Methodology/Principal Findings: DNA microarrays were used to measure mRNA steady-state levels in initiatio......C genes was increased at the non-permissive temperature in the respective mutant strains indicating auto-regulation of both genes. Induction of the SOS regulon was observed in dnaC2 cells at 38 degrees C and 42 degrees C. Flow cytometric analysis revealed that dnaC2 mutant cells at non......-permissive temperature had completed the early stages of chromosome replication initiation. Conclusion/Significance: We suggest that in dnaC2 cells the SOS response is triggered by persistent open-complex formation at oriC and/or by arrested forks that require DnaC for replication restart....

  18. Chromatin opening of DNA satellites by targeted sequence-specific drugs.

    Science.gov (United States)

    Janssen, S; Durussel, T; Laemmli, U K

    2000-11-01

    There are few tools available for dissecting and elucidating the functions of DNA satellites and other nongenic DNA. To address this, we have explored the experimental potential of DNA sequence-specific drugs containing pyrrole and imidazole amino acids (polyamides). Compounds were synthesized that target different Drosophila melanogaster satellites. Dimeric oligopyrroles were shown to target the AT-rich satellites I, III, and SARs (scaffold associated regions). One polyamide (P31) specifically binds the GAGAA satellite V. Specificity of targeting was established by footprinting, epifluorescence of nuclei, and polytene chromosomes stained with fluorescent derivatives. These polyamides were shown to mediate satellite-specific chromatin opening of the chromatin fiber. Remarkably, certain polyamides induced defined gain or loss-of-function phenotypes when fed to Drosophila melanogaster.

  19. ATPase-dependent auto-phosphorylation of the open condensin hinge diminishes DNA binding.

    Science.gov (United States)

    Akai, Yuko; Kanai, Ryuta; Nakazawa, Norihiko; Ebe, Masahiro; Toyoshima, Chikashi; Yanagida, Mitsuhiro

    2014-12-01

    Condensin, which contains two structural maintenance of chromosome (SMC) subunits and three regulatory non-SMC subunits, is essential for many chromosomal functions, including mitotic chromosome condensation and segregation. The ATPase domain of the SMC subunit comprises two termini connected by a long helical domain that is interrupted by a central hinge. The role of the ATPase domain has remained elusive. Here we report that the condensin SMC subunit of the fission yeast Schizosaccharomyces pombe is phosphorylated in a manner that requires the presence of the intact SMC ATPase Walker motif. Principal phosphorylation sites reside in the conserved, glycine-rich stretch at the hinge interface surrounded by the highly basic DNA-binding patch. Phosphorylation reduces affinity for DNA. Consistently, phosphomimetic mutants produce severe mitotic phenotypes. Structural evidence suggests that prior opening (though slight) of the hinge is necessary for phosphorylation, which is implicated in condensin's dissociation from and its progression along DNA.

  20. Genetic characterization of complete open reading frame of glycoprotein C gene of bovine herpesvirus 1

    Directory of Open Access Journals (Sweden)

    Saurabh Majumder

    2013-10-01

    Full Text Available Aim: To characterize one of the major glycoprotein genes viz., glycoprotein C (gC; UL44, unique long region 44 of bovineherpesvirus 1(BoHV1 of Indian origin at genetic and phylogenetic level.Materials and Methods: A bovine herpesvirus 1 isolate viz., (BoHV1/IBR 216 II/ 1976/ India maintained at Division ofVirology, IVRI, Mukteswar was used for the current study. The DNA was extracted using commercial kit and the completeORF of gC gene was amplified, cloned, and sequenced by conventional Sanger sequencing method. The sequence wasgenetically and phylogenetically analysed using various bioinformatic tools. The sequence was submitted in the Genbankwith accession number Kc756965.Results: The complete ORF of gC gene was amplified and sequenced. It showed 100% sequence homology with referencecooper strain of BoHV1 and divergence varied from 0% to 2.7% with other isolates of BoHV1. The isolate under study haddivergence of 9.2%, 13%, 26.6%, and 9.2% with BoHV5 (Bovine herpesvirus 5, CvHV1 (Cervid herpesvirus 1, CpHV1(Caprine herpesvirus 1, and BuHV1 (Bubaline herpesvirus 1, respectively.Conclusion: This is the first genetic characterization of complete open reading frame (ORF of glycoprotein C gene (UL44 ofIndian isolate of BoHV1. The gC gene of BoHV1 is highly conserved among all BoHV1 isolates and it can be used as a targetfor designing diagnostic primers for the specific detection of BoHV1.

  1. Promoter DNA hypermethylation and gene repression in undifferentiated Arabidopsis cells.

    Directory of Open Access Journals (Sweden)

    María Berdasco

    Full Text Available Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells.

  2. SIMAGE: simulation of DNA-microarray gene expression data

    Directory of Open Access Journals (Sweden)

    Kuipers Oscar P

    2006-04-01

    Full Text Available Abstract Background Simulation of DNA-microarray data serves at least three purposes: (i optimizing the design of an intended DNA microarray experiment, (ii comparing existing pre-processing and processing methods for best analysis of a given DNA microarray experiment, (iii educating students, lab-workers and other researchers by making them aware of the many factors influencing DNA microarray experiments. Results Our model has multiple layers of factors influencing the experiment. The relative influence of such factors can differ significantly between labs, experiments within labs, etc. Therefore, we have added a module to roughly estimate their parameters from a given data set. This guarantees that our simulated data mimics real data as closely as possible. Conclusion We introduce a model for the simulation of dual-dye cDNA-microarray data closely resembling real data and coin the model and its software implementation "SIMAGE" which stands for simulation of microarray gene expression data. The software is freely accessible at: http://bioinformatics.biol.rug.nl/websoftware/simage.

  3. Transcription without XPB Establishes a Unified Helicase-Independent Mechanism of Promoter Opening in Eukaryotic Gene Expression.

    Science.gov (United States)

    Alekseev, Sergey; Nagy, Zita; Sandoz, Jérémy; Weiss, Amélie; Egly, Jean-Marc; Le May, Nicolas; Coin, Frederic

    2017-02-02

    Transcription starts with the assembly of pre-initiation complexes on promoters followed by their opening. Current models suggest that class II gene transcription requires ATP and the TFIIH XPB subunit to open a promoter. Here, we observe that XPB depletion surprisingly leaves transcription virtually intact. In contrast, inhibition of XPB ATPase activity affects transcription, revealing that mRNA expression paradoxically accommodates the absence of XPB while being sensitive to the inhibition of its ATPase activity. The XPB-depleted TFIIH complex is recruited to active promoters and contributes to transcription. We finally demonstrate that the XPB ATPase activity is only used to relieve a transcription initiation block imposed by XPB itself. In the absence of this block, transcription initiation can take place without XPB ATPase activity. These results suggest that a helicase is dispensable for mRNA transcription, thereby unifying the mechanism of promoter DNA opening for the three eukaryotic RNA polymerases.

  4. Bio-reducible polycations from ring-opening polymerization as potential gene delivery vehicles.

    Science.gov (United States)

    Yu, Qing-Ying; Liu, Yan-Hong; Huang, Zheng; Zhang, Ji; Luan, Chao-Ran; Zhang, Qin-Fang; Yu, Xiao-Qi

    2016-07-01

    Synthetic polycations show great potential for the construction of ideal non-viral gene delivery systems. Several cationic polymers were synthesized by the epoxide ring-opening polymerization between diepoxide and various polyamines. Disulfide bonds were introduced to afford the polymers bio-reducibility, while the oxygen-rich structure might enhance the serum tolerance and biocompatibility. The polycations have much lower molecular weights than PEI 25 kDa, but still could well bind and condense DNA into nano-sized particles. DNA could be released from the polyplexes by addition of reductive DTT. Compared to PEI, the polycations have less cytotoxicity possibly due to their lower molecular weights and oxygen-rich structure. More significantly, these materials exhibit excellent serum tolerance than PEI, and up to 6 times higher transfection efficiency than PEI could be obtained in the presence of serum. The transfection mediated by was seldom affected even at a high concentration of serum. Much lower protein adsorption of polycations than PEI was proved by bovine serum albumin adsorption experiments. Flow cytometry also demonstrates their good serum resistance ability.

  5. Analysis of gene expression profile induced by EMP-1 in esophageal cancer cells using cDNA Microarray

    Institute of Scientific and Technical Information of China (English)

    Hai-Tao Wang; Jian-Ping Kong; Fang Ding; Xiu-Qin Wang; Ming-Rong Wang; Lian-Xin Liu; Min Wu; Zhi-Hua Liu

    2003-01-01

    AIM: To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1.METHODS: The authors first constructed pcDNA3.1/mychis expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes,classification was performed according to their function and cellular component.RESULTS: Human EMP-1 gene can be stably expressed in ECg706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion.CONCLUSION: Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved incell signaling, cell communication and adhesion regulators.

  6. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme of escherichia coli.

    Science.gov (United States)

    Burgers, P M; Kornberg, A; Sakakibara, Y

    1981-09-01

    An Escherichia coli mutant, dnaN59, stops DNA synthesis promptly upon a shift to a high temperature; the wild-type dnaN gene carried in a transducing phage encodes a polypeptide of about 41,000 daltons [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553; Yuasa, S. & Sakakibara, Y. (1980) Mol. Gen. Genet. 180, 267-273]. We now find that the product of dnaN gene is the beta subunit of DNA polymerase III holoenzyme, the principal DNA synthetic multipolypeptide complex in E. coli. The conclusion is based on the following observations: (i) Extracts from dnaN59 cells were defective in phage phi X174 and G4 DNA synthesis after the mutant cells had been exposed to the increased temperature. (ii) The enzymatic defect was overcome by addition of purified beta subunit but not by other subunits of DNA polymerase III holoenzyme or by other replication proteins required for phi X174 DNA synthesis. (iii) Partially purified beta subunit from the dnaN mutant, unlike that from the wild type, was inactive in reconstituting the holoenzyme when mixed with the other purified subunits. (iv) Increased dosage of the dnaN gene provided by a plasmid carrying the gene raised cellular levels of the beta subunit 5- to 6-fold.

  7. A Bacillus subtilis dnaG mutant harbours a mutation in a gene homologous to the dnaN gene of Escherichia coli.

    Science.gov (United States)

    Ogasawara, N; Moriya, S; Mazza, G; Yoshikawa, H

    1986-01-01

    A dnaG mutation of Bacillus subtilis, dnaG5, was found to be linked closely to recF. We have reported previously that two putative dna genes, 'dnaA' and 'dnaN', highly homologous to Escherichia coli's dnaA and dnaN, respectively, were located adjacent to recF [Ogasawara et al., EMBO J., 4 (1985) 3345-3350]. Transformation by various fragments cloned from the 'dnaA'-recF region of the wild-type cell revealed that a 532-bp AluI fragment containing 5'-portion of the 'dnaN' gene could transform the dnaG5 mutation. The nucleotide (nt) sequence of the same fragment cloned from the mutant cell shows a single nt change in the ORF of 'dnaN' which in turn causes a single amino acid alteration from Gly to Arg. The 'dnaN' gene is now proven to be a dna gene, mutations in which result in instant arrest of chromosomal replication.

  8. The prima donna of epigenetics: the regulation of gene expression by DNA methylation

    Directory of Open Access Journals (Sweden)

    K.F. Santos

    2005-10-01

    Full Text Available This review focuses on the mechanisms of DNA methylation, DNA methylation pattern formation and their involvement in gene regulation. Association of DNA methylation with imprinting, embryonic development and human diseases is discussed. Furthermore, besides considering changes in DNA methylation as mechanisms of disease, the role of epigenetics in general and DNA methylation in particular in transgenerational carcinogenesis, in memory formation and behavior establishment are brought about as mechanisms based on the cellular memory of gene expression patterns.

  9. Genes and the physics of the DNA double-helix.

    Science.gov (United States)

    Yeramian, E

    2000-09-19

    The processing of the genetic information stored in the double-helical DNA implies the separation of the two strands, the physics of which is described by the helix-coil transition model. Is there a relationship between genetic maps and DNA physical stability maps that plot the sequence-specific propensity for the thermal disruption of the double-helix? Here, with appropriate methodological formulations, such maps are derived for a large set of sequences, including complete genomes. The superposition of the two maps leads to a contrasted picture with correlations ranging between two extremes: from almost perfect (with the genes precisely delineated as stable regions) to more or less complete unrelatedness. The simplest explanation for the results is that the observed striking correlations correspond to the relics of a primeval organisation of the genetic message, with the physics of DNA playing a role in the delimitation of coding regions. In order to trace the evolutionary fate of this signal further, a detailed study of the yeast complete genome is performed. In this study, the superposition of the genetic and physical stability maps is examined in the light of information concerning gene duplication. On the basis of this analysis it is concluded that the 'signature' associated with the supposed archaic signal is in the process of being erased, most probably because the underlying feature is no longer under selective pressure. There are many evolutionary implications for the results presented and for their proposed interpretations, notably concerning models of mutational dynamics in relation to erasure processes.

  10. Polymorphism of the DNA Base Excision Repair Genes in Keratoconus

    Directory of Open Access Journals (Sweden)

    Katarzyna A. Wojcik

    2014-10-01

    Full Text Available Keratoconus (KC is a degenerative corneal disorder for which the exact pathogenesis is not yet known. Oxidative stress is reported to be associated with this disease. The stress may damage corneal biomolecules, including DNA, and such damage is primarily removed by base excision repair (BER. Variation in genes encoding BER components may influence the effectiveness of corneal cells to cope with oxidative stress. In the present work we genotyped 5 polymorphisms of 4 BER genes in 284 patients and 353 controls. The A/A genotype of the c.–1370T>A polymorphism of the DNA polymerase γ (POLG gene was associated with increased occurrence of KC, while the A/T genotype was associated with decreased occurrence of KC. The A/G genotype and the A allele of the c.1196A>G polymorphism of the X-ray repair cross-complementing group 1 (XRCC1 were associated with increased, and the G/G genotype and the G allele, with decreased KC occurrence. Also, the C/T and T as well as C/C genotypes and alleles of the c.580C>T polymorphism of the same gene displayed relationship with KC occurrence. Neither the g.46438521G>C polymorphism of the Nei endonuclease VIII-like 1 (NEIL1 nor the c.2285T>C polymorphism of the poly(ADP-ribose polymerase-1 (PARP-1 was associated with KC. In conclusion, the variability of the XRCC1 and POLG genes may play a role in KC pathogenesis and determine the risk of this disease.

  11. DNA Microspheres Coated with Bioavailable Polymer as an Efficient Gene Expression Agent in Yeasts

    Directory of Open Access Journals (Sweden)

    Irena Reytblat

    2016-01-01

    Full Text Available Gene delivery is one of the steps necessary for gene therapy and for genetic modification. However, delivering DNA into cells is challenging due to its negative charge that leads to repulsion by the negative cell membrane. In the current research, DNA spheres with a DNA encoding to a certain gene were coated with bioavailable polymers, polyethylene imine (PEI and polycaprolactone (PCL, in a short, one-step sonochemical reaction. The polymers were used in order to neutralize the negative charge of the DNA. Our study shows that the DNA nanospheres not only managed to penetrate the cell without causing it any damage, but also expressed the desired gene inside it.

  12. The Cloning of the Human Tumor Supressor Gene INGI: DNA Cloning into Plasmid Vector and DNA Analysis by Restriction Enzymes

    Directory of Open Access Journals (Sweden)

    Elza Ibrahim Auerkari

    2015-11-01

    Full Text Available DNA cloning is one of the most important techniques In the field of molecular biology, with a critical role in analyzing the structure and function of genes and their adjacent regulatory regions. DNA cloning is helpful in learning fundamental molecular biological techniques, since DNA cloning involves a series of them, such as polymerase chain reaction (PCR, DNA ligation, bacterial transformation, bacterial culture, plasmid DNA extraction, DNA digestion with restriction enzymes and agarose gel electrophoresis. In this paper the cloning of the human tumor suppressor gene INGI has been used to illustrate the methodology. The gene was amplified by PCR, cloned into a TA-cloning vectore, and restriction enzyme mapping was used to distinguish the sense INGI construct from the antisense INGI construct.

  13. How-to-Do-It: Recombinant DNA Made Easy II. Gene, Gene, Who's Got the Gene?

    Science.gov (United States)

    Thomson, Robert G.

    1989-01-01

    Described is an activity in which students are able to determine that DNA can be transferred between bacteria and should be able to predict the type of DNA transferred. Methods, materials, and results are discussed. (CW)

  14. Protection against heartwater by DNA immunisation with four Ehrlichia ruminantium open reading frames.

    Science.gov (United States)

    Pretorius, A; Collins, N E; Steyn, H C; van Strijp, F; van Kleef, M; Allsopp, B A

    2007-03-08

    We have reported previously that a recombinant DNA vaccine consisting of four Ehrlichia ruminantium (Welgevonden) open reading frames (ORFs) known as the 1H12 cocktail provided protection against a virulent E. ruminantium (Welgevonden) needle challenge in sheep. In this study, we have investigated the vaccine effectiveness of two other cocktails of E. ruminantium (Welgevonden) ORFs, as well as single ORFs from the 1H12 cocktail, to protect sheep against a virulent needle challenge with the homologous strain. Each individual 1H12 ORF provided protection, but all the animals vaccinated with the other cocktails succumbed to the challenge.

  15. Isolation of DNA-methyltransferase genes from strawberry (Fragaria x ananassa Duch.) and their expression in relation to micropropagation.

    Science.gov (United States)

    Chang, Linlin; Zhang, Zhihong; Han, Baiming; Li, He; Dai, Hongyan; He, Ping; Tian, Hongzhe

    2009-09-01

    DNA methylation can control gene expression and may also play a role in plant development. Methylation of cytosine residues in DNA is enzymatically catalyzed by DNA methyltransferases. In this study, full-length genomic genes and cDNAs of methyltransferase (MET1) and domain-rearranged methyltransferase (DRM) were isolated from strawberry (Fragaria x ananassa Duch.). Two genomic clones (FaMET1a and FaMET1b) encoding MET1 had open-reading frame of 4,695 and 4,671 nucleotides with two introns, respectively. Amino acid sequence comparison indicated high similarity (98.72% identity) of strawberry MET1 protein to other plant MET1 sequences. The full-length cDNA of strawberry DRM genes (FaDRMa, FaDRMb and FaDRMc) were 2,273, 2,282 and 2,288 bp, respectively. Ten introns with different sizes were dispersed in FaDRM genes. Similarly, FaDRMa, FaDRMb and FaDRMc had high-sequence similarity overall. Expressions of strawberry MET1 and DRM genes were compared among in vitro-micropropagated plants, generations of micropropagated plants and conventionally propagated plants. The transcriptional expressions of both FaMET1 and FaDRM genes were downregulated in micropropagated plants, and they were recovered in the first and second runner generations of micropropagated plants. However, there was a slighter difference in global DNA methylation rates between micropropagated plants and conventionally propagated plants. Therefore, there was no positive relation between global DNA methylation rates and the expression levels of MET1 and DRM genes.

  16. Hepatitis B virus DNA integration and transactivation of cellular genes

    Directory of Open Access Journals (Sweden)

    Vijay Kumar

    2007-02-01

    Full Text Available

    Chronic hepatitis B virus (HBV infection is etiologically related to human hepatocellular carcinoma (HCC. Most HCCs contain integrated HBV DNA in hepatocyte, suggesting that the integration may be involved in carcinogenesis. Available data on the integrants from human hepatocellular carcinomas seem to represent primary integrants as well as the products of secondary rearrangements. By means of structural analyses of the possible primary integrants, it has been observed that the replication intermediates of the viral genome are the preferred substrates for integration. The integrated HBV DNA and the target cellular DNA are invariably associated with deletions, possibly reflecting the substrate for, and the mechanism of, the integration reaction. The host DNA sequences as well as the target site of integration in chromosomes are selected randomly suggesting that HBV DNA integration should bring about random mutagenic effects. Analysis of the samples recovered from hepatocellular carcinomas show that the integrated HBV DNA can mediate secondary rearrangements of chromosomes, such as translocations, inversions, deletions and (possibly amplifications. The integration of HBV DNA into the host genome occurs at early steps of clonal tumor expansion. The integration has been shown in a number of cases to affect a variety of cancer-related genes and to exert insertional mutagenesis. However, in contrast to the woodchuck model, in which specific HBV-DNA integration is detectable in most cases, insertional activation or inactivation of cellular genes appears to be a rare event in man. The discovery of transactivating functions exerted by HBx and truncated HBs(urface proteins supports the notion that these could be relevant to hepatocarcinogenesis as these transactivator sequences have been found in a large number of HCC tumors or hepatoma-derived cell lines. The HBx

  17. DNA methylation and gene expression of HIF3A

    DEFF Research Database (Denmark)

    Main, Ailsa Maria; Gillberg, Linn; Jacobsen, Anna Louisa;

    2016-01-01

    from 48 families, from whom we had SAT and muscle biopsies. DNA methylation of four CpG sites in the HIF3A promoter was analyzed in the blood and SAT by pyrosequencing, and HIF3A gene expression was analyzed in SAT and muscle by qPCR. An index of whole-body insulin sensitivity was estimated from oral....... Interestingly, HIF3A expression in SAT, but not in muscle, associated negatively with BMI and whole-body insulin resistance. We found a significant effect of familiality on HIF3A methylation levels in the blood and HIF3A expression levels in skeletal muscle. CONCLUSIONS: Our findings are in line...... individuals, and whether HIF3A gene expression in SAT and skeletal muscle biopsies showed associations with BMI and insulin resistance. Furthermore, we aimed to investigate gender specificity and heritability of these traits. METHODS: We studied 137 first-degree relatives of type 2 diabetes (T2D) patients...

  18. Isolation of novel human cDNA (hGMF-gamma) homologous to Glia Maturation Factor-beta gene.

    Science.gov (United States)

    Asai, K; Fujita, K; Yamamoto, M; Hotta, T; Morikawa, M; Kokubo, M; Moriyama, A; Kato, T

    1998-03-13

    A novel full-length human cDNA homologous to Glia Maturation Factor-beta (GMF-beta) gene was isolated. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein sequence of 142 amino acid residues. The deduced amino acid sequences of its putative product is highly homologous to human GMF-beta (82% identity) and named for GMF-gamma. Northern blot analysis indicated that a message of 0.9 kb long, but not 4.1 kb of GMF-beta, is predominantly expressed in human lung, heart, and placenta.

  19. cDNA cloning and expression of an apoptosis-related gene, human TFAR15 gene

    Institute of Scientific and Technical Information of China (English)

    王玉刚; 刘洪涛; 张颖妹; 马大龙

    1999-01-01

    By means of cDNA-RDA method. some cDNA fragments were found to have high levels of expression during deprivation of GM-CSF (granulocyte macrophage-colony stimulating factor) in a human myeloid cell line, TF-1 cells. One of these tragments was identified as a novel gene. To get the full length of cDNA, rapid amplification of cDNA ends (RACE) and expressed sequence tags (EST) overlapping fragments assembling strategies were used. The novel gene was named TRAF15 (TF-1 cell apoptosis related gene-15), which consists of 1218 nueleotides and encodes 212 amino acids. The putative protein protein product of TFAR15 is partially homologous to C. elegans protein C14A4. 11. TFAR15 mRNA is expressed in fetal liver, kidney, spleen and lung. and also in some human myeloid cell lines. Both of the TFAR15 mRNA and protein were highly expressed in TF-(?) cells after GM-CSF withdrawal. In vitro analysis showed that the recombinant TFAR15 protein co(?)ld inhibit the natural cell death of 293 cells, an embryonic kidney cell

  20. Impact of prenatal polycyclic aromatic hydrocarbon exposure on behavior, cortical gene expression, and DNA methylation of the Bdnf gene

    Directory of Open Access Journals (Sweden)

    Rachel L. Miller

    2016-03-01

    Full Text Available Prenatal exposure to polycyclic aromatic hydrocarbons (PAH has been associated with sustained effects on the brain and behavior in offspring. However, the mechanisms have yet to be determined. We hypothesized that prenatal exposure to ambient PAH in mice would be associated with impaired neurocognition, increased anxiety, altered cortical expression of Bdnf and Grin2b, and greater DNA methylation of Bdnf. Our results indicated that during open-field testing, prenatal PAH–exposed offspring spent more time immobile and less time exploring. Females produced more fecal boli. Offspring prenatally exposed to PAH displayed modest reductions in overall exploration of objects. Further, prenatal PAH exposure was associated with lower cortical expression of Grin2b and Bdnf in males and greater Bdnf IV promoter methylation. Epigenetic differences within the Bdnf IV promoter correlated with Bdnf gene expression but not with the observed behavioral outcomes, suggesting that additional targets may account for these PAH-associated effects.

  1. DNA Isn't Destiny: Healthy Living Can Overcome Genes Linked to Heart Disease

    Science.gov (United States)

    ... page: https://medlineplus.gov/news/fullstory_161999.html DNA Isn't Destiny: Healthy Living Can Overcome Genes ... unhealthy lifestyle choices. "For heart attack at least, DNA is not destiny," Kathiresan said. "You have control ...

  2. Gene expression data (CEL files) - Open TG-GATEs | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available [ Credits ] BLAST Search Image Search Home About Archive Update History Contact us Open TG-GATEs Gene expres...sion data (CEL files) Data detail Data name Gene expression data (CEL files) Descri...Tab Separated Value (TSV) format is included. CEL is one of the file formats that expresses gene expression ...data (raw data) generated from Affymetrix GeneChip®. Data file File name: Gene expression data from rat samp...ze: 12.0GB total File name: Gene expression data from human samples File URL: ftp://ftp.biosciencedbc.jp/arc

  3. Clinical characteristics and prognosis of acute myeloid leukemia associated with DNA-methylation regulatory gene mutations.

    Science.gov (United States)

    Ryotokuji, Takeshi; Yamaguchi, Hiroki; Ueki, Toshimitsu; Usuki, Kensuke; Kurosawa, Saiko; Kobayashi, Yutaka; Kawata, Eri; Tajika, Kenji; Gomi, Seiji; Kanda, Junya; Kobayashi, Anna; Omori, Ikuko; Marumo, Atsushi; Fujiwara, Yusuke; Yui, Shunsuke; Terada, Kazuki; Fukunaga, Keiko; Hirakawa, Tsuneaki; Arai, Kunihito; Kitano, Tomoaki; Kosaka, Fumiko; Tamai, Hayato; Nakayama, Kazutaka; Wakita, Satoshi; Fukuda, Takahiro; Inokuchi, Koiti

    2016-09-01

    In recent years, it has been reported that the frequency of DNA-methylation regulatory gene mutations - mutations of the genes that regulate gene expression through DNA methylation - is high in acute myeloid leukemia. The objective of the present study was to elucidate the clinical characteristics and prognosis of acute myeloid leukemia with associated DNA-methylation regulatory gene mutation. We studied 308 patients with acute myeloid leukemia. DNA-methylation regulatory gene mutations were observed in 135 of the 308 cases (43.8%). Acute myeloid leukemia associated with a DNA-methylation regulatory gene mutation was more frequent in older patients (Pgene mutation was an unfavorable prognostic factor for overall survival in the whole cohort (P=0.0018), in patients aged ≤70 years, in patients with intermediate cytogenetic risk, and in FLT3-ITD-negative patients (P=0.0409). Among the patients with DNA-methylation regulatory gene mutations, 26.7% were found to have two or more such mutations and prognosis worsened with increasing number of mutations. In multivariate analysis DNA-methylation regulatory gene mutation was an independent unfavorable prognostic factor for overall survival (P=0.0424). However, patients with a DNA-methylation regulatory gene mutation who underwent allogeneic stem cell transplantation in first remission had a significantly better prognosis than those who did not undergo such transplantation (P=0.0254). Our study establishes that DNA-methylation regulatory gene mutation is an important unfavorable prognostic factor in acute myeloid leukemia.

  4. Interaction of DNA repair gene polymorphisms and aflatoxin B1 in the risk of hepatocellular carcinoma

    OpenAIRE

    2014-01-01

    Aflatoxin B1 (AFB1) is an important environmental carcinogen and can induce DNA damage and involve in the carcinogenesis of hepatocellular carcinoma (HCC). The deficiency of DNA repair capacity related to the polymorphisms of DNA repair genes might play a central role in the process of HCC tumorigenesis. However, the interaction of DNA repair gene polymorphisms and AFB1 in the risk of hepatocellular carcinoma has not been elucidated. In this study, we investigated whether six polymorphisms (i...

  5. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease

    Energy Technology Data Exchange (ETDEWEB)

    Dupuy, Aurélie [Laboratory of Genetic Instability and Oncogenesis UMR8200CNRS, Institut Gustave Roussy and University Paris-Sud, Villejuif (France); Sarasin, Alain, E-mail: alain.sarasin@gustaveroussy.fr [Laboratory of Genetic Instability and Oncogenesis UMR8200CNRS, Institut Gustave Roussy and University Paris-Sud, Villejuif (France); Service de Génétique, Institut Gustave Roussy (France)

    2015-06-15

    Graphical abstract: - Highlights: • Full correction of mutation in the XPC gene by engineered nucleases. • Meganucleases and TALENs are inhibited by 5-MeC for inducing double strand breaks. • Gene therapy of XP cells is possible using homologous recombination for DSB repair. - Abstract: Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients.

  6. IDENTIFICATION OF DIFFERENTIAL GENES IN OVARIAN CANCER USING REPRESENTATIONAL DIFFERENCE ANALYSIS OF cDNA

    Institute of Scientific and Technical Information of China (English)

    Hong Chen; Min Wang; Xin-yan Wang; Shan Gao; Jun Wang; Xiao-ming Guan

    2005-01-01

    Objective To identify differential genes between normal ovarian epithelium tissue and ovarian epithelial cancer using representational difference analysis of cDNA (cDNA-RDA). Methods cDNA-RDA was performed to identify the differentially expressed sequences between cDNAs from cancer tissue and cDNAs from normal ovarian tissue in the same patient who was in the early stage of ovarian serous cystadeno carcinoma. These differentially expressed fragments were cloned and analyzed, then sequenced and compared with known genes.Results Three differentially expressed cDNA fragments were isolated using cDNA from normal ovarian tissue as tester and cDNA from cancer tissue as driver amplicon by cDNA-RDA. DP Ⅲ-1 and DP Ⅲ-2 cDNA clone showed significant ho mology to the cDNA of alpha actin gene; DPⅢ-3 cDNA clone showed significant homology to the cDNA of transgelin gene. Conclusion cDNA-RDA can be used to sensitively identify the differentially expressed genes in ovarian serous cystadenocarcinoma. Ovarian serous cystadenocarcinoma involves alteration of multiple genes.

  7. The (PrS/HGF-pDNA) multilayer films for gene-eluting stent coating: Gene-protecting, anticoagulation, antibacterial properties, and in vivo antirestenosis evaluation.

    Science.gov (United States)

    Chang, Hao; Ren, Ke-feng; Zhang, He; Wang, Jin-lei; Wang, Bai-liang; Ji, Jian

    2015-02-01

    Vascular gene-eluting stents (GES) is a promising strategy for treatment of cardiovascular disease. Very recently, we have proved that the (protamine sulfate/plasmid DNA encoding hepatocyte growth factor) (PrS/HGF-pDNA) multilayer can serve as a powerful tool for enhancing competitiveness of endothelial cell over smooth muscle cell, which opens perspectives for the regulation of intercellular competitiveness in the field of interventional therapy. However, before the gene multilayer films could be used in vascular stents for real clinical application, the preservation of gene bioactivity during the industrial sterilization and the hemocompatibility of film should be taken into account. Actually, both are long been ignored issues in the field of gene coating for GES. In this study, we demonstrate that the (PrS/HGF-pDNA) multilayer film exhibits the good gene-protecting abilities, which is confirmed by using the industrial sterilizations (gamma irradiation and ethylene oxide) and a routine storage condition (dry state at 4°C for 30 days). Furthermore, hemocompatible measurements (such as platelet adhesion and whole blood coagulation) and antibacterial assays (bacteria adhesion and growth inhibition) indicate the good anticoagulation and antibacterial properties of the (PrS/HGF-pDNA) multilayer film. The in vivo preliminary data of angiography and histological analysis suggest that the (PrS/HGF-pDNA) multilayer coated stent can reduce the in-stent restenosis. This work reveals that the (PrS/HGF-pDNA) multilayer film could be a promising candidate as coating for GES, which is of great potential in future clinic application.

  8. Sequence analysis of a 9873 bp fragment of the left arm of yeast chromosome XV that contains the ARG8 and CDC33 genes, a putative riboflavin synthase beta chain gene, and four new open reading frames.

    Science.gov (United States)

    Casas, C; Aldea, M; Casamayor, A; Lafuente, M J; Gamo, F J; Gancedo, C; Ariño, J; Herrero, E

    1995-09-15

    The DNA sequence of a 9873 bp fragment located near the left telomere of chromosome XV has been determined. Sequence analysis reveals seven open reading frames. One is the ARG8 gene coding for N-acetylornithine aminotransferase. Another corresponds to CDC33, which codes for the initiation factor 4E or cap binding protein. The open reading frame AOE169 can be considered as the putative gene for the Saccharomyces cerevisiae riboflavin synthase beta chain, since its translation product shows strong homology with four prokaryotic riboflavin synthase beta chains.

  9. DNA Repair Gene Polymorphisms in Hereditary and Sporadic Breast Cancer

    Science.gov (United States)

    2006-03-01

    DNA polymerase beta, and DNA ligase 3. Alternatively, in long patch BER, few bases are excised and removed by FEN-1, including bases adjacent to...the damaged base, and incorporation of new nucleotides are mediated by PCNA, Polymerase delta or epsilon and DNA ligase I. 7 The nucleotide...requires the DNA-end-binding protein Ku, which binds free DNA ends and recruits DNA-PKcs. Xrcc4 is then recruited along with DNA ligase IV. The Rad50

  10. Complete gene expression profiling of Saccharopolyspora erythraea using GeneChip DNA microarrays

    Directory of Open Access Journals (Sweden)

    Bordoni Roberta

    2007-11-01

    Full Text Available Abstract Background The Saccharopolyspora erythraea genome sequence, recently published, presents considerable divergence from those of streptomycetes in gene organization and function, confirming the remarkable potential of S. erythraea for producing many other secondary metabolites in addition to erythromycin. In order to investigate, at whole transcriptome level, how S. erythraea genes are modulated, a DNA microarray was specifically designed and constructed on the S. erythraea strain NRRL 2338 genome sequence, and the expression profiles of 6494 ORFs were monitored during growth in complex liquid medium. Results The transcriptional analysis identified a set of 404 genes, whose transcriptional signals vary during growth and characterize three distinct phases: a rapid growth until 32 h (Phase A; a growth slowdown until 52 h (Phase B; and another rapid growth phase from 56 h to 72 h (Phase C before the cells enter the stationary phase. A non-parametric statistical method, that identifies chromosomal regions with transcriptional imbalances, determined regional organization of transcription along the chromosome, highlighting differences between core and non-core regions, and strand specific patterns of expression. Microarray data were used to characterize the temporal behaviour of major functional classes and of all the gene clusters for secondary metabolism. The results confirmed that the ery cluster is up-regulated during Phase A and identified six additional clusters (for terpenes and non-ribosomal peptides that are clearly regulated in later phases. Conclusion The use of a S. erythraea DNA microarray improved specificity and sensitivity of gene expression analysis, allowing a global and at the same time detailed picture of how S. erythraea genes are modulated. This work underlines the importance of using DNA microarrays, coupled with an exhaustive statistical and bioinformatic analysis of the results, to understand the transcriptional

  11. Comprehensive gene expression profiling following DNA vaccination of rainbow trout against infectious hematopoietic necrosis virus

    Science.gov (United States)

    Purcell, Maureen K.; Nichols, Krista M.; Winton, James R.; Kurath, Gael; Thorgaard, Gary H.; Wheeler, Paul; Hansen, John D.; Herwig, Russell P.; Park, Linda K.

    2006-01-01

    The DNA vaccine based on the glycoprotein gene of Infectious hematopoietic necrosis virus induces a non-specific anti-viral immune response and long-term specific immunity against IHNV. This study characterized gene expression responses associated with the early anti-viral response. Homozygous rainbow trout were injected intra-muscularly (I.M.) with vector DNA or the IHNV DNA vaccine. Gene expression in muscle tissue (I.M. site) was evaluated using a 16,008 feature salmon cDNA microarray. Eighty different genes were significantly modulated in the vector DNA group while 910 genes were modulated in the IHNV DNA vaccinate group relative to control group. Quantitative reverse-transcriptase PCR was used to examine expression of selected immune genes at the I.M. site and in other secondary tissues. In the localized response (I.M. site), the magnitudes of gene expression changes were much greater in the vaccinate group relative to the vector DNA group for the majority of genes analyzed. At secondary systemic sites (e.g. gill, kidney and spleen), type I IFN-related genes were up-regulated in only the IHNV DNA vaccinated group. The results presented here suggest that the IHNV DNA vaccine induces up-regulation of the type I IFN system across multiple tissues, which is the functional basis of early anti-viral immunity.

  12. Gypenosides causes DNA damage and inhibits expression of DNA repair genes of human oral cancer SAS cells.

    Science.gov (United States)

    Lu, Kung-Wen; Chen, Jung-Chou; Lai, Tung-Yuan; Yang, Jai-Sing; Weng, Shu-Wen; Ma, Yi-Shih; Tang, Nou-Ying; Lu, Pei-Jung; Weng, Jing-Ru; Chung, Jing-Gung

    2010-01-01

    Gypenosides (Gyp) are the major components of Gynostemma pentaphyllum Makino, a Chinese medical plant. Recently, Gyp has been shown to induce cell cycle arrest and apoptosis in many human cancer cell lines. However, there is no available information to address the effects of Gyp on DNA damage and DNA repair-associated gene expression in human oral cancer cells. Therefore, we investigated whether Gyp induced DNA damage and DNA repair gene expression in human oral cancer SAS cells. The results from flow cytometric assay indicated that Gyp-induced cytotoxic effects led to a decrease in the percentage of viable SAS cells. The results from comet assay revealed that the incubation of SAS cells with Gyp led to a longer DNA migration smear (comet tail) when compared with control and this effect was dose-dependent. The results from real-time PCR analysis indicated that treatment of SAS cells with 180 mug/ml of Gyp for 24 h led to a decrease in 14-3-3sigma, DNA-dependent serine/threonine protein kinase (DNAPK), p53, ataxia telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and breast cancer gene 1 (BRCA1) mRNA expression. These observations may explain the cell death caused by Gyp in SAS cells. Taken together, Gyp induced DNA damage and inhibited DNA repair-associated gene expressions in human oral cancer SAS cells in vitro.

  13. Tandem transcription termination sites in the dnaN gene of Escherichia coli.

    Science.gov (United States)

    Armengod, M E; García-Sogo, M; Pérez-Roger, I; Macián, F; Navarro-Aviñó, J P

    1991-10-15

    The dnaN gene of Escherichia coli encodes the beta-subunit of DNA polymerase III and maps between the dnaA and recF genes. We demonstrated previously that dnaN and recF constitute a transcriptional unit under control of the dnaN promoters. However, the recF gene has its own promoter region located in the middle of the dnaN structural gene. In this report, we use S1 mapping of mRNAs, transcriptional and translational fusions to the galK and lacZ genes, and in vitro mutagenesis to identify and characterize three tandem transcription termination sites responsible for transcriptional polarity in the dnaN-recF operon. These sites are located in the dnaN gene, downstream from the recF promoter region. Cumulatively, they terminate about 80% of the untranslated transcripts started at the recF promoters. As expected, they do not reduce transcription coming from the dnaN promoters unless dnaN translation was prematurely disrupted by the presence of a nonsense codon. The particular arrangement of regulatory elements (promoters and terminators) in the dnaN-recF region provides an exceptional in vivo system to confirm the latent termination site model of transcriptional polarity. In addition, our results contribute to the understanding of the complex regulation of the dnaA, dnaN, and recF genes. We propose that these three genes constitute an operon and that the terminators described in this work could be used to reduce expression of the distal genes of the operon under circumstances in which the dnaN translation happens to be slowed down.

  14. Characterization of swine stress gene by DNA testing using plucked hair as a source of DNA

    Directory of Open Access Journals (Sweden)

    Bastos Reginaldo Gaspar

    2000-01-01

    Full Text Available The swine stress gene (hal in recessive homozygotes (nn leads to porcine stress syndrome (PSS, and is associated with pale, soft, exudative pork (PSE. In heterozygosis (Nn it is linked to poor carcass quality. A total of 179 pigs (86 Large White, 69 Landrace, 12 Duroc and 12 Pietrain were characterized as normal homozygotes (NN, heterozygotes or recessive homozygotes following amplification of a target region of the hal gene using the polymerase chain reaction (PCR, followed by a restriction endonuclease assay. Plucked hair was used as a source of genomic DNA. The resulting PCR was digested with the restriction enzyme CfoI, followed by agarose gel electrophoresis. Of 179 animals tested, 70% were NN, 28% were Nn, and 2% were nn. The frequency of heterozygotes was higher (P < 0.05 in Landrace (0.43 for Nn than in Large White pigs (0.09 for Nn. Nine of the 12 Pietrain animals were Nn and three were nn, suggesting a high frequency for the n allele in this breed. These results may be related to the incidence of PSS and PSE in these two breeds, both of which are widely used in breeding programs. The utilization of plucked hair as the source of genomic DNA was a non-invasive and quick method to screen farm animals.

  15. Cloning and Characterization of Full Length cDNA of a CC-NBS-LRR Resistance Gene in Sweetpotato

    Institute of Scientific and Technical Information of China (English)

    CHEN Guan-shui; ZHOU Yi-fei; HOU Li-li; PAN Da-ren

    2009-01-01

    Conserved domain such as nucleotide binding site (NBS) was found in several cloned plant disease resistance genes.Based on the NBS domain,resistance gene analogues (RGAs) have been isolated.A full-length cDNA,SPRI was obtained by rapid amplification of cDNA ends (RACE) method.Sequence analysis indicated that the length of SPR1 was 3 066 bp,including a complete open reading frame of 2 667 bp encoding SPRI protein of 888 amino acids.Compared with known NBS-LRR genes,it presented relatively high amino acid sequence identity.The polypeptide has a typical structure of non TIR-NBS-LRR genes,with NB-ARC,CC,and LRR domains.The SPR1-related sequences belonged to multicopy gene family in sweetpotato genome according to the result of Southern blotting.Semi-quantitative RT-PCR analysis showed SPR1 expressed in all tested tissues.The cloning of putative resistance gene from sweetpotato provides a basis for studying the structure and function of sweetpotato disease-resistance relating genes and disease resistant genetic breeding in sweetpotato.The gene has been submitted to the GenBank database,and the accession number is EF428453.

  16. A new method of preparing fiber-optic DNA biosensor and its array for gene detection

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A new method of preparing fiber-optic DNA biosensor and its arrayfor the simultaneous detection of multiple genes is described. The optical fibers were first treated with poly-l-lysine, and then were made into fiber-optic DNA biosensors by adsorbing and immobilizing the oligonucleotide probe on its end. By assembling the fiber-optic DNA biosensors in a bundle in which each fiber carried a different DNA probe, the fiber-optic DNA biosensor array was well prepared. Hybridization of fluorescent- labeled cDNA of p53 gene, N-ras gene and Rb1 gene to the DNA array was monitored by CCD camera. A good result was achieved.

  17. Control of gene editing by manipulation of DNA repair mechanisms.

    Science.gov (United States)

    Danner, Eric; Bashir, Sanum; Yumlu, Saniye; Wurst, Wolfgang; Wefers, Benedikt; Kühn, Ralf

    2017-04-03

    DNA double-strand breaks (DSBs) are produced intentionally by RNA-guided nucleases to achieve genome editing through DSB repair. These breaks are repaired by one of two main repair pathways, classic non-homologous end joining (c-NHEJ) and homology-directed repair (HDR), the latter being restricted to the S/G2 phases of the cell cycle and notably less frequent. Precise genome editing applications rely on HDR, with the abundant c-NHEJ formed mutations presenting a barrier to achieving high rates of precise sequence modifications. Here, we give an overview of HDR- and c-NHEJ-mediated DSB repair in gene editing and summarize the current efforts to promote HDR over c-NHEJ.

  18. Widespread DNA hypomethylation and differential gene expression in Turner syndrome

    Science.gov (United States)

    Trolle, Christian; Nielsen, Morten Muhlig; Skakkebæk, Anne; Lamy, Philippe; Vang, Søren; Hedegaard, Jakob; Nordentoft, Iver; Ørntoft, Torben Falck; Pedersen, Jakob Skou; Gravholt, Claus Højbjerg

    2016-01-01

    Adults with 45,X monosomy (Turner syndrome) reflect a surviving minority since more than 99% of fetuses with 45,X monosomy die in utero. In adulthood 45,X monosomy is associated with increased morbidity and mortality, although strikingly heterogeneous with some individuals left untouched while others suffer from cardiovascular disease, autoimmune disease and infertility. The present study investigates the leukocyte DNAmethylation profile by using the 450K-Illumina Infinium assay and the leukocyte RNA-expression profile in 45,X monosomy compared with karyotypically normal female and male controls. We present results illustrating that genome wide X-chromosome RNA-expression profile, autosomal DNA-methylation profile, and the X-chromosome methylation profile clearly distinguish Turner syndrome from controls. Our results reveal genome wide hypomethylation with most differentially methylated positions showing a medium level of methylation. Contrary to previous studies, applying a single loci specific analysis at well-defined DNA loci, our results indicate that the hypomethylation extend to repetitive elements. We describe novel candidate genes that could be involved in comorbidity in TS and explain congenital urinary malformations (PRKX), premature ovarian failure (KDM6A), and aortic aneurysm formation (ZFYVE9 and TIMP1). PMID:27687697

  19. The DNA damage-inducible dinD gene of Escherichia coli is equivalent to orfY upstream of pyrE

    DEFF Research Database (Denmark)

    Lundegaard, Claus; Jensen, Kaj Frank

    1994-01-01

    The DNA damage-inducible gene dinD, originally identified by Kenyon and Walker (C. J. Kenyon and G. C. Walker, Proc. Natl. Acad. Sci. USA 77:2819-2823, 1980) by selection of the dinD::MudI (Ap lac) fusion, is shown here to be equivalent to the open reading frame orfY near pyrE. The evidence...

  20. Determining the effect of DNA methylation on gene expression in cancer cells.

    Science.gov (United States)

    Lee, Chai-Jin; Evans, Jared; Kim, Kwangsoo; Chae, Heejoon; Kim, Sun

    2014-01-01

    DNA methylation, a DNA modification by adding methyl group to cytosine, has an important role in the regulation of gene expression. DNA methylation is known to be associated with gene transcription by interfering with DNA-binding proteins, such as transcription factors. DNA methylation is closely related to tumorigenesis, and the methylation state of some genes can be used as a biomarker for tumorigenesis. Aberrant DNA methylation of genomic regions, including CpG islands, CpG shores, and first exons, is related to the altered gene expression pattern characteristics of all human cancers. Subheading 1 surveys recent developments on DNA methylation and gene expressions in cancer. Then we provide analysis of DNA methylation and gene expression in 30 breast cancer cell lines representing different tumor phenotypes. This study conducted an integrated analysis to identify the relationship between DNA methylation in various genomic regions and expression levels of downstream genes, using MethylCapseq data (affinity purification followed by next-generation sequencing of eluted DNA) and Affymetrix gene expression microarray data. The goal of this study was to assess genome-wide methylation profiles associated with different molecular subtypes of human breast cancer (luminal, basal A, and basal B) and to comprehensively investigate the effect of DNA methylation on gene expression in breast cancer phenotypes. This showed that methylation of genomic regions near transcription start sites, CpG island, CpG shore, and first exon was strongly associated with gene repression, and the effects of the regions on gene expression patterns were different for different molecular subtypes of breast cancer. The results further indicated that aberrant methylation of specific genomic regions was significantly associated with different breast cancer subtypes.

  1. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease.

    Science.gov (United States)

    Dupuy, Aurélie; Sarasin, Alain

    2015-06-01

    Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients.

  2. Cloning and cell cycle-dependent expression of DNA replication gene dnaC from Caulobacter crescentus.

    OpenAIRE

    1990-01-01

    Chromosome replication in the asymmetrically dividing bacteria Caulobacter crescentus is discontinuous with the new, motile swarmer cell undergoing an obligatory presynthetic gap period (G1 period) of 60 min before the initiation of DNA synthesis and stalk formation. To examine the regulation of the cell division cycle at the molecular level, we have cloned the DNA chain elongation gene dnaC from a genomic DNA library constructed in cosmid vector pLAFR1-7. To ensure that the cloned sequence c...

  3. Beryllium chloride-induced oxidative DNA damage and alteration in the expression patterns of DNA repair-related genes.

    Science.gov (United States)

    Attia, Sabry M; Harisa, Gamaleldin I; Hassan, Memy H; Bakheet, Saleh A

    2013-09-01

    Beryllium metal has physical properties that make its use essential for very specific applications, such as medical diagnostics, nuclear/fusion reactors and aerospace applications. Because of the widespread human exposure to beryllium metals and the discrepancy of the genotoxic results in the reported literature, detail assessments of the genetic damage of beryllium are warranted. Mice exposed to beryllium chloride at an oral dose of 23mg/kg for seven consecutive days exhibited a significant increase in the level of DNA-strand breaking and micronuclei formation as detected by a bone marrow standard comet assay and micronucleus test. Whereas slight beryllium chloride-induced oxidative DNA damage was detected following formamidopyrimidine DNA glycosylase digestion, digestion with endonuclease III resulted in considerable increases in oxidative DNA damage after the 11.5 and 23mg/kg/day treatment as detected by enzyme-modified comet assays. Increased 8-hydroxydeoxyguanosine was also directly correlated with increased bone marrow micronuclei formation and DNA strand breaks, which further confirm the involvement of oxidative stress in the induction of bone marrow genetic damage after exposure to beryllium chloride. Gene expression analysis on the bone marrow cells from beryllium chloride-exposed mice showed significant alterations in genes associated with DNA damage repair. Therefore, beryllium chloride may cause genetic damage to bone marrow cells due to the oxidative stress and the induced unrepaired DNA damage is probably due to the down-regulation in the expression of DNA repair genes, which may lead to genotoxicity and eventually cause carcinogenicity.

  4. Upstream promoter sequences and αCTD mediate stable DNA wrapping within the RNA polymerase–promoter open complex

    Science.gov (United States)

    Cellai, Sara; Mangiarotti, Laura; Vannini, Nicola; Naryshkin, Nikolai; Kortkhonjia, Ekaterine; Ebright, Richard H; Rivetti, Claudio

    2007-01-01

    We show that the extent of stable DNA wrapping by Escherichia coli RNA polymerase (RNAP) in the RNAP–promoter open complex depends on the sequence of the promoter and, in particular, on the sequence of the upstream region of the promoter. We further show that the extent of stable DNA wrapping depends on the presence of the RNAP α-subunit carboxy-terminal domain and on the presence and length of the RNAP α-subunit interdomain linker. Our results indicate that the extensive stable DNA wrapping observed previously in the RNAP–promoter open complex at the λ PR promoter is not a general feature of RNAP–promoter open complexes. PMID:17290289

  5. Upstream promoter sequences and alphaCTD mediate stable DNA wrapping within the RNA polymerase-promoter open complex.

    Science.gov (United States)

    Cellai, Sara; Mangiarotti, Laura; Vannini, Nicola; Naryshkin, Nikolai; Kortkhonjia, Ekaterine; Ebright, Richard H; Rivetti, Claudio

    2007-03-01

    We show that the extent of stable DNA wrapping by Escherichia coli RNA polymerase (RNAP) in the RNAP-promoter open complex depends on the sequence of the promoter and, in particular, on the sequence of the upstream region of the promoter. We further show that the extent of stable DNA wrapping depends on the presence of the RNAP alpha-subunit carboxy-terminal domain and on the presence and length of the RNAP alpha-subunit interdomain linker. Our results indicate that the extensive stable DNA wrapping observed previously in the RNAP-promoter open complex at the lambda P(R) promoter is not a general feature of RNAP-promoter open complexes.

  6. Alphavirus vectors: applications for DNA vaccine production and gene expression.

    Science.gov (United States)

    Lundstrom, K

    2000-01-01

    Replication-deficient alphavirus vectors have been developed for efficient high-level transgene expression. The broad host range of alphaviruses has allowed infection of a wide variety of mammalian cell lines and primary cultures. Particularly, G protein-coupled receptors have been expressed at high levels and subjected to binding and functional studies. Expression in suspension cultures has greatly facilitated production of large quantities of recombinant proteins for structural studies. Injection of recombinant alphavirus vectors into rodent brain resulted in local reporter gene expression. Highly neuron-specific expression was obtained in hippocampal slice cultures in vivo. Additionally, preliminary studies in animal models suggest that alphavirus vectors can be attractive candidates for gene therapy applications. Traditionally alphavirus vectors, either attenuated strains or replication-deficient particles, have been used to elicit efficient immune responses in animals. Recently, the application of alphaviruses has been extended to naked nucleic acids. Injection of DNA as well as RNA vectors has demonstrated efficient antigen production. In many cases, protection against lethal challenges has been obtained after immunization with alphavirus particles or nucleic acid vectors. Alphavirus vectors can therefore be considered as potentially promising vectors for vaccine production.

  7. Gene therapy of mitochondrial DNA mutations: a brief, biased history of allotopic expression in mammalian cells.

    Science.gov (United States)

    Zullo, S J

    2001-09-01

    Successful treatment of mitochondrial DNA (mtDNA) mutations might be possible by construction of mtDNA-encoded protein genes so that they can be inserted into the nuclear genome and the protein expressed in the mitochondria (allotopic expression). This technique would require individual assembly of all 13 mtDNA-encoded protein genes with an aminoterminal leader peptide that directs the cytoplasmic translated protein to the mitochondrial membrane. The 13 allotopic genes could be inserted into the nuclear genome of a patient's stem cell that had been "cured" of its nascent mtDNA via ethidium bromide treatment (rho-zero cell). The rho-zero cell would be a uridine auxotroph, and recovery from uridine auxotrophy would indicate successful transformation. The patient's own cells could then be returned to the patient's body. With a selective advantage of recovered oxidative phosphorylation, the transformed cells could replace cells with mtDNA mutations. Results of experiments by us on allotopically expressed CHO ATPase6 and of experiments by other workers suggest that there might be competition with endogenous mtDNA-encoded proteins if the particular protein gene is not removed from the endogenous mitochondrial genomes. Thus, it is likely that all 13 mtDNA-encoded protein genes will need to be allotopically expressed, with concomitant removal of all mtDNA genomes, in order for this form of mtDNA gene therapy to be successful.

  8. Gene structure, DNA methylation, and imprinted expression of the human SNRPN gene

    Energy Technology Data Exchange (ETDEWEB)

    Glenn, C.C.; Jong, T.C.; Filbrandt, M.M. [Univ. of Florida College of Medicine, Gainesville, FL (United States)] [and others

    1996-02-01

    The human SNRPN (small nuclear ribonucleoprotein polypeptide N) gene is one of a gene family that encode proteins involved in pre-mRNA splicing and maps to the smallest deletion region involved in the Prader-Willi syndrome (PWS) within chromosome 15q11-q13. Paternal only expression of SNRPN has previously been demonstrated by use of cell lines from PWS patients (maternal allele only) and Angelman syndrome (AS) patients (paternal allele only). We have characterized two previously unidentified 5{prime} exons of the SNRPN gene and demonstrate that exons -1 and 0 are included in the full-length transcript. This gene is expressed in a wide range of somatic tissues and at high, approximately equal levels in all regions of the brain. Both the first exon of SNRPN (exon -1) and the putative transcription start site are embedded within a CpG island. This CpG island is extensively methylated on the repressed maternal allele and is unmethylated on the expressed paternal allele, in a wide range of fetal and adult somatic cells. This provides a quick and highly reliable diagnostic assay for PWS and AS, which is based on DNA-methylation analysis that has been tested on >100 patients in a variety of tissues. Conversely, several CpG sites {approximately}22 kb downstream of the transcription start site in intron 5 are preferentially methylated on the expressed paternal allele in somatic tissues and male germ cells, whereas these same sites are unmethylated in fetal oocytes. These findings are consistent with a key role for DNA methylation in the imprinted inheritance and subsequent gene expression of the human SNRPN gene. 59 refs., 9 figs., 1 tab.

  9. Observation of intermittency in gene expression on cDNA microarrays

    CERN Document Server

    Peterson, L E

    2002-01-01

    We used scaled factorial moments to search for intermittency in the log expression ratios (LERs) for thousands of genes spotted on cDNA microarrays (gene chips). Results indicate varying levels of intermittency in gene expression. The observation of intermittency in the data analyzed provides a complimentary handle on moderately expressed genes, generally not tackled by conventional techniques.

  10. Genomic and cDNA cloning of a novel mouse lipoxygenase gene

    NARCIS (Netherlands)

    Willems van Dijk, K.; Steketee, K.; Havekes, L.; Frants, R.; Hofker, M.

    1995-01-01

    A novel 12- and 15-lipoxygenase related gene was isolated from a mouse strain 129 genomic phage library in a screen with a human 15-lipoxygenase cDNA probe. The complete genomic sequence revealed 14 exons and 13 introns covering 7.3 kb of DNA. The splice junctions were verified from the cDNA

  11. Tobacco smoking is associated with DNA methylation of diabetes susceptibility genes

    NARCIS (Netherlands)

    Ligthart, Symen; Steenaard, Rebecca V.; Peters, Marjolein J.; van Meurs, Joyce B. J.; Sijbrands, Eric J. G.; Uitterlinden, Andre G.; Bonder, Marc J.; Hofman, Albert; Franco, Oscar H.; Dehghan, Abbas

    Tobacco smoking, a risk factor for diabetes, is an established modifier of DNA methylation. We hypothesised that tobacco smoking modifies DNA methylation of genes previously identified for diabetes. We annotated CpG sites available on the Illumina Human Methylation 450K array to diabetes genes

  12. Analysis of DNA repair gene polymorphisms and survival in low-grade and anaplastic gliomas

    DEFF Research Database (Denmark)

    Berntsson, Shala Ghaderi; Wibom, Carl; Sjöström, Sara;

    2011-01-01

    The purpose of this study was to explore the variation in DNA repair genes in adults with WHO grade II and III gliomas and their relationship to patient survival. We analysed a total of 1,458 tagging single-nucleotide polymorphisms (SNPs) that were selected to cover DNA repair genes, in 81 grade ...

  13. Candidate luminal B breast cancer genes identified by genome, gene expression and DNA methylation profiling.

    Directory of Open Access Journals (Sweden)

    Stéphanie Cornen

    Full Text Available Breast cancers (BCs of the luminal B subtype are estrogen receptor-positive (ER+, highly proliferative, resistant to standard therapies and have a poor prognosis. To better understand this subtype we compared DNA copy number aberrations (CNAs, DNA promoter methylation, gene expression profiles, and somatic mutations in nine selected genes, in 32 luminal B tumors with those observed in 156 BCs of the other molecular subtypes. Frequent CNAs included 8p11-p12 and 11q13.1-q13.2 amplifications, 7q11.22-q34, 8q21.12-q24.23, 12p12.3-p13.1, 12q13.11-q24.11, 14q21.1-q23.1, 17q11.1-q25.1, 20q11.23-q13.33 gains and 6q14.1-q24.2, 9p21.3-p24,3, 9q21.2, 18p11.31-p11.32 losses. A total of 237 and 101 luminal B-specific candidate oncogenes and tumor suppressor genes (TSGs presented a deregulated expression in relation with their CNAs, including 11 genes previously reported associated with endocrine resistance. Interestingly, 88% of the potential TSGs are located within chromosome arm 6q, and seven candidate oncogenes are potential therapeutic targets. A total of 100 candidate oncogenes were validated in a public series of 5,765 BCs and the overexpression of 67 of these was associated with poor survival in luminal tumors. Twenty-four genes presented a deregulated expression in relation with a high DNA methylation level. FOXO3, PIK3CA and TP53 were the most frequent mutated genes among the nine tested. In a meta-analysis of next-generation sequencing data in 875 BCs, KCNB2 mutations were associated with luminal B cases while candidate TSGs MDN1 (6q15 and UTRN (6q24, were mutated in this subtype. In conclusion, we have reported luminal B candidate genes that may play a role in the development and/or hormone resistance of this aggressive subtype.

  14. Candidate luminal B breast cancer genes identified by genome, gene expression and DNA methylation profiling.

    Science.gov (United States)

    Cornen, Stéphanie; Guille, Arnaud; Adélaïde, José; Addou-Klouche, Lynda; Finetti, Pascal; Saade, Marie-Rose; Manai, Marwa; Carbuccia, Nadine; Bekhouche, Ismahane; Letessier, Anne; Raynaud, Stéphane; Charafe-Jauffret, Emmanuelle; Jacquemier, Jocelyne; Spicuglia, Salvatore; de The, Hugues; Viens, Patrice; Bertucci, François; Birnbaum, Daniel; Chaffanet, Max

    2014-01-01

    Breast cancers (BCs) of the luminal B subtype are estrogen receptor-positive (ER+), highly proliferative, resistant to standard therapies and have a poor prognosis. To better understand this subtype we compared DNA copy number aberrations (CNAs), DNA promoter methylation, gene expression profiles, and somatic mutations in nine selected genes, in 32 luminal B tumors with those observed in 156 BCs of the other molecular subtypes. Frequent CNAs included 8p11-p12 and 11q13.1-q13.2 amplifications, 7q11.22-q34, 8q21.12-q24.23, 12p12.3-p13.1, 12q13.11-q24.11, 14q21.1-q23.1, 17q11.1-q25.1, 20q11.23-q13.33 gains and 6q14.1-q24.2, 9p21.3-p24,3, 9q21.2, 18p11.31-p11.32 losses. A total of 237 and 101 luminal B-specific candidate oncogenes and tumor suppressor genes (TSGs) presented a deregulated expression in relation with their CNAs, including 11 genes previously reported associated with endocrine resistance. Interestingly, 88% of the potential TSGs are located within chromosome arm 6q, and seven candidate oncogenes are potential therapeutic targets. A total of 100 candidate oncogenes were validated in a public series of 5,765 BCs and the overexpression of 67 of these was associated with poor survival in luminal tumors. Twenty-four genes presented a deregulated expression in relation with a high DNA methylation level. FOXO3, PIK3CA and TP53 were the most frequent mutated genes among the nine tested. In a meta-analysis of next-generation sequencing data in 875 BCs, KCNB2 mutations were associated with luminal B cases while candidate TSGs MDN1 (6q15) and UTRN (6q24), were mutated in this subtype. In conclusion, we have reported luminal B candidate genes that may play a role in the development and/or hormone resistance of this aggressive subtype.

  15. Locus- and Site-Specific DNA Methylation of 19 kDa Zein Genes in Maize.

    Directory of Open Access Journals (Sweden)

    Jian-Hong Xu

    Full Text Available An interesting question in maize development is why only a single zein gene is highly expressed in each of the 19-kDa zein gene clusters (A and B types, z1A2-1 and z1B4, in the immature endosperm. For instance, epigenetic marks could provide a structural difference. Therefore, we investigated the DNA methylation of the arrays of gene copies in both promoter and gene body regions of leaf (non-expressing tissue as a control, normal endosperm, and cultured endosperm. Although we could show that expressed genes have much lower methylation levels in promoter regions than silent ones in both leaf and normal endosperm, there was surprisingly also a difference in the pattern of the z1A and z1B gene clusters. The expression of z1B gene is suppressed by increased DNA methylation and activated with reduced DNA methylation, whereas z1A gene expression is not. DNA methylation in gene coding regions is higher in leaf than in endosperm, whereas no significant difference is observed in gene bodies between expressed and non-expressed gene copies. A median CHG methylation (25-30% appears to be optimal for gene expression. Moreover, tissue-cultured endosperm can reset the DNA methylation pattern and tissue-specific gene expression. These results reveal that DNA methylation changes of the 19-kDa zein genes is subject to plant development and tissue culture treatment, but varies in different chromosomal locations, indicating that DNA methylation changes do not apply to gene expression in a uniform fashion. Because tissue culture is used to produce transgenic plants, these studies provide new insights into variation of gene expression of integrated sequences.

  16. Cloning chromosome specific genes by reciprocal probing of arrayed cDNA and cosmid libraries

    Energy Technology Data Exchange (ETDEWEB)

    Yazdani, A.; Lee, C.C.; Wehnert, M. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    A human gene map will greatly facilitate the association of genes to single locus diseases and provide candidates for genes involved in complex genetic traits. Given the estimated 100,000 human genes an integrated strategy with a high throughput approach for isolation and mapping of expressed sequences is needed to create such a gene map. We have developed an approach that allows high throughput gene isolation and mapping using arrayed genomic and cDNA lambda libraries. Reciprocal probing of the arrayed genomic and cDNA cosmic libraries can rapidly establish cDNA-cosmid associations. Fluorescence in situ hybridization (FISH) chromosomal mapping and expressed sequence tag/sequence tag site (EST/STS) primers generated from DNA sequence of PCR-based mapping using somatic hybrid cell line mapping panels were utilized to characterize further the hybridization-based cDNA cosmid association. We have applied this approach to chromosome 17 using a placental cDNA library and have identified a total of 30 genes out of which 11 are novel. Furthermore seven cDNAs were mapped to 17q21 in this study, providing novel candidate genes for BRCA-1 gene for early onset breast cancer. The results of our study clearly show that an integration of an expression map into physical and genetic maps can provide candidate genes for human diseases that have been mapped to specific regions. This approach combined with the current physical mapping efforts could efficiently provide a detailed human gene map.

  17. Non-invasive, neuron-specific gene therapy by focused ultrasound-induced blood-brain barrier opening in Parkinson's disease mouse model.

    Science.gov (United States)

    Lin, Chung-Yin; Hsieh, Han-Yi; Chen, Chiung-Mei; Wu, Shang-Rung; Tsai, Chih-Hung; Huang, Chiung-Yin; Hua, Mu-Yi; Wei, Kuo-Chen; Yeh, Chih-Kuang; Liu, Hao-Li

    2016-08-10

    Focused ultrasound (FUS)-induced with microbubbles (MBs) is a promising technique for noninvasive opening of the blood-brain barrier (BBB) to allow targeted delivery of therapeutic substances into the brain and thus the noninvasive delivery of gene vectors for CNS treatment. We have previously demonstrated that a separated gene-carrying liposome and MBs administration plus FUS exposure can deliver genes into the brain, with the successful expression of the reporter gene and glial cell line-derived neurotrophic factor (GDNF) gene. In this study, we further modify the delivery system by conjugating gene-carrying liposomes with MBs to improve the GDNF gene-delivery efficiency, and to verify the possibility of using this system to perform treatment in the 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced animal disease model. FUS-BBB opening was verified by contrast-enhanced MRI, and GFP gene expression was verified via in vivo imaging system (IVIS). Western blots as well as enzyme-linked immunosorbent assay (ELISA) were conducted to measure protein expression, and immunohistochemistry (IHC) was conducted to test the Tyrosine hydroxylase (TH)-neuron distribution. Dopamine (DA) and its metabolites as well as dopamine active transporter (DAT) were quantitatively analyzed to show dopaminergic neuronal dopamine secretion/activity/metabolism. Motor performance was evaluated by rotarod test weekly. Results demonstrated that the LpDNA-MBs (gene-liposome-MBs) complexes successfully serve as gene carrier and BBB-opening catalyst, and outperformed the separated LpDNA/MBs administration both in terms of gene delivery and expression. TH-positive IHC and measurement of DA and its metabolites DOPAC and HVA confirmed improved neuronal function, and the proposed system also provided the best neuroprotective effect to retard the progression of motor-related behavioral abnormalities. Immunoblotting and histological staining further confirmed the expression of reporter genes in

  18. Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips.

    Science.gov (United States)

    Kosuri, Sriram; Eroshenko, Nikolai; Leproust, Emily M; Super, Michael; Way, Jeffrey; Li, Jin Billy; Church, George M

    2010-12-01

    Development of cheap, high-throughput and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis. Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude, yet efforts to scale their use have been largely unsuccessful owing to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized gene assembly protocols and enzymatic error correction to develop a method for highly parallel gene synthesis. We tested our approach by assembling 47 genes, including 42 challenging therapeutic antibody sequences, encoding a total of ∼35 kilobase pairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ∼2.5 megabases of DNA, which is at least 50 times larger than in previously published attempts.

  19. Investigation of yeast genes possibly involved in mtDNA stability ...

    African Journals Online (AJOL)

    Phelim Isichei

    function and structure on mtDNA stability in yeast, our results did not support those ... most studied model organism for acquisition of basic ... RNA interference of genes involved in mtDNA replication ... polymerase, results in reduced mtDNA copy number but .... found that RNAi of 4 genes (M01E5.2, T27F6.5, T26A5.6.

  20. Construction of DNA damage response gene pprI function-deficient and function-complementary mutants in Deinococcus radiodurans

    Institute of Scientific and Technical Information of China (English)

    GAO Guanjun; LU Huiming; HUANG Lifen; HUA Yuejin

    2005-01-01

    PprI, a DNA damage response factor from the extraordinary radioresistant bacterium Deinococcus radiodurans, plays a central regulatory role in multiple DNA damage repair. In this study, a fusion DNA fragment carrying kanamycin resistance gene with the D. Radiodurans groEL promoter was cloned by PCR amplification and reversely inserted into the pprI locus in the genome of the wild-type strain R1. The resulting pprI-deficient strain, designated YR1, was very sensitive to ionizing radiation. Meanwhile, the re- combinant DNA fragment was cloned into the shuttle vector pRADZ3, and resulted in plasmid pRADK with kanamycin resistance in D. Radiodurans. The fragments containing complete pprI gene and 3'-terminal deletion pprI△ were cloned into plasmid pRADK. The resulted plasmids designated pRADKpprI and pRADKpprI△ were then transformed to YR1. Results show that YR1 carrying pRADKpprI was able to fully restore the extreme radioresistance to the same level as the wild-type D. Raiodurans R1, whereas YR1 pRADKpprI△ failed to do so. Construction of DNA repair switch PprI function-deficient and function-complementary mutants in D. Radiodurans is not only useful to elucidating the relationship between domains and functions of PprI protein, but also opens the door to the further studies of the biological functions of PprI protein in vivo.

  1. The human Bloom syndrome gene suppresses the DNA replication and repair defects of yeast dna2 mutants.

    Science.gov (United States)

    Imamura, Osamu; Campbell, Judith L

    2003-07-08

    Bloom syndrome is a disorder of profound and early cancer predisposition in which cells become hypermutable, exhibit high frequency of sister chromatid exchanges, and show increased micronuclei. BLM, the gene mutated in Bloom syndrome, has been cloned previously, and the BLM protein is a member of the RecQ family of DNA helicases. Many lines of evidence suggest that BLM is involved either directly in DNA replication or in surveillance during DNA replication, but its specific roles remain unknown. Here we show that hBLM can suppress both the temperature-sensitive growth defect and the DNA damage sensitivity of the yeast DNA replication mutant dna2-1. The dna2-1 mutant is defective in a helicase-nuclease that is required either to coordinate with the crucial Saccharomyces cerevisiae (sc) FEN1 nuclease in Okazaki fragment maturation or to compensate for scFEN1 when its activity is impaired. We show that human BLM interacts with both scDna2 and scFEN1 by using coimmunoprecipitation from yeast extracts, suggesting that human BLM participates in the same steps of DNA replication or repair as scFEN1 and scDna2.

  2. Hybridization of cloned Rhodopseudomonas capsulata photosynthesis genes with DNA from other photosynthetic bacteria.

    OpenAIRE

    Beatty, J T; Cohen, S N

    1983-01-01

    The homology of Rhodopseudomonas capsulata DNA segments carrying photosynthesis genes with sequences present in total DNA from certain other photosynthetic and non-photosynthetic bacterial species was determined by hybridization. R. capsulata DNA fragments that carry loci for production of peptide components of the photosynthetic reaction center and light-harvesting I antenna complex were found to hybridize to DNA from some photosynthetic species. However, fragments that carry carotenoid or b...

  3. DNA context represents transcription regulation of the gene in mouse embryonic stem cells

    Science.gov (United States)

    Ha, Misook; Hong, Soondo

    2016-04-01

    Understanding gene regulatory information in DNA remains a significant challenge in biomedical research. This study presents a computational approach to infer gene regulatory programs from primary DNA sequences. Using DNA around transcription start sites as attributes, our model predicts gene regulation in the gene. We find that H3K27ac around TSS is an informative descriptor of the transcription program in mouse embryonic stem cells. We build a computational model inferring the cell-type-specific H3K27ac signatures in the DNA around TSS. A comparison of embryonic stem cell and liver cell-specific H3K27ac signatures in DNA shows that the H3K27ac signatures in DNA around TSS efficiently distinguish the cell-type specific H3K27ac peaks and the gene regulation. The arrangement of the H3K27ac signatures inferred from the DNA represents the transcription regulation of the gene in mESC. We show that the DNA around transcription start sites is associated with the gene regulatory program by specific interaction with H3K27ac.

  4. The Second Subunit of DNA Polymerase Delta Is Required for Genomic Stability and Epigenetic Regulation1[OPEN

    Science.gov (United States)

    Cheng, Jinkui; Lai, Jinsheng; Gong, Zhizhong

    2016-01-01

    DNA polymerase δ plays crucial roles in DNA repair and replication as well as maintaining genomic stability. However, the function of POLD2, the second small subunit of DNA polymerase δ, has not been characterized yet in Arabidopsis (Arabidopsis thaliana). During a genetic screen for release of transcriptional gene silencing, we identified a mutation in POLD2. Whole-genome bisulfite sequencing indicated that POLD2 is not involved in the regulation of DNA methylation. POLD2 genetically interacts with Ataxia Telangiectasia-mutated and Rad3-related and DNA polymerase α. The pold2-1 mutant exhibits genomic instability with a high frequency of homologous recombination. It also exhibits hypersensitivity to DNA-damaging reagents and short telomere length. Whole-genome chromatin immunoprecipitation sequencing and RNA sequencing analyses suggest that pold2-1 changes H3K27me3 and H3K4me3 modifications, and these changes are correlated with the gene expression levels. Our study suggests that POLD2 is required for maintaining genome integrity and properly establishing the epigenetic markers during DNA replication to modulate gene expression. PMID:27208288

  5. Cloning and Sequence Analysis of the Full-length cDNA of a Novel yp05 Gene Associated With Citrinin Production in Monascus aurantiacus

    Institute of Scientific and Technical Information of China (English)

    YON-GHUA XIONG; YANG XU; WEI-HUA LAI; YAN-PIN LI; HUA WEI

    2007-01-01

    Objective To obtain the full-length cDNA of a novel gene (named yp05) associated with citrinin production-related genes in Monascus aurantiacus. Methods Total RNA was extracted from mycelium, 3' and 5' cDNA end of yp05 gene was amplified using smartTM trace cDNA amplification kit, and the full-length cDNA of a novel gene (named yp05) was obtained from the electronic assembly of 3'-RACE and 5'- RACE products. Results This yp05 gene was 787 bp including a 597 bp open reading frame (ORF) and encoded a deduced protein with 199 amino acid residues, and the amino acid sequence of this protein was found similar with the sequences of many fungal manganese-superoxide dismutases in the GenBank with the aid of BLASTp. The transcription of yp05 gene in Monascus strains was analyzed with the aid of Northern blotting. The transcription of yp05 gene was only detected in Monascus strains, provided that citrinin was produced. Conclusion The transcription of yp05 gene belongs to differential expression genes of citrinin yielded from Monascus and has no correlation with the biosynthesis pathway of red pigments.

  6. Analysis of MYOC gene mutation in a Chinese glaucoma family with primary open-angle glaucoma and primary congenital glaucoma

    Institute of Scientific and Technical Information of China (English)

    ZHUO Ye-hong; WANG Mei; WEI Yan-tao; HUANG Ya-lin; GE Jian

    2006-01-01

    Background Glaucoma is one of the leading causes of blindness in the world. Primary open-angle glaucoma (POAG) and primary congenital glaucoma (PCG) are subtypes of glaucoma. Myocillin is the first gene identified to be involved in POAG. Recently, myocillin mutation has been found in PCG In this context, we reported a special glaucoma pedigree, which was composed of both PCG and POAG patients, and analyzed the mutation of myocillin in this pedigree.Methods The family was composed of the parents, a son and a daughter. All members of the family underwent the complete ophthalmologic examinations. All coding exons 1-3 and flanking introns of myocilin gene were screened for sequence alterations by polymerase chain reaction and direct DNA sequencing.Results The son was the proband, who was diagnosed as PCG in both eyes. The father was diagnosed as POAG in the right eye, the left eye was still normal. Both the sister and the mother of the proband had normal intraocular pressure without glaucomatous optic disc changes. The mutations in intron 2 of myocilin gene were detected in the family. While the proband and the father were homozygous, the mother and the sister were heterozygous for the mutation.Conclusions Homozygous mutation in intron 2 of myocilin gene is involved in both POAG and PCG. It is suggested that the pathogenesis might be overlapping in POAG and PCG.

  7. Stationary phase induction of dnaN and recF, two genes of Escherichia coli involved in DNA replication and repair.

    OpenAIRE

    Villarroya, M; Pérez-Roger, I; Macián, F; Armengod, M E

    1998-01-01

    The beta subunit of DNA polymerase III holoenzyme, the Escherichia coli chromosomal replicase, is a sliding DNA clamp responsible for tethering the polymerase to DNA and endowing it with high processivity. The gene encoding beta, dnaN, maps between dnaA and recF, which are involved in initiation of DNA replication at oriC and resumption of DNA replication at disrupted replication forks, respectively. In exponentially growing cells, dnaN and recF are expressed predominantly from the dnaA promo...

  8. Hepatitis B virus DNA polymerase gene polymorphism based ...

    African Journals Online (AJOL)

    2017-09-03

    Sep 3, 2017 ... HBV is distributed into various genotypes based on nucleic acid sequence variation. ... compared to genotype B and higher incidence of HCC in genotype D ... DNA sequencing technology to sequence HBV DNA polymerase ...

  9. Transcript RNA supports precise repair of its own DNA gene.

    Science.gov (United States)

    Keskin, Havva; Meers, Chance; Storici, Francesca

    2016-01-01

    The transfer of genetic information from RNA to DNA is considered an extraordinary process in molecular biology. Despite the fact that cells transcribe abundant amount of RNA with a wide range of functions, it has been difficult to uncover whether RNA can serve as a template for DNA repair and recombination. An increasing number of experimental evidences suggest a direct role of RNA in DNA modification. Recently, we demonstrated that endogenous transcript RNA can serve as a template to repair a DNA double-strand break (DSB), the most harmful DNA lesion, not only indirectly via formation of a DNA copy (cDNA) intermediate, but also directly in a homology driven mechanism in budding yeast. These results point out that the transfer of genetic information from RNA to DNA is more general than previously thought. We found that transcript RNA is more efficient in repairing a DSB in its own DNA (in cis) than in a homologous but ectopic locus (in trans). Here, we summarize current knowledge about the process of RNA-driven DNA repair and recombination, and provide further data in support of our model of DSB repair by transcript RNA in cis. We show that a DSB is precisely repaired predominately by transcript RNA and not by residual cDNA in conditions in which formation of cDNA by reverse transcription is inhibited. Additionally, we demonstrate that defects in ribonuclease (RNase) H stimulate precise DSB repair by homologous RNA or cDNA sequence, and not by homologous DNA sequence carried on a plasmid. These results highlight an antagonistic role of RNase H in RNA-DNA recombination. Ultimately, we discuss several questions that should be addressed to better understand mechanisms and implications of RNA-templated DNA repair and recombination.

  10. Bacterial spores as particulate carriers for gene gun delivery of plasmid DNA.

    Science.gov (United States)

    Aps, Luana R M M; Tavares, Milene B; Rozenfeld, Julio H K; Lamy, M Teresa; Ferreira, Luís C S; Diniz, Mariana O

    2016-06-20

    Bacillus subtilis spores represent a suitable platform for the adsorption of proteins, enzymes and viral particles at physiological conditions. In the present work, we demonstrate that purified spores can also adsorb DNA on their surface after treatment with cationic molecules. In addition, we demonstrate that DNA-coated B. subtilis spores can be used as particulate carriers and act as an alternative to gold microparticles for the biolistic (gene gun) administration of plasmid DNA in mice. Gene gun delivery of spores pre-treated with DODAB (dioctadecyldimethylammonium bromide) allowed efficient plasmid DNA absorption and induced protein expression levels similar to those obtained with gold microparticles. More importantly, we demonstrated that a DNA vaccine adsorbed on spores can be loaded into biolistic cartridges and efficiently delivered into mice, which induced specific cellular and antibody responses. Altogether, these data indicate that B. subtilis spores represent a simple and low cost alternative for the in vivo delivery of DNA vaccines by the gene gun technology.

  11. Induction of somatic hypermutation in immunoglobulin genes is dependent on DNA polymerase iota.

    Science.gov (United States)

    Faili, Ahmad; Aoufouchi, Said; Flatter, Eric; Guéranger, Quentin; Reynaud, Claude-Agnès; Weill, Jean-Claude

    2002-10-31

    Somatic hypermutation of immunoglobulin genes is a unique, targeted, adaptive process. While B cells are engaged in germinal centres in T-dependent responses, single base substitutions are introduced in the expressed Vh/Vl genes to allow the selection of mutants with a higher affinity for the immunizing antigen. Almost every possible DNA transaction has been proposed to explain this process, but each of these models includes an error-prone DNA synthesis step that introduces the mutations. The Y family of DNA polymerases--pol eta, pol iota, pol kappa and rev1--are specialized for copying DNA lesions and have high rates of error when copying a normal DNA template. By performing gene inactivation in a Burkitt's lymphoma cell line inducible for hypermutation, we show here that somatic hypermutation is dependent on DNA polymerase iota.

  12. SIMAGE : simulation of DNA-microarray gene expression data

    NARCIS (Netherlands)

    Albers, Casper J.; Jansen, Ritsert C.; Kok, Jan; Kuipers, Oscar P.; Hijum, Sacha A.F.T. van

    2006-01-01

    Simulation of DNA-microarray data serves at least three purposes: (i) optimizing the design of an intended DNA microarray experiment, (ii) comparing existing pre-processing and processing methods for best analysis of a given DNA microarray experiment, (iii) educating students, lab-workers and other

  13. Gene Transfer into the Lung by Nanoparticle Dextran-Spermine/Plasmid DNA Complexes

    Directory of Open Access Journals (Sweden)

    Syahril Abdullah

    2010-01-01

    Full Text Available A novel cationic polymer, dextran-spermine (D-SPM, has been found to mediate gene expression in a wide variety of cell lines and in vivo through systemic delivery. Here, we extended the observations by determining the optimal conditions for gene expression of D-SPM/plasmid DNA (D-SPM/pDNA in cell lines and in the lungs of BALB/c mice via instillation delivery. In vitro studies showed that D-SPM could partially protect pDNA from degradation by nuclease and exhibited optimal gene transfer efficiency at D-SPM to pDNA weight-mixing ratio of 12. In the lungs of mice, the levels of gene expression generated by D-SPM/pDNA are highly dependent on the weight-mixing ratio of D-SPM to pDNA, amount of pDNA in the complex, and the assay time postdelivery. Readministration of the complex at day 1 following the first dosing showed no significant effect on the retention and duration of gene expression. The study also showed that there was a clear trend of increasing size of the complexes as the amount of pDNA was increased, where the sizes of the D-SPM/pDNA complexes were within the nanometer range.

  14. Comprehensive investigation of DNA methylation and gene expression in trisomy 21 placenta.

    Science.gov (United States)

    Lim, Ji Hyae; Kim, Shin Young; Han, Jung Yeol; Kim, Moon Young; Park, So Yeon; Ryu, Hyun Mee

    2016-06-01

    Trisomy 21 (T21) is the most common aneuploidy affecting humans and is caused by an extra copy of all or part of chromosome 21 (chr21). DNA methylation is an epigenetic event that plays an important role in human diseases via regulation of gene expression. However, the integrative association between DNA methylation and gene expression in T21 fetal placenta has yet to be determined. We profiled expression of 207 genes on chr21 and their DNA methylation patterns in placenta samples from normal and DS fetuses using microarray analysis and predicted the functions of differentially expressed genes using bioinformatics tools. We found 47 genes with significantly increased expression in the T21 placenta compared to the normal placenta. Hypomethylation of the 47 genes was observed in the T21 placenta. Most of hypomethylated DNA positions were intragenic regions, i.e. regions inside a gene. Moreover, gene expression and hypomethylated DNA position showed significantly positive associations. By analyzing the properties of the gene-disease network, we found that increased genes in the T21 placenta were significantly associated with T21 and T21 complications such as mental retardation, neurobehavioral manifestations, and congenital abnormalities. To our knowledge, this is the first study to comprehensively survey the association between gene expression and DNA methylation in chr21 of the T21 fetal placenta. Our findings provide a broad overview of the relationships between gene expression and DNA methylation in the placentas of fetuses with T21 and could contribute to future research efforts concerning genes involvement in disease pathogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. DNA Methylation Profiling Reveals Correlation of Differential Methylation Patterns with Gene Expression in Human Epilepsy.

    Science.gov (United States)

    Wang, Liang; Fu, Xinwei; Peng, Xi; Xiao, Zheng; Li, Zhonggui; Chen, Guojun; Wang, Xuefeng

    2016-05-01

    DNA methylation plays important roles in regulating gene expression and has been reported to be related with epilepsy. This study aimed to define differential DNA methylation patterns in drug-refractory epilepsy patients and to investigate the role of DNA methylation in human epilepsy. We performed DNA methylation profiling in brain tissues from epileptic and control patients via methylated-cytosine DNA immunoprecipitation microarray chip. Differentially methylated loci were validated by bisulfite sequencing PCR, and the messenger RNA (mRNA) levels of candidate genes were evaluated by reverse transcriptase PCR. We found 224 genes that showed differential DNA methylation between epileptic patients and controls. Among the seven candidate genes, three genes (TUBB2B, ATPGD1, and HTR6) showed relative transcriptional regulation by DNA methylation. TUBB2B and ATPGD1 exhibited hypermethylation and decreased mRNA levels, whereas HTR6 displayed hypomethylation and increased mRNA levels in the epileptic samples. Our findings suggest that certain genes become differentially regulated by DNA methylation in human epilepsy.

  16. Natural variation in DNA methylation in ribosomal RNA genes of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Richards Eric J

    2008-09-01

    Full Text Available Abstract Background DNA methylation is an important biochemical mark that silences repetitive sequences, such as transposons, and reinforces epigenetic gene expression states. An important class of repetitive genes under epigenetic control in eukaryotic genomes encodes ribosomal RNA (rRNA transcripts. The ribosomal genes coding for the 45S rRNA precursor of the three largest eukaryotic ribosomal RNAs (18S, 5.8S, and 25–28S are found in nucleolus organizer regions (NORs, comprised of hundreds to thousands of repeats, only some of which are expressed in any given cell. An epigenetic switch, mediated by DNA methylation and histone modification, turns rRNA genes on and off. However, little is known about the mechanisms that specify and maintain the patterns of NOR DNA methylation. Results Here, we explored the extent of naturally-occurring variation in NOR DNA methylation among accessions of the flowering plant Arabidopsis thaliana. DNA methylation in coding regions of rRNA genes was positively correlated with copy number of 45S rRNA gene and DNA methylation in the intergenic spacer regions. We investigated the inheritance of NOR DNA methylation patterns in natural accessions with hypomethylated NORs in inter-strain crosses and defined three different categories of inheritance in F1 hybrids. In addition, subsequent analysis of F2 segregation for NOR DNA methylation patterns uncovered different patterns of inheritance. We also revealed that NOR DNA methylation in the Arabidopsis accession Bor-4 is influenced by the vim1-1 (variant in methylation 1-1 mutation, but the primary effect is specified by the NORs themselves. Conclusion Our results indicate that the NORs themselves are the most significant determinants of natural variation in NOR DNA methylation. However, the inheritance of NOR DNA methylation suggests the operation of a diverse set of mechanisms, including inheritance of parental methylation patterns, reconfiguration of parental NOR DNA

  17. Identification of novel gene targets and functions of p21-activated kinase 1 during DNA damage by gene expression profiling.

    Directory of Open Access Journals (Sweden)

    Mona Motwani

    Full Text Available P21-activated kinase 1 (PAK1, a serine/threonine protein kinase, modulates many cellular processes by phosphorylating its downstream substrates. In addition to its role in the cytoplasm, PAK1 also affects gene transcription due to its nuclear localization and association with chromatin. It is now recognized that PAK1 kinase activity and its nuclear translocation are rapidly stimulated by ionizing radiation (IR, and that PAK1 activation is a component of the DNA damage response. Owing to the role of PAK1 in the cell survival, its association with the chromatin, and now, stimulation by ionizing radiation, we hypothesize that PAK1 may be contributing to modulation of genes with roles in cellular processes that might be important in the DNA damage response. The purpose of this study was to identify new PAK1 targets in response to ionizing radiation with putative role in the DNA damage response. We examined the effect of IR on the gene expression patterns in the murine embryonic fibroblasts with or without Pak1 using microarray technology. Differentially expressed transcripts were identified using Gene Spring GX 10.0.2. Pathway, network, functional analyses and gene family classification were carried out using Kyoto Encyclopedia of Genes and Genomes (KEGG, Ingenuity Pathway, Gene Ontology and PANTHER respectively. Selective targets of PAK1 were validated by RT-qPCR. For the first time, we provide a genome-wide analysis of PAK1 and identify its targets with potential roles in the DNA damage response. Gene Ontology analysis identified genes in the IR-stimulated cells that were involved in cell cycle arrest and cell death. Pathway analysis revealed p53 pathway being most influenced by IR responsive, PAK1 targets. Gene family of transcription factors was over represented and gene networks involved in DNA replication, repair and cellular signaling were identified. In brief, this study identifies novel PAK1 dependent IR responsive genes which reveal new

  18. Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction.

    Science.gov (United States)

    Schmidt, Florian; Gasparoni, Nina; Gasparoni, Gilles; Gianmoena, Kathrin; Cadenas, Cristina; Polansky, Julia K; Ebert, Peter; Nordström, Karl; Barann, Matthias; Sinha, Anupam; Fröhler, Sebastian; Xiong, Jieyi; Dehghani Amirabad, Azim; Behjati Ardakani, Fatemeh; Hutter, Barbara; Zipprich, Gideon; Felder, Bärbel; Eils, Jürgen; Brors, Benedikt; Chen, Wei; Hengstler, Jan G; Hamann, Alf; Lengauer, Thomas; Rosenstiel, Philip; Walter, Jörn; Schulz, Marcel H

    2017-01-09

    The binding and contribution of transcription factors (TF) to cell specific gene expression is often deduced from open-chromatin measurements to avoid costly TF ChIP-seq assays. Thus, it is important to develop computational methods for accurate TF binding prediction in open-chromatin regions (OCRs). Here, we report a novel segmentation-based method, TEPIC, to predict TF binding by combining sets of OCRs with position weight matrices. TEPIC can be applied to various open-chromatin data, e.g. DNaseI-seq and NOMe-seq. Additionally, Histone-Marks (HMs) can be used to identify candidate TF binding sites. TEPIC computes TF affinities and uses open-chromatin/HM signal intensity as quantitative measures of TF binding strength. Using machine learning, we find low affinity binding sites to improve our ability to explain gene expression variability compared to the standard presence/absence classification of binding sites. Further, we show that both footprints and peaks capture essential TF binding events and lead to a good prediction performance. In our application, gene-based scores computed by TEPIC with one open-chromatin assay nearly reach the quality of several TF ChIP-seq data sets. Finally, these scores correctly predict known transcriptional regulators as illustrated by the application to novel DNaseI-seq and NOMe-seq data for primary human hepatocytes and CD4+ T-cells, respectively.

  19. Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction

    Science.gov (United States)

    Schmidt, Florian; Gasparoni, Nina; Gasparoni, Gilles; Gianmoena, Kathrin; Cadenas, Cristina; Polansky, Julia K.; Ebert, Peter; Nordström, Karl; Barann, Matthias; Sinha, Anupam; Fröhler, Sebastian; Xiong, Jieyi; Dehghani Amirabad, Azim; Behjati Ardakani, Fatemeh; Hutter, Barbara; Zipprich, Gideon; Felder, Bärbel; Eils, Jürgen; Brors, Benedikt; Chen, Wei; Hengstler, Jan G.; Hamann, Alf; Lengauer, Thomas; Rosenstiel, Philip; Walter, Jörn; Schulz, Marcel H.

    2017-01-01

    The binding and contribution of transcription factors (TF) to cell specific gene expression is often deduced from open-chromatin measurements to avoid costly TF ChIP-seq assays. Thus, it is important to develop computational methods for accurate TF binding prediction in open-chromatin regions (OCRs). Here, we report a novel segmentation-based method, TEPIC, to predict TF binding by combining sets of OCRs with position weight matrices. TEPIC can be applied to various open-chromatin data, e.g. DNaseI-seq and NOMe-seq. Additionally, Histone-Marks (HMs) can be used to identify candidate TF binding sites. TEPIC computes TF affinities and uses open-chromatin/HM signal intensity as quantitative measures of TF binding strength. Using machine learning, we find low affinity binding sites to improve our ability to explain gene expression variability compared to the standard presence/absence classification of binding sites. Further, we show that both footprints and peaks capture essential TF binding events and lead to a good prediction performance. In our application, gene-based scores computed by TEPIC with one open-chromatin assay nearly reach the quality of several TF ChIP-seq data sets. Finally, these scores correctly predict known transcriptional regulators as illustrated by the application to novel DNaseI-seq and NOMe-seq data for primary human hepatocytes and CD4+ T-cells, respectively. PMID:27899623

  20. Evolutionary Fates and Dynamic Functionalization of Young Duplicate Genes in Arabidopsis Genomes1[OPEN

    Science.gov (United States)

    Wang, Jun; Tao, Feng; Marowsky, Nicholas C.; Fan, Chuanzhu

    2016-01-01

    Gene duplication is a primary means to generate genomic novelties, playing an essential role in speciation and adaptation. Particularly in plants, a high abundance of duplicate genes has been maintained for significantly long periods of evolutionary time. To address the manner in which young duplicate genes were derived primarily from small-scale gene duplication and preserved in plant genomes and to determine the underlying driving mechanisms, we generated transcriptomes to produce the expression profiles of five tissues in Arabidopsis thaliana and the closely related species Arabidopsis lyrata and Capsella rubella. Based on the quantitative analysis metrics, we investigated the evolutionary processes of young duplicate genes in Arabidopsis. We determined that conservation, neofunctionalization, and specialization are three main evolutionary processes for Arabidopsis young duplicate genes. We explicitly demonstrated the dynamic functionalization of duplicate genes along the evolutionary time scale. Upon origination, duplicates tend to maintain their ancestral functions; but as they survive longer, they might be likely to develop distinct and novel functions. The temporal evolutionary processes and functionalization of plant duplicate genes are associated with their ancestral functions, dynamic DNA methylation levels, and histone modification abundances. Furthermore, duplicate genes tend to be initially expressed in pollen and then to gain more interaction partners over time. Altogether, our study provides novel insights into the dynamic retention processes of young duplicate genes in plant genomes. PMID:27485883

  1. Quantitative analysis of EDC-condensed DNA on vertically aligned carbon nanofiber gene delivery arrays

    Energy Technology Data Exchange (ETDEWEB)

    Mann, David G. J. [Univ. of Tennessee, Knoxville, TN (United States). Center for Environmental Biotechnology; McKnight, Timothy E. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Engineering Science and Technology Division; Melechko, Anatoli V. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Nanophase Materials Science (CNMS); Simpson, Michael L. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Nanophase Materials Science (CNMS); Sayler, Gary S. [Univ. of Tennessee, Knoxville, TN (United States). Center for Environmental Biotechnology

    2006-12-08

    Vertically aligned carbon nanofibers (VACNFs) with immobilized DNA have been developed as a novel tool for direct physical introduction and expression of exogenous genes in mammalian cells. Immobilization of DNA base amines to the carboxylic acids on nanofibers can influence the accessibility and transcriptional activity of the DNA template, making it necessary to determine the number of accessible gene copies on nanofiber arrays. We used polymerase chain reaction (PCR) and in vitro transcription (IVT) to investigate the transcriptional accessibility of DNA tethered to VACNFs by correlating the yields of both IVT and PCR to that of non-tethered, free DNA. Yields of the promoter region and promoter/gene region of bound DNA plasmid were high. Amplification using primers designed to cover 80% of the plasmid failed to yield any product. These results are consistent with tethered, longer DNA sequences having a higher probability of interfering with the activity of DNA and RNA polymerases. Quantitative PCR (qPCR) was used to quantify the number of accessible gene copies tethered to nanofiber arrays. Copy numbers of promoters and reporter genes were quantified and compared to non-tethered DNA controls. In subsequent reactions of the same nanofiber arrays, DNA yields decreased dramatically in the non-tethered control, while the majority of tethered DNA was retained on the arrays. This decrease could be explained by the presence of DNA which is non-tethered to all samples and released during the assay. In conclusion,this investigation shows the applicability of these methods for monitoring DNA immobilization techniques.

  2. Chitosan-graft-polyethylenimine/DNA nanoparticles as novel non-viral gene delivery vectors targeting osteoarthritis.

    Science.gov (United States)

    Lu, Huading; Dai, Yuhu; Lv, Lulu; Zhao, Huiqing

    2014-01-01

    The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes.

  3. DNA tagging of blast resistant gene(s in three Brazilian rice cultivars

    Directory of Open Access Journals (Sweden)

    S.S. Sandhu

    2003-12-01

    Full Text Available Rice blast is the most important fungal disease of rice and is caused by Pyricularia oryzae Sacc. (Telomorph Magnoporthe grisea Barr.. Seven randomly amplified polymorphic DNA (RAPD markers OPA5, OPG17, OPG18, OPG19, OPF9, OPF17 and OPF19 showed very clear polymorphism in resistant cultivar lines which differed from susceptible lines. By comparing different susceptible lines, nine DNA amplifications of seven primers (OPA5(1000, OPA5(1200, OPG17(700, OPG18(850, OPG19(500, OPG19(600, OPF9(600, OPF17(1200 and OPF19(600 were identified as dominant markers for the blast resistant gene in resistant cultivar lines. These loci facilitate the indirect scoring of blast resistant and blast susceptible genotypes. The codomine RAPDs markers will facilitate marker-assisted selection of the blast resistant gene in two blast resistant genotypes of rice (Labelle and Line 11 and will be useful in rice breeding programs.

  4. Transcriptional organization of the dnaN and recF genes of Escherichia coli K-12.

    Science.gov (United States)

    Armengod, M E; García-Sogo, M; Lambíes, E

    1988-08-25

    The dnaN gene of Escherichia coli determines the beta subunit of DNA polymerase III, a multisubunit enzyme responsible for most of the replicative DNA synthesis. The dnaN gene maps between the dnaA and recF genes. We have characterized the regulatory region of the dnaN gene by screening DNA restriction fragments for promoter activity, S1 mapping of mRNAs, deletion analysis, and in vivo dnaN complementation tests. There are at least three dnaN promoters located in the second half of the dnaA coding region. The one closest to the dnaN structural gene is the weakest, but it provides sufficient dnaN expression for complementation when the gene is present on a multicopy plasmid. Deletion of sequences needed for initiation of dnaN translation or introduction of nonsense codons into dnaN causes reduction of recF expression. However, a deletion inactivating dnaN without changing the reading frame of the gene does not affect expression of the recF gene. These results indicate that the dnaN and recF genes are organized in an operon. We have previously shown the presence of termination signals within the dnaN coding region (Armengod, M.E., and Lambíes, E. (1986) Gene (Amst.) 43, 183-196). Therefore, we propose that the polarity produced by nonsense mutations in dnaN is primarily transcriptional. The uncoupling of transcription and translation of the dnaN gene (when translation is interrupted by premature nonsense codons or by other mechanisms) probably results in transcription termination at termination signals in dnaN.

  5. High levels of gene expression in the hepatocytes of adult mice, neonatal mice and tree shrews via retro-orbital sinus hydrodynamic injections of naked plasmid DNA.

    Science.gov (United States)

    Yan, Shaoduo; Fu, Qiuxia; Zhou, Yong; Wang, Jidong; Liu, Ying; Duan, Xiangguo; Jia, Shuaizheng; Peng, Jianchun; Gao, Bo; Du, Juan; Zhou, Qianqian; Li, Yuan; Wang, Xiaohui; Zhan, Linsheng

    2012-08-10

    Hydrodynamic-based gene delivery has emerged as an efficient and simple method for the intracellular transfection of naked plasmid DNA (pDNA) in vivo. In this system, a hydrodynamic injection via the tail vein is the most effective non-viral method of liver-targeted gene delivery. However, this injection is often technically challenging when used in animals whose tail veins are difficult to visualize or too small to operate on. To overcome this limitation, an alternative in vivo gene delivery method, the rapid injection of large volume of pDNA solution through retro-orbital sinus, was established. Using this technique, we successfully delivered pDNA to the tissue of adult mice, neonatal mice and tree shrews. The efficient expression of exogenous genes was specifically detected in the liver of test animals treated with this gene delivery method. This study demonstrates for the first time that the hydrodynamic gene delivery via the retro-orbital sinus can not only reach the same transgene efficiency as a tradition hydrodynamic-based intravascular injection but also be used in animals that are difficult to inject via the tail vein. This method could open up new areas in gene function studies and gene therapy disease treatment.

  6. Variants in the ASB10 Gene Are Associated with Primary Open Angle Glaucoma

    NARCIS (Netherlands)

    Micheal, S.; Ayub, H.; Islam, F.; Siddiqui, S.N.; Khan, W.A.; Akhtar, F.; Qamar, R.; Khan, M.I.; Hollander, A.I. den

    2015-01-01

    BACKGROUND: Recently nonsynonymous coding variants in the ankyrin repeats and suppressor of cytokine signaling box-containing protein 10 (ASB10) gene were found to be associated with primary open angle glaucoma (POAG) in cohorts from Oregon and Germany, but this finding was not confirmed in an indep

  7. Evaluation of Optineurin as a candidate gene in Indian patients with primary open angle glaucoma.

    NARCIS (Netherlands)

    Mukhopadhyay, A.; Komatireddy, S.; Acharya, M.; Bhattacharjee, A.; Mandal, A.K.; Thakur, S.K.D.; Chandrasekhar, G.; Banerjee, A.; Thomas, R.; Chakrabarti, S.; Ray, K.

    2005-01-01

    PURPOSE: To evaluate the role of the optineurin gene (OPTN) in Indian primary open angle glaucoma (POAG) patients from different parts of the country. METHODS: Two hundred patients with POAG and 200 ethnically matched normal controls were recruited from various parts of India for the study. The enti

  8. DNA Methyltransferase Gene dDnmt2 and Longevity of Drosophila

    Institute of Scientific and Technical Information of China (English)

    Meng-JauLin; Lin-YaTang; M.NarsaReddy; C.K.JamesShen

    2005-01-01

    The DNA methylation program of the fruit fly Drosophila melanogaster is carried out by the single DNA methyltransferase gene dDnmt2, the function of which is unknown before. We present evidence that intactness of the gene is required for maintenance of the normal life span of the fruit flies. In contrast, overexpression of dDnmt2 could extend Drosophila life span. The study links the Drosophila DNA methylation program with the small heatshock proteins and longevity/aging and has interesting implication on the eukaryotic DNA methyl-ation programs in general.

  9. Transcription of ncDNA: Many roads lead to local gene regulation

    OpenAIRE

    Hainer, Sarah J; Martens, Joseph A

    2011-01-01

    Transcription of ncDNA occurs throughout eukaryotic genomes, generating a wide array of ncRNAs. One large class of ncRNAs includes those transcribed over the promoter regions of nearby protein coding genes. Recent studies, primarily focusing on individual genes have uncovered multiple mechanisms by which promoter-associated transcriptional activity locally alters gene expression.

  10. Fact or fiction: updates on how protein-coding genes might emerge de novo from previously non-coding DNA

    Science.gov (United States)

    Schmitz, Jonathan F; Bornberg-Bauer, Erich

    2017-01-01

    Over the last few years, there has been an increasing amount of evidence for the de novo emergence of protein-coding genes, i.e. out of non-coding DNA. Here, we review the current literature and summarize the state of the field. We focus specifically on open questions and challenges in the study of de novo protein-coding genes such as the identification and verification of de novo-emerged genes. The greatest obstacle to date is the lack of high-quality genomic data with very short divergence times which could help precisely pin down the location of origin of a de novo gene. We conclude that, while there is plenty of evidence from a genetics perspective, there is a lack of functional studies of bona fide de novo genes and almost no knowledge about protein structures and how they come about during the emergence of de novo protein-coding genes. We suggest that future studies should concentrate on the functional and structural characterization of de novo protein-coding genes as well as the detailed study of the emergence of functional de novo protein-coding genes.

  11. A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs

    Directory of Open Access Journals (Sweden)

    Gong Shiaochin

    2009-03-01

    Full Text Available Abstract Background Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available. Results We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1 subclone genes of interest into BAC linking vectors, (2 insert desired reporter genes into respective genes and (3 link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts. Conclusion The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

  12. Differential DNA methylation of genes involved in fibrosis progression in non-alcoholic fatty liver disease and alcoholic liver disease.

    OpenAIRE

    Zeybel, Müjdat; Hardy, Timothy; Robinson, Stuart M.; Fox, Christopher; Anstee, Quentin M.; Ness, Thomas; Masson, Steven; Masson, Steven; French, Jeremy; White, Steve; Mann, Jelena

    2015-01-01

    RESEARCH Open Access Differential DNA methylation of genes involved in fibrosis progression in non-alcoholic fatty liver disease and alcoholic liver disease Müjdat Zeybel1, Timothy Hardy1, Stuart M Robinson1, Christopher Fox1, Quentin M Anstee1, Thomas Ness2, Steven Masson1, John C Mathers1, Jeremy French1, Steve White1 and Jelena Mann1* Abstract Background: Chronic liver injury can lead to the development of liver fibrosis and cirrhosis but only in a minority of patie...

  13. Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of Shigella flexneri 2a

    Institute of Scientific and Technical Information of China (English)

    张继瑜; 刘红; 张笑兵; 杨剑; 杨帆; 杨国威; 沈岩; 侯云德; 金奇

    2003-01-01

    The complete nucleotide sequence and organization of the large virulence plasmid pCP301 (termed by us) of Shigella flexneri 2a strain 301 were determined and analyzed. The result showed that the entire DNA sequence of pCP301 is composed of 221618 bp which form a circular plasmid. Sequence analysis identified 272 open reading frames (ORFs), among which, 194 correspond to the proteins described previously, 61 have low identity (<60%) to known proteins and the rest 17 have no regions of significant homology with proteins in database. The genes of pCP301 mainly include the genes associated with bacterial virulence, the genes associated with regulation and the genes relating to plasmid maintenance, stability and DNA metabolism. Insertion sequence (IS) elements are 68 kb in length and account for 30 percent of complete sequence of the plasmid which indicates that gene multiple rearrangements of the pCP301 have taken place in Shigella flexneri evolution history. The research result is helpful for interpreting the pathogenesis of Shigella, as well as the genetics and evolution of the plasmid.

  14. Full length cDNA cloning and expression analysis of annexinA2 gene from deer antler tissue

    Institute of Scientific and Technical Information of China (English)

    Li Hao; Xianghong Xiao; Heping Li

    2014-01-01

    ANXA2(AnnexinA2), a calcium-dependent phospholipid bind-ing protein, is involved in various Ca2+-related biological activities. In the present study, full-length cDNA of ANXA2 was isolated from the velvet antler tip tissue of sika deer (Cervus nippon hortulorum);the amino acid sequence and gene expression was analyzed by using bioinformatics and real-time reverse transcriptase polymerase chain reaction (RT-PCR) techniques. Nucleotide sequence analysis reveals that the full-length cDNA of the ANXA2 gene was 1372 bp, of which 1020 bp was in the open-reading frame (ORF) encoding 339 amino acids; its relative mo-lecular weight was 38.3 kDa; and isoelectric point was 6.72. Sequence analysis indicates that the protein includes four conserved tan-dem-duplication ANX domains. The gene-accession nucleotide sequence number in GenBank is JX315571. Expression analysis by RT-PCR re-veals that ANXA2 gene expression has a significant positive correlation with the antler-tissue mineralization process, indicating that this gene may play an important role in the regulation of antler-tissue mineraliza-tion.

  15. An "instant gene bank" method for heterologous gene cloning: complementation of two Aspergillus nidulans mutants with Gaeumannomyces graminis DNA.

    Science.gov (United States)

    Bowyer, P; Osbourn, A E; Daniels, M J

    1994-02-01

    We present a novel technique for gene cloning by complementation of mutations in Aspergillus nidulans with DNA from a heterologous organism, Gaeumannomyces graminis. This technique bypasses the time-consuming and difficult construction of gene libraries, making it both rapid and simple. The method relies on recombination between a fungal replicating vector pHELP1 and linear G. graminis genomic DNA during co-transformation. We were able to complement two out of seven A. nidulans mutants tested and to rescue transforming DNA from both in Escherichia coli. Complementation of the A. nidulans argB mutation resulted from integration of 8-10 kb segments of G. graminis DNA into pHELP1. The complementation of the A. nidulans pyrG mutation resulted from a complex rearrangement. Complementing DNA was shown to originate from G. graminis, and was capable of retransforming the original mutants to give the expected phenotype.

  16. Isolation and characterisation of the human lung NK-2 receptor gene using rapid amplification of cDNA ends.

    Science.gov (United States)

    Graham, A; Hopkins, B; Powell, S J; Danks, P; Briggs, I

    1991-05-31

    Functional cDNA clones for human NK-2 receptor were isolated from human lung RNA using a polymerase chain reaction (PCR) based method (RACE-PCR). In this method the cDNA was isolated as 5' end and 3'-end fragments; the entire cDNA was obtained by RNA-PCR. The sequence derived was 398 amino acids in length encoding an open-reading frame that was highly homologous to both the bovine and rat NK-2 receptor. The entire human cDNA sequence was cloned into a mammalian expression vector and mRNA was synthesised by in vitro transcription. Applications of tachykinins caused membrane current responses in Xenopus oocytes injected with the in vitro synthesised mRNA. The most potent of the three tachykinin peptides tested was neurokinin A. We have screened a human cosmid library and isolated a clone which contains the entire NK-2 receptor gene. The gene contains five exons and we have determined the complete sequence of the exons and the intron-exon junctions.

  17. Nucleotide sequence of the DNA polymerase gene of herpes simplex virus type 2 and comparison with the type 1 counterpart.

    Science.gov (United States)

    Tsurumi, T; Maeno, K; Nishiyama, Y

    1987-01-01

    The complete nucleotide sequence of the DNA polymerase gene of herpes simplex virus (HSV) type 2 strain 186 has been determined. The gene included a 3720-bp major open reading frame capable of encoding 1240 amino acids. The predicted primary translation product had an Mr of 137,354, which was slightly larger than its HSV-1 counterpart. A comparison of the predicted functional amino acid sequences of the HSV-1 and HSV-2 DNA polymerases revealed 95.5% overall amino acid homology, the value of which was the highest among those of the other known polypeptides encoded by HSV-1 and HSV-2. The functional amino acid changes were spread in the N-terminal one-third of the protein, whereas the C-terminal two-third was almost identical between the two types except a particular hydrophilic region. A highly conserved sequence of 6 aa, YGDTDS, which has been observed in DNA polymerases of HSV-1, Epstein-Barr virus, adenovirus, and vaccinia virus, was also present at positions 889 to 894 in the C-terminal region of HSV-2 DNA polymerase.

  18. DNA-dependent protein kinase inhibits AID-induced antibody gene conversion.

    Directory of Open Access Journals (Sweden)

    Adam J L Cook

    2007-04-01

    Full Text Available Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID-dependent hypermutation of Ig V(DJ rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(DJ regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(DJ hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs. Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion.

  19. SETDB1 is involved in postembryonic DNA methylation and gene silencing in Drosophila.

    Directory of Open Access Journals (Sweden)

    Dawei Gou

    Full Text Available DNA methylation is fundamental for the stability and activity of genomes. Drosophila melanogaster and vertebrates establish a global DNA methylation pattern of their genome during early embryogenesis. Large-scale analyses of DNA methylation patterns have uncovered revealed that DNA methylation patterns are dynamic rather than static and change in a gene-specific fashion during development and in diseased cells. However, the factors and mechanisms involved in dynamic, postembryonic DNA methylation remain unclear. Methylation of lysine 9 in histone H3 (H3-K9 by members of the Su(var3-9 family of histone methyltransferases (HMTs triggers embryonic DNA methylation in Arthropods and Chordates. Here, we demonstrate that Drosophila SETDB1 (dSETDB1 can mediate DNA methylation and silencing of genes and retrotransposons. We found that dSETDB1 tri-methylates H3-K9 and binds methylated CpA motifs. Tri-methylation of H3-K9 by dSETDB1 mediates recruitment of DNA methyltransferase 2 (Dnmt2 and Su(var205, the Drosophila ortholog of mammalian "Heterochromatin Protein 1", to target genes for dSETDB1. By enlisting Dnmt2 and Su(var205, dSETDB1 triggers DNA methylation and silencing of genes and retrotransposons in Drosophila cells. DSETDB1 is involved in postembryonic DNA methylation and silencing of Rt1b{} retrotransposons and the tumor suppressor gene retinoblastoma family protein 1 (Rb in imaginal discs. Collectively, our findings implicate dSETDB1 in postembryonic DNA methylation, provide a model for silencing of the tumor suppressor Rb, and uncover a role for cell type-specific DNA methylation in Drosophila development.

  20. Evolutionary Transition of Promoter and Gene Body DNA Methylation across Invertebrate-Vertebrate Boundary.

    Science.gov (United States)

    Keller, Thomas E; Han, Priscilla; Yi, Soojin V

    2016-04-01

    Genomes of invertebrates and vertebrates exhibit highly divergent patterns of DNA methylation. Invertebrate genomes tend to be sparsely methylated, and DNA methylation is mostly targeted to a subset of transcription units (gene bodies). In a drastic contrast, vertebrate genomes are generally globally and heavily methylated, punctuated by the limited local hypo-methylation of putative regulatory regions such as promoters. These genomic differences also translate into functional differences in DNA methylation and gene regulation. Although promoter DNA methylation is an important regulatory component of vertebrate gene expression, its role in invertebrate gene regulation has been little explored. Instead, gene body DNA methylation is associated with expression of invertebrate genes. However, the evolutionary steps leading to the differentiation of invertebrate and vertebrate genomic DNA methylation remain unresolved. Here we analyzed experimentally determined DNA methylation maps of several species across the invertebrate-vertebrate boundary, to elucidate how vertebrate gene methylation has evolved. We show that, in contrast to the prevailing idea, a substantial number of promoters in an invertebrate basal chordate Ciona intestinalis are methylated. Moreover, gene expression data indicate significant, epigenomic context-dependent associations between promoter methylation and expression in C. intestinalis. However, there is no evidence that promoter methylation in invertebrate chordate has been evolutionarily maintained across the invertebrate-vertebrate boundary. Rather, body-methylated invertebrate genes preferentially obtain hypo-methylated promoters among vertebrates. Conversely, promoter methylation is preferentially found in lineage- and tissue-specific vertebrate genes. These results provide important insights into the evolutionary origin of epigenetic regulation of vertebrate gene expression. © The Author(s) 2015. Published by Oxford University Press on behalf

  1. DNA capture reveals transoceanic gene flow in endangered river sharks.

    Science.gov (United States)

    Li, Chenhong; Corrigan, Shannon; Yang, Lei; Straube, Nicolas; Harris, Mark; Hofreiter, Michael; White, William T; Naylor, Gavin J P

    2015-10-27

    For over a hundred years, the "river sharks" of the genus Glyphis were only known from the type specimens of species that had been collected in the 19th century. They were widely considered extinct until populations of Glyphis-like sharks were rediscovered in remote regions of Borneo and Northern Australia at the end of the 20th century. However, the genetic affinities between the newly discovered Glyphis-like populations and the poorly preserved, original museum-type specimens have never been established. Here, we present the first (to our knowledge) fully resolved, complete phylogeny of Glyphis that includes both archival-type specimens and modern material. We used a sensitive DNA hybridization capture method to obtain complete mitochondrial genomes from all of our samples and show that three of the five described river shark species are probably conspecific and widely distributed in Southeast Asia. Furthermore we show that there has been recent gene flow between locations that are separated by large oceanic expanses. Our data strongly suggest marine dispersal in these species, overturning the widely held notion that river sharks are restricted to freshwater. It seems that species in the genus Glyphis are euryhaline with an ecology similar to the bull shark, in which adult individuals live in the ocean while the young grow up in river habitats with reduced predation pressure. Finally, we discovered a previously unidentified species within the genus Glyphis that is deeply divergent from all other lineages, underscoring the current lack of knowledge about the biodiversity and ecology of these mysterious sharks.

  2. The physics of protein-DNA interaction networks in the control of gene expression

    Science.gov (United States)

    Saiz, Leonor

    2012-05-01

    Protein-DNA interaction networks play a central role in many fundamental cellular processes. In gene regulation, physical interactions and reactions among the molecular components together with the physical properties of DNA control how genes are turned on and off. A key player in all these processes is the inherent flexibility of DNA, which provides an avenue for long-range interactions between distal DNA elements through DNA looping. Such versatility enables multiple interactions and results in additional complexity that is remarkably difficult to address with traditional approaches. This topical review considers recent advances in statistical physics methods to study the assembly of protein-DNA complexes with loops, their effects in the control of gene expression, and their explicit application to the prototypical lac operon genetic system of the E. coli bacterium. In the last decade, it has been shown that the underlying physical properties of DNA looping can actively control transcriptional noise, cell-to-cell variability, and other properties of gene regulation, including the balance between robustness and sensitivity of the induction process. These physical properties are largely dependent on the free energy of DNA looping, which accounts for DNA bending and twisting effects. These new physical methods have also been used in reverse to uncover the actual in vivo free energy of looping double-stranded DNA in living cells, which was not possible with existing experimental techniques. The results obtained for DNA looping by the lac repressor inside the E. coli bacterium showed a more malleable DNA than expected as a result of the interplay of the simultaneous presence of two distinct conformations of looped DNA.

  3. Gene for the catalytic subunit of mouse DNA-dependent protein kinase maps to the scid locus.

    Science.gov (United States)

    Miller, R D; Hogg, J; Ozaki, J H; Gell, D; Jackson, S P; Riblet, R

    1995-01-01

    The gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) has been proposed recently as a candidate gene for the mouse severe combined immune deficiency (scid) locus. We have used a partial cDNA clone for human DNA-PKcs to map the mouse homologue using a large interspecific backcross panel. We found that the mouse gene for DNA-PKcs does not recombine with scid, consistent with the hypothesis that scid is a mutation in the mouse gene for DNA-PKcs. Images Fig. 3 PMID:7479885

  4. Telomeric DNA quantity, DNA damage, and heat shock protein gene expression as physiological stress markers in chickens.

    Science.gov (United States)

    Sohn, S H; Subramani, V K; Moon, Y S; Jang, I S

    2012-04-01

    In this longitudinal study with Single Comb White Leghorn chickens, we investigated the effects of stress conditions in birds that were subjected to a high stocking density with feed restrictions on the quantity of telomeric DNA, the rate of DNA damage, and the expression levels of heat shock proteins (HSP) and hydroxyl-3-methyl-glutaryl coenzyme A reductase (HMGCR) genes. The telomere length and telomere-shortening rates were analyzed by quantitative fluorescence in situ hybridization on the nuclei of lymphocytes. The DNA damage rate of lymphocytes was quantified by the comet assay. The expression levels of HSP70, HSP90, and HMGCR genes were measured by quantitative real-time PCR in lymphocytes. The telomere-shortening rate of the lymphocytes was significantly higher in the stress group than in the control. The DNA damage also increased in birds raised under stress conditions, as compared with the control group. The stress conditions had a significant effect on the expressions of HMGCR and HSP90α in lymphocytes but had no significance on HSP70 and HSP90β in blood. We conclude that the telomere length, especially the telomere-shortening rates, the quantification of total DNA damage, and the expression levels of the HMGCR and HSP90α genes can be used as sensitive physiological stress markers in chickens.

  5. Cloning and expression of dnaK gene from Bacillus pumilus of hot water spring origin

    Directory of Open Access Journals (Sweden)

    Murugan Kumar

    2014-03-01

    Full Text Available A set of thermotolerant strains isolated from hot springs of Manikaran and Bakreshwar (India were selected with an aim to isolate dnak gene which encodes DnaK protein. The gene dnaK along with its flanking region was successfully amplified from 5 different strains (4 from Bakreshwar and one from Manikaran. Restriction fragment length polymorphism (RFLP revealed that amplicons were almost identical in sequence. The dnak gene from one representative, Bacillus pumilus strain B3 isolated from Bakreshwar hot springs was successfully cloned and sequenced. The dnaK gene was flanked by gene grpE on one side. The dnaK gene was 1842 bp in length encoding a polypeptide of 613 amino acid residues. Calculated molecular weight and pI of the protein were 66,128.36 Da and 4.72 respectively. The deduced amino acid sequence of this gene shared high sequence homology with other DnaK proteins and its homologue Hsp 70 from other microorganisms, but possessed 36 substitutions and two insertions, as compared to DnaK protein of Bacillus subtilis. The dnaK gene of B. pumilus was successfully expressed in Escherichia coli BL 21 (DE3 using pET expression systems. Heterologous expression of dnaK of B. pumilus in E. coli BL 21 (DE3 allowed for the growth of E. coli up to 50 °C and survival up to 60 °C for 16 h, suggesting that dnak from B. pumilus imparts tolerance to host cells under high temperature. This novel gene can be an important component for possible utilization in abiotic stress management of plants.

  6. A DNA nanocapsule with aptamer-controlled open-closure function for targeted delivery

    DEFF Research Database (Denmark)

    Bentin, Thomas

    2012-01-01

    A DNA capsule fitted with aptamer controlled target sensing has been "woven" using a 7308-base single-stranded DNA "thread" and 196 staple oligonucleotides. The capsule enables logic-gated molecular cargo delivery to targeted cell surfaces.......A DNA capsule fitted with aptamer controlled target sensing has been "woven" using a 7308-base single-stranded DNA "thread" and 196 staple oligonucleotides. The capsule enables logic-gated molecular cargo delivery to targeted cell surfaces....

  7. PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms

    Energy Technology Data Exchange (ETDEWEB)

    Binkova, Blanka [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Chvatalova, Irena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Lnenickova, Zdena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Milcova, Alena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Tulupova, Elena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester (United Kingdom); Farmer, Peter B. [Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester (United Kingdom); Sram, Radim J. [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic)]. E-mail: sram@biomed.cas.cz

    2007-07-01

    Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair gene polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N = 53, males, aged 22-50 years) working outdoors in the downtown area of Prague and in matched 'unexposed' controls (CON, N = 52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by {sup 32}P-postlabeling assay. Polymorphisms of metabolizing (GSTM1, GSTP1, GSTT1, EPHX1, CYP1A1-MspI) and DNA repair (XRCC1, XPD) genes were determined by PCR-based RFLP assays. As potential modifiers and/or cofounders, urinary cotinine levels were analyzed by radioimmunoassay, plasma levels of vitamins A, C, E and folates by HPLC, cholesterol and triglycerides using commercial kits. During the sampling period ambient particulate air pollution was as follows: PM10 32-55 {mu}g/m{sup 3}, PM2.5 27-38 {mu}g/m{sup 3}, c-PAHs 18-22 ng/m{sup 3}; personal exposure to c-PAHs: 9.7 ng/m{sup 3} versus 5.8 ng/m{sup 3} (P < 0.01) for EXP and CON groups, respectively. The total DNA adduct levels did not significantly differ between EXP and CON groups (0.92 {+-} 0.28 adducts/10{sup 8} nucleotides versus 0.82 {+-} 0.23 adducts/10{sup 8} nucleotides, P = 0.065), whereas the level of the B[a]P-'like' adduct was significantly higher in exposed group (0.122 {+-} 0.036 adducts/10{sup 8} nucleotides versus 0.099 {+-} 0.035 adducts/10{sup 8} nucleotides, P = 0

  8. Radioactive cDNA microarray (II): Gene expression profiling of antidepressant treatment by human cDNA microarray

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji Hye; Kang, Rhee Hun; Ham, Byung Joo; Lee, Min Su; Shin, Kyung Ho; Choe, Jae Gol; Kim, Meyoung Kon [College of Medicine, Univ. of Korea, Seoul (Korea, Republic of)

    2003-07-01

    Major depressive disorder is a prevalent psychiatric disorder in primary care, associated with impaired patient functioning and well-being. Fluoxetine is a selective serotonin-reuptake inhibitors (SSRIs) and is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Objectives ; the aims of this study were two-fold: (1) to determine the usefulness for investigation of the transcription profiles in depression patients, and (2) to assess the differences in gene expression profiles between positive response group and negative response groups by fluoxetine treatment. This study included 53 patients with major depression (26 in positive response group with antidepressant treatment, 27 in negative response group with antidepressant treatment), and 53 healthy controls. To examine the difference of gene expression profile in depression patients, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 genes in total. Using 33p-labeled probes, this method provided highly sensitive gene expression profiles including brain receptors, drug metabolism, and cellular signaling. Gene transcription profiles were classified into several categories in accordance with the antidepressant gene-regulation. The gene profiles were significantly up-(22 genes) and down-(16 genes) regulated in the positive response group when compared to the control group. Also, in the negative response group, 35 genes were up-regulated and 8 genes were down-regulated when compared to the control group. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology.

  9. How to Open the Treasure Chest? Optimising DNA Extraction from Herbarium Specimens

    NARCIS (Netherlands)

    Särkinen, T.; Staats, M.; Richardson, J.E.; Cowan, R.S.; Bakker, F.T.

    2012-01-01

    Herbarium collections are potentially an enormous resource for DNA studies, but the use of herbarium specimens in molecular studies has thus far been slowed down by difficulty in obtaining amplifiable DNA. Here we compare a set of commercially available DNA extraction protocols and their performance

  10. Using a cDNA microarray to study cellular gene expression altered by Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    徐永忠; 谢建平; 李瑶; 乐军; 陈建平; 淳于利娟; 王洪海

    2003-01-01

    Objective To examine the global effects of Mycobacterium tuberculosis (M.tuberculosis) infection on macrophages. Methods The gene expression profiling of macrophage U937, in response to infection with M.tuberculosis H37Ra, was monitored using a high-density cDNA microarray. Results M.tuberculosis infection caused 463 differentially expressed genes, of which 366 genes are known genes registered in the Gene Bank. These genes function in various cellular processes including intracellular signalling, cytoskeletal rearrangement, apoptosis, transcriptional regulation, cell surface receptors, cell-mediated immunity as well as a variety of cellular metabolic pathways, and may play key roles in M.tuberculosis infection and intracellular survival. Conclusions M.tuberculosis infection alters the expression of host-cell genes, and these genes will provide a foundation for understanding the infection process of M.tuberculosis. The cDNA microarray is a powerful tool for studying pathogen-host cell interaction.

  11. Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones

    Energy Technology Data Exchange (ETDEWEB)

    Imanishi, Tadashi; Itoh, Takeshi; Suzuki, Yutaka; O' Donovan, Claire; Fukuchi, Satoshi; Koyanagi, Kanako O.; Barrero, Roberto A.; Tamura, Takuro; Yamaguchi-Kabata, Yumi; Tanino, Motohiko; Yura, Kei; Miyazaki, Satoru; Ikeo, Kazuho; Homma, Keiichi; Kasprzyk, Arek; Nishikawa, Tetsuo; Hirakawa, Mika; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Ashurst, Jennifer; Jia, Libin; Nakao, Mitsuteru; Thomas, Michael A.; Mulder, Nicola; Karavidopoulou, Youla; Jin, Lihua; Kim, Sangsoo; Yasuda, Tomohiro; Lenhard, Boris; Eveno, Eric; Suzuki, Yoshiyuki; Yamasaki, Chisato; Takeda, Jun-ichi; Gough, Craig; Hilton, Phillip; Fujii, Yasuyuki; Sakai, Hiroaki; Tanaka, Susumu; Amid, Clara; Bellgard, Matthew; de Fatima Bonaldo, Maria; Bono Hidemasa; Bromberg, Susan K.; Brookes, Anthony J.; Bruford, Elspeth; Carninci Piero; Chelala, Claude; Couillault, Christine; de Souza, Sandro J.; Debily, Marie-Anne; Devignes, Marie-Dominique; Dubchak, Inna; Endo, Toshinori; Estreicher, Anne; Eyras, Eduardo; Fukami-Kobayashi, Kaoru; Gopinath, Gopal R.; Graudens, Esther; Hahn, Yoonsoo; Han, Michael; Han, Ze-Guang; Hanada, Kousuke; Hanaoka, Hideki; Harada, Erimi; Hashimoto, Katsuyuki; Hinz, Ursula; Hirai, Momoki; Hishiki, Teruyoshi; Hopkinson, Ian; Imbeaud, Sandrine; Inoko, Hidetoshi; Kanapin, Alexander; Kaneko, Yayoi; Kasukawa, Takeya; Kelso, Janet; Kersey, Paul; Kikuno Reiko; Kimura, Kouichi; Korn, Bernhard; Kuryshev, Vladimir; Makalowska, Izabela; Makino Takashi; Mano, Shuhei; Mariage-Samson, Regine; Mashima, Jun; Matsuda, Hideo; Mewes, Hans-Werner; Minoshima, Shinsei; Nagai, Keiichi; Nagasaki, Hideki; Nagata, Naoki; Nigam, Rajni; Ogasawara, Osamu; Ohara, Osamu; Ohtsubo, Masafumi; Okada, Norihiro; Okido, Toshihisa; Oota, Satoshi; Ota, Motonori; Ota, Toshio; Otsuki, Tetsuji; Piatier-Tonneau, Dominique; Poustka, Annemarie; Ren, Shuang-Xi; Saitou, Naruya; Sakai, Katsunaga; Sakamoto, Shigetaka; Sakate, Ryuichi; Schupp, Ingo; Servant, Florence; Sherry, Stephen; Shiba Rie; et al.

    2004-01-15

    The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4 percent of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5 percent of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for nonprotein-coding RNA

  12. Integrative annotation of 21,037 human genes validated by full-length cDNA clones.

    Directory of Open Access Journals (Sweden)

    Tadashi Imanishi

    2004-06-01

    Full Text Available The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/. It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs, identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA

  13. Identifying Intracellular pDNA Losses From a Model of Nonviral Gene Delivery.

    Science.gov (United States)

    Martin, Timothy Michael; Wysocki, Beata Joanna; Wysocki, Tadeusz Antoni; Pannier, Angela K

    2015-06-01

    Nonviral gene delivery systems are a type of nanocommunication system that transmit plasmid packets (i.e., pDNA packets) that are programmed at the nanoscale to biological systems at the microscopic cellular level. This engineered nanocommunication system suffers large pDNA losses during transmission of the genetically encoded information, preventing its use in biotechnological and medical applications. The pDNA losses largely remain uncharacterized, and the ramifications of reducing pDNA loss from newly designed gene delivery systems remain difficult to predict. Here, the pDNA losses during primary and secondary transmission chains were identified utilizing a MATLAB model employing queuing theory simulating delivery of pEGFPLuc transgene to HeLa cells carried by Lipofectamine 2000 nonviral DNA carrier. Minimizing pDNA loss during endosomal escape of the primary transmission process results in increased number of pDNA in the nucleus with increased transfection, but with increased probability of cell death. The number of pDNA copies in the nucleus and the amount of time the pDNAs are in the nucleus directly correlates to improved transfection efficiency. During secondary transmission, pDNAs are degraded during distribution to daughter cells. Reducing pDNA losses improves transfection, but a balance in quantity of nuclear pDNA, mitosis, and toxicity must be considered in order to achieve therapeutically relevant transfection levels.

  14. cDNA macroarray for analysis of gene expression profiles in prostate cancer

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Background Early diagnosis and timely treatment are important for improving therapeutic efficiency of prostate cancer. DNA array is a new bio-technology for disease diagnosis. This study was conducted to diagnose prostate cancer with cDNA macroarray and analysis gene expression profiles of some selective genes in prostate cancer.Methods Total RNA was isolated from patients with prostate cancer and from normal people, and poly(A) RNA was further purified. Then it was analyzed for differentially expressed genes in prostate cancer and normal prostate by cDNA macroarray system.Results There were different expressions in the nine prostate-associated specific genes in prostate cancer as compared with normal prostate, in which, 7 were significantly upregulated and 2 were down-regulated.Conclusion As a diagnostic approach at molecular level, the cDNA macroarray is an effectively diagnostic method for prostate cancer.

  15. Identification of a DNA binding protein that recognizes the nonamer recombinational signal sequence of immunoglobulin genes.

    Science.gov (United States)

    Halligan, B D; Desiderio, S V

    1987-10-01

    Extracts of nuclei from B- and T-lymphoid cells contain a protein that binds specifically to the conserved nonamer DNA sequence within the recombinational signals of immunoglobulin genes. Complexes with DNA fragments from four kappa light-chain joining (J) segments have the same electrophoretic mobility. Nonamer-containing DNA fragments from heavy-chain and light-chain genes compete for binding. Within the 5'-flanking DNA of the J kappa 4 gene segment, the binding site has been localized to a 27-base-pair interval spanning the nonamer region. The binding activity is recovered as a single peak after ion-exchange chromatography. The site of binding of the protein and its presence in nuclei of lymphoid cells suggest that it may function in the assembly of immunoglobulin genes.

  16. SPERM RNA AMPLIFICATION FOR GENE EXPRESSION PROFILING BY DNA MICROARRAY TECHNOLOGY

    Science.gov (United States)

    Sperm RNA Amplification for Gene Expression Profiling by DNA Microarray TechnologyHongzu Ren, Kary E. Thompson, Judith E. Schmid and David J. Dix, Reproductive Toxicology Division, NHEERL, Office of Research and Development, US Environmental Protection Agency, Research Triang...

  17. Methods for the identification of mutations in the human phenylalanine hydroxylase gene using DNA probes

    Energy Technology Data Exchange (ETDEWEB)

    Woo, S.L.C.; Dilella, A.G.

    1990-10-23

    This patent describes a method of detecting a mutation in a phenylalanine hydroxylase gene of human genomic DNA. Also described is an automated method of detecting PKU affected, PKU helerozgotes and normals in fetal to adult human samples.

  18. PEG stabilized DNA-poly(ferrocenylsilane) polyplexes for gene delivery

    NARCIS (Netherlands)

    Zoetebier, B.; Sohrabi, A.; Lou, B.; Hempenius, M. A.; Hennink, W. E.; Vancso, G. J.

    2016-01-01

    Polycationic poly(ferrocenylsilane)s (PFS) with tunable amounts of PEG side chains were used for the condensation of DNA into polyplexes of 110 nm in 5.0 mM HEPES. The PFS-PEG/DNA polyplexes showed negligible aggregation, a strongly reduced protein adsorption, transfection activities comparable with

  19. Requirements in screening cDNA libraries for new genes and solutions offered by SBH technology

    Energy Technology Data Exchange (ETDEWEB)

    Drmanac, R.; Drmanac, S.; Labat, I.; Stavropoulos, N.

    1993-12-31

    Under different assumptions about the total number of genes, the number of housekeeping and tissue-specific genes, and the difference in the number of mRNAs per cell for functional and nonfunctional genes, significantly different results can be expected from screening random cDNA clones. We have developed gene expression models as a guide for interpretation of experimental results. For statistical, biological, and technical reasons, the search for 100,000 plus genes and discrimination between nonfunctional, housekeeping, and tissue-specific genes requires the analysis of up to 10 million clones from 20 to 50 tissues. Oligonucleotide hybridization of dense clone blots is an inexpensive and fast way to screen such large clone sets. Our preliminary results on control clones and thousands of cDNA clones from an infant brain library demonstrate the feasibility of the method. We present several models of gene expression and analyze the main factors which can influence the hunt for new genes via the screening of random cDNA libraries. The basic steps in the preparation and use of dense DNA dot arrays are described, and some results that demonstrate the feasibility and efficiency of gene inventorying by oligonucleotide hybridization are presented. Furthermore, partial SBH and single-pass gel sequencing are compared and a gene analysis scheme that combines the two approaches is discussed.

  20. Lineage-Specific Genes Are Prominent DNA Damage Hotspots during Leukemic Transformation of B Cell Precursors

    Directory of Open Access Journals (Sweden)

    Bryant Boulianne

    2017-02-01

    Full Text Available In human leukemia, lineage-specific genes represent predominant targets of deletion, with lymphoid-specific genes frequently affected in lymphoid leukemia and myeloid-specific genes in myeloid leukemia. To investigate the basis of lineage-specific alterations, we analyzed global DNA damage in primary B cell precursors expressing leukemia-inducing oncogenes by ChIP-seq. We identified more than 1,000 sensitive regions, of which B lineage-specific genes constitute the most prominent targets. Identified hotspots at B lineage genes relate to DNA-DSBs, affect genes that harbor genomic lesions in human leukemia, and associate with ectopic deletion in successfully transformed cells. Furthermore, we show that most identified regions overlap with gene bodies of highly expressed genes and that induction of a myeloid lineage phenotype in transformed B cell precursors promotes de novo DNA damage at myeloid loci. Hence, we demonstrate that lineage-specific transcription predisposes lineage-specific genes in transformed B cell precursors to DNA damage, which is likely to promote the frequent alteration of lineage-specific genes in human leukemia.

  1. Discovery and analysis of pancreatic adenocarcinoma genes using cDNA microarrays

    Institute of Scientific and Technical Information of China (English)

    Gang Jin; Xian-Gui Hu; Kang Ying; Yan Tang; Rui Liu; Yi-Jie Zhang; Zai-Ping Jing; Yi Xie; Yu-Min Mao

    2005-01-01

    AIM: To study the pathogenetic processes and the role of gene expression by microarray analyses in expediting our understanding of the molecular pathophysiology of pancreatic adenocarcinoma, and to identify the novel cancer-associated genes.METHODS: Nine histologically defined pancreatic head adenocarcinoma specimens associated with clinical data were studied. Total RNA and mRNA were isolated and labeled by reverse transcription reaction with Cy5 and Cy3 for cDNA probe. The cDNA microarrays that represent a set of 4 096 human genes were hybridized with labeled cDNA probe and screened for molecular profiling analyses.RESULTS: Using this methodology, 184 genes were screened out for differences in gene expression level after nine couples of hybridizations. Of the 184 genes,87 were upregulated and 97 downregulated, including 11 novel human genes. In pancreatic adenocarcinoma tissue, several invasion and metastasis related genes showed their high expression levels, suggesting that poor prognosis of pancreatic adenocarcinoma might have a solid molecular biological basis.CONCLUSION: The application of cDNA microarray technique for analysis of gene expression patterns is a powerful strategy to identify novel cancer-associated genes, and to rapidly explore their role in clinical pancreatic adenocarcinoma. Microarray profiles provide us new insights into the carcinogenesis and invasive process of pancreatic adenocarcinoma. Our results suggest that a highly organized and structured process of tumor invasion exists in the pancreas.

  2. Mutagen sensitivity and DNA repair of the EGFR gene in oropharyngeal cancer.

    Science.gov (United States)

    Reiter, Maximilian; Welz, Christian; Baumeister, Philipp; Schwenk-Zieger, Sabina; Harréus, Ulrich

    2010-07-01

    Epidermal growth factor receptor (EGFR) is overexpressed in several epithelial malignancies, including head and neck squamous cell cancer. Up to 90% of the tumour cases in this area exhibit EGFR overexpression. The reasons for overexpression are still not clear. Mutagen sensitivity, pre-existing conditions for genotoxic damage, gene amplification, and reduced DNA repair of the EGFR gene are possible causes for EGFR protein overexpression. DNA damage in macroscopically healthy pharyngeal mucosal tissue of 30 patients with (15) and without cancer (15) of the oropharynx was evaluated after incubation with Benz[a]pyren-7,8-diol-9,10-epoxid (BPDE), a tobacco-associated carcinogen. Emerging DNA fragmentation of the EGFR gene located on chromosome 7 was evaluated. The centromere of the chromosome served as a reference gene. Comet FISH was applied to assess the mutagen sensitivity in these regions. The extent of DNA repair was evaluated in the same samples after a 24-h repair-period. Differences in gene amplification and protein expression between the two groups were analysed by Interphase-FISH (I-FISH) and immunohistochemistry (IHC), respectively. BPDE caused significant DNA damage compared to the negative control in oropharyngeal mucosa cells of patients with- and without carcinoma. DNA fragmentation of the EGFR gene in the two groups was comparable. Mutagen sensitivity was significantly higher in the EGFR gene than in the reference gene, but fragmentation of the EGFR gene was not enhanced compared to the DNA damage of the entire DNA. The DNA repair period led to a significant reduction in DNA damage levels in all groups, without preference for any of the groups or genes. EGFR amplification was found in 7.7% of the tumour patients but not in control patients. Of the patients with oropharyngeal carcinoma, 66.6% showed enhanced expression of EGFR protein (grades 2 and 3), whereas only 13% of tumour-free patients showed such protein expression. No significant differences in

  3. Application of an improved cDNA competition technique to identify prostate cancer-associated gene.

    Science.gov (United States)

    Rinaldy, A R; Steiner, M S

    1999-11-01

    A technique to improve cDNA library screening was developed by using mixed probes derived from two closely related cDNA populations of high-metastatic MAT-LyLu and low-metastatic AT-1 Dunning R3227 rat prostate cancer sublines. The technique required the generation of a cDNA library from each subline followed by polymerase chain reaction (PCR) amplification of the cDNA insert population. The PCR products derived from the first library were radiolabeled and mixed with an excess amount of PCR products from the second library. The mixture and an excess amount of both the lambda and pBluescript DNA were used as a probe to screen the first cDNA library. This mixed probe (designated the competition probe) differentially cross-hybridized with the plaque lift of the screened first cDNA library. Weak radioactive signals indicated the cross-hybridization of cDNA sequences common to the competition probe mixture and the first cDNA library, whereas strong signals implied unhybridized unique or abundant cDNA sequences in the first cDNA library. The reproducibility of this technique was confirmed by showing that the full-length cDNA clones were associated with the phenotype of the screened first cell line. The isolated clones were characterized as rat nucleolar protein, rat mitochondrial genes coding for 16S and 12S rRNAs, and rat tRNAs specific for valine and phenyl-alanine. This result is consistent with the fact that the first cell line, MAT-LyLu, is metabolically more active than are AT-1 cells because of higher gene dosage or amplification of nucleolar and mitochondrial RNA and its associated genes. Another clone which had a strong signal represented a novel gene associated with the MAT-LyLu cancer phenotype.

  4. Pancreatic cancer patient survival correlates with DNA methylation of pancreas development genes.

    Science.gov (United States)

    Thompson, Michael J; Rubbi, Liudmilla; Dawson, David W; Donahue, Timothy R; Pellegrini, Matteo

    2015-01-01

    DNA methylation is an epigenetic mark associated with regulation of transcription and genome structure. These markers have been investigated in a variety of cancer settings for their utility in differentiating normal tissue from tumor tissue. Here, we examine the direct correlation between DNA methylation and patient survival. We find that changes in the DNA methylation of key pancreatic developmental genes are strongly associated with patient survival.

  5. Tobacco smoking is associated with DNA methylation of diabetes susceptibility genes

    NARCIS (Netherlands)

    S. Ligthart (Symen); R.V. Steenaard (Rebecca); M.J. Peters (Marjolein); J.B.J. van Meurs (Joyce); E.J.G. Sijbrands (Eric); A.G. Uitterlinden (André); M.J. Bonder (Marc); A. Hofman (Albert); O.H. Franco (Oscar); A. Dehghan (Abbas); B.T. Heijman (Bastiaan); P.A.C. 't Hoen (Peter); J.B.J. van Meurs (Joyce); A.J. Isaacs (Aaron); R. Jansen; L. Franke (Lude); D.I. Boomsma (Dorret); R. Pool (Reńe); J. van Dongen (Jenny); J.J. Hottenga (Jouke Jan); M.M. van Greevenbroek; C.D.A. Stehouwer (Coen D.A.); C.J. van der Kallen; C.G. Schalkwijk (Casper); C. Wijmenga (Cisca); A. Zhernakova (Alexandra); E.F. Tigchelaar (Ettje F.); P. Eline Slagboom; M. Beekman (Marian); J. Deelen (Joris); D. van Heemst (Diana); J.H. Veldink (Jan); L.H. van den Berg (Leonard); C.M. van Duijn (Cock); A. Hofman (Albert); A.G. Uitterlinden (André); P. Mila Jhamai; M.M.P.J. Verbiest (Michael); H. Eka; D. Suchiman; M. Verkerk (Marijn); R. van der Breggen (Ruud); J. van Rooij (Jeroen); N. Lakenberg (Nico); H. Mei (Hailiang); M. van Iterson; M. Van Galen (Michiel); J.J. Bot (Jan); P. van 't Hof (Peter); P. Deelen (Patrick); I. Nooren (Irene); H. Moed (Heleen); M. Vermaat (Martijn); D.V. Zhernakova (Dasha V.); R. Luijk (René); M.J. Bonder (Marc Jan); F. van Dijk (F.); W. Arindrarto (Wibowo); S.M. Kielbasa (Szymon M.); M. Swertz (Morris); E.W. van Zwet (Erik)

    2016-01-01

    textabstractAims/hypothesis: Tobacco smoking, a risk factor for diabetes, is an established modifier of DNA methylation. We hypothesised that tobacco smoking modifies DNA methylation of genes previously identified for diabetes. Methods: We annotated CpG sites available on the Illumina Human

  6. DNA polymorphism of HLA class II genes in pauciarticular juvenile rheumatoid arthritis

    DEFF Research Database (Denmark)

    Morling, N; Friis, J; Fugger, L;

    1991-01-01

    We investigated the DNA restriction fragment length polymorphism (RFLP) of the major histocompatibility complex (MHC) class II genes: HLA-DRB, -DQA, -DQB, DPA, and -DPB in 54 patients with pauciarticular juvenile rheumatoid arthritis (PJRA) and in healthy Danes. The frequencies of DNA fragments a...

  7. Tobacco smoking is associated with DNA methylation of diabetes susceptibility genes

    NARCIS (Netherlands)

    S. Ligthart (Symen); R.V. Steenaard (Rebecca V.); M.J. Peters (Marjolein); J.B.J. van Meurs (Joyce); E.J.G. Sijbrands (Eric); A.G. Uitterlinden (André); M.J. Bonder (Marc Jan); A. Hofman (Albert); O.H. Franco (Oscar); A. Dehghan (Abbas); B.T. Heijman (Bastiaan); P.A.C. 't Hoen (Peter); J.B.J. van Meurs (Joyce); A.J. Isaacs (Aaron); R. Jansen; L. Franke (Lude); D.I. Boomsma (Dorret); R. Pool (Reńe); J. van Dongen (Jenny); J.J. Hottenga (Jouke Jan); M.M. van Greevenbroek; C.D.A. Stehouwer (Coen D.A.); C.J. van der Kallen; C.G. Schalkwijk (Casper); C. Wijmenga (Cisca); A. Zhernakova (Alexandra); E.F. Tigchelaar (Ettje F.); P. Eline Slagboom; M. Beekman (Marian); J. Deelen (Joris); D. van Heemst (Diana); J.H. Veldink (Jan); L.H. van den Berg (Leonard); C.M. van Duijn (Cock); A. Hofman (Albert); A.G. Uitterlinden (Andre G.); P. Mila Jhamai; M.M.P.J. Verbiest (Michael); H. Eka; D. Suchiman; M. Verkerk (Marijn); R. van der Breggen (Ruud); J. van Rooij (Jeroen); N. Lakenberg (Nico); H. Mei (Hailiang); M. van Iterson; M. Van Galen (Michiel); J.J. Bot (Jan); P. van 't Hof (Peter); P. Deelen (Patrick); I. Nooren (Irene); H. Moed (Heleen); M. Vermaat (Martijn); D.V. Zhernakova (Dasha V.); R. Luijk (René); M.J. Bonder (Marc Jan); F. van Dijk (F.); W. Arindrarto (Wibowo); S.M. Kielbasa (Szymon M.); M. Swertz (Morris); E.W. van Zwet (Erik)

    2016-01-01

    textabstractAims/hypothesis: Tobacco smoking, a risk factor for diabetes, is an established modifier of DNA methylation. We hypothesised that tobacco smoking modifies DNA methylation of genes previously identified for diabetes. Methods: We annotated CpG sites available on the Illumina Human Methylat

  8. Gene expression analysis of strawberry achene and receptacle maturation using DNA microarrays

    NARCIS (Netherlands)

    Aharoni, A.; O'Connell, A.P.

    2002-01-01

    Large-scale, single pass sequencing and parallel gene expression analysis using DNA microarrays were employed for the comprehensive investigation of ripening in strawberry fruit. A total of 1701 cDNA clones (comprising 1100 strawberry ESTs and 601 unsequenced cDNAs) obtained from a strawberry (Fraga

  9. Comprehensive analysis of DNA repair gene variants and risk of meningioma

    DEFF Research Database (Denmark)

    Bethke, L.; Murray, A.; Webb, E.

    2008-01-01

    of meningioma and exposure to ionizing radiation is also well known and led us to examine whether variants in DNA repair genes contribute to disease susceptibility. METHODS: We analyzed 1127 tagging single-nucleotide polymorphisms (SNPs) that were selected to capture most of the common variation in 136 DNA...

  10. An automated annotation tool for genomic DNA sequences using GeneScan and BLAST

    Indian Academy of Sciences (India)

    Andrew M. Lynn; Chakresh Kumar Jain; K. Kosalai; Pranjan Barman; Nupur Thakur; Harish Batra; Alok Bhattacharya

    2001-04-01

    Genomic sequence data are often available well before the annotated sequence is published. We present a method for analysis of genomic DNA to identify coding sequences using the GeneScan algorithm and characterize these resultant sequences by BLAST. The routines are used to develop a system for automated annotation of genome DNA sequences.

  11. Therapeutic modulation of endogenous gene function by agents with designed DNA-sequence specificities

    NARCIS (Netherlands)

    Uil, T.G.; Haisma, H.J.; Rots, Marianne

    2003-01-01

    Designer molecules that can specifically target pre-determined DNA sequences provide a means to modulate endogenous gene function. Different classes of sequence-specific DNA-binding agents have been developed, including triplex-forming molecules, synthetic polyamides and designer zinc finger protein

  12. Insulin regulation of the glucagon gene is mediated by an insulin-responsive DNA element.

    OpenAIRE

    1991-01-01

    Diabetes mellitus is characterized by insulin deficiency and high plasma glucagon levels, which can be normalized by insulin replacement. It has previously been reported that glucagon gene expression is negatively regulated by insulin at the transcriptional level. By transfection studies, I have now localized a DNA control element that mediates insulin effects on glucagon gene transcription. This element also confers insulin responsiveness to a heterologous promoter. DNA-binding proteins that...

  13. Distinct co-evolution patterns of genes associated to DNA polymerase III DnaE and PolC

    Directory of Open Access Journals (Sweden)

    Engelen Stefan

    2012-02-01

    Full Text Available Abstract Background Bacterial genomes displaying a strong bias between the leading and the lagging strand of DNA replication encode two DNA polymerases III, DnaE and PolC, rather than a single one. Replication is a highly unsymmetrical process, and the presence of two polymerases is therefore not unexpected. Using comparative genomics, we explored whether other processes have evolved in parallel with each polymerase. Results Extending previous in silico heuristics for the analysis of gene co-evolution, we analyzed the function of genes clustering with dnaE and polC. Clusters were highly informative. DnaE co-evolves with the ribosome, the transcription machinery, the core of intermediary metabolism enzymes. It is also connected to the energy-saving enzyme necessary for RNA degradation, polynucleotide phosphorylase. Most of the proteins of this co-evolving set belong to the persistent set in bacterial proteomes, that is fairly ubiquitously distributed. In contrast, PolC co-evolves with RNA degradation enzymes that are present only in the A+T-rich Firmicutes clade, suggesting at least two origins for the degradosome. Conclusion DNA replication involves two machineries, DnaE and PolC. DnaE co-evolves with the core functions of bacterial life. In contrast PolC co-evolves with a set of RNA degradation enzymes that does not derive from the degradosome identified in gamma-Proteobacteria. This suggests that at least two independent RNA degradation pathways existed in the progenote community at the end of the RNA genome world.

  14. Physiological consequences of blocked Caulobacter crescentus dnaA expression, an essential DNA replication gene.

    Science.gov (United States)

    Gorbatyuk, B; Marczynski, G T

    2001-04-01

    Caulobacter crescentus chromosome replication is precisely coupled to a developmental cell cycle. Like most eubacteria, C. crescentus has a DnaA homologue that is presumed to initiate chromosome replication. However, the C. crescentus replication origin (Cori) lacks perfect consensus Escherichia coli DnaA boxes. Instead, the Cori strong transcription promoter (Ps) may regulate chromosome replication through the CtrA cell cycle response regulator. We therefore created a conditional dnaA C. crescentus strain. Blocking dnaA expression immediately decreased DNA synthesis, which stopped after approximately one doubling period. Fluorescent flow cytometry confirmed that DNA synthesis is blocked at the initiation stage. Cell division also stopped, but not swarmer to stalked cell differentiation. All cells became stalked cells that grew as long filaments. Therefore, general transcription and protein synthesis continued, whereas DNA synthesis stopped. However, transcription was selectively blocked from the flagellar fliQ and fliL and methyltransferase ccrM promoters, which require CtrA and are blocked by different DNA synthesis inhibitors. Interestingly, transcription from Cori Ps continued unaltered. Therefore, Ps transcription is not sufficient for chromosome replication. Approximately 6-8 h after blocked dnaA expression, cells lost viability exponentially. Coincidentally, beta-galactosidase was induced from one transcription reporter, suggesting an altered physiology. We conclude that C. crescentus DnaA is essential for chromosome replication initiation, and perhaps also has a wider role in cell homeostasis.

  15. Association Between Polymorphisms of DNA Repair Gene XRCC1 and DNA Damage in Asbestos-Exposed Workers

    Institute of Scientific and Technical Information of China (English)

    XIAO-HONG ZHAO; GUANG JIA; YONG-QUAN LIU; SHAO-WEI LIU; LEI YAN; YU JIN; NIAN LIU

    2006-01-01

    Objective To compare the asbestos-induced DNA damage and repair capacities of DNA damage between 104 asbestos exposed workers and 101 control workers in Qingdao City of China and to investigate the possible association between polymorphisms in codon 399 of XRCC1 and susceptibility to asbestosis. Methods DNA damage levels in peripheral bloodlymphocytes were determined by comet assay, and XRCC 1 genetic polymorphisms of DNA samples from 51 asbestosis cases and 53 non-asbestosis workers with a similar asbestos exposure history were analyzed by PCR/RFLP. Results The basal comet scores (3.95±2.95) were significantly higher in asbestos-exposed workers than in control workers (0.10±0.28). After 1 h H2O2 stimulation, DNA damage of lymphocytes exhibited different increases. After a 4 h repair period, the comet scores were 50.98±19.53 in asbestos-exposed workers and 18.32±12.04 in controls. The residual DNA damage (RD) was significantly greater (P<0.01) in asbestos-exposed workers (35.62%) than in controls (27.75%). XRCC1 genetic polymorphism in 104 asbestos-exposed workers was not associated with increased risk of asbestosis. But compared with polymorphisms in the DNA repair gene XRCC1 (polymorphisms in codon 399) and the DNA damage induced by asbestos, the comet scores in asbestosis cases with Gln/Gln, Gln/Arg, and Arg/Arg were 40.26±18.94, 38.03±28.22, and 32.01±11.65, respectively, which were higher than those in non-asbestosis workers with the same genotypes (25.58±11.08, 37.08±14.74, and 29.38±10.15). There were significant differences in the comet scores between asbestosis cases and non-asbestosis workers with Gln/Gln by Student's t-test (P<0.05 or 0.01). The comet scores were higher in asbestosis workers with Gln/Gln than in those with Arg/Arg and in non-asbestosis workers exposed to asbestos, but without statistically significant difference. Conclusions Exposure to asbestos may be related to DNA damage or the capacity of cells to repair H2O2-induced

  16. DNA Methylation and Gene Expression Profiling of Ewing Sarcoma Primary Tumors Reveal Genes That Are Potential Targets of Epigenetic Inactivation

    Directory of Open Access Journals (Sweden)

    Nikul Patel

    2012-01-01

    Full Text Available The role of aberrant DNA methylation in Ewing sarcoma is not completely understood. The methylation status of 503 genes in 52 formalin-fixed paraffin-embedded EWS tumors and 3 EWS cell lines was compared to human mesenchymal stem cell primary cultures (hMSCs using bead chip methylation analysis. Relative expression of methylated genes was assessed in 5-Aza-2-deoxycytidine-(5-AZA-treated EWS cell lines and in a cohort of primary EWS samples and hMSCs by gene expression and quantitative RT-PCR. 129 genes demonstrated statistically significant hypermethylation in EWS tumors compared to hMSCs. Thirty-six genes were profoundly methylated in EWS and unmethylated in hMSCs. 5-AZA treatment of EWS cell lines resulted in upregulation of expression of hundreds of genes including 162 that were increased by at least 2-fold. The expression of 19 of 36 candidate hypermethylated genes was increased following 5-AZA. Analysis of gene expression from an independent cohort of tumors confirmed decreased expression of six of nineteen hypermethylated genes (AXL, COL1A1, CYP1B1, LYN, SERPINE1, and VCAN. Comparing gene expression and DNA methylation analyses proved to be an effective way to identify genes epigenetically regulated in EWS. Further investigation is ongoing to elucidate the role of these epigenetic alterations in EWS pathogenesis.

  17. Identification of Bicarbonate as a Trigger and Genes Involved with Extracellular DNA Export in Mycobacterial Biofilms.

    Science.gov (United States)

    Rose, Sasha J; Bermudez, Luiz E

    2016-12-06

    Extracellular DNA (eDNA) is an integral biofilm matrix component of numerous pathogens, including nontuberculous mycobacteria (NTM). Cell lysis is the source of eDNA in certain bacteria, but the source of eDNA remains unidentified for NTM, as well as for other eDNA-containing bacterial species. In this study, conditions affecting eDNA export were examined, and genes involved with the eDNA export mechanism were identified. After a method for monitoring eDNA in real time in undisturbed biofilms was established, different conditions affecting eDNA were investigated. Bicarbonate positively influenced eDNA export in a pH-independent manner in Mycobacterium avium, M. abscessus, and M. chelonae The surface-exposed proteome of M. avium in eDNA-containing biofilms revealed abundant carbonic anhydrases. Chemical inhibition of carbonic anhydrases with ethoxzolamide significantly reduced eDNA export. An unbiased transposon mutant library screen for eDNA export in M. avium identified many severely eDNA-attenuated mutants, including one not expressing a unique FtsK/SpoIIIE-like DNA-transporting pore, two with inactivation of carbonic anhydrases, and nine with inactivation of genes belonging to a unique genomic region, as well as numerous mutants involved in metabolism and energy production. Complementation of nine mutants that included the FtsK/SpoIIIE and carbonic anhydrase significantly restored eDNA export. Interestingly, several attenuated eDNA mutants have mutations in genes encoding proteins that were found with the surface proteomics, and many more mutations are localized in operons potentially encoding surface proteins. Collectively, our data strengthen the evidence of eDNA export being an active mechanism that is activated by the bacterium responding to bicarbonate. Many bacteria contain extracellular DNA (eDNA) in their biofilm matrix, as it has various biological and physical functions. We recently reported that nontuberculous mycobacteria (NTM) can contain

  18. Selection for the G4 DNA motif at the 5' end of human genes.

    Science.gov (United States)

    Eddy, Johanna; Maizels, Nancy

    2009-04-01

    Formation of G4 DNA may occur in the course of replication and transcription, and contribute to genomic instability. We have quantitated abundance of G4 motifs and potential for G4 DNA formation of the nontemplate strand of 5' exons and introns of transcripts of human genes. We find that, for all human genes, G4 motifs are enriched in 5' regions of transcripts relative to downstream regions; and in 5' regulatory regions relative to coding regions. Notably, although tumor suppressor genes are depleted and proto-oncogenes enriched in G4 motifs, abundance of G4 motifs in the 5' regions of transcripts of genes in these categories does not differ. These results support the hypothesis that G4 motifs are under selection in the human genome. They further show that for tumor suppressor genes and proto-oncogenes, independent selection determines potential for G4 DNA formation of 5' regulatory regions of transcripts and downstream coding regions.

  19. GeneLab: NASA's Open Access, Collaborative Platform for Systems Biology and Space Medicine

    Science.gov (United States)

    Berrios, Daniel C.; Thompson, Terri G.; Fogle, Homer W.; Rask, Jon C.; Coughlan, Joseph C.

    2015-01-01

    NASA is investing in GeneLab1 (http:genelab.nasa.gov), a multi-year effort to maximize utilization of the limited resources to conduct biological and medical research in space, principally aboard the International Space Station (ISS). High-throughput genomic, transcriptomic, proteomic or other omics analyses from experiments conducted on the ISS will be stored in the GeneLab Data Systems (GLDS), an open-science information system that will also include a biocomputation platform with collaborative science capabilities, to enable the discovery and validation of molecular networks.

  20. Identification of brassinosteroid responsive genes in Arabidopsis by cDNA array

    Institute of Scientific and Technical Information of China (English)

    胡玉欣; 汪政科; 王永红; 包方; 李凝; 彭镇华; 李家洋

    2001-01-01

    We have systematically monitored brassinosteroid (BR) responsive genes in a BR-deficient mutant det2 suspension culture of Arabidopsis by using a cDNA array approach. Among 13000 cDNA clones arrayed on filters, 53 BR responsive clones were identified and designated BRR1-BRR53. Sequence analysis of 43 clones showed that 19 clones are novel genes, 3 clones are genes involved in the control of cell division, 4 clones are genes related to plant stress responses, 4 clones are transcriptional factor or signal transduction component genes, and 3 clones are genes involved in RNA splicing or structure forming. In addition, we also found that BR regulated the transcription of genes related to many physiological processes, such as photoreaction, ion transportation and some metabolic processes. These findings present molecular evidence that BR plays an essential role in plant growth and development.

  1. Genome-Wide Targets Regulated by the OsMADS1 Transcription Factor Reveals Its DNA Recognition Properties1[OPEN

    Science.gov (United States)

    Khanday, Imtiyaz; Das, Sanjukta; Chongloi, Grace L; Vijayraghavan, Usha

    2016-01-01

    OsMADS1 controls rice (Oryza sativa) floral fate and organ development. Yet, its genome-wide targets and the mechanisms underlying its role as a transcription regulator controlling developmental gene expression are unknown. We identify 3112 gene-associated OsMADS1-bound sites in the floret genome. These occur in the vicinity of transcription start sites, within gene bodies, and in intergenic regions. Majority of the bound DNA contained CArG motif variants or, in several cases, only A-tracts. Sequences flanking the binding peak had a higher AT nucleotide content, implying that broader DNA structural features may define in planta binding. Sequences for binding by other transcription factor families like MYC, AP2/ERF, bZIP, etc. are enriched in OsMADS1-bound DNAs. Target genes implicated in transcription, chromatin remodeling, cellular processes, and hormone metabolism were enriched. Combining expression data from OsMADS1 knockdown florets with these DNA binding data, a snapshot of a gene regulatory network was deduced where targets, such as AP2/ERF and bHLH transcription factors and chromatin remodelers form nodes. We show that the expression status of these nodal factors can be altered by inducing the OsMADS1-GR fusion protein and present a model for a regulatory cascade where the direct targets of OsMADS1, OsbHLH108/SPT, OsERF034, and OsHSF24, in turn control genes such as OsMADS32 and OsYABBY5. This cascade, with other similar relationships, cumulatively contributes to floral organ development. Overall, OsMADS1 binds to several regulatory genes and, probably in combination with other factors, controls a gene regulatory network that ensures rice floret development. PMID:27457124

  2. Construction of cDNA Library from NPC Tissue and Screening of Antigenic Genes

    Institute of Scientific and Technical Information of China (English)

    Jun Shu; Xiaojuan He; Guancheng Li

    2006-01-01

    To construct cDNA library of nasopharyngeal carcinoma (NPC) and obtain the NPC associated or specific antigens from it, we used a powerful new method to identify the antigens eliciting humoral immune response, which is SEREX (serological identification of antigen by recombinant cDNA expression library). Autologous serum of NPC patient was used to screen the reactive clones in the human NPC tissue cDNA library consisted of 3.64×106 recombinants. The 23 exact positive clones were subcloned to monoclonality and the size of cDNA inserts was identified by PCR. Then the nucleotide sequence of cDNA inserts was determined, and the sequence alignments were performed with BLAST software on GenBank database. They represented 16 different antigens. A detailed sequence analysis showed that 10 of 16 genes were high homologous to genes known in GenBank, such as RPL31,S100 A2, MT2A, etc. However, there were also 6 genes with low homology to genes in GenBank. Furthermore, 3 of 6 genes may be novel genes. The associations of these genes to NPC and the roles that they played in the occurrence and development of NPC should be further revealed.

  3. DNA methylation patterns of candidate genes regulated by thymine DNA glycosylase in patients with TP53 germline mutations

    Directory of Open Access Journals (Sweden)

    F.P. Fortes

    2015-07-01

    Full Text Available Li-Fraumeni syndrome (LFS is a rare, autosomal dominant, hereditary cancer predisposition disorder. In Brazil, the p.R337H TP53 founder mutation causes the variant form of LFS, Li-Fraumeni-like syndrome. The occurrence of cancer and age of disease onset are known to vary, even in patients carrying the same mutation, and several mechanisms such as genetic and epigenetic alterations may be involved in this variability. However, the extent of involvement of such events has not been clarified. It is well established that p53 regulates several pathways, including the thymine DNA glycosylase (TDG pathway, which regulates the DNA methylation of several genes. This study aimed to identify the DNA methylation pattern of genes potentially related to the TDG pathway (CDKN2A, FOXA1, HOXD8, OCT4, SOX2, and SOX17 in 30 patients with germline TP53 mutations, 10 patients with wild-type TP53, and 10 healthy individuals. We also evaluated TDG expression in patients with adrenocortical tumors (ADR with and without the p.R337H TP53 mutation. Gene methylation patterns of peripheral blood DNA samples assessed by pyrosequencing revealed no significant differences between the three groups. However, increased TDG expression was observed by quantitative reverse transcription PCR in p.R337H carriers with ADR. Considering the rarity of this phenotype and the relevance of these findings, further studies using a larger sample set are necessary to confirm our results.

  4. DNA methylation patterns of candidate genes regulated by thymine DNA glycosylase in patients with TP53 germline mutations

    Energy Technology Data Exchange (ETDEWEB)

    Fortes, F.P. [CIPE, Laboratrio de Oncogentica Molecular, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Kuasne, H. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Urologia, Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, SP (Brazil); Marchi, F.A. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Programa Inter-Institucional em Bioinformtica, Instituto de Matemtica e Estatstica, Universidade So Paulo, So Paulo, SP (Brazil); Miranda, P.M. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Rogatto, S.R. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Urologia, Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, SP (Brazil); Achatz, M.I. [CIPE, Laboratrio de Oncogentica Molecular, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Oncogentica, A.C. Camargo Cancer Center, So Paulo, SP (Brazil)

    2015-04-28

    Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant, hereditary cancer predisposition disorder. In Brazil, the p.R337H TP53 founder mutation causes the variant form of LFS, Li-Fraumeni-like syndrome. The occurrence of cancer and age of disease onset are known to vary, even in patients carrying the same mutation, and several mechanisms such as genetic and epigenetic alterations may be involved in this variability. However, the extent of involvement of such events has not been clarified. It is well established that p53 regulates several pathways, including the thymine DNA glycosylase (TDG) pathway, which regulates the DNA methylation of several genes. This study aimed to identify the DNA methylation pattern of genes potentially related to the TDG pathway (CDKN2A, FOXA1, HOXD8, OCT4, SOX2, and SOX17) in 30 patients with germline TP53mutations, 10 patients with wild-type TP53, and 10 healthy individuals. We also evaluated TDG expression in patients with adrenocortical tumors (ADR) with and without the p.R337H TP53 mutation. Gene methylation patterns of peripheral blood DNA samples assessed by pyrosequencing revealed no significant differences between the three groups. However, increased TDG expression was observed by quantitative reverse transcription PCR in p.R337H carriers with ADR. Considering the rarity of this phenotype and the relevance of these findings, further studies using a larger sample set are necessary to confirm our results.

  5. Analysis of gene expression profile of aspermia using cDNA microarray

    Institute of Scientific and Technical Information of China (English)

    杨波; 高晓康; 王禾; 刘贺亮; 陈宝琦; 秦荣良; 康福霞; 邵国兴; 邵晨

    2003-01-01

    Objective: To identify the differential gene expression profiles between the normal and aspermia human testes utilizing cDNA microarray. Methods: cDNA probes were prepared by labeling mRNA of aspermia testes tissues with Cy5-dUTP and mRNA of normal testes tissues with Cy3-dUTP respectively through reverse transcription. The mixed cDNA probes were then hybridized with 4096 cDNA arrays (4096 unique human cDNA sequences), and the fluorescent signals were scanned by ScanArray 3000 scanner (General Scanning, Inc.). The values of Cy5-dUTP and Cy3-dUTP on each spot were analyzed and calculated by ImaGene 3.0 software (BioDiscovery, Inc.). Differentially expressed genes were screened according to the criterion that the absolute value of natural logarithm of the ratio of Cy5-dUTP to Cy3-dUTP was greater-than 2.0 or less-than 0.5. A randomly chosen gene RAP1A was studied by in situ hybridization to evaluate the accuracy of the results. Results: 623 differential expressed genes related to aspermia were found. There were 303 up-expressed genes and 320 down-expressed genes. A distinct up-expressed gene RAP1A was confirmed by in situ hybridization. Conclusions: Screening the differential gene expression profiles between the normal and aspermia human testis by cDNA microarray can be used in the study of aspermia-related genes and the further research due to its properties, RAP1A may play some roles in the development and progression of aspermia.

  6. Prenatal diagnosis of common single gene disorders by DNA technology

    OpenAIRE

    1997-01-01

    Using the new DNA technology, it is now possible to offer prenatal diagnosis or presymptomatic testing for many genetic diseases. For prenatal diagnosis, foetal tissue is obtained y chorionic villus sampling at 9 to 11 weeks gestation or amniocentesis at 18 weeks. The programme in Hong Kong, which started in 1982, is reviewed here and now included alpha and beta thalassaemia, haemophilia A and B, Duchenne muscular dystrophy, Huntington's diseases, and spinal muscular atrophy. DNA diagnosis ca...

  7. Stationary phase induction of dnaN and recF, two genes of Escherichia coli involved in DNA replication and repair.

    Science.gov (United States)

    Villarroya, M; Pérez-Roger, I; Macián, F; Armengod, M E

    1998-03-16

    The beta subunit of DNA polymerase III holoenzyme, the Escherichia coli chromosomal replicase, is a sliding DNA clamp responsible for tethering the polymerase to DNA and endowing it with high processivity. The gene encoding beta, dnaN, maps between dnaA and recF, which are involved in initiation of DNA replication at oriC and resumption of DNA replication at disrupted replication forks, respectively. In exponentially growing cells, dnaN and recF are expressed predominantly from the dnaA promoters. However, we have found that stationary phase induction of the dnaN promoters drastically changes the expression pattern of the dnaA operon genes. As a striking consequence, synthesis of the beta subunit and RecF protein increases when cell metabolism is slowing down. Such an induction is dependent on the stationary phase sigma factor, RpoS, although the accumulation of this factor alone is not sufficient to activate the dnaN promoters. These promoters are located in DNA regions without static bending, and the -35 hexamer element is essential for their RpoS-dependent induction. Our results suggest that stationary phase-dependent mechanisms have evolved in order to coordinate expression of dnaN and recF independently of the dnaA regulatory region. These mechanisms might be part of a developmental programme aimed at maintaining DNA integrity under stress conditions.

  8. Enhanced brain targeting efficiency of intranasally administered plasmid DNA: an alternative route for brain gene therapy.

    Science.gov (United States)

    Han, In-Kwon; Kim, Mi Young; Byun, Hyang-Min; Hwang, Tae Sun; Kim, Jung Mogg; Hwang, Kwang Woo; Park, Tae Gwan; Jung, Woon-Won; Chun, Taehoon; Jeong, Gil-Jae; Oh, Yu-Kyoung

    2007-01-01

    Recently, nasal administration has been studied as a noninvasive route for delivery of plasmid DNA encoding therapeutic or antigenic genes. Here, we examined the brain targeting efficiency and transport pathways of intranasally administered plasmid DNA. Quantitative polymerase chain reaction (PCR) measurements of plasmid DNA in blood and brain tissues revealed that intranasally administered pCMVbeta (7.2 kb) and pN2/CMVbeta (14.1 kb) showed systemic absorption and brain distribution. Following intranasal administration, the beta-galactosidase protein encoded by these plasmids was significantly expressed in brain tissues. Kinetic studies showed that intranasally administered plasmid DNA reached the brain with a 2,595-fold higher efficiency than intravenously administered plasmid DNA did, 10 min post-dose. Over 1 h post-dose, the brain targeting efficiencies were consistently higher for intranasally administered plasmid DNA than for intravenously administered DNA. To examine how plasmid DNA enters the brain and moves to the various regions, we examined tissues from nine brain regions, at 5 and 10 min after intranasal or intravenous administration of plasmid DNA. Intravenously administered plasmid DNA displayed similar levels of plasmid DNA in the nine different regions, whereas, intranasally administered plasmid DNA exhibited different levels of distribution among the regions, with the highest plasmid DNA levels in the olfactory bulb. Moreover, plasmid DNA was mainly detected in the endothelial cells, but not in glial cells. Our results suggest that intranasally applied plasmid DNA may reach the brain through a direct route, possibly via the olfactory bulb, and that the nasal route might be an alternative method for efficiently delivering plasmid DNA to the brain.

  9. Recharging cationic DNA complexes with highly charged polyanions for in vitro and in vivo gene delivery.

    Science.gov (United States)

    Trubetskoy, V S; Wong, S C; Subbotin, V; Budker, V G; Loomis, A; Hagstrom, J E; Wolff, J A

    2003-02-01

    The intravenous delivery of plasmid DNA complexed with either cationic lipids (CL) or polyethyleneimine (PEI) enables high levels of foreign gene expression in lung. However, these cationic DNA complexes cause substantial toxicity. The present study found that the inclusion of polyacrylic acid (pAA) with DNA/polycation and DNA/CL complexes prevented the serum inhibition of the transfection complexes in cultured cells. The mechanism mediating this increase seems to involve both particle size enlargement due to flocculation and electrostatic shielding from opsonizing serum proteins. The use of pAA also increased the levels of lung expression in mice in vivo substantially above the levels achieved with just binary complexes of DNA and linear PEI (lPEI) or CL and reduced their toxicity. Also, the use of a "chaser" injection of pAA 30 min after injection of the ternary DNA/lPEI/pAA complexes further aided this effort to reduce toxicity while not affecting foreign gene expression. By optimizing the amount of pAA, lPEI, and DNA within the ternary complexes and using the "chaser" injection, substantial levels of lung expression were obtained while avoiding adverse effects in lung or liver. These developments will aid the use of cationic DNA complexes in animals and for eventual human gene therapy.

  10. Cloning and bioinformatics analysis of cDNA encoding cattle Smad4 gene

    Institute of Scientific and Technical Information of China (English)

    Xiaohui ZHANG; Shangzhong XU; Xue GAO; Hongyan REN; Jinbao CHEN

    2008-01-01

    The cDNA of cattle Smad4 gene was cloned by RT-PCR, 3' RACE and 5' RACE and got a 3503-bp full-long cDNA sequence. The cloned cattle Smad4 cDNA sequence had been send to GenBank and got an accession number: DQ494856. Cattle Smad4 gene consists of 12 exons and codes 553 amino acids. Cattle Smad4 cDNA shares 99%, 96%, 95%, 91% and 91% similarity in nucleic acid sequences, and 99%, 98%, 98%, 99% and 98% sim-ilarity in amino acid sequences with sheep, pig, human, rat and mouse, respectively. Smad4 cDNA was found in the testes, pancreas, liver, small intestine, ovary, lymph, car-diac muscle, skeleton muscle and thymus gland, which indicated that Smad4 was broadly expressed in cattle.

  11. miRNA gene promoters are frequent targets of aberrant DNA methylation in human breast cancer.

    Science.gov (United States)

    Vrba, Lukas; Muñoz-Rodríguez, José L; Stampfer, Martha R; Futscher, Bernard W

    2013-01-01

    miRNAs are important regulators of gene expression that are frequently deregulated in cancer, with aberrant DNA methylation being an epigenetic mechanism involved in this process. We previously identified miRNA promoter regions active in normal mammary cell types and here we analyzed which of these promoters are targets of aberrant DNA methylation in human breast cancer cell lines and breast tumor specimens. Using 5-methylcytosine immunoprecipitation coupled to miRNA tiling microarray hybridization, we performed comprehensive evaluation of DNA methylation of miRNA gene promoters in breast cancer. We found almost one third (55/167) of miRNA promoters were targets for aberrant methylation in breast cancer cell lines. Breast tumor specimens displayed DNA methylation of majority of these miRNA promoters, indicating that these changes in DNA methylation might be clinically relevant. Aberrantly methylated miRNA promoters were, similar to protein coding genes, enriched for promoters targeted by polycomb in normal cells. Detailed analysis of selected miRNA promoters revealed decreased expression of miRNA linked to increased promoter methylation for mir-31, mir-130a, let-7a-3/let-7b, mir-155, mir-137 and mir-34b/mir-34c genes. The proportion of miRNA promoters we found aberrantly methylated in breast cancer is several fold larger than that observed for protein coding genes, indicating an important role of DNA methylation in miRNA deregulation in cancer.

  12. DNA-Binding Kinetics Determines the Mechanism of Noise-Induced Switching in Gene Networks.

    Science.gov (United States)

    Tse, Margaret J; Chu, Brian K; Roy, Mahua; Read, Elizabeth L

    2015-10-20

    Gene regulatory networks are multistable dynamical systems in which attractor states represent cell phenotypes. Spontaneous, noise-induced transitions between these states are thought to underlie critical cellular processes, including cell developmental fate decisions, phenotypic plasticity in fluctuating environments, and carcinogenesis. As such, there is increasing interest in the development of theoretical and computational approaches that can shed light on the dynamics of these stochastic state transitions in multistable gene networks. We applied a numerical rare-event sampling algorithm to study transition paths of spontaneous noise-induced switching for a ubiquitous gene regulatory network motif, the bistable toggle switch, in which two mutually repressive genes compete for dominant expression. We find that the method can efficiently uncover detailed switching mechanisms that involve fluctuations both in occupancies of DNA regulatory sites and copy numbers of protein products. In addition, we show that the rate parameters governing binding and unbinding of regulatory proteins to DNA strongly influence the switching mechanism. In a regime of slow DNA-binding/unbinding kinetics, spontaneous switching occurs relatively frequently and is driven primarily by fluctuations in DNA-site occupancies. In contrast, in a regime of fast DNA-binding/unbinding kinetics, switching occurs rarely and is driven by fluctuations in levels of expressed protein. Our results demonstrate how spontaneous cell phenotype transitions involve collective behavior of both regulatory proteins and DNA. Computational approaches capable of simulating dynamics over many system variables are thus well suited to exploring dynamic mechanisms in gene networks.

  13. Gene-gun DNA vaccination aggravates respiratory syncytial virus-induced pneumonitis

    DEFF Research Database (Denmark)

    Bartholdy, Christina; Olszewska, Wieslawa; Stryhn, Anette

    2004-01-01

    elicited with recombinant vaccinia virus expressing the complete RSV M2 protein, but stronger than those induced by a similar DNA construct without the beta2m gene. DNA vaccination led to enhanced pulmonary disease after RSV challenge, with increased weight loss and cell recruitment to the lung. Depletion......A CD8+ T-cell memory response to respiratory syncytial virus (RSV) was generated by using a DNA vaccine construct encoding the dominant Kd-restricted epitope from the viral transcription anti-terminator protein M2 (M2(82-90)), linked covalently to human beta2-microglobulin (beta2m). Cutaneous gene......+ T-cell responses were not induced. Thus, in addition to specific CD8+ T cell-mediated immunopathology, gene-gun DNA vaccination causes non-specific enhancement of RSV disease without affecting virus clearance....

  14. Satellite DNA Modulates Gene Expression in the Beetle Tribolium castaneum after Heat Stress.

    Science.gov (United States)

    Feliciello, Isidoro; Akrap, Ivana; Ugarković, Đurđica

    2015-08-01

    Non-coding repetitive DNAs have been proposed to perform a gene regulatory role, however for tandemly repeated satellite DNA no such role was defined until now. Here we provide the first evidence for a role of satellite DNA in the modulation of gene expression under specific environmental conditions. The major satellite DNA TCAST1 in the beetle Tribolium castaneum is preferentially located within pericentromeric heterochromatin but is also dispersed as single repeats or short arrays in the vicinity of protein-coding genes within euchromatin. Our results show enhanced suppression of activity of TCAST1-associated genes and slower recovery of their activity after long-term heat stress relative to the same genes without associated TCAST1 satellite DNA elements. The level of gene suppression is not influenced by the distance of TCAST1 elements from the associated genes up to 40 kb from the genes' transcription start sites, but it does depend on the copy number of TCAST1 repeats within an element, being stronger for the higher number of copies. The enhanced gene suppression correlates with the enrichment of the repressive histone marks H3K9me2/3 at dispersed TCAST1 elements and their flanking regions as well as with increased expression of TCAST1 satellite DNA. The results reveal transient, RNAi based heterochromatin formation at dispersed TCAST1 repeats and their proximal regions as a mechanism responsible for enhanced silencing of TCAST1-associated genes. Differences in the pattern of distribution of TCAST1 elements contribute to gene expression diversity among T. castaneum strains after long-term heat stress and might have an impact on adaptation to different environmental conditions.

  15. Satellite DNA Modulates Gene Expression in the Beetle Tribolium castaneum after Heat Stress.

    Directory of Open Access Journals (Sweden)

    Isidoro Feliciello

    2015-08-01

    Full Text Available Non-coding repetitive DNAs have been proposed to perform a gene regulatory role, however for tandemly repeated satellite DNA no such role was defined until now. Here we provide the first evidence for a role of satellite DNA in the modulation of gene expression under specific environmental conditions. The major satellite DNA TCAST1 in the beetle Tribolium castaneum is preferentially located within pericentromeric heterochromatin but is also dispersed as single repeats or short arrays in the vicinity of protein-coding genes within euchromatin. Our results show enhanced suppression of activity of TCAST1-associated genes and slower recovery of their activity after long-term heat stress relative to the same genes without associated TCAST1 satellite DNA elements. The level of gene suppression is not influenced by the distance of TCAST1 elements from the associated genes up to 40 kb from the genes' transcription start sites, but it does depend on the copy number of TCAST1 repeats within an element, being stronger for the higher number of copies. The enhanced gene suppression correlates with the enrichment of the repressive histone marks H3K9me2/3 at dispersed TCAST1 elements and their flanking regions as well as with increased expression of TCAST1 satellite DNA. The results reveal transient, RNAi based heterochromatin formation at dispersed TCAST1 repeats and their proximal regions as a mechanism responsible for enhanced silencing of TCAST1-associated genes. Differences in the pattern of distribution of TCAST1 elements contribute to gene expression diversity among T. castaneum strains after long-term heat stress and might have an impact on adaptation to different environmental conditions.

  16. Failure to induce a DNA repair gene, RAD54, in Saccharomyces cerevisiae does not affect DNA repair or recombination phenotypes

    Energy Technology Data Exchange (ETDEWEB)

    Cole, G.M.; Mortimer, R.K. (Univ. of California, Berkeley (USA))

    1989-08-01

    The Saccharomyces cerevisiae RAD54 gene is transcriptionally regulated by a broad spectrum of DNA-damaging agents. Induction of RAD54 by DNA-damaging agents is under positive control. Sequences responsible for DNA damage induction (the DRS element) lie within a 29-base-pair region from -99 to -70 from the most proximal transcription start site. This inducible promoter element is functionally separable from a poly(dA-dT) region immediately downstream which is required for constitutive expression. Deletions which eliminate induction of RAD54 transcription by DNA damage but do not affect constitutive expression have no effect on growth or survival of noninducible strains relative to wild-type strains in the presence of DNA-damaging agents. The DRS element is also not required for homothallic mating type switching, transcriptional induction of RAD54 during meiosis, meiotic recombination, or spontaneous or X-ray-induced mitotic recombination. We find no phenotype for a lack of induction of RAD54 message via the damage-inducible DRS, which raises significant questions about the physiology of DNA damage induction in S. cerevisiae.

  17. DNA microarray analysis of genes differentially expressed in adipocyte differentiation

    Indian Academy of Sciences (India)

    Chunyan Yin; Yanfeng Xiao; Wei Zhang; Erdi Xu; Weihua Liu; Xiaoqing Yi; Ming Chang

    2014-06-01

    In the present study, the human liposarcoma cell line SW872 was used to identify global changes in gene expression profiles occurring during adipogenesis. We further explored some of the genes expressed during the late phase of adipocyte differentiation. These genes may play a major role in promoting excessive proliferation and accumulation of lipid droplets, which contribute to the development of obesity. By using microarray-based technology, we examined differential gene expression in early differentiated adipocytes and late differentiated adipocytes. Validated genes exhibited a ≥ 10-fold increase in the late phase of adipocyte differentiation by polymerase chain reaction (RT-PCR). Compared with undifferentiated preadipocytes, we found that 763 genes were increased in early differentiated adipocytes, and 667 genes were increased in later differentiated adipocytes. Furthermore, 21 genes were found being expressed 10-fold higher in the late phase of adipocyte differentiation. The results were in accordance with the RT-PCR test, which validated 11 genes, namely, CIDEC, PID1, LYRM1, ADD1, PPAR2, ANGPTL4, ADIPOQ, ACOX1, FIP1L1, MAP3K2 and PEX14. Most of these genes were found being expressed in the later phase of adipocyte differentiation involved in obesity-related diseases. The findings may help to better understand the mechanism of obesity and related diseases.

  18. DNA microarray analysis of genes differentially expressed in adipocyte differentiation.

    Science.gov (United States)

    Yin, Chunyan; Xiao, Yanfeng; Zhang, Wei; Xu, Erdi; Liu, Weihua; Yi, Xiaoqing; Chang, Ming

    2014-06-01

    In the present study, the human liposarcoma cell line SW872 was used to identify global changes in gene expression profiles occurring during adipogenesis. We further explored some of the genes expressed during the late phase of adipocyte differentiation. These genes may play a major role in promoting excessive proliferation and accumulation of lipid droplets, which contribute to the development of obesity. By using microarray-based technology, we examined differential gene expression in early differentiated adipocytes and late differentiated adipocytes. Validated genes exhibited a greater than or equal to 10-fold increase in the late phase of adipocyte differentiation by polymerase chain reaction (RT-PCR). Compared with undifferentiated preadipocytes, we found that 763 genes were increased in early differentiated adipocytes, and 667 genes were increased in later differentiated adipocytes. Furthermore, 21 genes were found being expressed 10-fold higher in the late phase of adipocyte differentiation. The results were in accordance with the RTPCR test, which validated 11 genes, namely, CIDEC, PID1, LYRM1, ADD1, PPAR?2, ANGPTL4, ADIPOQ, ACOX1, FIP1L1, MAP3K2 and PEX14. Most of these genes were found being expressed in the later phase of adipocyte differentiation involved in obesity-related diseases. The findings may help to better understand the mechanism of obesity and related diseases.

  19. Phylogenetic analysis of dermatophyte species using DNA sequence polymorphism in calmodulin gene

    NARCIS (Netherlands)

    Ahmadi, Bahram; Mirhendi, Hossein; Makimura, Koichi; de Hoog, G Sybren; Shidfar, Mohammad Reza; Nouripour-Sisakht, Sadegh; Jalalizand, Niloofar

    2016-01-01

    Use of phylogenetic species concepts based on rDNA internal transcribe spacer (ITS) regions have improved the taxonomy of dermatophyte species; however, confirmation and refinement using other genes are needed. Since the calmodulin gene has not been systematically used in dermatophyte taxonomy, we e

  20. Assignment of ten DNA repair genes from Schizosaccharomyces pombe to chromosomal NotI restriction fragments

    NARCIS (Netherlands)

    B.C. Broughton; N.C. Barbet; J. Murray (Johanne); F.Z. Watts (Felicity); M.H.M. Koken (Marcel); A.R. Lehmann (Alan); A.M. Carr (Anthony)

    1991-01-01

    textabstractTen DNA repair (rad) genes from the fission yeast, Schizosaccharomyces pombe were mapped to the 17 NotI fragments of the three chromosomes. Nine of the genes map to chromosome I, but there is no evidence for significant clustering.

  1. Quantitative studies of DNA methylation and gene expression in neuropsychiatric traits

    NARCIS (Netherlands)

    van Eijk, K.R.

    2014-01-01

    Research has shown that besides genes and environment, epigenetic factors are also playing a role in the development of traits and diseases. Epigenetic changes do not affect the underlying DNA sequence, but affect its structure and is thought to play a major role in regulation of gene expression. DN

  2. Improvement of Hydrodynamics-Based Gene Transfer of Nonviral DNA Targeted to Murine Hepatocytes

    Directory of Open Access Journals (Sweden)

    Shingo Nakamura

    2013-01-01

    Full Text Available The liver is an important organ for supporting the life of an individual. Gene transfer toward this organ has been attempted in many laboratories to date; however, there have been few reports on improved liver-targeted gene delivery by using a nonviral vector. In this study, we examined the effect of various types of gene delivery carriers on enhancing the uptake and gene expression of exogenous DNA in murine hepatocytes when a hydrodynamics-based gene delivery (HGD is performed via tail-vein injection. Mice were singly injected with a large amount of phosphate-buffered saline containing reporter plasmid DNA and/or with a gene delivery carrier. One day after the gene delivery, the animals' livers were dissected and subjected to biochemical, histochemical, and molecular biological analyses. The strongest signal from the reporter plasmid DNA was observed when the DNA was mixed with a polyethylenimine- (PEI- based reagent. Coinjection with pCRTEIL (a loxP-floxed reporter construct and pTR/NCre (a liver-specific Cre expression vector resulted in the liver-specific recombination of pCRTEIL. The combination of PEI with HGD would thus be a valuable tool for liver-specific manipulation to examine the function of a gene of interest in the liver and for creating liver disease models.

  3. Absolute Quantitation of DNA Methylation of 28 Candidate Genes in Prostate Cancer Using Pyrosequencing

    Directory of Open Access Journals (Sweden)

    Nataڑa Vasiljeviš

    2011-01-01

    Full Text Available Aberrant DNA methylation plays a pivotal role in carcinogenesis and its mapping is likely to provide biomarkers for improved diagnostic and risk assessment in prostate cancer (PCa. We quantified and compared absolute methylation levels among 28 candidate genes in 48 PCa and 29 benign prostate hyperplasia (BPH samples using the pyrosequencing (PSQ method to identify genes with diagnostic and prognostic potential.

  4. The nucleotide sequence of a 39 kb segment of yeast chromosome IV: 12 new open reading frames, nine known genes and one genes for Gly-tRNA.

    Science.gov (United States)

    Bahr, A; Möller-Rieker, S; Hankeln, T; Kraemer, C; Protin, U; Schmidt, E R

    1997-02-01

    The complete nucleotide sequence of a 39,090 bp segment from the left arm of yeast chromosome IV was determined. Twenty-one open reading frames (ORFs) longer than 100 amino acids and a Gly-tRNA gene were discovered. Nine of the 21 ORFs (D0892, D1022, D1037, D1045, D1057, D1204, D1209, D1214, D1219) correspond to the previously sequenced Saccharomyces cerevisiae genes for the NAD-dependent glutamate dehydrogenase (GDH), the secretory component (SHR3), the GABA transport protein (UGA4), the high mobility group-like protein (NHP2), the hydroxymethylbilane synthase (HEM3), the methylated DNA protein-cysteine S-methyltransferase (MGT1), a putative sugar transport protein, the Shm1 protein (SHM1) and the anti-silencing protein (ASF2). The inferred amino acid sequences of 11 ORFs show significant similarity with known proteins from various organisms, whereas the remaining ORF does not share any similarity with known proteins.

  5. Versatile types of polysaccharide-based supramolecular polycation/pDNA nanoplexes for gene delivery

    Science.gov (United States)

    Hu, Yang; Zhao, Nana; Yu, Bingran; Liu, Fusheng; Xu, Fu-Jian

    2014-06-01

    Different polysaccharide-based supramolecular polycations were readily synthesized by assembling multiple β-cyclodextrin-cored star polycations with an adamantane-functionalized dextran via host-guest interaction in the absence or presence of bioreducible linkages. Compared with nanoplexes of the starting star polycation and pDNA, the supramolecular polycation/pDNA nanoplexes exhibited similarly low cytotoxicity, improved cellular internalization and significantly higher gene transfection efficiencies. The incorporation of disulfide linkages imparted the supramolecular polycation/pDNA nanoplexes with the advantage of intracellular bioreducibility, resulting in better gene delivery properties. In addition, the antitumor properties of supramolecular polycation/pDNA nanoplexes were also investigated using a suicide gene therapy system. The present study demonstrates that the proper assembly of cyclodextrin-cored polycations with adamantane-functionalized polysaccharides is an effective strategy for the production of new nanoplex delivery systems.Different polysaccharide-based supramolecular polycations were readily synthesized by assembling multiple β-cyclodextrin-cored star polycations with an adamantane-functionalized dextran via host-guest interaction in the absence or presence of bioreducible linkages. Compared with nanoplexes of the starting star polycation and pDNA, the supramolecular polycation/pDNA nanoplexes exhibited similarly low cytotoxicity, improved cellular internalization and significantly higher gene transfection efficiencies. The incorporation of disulfide linkages imparted the supramolecular polycation/pDNA nanoplexes with the advantage of intracellular bioreducibility, resulting in better gene delivery properties. In addition, the antitumor properties of supramolecular polycation/pDNA nanoplexes were also investigated using a suicide gene therapy system. The present study demonstrates that the proper assembly of cyclodextrin-cored polycations

  6. Candidate genes for cross-resistance against DNA-damaging drugs

    DEFF Research Database (Denmark)

    Wittig, Rainer; Nessling, Michelle; Will, Rainer D

    2002-01-01

    Drug resistance of tumor cells leads to major drawbacks in the treatment of cancer. To identify candidate genes for drug resistance, we compared the expression patterns of the drug-sensitive human malignant melanoma cell line MeWo and three derived sublines with acquired resistance to the DNA...... as several apoptosis-related genes, in particular STK17A and CRYAB. As MPP1 and CRYAB are also among the 14 genes differentially expressed in all three of the drug-resistant sublines, they represent the strongest candidates for resistance against DNA-damaging drugs....

  7. [Problems and prospects of gene therapeutics and DNA vaccines development and application].

    Science.gov (United States)

    Kibirev, Ia A; Drobkov, B I; Marakulin, I V

    2010-01-01

    The review is summarized foreign publications devoted to different aspects of DNA vaccines and gene therapeutics' biological safety. In spite of incomprehension in their action, numerous prototype DNA-based biopharmaceuticals are in advanced stages of human clinical trials. This review is focused on some safety concerns of gene formulations vaccines relate to toxic effects, vertical transmission possibility, genome integration complications, immunologic and immunopathologic effects and environmental spread. It is noted that necessity of national regulatory documents development related to gene therapy medicinal products is significant condition of their application to medical practice.

  8. Obesity is associated with depot-specific alterations in adipocyte DNA methylation and gene expression

    DEFF Research Database (Denmark)

    Sonne, Si Brask; Yadav, Rachita; Yin, Guangliang

    2017-01-01

    The present study aimed to identify genes exhibiting concomitant obesity-dependent changes in DNA methylation and gene expression in adipose tissues in the mouse using diet-induced obese (DIO) C57BL/6J and genetically obese ob/ob mice as models. Mature adipocytes were isolated from epididymal...... and inguinal adipose tissues of ob/ob and DIO C57BL/6J mice. DNA methylation was analyzed by MeDIP-sequencing and gene expression by microarray analysis. The majority of differentially methylated regions (DMRs) were hypomethylated in obese mice. Global methylation of long interspersed elements indicated...... and expression of 9 genes in epididymal adipocytes, including the known obesity-associated genes, Ehd2 and Kctd15, and a novel candidate gene, Irf8, possibly involved in immune type 1/type2 balance. The use of 2 obesity models enabled us to dissociate changes associated with high fat feeding from those...

  9. Ionizing and ultraviolet radiation enhances the efficiency of DNA mediated gene transfer in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Perez, C.F.

    1984-08-01

    The enhancement effects of ionizing and non-ionizing radiation on the efficiency of DNA mediated gene transfer were studied. Confluent Rat-2 cells were transfected with purified SV40 viral DNA, irradiated with either X-rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were non-linear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X-rays or 330 MeV/amu argon particles at the Berkeley Bevalac showed a higher frequency of HAT/sup +/ colonies/survivor than unirradiated transfected cells. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK-transformation by both X-rays and ultraviolet radiation. The results demonstrate that radiation enhancement of the efficiency of DNA mediated gene transfer is not explained by increased nuclear uptake of the transfected DNA. Radiation increases the competence of the transfected cell population for genetic transformation. Three models for this increased competence are presented. The targeted integration model, the inducible recombination model, the partition model, and the utilization of DNA mediated gene transfer for DNA repair studies are discussed. 465 references.

  10. GENE EXPRESSION PROFILING OF GANGLIOGLIOMA MALIGNANT PROGRESSION BY cDNA ARRAY

    Institute of Scientific and Technical Information of China (English)

    ZHANG Quan-bin; HUANG Qiang; DONG Jun; WANG Ai-dong; SUN Ji-yong; LAN Qing; HU Geng-xi

    2005-01-01

    Objective: To establish gene expression profiles associated with malignant progression of ganglioglioma. Methods: The primary and two recurrent glioma specimens were collected intraoperatively from the same patient who experienced tumor transformation into anaplastic astrocytoma and glioblastoma multiform for the first and second recurrence respectively. Gene expression was assayed through cDNA array and bioinformatics analysis. Results: A total of 197 differentially expressed genes with differential ratio value more than 3 compared with normal brain tissue were obtained. Among 109 functionally denned genes, those associated with development ranked the first by frequency, followed by genes associated with metabolism, differentiation, signal transduction and so on. As a result of cluster analysis among 368 genes, eleven genes were up regulated with malignant progression, while six genes were down regulated. Conclusion: Gene expression profiles associated with malignant progression of glioma were successfully established, which provides a powerful tool for research on molecular mechanisms of malignant progression of gliomas.

  11. Mapping a gene for adult-onset primary open-angle glaucoma to chromosome 3q

    Energy Technology Data Exchange (ETDEWEB)

    Wirtz, M.K.; Samples, J.R.; Kramer, P.L. [Oregon Health Sciences Univ., Portland, OR (United States)] [and others

    1997-02-01

    Glaucoma is the third-leading cause of blindness in the world, affecting >13.5 million people. Adult-on-set primary open-angle glaucoma (POAG) is the most common form of glaucoma in the United States. We present a family in which adult-onset POAG is inherited as an autosomal dominant trait. Twelve affected family members were identified from 44 at-risk individuals. The disease-causing gene was mapped to chromosome 3q21-24, with analysis of recombinant haplotypes suggesting a total inclusion region of 11.1 cM between markers D3S3637 and D3S1744. This is the first report of mapping of an adult-onset POAG gene to chromosome 3q, gene symbol GLC1C. 57 refs., 3 figs., 3 tabs.

  12. The human insulin gene is part of a large open chromatin domain specific for human islets.

    Science.gov (United States)

    Mutskov, Vesco; Felsenfeld, Gary

    2009-10-13

    Knowledge of how insulin (INS) gene expression is regulated will lead to better understanding of normal and abnormal pancreatic beta cell function. We have mapped histone modifications over the INS region, coupled with an expression profile, in freshly isolated islets from multiple human donors. Unlike many other human genes, in which active modifications tend to be concentrated within 1 kb around the transcription start site, these marks are distributed over the entire coding region of INS as well. Moreover, a region of approximately 80 kb around the INS gene, which contains the {tyrosine hydroxylase (TH)-(INS)-insulin-like growth factor 2 antisense (IGF2AS)-insulin-like growth factor 2 (IGF2)} gene cluster, unusually is marked by almost uniformly elevated levels of histone acetylation and H3K4 dimethylation, extending both downstream into IGF2 and upstream beyond the TH gene. This is accompanied by islet specific coordinate expression with INS of the neighboring TH and IGF2 genes. The presence of islet specific intergenic transcripts suggests their possible function in the maintenance of this unusual large open chromatin domain.

  13. Approaches to diagnose DNA mismatch repair gene defects in cancer

    DEFF Research Database (Denmark)

    Peña-Diaz, Javier; Rasmussen, Lene Juel

    2016-01-01

    The DNA repair pathway mismatch repair (MMR) is responsible for the recognition and correction of DNA biosynthetic errors caused by inaccurate nucleotide incorporation during replication. Faulty MMR leads to failure to address the mispairs or insertion deletion loops (IDLs) left behind...... already been well defined and their pathogenicity assessed. Despite this substantial wealth of knowledge, the effects of a large number of alterations remain uncharacterized (variants of uncertain significance, VUSs). The advent of personalized genomics is likely to increase the list of VUSs found in MMR...

  14. Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its histone recognition

    Science.gov (United States)

    Fang, Jian; Cheng, Jingdong; Wang, Jiaolong; Zhang, Qiao; Liu, Mengjie; Gong, Rui; Wang, Ping; Zhang, Xiaodan; Feng, Yangyang; Lan, Wenxian; Gong, Zhou; Tang, Chun; Wong, Jiemin; Yang, Huirong; Cao, Chunyang; Xu, Yanhui

    2016-04-01

    UHRF1 is an important epigenetic regulator for maintenance DNA methylation. UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. Hm-DNA impairs the intramolecular interactions and promotes H3K9me3 recognition by TTD-PHD. The Spacer also facilitates UHRF1-DNMT1 interaction and enhances hm-DNA-binding affinity of the SRA. When TTD-PHD binds to H3K9me3, SRA-Spacer may exist in a dynamic equilibrium: either recognizes hm-DNA or recruits DNMT1 to chromatin. Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by URHF1.

  15. Exercise-associated DNA methylation change in skeletal muscle and the importance of imprinted genes: a bioinformatics meta-analysis.

    Science.gov (United States)

    Brown, William M

    2015-12-01

    Epigenetics is the study of processes--beyond DNA sequence alteration--producing heritable characteristics. For example, DNA methylation modifies gene expression without altering the nucleotide sequence. A well-studied DNA methylation-based phenomenon is genomic imprinting (ie, genotype-independent parent-of-origin effects). We aimed to elucidate: (1) the effect of exercise on DNA methylation and (2) the role of imprinted genes in skeletal muscle gene networks (ie, gene group functional profiling analyses). Gene ontology (ie, gene product elucidation)/meta-analysis. 26 skeletal muscle and 86 imprinted genes were subjected to g:Profiler ontology analysis. Meta-analysis assessed exercise-associated DNA methylation change. g:Profiler found four muscle gene networks with imprinted loci. Meta-analysis identified 16 articles (387 genes/1580 individuals) associated with exercise. Age, method, sample size, sex and tissue variation could elevate effect size bias. Only skeletal muscle gene networks including imprinted genes were reported. Exercise-associated effect sizes were calculated by gene. Age, method, sample size, sex and tissue variation were moderators. Six imprinted loci (RB1, MEG3, UBE3A, PLAGL1, SGCE, INS) were important for muscle gene networks, while meta-analysis uncovered five exercise-associated imprinted loci (KCNQ1, MEG3, GRB10, L3MBTL1, PLAGL1). DNA methylation decreased with exercise (60% of loci). Exercise-associated DNA methylation change was stronger among older people (ie, age accounted for 30% of the variation). Among older people, genes exhibiting DNA methylation decreases were part of a microRNA-regulated gene network functioning to suppress cancer. Imprinted genes were identified in skeletal muscle gene networks and exercise-associated DNA methylation change. Exercise-associated DNA methylation modification could rewind the 'epigenetic clock' as we age. CRD42014009800. Published by the BMJ Publishing Group Limited. For permission to use (where

  16. How to open the treasure chest? Optimising DNA extraction from herbarium specimens.

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    Tiina Särkinen

    Full Text Available Herbarium collections are potentially an enormous resource for DNA studies, but the use of herbarium specimens in molecular studies has thus far been slowed down by difficulty in obtaining amplifiable DNA. Here we compare a set of commercially available DNA extraction protocols and their performance in terms of DNA purity and yield, and PCR amplification success as measured by using three differentially sized markers, the rbcL barcoding marker (cpDNA, the LEAFY exon 3 (nrDNA, and the trnL((UAA P6 loop (cpDNA. Results reveal large differences between extraction methods, where DNA purity rather than yield is shown to be strongly correlated with PCR success. Amplicon size shows similarly strong correlation with PCR success, with the shortest fragment showing the highest success rate (78%, P6 loop, 10-143 base pairs (bp and the largest fragment the lowest success (10%, rbcL, 670 bp. The effect of specimen preparation method on PCR success was also tested. Results show that drying method strongly affects PCR success, especially the availability of fragments longer than 250 bp, where longer fragments are more available for PCR amplification in air dried material compared to alcohol dried specimens. Results from our study indicate that projects relying on poor-quality starting material such as herbarium or scat samples should focus on extracting pure DNA and aim to amplify short target regions (<200-300 bp in order to maximise outcomes. Development of shorter barcoding regions, or mini-barcodes within existing ones should be of high importance as only a few options are currently available; this is particularly important if we hope to incorporate the millions of herbarium samples available into barcoding initiatives and other molecular studies.

  17. How to Open the Treasure Chest? Optimising DNA Extraction from Herbarium Specimens

    Science.gov (United States)

    Särkinen, Tiina; Staats, Martijn; Richardson, James E.; Cowan, Robyn S.; Bakker, Freek T.

    2012-01-01

    Herbarium collections are potentially an enormous resource for DNA studies, but the use of herbarium specimens in molecular studies has thus far been slowed down by difficulty in obtaining amplifiable DNA. Here we compare a set of commercially available DNA extraction protocols and their performance in terms of DNA purity and yield, and PCR amplification success as measured by using three differentially sized markers, the rbcL barcoding marker (cpDNA), the LEAFY exon 3 (nrDNA), and the trnL(UAA) P6 loop (cpDNA). Results reveal large differences between extraction methods, where DNA purity rather than yield is shown to be strongly correlated with PCR success. Amplicon size shows similarly strong correlation with PCR success, with the shortest fragment showing the highest success rate (78%, P6 loop, 10–143 base pairs (bp)) and the largest fragment the lowest success (10%, rbcL, 670 bp). The effect of specimen preparation method on PCR success was also tested. Results show that drying method strongly affects PCR success, especially the availability of fragments longer than 250 bp, where longer fragments are more available for PCR amplification in air dried material compared to alcohol dried specimens. Results from our study indicate that projects relying on poor-quality starting material such as herbarium or scat samples should focus on extracting pure DNA and aim to amplify short target regions (<200–300 bp) in order to maximise outcomes. Development of shorter barcoding regions, or mini-barcodes within existing ones should be of high importance as only a few options are currently available; this is particularly important if we hope to incorporate the millions of herbarium samples available into barcoding initiatives and other molecular studies. PMID:22952770

  18. Sequence and stress-response analyses of the DNA mismatch repair gene hexA in Lactococcus lactis.

    Science.gov (United States)

    Ren, J; Park, J H; Dunn, N W; Kim, W S

    2001-10-01

    The DNA mismatch repair gene hexA was identified in Lactococcus lactis by PCR amplification by using a pair of primers homologous to the DNA-binding Dps protein. The gene in its entirety, including the regulatory regions, was sequenced, by using a strategy of chromosomal walking based on two PCR protocols. The open reading frame of 2526 bp was preceded by a strong ribosome-binding site (AGGAAG) and was followed by a potential transcription terminator (hairpin loop structure). The 5' terminus of the hexA mRNA was located 135 bp upstream of the start codon, and putative -10 and -35 regions were identified. The deduced amino acid sequence revealed two motifs, the ATP/GTP-binding site (P-loop) and the "MutS family signature". The hexA promoter was cloned into pMU1327, which contained a promoter-less CAT reporter gene, and the promoter activity was examined under oxidative-stress conditions. It appears that the promoter activity is down-shifted by H2O2 at 4 mM.

  19. Significance of RNA reference in tumour-related gene expression analyses by cDNA array.

    Science.gov (United States)

    Laytragoon-Lewin, Nongnit; Lagerlund, Magnus; Lundgren, Jan; Nordlander, Britt; Elmberger, Göran; Södergren, Towe; Lagerros, Christofer; Rutqvist, Lars Erik; Lewin, Freddi

    2005-01-01

    The cDNA array technique is an efficient approach for studying the expression of a large number of genes in a single experiment. The cDNA array analysis indicates the relative level of corresponding gene expression from a specimen and a reference. Our investigation was performed to address the significance of reference RNA on the outcome of the cancer-related gene expression profile obtained from cDNA array analysis. Human head and neck squamous cell carcinoma (HNSCC) biopsies and 5 sources of RNA reference were used for this purpose. In these biopsies, each individual patient expressed a unique set of genes both in normal and tumour tissue. It is important to note that 5 striking patterns of tumour-related gene expression were obtained according to the 5 references used. Significant differences in 60%, 16%, 15% and 15% of the genes expressed were shown when autologous normal matched tissue biopsy references were compared to pooled cell lines, allogenic normal mixed cell types, tumours or allogenic normal matched cell type references, respectively. Thus, theoretically and our study suggested that patient autologous normal cells matching with the tumour type should be the most suitable reference in cDNA array for the identification of individual tumour gene profiles with clinical purpose.

  20. Genome-wide DNA methylation indicates silencing of tumor suppressor genes in uterine leiomyoma.

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    Antonia Navarro

    Full Text Available BACKGROUND: Uterine leiomyomas, or fibroids, represent the most common benign tumor of the female reproductive tract. Fibroids become symptomatic in 30% of all women and up to 70% of African American women of reproductive age. Epigenetic dysregulation of individual genes has been demonstrated in leiomyoma cells; however, the in vivo genome-wide distribution of such epigenetic abnormalities remains unknown. PRINCIPAL FINDINGS: We characterized and compared genome-wide DNA methylation and mRNA expression profiles in uterine leiomyoma and matched adjacent normal myometrial tissues from 18 African American women. We found 55 genes with differential promoter methylation and concominant differences in mRNA expression in uterine leiomyoma versus normal myometrium. Eighty percent of the identified genes showed an inverse relationship between DNA methylation status and mRNA expression in uterine leiomyoma tissues, and the majority of genes (62% displayed hypermethylation associated with gene silencing. We selected three genes, the known tumor suppressors KLF11, DLEC1, and KRT19 and verified promoter hypermethylation, mRNA repression and protein expression using bisulfite sequencing, real-time PCR and western blot. Incubation of primary leiomyoma smooth muscle cells with a DNA methyltransferase inhibitor restored KLF11, DLEC1 and KRT19 mRNA levels. CONCLUSIONS: These results suggest a possible functional role of promoter DNA methylation-mediated gene silencing in the pathogenesis of uterine leiomyoma in African American women.

  1. Intragenic vectors for gene transfer without foreign DNA

    NARCIS (Netherlands)

    Conner, A.J.; Barrell, P.J.; Baldwin, S.J.; Lokerse, A.S.; Cooper, P.A.; Erasmuson, A.K.; Nap, J.P.H.; Jacobs, J.M.E.

    2007-01-01

    The intragenic vector system involves identifying functional equivalents of vector components from the genome of a specific crop species (or related species to which it can be hybridised) and using these DNA sequences to assemble vectors for transformation of that plant species. This system offers a

  2. Electromobility of plasmid DNA in tumor tissues during electric field-mediated gene delivery.

    Science.gov (United States)

    Zaharoff, D A; Barr, R C; Li, C-Y; Yuan, F

    2002-10-01

    Interstitial transport is a crucial step in plasmid DNA-based gene therapy. However, interstitial diffusion of large nucleic acids is prohibitively slow. Therefore, we proposed to facilitate interstitial transport of DNA via pulsed electric fields. To test the feasibility of this approach to gene delivery, we developed an ex vivo technique to quantify the magnitude of DNA movement due to pulsed electric fields in two tumor tissues: B16.F10 (a mouse melanoma) and 4T1 (a mouse mammary carcinoma). When the pulse duration and strength were 50 ms and 233 V/cm, respectively, we found that the average plasmid DNA movements per 10 pulses were 1.47 microm and 0.35 microm in B16.F10 and 4T1 tumors, respectively. The average plasmid DNA movements could be approximately tripled, ie to reach 3.69 microm and 1.01 microm, respectively, when the pulse strength was increased to 465 V/cm. The plasmid DNA mobility was correlated with the tumor collagen content, which was approximately eight times greater in 4T1 than in B16.F10 tumors. These data suggest that electric field can be a powerful driving force for improving interstitial transport of DNA during gene delivery.

  3. Genetic variants of the DNA repair genes from Exome Aggregation Consortium (EXAC) database: significance in cancer.

    Science.gov (United States)

    Das, Raima; Ghosh, Sankar Kumar

    2017-04-01

    DNA repair pathway is a primary defense system that eliminates wide varieties of DNA damage. Any deficiencies in them are likely to cause the chromosomal instability that leads to cell malfunctioning and tumorigenesis. Genetic polymorphisms in DNA repair genes have demonstrated a significant association with cancer risk. Our study attempts to give a glimpse of the overall scenario of the germline polymorphisms in the DNA repair genes by taking into account of the Exome Aggregation Consortium (ExAC) database as well as the Human Gene Mutation Database (HGMD) for evaluating the disease link, particularly in cancer. It has been found that ExAC DNA repair dataset (which consists of 228 DNA repair genes) comprises 30.4% missense, 12.5% dbSNP reported and 3.2% ClinVar significant variants. 27% of all the missense variants has the deleterious SIFT score of 0.00 and 6% variants carrying the most damaging Polyphen-2 score of 1.00, thus affecting the protein structure and function. However, as per HGMD, only a fraction (1.2%) of ExAC DNA repair variants was found to be cancer-related, indicating remaining variants reported in both the databases to be further analyzed. This, in turn, may provide an increased spectrum of the reported cancer linked variants in the DNA repair genes present in ExAC database. Moreover, further in silico functional assay of the identified vital cancer-associated variants, which is essential to get their actual biological significance, may shed some lights in the field of targeted drug development in near future. Copyright © 2017. Published by Elsevier B.V.

  4. Encapsulation of plasmid DNA in calcium phosphate nanoparticles: stem cell uptake and gene transfer efficiency

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    Cao X

    2011-12-01

    Full Text Available Xia Cao*, Wenwen Deng*, Yuan Wei*, Weiyan Su, Yan Yang, Yawei Wei, Jiangnan Yu, Ximing XuDepartment of Pharmaceutics, School of Pharmacy, and Center for Nano Drug/Gene Delivery and Tissue Engineering, Jiangsu University, Jingkou District, Zhenjiang, People's Republic of China*These authors contributed equally to this workBackground: The purpose of this study was to develop calcium phosphate nanocomposite particles encapsulating plasmid DNA (CP-pDNA nanoparticles as a nonviral vector for gene delivery.Methods: CP-pDNA nanoparticles employing plasmid transforming growth factor beta 1 (TGF-β1 were prepared and characterized. The transfection efficiency and cell viability of the CP-pDNA nanoparticles were evaluated in mesenchymal stem cells, which were identified by immunofluorescence staining. Cytotoxicity of plasmid TGF-β1 and calcium phosphate to mesenchymal stem cells were evaluated by MTT assay.Results: The integrity of TGF-β1 encapsulated in the CP-pDNA nanoparticles was maintained. The well dispersed CP-pDNA nanoparticles exhibited an ultralow particle size (20–50 nm and significantly lower cytotoxicity than Lipofectamine™ 2000. Immunofluorescence staining revealed that the cultured cells in this study were probably mesenchymal stem cells. The cellular uptake and transfection efficiency of the CP-pDNA nanoparticles into the mesenchymal stem cells were higher than that of needle-like calcium phosphate nanoparticles and a standard calcium phosphate transfection kit. Furthermore, live cell imaging and confocal laser microscopy vividly showed the transportation process of the CP-pDNA nanoparticles in mesenchymal stem cells. The results of a cytotoxicity assay found that both plasmid TGF-β1 and calcium phosphate were not toxic to mesenchymal stem cells.Conclusion: CP-pDNA nanoparticles can be developed into an effective alternative as a nonviral gene delivery system that is highly efficient and has low cytotoxicity.Keywords: calcium

  5. The novel quinolone CHM-1 induces DNA damage and inhibits DNA repair gene expressions in a human osterogenic sarcoma cell line.

    Science.gov (United States)

    Chen, Hung-Yi; Lu, Hsu-Feng; Yang, Jai-Sing; Kuo, Sheng-Chu; Lo, Chyi; Yang, Mei-Due; Chiu, Tsan-Hung; Chueh, Fu-Shin; Ho, Heng-Chien; Ko, Yang-Ching; Chung, Jing-Gung

    2010-10-01

    20-Fluoro-6,7-methylenedioxy-2-phenyl-4-quino-lone (CHM-1) has been reported to induce cell cycle arrest and apoptosis in many types of cancer cells. However, there is no available information to show CHM-1 affecting DNA damage and expression of associated repair genes. Herein, we investigated whether or not CHM-1 induced DNA damage and affected DNA repair gene expression in U-2 OS human osterogenic sarcoma cells. The comet assay showed that incubation of U-2 OS cells with 0, 0.75, 1.5, 3 and 6 μM of CHM-1 led to a longer DNA migration smear (comet tail). DNA gel electrophoresis showed that 3 μM of CHM-1 for 24 and 48 h treatment induced DNA fragmentation in U-2 OS cells. Real-time PCR analysis showed that treatment with 3 μM of CHM-1 for 24 h reduced the mRNA expression levels of ataxia telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR), breast cancer 1, early onset (BRCA1), 14-3-3sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK) and O(6)-methylguanine-DNA methyltransferase (MGMT) genes in a time-dependent manner. Taken together, the results indicate that CHM-1 caused DNA damage and reduced DNA repair genes in U-2 OS cells, which may be the mechanism for CHM-1-inhibited cell growth and induction of apoptosis.

  6. A nuclear ribosomal DNA pseudogene in triatomines opens a new research field of fundamental and applied implications in Chagas disease

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    María Angeles Zuriaga

    2015-05-01

    Full Text Available A pseudogene, designated as "ps(5.8S+ITS-2", paralogous to the 5.8S gene and internal transcribed spacer (ITS-2 of the nuclear ribosomal DNA (rDNA, has been recently found in many triatomine species distributed throughout North America, Central America and northern South America. Among characteristics used as criteria for pseudogene verification, secondary structures and free energy are highlighted, showing a lower fit between minimum free energy, partition function and centroid structures, although in given cases the fit only appeared to be slightly lower. The unique characteristics of "ps(5.8S+ITS-2" as a processed or retrotransposed pseudogenic unit of the ghost type are reviewed, with emphasis on its potential functionality compared to the functionality of genes and spacers of the normal rDNA operon. Besides the technical problem of the risk for erroneous sequence results, the usefulness of "ps(5.8S+ITS-2" for specimen classification, phylogenetic analyses and systematic/taxonomic studies should be highlighted, based on consistence and retention index values, which in pseudogenic sequence trees were higher than in functional sequence trees. Additionally, intraindividual, interpopulational and interspecific differences in pseudogene amount and the fact that it is a pseudogene in the nuclear rDNA suggests a potential relationships with fitness, behaviour and adaptability of triatomine vectors and consequently its potential utility in Chagas disease epidemiology and control.

  7. Antibiotic resistance genes in the bacteriophage DNA fraction of environmental samples.

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    Marta Colomer-Lluch

    Full Text Available Antibiotic resistance is an increasing global problem resulting from the pressure of antibiotic usage, greater mobility of the population, and industrialization. Many antibiotic resistance genes are believed to have originated in microorganisms in the environment, and to have been transferred to other bacteria through mobile genetic elements. Among others, β-lactam antibiotics show clinical efficacy and low toxicity, and they are thus widely used as antimicrobials. Resistance to β-lactam antibiotics is conferred by β-lactamase genes and penicillin-binding proteins, which are chromosomal- or plasmid-encoded, although there is little information available on the contribution of other mobile genetic elements, such as phages. This study is focused on three genes that confer resistance to β-lactam antibiotics, namely two β-lactamase genes (blaTEM and blaCTX-M9 and one encoding a penicillin-binding protein (mecA in bacteriophage DNA isolated from environmental water samples. The three genes were quantified in the DNA isolated from bacteriophages collected from 30 urban sewage and river water samples, using quantitative PCR amplification. All three genes were detected in the DNA of phages from all the samples tested, in some cases reaching 104 gene copies (GC of blaTEM or 102 GC of blaCTX-M and mecA. These values are consistent with the amount of fecal pollution in the sample, except for mecA, which showed a higher number of copies in river water samples than in urban sewage. The bla genes from phage DNA were transferred by electroporation to sensitive host bacteria, which became resistant to ampicillin. blaTEM and blaCTX were detected in the DNA of the resistant clones after transfection. This study indicates that phages are reservoirs of resistance genes in the environment.

  8. Antibiotic resistance genes in the bacteriophage DNA fraction of environmental samples.

    Science.gov (United States)

    Colomer-Lluch, Marta; Jofre, Juan; Muniesa, Maite

    2011-03-03

    Antibiotic resistance is an increasing global problem resulting from the pressure of antibiotic usage, greater mobility of the population, and industrialization. Many antibiotic resistance genes are believed to have originated in microorganisms in the environment, and to have been transferred to other bacteria through mobile genetic elements. Among others, β-lactam antibiotics show clinical efficacy and low toxicity, and they are thus widely used as antimicrobials. Resistance to β-lactam antibiotics is conferred by β-lactamase genes and penicillin-binding proteins, which are chromosomal- or plasmid-encoded, although there is little information available on the contribution of other mobile genetic elements, such as phages. This study is focused on three genes that confer resistance to β-lactam antibiotics, namely two β-lactamase genes (blaTEM and blaCTX-M9) and one encoding a penicillin-binding protein (mecA) in bacteriophage DNA isolated from environmental water samples. The three genes were quantified in the DNA isolated from bacteriophages collected from 30 urban sewage and river water samples, using quantitative PCR amplification. All three genes were detected in the DNA of phages from all the samples tested, in some cases reaching 104 gene copies (GC) of blaTEM or 102 GC of blaCTX-M and mecA. These values are consistent with the amount of fecal pollution in the sample, except for mecA, which showed a higher number of copies in river water samples than in urban sewage. The bla genes from phage DNA were transferred by electroporation to sensitive host bacteria, which became resistant to ampicillin. blaTEM and blaCTX were detected in the DNA of the resistant clones after transfection. This study indicates that phages are reservoirs of resistance genes in the environment.

  9. Widespread Horizontal Gene Transfer from Circular Single-stranded DNA Viruses to Eukaryotic Genomes

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    Xie Jiatao

    2011-09-01

    Full Text Available Abstract Background In addition to vertical transmission, organisms can also acquire genes from other distantly related species or from their extra-chromosomal elements (plasmids and viruses via horizontal gene transfer (HGT. It has been suggested that phages represent substantial forces in prokaryotic evolution. In eukaryotes, retroviruses, which can integrate into host genome as an obligate step in their replication strategy, comprise approximately 8% of the human genome. Unlike retroviruses, few members of other virus families are known to transfer genes to host genomes. Results Here we performed a systematic search for sequences related to circular single-stranded DNA (ssDNA viruses in publicly available eukaryotic genome databases followed by comprehensive phylogenetic analysis. We conclude that the replication initiation protein (Rep-related sequences of geminiviruses, nanoviruses and circoviruses have been frequently transferred to a broad range of eukaryotic species, including plants, fungi, animals and protists. Some of the transferred viral genes were conserved and expressed, suggesting that these genes have been coopted to assume cellular functions in the host genomes. We also identified geminivirus-like and parvovirus-like transposable elements in genomes of fungi and lower animals, respectively, and thereby provide direct evidence that eukaryotic transposons could derive from ssDNA viruses. Conclusions Our discovery extends the host range of circular ssDNA viruses and sheds light on the origin and evolution of these viruses. It also suggests that ssDNA viruses act as an unforeseen source of genetic innovation in their hosts.

  10. Relationship between mutations of mitochondrial DNA ND1 gene and type 2 diabetes

    Institute of Scientific and Technical Information of China (English)

    于珮; 于德民; 刘德敏; 王琨; 汤新之

    2004-01-01

    Background Recent studies have indicated that many mutations in mitochondrial (mt)DNA NDI gene region are related to diabetes mellitus. In this study we explored the relationship between various mtDNA ND1 gene mutations and type 2 diabetes mellitus (DM) among Chinese. Methods Using PCR restriction fragment length polymorphism (PCR-RFLP) analysis and gene sequencing, 4 spots of mtDNA (nt3243, nt3316, nt3394, nt3426) were screened in 478 diabetics and 430 non-diabetic subjects.Results In diabetic group, there were 13 carriers (2.72%)of 3316 G→A mutation,12 (2.51%) of 3394 T→C mutation and 2 (0.42%) of 3426A→G mutation. In controls, only 3394 T→C mutation was observed in 2 subjects (0.47%). There was significant difference in the frequency of 3316 and 3394 mutation between two groups (P<0.05, respectively). More subjects with mitochondrial DNA ND1 gene mutations had DM family history and greater tendency of maternal inheritance when compared to those patients without mutation in diabetic group(P<0.01). A 3426 mutation diabetic pedigree was studied, and we found 12 maternal members in the family had the same mutation. Conclusion mtDNA ND1 gene mutations at nt3316 (G→A), nt3394 (T→C) and 3426 (A→G) might contribute to the pathogenesis of DM with other genetic factors and environment factors.

  11. Multiplex cDNA quantification method that facilitates the standardization of gene expression data

    Science.gov (United States)

    Gotoh, Osamu; Murakami, Yasufumi; Suyama, Akira

    2011-01-01

    Microarray-based gene expression measurement is one of the major methods for transcriptome analysis. However, current microarray data are substantially affected by microarray platforms and RNA references because of the microarray method can provide merely the relative amounts of gene expression levels. Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations. These requirements impose limitations on the extensive comparison of gene expression data. Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays. We have developed a multiplex cDNA quantification method called GEP-DEAN (Gene expression profiling by DCN-encoding-based analysis). The method was validated by using chemically synthesized DNA strands of known quantities and cDNA samples prepared from mouse liver, demonstrating that the absolute amounts of cDNA strands were successfully measured with a sensitivity of 18 zmol in a highly multiplexed manner in 7 h. PMID:21415008

  12. A Novel Self-Assembling DNA Nano Chip for Rapid Detection of Human Papillomavirus Genes

    Science.gov (United States)

    Li, Xin; Li, Yanbo; Hong, Li

    2016-01-01

    Rapid detection of tumor-associated DNA such as Human Papillomavirus (HPV) has important clinical value for the early screening of tumors. By attaching oligonucleotides or cDNA onto the chip surface, DNA chip technology provides a rapid method to analyze gene expression. However, challenges remain regarding increasing probe density and improving detection time. To address these challenges, we proposed a DNA chip that was self-assembled from single stranded DNA in combination with high probe density and a rapid detection method. Over 200 probes could be attached to the surface of this 100-nm diameter DNA chip. For detection, the chips were adsorbed onto a mica surface and then incubated for ten minutes with HPV-DNA; the results were directly observable using atomic force microscopy (AFM). This bottom-up fabricated DNA nano chip combined with high probe density and direct AFM detection at the single molecule level will likely have numerous potential clinical applications for gene screening and the early diagnosis of cancer. PMID:27706184

  13. A Novel Self-Assembling DNA Nano Chip for Rapid Detection of Human Papillomavirus Genes.

    Science.gov (United States)

    Li, Xin; Li, Yanbo; Hong, Li

    2016-01-01

    Rapid detection of tumor-associated DNA such as Human Papillomavirus (HPV) has important clinical value for the early screening of tumors. By attaching oligonucleotides or cDNA onto the chip surface, DNA chip technology provides a rapid method to analyze gene expression. However, challenges remain regarding increasing probe density and improving detection time. To address these challenges, we proposed a DNA chip that was self-assembled from single stranded DNA in combination with high probe density and a rapid detection method. Over 200 probes could be attached to the surface of this 100-nm diameter DNA chip. For detection, the chips were adsorbed onto a mica surface and then incubated for ten minutes with HPV-DNA; the results were directly observable using atomic force microscopy (AFM). This bottom-up fabricated DNA nano chip combined with high probe density and direct AFM detection at the single molecule level will likely have numerous potential clinical applications for gene screening and the early diagnosis of cancer.

  14. cDNA, genomic sequence cloning and overexpression of ribosomal protein S25 gene (RPS25) from the Giant Panda.

    Science.gov (United States)

    Hao, Yan-Zhe; Hou, Wan-Ru; Hou, Yi-Ling; Du, Yu-Jie; Zhang, Tian; Peng, Zheng-Song

    2009-11-01

    RPS25 is a component of the 40S small ribosomal subunit encoded by RPS25 gene, which is specific to eukaryotes. Studies in reference to RPS25 gene from animals were handful. The Giant Panda (Ailuropoda melanoleuca), known as a "living fossil", are increasingly concerned by the world community. Studies on RPS25 of the Giant Panda could provide scientific data for inquiring into the hereditary traits of the gene and formulating the protective strategy for the Giant Panda. The cDNA of the RPS25 cloned from Giant Panda is 436 bp in size, containing an open reading frame of 378 bp encoding 125 amino acids. The length of the genomic sequence is 1,992 bp, which was found to possess four exons and three introns. Alignment analysis indicated that the nucleotide sequence of the coding sequence shows a high homology to those of Homo sapiens, Bos taurus, Mus musculus and Rattus norvegicus as determined by Blast analysis, 92.6, 94.4, 89.2 and 91.5%, respectively. Primary structure analysis revealed that the molecular weight of the putative RPS25 protein is 13.7421 kDa with a theoretical pI 10.12. Topology prediction showed there is one N-glycosylation site, one cAMP and cGMP-dependent protein kinase phosphorylation site, two Protein kinase C phosphorylation sites and one Tyrosine kinase phosphorylation site in the RPS25 protein of the Giant Panda. The RPS25 gene was overexpressed in E. coli BL21 and Western Blotting of the RPS25 protein was also done. The results indicated that the RPS25 gene can be really expressed in E. coli and the RPS25 protein fusioned with the N-terminally his-tagged form gave rise to the accumulation of an expected 17.4 kDa polypeptide. The cDNA and the genomic sequence of RPS25 were cloned successfully for the first time from the Giant Panda using RT-PCR technology and Touchdown-PCR, respectively, which were both sequenced and analyzed preliminarily; then the cDNA of the RPS25 gene was overexpressed in E. coli BL21 and immunoblotted, which is the first

  15. Ternary nanoparticles composed of cationic solid lipid nanoparticles, protamine, and DNA for gene delivery

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    He SN

    2013-08-01

    Full Text Available Sai-Nan He,1 Yun-Long Li,1,2 Jing-Jing Yan,2 Wei Zhang,2 Yong-Zhong Du,2 He-Yong Yu,1 Fu-Qiang Hu,2 Hong Yuan21Women’s Hospital, 2College of Pharmaceutical Sciences, School of Medicine, Zhejiang University, Hangzhou, People’s Republic of ChinaBackground: The objective of this research was to design an effective gene delivery system composed of cationic solid lipid nanoparticles (SLNs, protamine, and Deoxyribonucleic acid DNA.Methods: Cationic SLNs were prepared using an aqueous solvent diffusion method with octadecylamine as the cationic lipid material. First, protamine was combined with DNA to form binary protamine/DNA nanoparticles, and the ternary nanoparticle gene delivery system was then obtained by combining binary protamine/DNA nanoparticles with cationic SLNs. The size, zeta potential, and ability of the binary and ternary nanoparticles to compact and protect DNA were characterized. The effect of octadecylamine content in SLNs and the SLNS/DNA ratios on transfection efficiency, cellular uptake and cytotoxicity of the ternary nanoparticles were also assessed using HEK293 cells.Results: When the weight ratio of protamine to DNA reached 1.5:1, the plasmid DNA could be effectively compacted and protected. The average hydrodynamic diameter of the ternary nanoparticles when combined with protamine increased from 188.50 ± 0.26 nm to 259.33 ± 3.44 nm, and the zeta potential increased from 25.50 ± 3.30 mV to 33.40 ± 2.80 mV when the weight ratio of SLNs to DNA increased from 16/3 to 80/3. The ternary nanoparticles showed high gene transfection efficiency compared with LipofectamineTM 2000/DNA nanoparticles. Several factors that might affect gene transfection efficiency, such as content and composition of SLNs, post-transfection time, and serum were examined. The ternary nanoparticles composed of SLNs with 15 wt% octadecylamine (50/3 weight ratio of SLNs to DNA showed the best transfection efficiency (26.13% ± 5.22% in the presence of

  16. Genomic survey and expression analysis of DNA repair genes in the genus Leptospira.

    Science.gov (United States)

    Martins-Pinheiro, Marinalva; Schons-Fonseca, Luciane; da Silva, Josefa B; Domingos, Renan H; Momo, Leonardo Hiroyuki Santos; Simões, Ana Carolina Quirino; Ho, Paulo Lee; da Costa, Renata M A

    2016-04-01

    Leptospirosis is an emerging zoonosis with important economic and public health consequences and is caused by pathogenic leptospires. The genus Leptospira belongs to the order Spirochaetales and comprises saprophytic (L. biflexa), pathogenic (L. interrogans) and host-dependent (L. borgpetersenii) members. Here, we present an in silico search for DNA repair pathways in Leptospira spp. The relevance of such DNA repair pathways was assessed through the identification of mRNA levels of some genes during infection in animal model and after exposition to spleen cells. The search was performed by comparison of available Leptospira spp. genomes in public databases with known DNA repair-related genes. Leptospires exhibit some distinct and unexpected characteristics, for instance the existence of a redundant mechanism for repairing a chemically diverse spectrum of alkylated nucleobases, a new mutS-like gene and a new shorter version of uvrD. Leptospira spp. shares some characteristics from Gram-positive, as the presence of PcrA, two RecQ paralogs and two SSB proteins; the latter is considered a feature shared by naturally competent bacteria. We did not find a significant reduction in the number of DNA repair-related genes in both pathogenic and host-dependent species. Pathogenic leptospires were enriched for genes dedicated to base excision repair and non-homologous end joining. Their evolutionary history reveals a remarkable importance of lateral gene transfer events for the evolution of the genus. Up-regulation of specific DNA repair genes, including components of SOS regulon, during infection in animal model validates the critical role of DNA repair mechanisms for the complex interplay between host/pathogen.

  17. Horizontal gene transfer regulation in bacteria as a "spandrel" of DNA repair mechanisms.

    Directory of Open Access Journals (Sweden)

    Saliou Fall

    Full Text Available Horizontal gene transfer (HGT is recognized as the major force for bacterial genome evolution. Yet, numerous questions remain about the transferred genes, their function, quantity and frequency. The extent to which genetic transformation by exogenous DNA has occurred over evolutionary time was initially addressed by an in silico approach using the complete genome sequence of the Ralstonia solanacearum GMI1000 strain. Methods based on phylogenetic reconstruction of prokaryote homologous genes families detected 151 genes (13.3% of foreign origin in the R. solanacearum genome and tentatively identified their bacterial origin. These putative transfers were analyzed in comparison to experimental transformation tests involving 18 different genomic DNA positions in the genome as sites for homologous or homeologous recombination. Significant transformation frequency differences were observed among these positions tested regardless of the overall genomic divergence of the R. solanacearum strains tested as recipients. The genomic positions containing the putative exogenous DNA were not systematically transformed at the highest frequencies. The two genomic "hot spots", which contain recA and mutS genes, exhibited transformation frequencies from 2 to more than 4 orders of magnitude higher than positions associated with other genes depending on the recipient strain. These results support the notion that the bacterial cell is equipped with active mechanisms to modulate acquisition of new DNA in different genomic positions. Bio-informatics study correlated recombination "hot-spots" to the presence of Chi-like signature sequences with which recombination might be preferentially initiated. The fundamental role of HGT is certainly not limited to the critical impact that the very rare foreign genes acquired mainly by chance can have on the bacterial adaptation potential. The frequency to which HGT with homologous and homeologous DNA happens in the environment

  18. Genome-wide age-related changes in DNA methylation and gene expression in human PBMCs.

    Science.gov (United States)

    Steegenga, Wilma T; Boekschoten, Mark V; Lute, Carolien; Hooiveld, Guido J; de Groot, Philip J; Morris, Tiffany J; Teschendorff, Andrew E; Butcher, Lee M; Beck, Stephan; Müller, Michael

    2014-06-01

    Aging is a progressive process that results in the accumulation of intra- and extracellular alterations that in turn contribute to a reduction in health. Age-related changes in DNA methylation have been reported before and may be responsible for aging-induced changes in gene expression, although a causal relationship has yet to be shown. Using genome-wide assays, we analyzed age-induced changes in DNA methylation and their effect on gene expression with and without transient induction with the synthetic transcription modulating agent WY14,643. To demonstrate feasibility of the approach, we isolated peripheral blood mononucleated cells (PBMCs) from five young and five old healthy male volunteers and cultured them with or without WY14,643. Infinium 450K BeadChip and Affymetrix Human Gene 1.1 ST expression array analysis revealed significant differential methylation of at least 5 % (ΔYO > 5 %) at 10,625 CpG sites between young and old subjects, but only a subset of the associated genes were also differentially expressed. Age-related differential methylation of previously reported epigenetic biomarkers of aging including ELOVL2, FHL2, PENK, and KLF14 was confirmed in our study, but these genes did not display an age-related change in gene expression in PBMCs. Bioinformatic analysis revealed that differentially methylated genes that lack an age-related expression change predominantly represent genes involved in carcinogenesis and developmental processes, and expression of most of these genes were silenced in PBMCs. No changes in DNA methylation were found in genes displaying transiently induced changes in gene expression. In conclusion, aging-induced differential methylation often targets developmental genes and occurs mostly without change in gene expression.

  19. Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes

    Directory of Open Access Journals (Sweden)

    Fu Juanjuan

    2011-07-01

    Full Text Available Abstract To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP or Gaussia luciferase (G.luc were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

  20. Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes.

    Science.gov (United States)

    Zhu, Wuyang; Li, Jiangjiao; Tang, Li; Wang, Huanqin; Li, Jia; Fu, Juanjuan; Liang, Guodong

    2011-07-10

    To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV) promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP) or Gaussia luciferase (G.luc) were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

  1. SiGN-SSM: open source parallel software for estimating gene networks with state space models.

    Science.gov (United States)

    Tamada, Yoshinori; Yamaguchi, Rui; Imoto, Seiya; Hirose, Osamu; Yoshida, Ryo; Nagasaki, Masao; Miyano, Satoru

    2011-04-15

    SiGN-SSM is an open-source gene network estimation software able to run in parallel on PCs and massively parallel supercomputers. The software estimates a state space model (SSM), that is a statistical dynamic model suitable for analyzing short time and/or replicated time series gene expression profiles. SiGN-SSM implements a novel parameter constraint effective to stabilize the estimated models. Also, by using a supercomputer, it is able to determine the gene network structure by a statistical permutation test in a practical time. SiGN-SSM is applicable not only to analyzing temporal regulatory dependencies between genes, but also to extracting the differentially regulated genes from time series expression profiles. SiGN-SSM is distributed under GNU Affero General Public Licence (GNU AGPL) version 3 and can be downloaded at http://sign.hgc.jp/signssm/. The pre-compiled binaries for some architectures are available in addition to the source code. The pre-installed binaries are also available on the Human Genome Center supercomputer system. The online manual and the supplementary information of SiGN-SSM is available on our web site. tamada@ims.u-tokyo.ac.jp.

  2. Identification of auxin responsive genes in Arabidopsis by cDNA array

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The plant hormone auxin influences a variety of developmental and physiological processes. But the mechanism of its action is quite unclear. In order to identify and analyze the expression of auxin responsive genes, a cDNA array approach was used to screen for genes with altered expression from Arabidopsis suspension culture after IAA treatment and was identified 50 differentially expressed genes from 13824 cDNA clones. These genes were related to signal transduction, stress responses, senescence, photosynthesis, protein biosynthesis and transportation. The results provide the molecular evidence that auxin influences a variety of physiological processes and pave a way for further investigation of the mechanism of auxin action. Furthermore,we found that the expression of a ClpC (regulation subunit of Clp protease) was repressed by exogenous auxin, but increased in dark-induced senescing leaves. This suggests that ClpC may be a senescence-associated gene and can be regulated by auxin.

  3. Comparative molecular dynamics studies of heterozygous open reading frames of DNA polymerase eta (η) in pathogenic yeast Candida albicans

    Science.gov (United States)

    Satpati, Suresh; Manohar, Kodavati; Acharya, Narottam; Dixit, Anshuman

    2017-01-01

    Genomic instability in Candida albicans is believed to play a crucial role in fungal pathogenesis. DNA polymerases contribute significantly to stability of any genome. Although Candida Genome database predicts presence of S. cerevisiae DNA polymerase orthologs; functional and structural characterizations of Candida DNA polymerases are still unexplored. DNA polymerase eta (Polη) is unique as it promotes efficient bypass of cyclobutane pyrimidine dimers. Interestingly, C. albicans is heterozygous in carrying two Polη genes and the nucleotide substitutions were found only in the ORFs. As allelic differences often result in functional differences of the encoded proteins, comparative analyses of structural models and molecular dynamic simulations were performed to characterize these orthologs of DNA Polη. Overall structures of both the ORFs remain conserved except subtle differences in the palm and PAD domains. The complementation analysis showed that both the ORFs equally suppressed UV sensitivity of yeast rad30 deletion strain. Our study has predicted two novel molecular interactions, a highly conserved molecular tetrad of salt bridges and a series of π-π interactions spanning from thumb to PAD. This study suggests these ORFs as the homologues of yeast Polη, and due to its heterogeneity in C. albicans they may play a significant role in pathogenicity.

  4. Identification of novel endogenous antisense transcripts by DNA microarray analysis targeting complementary strand of annotated genes

    Directory of Open Access Journals (Sweden)

    Kohama Chihiro

    2009-08-01

    Full Text Available Abstract Background Recent transcriptomic analyses in mammals have uncovered the widespread occurrence of endogenous antisense transcripts, termed natural antisense transcripts (NATs. NATs are transcribed from the opposite strand of the gene locus and are thought to control sense gene expression, but the mechanism of such regulation is as yet unknown. Although several thousand potential sense-antisense pairs have been identified in mammals, examples of functionally characterized NATs remain limited. To identify NAT candidates suitable for further functional analyses, we performed DNA microarray-based NAT screening using mouse adult normal tissues and mammary tumors to target not only the sense orientation but also the complementary strand of the annotated genes. Results First, we designed microarray probes to target the complementary strand of genes for which an antisense counterpart had been identified only in human public cDNA sources, but not in the mouse. We observed a prominent expression signal from 66.1% of 635 target genes, and 58 genes of these showed tissue-specific expression. Expression analyses of selected examples (Acaa1b and Aard confirmed their dynamic transcription in vivo. Although interspecies conservation of NAT expression was previously investigated by the presence of cDNA sources in both species, our results suggest that there are more examples of human-mouse conserved NATs that could not be identified by cDNA sources. We also designed probes to target the complementary strand of well-characterized genes, including oncogenes, and compared the expression of these genes between mammary cancerous tissues and non-pathological tissues. We found that antisense expression of 95 genes of 404 well-annotated genes was markedly altered in tumor tissue compared with that in normal tissue and that 19 of these genes also exhibited changes in sense gene expression. These results highlight the importance of NAT expression in the regulation

  5. Structural analysis of DNA sequence: evidence for lateral gene transfer in Thermotoga maritima

    DEFF Research Database (Denmark)

    Worning, Peder; Jensen, Lars Juhl; Nelson, K. E.

    2000-01-01

    The recently published complete DNA sequence of the bacterium Thermotoga maritima provides evidence, based on protein sequence conservation, for lateral gene transfer between Archaea and Bacteria. We introduce a new method of periodicity analysis of DNA sequences, based on structural parameters......, which brings independent evidence for the lateral gene transfer in the genome of T.maritima, The structural analysis relates the Archaea-like DNA sequences to the genome of Pyrococcus horikoshii. Analysis of 24 complete genomic DNA sequences shows different periodicity patterns for organisms...... of different origin, The typical genomic periodicity for Bacteria is 11 bp whilst it is 10 bp for Archaea, Eukaryotes have more complex spectra but the dominant period in the yeast Saccharomyces cerevisiae is 10.2 bp. These periodicities are most likely reflective of differences in chromatin structure....

  6. Anthracyclines induce double-strand DNA breaks at active gene promoters.

    Science.gov (United States)

    Yang, Fan; Kemp, Christopher J; Henikoff, Steven

    2015-03-01

    Doxorubicin is a widely used chemotherapeutic drug that intercalates between DNA base-pairs and poisons Topoisomerase II, although the mechanistic basis for cell killing remains speculative. Doxorubicin and related anthracycline compounds have been shown to increase nucleosome turnover and/or eviction around promoters, which suggests that the resulting enhanced exposure of DNA might underlie cell killing. Previously, we showed that low doses of anthracyclines increase nucleosome turnover around active gene promoters, which suggests that loss of nucleosomes might contribute to cancer cell killing. Here we apply a genome-wide method to precisely map DNA double-strand breaks (DSBs) in cancer cells. We find that spontaneous DSBs occur preferentially around promoters of active genes, and that both anthracyclines and etoposide, a Topoisomerase II poison, increase DSBs around promoters, although CpG islands are conspicuously protected from DSBs. We propose that torsion-based enhancement of nucleosome turnover by anthracyclines exposes promoter DNA, ultimately causing DSBs around promoters.

  7. DNA capture reveals transoceanic gene flow in endangered river sharks

    OpenAIRE

    Li, Chenhong; Corrigan, Shannon; Yang, Lei; Straube, Nicolas; Harris, Mark; Hofreiter, Michael; White, William T.; Naylor, Gavin J. P.

    2015-01-01

    The river sharks of the genus Glyphis, widely feared as man-eaters throughout India, remain very poorly known to science. The group constitutes five described species, all of which are considered highly endangered and restricted to freshwater systems in Australasia and Southeast Asia. DNA sequence data derived from 19th-century dried museum material augmented with contemporary samples indicates that only three of the five currently described species are valid; that there is a genetically dist...

  8. Structural Rearrangements in DNA Repair Genes in Breast Cancer

    Science.gov (United States)

    2013-10-01

    As this eliminates the mRNA polyA tail, we believed this would result in an unstable mRNA that is rapidly degraded, thus resulting in reduced mRNA...replaced by non-genic DNA. As this eliminates the mRNA polyA tail we believed this would result in an unstable mRNA that is rapidly degraded

  9. Improvement in efficacy of DNA vaccine encoding HIV-1 Vif by LIGHT gene adjuvant.

    Science.gov (United States)

    Du, Jiani; Wu, Xiaoyu; Long, Fengying; Wen, Jiejun; Hao, Wenli; Chen, Ran; Kong, Xiaobo; Qian, Min; Jiang, Wenzheng

    2013-02-01

    DNA vaccine can induce the prolonged immune responses against the encoded antigen with the appropriate adjuvant. To study the immunogenicity of the HIV-1 vif DNA vaccine in inducing the humoral and cellular immune responses and the immunoadjuvant effect of LIGHT, which is a member of TNF superfamily and can stimulate the proliferation of naïve T cells as a co-stimulatory molecule, DNA vaccine plasmid pcDNA-Vif was constructed by inserting HIV-1 vif gene into the downstream of CMV promoter in eukaryotic expression vector pcDNA3.1(+). In vitro expression of HIV-1 Vif in pcDNA-Vif-transfected HeLa cells was confirmed in transcriptional and protein level by RT-PCR and Western blot, respectively. After BALB/c mice were injected muscularly with DNA vaccines for three times, the specific immune responses were analyzed. The data showed that anti-Vif antibody response, Vif-specific T cell proliferation, and CTL activities were induced in the mice that were inoculated with HIV-1 vif DNA vaccine plasmid. Interestingly, stronger humoral and cellular immune responses were detected in mice that were immunized with plasmid pcDNA-Vif and pcDNA-LIGHT together compared to the single immunization with plasmid pcDNA-Vif alone. Together, the results of the study suggest that candidate HIV-1 DNA vaccine can elicit HIV-1 Vif-specific immune responses in mice and that LIGHT plays the role of immunoadjuvant in co-immunization with DNA vaccine.

  10. Production of DNA microarray and expression analysis of genes from Xylella fastidiosa in different culture media

    Directory of Open Access Journals (Sweden)

    Regiane de Fátima Travensolo

    2009-06-01

    Full Text Available DNA Microarray was developed to monitor the expression of many genes from Xylella fastidiosa, allowing the side by-side comparison of two situations in a single experiment. The experiments were performed using X. fastidiosa cells grown in two culture media: BCYE and XDM2. The primers were synthesized, spotted onto glass slides and the array was hybridized against fluorescently labeled cDNAs. The emitted signals were quantified, normalized and the data were statistically analyzed to verify the differentially expressed genes. According to the data, 104 genes were differentially expressed in XDM2 and 30 genes in BCYE media. The present study showed that DNA microarray technique efficiently differentiate the expressed genes under different conditions.DNA Microarray foi desenvolvida para monitorar a expressão de muitos genes de Xylella fastidiosa, permitindo a comparação de duas situações distintas em um único experimento. Os experimentos foram feitos utilizando células de X. fastidiosa cultivada em dois meios de cultura: BCYE e XDM2. Pares de oligonucleotídeos iniciadores foram sintetizados, depositados em lâminas de vidro e o arranjo foi hibridizado contra cDNAs marcados fluorescentemente. Os sinais emitidos foram quantificados, normalizados e os dados foram estatisticamente analisados para verificar os genes diferencialmente expressos. De acordo com nossos dados, 104 genes foram diferencialmente expressos para o meio de cultura XDM2 e 30 genes para o BCYE. No presente estudo, nós demonstramos que a técnica de DNA microarrays eficientemente diferencia genes expressos sob diferentes condições de cultivo.

  11. Tank-Binding Kinase 1 (TBK1) Gene and Open-Angle Glaucomas (An American Ophthalmological Society Thesis).

    Science.gov (United States)

    Fingert, John H; Robin, Alan L; Scheetz, Todd E; Kwon, Young H; Liebmann, Jeffrey M; Ritch, Robert; Alward, Wallace L M

    2016-08-01

    To investigate the role of TANK-binding kinase 1 (TBK1) gene copy-number variations (ie, gene duplications and triplications) in the pathophysiology of various open-angle glaucomas. In previous studies, we discovered that copy-number variations in the TBK1 gene are associated with normal-tension glaucoma. Here, we investigated the prevalence of copy-number variations in cohorts of patients with other open-angle glaucomas-juvenile-onset open-angle glaucoma (n=30), pigmentary glaucoma (n=209), exfoliation glaucoma (n=225), and steroid-induced glaucoma (n=79)-using a quantitative polymerase chain reaction assay. No TBK1 gene copy-number variations were detected in patients with juvenile-onset open-angle glaucoma, pigmentary glaucoma, or steroid-induced glaucoma. A TBK1 gene duplication was detected in one (0.44%) of the 225 exfoliation glaucoma patients. TBK1 gene copy-number variations (gene duplications and triplications) have been previously associated with normal-tension glaucoma. An exploration of other open-angle glaucomas detected a TBK1 copy-number variation in a patient with exfoliation glaucoma, which is the first example of a TBK1 mutation in a glaucoma patient with a diagnosis other than normal-tension glaucoma. A broader phenotypic range may be associated with TBK1 copy-number variations, although mutations in this gene are most often detected in patients with normal-tension glaucoma.

  12. SNP genotyping by DNA photoligation: application to SNP detection of genes from food crops

    Directory of Open Access Journals (Sweden)

    Yoshinaga Yoshimura, Tomoko Ohtake, Hajime Okada, Takehiro Ami, Tadashi Tsukaguchi and Kenzo Fujimoto

    2009-01-01

    Full Text Available We describe a simple and inexpensive single-nucleotide polymorphism (SNP typing method, using DNA photoligation with 5-carboxyvinyl-2'-deoxyuridine and two fluorophores. This SNP-typing method facilitates qualitative determination of genes from indica and japonica rice, and showed a high degree of single nucleotide specificity up to 10 000. This method can be used in the SNP typing of actual genomic DNA samples from food crops.

  13. SNP genotyping by DNA photoligation: application to SNP detection of genes from food crops

    Energy Technology Data Exchange (ETDEWEB)

    Yoshimura, Yoshinaga; Ohtake, Tomoko; Okada, Hajime; Fujimoto, Kenzo [School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292 (Japan); Ami, Takehiro [Innovation Plaza Ishikawa, Japan Science and Technology Agency, 2-13 Asahidai, Nomi, Ishikawa 923-1211 (Japan); Tsukaguchi, Tadashi, E-mail: kenzo@jaist.ac.j [Faculty of Bioresources and Environmental Sciences, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi, Ishikawa 921-8836 (Japan)

    2009-06-15

    We describe a simple and inexpensive single-nucleotide polymorphism (SNP) typing method, using DNA photoligation with 5-carboxyvinyl-2'-deoxyuridine and two fluorophores. This SNP-typing method facilitates qualitative determination of genes from indica and japonica rice, and showed a high degree of single nucleotide specificity up to 10 000. This method can be used in the SNP typing of actual genomic DNA samples from food crops.

  14. Multidrug Resistance 1 Gene Variants, Pesticide Exposure, and Increased Risk of DNA Damage

    OpenAIRE

    Chun-Chieh Chen; Chun-Huang Huang; Man-Tzu Marcie Wu; Chia-Hsuan Chou; Chia-Chen Huang; Tzu-Yen Tseng; Fang-Yu Chang; Ying-Ti Li; Chun-Cheng Tsai; Tsung-Shing Wang; Ruey-Hong Wong

    2014-01-01

    The P-glycoprotein, encoded by the multidrug resistance (MDR)1 gene, extrudes fat-soluble compounds to the extracellular environment. However, the DNA damage of pesticides in subjects with genetic variation in MDR1 has not been investigated. In this study, the comet assay was applied to examine the extent of DNA damage in the peripheral blood of 195 fruit growers who had been exposed to pesticides and 141 unexposed controls. The MDR1 polymorphisms were identified. Questionnaires were administ...

  15. ThioFinder: a web-based tool for the identification of thiopeptide gene clusters in DNA sequences.

    Directory of Open Access Journals (Sweden)

    Jing Li

    Full Text Available Thiopeptides are a growing class of sulfur-rich, highly modified heterocyclic peptides that are mainly active against Gram-positive bacteria including various drug-resistant pathogens. Recent studies also reveal that many thiopeptides inhibit the proliferation of human cancer cells, further expanding their application potentials for clinical use. Thiopeptide biosynthesis shares a common paradigm, featuring a ribosomally synthesized precursor peptide and conserved posttranslational modifications, to afford a characteristic core system, but differs in tailoring to furnish individual members. Identification of new thiopeptide gene clusters, by taking advantage of increasing information of DNA sequences from bacteria, may facilitate new thiopeptide discovery and enrichment of the unique biosynthetic elements to produce novel drug leads by applying the principle of combinatorial biosynthesis. In this study, we have developed a web-based tool ThioFinder to rapidly identify thiopeptide biosynthetic gene cluster from DNA sequence using a profile Hidden Markov Model approach. Fifty-four new putative thiopeptide biosynthetic gene clusters were found in the sequenced bacterial genomes of previously unknown producing microorganisms. ThioFinder is fully supported by an open-access database ThioBase, which contains the sufficient information of the 99 known thiopeptides regarding the chemical structure, biological activity, producing organism, and biosynthetic gene (cluster along with the associated genome if available. The ThioFinder website offers researchers a unique resource and great flexibility for sequence analysis of thiopeptide biosynthetic gene clusters. ThioFinder is freely available at http://db-mml.sjtu.edu.cn/ThioFinder/.

  16. Triple Negative Breast Cancers Have a Reduced Expression of DNA Repair Genes

    Science.gov (United States)

    Andreis, Daniele; Bertoni, Ramona; Giardini, Roberto; Fox, Stephen B.; Broggini, Massimo; Bottini, Alberto; Zanoni, Vanessa; Bazzola, Letizia; Foroni, Chiara; Generali, Daniele; Damia, Giovanna

    2013-01-01

    DNA repair is a key determinant in the cellular response to therapy and tumor repair status could play an important role in tailoring patient therapy. Our goal was to evaluate the mRNA of 13 genes involved in different DNA repair pathways (base excision, nucleotide excision, homologous recombination, and Fanconi anemia) in paraffin embedded samples of triple negative breast cancer (TNBC) compared to luminal A breast cancer (LABC). Most of the genes involved in nucleotide excision repair and Fanconi Anemia pathways, and CHK1 gene were significantly less expressed in TNBC than in LABC. PARP1 levels were higher in TNBC than in LABC. In univariate analysis high level of FANCA correlated with an increased overall survival and event free survival in TNBC; however multivariate analyses using Cox regression did not confirm FANCA as independent prognostic factor. These data support the evidence that TNBCs compared to LABCs harbour DNA repair defects. PMID:23825533

  17. DNA polymorphism at locus-2 of growth hormone gene of Madura cattle

    Directory of Open Access Journals (Sweden)

    NITA ETIKAWATI

    2003-01-01

    Full Text Available The objectives of the research were to detect DNA polymorphism at locus 2 of bovine growth hormone gene of Madura cattle and to know its genetic diversity. DNA polymorphisms and their effect on phenotypic traits have been studied widely in dairy cattle but not for beef cattle, especially for Indonesian local cattle. Polymorphism was detected using PCR-RFLP using primer GH-5 and GH-6 for amplifying locus 2 of growth hormone gene. Genetic diversity was analyzed based on the formula of Nei (1973, 1975. DNA polymorphism was found on locus 2 of growth hormone gene using MspI restriction enzyme. This polymorphism may be caused the lost of restriction MspI site. The genetic diversity was 0.4422.

  18. Profiling gene expression patterns of nasopharyngeal carcinoma and normal nasopharynx tissues with cDNA microarray

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    5 μg of total RNAs from normal nasopharynx and nasopharyngeal carcinoma tissue have been labeled with α-32P-dCTP during reverse transcription. The synthesized cDNA probes have been hybridized to high-density cDNA microarray containing 5184 genes or expression sequence tags (ESTs). Then image analysis software has been applied to comparing their expression profiles. Results show that 187 ESTs were of density value above 200 in nasopharyngeal carcinoma tissue while there were 307 such ESTs in normal nasopharynx tissue; 38 ESTs were strongly expressed in nasopharynx, but weakly expressed in nasopharyngeal carcinoma; 48 ESTs were strongly expressed in nasopharyngeal carcinoma, but weakly expressed in normal nasopharynx. These results suggest that there may exist some new differentially expressed genes involved in nasopharyngeal carcinoma development. Furthermore, the results strongly indicate that high-density cDNA microarray is a powerful and efficient tool for large-scale screening differentially expressed genes.

  19. Biological characterization of liver fatty acid binding gene from miniature pig liver cDNA library.

    Science.gov (United States)

    Gao, Y H; Wang, K F; Zhang, S; Fan, Y N; Guan, W J; Ma, Y H

    2015-01-01

    Liver fatty acid binding proteins (L-FABP) are a family of small, highly conserved, cytoplasmic proteins that bind to long-chain fatty acids and other hydrophobic ligands. In this study, a full-length enriched cDNA library was successfully constructed from Wuzhishan miniature pig, and then the L-FABP gene was cloned from this cDNA library and an expression vector (pEGFP-N3-L-FABP) was constructed in vitro. This vector was transfected into hepatocytes to test its function. The results of western blotting analysis demonstrated that the L-FABP gene from our full-length enriched cDNA library regulated downstream genes, including the peroxisome proliferator-activated receptor family in hepatocytes. This study provides a theoretical basis and experimental evidence for the application of L-FABP for the treatment of liver injury.

  20. Real-Time Imaging of Gene Delivery and Expression with DNA Nanoparticle Technologies

    Science.gov (United States)

    Sun, Wenchao; Ziady, Assem G.

    The construction of safe, efficient, and modifiable synthetic DNA nanoparticles is an emerging technology that has achieved important milestones of success in the past 5 years. Advances in chemical conjugation, purification, and controlled synthesis have allowed researchers to produce uniform and stable particles, whose physical characteristics can be well characterized and monitored. As a result of these improvements, DNA nanoparticles have now been cleared for clinical testing, and show good potential for human gene therapy. A very important recent development in the study of DNA nanoparticles is the use of small-animal imaging. Real-time imaging has become a valuable technique for tracking particle biodistribution and gene transfer efficacy. In this chapter, we discuss how bioluminescent, positron emission tomography, and magnetic resonance imaging can be used separately or in concert to study particle delivery, localization, and magnitude of gene expression in vivo.

  1. cDNA sequence, gene structure, and in vitro expression of ace-1, the gene encoding acetylcholinesterase of class A in the nematode Caenorhabditis elegans.

    Science.gov (United States)

    Arpagaus, M; Fedon, Y; Cousin, X; Chatonnet, A; Bergé, J B; Fournier, D; Toutant, J P

    1994-04-01

    Three genes, ace-1, ace-2, and ace-3, encode three acetylcholinesterase classes (A, B, and C) in the nematode Caenorhabditis elegans. A fragment of genomic DNA was amplified by a polymerase chain reaction (PCR) using degenerate oligonucleotides based on sequences conserved in the cholinesterase family. This fragment mapped to chromosome X at a position that perfectly matched the location of ace-1 previously determined by genetic methods. Comparison of genomic and cDNA sequences showed that the open reading frame was interrupted by eight introns. The product of ace-1 (ACE-1, 620 amino acids) presented 42% identity with Torpedo and human acetylcholinesterases, 41% with human butyrylcholinesterase, and 35% with Drosophila acetylcholinesterase. The overall structure of cholinesterases was conserved in ACE-1 as indicated by the conserved sequence positions of Ser-216, His-468, and Glu-346 (S200, H440, E327 in Torpedo (AChE) as components of the catalytic triad, of the six cysteines which form three intrachain disulfide bonds, and of Trp-99(84), a critical side chain in the choline binding site. Spodoptera Sf9 cells were infected by a recombinant baculovirus containing ace-1 cDNA. The secreted enzyme was active and existed as hydrophilic 5 and 11.5 S molecular forms. It hydrolyzed both acetylthiocholine and butyrylthiocholine and was inhibited by acetylthiocholine above 10 mM.

  2. Studying Closed Hydrodynamic Models of "In Vivo" DNA Perfusion in Pig Liver for Gene Therapy Translation to Humans

    National Research Council Canada - National Science Library

    Luis Sendra; Antonio Miguel; Daniel Pérez-Enguix; María José Herrero; Eva Montalvá; María Adelaida García-Gimeno; Inmaculada Noguera; Ana Díaz; Judith Pérez; Pascual Sanz; Rafael López-Andújar; Luis Martí-Bonmatí; Salvador F Aliño

    2016-01-01

    .... Tail vein DNA injection is an efficient procedure for gene transfer in murine liver. Hydrodynamic procedures in large animals require organ targeting, and improve with liver vascular exclusion...

  3. Corruption of the intra-gene DNA methylation architecture is a hallmark of cancer.

    Science.gov (United States)

    Bartlett, Thomas E; Zaikin, Alexey; Olhede, Sofia C; West, James; Teschendorff, Andrew E; Widschwendter, Martin

    2013-01-01

    Epigenetic processes--including DNA methylation--are increasingly seen as having a fundamental role in chronic diseases like cancer. It is well known that methylation levels at particular genes or loci differ between normal and diseased tissue. Here we investigate whether the intra-gene methylation architecture is corrupted in cancer and whether the variability of levels of methylation of individual CpGs within a defined gene is able to discriminate cancerous from normal tissue, and is associated with heterogeneous tumour phenotype, as defined by gene expression. We analysed 270985 CpGs annotated to 18272 genes, in 3284 cancerous and 681 normal samples, corresponding to 14 different cancer types. In doing so, we found novel differences in intra-gene methylation pattern across phenotypes, particularly in those genes which are crucial for stem cell biology; our measures of intra-gene methylation architecture are a better determinant of phenotype than measures based on mean methylation level alone (K-S test [Formula: see text] in all 14 diseases tested). These per-gene methylation measures also represent a considerable reduction in complexity, compared to conventional per-CpG beta-values. Our findings strongly support the view that intra-gene methylation architecture has great clinical potential for the development of DNA-based cancer biomarkers.

  4. Corruption of the Intra-Gene DNA Methylation Architecture Is a Hallmark of Cancer

    Science.gov (United States)

    Bartlett, Thomas E.; Zaikin, Alexey; Olhede, Sofia C.; West, James; Teschendorff, Andrew E.; Widschwendter, Martin

    2013-01-01

    Epigenetic processes - including DNA methylation - are increasingly seen as having a fundamental role in chronic diseases like cancer. It is well known that methylation levels at particular genes or loci differ between normal and diseased tissue. Here we investigate whether the intra-gene methylation architecture is corrupted in cancer and whether the variability of levels of methylation of individual CpGs within a defined gene is able to discriminate cancerous from normal tissue, and is associated with heterogeneous tumour phenotype, as defined by gene expression. We analysed 270985 CpGs annotated to 18272 genes, in 3284 cancerous and 681 normal samples, corresponding to 14 different cancer types. In doing so, we found novel differences in intra-gene methylation pattern across phenotypes, particularly in those genes which are crucial for stem cell biology; our measures of intra-gene methylation architecture are a better determinant of phenotype than measures based on mean methylation level alone (K-S test in all 14 diseases tested). These per-gene methylation measures also represent a considerable reduction in complexity, compared to conventional per-CpG beta-values. Our findings strongly support the view that intra-gene methylation architecture has great clinical potential for the development of DNA-based cancer biomarkers. PMID:23874574

  5. Unmasking risk loci: DNA methylation illuminates the biology of cancer predisposition: analyzing DNA methylation of transcriptional enhancers reveals missed regulatory links between cancer risk loci and genes.

    Science.gov (United States)

    Aran, Dvir; Hellman, Asaf

    2014-02-01

    Paradoxically, DNA sequence polymorphisms in cancer risk loci rarely correlate with the expression of cancer genes. Therefore, the molecular mechanism underlying an individual's susceptibility to cancer has remained largely unknown. However, recent evaluations of the correlations between DNA methylation and gene expression levels across healthy and cancerous genomes have revealed enrichment of disease-related DNA methylation variations within disease-associated risk loci. Moreover, it appears that transcriptional enhancers embedded in cancer risk loci often contain DNA methylation sites that closely define the expression of prominent cancer genes, despite the lack of significant correlations between gene expression levels and the surrounding disease-associated polymorphic sequences. We suggest that DNA methylation variations may obscure the effect of co-residing risk sequence alleles. Analysis of enhancer methylation data may help to reveal the regulatory circuits underlying predisposition to cancers and other common diseases.

  6. Racial Differences in DNA-Methylation of CpG Sites Within Preterm-Promoting Genes and Gene Variants.

    Science.gov (United States)

    Salihu, H M; Das, R; Morton, L; Huang, H; Paothong, A; Wilson, R E; Aliyu, M H; Salemi, J L; Marty, P J

    2016-08-01

    Objective To evaluate the role DNA methylation may play in genes associated with preterm birth for higher rates of preterm births in African-American women. Methods Fetal cord blood samples from births collected at delivery and maternal demographic and medical information were used in a cross-sectional study to examine fetal DNA methylation of genes implicated in preterm birth among black and non-black infants. Allele-specific DNA methylation analysis was performed using a methylation bead array. Targeted maximum likelihood estimation was applied to examine the relationship between race and fetal DNA methylation of candidate preterm birth genes. Receiver-operating characteristic analyses were then conducted to validate the CpG site methylation marker within the two racial groups. Bootstrapping, a method of validation and replication, was employed. Results 42 CpG sites were screened within 20 candidate gene variants reported consistently in the literature as being associated with preterm birth. Of these, three CpG sites on TNFAIP8 and PON1 genes (corresponding to: cg23917399; cg07086380; and cg07404485, respectively) were significantly differentially methylated between black and non-black individuals. The three CpG sites showed lower methylation status among infants of black women. Bootstrapping validated and replicated results. Conclusion for Practice Our study identified significant differences in levels of methylation on specific genes between black and non-black individuals. Understanding the genetic/epigenetic mechanisms that lead to preterm birth may lead to enhanced prevention strategies to reduce morbidity and mortality by eventually providing a means to identify individuals with a genetic predisposition to preterm labor.

  7. DNA replication factor C1 mediates genomic stability and transcriptional gene silencing in Arabidopsis

    KAUST Repository

    Liu, Qian

    2010-07-01

    Genetic screening identified a suppressor of ros1-1, a mutant of REPRESSOR OF SILENCING1 (ROS1; encoding a DNA demethylation protein). The suppressor is a mutation in the gene encoding the largest subunit of replication factor C (RFC1). This mutation of RFC1 reactivates the unlinked 35S-NPTII transgene, which is silenced in ros1 and also increases expression of the pericentromeric Athila retrotransposons named transcriptional silent information in a DNA methylationindependent manner. rfc1 is more sensitive than the wild type to the DNA-damaging agent methylmethane sulphonate and to the DNA inter- and intra- cross-linking agent cisplatin. The rfc1 mutant constitutively expresses the G2/M-specific cyclin CycB1;1 and other DNA repair-related genes. Treatment with DNA-damaging agents mimics the rfc1 mutation in releasing the silenced 35S-NPTII, suggesting that spontaneously induced genomic instability caused by the rfc1 mutation might partially contribute to the released transcriptional gene silencing (TGS). The frequency of somatic homologous recombination is significantly increased in the rfc1 mutant. Interestingly, ros1 mutants show increased telomere length, but rfc1 mutants show decreased telomere length and reduced expression of telomerase. Our results suggest that RFC1 helps mediate genomic stability and TGS in Arabidopsis thaliana. © 2010 American Society of Plant Biologists.

  8. Chromosomal Aberrations and DNA Repair Gene Variants in a Radon-exposed Population

    Energy Technology Data Exchange (ETDEWEB)

    Kiuru, A.; Lindholm, C.; Koivistoinen, A.; Salomaa, S.

    2004-07-01

    Polymorphisms of XRCC1 (X-ray repair cross-complementing group 1), XRCC3 (X-ray repair cross-complementing group 3), and hOGG1 (the human homologue of the yeast OGG1 gene) DNA repair genes have been associated with altered DNA repair capacity and risk of various cancers. In the present study our goal was to clarify the influence of various DNA repair gene variants on the frequency of chromosomal aberrations (CA) in subjects exposed to residential radon. The study group of 84 non-smoking, healthy individuals exposed to domestic radon were analysed using the fluorescence in-situ hybridization (FISH) technique. No association between radon concentration and CA frequencies was observed. However, a significant increase with age was shown as well as a large variability in translocation frequencies between individuals within the same age group. In order to investigate the role of individual susceptibility to this variation genotypes of DNA repair genes XRCC1 (codons 194, 280 and 399), XRCC3 (codon 241) and hOGG1 (codon 326) were determined from leukocyte DNA using methods based on polymerase chain reaction. Multiple regression analysis was applied to evaluate the effect of the polymorphisms and the other confounding factors (age, exposure to randon etc) to the frequency of CA. The preliminary statistical analyses showed that the different gene appeared not to be related to a pronounced increase in chromosome aberration frequencies observed by FISH painting. However, the analysis indicated that the homozygous variant of XRCC3 codon 241 was associated (P<0.05) with two-ways translocations in conjunction with age. Larger studies, both with regard to the cohort and the number of gene variants are needed to elucidate the influence of other DNA repair variants to the yield of chromosomal aberrations. The results indicate that the chromosomal translocations accumulated by age (spontaneous background) may be partly explained by defects in homologous recombination repair. (Author

  9. Gene-specific DNA methylation of DNMT3B and MTHFR and colorectal adenoma risk.

    Science.gov (United States)

    Ho, Vikki; Ashbury, Janet E; Taylor, Sherryl; Vanner, Stephen; King, Will D

    2015-12-01

    DNA methyltransferase 3B (DNMT3B) and methylenetetrahydrofolate reductase (MTHFR) are genes which encode enzymes critical to one-carbon metabolism. Polymorphisms in these genes have been implicated in colorectal cancer etiology; however, epigenetic modifications such as gene-specific DNA methylation also affect gene expression. DNA methylation of DNMT3B and MTHFR was quantified in blood leukocytes using Sequenom EpiTYPER® among 272 participants undergoing a screening colonoscopy. DNA methylation was quantified in 66 and 28CpG sites of DNMT3B and MTHFR respectively, and conceptualized using two approaches. First, measures representing average methylation across all CpG sites were created. Second, unsupervised principal component (PC) analysis was used to identify summary variables representing methylation around the transcription start site and in the gene-coding area for both DNMT3B and MTHFR. Logistic regression was used to compare methylation levels between participants diagnosed with colorectal adenoma(s) versus those with a normal colonoscopy via the estimation of odds ratios (ORs) and 95% confidence intervals (95% CIs) for the risk of colorectal adenomas. No association was observed between average DNA methylation of either DNMT3B or MTHFR and colorectal adenoma risk. For DNMT3B, increasing DNA methylation of CpG sites in the gene-coding area was associated with a higher risk of colorectal adenomas (OR=1.34; 95% CI: 1.01-1.79 per SD). This research provides preliminary evidence that methylation of DNMT3B may have functional significance with respect to colorectal adenomas, precursors to the vast majority of colorectal cancers.

  10. THE CONSTRUCTION AND PRELIMINARY APPRAISEMENT OF HSV-2gD GENE DNA VACCINE

    Institute of Scientific and Technical Information of China (English)

    王军阳; 范桂香; 胜利; 袁育康

    2002-01-01

    Objective To prevent infection from herpes simplex virus type 2(HSV-2),and make the foundation for the construction of multi-valent DNA vaccine. Methods The complete DNA sequence,which encoded the amino acid sequence of the viral glycoprotein D(gD),was obtained from the HSV-2 genome by polymerase chain reaction(PCR).The fragment was inserted into the lower stream of Cytomegalovirus(CMV) promoter in the eukaryotic expression plasmid pcDNA3.1(+), then immunized mice by bilateral intramuscular injection into the rear legs with this recombinant plasmid and tested the specific antibodies against glycoprotein D by ELISA. Results Animal experiment have demonstrated that the recombinant plasmid(pcDNA-gD2)inoculated into mice could induce the production of specific antibodies against glycoprotein D. Conclusion The eukaryotic plasmid pcDNA-gD2 constructed by us could correctly express gD gene and induce the production of specific antibodies.

  11. Conserved Organisation of 45S rDNA Sites and rDNA Gene Copy Number among Major Clades of Early Land Plants.

    Science.gov (United States)

    Rosato, Marcela; Kovařík, Aleš; Garilleti, Ricardo; Rosselló, Josep A

    2016-01-01

    Genes encoding ribosomal RNA (rDNA) are universal key constituents of eukaryotic genomes, and the nuclear genome harbours hundreds to several thousand copies of each species. Knowledge about the number of rDNA loci and gene copy number provides information for comparative studies of organismal and molecular evolution at various phylogenetic levels. With the exception of seed plants, the range of 45S rDNA locus (encoding 18S, 5.8S and 26S rRNA) and gene copy number variation within key evolutionary plant groups is largely unknown. This is especially true for the three earliest land plant lineages Marchantiophyta (liverworts), Bryophyta (mosses), and Anthocerotophyta (hornworts). In this work, we report the extent of rDNA variation in early land plants, assessing the number of 45S rDNA loci and gene copy number in 106 species and 25 species, respectively, of mosses, liverworts and hornworts. Unexpectedly, the results show a narrow range of ribosomal locus variation (one or two 45S rDNA loci) and gene copies not present in vascular plant lineages, where a wide spectrum is recorded. Mutation analysis of whole genomic reads showed higher (3-fold) intragenomic heterogeneity of Marchantia polymorpha (Marchantiophyta) rDNA compared to Physcomitrella patens (Bryophyta) and two angiosperms (Arabidopsis thaliana and Nicotiana tomentosifomis) suggesting the presence of rDNA pseudogenes in its genome. No association between phylogenetic position, taxonomic adscription and the number of rDNA loci and gene copy number was found. Our results suggest a likely evolutionary rDNA stasis during land colonisation and diversification across 480 myr of bryophyte evolution. We hypothesise that strong selection forces may be acting against ribosomal gene locus amplification. Despite showing a predominant haploid phase and infrequent meiosis, overall rDNA homogeneity is not severely compromised in bryophytes.

  12. Conserved Organisation of 45S rDNA Sites and rDNA Gene Copy Number among Major Clades of Early Land Plants

    Science.gov (United States)

    Rosato, Marcela; Kovařík, Aleš; Garilleti, Ricardo; Rosselló, Josep A.

    2016-01-01

    Genes encoding ribosomal RNA (rDNA) are universal key constituents of eukaryotic genomes, and the nuclear genome harbours hundreds to several thousand copies of each species. Knowledge about the number of rDNA loci and gene copy number provides information for comparative studies of organismal and molecular evolution at various phylogenetic levels. With the exception of seed plants, the range of 45S rDNA locus (encoding 18S, 5.8S and 26S rRNA) and gene copy number variation within key evolutionary plant groups is largely unknown. This is especially true for the three earliest land plant lineages Marchantiophyta (liverworts), Bryophyta (mosses), and Anthocerotophyta (hornworts). In this work, we report the extent of rDNA variation in early land plants, assessing the number of 45S rDNA loci and gene copy number in 106 species and 25 species, respectively, of mosses, liverworts and hornworts. Unexpectedly, the results show a narrow range of ribosomal locus variation (one or two 45S rDNA loci) and gene copies not present in vascular plant lineages, where a wide spectrum is recorded. Mutation analysis of whole genomic reads showed higher (3-fold) intragenomic heterogeneity of Marchantia polymorpha (Marchantiophyta) rDNA compared to Physcomitrella patens (Bryophyta) and two angiosperms (Arabidopsis thaliana and Nicotiana tomentosifomis) suggesting the presence of rDNA pseudogenes in its genome. No association between phylogenetic position, taxonomic adscription and the number of rDNA loci and gene copy number was found. Our results suggest a likely evolutionary rDNA stasis during land colonisation and diversification across 480 myr of bryophyte evolution. We hypothesise that strong selection forces may be acting against ribosomal gene locus amplification. Despite showing a predominant haploid phase and infrequent meiosis, overall rDNA homogeneity is not severely compromised in bryophytes. PMID:27622766

  13. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes.

    Science.gov (United States)

    Yang, G P; Ross, D T; Kuang, W W; Brown, P O; Weigel, R J

    1999-03-15

    Comparing patterns of gene expression in cell lines and tissues has important applications in a variety of biological systems. In this study we have examined whether the emerging technology of cDNA microarrays will allow a high throughput analysis of expression of cDNA clones generated by suppression subtractive hybridization (SSH). A set of cDNA clones including 332 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast cancer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysis confirmed over-expression of these 10 cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-transferase mu-3. Of the remaining six cDNA clones, four clones matched EST sequences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expressed in the ER-positive cell lines. We conclude that SSH and microarray technology can be successfully applied to identify differentially expressed genes. This approach allowed the identification of differentially expressed genes without the need to obtain previously cloned cDNAs.

  14. Structure-function investigations of DNA condensing agents with application to gene delivery

    Science.gov (United States)

    Evans, Heather Marie

    Lipid-based systems are notoriously poor for gene delivery, and their use has been primarily empirical. In order to improve these systems, it is imperative to obtain a greater understanding of molecular interactions between DNA and positively charged molecules. A variety of cationic molecules have been studied with DNA, in an attempt to correlate structural properties of these assemblies (using x-ray diffraction) with their efficiency as DNA carriers for gene delivery (using a luciferase assay). Several systems have been studied, some of which use the same charged amine moieties presented in three distinct morphologies: the multivalent salts spermine and spermidine, dendrimers, and dendrimeric lipids. The dendrimers somewhat approximate the properties of histories, cylindrical proteins that condense intracellular DNA. Structural studies of histone and DNA have also been conducted in order to better understand these interactions and their possible relevance to the gene delivery pathway. In addition, empirical evidence suggests that for successful in vivo gene delivery, cholesterol should be used as a helper lipid. The delivery efficiency and structural behavior of cholesterol and other sterol molecules have been studied in ternary lipid mixtures.

  15. Arsenic exposure is associated with DNA hypermethylation of the tumor suppressor gene p16.

    Science.gov (United States)

    Lu, Guangming; Xu, Huiwen; Chang, De; Wu, Zhenglai; Yao, Xiaoyuan; Zhang, Shiying; Li, Zhenlong; Bai, Jieben; Cai, Qing; Zhang, Wen

    2014-01-01

    Occupational and environmental exposure to inorganic arsenic leads to development of cancer and represents a significant health hazard in more than 70 countries. The underlying mechanism for arsenic-induced carcinogenesis remains unclear. Laboratory studies suggest that arsenic is a poor mutagen but may cause epigenetic silencing of key tumor suppressor genes such as p16 through DNA hypermethylation. However, the evidence for an association between human arsenic exposure and abnormal DNA methylation of tumor suppressor genes is lacking. Paired case-control studies were conducted involving 40 individuals with high arsenic exposure and arsenicosis, 40 individuals with similarly high exposure to arsenic but without arsenicosis, and 40 individuals with normal exposure to arsenic. DNA methylation status of p16 was determined using methylation-specific PCR. Conditional logistic regression analysis showed that DNA hypermethylation of p16 gene was significantly associated with high arsenic exposure (Odds Ratio = 10.0, P = 0.0019) independently of the development of arsenicosis (Odds Ratio = 2.0, P = 0.1343). High exposure of arsenic in human is positively linked to DNA hypermethylation of p16 gene, suggesting that epigenetic silencing of key tumor suppressor may be an important mechanism by which arsenic promotes cancer initiation.

  16. Polymorphisms in human DNA repair genes and head and neck squamous cell carcinoma

    Indian Academy of Sciences (India)

    Rim Khlifi; Ahmed Rebai; Amel Hamza-Chaffai

    2012-12-01

    Genetic polymorphisms in some DNA repair proteins are associated with a number of malignant transformations like head and neck squamous cell carcinoma (HNSCC). Xeroderma pigmentosum group D (XPD) and X-ray repair cross-complementing proteins 1 (XRCC1) and 3 (XRCC3) genes are involved in DNA repair and were found to be associated with HNSCC in numerous studies. To establish our overall understanding of possible relationships between DNA repair gene polymorphisms and development of HNSCC, we surveyed the literature on epidemiological studies that assessed potential associations with HNSCC risk in terms of gene–environment interactions, genotype-induced functional defects in enzyme activity and/or protein expression, and the influence of ethnic origin on these associations.We conclude that large, well-designed studies of common polymorphisms in DNA repair genes are needed. Such studies may benefit from analysis of multiple genes or polymorphisms and from the consideration of relevant exposures that may influence the likelihood of HNSCC when DNA repair capacity is reduced.

  17. Cloning of the PYR3 gene of Ustilago maydis and its use in DNA transformation

    Energy Technology Data Exchange (ETDEWEB)

    Banks, G.R.; Taylor, S.Y. (National Institute for Medical Research, London (England))

    1988-12-01

    The Ustilago maydis PYR3 gene encoding dihydroorotase activity was cloned by direct complementation of Escherichia coli pyrC mutations. PYR3 transformants of E. coli pyrC mutants expressed homologous transcripts of a variety of sizes and regained dihydroorotase activity. PYR3 also complemented Saccharomyces cerevisiae ura4 mutations, and again multiple transcripts were expressed in transformants, and enzyme activity was regained. A 1.25-kilobase poly(rA)+ PYR3 transcript was detected in U. maydis itself. Linear DNA carrying the PYR3 gene transformed a U. maydis pyr3-1 pyrimidine auxotroph to prototrophy. Hybridization analysis revealed that three different types of transformants could be generated, depending on the structure of the transforming DNA used. The first type involved exchange of chromosomal mutant gene sequences with the cloned wild-type plasmid sequences. A second type had integrated linear transforming DNA at the chromosomal PYR3 locus, probably via a single crossover event. The third type had integrated transforming DNA sequences at multiple sites in the U. maydis genome. In the last two types, tandemly reiterated copies of the transforming DNA were found to have been integrated. All three types had lost the sensitivity of the parental pyr3-1 mutant to UV irradiation. They had also regained dihydroorotase activity, although its level did not correlate with the PYR3 gene copy number.

  18. Frequency and character of alternative somatic recombination fates of paralogous genes during T-DNA integration.

    Science.gov (United States)

    Jelesko, John G; Carter, Kristy; Kinoshita, Yuki; Gruissem, Wilhelm

    2005-09-01

    A synthetic RBCSB gene cluster was transformed into Arabidopsis in order to simultaneously evaluate the frequency and character of somatic illegitimate recombination, homologous recombination, and targeted gene replacement events associated with T-DNA-mediated transformation. The most frequent type of recombination event observed was illegitimate integration of the T-DNA without activation of the silent DeltaRBCS1B: LUC transgene. Sixteen luc(+) (firefly luciferase positive) T1 plants were isolated. Six of these were due to illegitimate recombination events resulting in a gene trapping effect. Nine resulted from homologous recombination between paralogous RBCSB sequences associated with T-DNA integration. The frequency of somatic homologous recombination associated with T-DNA integration was almost 200 times higher than previously reported rates of meiotic homologous recombination with the same genes. The distribution of (somatic homologous) recombination resolution sites generally fits a fractional interval length model. However, a small region adjacent to an indel showed a significant over-representation of resolution sites, suggesting that DNA mismatch recognition may also play an important role in the positioning of somatic resolution sites. The frequency of somatic resolution within exon-2 was significantly different from that previously observed during meiotic recombination.

  19. DNMT3B gene amplification predicts resistance to DNA demethylating drugs.

    Science.gov (United States)

    Simó-Riudalbas, Laia; Melo, Sónia A; Esteller, Manel

    2011-07-01

    Disruption of the DNA methylation landscape is one of the most common features of human tumors. However, genetic alterations of DNA methyltransferases (DNMTs) have not been described in carcinogenesis. Herein, we show that pancreatic and breast cancer cells undergo gene amplification of the DNA methyltransferase 3B (DNMT3B). The presence of extra copies of the DNMT3B gene is linked to higher levels of the corresponding mRNA and protein. Most importantly, the elevated gene dosage of DNMT3B is associated with increased resistance to the growth-inhibitory effect mediated by DNA demethylating agents. In particular, cancer cells harboring DNMT3B gene amplification are less sensitive to the decrease in cell viability caused by 5-azacytidine (Vidaza), 5-aza-2-deoxycytidine (Decitabine), and SGI-1027. Overall, the data confirm DNMT3B as a bona fide oncogene in human cancer and support the incorporation of the DNMT3B copy number assay into current clinical trials assessing the efficacy of DNA demethylating drugs in solid tumors.

  20. Caffeine inhibits gene conversion by displacing Rad51 from ssDNA

    Science.gov (United States)

    Tsabar, Michael; Mason, Jennifer M.; Chan, Yuen-Ling; Bishop, Douglas K.; Haber, James E.

    2015-01-01

    Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1ATR/Tel1ATM-dependent DNA damage response or caffeine's inhibition of 5′ to 3′ resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments. PMID:26019181

  1. Preferential Nucleosome Assembly at DNA Triplet Repeats from the Myotonic Dystrophy Gene

    Science.gov (United States)

    Wang, Yuh-Hwa; Amirhaeri, Sorour; Kang, Seongman; Wells, Robert D.; Griffith, Jack D.

    1994-07-01

    The expansion of CTG repeats in DNA occurs in or near genes involved in several human diseases, including myotonic dystrophy and Huntington's disease. Nucleosomes, the basic structural element of chromosomes, consist of 146 base pairs of DNA coiled about an octamer of histone proteins and mediate general transcriptional repression. Electron microscopy was used to examine in vitro the nucleosome assembly of DNA containing repeating CTG triplets. The efficiency of nucleosome formation increased with expanded triplet blocks, suggesting that such blocks may repress transcription through the creation of stable nucleosomes.

  2. Spermatozoal cell death-inducing DNA fragmentation factor-α-like effector A (CIDEA) gene expression and DNA fragmentation in infertile men with metabolic syndrome and normal seminogram.

    Science.gov (United States)

    Elsamanoudy, Ayman Z; Abdalla, Hussein Abdelaziz; Hassanien, Mohammed; Gaballah, Mohammad A

    2016-01-01

    This is the first study to investigate spermatozoal cell death-inducing DNA fragmentation factor-α-like effector A (CIDEA) gene expression and DNA fragmentations in the spermatozoa of men diagnosed with metabolic syndrome (MS) who have normal seminograms with unexplained infertility, and to correlate these parameters with seminal glucose concentration. This study included 120 participants: 75 male subjects with MS (38 fertile and 37 infertile), and a control group of 45 fertile males without MS. HOMA-IR, semen analysis, and biochemical measurement of seminal plasma insulin and glucose levels were carried out. Spermatozoal insulin gene and CIDEA gene expressions were performed by the RT-PCR method. The percentage of spermatozoal DNA fragmentation was also estimated. The spermatozoal insulin and CIDEA gene expression, as well as the DNA fragmentation, were significantly higher in the infertile MS group than in the fertile MS group, and significantly higher in both the MS groups than in the control group. Seminal glucose concentration showed significant positive correlations with seminal insulin level, spermatozoa insulin, CIDEA gene expression, and DNA fragmentation. Moreover, there was a positive correlation between spermatozoa CIDEA gene expression and DNA fragmentation. It can be concluded that MS may affect male fertility at the molecular level, through its possible inducing effect of spermatozoa CIDEA and insulin gene expression, DNA fragmentation, and increased seminal glucose.

  3. Construction of cDNA representational difference analysis based on two cDNA libraries and identification of garlic inducible expression genes in human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yong Li; Lin Yang; Jian-Tao Cui; Wen-Mei Li; Rui-Fang Guo; You-Yong Lu

    2002-01-01

    AIM: To elucidate molecular mechanism of chemopreventiveefficacies of garlic against human gastric cancer (HGC):METHODS: HGC cell line BGC823 was treated with Allitridi (akind of garlic extract) and Allitridi-treated and parentalBGC823 cDNA librarles were constructed respectively byusing λZAP Ⅱ vector. cDNA Representatinal DifferenceAnalysis (cDNA RDA) was perfonmed using BamH Ⅰ cutting-site and abundant ~DNA messages provided by the Iibrarles.Northern blot analysls was applied to identifythe obtaineddifference prnducts.RESULTS: Two specific cDNA fragments were obtained andcharacterized to be derived from homo sapiens folatereceptorα (FRα) gene and calcyclin gene respectively.Northern blot results showed a 4-fold increase in FRα geneexpression level and 9-fold increase in calcyclin mRNA levelin BGC823 cells after Allilridi treatment for 72 h.CONCLUSION: The method of cDNA RDA based on cDNAlibraries combines the high specificity of cDNA RDA withabundant cDNA messages in cDNA library; this expands theapplication of cDNA library and increases the specificity ofcDNA RDA. Up-regulstion of FRα gene and calcyclin geneexpressions induced by Allitridi provide valuable molecularevidence for theefficacy of garlic in treating HGC as well asother diseases.

  4. Identification of Mutations in Myocilin and Beta-1,4-galactosyltransferase 3 Genes in a Chinese Family with Primary Open-angle Glaucoma.

    Science.gov (United States)

    Liao, Rong-Feng; Zhong, Zi-Lin; Ye, Min-Jie; Han, Li-Yun; Ye, Dong-Qing; Chen, Jian-Jun

    2016-12-05

    Glaucoma is a major cause of irreversible blindness worldwide. There is evidence showing that a subset of the disease is genetically determined. In this study, we screened for mutations in chromosome 1q-linked open-angle glaucoma (GLC1A) in a Chinese family with primary open-angle glaucoma (POAG). A total of 23 members from five generations of a family were enrolled and underwent thorough ophthalmologic examinations. In addition, 200 unrelated healthy Chinese controls were also recruited as normal control. GLC1A gene was amplified by polymerase chain reaction, and DNA sequencing was performed to screen for mutations. Six members were diagnosed as POAG, with severe clinical manifestations, and history of high intraocular pressures. The mean age of disease onset was 26.3 years. However, the others were asymptomatic. In six affected and three asymptomatic members, gene sequencing revealed a mutation c.C1456T in exon 3 of myocilin gene (MYOC). Furthermore, we also identified a novel mutation c.G322A in beta-1,4-galactosyltransferase 3 (B4GALT3) gene in all six affected and three asymptomatic members, which was not reported previously in POAG patients. The two newly identified variants were absent in other family members as well as controls. The mutations c.1456C family.

  5. New conjugated polymers for photoinduced unwinding of DNA supercoiling and gene regulation.

    Science.gov (United States)

    Yang, Gaomai; Yuan, Huanxiang; Zhu, Chunlei; Liu, Libing; Yang, Qiong; Lv, Fengting; Wang, Shu

    2012-05-01

    Three cationic polythiophene derivatives (P1, P2, P3) were synthesized and characterized. Under white light irradiation (400-800 nm), they sensitize oxygen molecule in the surrounding to generate reactive oxygen species (ROS) that can efficiently unwind the supercoiled DNA in vitro. Further study shows that this relaxation of the DNA supercoiling results in the decrease of gene (pCX-EGFP plasmid) expression level. The ability of these conjugated polymers for regulating gene expression will add a new dimension to the function of conjugated polymers.

  6. Exercise training alters DNA methylation patterns in genes related to muscle growth and differentiation in mice.

    Science.gov (United States)

    Kanzleiter, Timo; Jähnert, Markus; Schulze, Gunnar; Selbig, Joachim; Hallahan, Nicole; Schwenk, Robert Wolfgang; Schürmann, Annette

    2015-05-15

    The adaptive response of skeletal muscle to exercise training is tightly controlled and therefore requires transcriptional regulation. DNA methylation is an epigenetic mechanism known to modulate gene expression, but its contribution to exercise-induced adaptations in skeletal muscle is not well studied. Here, we describe a genome-wide analysis of DNA methylation in muscle of trained mice (n = 3). Compared with sedentary controls, 2,762 genes exhibited differentially methylated CpGs (P 5%, coverage >10) in their putative promoter regions. Alignment with gene expression data (n = 6) revealed 200 genes with a negative correlation between methylation and expression changes in response to exercise training. The majority of these genes were related to muscle growth and differentiation, and a minor fraction involved in metabolic regulation. Among the candidates were genes that regulate the expression of myogenic regulatory factors (Plexin A2) as well as genes that participate in muscle hypertrophy (Igfbp4) and motor neuron innervation (Dok7). Interestingly, a transcription factor binding site enrichment study discovered significantly enriched occurrence of CpG methylation in the binding sites of the myogenic regulatory factors MyoD and myogenin. These findings suggest that DNA methylation is involved in the regulation of muscle adaptation to regular exercise training.

  7. Hepatocyte gene transfer mediated by stable polyplexes based on MPP-containing DNA complexes

    Institute of Scientific and Technical Information of China (English)

    Bao-Feng Yu; Wan-I Li; Xiao-Nian Hu; Yue-Hong Zhang; Bo Niu; Jun Xie

    2009-01-01

    BACKGROUND: In the field of gene therapy, viral vectors as delivery tools have a number of disadvantages for medical application. This study aimed to explore a novel nonviral vector as a vehicle for gene therapy. METHODS: Transvector-rpE-MPP and EGFP (enhanced green fluorescent protein) were used as the gene transfer carrier and the reporter gene, respectively. Polyplexes which integrate transvector-rpE-MPP, the object gene, and EGFP were formed. The optimal charge ratio, stability, and transduction capacity of the polyplexes in mouse hepatocytes in vitro and in mouse liver in vivo were investigated. The polyplexes of transvector-rpE-MPP and pcDNA3-EGFP, with charge ratios of 0, 0.25, 0.5, 0.75, 1 and 1.5 were compared to determine the optimal charge ratio. RESULTS:  Polyplexes with charge ratios of 1∶1 were most stable; pcDNA3-EGFP in these complexes resisted digestion by DNase Ⅰ and blood plasma. On the other hand, pcDNA3-EGFP alone was digested. Fluorescence analysis indicated that transvector-rpE-MPP successfully delivered the reporter gene EGFP into hepatocytes and that EGFP expression was detected in hepatocyte cultures and in liver tissue. CONCLUSION: These results have laid a foundation for further study of a novel nonviral gene delivery system.

  8. Nucleotide sequence of the hexA gene for DNA mismatch repair in Streptococcus pneumoniae and homology of hexA to mutS of Escherichia coli and Salmonella typhimurium.

    OpenAIRE

    Priebe, S D; Hadi, S M; Greenberg, B; Lacks, S A

    1988-01-01

    The Hex system of heteroduplex DNA base mismatch repair operates in Streptococcus pneumoniae after transformation and replication to correct donor and nascent DNA strands, respectively. A functionally similar system, called Mut, operates in Escherichia coli and Salmonella typhimurium. The nucleotide sequence of a 3.8-kilobase segment from the S. pneumoniae chromosome that includes the 2.7-kilobase hexA gene was determined. An open reading frame that could encode a 17-kilodalton polypeptide (O...

  9. DNA methylation patterns of protein coding genes and long noncoding RNAs in female schizophrenic patients.

    Science.gov (United States)

    Liao, Qi; Wang, Yunliang; Cheng, Jia; Dai, Dongjun; Zhou, Xingyu; Zhang, Yuzheng; Gao, Shugui; Duan, Shiwei

    2015-02-01

    Schizophrenia (SCZ) is a complex mental disorder contributed by both genetic and epigenetic factors. Long noncoding RNAs (lncRNAs) was recently found playing an important regulatory role in mental disorders. However, little was known about the DNA methylation of lncRNAs, although numerous SCZ studies have been performed on genetic polymorphisms or epigenetic marks in protein coding genes. We presented a comprehensive genome wide DNA methylation study of both protein coding genes and lncRNAs in female patients with paranoid and undifferentiated SCZ. Using the methyl-CpG binding domain (MBD) protein-enriched genome sequencing (MBD-seq), 8,163 and 764 peaks were identified in paranoid and undifferentiated SCZ, respectively (p Gene ontology analysis showed that the hypermethylated regions were enriched in the genes related to neuron system and brain for both paranoid and undifferentiated SCZ (p gene promoter regions that might affect gene expression and influence the SCZ related pathways. Interestingly, DNA methylation of 136 and 23 known lncRNAs in Refseq database were identified in paranoid and undifferentiated SCZ, respectively. In addition, ∼20% of intergenic peaks annotated based on Refseq genes were overlapped with lncRNAs in UCSC and gencode databases. In order to show the results well for most biological researchers, we created an online database to display and visualize the information of DNA methyation peaks in both types of SCZ (http://www.bioinfo.org/scz/scz.htm). Our results showed that the aberrant DNA methylation of lncRNAs might be another important epigenetic factor for SCZ.

  10. DNA-repair gene variants are associated with glioblastoma survival

    DEFF Research Database (Denmark)

    Wibom, Carl; Sjöström, Sara; Henriksson, Roger

    2012-01-01

    genes, in 138 glioblastoma samples from Sweden and Denmark. We confirmed our findings in an independent cohort of 121 glioblastoma patients from the UK. Our analysis revealed nine SNPs annotating MSH2, RAD51L1 and RECQL4 that were significantly (p

  11. Biased distribution of DNA uptake sequences towards genome maintenance genes

    DEFF Research Database (Denmark)

    Davidsen, T.; Rodland, E.A.; Lagesen, K.

    2004-01-01

    in these organisms. Pasteurella multocida also displayed high frequencies of a putative DUS identical to that previously identified in H. influenzae and with a skewed distribution towards genome maintenance genes, indicating that this bacterium might be transformation competent under certain conditions....

  12. Quantitative DNA methylation analysis of selected genes in endometrial carcinogenesis

    Directory of Open Access Journals (Sweden)

    Ying-Chieh Chen

    2015-10-01

    Conclusion: Promoter methylation of ZNF177, COL14A1, HOXA9, DPYSL4, and TMEFF2 genes is a frequent epigenetic event in EC. Furthermore, the epigenetic hypermethylation of TMEFF2 may be a valuable marker for identifying undetected EC within endometrial hyperplasia.

  13. Plasmid containing a DNA ligase gene from Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.; Griffin, K.; Setlow, J.K.

    1984-05-01

    A ligase gene from Haemophilus influenzae was cloned into the shuttle vector pDM2. Although the plasmid did not affect X-ray sensitivity, it caused an increase in UV sensitivity of the wild-type but not excision-defective H. influenzae and a decrease in UV sensitivity of the rec-1 mutant. 14 references, 2 figures.

  14. Flatworms have lost the right open reading frame kinase 3 gene during evolution.

    Science.gov (United States)

    Breugelmans, Bert; Ansell, Brendan R E; Young, Neil D; Amani, Parisa; Stroehlein, Andreas J; Sternberg, Paul W; Jex, Aaron R; Boag, Peter R; Hofmann, Andreas; Gasser, Robin B

    2015-05-15

    All multicellular organisms studied to date have three right open reading frame kinase genes (designated riok-1, riok-2 and riok-3). Current evidence indicates that riok-1 and riok-2 have essential roles in ribosome biosynthesis, and that the riok-3 gene assists this process. In the present study, we conducted a detailed bioinformatic analysis of the riok gene family in 25 parasitic flatworms (platyhelminths) for which extensive genomic and transcriptomic data sets are available. We found that none of the flatworms studied have a riok-3 gene, which is unprecedented for multicellular organisms. We propose that, unlike in other eukaryotes, the loss of RIOK-3 from flatworms does not result in an evolutionary disadvantage due to the unique biology and physiology of this phylum. We show that the loss of RIOK-3 coincides with a loss of particular proteins associated with essential cellular pathways linked to cell growth and apoptosis. These findings indicate multiple, key regulatory functions of RIOK-3 in other metazoan species. Taking advantage of a known partial crystal structure of human RIOK-1, molecular modelling revealed variability in nucleotide binding sites between flatworm and human RIOK proteins.

  15. Cloning of Thermostable DNA Polymerase Gene from a Thermophilic Brevibacillus sp. Isolated from Sikidang Crater, Dieng Plateu, Central Java

    Directory of Open Access Journals (Sweden)

    Lucia Dhiantika Witasari

    2015-11-01

    Full Text Available Thermostable DNA polymerase has an important role for amplifying small amount of DNA through polymerase chain reaction (PCR. Thermophillic bacteria Brevibacillus sp. was isolated from Sikidang Crater, Dieng Plateu, Central Java. Previous study showed that crude protein of the isolate could be used in PCR. Unfortunately, like most native thermostable enzymes, the thermostable DNA polymerase of the isolate is synthesized in a very low level and therefore is cumbersome to purify. The purpose of this research is to clone thermostable DNA polymerase gene of the isolate. The DNA polymerase gene was amplified by means of PCR using spesific primers. The amplified fragment was then isolated, purified, and ligated into the pGEM-T cloning vector. The recombinant plasmid was then transformed to competent E. coli JM109 cells using heat shock method. The cloned thermostable DNA polymerase gene from the thermophilic isolate was then characterized for its nucleotide base sequence. The result showed that the DNA Pol I gene was successfully be amplified from the isolate DNA genom, resulting in ± 2,7 kb DNA fragment in length. Sequence analysis of segment of targeted gene showed high similarity to that of thermostable DNA polymerase genes from other Bacillus.Key words : Thermostable DNA Pol I, Brevibacillus sp., PCR, cloning

  16. Targeting CTCF to Control Virus Gene Expression: A Common Theme amongst Diverse DNA Viruses

    Directory of Open Access Journals (Sweden)

    Ieisha Pentland

    2015-07-01

    Full Text Available All viruses target host cell factors for successful life cycle completion. Transcriptional control of DNA viruses by host cell factors is important in the temporal and spatial regulation of virus gene expression. Many of these factors are recruited to enhance virus gene expression and thereby increase virus production, but host cell factors can also restrict virus gene expression and productivity of infection. CCCTC binding factor (CTCF is a host cell DNA binding protein important for the regulation of genomic chromatin boundaries, transcriptional control and enhancer element usage. CTCF also functions in RNA polymerase II regulation and in doing so can influence co-transcriptional splicing events. Several DNA viruses, including Kaposi’s sarcoma-associated herpesvirus (KSHV, Epstein-Barr virus (EBV and human papillomavirus (HPV utilize CTCF to control virus gene expression and many studies have highlighted a role for CTCF in the persistence of these diverse oncogenic viruses. CTCF can both enhance and repress virus gene expression and in some cases CTCF increases the complexity of alternatively spliced transcripts. This review article will discuss the function of CTCF in the life cycle of DNA viruses in the context of known host cell CTCF functions.

  17. DNA repair gene polymorphisms in relation to chromosome aberration frequencies in retired radiation workers

    Energy Technology Data Exchange (ETDEWEB)

    Wilding, Craig S. [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom)]. E-mail: craig.wilding@westlakes.ac.uk; Relton, Caroline L. [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom); Paediatric and Lifecourse Epidemiology Research Group, School of Clinical Medical Sciences (Child Health), Newcastle University, Sir James Spence Institute, Royal Victoria Infirmary, Newcastle-upon-Tyne NE1 4LP (United Kingdom); Rees, Gwen S. [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom); Tarone, Robert E. [International Epidemiology Institute, 1455 Research Boulevard, Suite 550, Rockville, MD 20850 (United States); Whitehouse, Caroline A. [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom); Tawn, E. Janet [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom)

    2005-02-15

    Polymorphic variation in DNA repair genes was examined in a group of retired workers from the British Nuclear Fuels plc facility at Sellafield in relation to previously determined translocation frequencies in peripheral blood lymphocytes. Variation at seven polymorphisms in four genes involved in the base excision repair (XRCC1 R194W, R399Q and a [AC]{sub n} microsatellite in the 3' UTR) and double strand break repair (XRCC3 T241M and a [AC]{sub n} microsatellite in intron 3 of XRCC3, XRCC4 I134T, and a GACTAn microsatellite located 120kb 5' of XRCC5) pathways was determined for 291 retired radiation workers who had received cumulative occupational external radiation doses of between 0 and 1873mSv. When the interaction between radiation dose and each DNA repair gene polymorphism was examined in relation to translocation frequency there was no evidence for any of the polymorphisms studied influencing the response to occupational exposure. A positive interaction observed between genotype (individuals with at least one allele >=20 repeat units) at a microsatellite locus in the XRCC3 gene and smoking status should be interpreted cautiously because interactions were investigated for seven polymorphisms and two exposures. Nonetheless, further research is warranted to examine whether this DNA repair gene variant might be associated with a sub-optimal repair response to smoking-induced DNA damage and hence an increased frequency of translocations.

  18. Targeting CTCF to Control Virus Gene Expression: A Common Theme amongst Diverse DNA Viruses.

    Science.gov (United States)

    Pentland, Ieisha; Parish, Joanna L

    2015-07-06

    All viruses target host cell factors for successful life cycle completion. Transcriptional control of DNA viruses by host cell factors is important in the temporal and spatial regulation of virus gene expression. Many of these factors are recruited to enhance virus gene expression and thereby increase virus production, but host cell factors can also restrict virus gene expression and productivity of infection. CCCTC binding factor (CTCF) is a host cell DNA binding protein important for the regulation of genomic chromatin boundaries, transcriptional control and enhancer element usage. CTCF also functions in RNA polymerase II regulation and in doing so can influence co-transcriptional splicing events. Several DNA viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and human papillomavirus (HPV) utilize CTCF to control virus gene expression and many studies have highlighted a role for CTCF in the persistence of these diverse oncogenic viruses. CTCF can both enhance and repress virus gene expression and in some cases CTCF increases the complexity of alternatively spliced transcripts. This review article will discuss the function of CTCF in the life cycle of DNA viruses in the context of known host cell CTCF functions.

  19. Open source tools to exploit DNA sequence data from livestock species

    Science.gov (United States)

    Next-Generation Sequencing (NGS) is a recent technological development that allows researchers to rapidly determine the DNA sequence of an individual. The decrease in cost of NGS has brought the technology into the realm of practical applications in livestock genomics, where it can be used to genera...

  20. Difference in gene expression of macrophage between normal spleen and portal hypertensive spleen idendified by cDNA microarray

    OpenAIRE

    2007-01-01

    AIM: To identify the difference in gene expression of microphage (Mφ) between normal spleen and portal hypertensive spleen using cDNA microarrays and find new gene functions associated with hypersplenism in portal hypertension.

  1. Effective inhibition of human cytomegalovirus gene expression by DNA-based external guide sequences

    Institute of Scientific and Technical Information of China (English)

    Zhifeng Zeng; Hongjian Li; Yueqing Li; Yanwei Cui; Qi Zhou; Yi Zou; Guang Yang; Tianhong Zhou

    2009-01-01

    To investigate whether a 12 nucleotide DNA-based miniEGSs can silence the expression of human cytomegalovirus(HCMV)UL49 gene efficiently,A HeLa cell line stably expressing UL49 gene was constructed and the putative miniEGSs(UL49-miniEGSs)were assayed in the stable cell line.Quantitative RT-PCR and western blot resuits showed a reduction of 67%in UL49expression level in HeLa cells that were transfected with UL49-miniEGSs.It was significantly different from that of mock and control miniEGSs(TK-miniEGSs)which were 1 and 7%,respectively.To further confirm the gene silence directed by UL49-miniEGSs with human RNase P,a mutant of UL49-miniEGSs was constructedand a modified 5'RACE was carried out.Data showed that the inhibition of UL49 gene expression directed by UL49-miniEGSs was RNase P-dependent and the clea vage of UL49 mRNA by RNase P was site specific.As a result,the length of DNA-based miniEGSs that could silence gene expression efficiently was only 12 nt.That is significantly less than any other Oligonucleotide-based method of gene inactivation known SO far.MiniEGSs may represent novel gene-targeting agents for the inhibition of viral genes and other human disease reiated gene expression.

  2. cDNA, genomic sequence and overexpression of crystallin alpha-B Gene (CRYAB) of the Giant Panda

    Science.gov (United States)

    Hou, Yi-ling; Hou, Wan-ru; Ren, Zheng-long; Hao, Yan-zhe; Zhang, Tian

    2008-01-01

    αB-crystallin, a small heat-shock protein, has been shown to prevent the aggregation of other proteins under various stress conditions. Here we have cloned the cDNA and the genomic sequence of CRYAB gene from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology and Touchdown-PCR, respectively. The length of cDNA fragment cloned contains an open reading frame of 528bp encoding 175 amino acids and the length of the genomic sequence is 3189bp, containing three exons and two introns. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other four species studied, including Homo sapiens, Mus musculus, Rattus norvegicus and Bos taurus. The homologies for nucleotide sequences of Giant Panda CRYAB to that of these species are 93.9%, 91.5%, 91.5% and 95.3%, respectively, and the homologies for amino acid sequences are 98.3%, 97.1%,97.7% and 99.4%, respectively. Topology prediction shows that there are only four Casein kinase II phosphorylation sites in the CRYAB protein of the Giant Panda. The cDNA of CRYAB was transfected into E. coli, and the CRYAB fused with the N-terminally His-tagged protein gave rise to the accumulation of an expected 24KDa polypeptide, which accorded with the predicted protein. The expression product obtained could be used for purification and study of its function further. PMID:19043608

  3. DNA copy-number alterations underlie gene expression differences between microsatellite stable and unstable colorectal cancers

    DEFF Research Database (Denmark)

    Jorissen, Robert N; Lipton, Lara; Gibbs, Peter

    2008-01-01

    Purpose: About 15% of colorectal cancers harbor microsatellite instability (MSI). MSI-associated gene expression changes have been identified in colorectal cancers, but little overlap exists between signatures hindering an assessment of overall consistency. Little is known about the causes...... and downstream effects of differential gene expression. Experimental Design: DNA microarray data on 89 MSI and 140 microsatellite-stable (MSS) colorectal cancers from this study and 58 MSI and 77 MSS cases from three published reports were randomly divided into test and training sets. MSI-associated gene...... expression changes were assessed for cross-study consistency using training samples and validated as MSI classifier using test samples. Differences in biological pathways were identified by functional category analysis. Causation of differential gene expression was investigated by comparison to DNA copy...

  4. Comparative analysis of DNA nanoparticles and AAVs for ocular gene delivery.

    Directory of Open Access Journals (Sweden)

    Zongchao Han

    Full Text Available Gene therapy is a critical tool for the treatment of monogenic retinal diseases. However, the limited vector capacity of the current benchmark delivery strategy, adeno-associated virus (AAV, makes development of larger capacity alternatives, such as compacted DNA nanoparticles (NPs, critical. Here we conduct a side-by-side comparison of self-complementary AAV and CK30PEG NPs using matched ITR plasmids. We report that although AAVs are more efficient per vector genome (vg than NPs, NPs can drive gene expression on a comparable scale and longevity to AAV. We show that subretinally injected NPs do not leave the eye while some of the AAV-injected animals exhibited vector DNA and GFP expression in the visual pathways of the brain from PI-60 onward. As a result, these NPs have the potential to become a successful alternative for ocular gene therapy, especially for the multitude of genes too large for AAV vectors.

  5. [Duplication of DNA--a mechanism for the development of new functionality of genes].

    Science.gov (United States)

    Maślanka, Roman; Zadrąg-Tęcza, Renata

    2015-01-01

    The amplification of DNA is considered as a mechanism for rapid evolution of organisms. Duplication can be especially advantageous in the case of changing environmental conditions. Whole genome duplication maintains the proper balance between gene expression. This seems to be the main reason why WGD is more favorable than duplication of the fragments of DNA. The polyploidy status disappear as a result of the loss of the majority of duplicated genes. The preservation of duplicated genes is associated with the development of their new functions. Polyploidization is often noted for plants. However due to sequencing technique, the duplications episodes are more frequently reports also for the other systematic taxa, including animals. The occurrence of ancient genome duplication is also considered for yeast Saccharomyces cerevisiae. The existence of two active copies of ribosomal protein genes can be a confirmation of this process. Development of the fermentation process might be one of the probable causes of the yeast genome duplication.

  6. [Association between the canine monoamine oxidase B (MAOB) gene polymorphisms and behavior of puppies in open-field test].

    Science.gov (United States)

    Li, Xiao-Hui; Xu, Han-Kun; Mao, Da-Gan; Ma, Da-Jun; Chen, Peng; Yang, Li-Guo

    2006-11-01

    Excitability, activity and exploration behavior of puppies in a novel open-field were tested in a total of 204 two-month-old German shepherd dog, labrador retriever or English springer spaniel puppies. The polymorphisms of monoamine oxidase B gene (MAOB) were detected by PCR-RFLP. Statistics analysis indicated that genotype and allele frequencies of the polymorphisms were significantly different among three breeds (P open-field test. The results showed that MAOB gene polymorphisms had a significant effect on walking time, squares crossed, lying time, the times of standing up against walls(P open-field test and TT genotype has favorable effects in these behavior traits.

  7. DNA methylation analysis of chromosome 21 gene promoters at single base pair and single allele resolution.

    Directory of Open Access Journals (Sweden)

    Yingying Zhang

    2009-03-01

    Full Text Available Differential DNA methylation is an essential epigenetic signal for gene regulation, development, and disease processes. We mapped DNA methylation patterns of 190 gene promoter regions on chromosome 21 using bisulfite conversion and subclone sequencing in five human cell types. A total of 28,626 subclones were sequenced at high accuracy using (long-read Sanger sequencing resulting in the measurement of the DNA methylation state of 580427 CpG sites. Our results show that average DNA methylation levels are distributed bimodally with enrichment of highly methylated and unmethylated sequences, both for amplicons and individual subclones, which represent single alleles from individual cells. Within CpG-rich sequences, DNA methylation was found to be anti-correlated with CpG dinucleotide density and GC content, and methylated CpGs are more likely to be flanked by AT-rich sequences. We observed over-representation of CpG sites in distances of 9, 18, and 27 bps in highly methylated amplicons. However, DNA sequence alone is not sufficient to predict an amplicon's DNA methylation status, since 43% of all amplicons are differentially methylated between the cell types studied here. DNA methylation in promoter regions is strongly correlated with the absence of gene expression and low levels of activating epigenetic marks like H3K4 methylation and H3K9 and K14 acetylation. Utilizing the single base pair and single allele resolution of our data, we found that i amplicons from different parts of a CpG island frequently differ in their DNA methylation level, ii methylation levels of individual cells in one tissue are very similar, and iii methylation patterns follow a relaxed site-specific distribution. Furthermore, iv we identified three cases of allele-specific DNA methylation on chromosome 21. Our data shed new light on the nature of methylation patterns in human cells, the sequence dependence of DNA methylation, and its function as epigenetic signal in gene

  8. A simple and rapid method for the preparation of homologous DNA oligonucleotide hybridization probes from heterologous gene sequences and probes.

    Science.gov (United States)

    Maxwell, E S; Sarge, K D

    1988-11-30

    We describe a simple and rapid method for the preparation of homologous DNA oligonucleotide probes for hybridization analysis and/or cDNA/genomic library screening. With this method, a synthetic DNA oligonucleotide derived from a known heterologous DNA/RNA/protein sequence is annealed to an RNA preparation containing the gene transcript of interest. Any unpaired 3'-terminal oligonucleotides of the heterologous DNA primer are then removed using the 3' exonuclease activity of the DNA Polymerase I Klenow fragment before primer extension/dideoxynucleotide sequencing of the annealed RNA species with AMV reverse transcriptase. From the determined RNA sequence, a completely homologous DNA oligonucleotide probe is then prepared. This approach has been used to prepare a homologous DNA oligonucleotide probe for the successful library screening of the yeast hybRNA gene starting with a heterologous mouse hybRNA DNA oligonucleotide probe.

  9. Altered regulation of DNA ligase IV activity by aberrant promoter DNA methylation and gene amplification in colorectal cancer.

    Science.gov (United States)

    Kuhmann, Christine; Li, Carmen; Kloor, Matthias; Salou, Mariam; Weigel, Christoph; Schmidt, Christopher R; Ng, Linda W C; Tsui, Wendy W Y; Leung, Suet Y; Yuen, Siu T; Becker, Natalia; Weichenhan, Dieter; Plass, Christoph; Schmezer, Peter; Chan, Tsun L; Popanda, Odilia

    2014-04-15

    Colorectal cancer (CRC) presents as a very heterogeneous disease which cannot sufficiently be characterized with the currently known genetic and epigenetic markers. To identify new markers for CRC we scrutinized the methylation status of 231 DNA repair-related genes by methyl-CpG immunoprecipitation followed by global methylation profiling on a CpG island microarray, as altered expression of these genes could drive genomic and chromosomal instability observed in these tumors. We show for the first time hypermethylation of MMP9, DNMT3A and LIG4 in CRC which was confirmed in two CRC patient groups with different ethnicity. DNA ligase IV (LIG4) showed strong differential promoter methylation (up to 60%) which coincided with downregulation of mRNA in 51% of cases. This functional association of LIG4 methylation and gene expression was supported by LIG4 re-expression in 5-aza-2'-deoxycytidine-treated colon cancer cell lines, and reduced ligase IV amounts and end-joining activity in extracts of tumors with hypermethylation. Methylation of LIG4 was not associated with other genetic and epigenetic markers of CRC in our study. As LIG4 is located on chromosome 13 which is frequently amplified in CRC, two loci were tested for gene amplification in a subset of 47 cases. Comparison of amplification, methylation and expression data revealed that, in 30% of samples, the LIG4 gene was amplified and methylated, but expression was not changed. In conclusion, hypermethylation of the LIG4 promoter is a new mechanism to control ligase IV expression. It may represent a new epigenetic marker for CRC independent of known markers.

  10. The genome of the stick insect Medauroidea extradentata is strongly methylated within genes and repetitive DNA.

    Directory of Open Access Journals (Sweden)

    Veiko Krauss

    Full Text Available BACKGROUND: Cytosine DNA methylation has been detected in many eukaryotic organisms and has been shown to play an important role in development and disease of vertebrates including humans. Molecularly, DNA methylation appears to be involved in the suppression of initiation or of elongation of transcription. Resulting organismal functions are suggested to be the regulation of gene silencing, the suppression of transposon activity and the suppression of initiation of transcription within genes. However, some data concerning the distribution of methylcytosine in insect species appear to contradict such roles. PRINCIPAL FINDINGS: By comparison of MspI and HpaII restriction patterns in genomic DNA of several insects we show that stick insects (Phasmatodea have highly methylated genomes. We isolated methylated DNA fragments from the Vietnamese Walking Stick Medauroidea extradentata (formerly known as Baculum extradentatum and demonstrated that most of the corresponding sequences are repetitive. Bisulfite sequencing of one of these fragments and of parts of conserved protein-coding genes revealed a methylcytosine content of 12.6%, mostly found at CpG, but also at CpT and CpA dinucleotides. Corresponding depletions of CpG and enrichments of TpG and CpA dinucleotides in some highly conserved protein-coding genes of Medauroidea reach a similar degree as in vertebrates and show that CpG methylation has occurred in the germline of these insects. CONCLUSIONS: Using four different methods, we demonstrate that the genome of Medauroidea extradentata is strongly methylated. Both repetitive DNA and coding genes appear to contain high levels of methylcytosines. These results argue for similar functions of DNA methylation in stick insects as those already known for vertebrates.

  11. 'Hide-then-hit' to explain the importance of genotypic polymorphism of DNA repair genes in determining susceptibility to cancer

    Institute of Scientific and Technical Information of China (English)

    Pei-Ei Wu; Chen-Yang Shen

    2011-01-01

    Interindividual variations in DNA repair capacity/efficiency linked to the presence of polymorphisms in DNA repair-related genes have been suggested to account for different risk of developing cancers. In this review article, on the basis of breast cancer formation as a model, we propose a 'hide-then-hit' hypothesis indicating the importance of escaping checkpoint surveillance for sub-optimal DNA repair variants to cause cancer. Therefore, only cells with subtle defects in repair capacity arising from low-penetrance variants of DNA repair genes would have the opportunity to grow and accumulate the genetic changes needed for cancer formation, without triggering cell-cycle checkpoint surveillance. Furthermore, distinct from high-penetrance alleles, these polymorphic alleles of DNA repair genes would predispose carriers to a higher risk of developing cancer but would not necessarily cause cancer. To examine this,we simultaneously genotyped multiple SNPs of cell-cycle checkpoint genes and the DNA repair genes. Support for the hypothesis came from observations that breast cancer risk associated with variant genotypes of DNA repair genes became more significant in be confirmed by biological evidence in which a cause-effect relationship has to be established. However, based on this, possible gene-gene interaction is considered to play an important role in modifying the cancer risk associated with genotypic polymorphism of DNA repair gene in different study populations.

  12. Neural network predicts sequence of TP53 gene based on DNA chip

    DEFF Research Database (Denmark)

    Spicker, J.S.; Wikman, F.; Lu, M.L.;

    2002-01-01

    We have trained an artificial neural network to predict the sequence of the human TP53 tumor suppressor gene based on a p53 GeneChip. The trained neural network uses as input the fluorescence intensities of DNA hybridized to oligonucleotides on the surface of the chip and makes between zero...... and four errors in the predicted 1300 bp sequence when tested on wild-type TP53 sequence....

  13. The evolution of CHROMOMETHYLASES and gene body DNA methylation in plants.

    Science.gov (United States)

    Bewick, Adam J; Niederhuth, Chad E; Ji, Lexiang; Rohr, Nicholas A; Griffin, Patrick T; Leebens-Mack, Jim; Schmitz, Robert J

    2017-05-01

    The evolution of gene body methylation (gbM), its origins, and its functional consequences are poorly understood. By pairing the largest collection of transcriptomes (>1000) and methylomes (77) across Viridiplantae, we provide novel insights into the evolution of gbM and its relationship to CHROMOMETHYLASE (CMT) proteins. CMTs are evolutionary conserved DNA methyltransferases in Viridiplantae. Duplication events gave rise to what are now referred to as CMT1, 2 and 3. Independent losses of CMT1, 2, and 3 in eudicots, CMT2 and ZMET in monocots and monocots/commelinids, variation in copy number, and non-neutral evolution suggests overlapping or fluid functional evolution of this gene family. DNA methylation within genes is widespread and is found in all major taxonomic groups of Viridiplantae investigated. Genes enriched with methylated CGs (mCG) were also identified in species sister to angiosperms. The proportion of genes and DNA methylation patterns associated with gbM are restricted to angiosperms with a functional CMT3 or ortholog. However, mCG-enriched genes in the gymnosperm Pinus taeda shared some similarities with gbM genes in Amborella trichopoda. Additionally, gymnosperms and ferns share a CMT homolog closely related to CMT2 and 3. Hence, the dependency of gbM on a CMT most likely extends to all angiosperms and possibly gymnosperms and ferns. The resulting gene family phylogeny of CMT transcripts from the most diverse sampling of plants to date redefines our understanding of CMT evolution and its evolutionary consequences on DNA methylation. Future, functional tests of homologous and paralogous CMTs will uncover novel roles and consequences to the epigenome.

  14. Identification of smut-responsive genes in sugarcane using cDNA-SRAP.

    Science.gov (United States)

    Huang, N; Zhang, Y Y; Xiao, X H; Huang, L; Wu, Q B; Que, Y X; Xu, L P

    2015-06-18

    Sugarcane smut, caused by the fungus Sporisorium scitamineum, is one of the main diseases that affect sugarcane worldwide. In the present study, the cDNA-SRAP technique was used to identify genes that are likely to be involved in the response of sugarcane to S. scitamineum infection. In total, 21 bands with significant differential expression during cDNA-SRAP analysis were cloned and sequenced. Real-time qPCR confirmation demonstrated that expression of 19 of these 21 differential bands was consistent with the expression observed during cDNA-SRAP analysis, with a deduced false positive rate of 9.5%. Sequence alignment indicated that 18 of 19 differentially expressed genes showed homologies from 19% to 100% to certain genes in GenBank, including the following genes: topoisomerase (EU048780), ethylene insensitive (EU048778), and tetraspanin (EU048770). A real-time qPCR assay showed that during 0-72 h after pathogen infection, expression of the topoisomerase and the ethylene insensitive genes was upregulated, whereas expression of the tetraspanin gene was downregulated, identical to the expression patterns observed under salicylic acid treatment. Therefore, all three genes are thought to play a role during S. scitamineum challenge, but with different functions. To our knowledge, this is the first report on the application of cDNA-SRAP in differential gene expression analysis of sugarcane during a sugarcane-S. scitamineum interaction. The results obtained also contribute to a better understanding of the molecular mechanisms associated with sugarcane-S. scitamineum interactions.

  15. Trypanosome Surface Antigen Genes: Analysis Using Recombinant DNA.

    Science.gov (United States)

    1984-06-15

    different VATs. These cDNAs were used to screen the genomic libraries . The cloned probes were examined by nucleotide sequence analysis and used to...examination of the consequence of - antigenic variation on the structure of the 1.1, 1.D and 1.11 VSG -, gene sequences. In addition, all genomic libraries have...immunoprecipitated by heterologous VAt specific antisera (preprint 2, figure 3; preprint 3, figure 1). These data presented in preprints 2 and Genomic

  16. Mitochondrial transcription termination factor 2 binds to entire mitochondrial DNA and negatively regulates mitochondrial gene expression

    Institute of Scientific and Technical Information of China (English)

    Weiwei Huang; Min Yu; Yang Jiao; Jie Ma; Mingxing Ma; Zehua Wang; Hong Wu; Deyong Tan

    2011-01-01

    Mitochondrial transcription termination factor 2 (mTERF2) is a mitochondriai matrix protein that binds to the mitochondriai DNA.Previous studies have shown that overexpression of mTERF2 can inhibit cell proliferation, but the mechanism has not been well defined so far.This study aimed to present the binding pattern of mTERF2 to the mitochondrial DNA (mtDNA) in vivo, and investigated the biological function of mTERF2 on the replication of mtDNA, mRNA transcription, and protein translation.The mTERF2 binding to entire mtDNA was identified via the chromatin immunoprecipitation analysis.The mtDNA replication efficiency and expression levels of mitochondria genes were significantly inhibited when the mTERF2 was overexpressed in HeLa cells.The inhibition level of mtDNA content was the same with the decreased levels of mRNA and mitochondrial protein expression.Overall, the mTERF2 might be a cell growth inhibitor based on its negative effect on mtDNA replication, which eventually own-regulated all of the oxidative phosphorylation components in the mitochondria that were essential for the cell's energy metabolism.

  17. Regulation of the Saccharomyces cerevisiae DNA repair gene RAD16.

    Science.gov (United States)

    Bang, D D; Timmermans, V; Verhage, R; Zeeman, A M; van de Putte, P; Brouwer, J

    1995-05-25

    The RAD16 gene product has been shown to be essential for the repair of the silenced mating type loci [Bang et al. (1992) Nucleic Acids Res. 20, 3925-3931]. More recently we demonstrated that the RAD16 and RAD7 proteins are also required for repair of non-transcribed strands of active genes in Saccharomyces cerevisiae [Waters et al. (1993) Mol. Gen. Genet. 239, 28-32]. We have studied the regulation of the RAD16 gene and found that the RAD16 transcript levels increased up to 7-fold upon UV irradiation. Heat shock at 42 degrees C also results in elevated levels of RAD16 mRNA. In sporulating MAT alpha/MATa diploid cells RAD16 mRNA is also induced. The basal level of the RAD16 transcript is constant during the mitotic cell cycle. G1-arrested cells show normal induction of RAD16 mRNA upon UV irradiation demonstrating that the induction is not a secondary consequence of G2 cell cycle arrest following UV irradiation. However, in cells arrested in G1 the induction of RAD16 mRNA after UV irradiation is not followed by a rapid decline as occurs in normal growing cells suggesting that the down regulation of RAD16 transcription is dependent on progression into the cell cycle.

  18. OS052. Preeclampsia candidate genes differentially methylated in maternal leukocyte DNA.

    Science.gov (United States)

    White, W; Brost, B; O'Brien, J; Rose, C; Davies, N; Sun, Z; Turner, S; Garovic, V

    2012-07-01

    Altered gene expression in biomarkers associated with preeclampsia/ eclampsia (PE) could be explained in part by epigenetic phenomena such as variable methylation We sought to characterize the methylation profiles of candidate genes known to be associated with the preeclampsia phenotype in maternal leukocyte DNA in preeclamptic cases and normotensive controls at the time of delivery. Methylation profiles of maternal leukocyte DNA were evaluated in 14 PE cases and 14 normotensive controls. Subjects were nulliparous, non-smokers, age and BMI matched. Genomic DNA was run on a commercially available beadchip human methylation assay. Mean methylation at sites in genes from a well-defined preeclampsia gene set present on our platform were compared using a t-test. QC confirmed high correlation of replicates and detection p values >95%. Of the 39 genes in the "preeclampsia gene set", 34 were present on our platform with 73 CpG sites. Seven out of 34 tested in this gene set had differential methylation with p value +4%;p=0.001) forms calcitonin-gene related peptide, a potent vasodilator decreased in the PE . F5 (+1%;p=0.016), coagulation Factor V, is a target of activated protein C, and increased resistance related to genetic variants (Factor V Leiden) or pregnancy have been associated with PE. MTHFR (+3%;p=0.041) regulates homocysteine; high levels are associated with a 20X increase in risk for PE. POMC (+4%;p=0.014) produces beta endorphin and through ACTH stimulates aldosterone, both decreased in PE. PTGS2 (+3%;p=0.03) is part of the COX 2 prostaglandin pathway involved in inflammation. Differential methylation of these 7 genes may affect transcription and explain known alterations in gene product associated with PE. Copyright © 2012. Published by Elsevier B.V.

  19. Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase.

    Science.gov (United States)

    Collis, C M; Hall, R M

    1992-01-01

    Deletion of individual antibiotic resistance genes found within the variable region of integrons is demonstrated. Evidence for gene duplications and rearrangements resulting from the insertion of gene units at new locations is also presented. Deletion, duplication, and rearrangement occur only in the presence of the integron-encoded DNA integrase. These events are precise and involve loss or gain of one or more complete insert units or gene cassettes. This confirms the recent definition of gene cassettes as consisting of the gene coding sequences, all except the last 7 bases of the 59-base element found at the 3' end of the gene, and the core site located 5' to the gene (Hall et al., Mol. Microbiol. 5:1941-1959, 1991) and demonstrates that individual gene cassettes are functional units which can be independently mobilized. Both deletions and duplications can be generated by integrase-mediated cointegrate formation followed by integrase-mediated resolution involving a different pair of sites. However, deletion occurs 10 times more frequently than duplication, and we propose that the majority of deletion events are likely to involve integrase-dependent excision of the gene unit to generate a circular gene cassette. The implications of these findings in understanding the evolution of integrons and the spread of antibiotic resistance genes in bacterial populations is discussed. Images PMID:1311297

  20. Comparison of different protocols for DNA preparation and PCR amplification of mitochondrial genes of tardigrades

    Directory of Open Access Journals (Sweden)

    Ralph O. SCHILL

    2007-09-01

    Full Text Available Phylogenetic relationships and molecular taxonomy within the Tardigrada have been given a lot of attention in recent years. Here I present the first comparison of different protocols for DNA preparation by investigating six commercial available DNA extraction kits and the CTAB method. Successful extraction of DNA from tardigrades depends strongly on the life-stage (embryo, adult, and on the condition of the specimens, respectively on the preservation (anhydrobiotic, ethanol. Although the extraction kits showed differences in the amount of extracted DNA, in all cases fresh tissue of live animals or embryos resulted in the best quality and quantity of DNA. A lesser amount of DNA was extractable from anhydrobiotic animals and embryos and the results of specimens fixed in ethanol were unsatisfactory. All used commercially available DNA extraction kits and PCR cocktails have been focused on vertebrate tissues, blood, cultured cells, bacteria and yeast. However, I used successfully the kits according to the manufacturer’s instruction without changes in the protocols for DNA extraction of tardigrades. Commercial kits provide a simple and convenient way to isolate pure genomic DNA of high-quality from tardigrades. Furthermore I tested eight different Taq polymerase enzymes for PCR amplification of mitochondrial genes of tardigrades. Each of the enzymes resulted in a PCR product, and even if the amount of the PCR products was quite different, it was possible to use it successful for direct sequencing. Summarizing, the successful PCR of the target DNA depends on the purity and quality of the DNA template and for this the species preservation is more critical than the extraction method or the PCR cocktail which can be optimized.

  1. Polymorphisms in DNA Repair Genes and MDR1 and the Risk for Non-Hodgkin Lymphoma

    Directory of Open Access Journals (Sweden)

    Hee Nam Kim

    2014-04-01

    Full Text Available The damage caused by oxidative stress and exposure to cigarette smoke and alcohol necessitate DNA damage repair and transport by multidrug resistance-1 (MDR1. To explore the association between polymorphisms in these genes and non-Hodgkin lymphoma risk, we analyzed 15 polymorphisms of 12 genes in a population-based study in Korea (694 cases and 1700 controls. Four genotypes of DNA repair pathway genes (XRCC1 399 GA, OGG1 326 GG, BRCA1 871 TT, and WRN 787 TT were associated with a decreased risk for NHL [odds ratio (ORXRCC1 GA = 0.80, p = 0.02; OROGG1 GG = 0.70, p = 0.008; ORBRCA1 TT = 0.71, p = 0.048; ORWRN TT = 0.68, p = 0.01]. Conversely, the MGMT 115 CT genotype was associated with an increased risk for NHL (OR = 1.25, p = 0.04. In the MDR1 gene, the 1236 CC genotype was associated with a decreased risk for NHL (OR = 0.74, p = 0.04, and the 3435 CT and TT genotypes were associated with an increased risk (OR3435CT = 1.50, p < 0.0001; OR3435TT = 1.43, p = 0.02. These results suggest that polymorphisms in the DNA repair genes XRCC1, OGG1, BRCA1, WRN1, and MGMT and in the MDR1 gene may affect the risk for NHL in Korean patients.

  2. Clinical relevance of the glucocorticoid receptor gene polymorphisms in glucocorticoid-induced ocular hypertension and primary open angle glaucoma

    Institute of Scientific and Technical Information of China (English)

    Xiu-Qing; Wang; Zhao-Xia; Duan; Xiang-Ge; He; Xi-Yuan; Zhou

    2015-01-01

    AIM: To avoid the side effects of ocular hypertension of glucocorticoid(GC) usage in eye, we must identify susceptible individuals, which exists in about one-third of all population. Further, the majority of all primary open angle glaucoma(POAG) patients show this phenotype.Glucocorticoid receptor(GR) regulates C responsiveness in trabecular meshwork(TM) cells. In this study, single nucleotide polymorphism(SNP) genotyping was used to determine whether there are differences in the Bcl I(rs41423247) and N363S(rs6195) polymorphisms of the GR gene in healthy and POAG patients, and glucocorticoid-induced ocular hypertension(GIOH)populations.METHODS: Three hundred and twenty-seven unrelated Chinese adults, including 111 normal controls, 117 GIOH subjects and 99 POAG patients, were recruited. DNA samples were prepared and the Bcl I and N363 S polymorphisms were screened using real-time polymerase chain reaction(RT-PCR)-restriction fragment length polymorphism(RFLP) analysis. Frequencies of the Bcl I and N363 S polymorphisms were determined and compared using Fisher’s exact test and the Chi-squared test.RESULTS: Only the Bcl I polymorphism was identified in the Chinese Han population. The frequency of the G allele was 21.6 % in normal controls, 18.3% in GIOH patients, and 13.64% in the POAG patients. There was no significant difference in polymorphism or allele frequency in the 3 groups. Furthermore, no N363 S polymorphism was found in the study subjects.CONCLUSION: The Bcl I polymorphisms in GR gene had no association with GIOH and POAG patients, and N363 S polymorphism might not exist in the Chinese Han population. Therefore, the Bcl I polymorphism might not be responsible for the development of GC-induced ocular hypertension or POAG.

  3. Genes of the unfolded protein response pathway harbor risk alleles for primary open angle glaucoma.

    Directory of Open Access Journals (Sweden)

    Mary Anna Carbone

    Full Text Available The statistical power of genome-wide association (GWA studies to detect risk alleles for human diseases is limited by the unfavorable ratio of SNPs to study subjects. This multiple testing problem can be surmounted with very large population sizes when common alleles of large effects give rise to disease status. However, GWA approaches fall short when many rare alleles may give rise to a common disease, or when the number of subjects that can be recruited is limited. Here, we demonstrate that this multiple testing problem can be overcome by a comparative genomics approach in which an initial genome-wide screen in a genetically amenable model organism is used to identify human orthologues that may harbor risk alleles for adult-onset primary open angle glaucoma (POAG. Glaucoma is a major cause of blindness, which affects over 60 million people worldwide. Several genes have been associated with juvenile onset glaucoma, but genetic factors that predispose to adult onset primary open angle glaucoma (POAG remain largely unknown. Previous genome-wide analysis in a Drosophila ocular hypertension model identified transcripts with altered regulation and showed induction of the unfolded protein response (UPR upon overexpression of transgenic human glaucoma-associated myocilin (MYOC. We selected 16 orthologous genes with 62 polymorphic markers and identified in two independent human populations two genes of the UPR that harbor POAG risk alleles, BIRC6 and PDIA5. Thus, effectiveness of the UPR in response to accumulation of misfolded or aggregated proteins may contribute to the pathogenesis of POAG and provide targets for early therapeutic intervention.

  4. Transcriptional analysis of the DNA polymerase gene of shrimp white spot syndrome virus.

    Science.gov (United States)

    Chen, Li-Li; Wang, Han-Ching; Huang, Chiu-Jung; Peng, Shao-En; Chen, Yen-Gu; Lin, Shin-Jen; Chen, Wei-Yu; Dai, Chang-Feng; Yu, Hon-Tsen; Wang, Chung-Hsiung; Lo, Chu-Fang; Kou, Guang-Hsiung

    2002-09-15

    The white spot syndrome virus DNA polymerase (DNA pol) gene (WSSV dnapol) has already been tentatively identified based on the presence of highly conserved motifs, but it shows low overall homology with other DNA pols and is also much larger (2351 amino acid residues vs 913-1244 aa). In the present study we perform a transcriptional analysis of the WSSV dnapol gene using the total RNA isolated from WSSV-infected shrimp at different times after infection. Northern blot analysis with a WSSV dnapol-specific riboprobe found a major transcript of 7.5 kb. 5'-RACE revealed that the major transcription start point is located 27 nucleotides downstream of the TATA box, at the nucleotide residue A within a CAGT motif, one of the initiator (Inr) motifs of arthropods. In a temporal expression analysis using differential RT-PCR, WSSV dnapol transcripts were detected at low levels at 2-4 h.p.i., increased at 6 h.p.i., and remained fairly constant thereafter. This is similar to the previously reported transcription patterns for genes encoding the key enzyme of nucleotide metabolism, ribonucleotide reductase. Phylogenetic analysis showed that the DNA pols from three different WSSV isolates form an extremely tight cluster. In addition, similar to an earlier phylogenetic analysis of WSSV protein kinase, the phylogenetic tree of viral DNA pols further supports the suggestion that WSSV is a distinct virus (likely at the family level) that does not belong to any of the virus families that are currently recognized.

  5. The Eukaryotic DNMT2 Genes Encode a New Class of Cytosine-5 DNA Methyltransferases

    Institute of Scientific and Technical Information of China (English)

    Lin-YaTang; M.NarsaReddy; VanyaRasheva; Tai-LinLee; Meng-JauLin; Ming-ShiuHung; C.-K.JamesShen

    2005-01-01

    DNMT2 is a subgroup of the eukaryotic cytosine-5 DNA methyltransferase gene family. Unlike the other family members, proteins encoded by DNMT2 genes were not known before to possess DNA methyltransferase activities. Most recently, we have showm that thegenome of Drosophila S2 cells stably expressing an exogenous Drosophila dDNMT2 cDNA became anoma-lously methylated at the 5'-positions of cytosines(Reddy, M. N., Tang, L. Y., Lee, T. L., and Shen, C.-K. J.(2003) Oncogene, in press). We present evidence here that the genomes of transgenic flies overexpressing the dDnmt2 protein also became hypermethylated at specific regions. Furthermore, transient transfection studies in combination with sodium bisulfite sequencing demonstrated that dDnmt2 as well as its mousc ortholog, mDnmt2, are capable of methylating a cotrans-fected plasmid DNA. These data provide solid evidence that the fly and mouse DNMT2 gene products are genuine cytosine-5 DNA methyltransferases.

  6. Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing.

    Directory of Open Access Journals (Sweden)

    Olivier J Becherel

    2013-04-01

    Full Text Available Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2, plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI. Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops, and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

  7. Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing.

    Directory of Open Access Journals (Sweden)

    Olivier J Becherel

    2013-04-01

    Full Text Available Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2, plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI. Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops, and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

  8. Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing.

    Science.gov (United States)

    Becherel, Olivier J; Yeo, Abrey J; Stellati, Alissa; Heng, Evelyn Y H; Luff, John; Suraweera, Amila M; Woods, Rick; Fleming, Jean; Carrie, Dianne; McKinney, Kristine; Xu, Xiaoling; Deng, Chuxia; Lavin, Martin F

    2013-04-01

    Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

  9. Electroporation of proviral RCAS DNA alters gene expression in the embryonic chick hindbrain.

    Science.gov (United States)

    Hermann, Petra M; Logan, C Cairine

    2003-11-01

    Gene transfer by means of electroporation is an effective method for delivering DNA into cells. Expression vectors encoding green fluorescent protein (GFP) are routinely used as a control for this technique and are also regularly used to indirectly or directly monitor the expression of introduced transgenes. However, recent studies suggest that GFP may have nonspecific and/or cytotoxic side effects. In this study, we investigated the effects of enhanced GFP (EGFP) expression delivered by means of electroporation of proviral RCASBP(B)-EGFP DNA on gene expression in the hindbrain of chick embryos. We examined, via whole-mount in situ hybridization, the expression of a number of transcription factors. We found that Tlx-1 was ectopically expressed following electroporation of proviral RCASBP(B)-EGFP DNA. In contrast, the number of cells expressing Tlx-3, Phox2a, and Phox2b were reduced. Intriguingly, these effects could be mimicked by electroporation of wild-type proviral RCASBP(B) DNA (i.e., lacking the GFP insert). However, neither delivery of the EGFP transgene by means of viral infection nor electroporation alone yielded aberrant expression patterns. Together our data indicate that alterations of gene expression patterns are not directly due to the expression of EGFP but instead reflect a confounding effect of electroporating proviral DNA.

  10. Self-entanglement of long linear DNA vectors using transient non-B-DNA attachment points: a new concept for improvement of non-viral therapeutic gene delivery.

    Science.gov (United States)

    Tolmachov, Oleg E

    2012-05-01

    The cell-specific and long-term expression of therapeutic transgenes often requires a full array of native gene control elements including distal enhancers, regulatory introns and chromatin organisation sequences. The delivery of such extended gene expression modules to human cells can be accomplished with non-viral high-molecular-weight DNA vectors, in particular with several classes of linear DNA vectors. All high-molecular-weight DNA vectors are susceptible to damage by shear stress, and while for some of the vectors the harmful impact of shear stress can be minimised through the transformation of the vectors to compact topological configurations by supercoiling and/or knotting, linear DNA vectors with terminal loops or covalently attached terminal proteins cannot be self-compacted in this way. In this case, the only available self-compacting option is self-entangling, which can be defined as the folding of single DNA molecules into a configuration with mutual restriction of molecular motion by the individual segments of bent DNA. A negatively charged phosphate backbone makes DNA self-repulsive, so it is reasonable to assume that a certain number of 'sticky points' dispersed within DNA could facilitate the entangling by bringing DNA segments into proximity and by interfering with the DNA slipping away from the entanglement. I propose that the spontaneous entanglement of vector DNA can be enhanced by the interlacing of the DNA with sites capable of mutual transient attachment through the formation of non-B-DNA forms, such as interacting cruciform structures, inter-segment triplexes, slipped-strand DNA, left-handed duplexes (Z-forms) or G-quadruplexes. It is expected that the non-B-DNA based entanglement of the linear DNA vectors would consist of the initial transient and co-operative non-B-DNA mediated binding events followed by tight self-ensnarement of the vector DNA. Once in the nucleoplasm of the target human cells, the DNA can be disentangled by type II

  11. Cloning, DNA sequencing and heterologous expression of the gene for thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60 in Escherichia coli.

    Science.gov (United States)

    Tokuyama, S; Hatano, K

    1995-03-01

    The gene encoding the novel enzyme N-acylamino acid racemase (AAR) was cloned in recombinant phage lambda-4 from the DNA library of Amycolatopsis sp. TS-1-60, a rare actinomycete, using antiserum against the enzyme. The cloned gene was subcloned and transformed in Escherichia coli JM105 using pUC118 as a vector. The AAR gene consists of an open-reading frame of 1104 nucleotides, which specifies a 368-amino-acid protein with a molecular mass of 39411Da. The molecular mass deduced from the AAR gene is in good agreement with the subunit molecular mass (40kDa) of AAR from Amycolatopsis sp. TS-1-60. The guanosine plus cytosine content of the AAR gene was about 70%. Although the AAR gene uses the unusual initiation codon GTG, the gene was expressed in Escherichia coli using the lac promoter of pUC118. The amount of the enzyme produced by the transformant was 16 times that produced by Amycolatopsis sp. TS-1-60. When the unusual initiation codon GTG was changed to ATG, the enzyme productivity of the transformant increased to more than 37 times that of Amycolatopsis sp. TS-1-60. In the comparison of the DNA sequence and the deduced amino acid sequence of AAR with those of known racemases and epimerases in data bases, no significant sequence homology was found. However, AAR resembles mandelate racemase in that requires metal ions for enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. DNA methylation profiling of transcription factor genes in normal lymphocyte development and lymphomas.

    Science.gov (United States)

    Ivascu, Claudia; Wasserkort, Reinhold; Lesche, Ralf; Dong, Jun; Stein, Harald; Thiel, Andreas; Eckhardt, Florian

    2007-01-01

    Transcription factors play a crucial role during hematopoiesis by orchestrating lineage commitment and determining cellular fate. Although tight regulation of transcription factor expression appears to be essential, little is known about the epigenetic mechanisms involved in transcription factor gene regulation. We have analyzed DNA methylation profiles of 13 key transcription factor genes in primary cells of the hematopoietic cascade, lymphoma cell lines and lymph node biopsies of diffuse large B-cell- and T-cell-non-Hodgkin lymphoma patients. Several of the transcription factor genes (SPI1, GATA3, TCF-7, Etv5, c-maf and TBX21) are differentially methylated in specific cell lineages and stages of the hematopoietic cascade. For some genes, such as SPI1, Etv5 and Eomes, we found an inverse correlation between the methylation of the 5' untranslated region and expression of the associated gene suggesting that these genes are regulated by DNA methylation. Differential methylation is not limited to cells of the healthy hematopoietic cascade, as we observed aberrant methylation of c-maf, TCF7, Eomes and SPI1 in diffuse large B-cell lymphomas. Our results suggest that epigenetic remodelling of transcription factor genes is a frequent mechanism during hematopoietic development. Aberrant methylation of transcription factor genes is frequently observed in diffuse large B-cell lymphomas and might have a functional role during tumorigenesis.

  13. Horizontal gene transfer and nucleotide compositional anomaly in large DNA viruses

    Directory of Open Access Journals (Sweden)

    Ogata Hiroyuki

    2007-12-01

    Full Text Available Abstract Background DNA viruses have a wide range of genome sizes (5 kb up to 1.2 Mb, compared to 0.16 Mb to 1.5 Mb for obligate parasitic bacteria that do not correlate with their virulence or the taxonomic distribution of their hosts. The reasons for such large variation are unclear. According to the traditional view of viruses as gifted "gene pickpockets", large viral genome sizes could originate from numerous gene acquisitions from their hosts. We investigated this hypothesis by studying 67 large DNA viruses with genome sizes larger than 150 kb, including the recently characterized giant mimivirus. Given that horizontally transferred DNA often have anomalous nucleotide compositions differing from the rest of the genome, we conducted a detailed analysis of the inter- and intra-genome compositional properties of these viruses. We then interpreted their compositional heterogeneity in terms of possible causes, including strand asymmetry, gene function/expression, and horizontal transfer. Results We first show that the global nucleotide composition and nucleotide word usage of viral genomes are species-specific and distinct from those of their hosts. Next, we identified compositionally anomalous (cA genes in viral genomes, using a method based on Bayesian inference. The proportion of cA genes is highly variable across viruses and does not exhibit a significant correlation with genome size. The vast majority of the cA genes were of unknown function, lacking homologs in the databases. For genes with known homologs, we found a substantial enrichment of cA genes in specific functional classes for some of the viruses. No significant association was found between cA genes and compositional strand asymmetry. A possible exogenous origin for a small fraction of the cA genes could be confirmed by phylogenetic reconstruction. Conclusion At odds with the traditional dogma, our results argue against frequent genetic transfers to large DNA viruses from their

  14. Comparative analysis of DNA methyltransferase gene family in fungi: a focus on Basidiomycota

    Directory of Open Access Journals (Sweden)

    Ruirui Huang

    2016-10-01

    Full Text Available DNA methylation plays a crucial role in the regulation of gene expression in eukaryotes. Mushrooms belonging to the phylum Basidiomycota are highly valued for both nutritional and pharmaceutical uses. A growing number of studies have demonstrated the significance of DNA methylation in the development of plants and animals. However, our understanding of DNA methylation in mushrooms is limited. In this study, we identified and conducted comprehensive analyses on DNA methyltransferases (DNMtases in representative species from Basidiomycota and Ascomycota, and obtained new insights into their classification and characterization in fungi. Our results revealed that DNMtases in basidiomycetes can be divided into two classes, the Dnmt1 class and the newly defined Rad8 class. We also demonstrated that the fusion event between the characteristic domains of the DNMtases family and Snf2 family in the Rad8 class is fungi-specific, possibly indicating a functional novelty of Rad8 DNMtases in fungi. Additionally, expression profiles of DNMtases in the edible mushroom Pleurotus ostreatus revealed diverse expression patterns in various organs and developmental stages. For example, DNMtase genes displayed higher expression levels in dikaryons than in monokaryons. Consistent with the expression profiles, we found that dikaryons are more susceptible to the DNA methyltransferase inhibitor 5-azacytidine. Taken together, our findings pinpoint an important role of DNA methylation during the growth of mushrooms and provide a foundation for understanding of DNMtases in basidiomycetes.

  15. Comparative Analysis of DNA Methyltransferase Gene Family in Fungi: A Focus on Basidiomycota

    Science.gov (United States)

    Huang, Ruirui; Ding, Qiangqiang; Xiang, Yanan; Gu, Tingting; Li, Yi

    2016-01-01

    DNA methylation plays a crucial role in the regulation of gene expression in eukaryotes. Mushrooms belonging to the phylum Basidiomycota are highly valued for both nutritional and pharmaceutical uses. A growing number of studies have demonstrated the significance of DNA methylation in the development of plants and animals. However, our understanding of DNA methylation in mushrooms is limited. In this study, we identified and conducted comprehensive analyses on DNA methyltransferases (DNMtases) in representative species from Basidiomycota and Ascomycota, and obtained new insights into their classification and characterization in fungi. Our results revealed that DNMtases in basidiomycetes can be divided into two classes, the Dnmt1 class and the newly defined Rad8 class. We also demonstrated that the fusion event between the characteristic domains of the DNMtases family and Snf2 family in the Rad8 class is fungi-specific, possibly indicating a functional novelty of Rad8 DNMtases in fungi. Additionally, expression profiles of DNMtases in the edible mushroom Pleurotus ostreatus revealed diverse expression patterns in various organs and developmental stages. For example, DNMtase genes displayed higher expression levels in dikaryons than in monokaryons. Consistent with the expression profiles, we found that dikaryons are more susceptible to the DNA methyltransferase inhibitor 5-azacytidine. Taken together, our findings pinpoint an important role of DNA methylation during the growth of mushrooms and provide a foundation for understanding of DNMtases in basidiomycetes. PMID:27818666

  16. Immune response to dna vaccine expressing transferrin binding protein a gene of Pasteurella multocida

    Directory of Open Access Journals (Sweden)

    Satparkash Singh

    2011-06-01

    Full Text Available Haemorrhagic Septicaemia (HS, an acute and fatal disease of cattle and buffalo is primarily caused by serotype B:2 or E:2 of Pasteurella multocida. The transferrin binding protein A (TbpA has been found to act as immunogen and potent vaccine candidate in various Gram negative bacteria including P. multocida. The present study was carried out to evaluate the potential of this antigen as a DNA vaccine against HS in mice model. The tbpA gene of P. multocida serotype B:2 was cloned in a mammalian expression vector alone and along with murine IL2 gene as immunological adjuvant to produce monocistronic and bicistronic DNA vaccine constructs, respectively. The immune response to DNA vaccines was evaluated based on serum antibody titres and lymphocyte proliferation assay. A significant increase in humoral and cell mediated immune responses was observed in mice vaccinated with DNA vaccines as compared to non immunized group. Additionally, the bicistronic DNA vaccine provided superior immune response and protection level following challenge as compared to monocistronic construct. The study revealed that DNA vaccine presents a promising approach for the prevention of HS.

  17. Immune response to dna vaccine expressing transferrin binding protein a gene of Pasteurella multocida.

    Science.gov (United States)

    Singh, Satparkash; Singh, Vijendra Pal; Cheema, Pawanjit Singh; Sandey, Maninder; Ranjan, Rajeev; Gupta, Santosh Kumar; Sharma, Bhaskar

    2011-04-01

    Haemorrhagic Septicaemia (HS), an acute and fatal disease of cattle and buffalo is primarily caused by serotype B:2 or E:2 of Pasteurella multocida. The transferrin binding protein A (TbpA) has been found to act as immunogen and potent vaccine candidate in various Gram negative bacteria including P. multocida. The present study was carried out to evaluate the potential of this antigen as a DNA vaccine against HS in mice model. The tbpA gene of P. multocida serotype B:2 was cloned in a mammalian expression vector alone and along with murine IL2 gene as immunological adjuvant to produce monocistronic and bicistronic DNA vaccine constructs, respectively. The immune response to DNA vaccines was evaluated based on serum antibody titres and lymphocyte proliferation assay. A significant increase in humoral and cell mediated immune responses was observed in mice vaccinated with DNA vaccines as compared to non immunized group. Additionally, the bicistronic DNA vaccine provided superior immune response and protection level following challenge as compared to monocistronic construct. The study revealed that DNA vaccine presents a promising approach for the prevention of HS.

  18. DNA methylation and gene expression regulation associated with vascularization in Sorghum bicolor.

    Science.gov (United States)

    Turco, Gina M; Kajala, Kaisa; Kunde-Ramamoorthy, Govindarajan; Ngan, Chew-Yee; Olson, Andrew; Deshphande, Shweta; Tolkunov, Denis; Waring, Barbara; Stelpflug, Scott; Klein, Patricia; Schmutz, Jeremy; Kaeppler, Shawn; Ware, Doreen; Wei, Chia-Lin; Etchells, J Peter; Brady, Siobhan M

    2017-05-01

    Plant secondary cell walls constitute the majority of plant biomass. They are predominantly found in xylem cells, which are derived from vascular initials during vascularization. Little is known about these processes in grass species despite their emerging importance as biomass feedstocks. The targeted biofuel crop Sorghum bicolor has a sequenced and well-annotated genome, making it an ideal monocot model for addressing vascularization and biomass deposition. Here we generated tissue-specific transcriptome and DNA methylome data from sorghum shoots, roots and developing root vascular and nonvascular tissues. Many genes associated with vascular development in other species show enriched expression in developing vasculature. However, several transcription factor families varied in vascular expression in sorghum compared with Arabidopsis and maize. Furthermore, differential expression of genes associated with DNA methylation were identified between vascular and nonvascular tissues, implying that changes in DNA methylation are a feature of sorghum root vascularization, which we confirmed using tissue-specific DNA methylome data. Roots treated with a DNA methylation inhibitor also showed a significant decrease in root length. Tissues and organs can be discriminated based on their genomic methylation patterns and methylation context. Consequently, tissue-specific changes in DNA methylation are part of the normal developmental process. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  19. Comparative Analysis of DNA Methyltransferase Gene Family in Fungi: A Focus on Basidiomycota.

    Science.gov (United States)

    Huang, Ruirui; Ding, Qiangqiang; Xiang, Yanan; Gu, Tingting; Li, Yi

    2016-01-01

    DNA methylation plays a crucial role in the regulation of gene expression in eukaryotes. Mushrooms belonging to the phylum Basidiomycota are highly valued for both nutritional and pharmaceutical uses. A growing number of studies have demonstrated the significance of DNA methylation in the development of plants and animals. However, our understanding of DNA methylation in mushrooms is limited. In this study, we identified and conducted comprehensive analyses on DNA methyltransferases (DNMtases) in representative species from Basidiomycota and Ascomycota, and obtained new insights into their classification and characterization in fungi. Our results revealed that DNMtases in basidiomycetes can be divided into two classes, the Dnmt1 class and the newly defined Rad8 class. We also demonstrated that the fusion event between the characteristic domains of the DNMtases family and Snf2 family in the Rad8 class is fungi-specific, possibly indicating a functional novelty of Rad8 DNMtases in fungi. Additionally, expression profiles of DNMtases in the edible mushroom Pleurotus ostreatus revealed diverse expression patterns in various organs and developmental stages. For example, DNMtase genes displayed higher expression levels in dikaryons than in monokaryons. Consistent with the expression profiles, we found that dikaryons are more susceptible to the DNA methyltransferase inhibitor 5-azacytidine. Taken together, our findings pinpoint an important role of DNA methylation during the growth of mushrooms and provide a foundation for understanding of DNMtases in basidiomycetes.

  20. Optimization of ectopic gene expression in skeletal muscle through DNA transfer by electroporation

    Directory of Open Access Journals (Sweden)

    Latour Mickey

    2004-05-01

    Full Text Available Abstract Background Electroporation (EP is a widely used non-viral gene transfer method. We have attempted to develop an exact protocol to maximize DNA expression while minimizing tissue damage following EP of skeletal muscle in vivo. Specifically, we investigated the effects of varying injection techniques, electrode surface geometry, and plasmid mediums. Results We found that as the amount of damage increased in skeletal muscle in response to EP, the level of β-galactosidase (β-gal expression drastically decreased and that there was no evidence of β-gal expression in damaged fibers. Two specific types of electrodes yielded the greatest amount of expression. We also discovered that DNA uptake in skeletal muscle following intra-arterial injection of DNA was significantly enhanced by EP. Finally, we found that DMSO and LipoFECTAMINE™, common enhancers of DNA electroporation in vitro, had no positive effect on DNA electroporation in vivo. Conclusions When injecting DNA intramuscularly, a flat plate electrode without any plasmid enhancers is the best method to achieve high levels of gene expression.

  1. Structural and functional conservation of two human homologs of the yeast DNA repair gene RAD6.

    NARCIS (Netherlands)

    M.H.M. Koken (Marcel); P. Reynolds (Paul); I. Jaspers-Dekker (Iris); L. Prakash; S. Prakash; D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1991-01-01

    textabstractThe RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme (E2) that is required for DNA repair, damage-induced mutagenesis, and sporulation. We have cloned the two human RAD6 homologs, designated HHR6A and HHR6B. The two 152-amino acid human proteins share 95% sequ

  2. Establishing a Gene Trap System Mediated by T-DNA(GUS) in Rice

    Institute of Scientific and Technical Information of China (English)

    Shi-Yan Chen; Ai-Min Wang; Wei Li; Zong-Yang Wang; Xiu-Ling Cai

    2008-01-01

    Two plasmids, p13GUS and p13GUS2, were constructed to create a gene trap system containing the promoterless β-glucuronidase (GUS) reporter gene in the T-DNA region. Transformation of these two plasmids into the rice variety Zhonghua 11 (Oryza sativa ssp. japonica cv.), mediated by Agrobacterium tumefaciens, resulted in 942 independent transgenic lines. Histochemical GUS assays revealed that 31 To plants had various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. Hygromycin-resistant (hygr) homozygotes were screened and the copy number of the T-DNA inserts was determined in the GUS-positivs transgenic plants. The flanking sequences of the T-DNA were isolated by inverse-polymerase chain reaction and the insert positions on the rice genome of T-DNA were determined by a basic local alignment search tool in the GUS-positive transgenic plants transformed with plasmid p13GUS. Moreover, calii induced from the seeds of the T1 generation of 911 GUS-negative transgenic lines were subjected to stress and hormone treatments. Histochemical GUS assays were carried out on the calli before and after treatment. The results revealed that calli from 21 lines displayed differential GUS expression after treatment. All of these data demonstrated that this trap system is suitable for identifying rice genes, including those that are sensitive to induction.

  3. Benzo[a]pyrene decreases global and gene specific DNA methylation during zebrafish development

    Science.gov (United States)

    DNA methylation is important for gene regulation and is vulnerable to early-life exposure to environmental contaminants. We found that direct waterborne benzo[a]pyrene (BaP) exposure at 24 'g/L from 2.5 to 96 hours post fertilization (hpf) to zebrafish embryos significantly decreased global cytosine...

  4. DNA repair in human cells: from genetic complementation to isolation of genes.

    NARCIS (Netherlands)

    D. Bootsma (Dirk); A. Westerveld (Andries); J.H.J. Hoeijmakers (Jan)

    1988-01-01

    textabstractThe genetic disease xeroderma pigmentosum (XP) demonstrates the association between defective repair of DNA lesions and cancer. Complementation analysis performed on XP cell strains and on repair deficient rodent cell lines has revealed that at least nine and possibly more than 13 genes

  5. Cloning and characterization of the human DNA-excision repair gene ERCC-1

    NARCIS (Netherlands)

    M. van Duin (Michel)

    1988-01-01

    textabstractIt is the aim of the work described in this thesis to isolate and characterize human genes involved DNA excision repair. This will facilitate the understanding of the mechanism of this repair process whereas it also provides an important step to better understand the relationship

  6. Candidate genes for cross-resistance against DNA-damaging drugs

    DEFF Research Database (Denmark)

    Wittig, Rainer; Nessling, Michelle; Will, Rainer D

    2002-01-01

    Drug resistance of tumor cells leads to major drawbacks in the treatment of cancer. To identify candidate genes for drug resistance, we compared the expression patterns of the drug-sensitive human malignant melanoma cell line MeWo and three derived sublines with acquired resistance to the DNA-dam...

  7. Cloning and characterization of the human DNA-excision repair gene ERCC-1

    NARCIS (Netherlands)

    M. van Duin (Michel)

    1988-01-01

    textabstractIt is the aim of the work described in this thesis to isolate and characterize human genes involved DNA excision repair. This will facilitate the understanding of the mechanism of this repair process whereas it also provides an important step to better understand the relationship between

  8. DNA repair in human cells: from genetic complementation to isolation of genes.

    NARCIS (Netherlands)

    D. Bootsma (Dirk); A. Westerveld (Andries); J.H.J. Hoeijmakers (Jan)

    1988-01-01

    textabstractThe genetic disease xeroderma pigmentosum (XP) demonstrates the association between defective repair of DNA lesions and cancer. Complementation analysis performed on XP cell strains and on repair deficient rodent cell lines has revealed that at least nine and possibly more than 13 genes

  9. Complex Oncogenic Translocations with Gene Amplification are Initiated by Specific DNA Breaks in Lymphocytes

    OpenAIRE

    2009-01-01

    Chromosomal instability is a hallmark of many tumor types. Complex chromosomal rearrangements with associated gene amplification, known as complicons, characterize many hematologic and solid cancers. While chromosomal aberrations, including complicons, are useful diagnostic and prognostic cancer markers, their molecular origins are not known. Although accumulating evidence has implicated DNA double strand break repair in suppression of oncogenic genome instability, the genomic elements requir...

  10. DNA polymorphism of HLA class II genes in systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Cowland, J B; Andersen, V; Halberg, P

    1994-01-01

    We investigated the DNA restriction fragment length polymorphism (RFLP) of the major histocompatibility complex (MHC) genes: HLA-DRB, -DQA, -DQB, -DPB in 24 Danish patients with systemic lupus erythematosus (SLE) and in 102 healthy Danes. A highly significant increase of the frequency of the DR3...

  11. Characterization of mouse Clpp protease cDNA, gene, and protein

    DEFF Research Database (Denmark)

    Andresen, B S; Corydon, T J; Wilsbech, M

    2000-01-01

    characterized the mouse Clpp cDNA sequence, the organization of the mouse gene, the chromosomal localization, and the tissue-specific expression pattern. Moreover. the cellular localization and processing of mouse Clpp was studied by overexpression in transfected eukaryotic cells. Our results indicate...

  12. DEMETER DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation.

    Science.gov (United States)

    Gehring, Mary; Huh, Jin Hoe; Hsieh, Tzung-Fu; Penterman, Jon; Choi, Yeonhee; Harada, John J; Goldberg, Robert B; Fischer, Robert L

    2006-02-10

    MEDEA (MEA) is an Arabidopsis Polycomb group gene that is imprinted in the endosperm. The maternal allele is expressed and the paternal allele is silent. MEA is controlled by DEMETER (DME), a DNA glycosylase required to activate MEA expression, and METHYLTRANSFERASE I (MET1), which maintains CG methylation at the MEA locus. Here we show that DME is responsible for endosperm maternal-allele-specific hypomethylation at the MEA gene. DME can excise 5-methylcytosine in vitro and when expressed in E. coli. Abasic sites opposite 5-methylcytosine inhibit DME activity and might prevent DME from generating double-stranded DNA breaks. Unexpectedly, paternal-allele silencing is not controlled by DNA methylation. Rather, Polycomb group proteins that are expressed from the maternal genome, including MEA, control paternal MEA silencing. Thus, DME establishes MEA imprinting by removing 5-methylcytosine to activate the maternal allele. MEA imprinting is subsequently maintained in the endosperm by maternal MEA silencing the paternal allele.

  13. DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

    DEFF Research Database (Denmark)

    Sander, Adam F.; Lavstsen, Thomas; Rask, Thomas Salhøj

    2014-01-01

    -wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum......-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers...... of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens....

  14. In-channel printing-device opening assay for micropatterning multiple cells and gene analysis.

    Science.gov (United States)

    Zhou, Hao; Zhao, Liang; Zhang, Xueji

    2015-02-17

    Herein we report an easy but versatile method for patterning different cells on a single substrate by using a microfluidic approach that allows not only spatial and temporal control of multiple microenvironments but also retrieval of specific treated cells to profile their expressed genetic information at around 10-cell resolution. By taking advantages of increased surface area of gold nanoparticles on a poly(dimethylsiloxane) (PDMS) coated substrate, cell adhesive-promotive protein, human fibronectin (hFN) can be significantly accumulated on designed regions where cells can recognize the protein and spread out. Moreover, the whole device can be easily opened by hand without any loss of patterned cells which could be retrieved by mouth-pipet. Consequently, we demonstrate the possibility of analyzing the difference of gene expression patterns between wild type MCF-7 cell and MCF/Adr (drug-resistant cell line) from less than 400 cells in total for a single comprehensive assay, including parallel experiments, controls, and multiple dose treatments. Certain genes, especially the P-glycoprotein coding gene (ABCB1), show high expression level in resistant cells compared with the wild type, suggesting a possible pathway that may contribute to the antidrug mechanism.

  15. Gene expression regulation by upstream open reading frames and human disease.

    Directory of Open Access Journals (Sweden)

    Cristina Barbosa

    Full Text Available Upstream open reading frames (uORFs are major gene expression regulatory elements. In many eukaryotic mRNAs, one or more uORFs precede the initiation codon of the main coding region. Indeed, several studies have revealed that almost half of human transcripts present uORFs. Very interesting examples have shown that these uORFs can impact gene expression of the downstream main ORF by triggering mRNA decay or by regulating translation. Also, evidence from recent genetic and bioinformatic studies implicates disturbed uORF-mediated translational control in the etiology of many human diseases, including malignancies, metabolic or neurologic disorders, and inherited syndromes. In this review, we will briefly present the mechanisms through which uORFs regulate gene expression and how they can impact on the organism's response to different cell stress conditions. Then, we will emphasize the importance of these structures by illustrating, with specific examples, how disturbed uORF-mediated translational control can be involved in the etiology of human diseases, giving special importance to genotype-phenotype correlations. Identifying and studying more cases of uORF-altering mutations will help us to understand and establish genotype-phenotype associations, leading to advancements in diagnosis, prognosis, and treatment of many human disorders.

  16. Regulated expression of the Saccharomyces cerevisiae DNA repair gene RAD7 in response to DNA damage and during sporulation.

    Science.gov (United States)

    Jones, J S; Prakash, L; Prakash, S

    1990-06-11

    The RAD7 gene of Saccharomyces cerevisiae affects the proficiency of excision repair of DNA damaged by UV light. Here, we report our studies on the regulation of the RAD7 gene in response to UV irradiation and during sporulation. RAD7 transcript levels increased 6-fold within 40 min of exposure of cells to 37 J/m2 of UV light. Higher UV doses also elicited rapid increases in the level of RAD7 mRNA. RAD7 mRNA levels increased in sporulating MATa/MAT alpha diploid cells, but not in the asporogenous MATa/MATa strain exposed to sporulation conditions. The increase in RAD7 mRNA level in MATa/MAT alpha cells was 15-fold after 6 h and 9-fold after 7 h in sporulation medium; thereafter, RAD7 mRNA levels declined. Periodic transcription of RAD7 during sporulation suggests a role for RAD7 in this process.

  17. Phylogenetic analysis and possible function of bro-like genes, a multigene family widespread among large double-stranded DNA viruses of invertebrates and bacteria.

    Science.gov (United States)

    Bideshi, Dennis K; Renault, Sylvaine; Stasiak, Karine; Federici, Brian A; Bigot, Yves

    2003-09-01

    Baculovirus repeated open reading frame (bro) genes and their relatives constitute a multigene family, typically with multiple copies per genome, known to occur among certain insect dsDNA viruses and bacteriophages. Little is known about the evolutionary history and function of the proteins encoded by these genes. Here we have shown that bro and bro-like (bro-l) genes occur among viruses of two additional invertebrate viral families, Ascoviridae and Iridoviridae, and in prokaryotic class II transposons. Analysis of over 100 sequences showed that the N-terminal region, consisting of two subdomains, is the most conserved region and contains a DNA-binding motif that has been characterized previously. Phylogenetic analysis indicated that these proteins are distributed among eight groups, Groups 1-7 consisting of invertebrate virus proteins and Group 8 of proteins in bacteriophages and bacterial transposons. No bro genes were identified in databases of invertebrate or vertebrate genomes, vertebrate viruses and transposons, nor in prokaryotic genomes, except in prophages or transposons of the latter. The phylogenetic relationship between bro genes suggests that they have resulted from recombination of viral genomes that allowed the duplication and loss of genes, but also the acquisition of genes by horizontal transfer over evolutionary time. In addition, the maintenance and diversity of bro-l genes in different types of invertebrate dsDNA viruses, but not in vertebrate viruses, suggests that these proteins play an important role in invertebrate virus biology. Experiments with the unique orf2 bro gene of Autographa californica multicapsid nucleopolyhedrovirus showed that it is not required for replication, but may enhance replication during the occlusion phase of reproduction.

  18. p21WAF1/CIP1 gene DNA sequencing and its expression in human osteosarcoma

    Institute of Scientific and Technical Information of China (English)

    廖威明; 张春林; 李佛保; 曾炳芳; 曾益新

    2004-01-01

    Background Mutation and expression change of p21WAF1/CIP1 may play a role in the growth of osteosarcoma. This study was to investigate the expression of the p21WAF1/CIP1 gene in human osteosarcoma, p21WAF1/CIP1 gene DNA sequence change and their relationships with the phenotype and clinical prognosis.Methods p21WAF1/CIP1 gene in 10 normal people and the tumours of 45 osteosarcoma patients were examined using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) with silver staining. The PCR product with an abnormal strand was sequenced directly. The p21WAF1/CIP1 gene mRNA and P21 protein of 45 cases of osteosarcoma were investigated by using in situ hybridization and immunohistochemistry, respectively. Results The occurrence of P21 protein in osteosarcoma was 17.78% (8/45), and p21WAF1/CIP1 mRNA expression in osteosarcoma was 42.22% (19/45). The p21WAF1/CIP1 gene DNA sequencing of amplified production showed that in p21WAF1/CIP1 gene exon 3 of 36 cases of human osteosarcoma, there were 17 cases (47.22%) with C→T at position 609; 10 normal blood samples' DNA sequence analysis yielded 8 cases (80.00%) with C→T at the same position. Conclusions Along with the increase of malignancy, the expression of p21WAF1/CIP1mRNA and P21 protein in osteosarcoma tends to decrease. It is uncommon for the p21WAF1/CIP1 gene mutation to occur in human osteosarcoma. As a result, the possible existence of tumour subtypes of p21WAF1/CIP1 gene mutation should be investigated. Our research leads to the location of p21WAF1/CIP1 gene polymorphism of Chinese osteosarcoma patients, which can provide a basis for further research.

  19. ANALYSIS OF GENES ASSOCIATED WITH LYMPHATIC METASTASIS IN PANCREATIC CARCINOMA USING cDNA MICROARRAY

    Institute of Scientific and Technical Information of China (English)

    谭志军; 胡先贵; 曹贵松; 唐岩

    2003-01-01

    Objective: To identify new markers for prediction of lymph node metastasis. Methods: cDNA probes were prepared by labeling mRNA from samples of four pancreatic carcinoma tissues with Cy5-dUTP and mRNA from adjacent normal tissues with Cy3-dUTP respectively through reverse transcription. The mixed probes of each sample were then hybridized with 4,096 cDNA arrays (4,000 unique human cDNA sequences), and the fluorescent signals were scanned by ScanArray 3000 scanner (General Scanning, Inc.). The values of Cy5-dUTP and Cy3-dUTP on each spot were analyzed and calculated by ImaGene 3.0 software (BioDiscovery, Inc.). Genes that differentially expresses in each cancerous tissue were sought out according to the standard that the absolute value of natural logarithm of the ratio of Cy5 to Cy3 is greater than 0.69, i. e., more than 2 times change of gene expression, and the signal value of either Cy3 and Cy5 need to be greater than 600. Then, the genes differently expressed in cancer with and without lymphatic metastasis were screened out for further analysis. Results: Among 2 samples with lymphatic metastasis and 2 samples without metastasis, 56 genes, which accounted for 1.40% of genes on the microarray slides, exhibited differentially expression in cancerous tissues with lymphatic metastasis. There were 32 over-expressed genes including 11 having been registered in Genebank, and 24 under-expressed genes including 3 in Genebank. Conclusion: Microarray analysis may provide invaluable information to identify specific gene expression profile of lymphatic metastasis in pancreatic cancer.

  20. Cloning large natural product gene clusters from the environment: Piecing environmental DNA gene clusters back together with TAR

    OpenAIRE

    Kim, Jeffrey H.; Feng, Zhiyang; Bauer, John D.; Kallifidas, Dimitris; Calle, Paula Y.; Brady, Sean F

    2010-01-01

    A single gram of soil can contain thousands of unique bacterial species, of which only a small fraction is regularly cultured in the laboratory. Although the fermentation of cultured microorganisms has provided access to numerous bioactive secondary metabolites, with these same methods it is not possible to characterize the natural products encoded by the uncultured majority. The heterologous expression of biosynthetic gene clusters cloned from DNA extracted directly from environmental sample...

  1. Evaluation of OPEN zinc finger nucleases for direct gene targeting of the ROSA26 locus in mouse embryos.

    Directory of Open Access Journals (Sweden)

    Mario Hermann

    Full Text Available Zinc finger nucleases (ZFNs enable precise genome modification in a variety of organisms and cell types. Commercial ZFNs were reported to enhance gene targeting directly in mouse zygotes, whereas similar approaches using publicly available resources have not yet been described. Here we report precise targeted mutagenesis of the mouse genome using Oligomerized Pool Engineering (OPEN ZFNs. OPEN ZFN can be constructed using publicly available resources and therefore provide an attractive alternative for academic researchers. Two ZFN pairs specific to the mouse genomic locus gt(ROSA26Sor were generated by OPEN selections and used for gene disruption and homology-mediated gene replacement in single cell mouse embryos. One specific ZFN pair facilitated non-homologous end joining (NHEJ-mediated gene disruption when expressed in mouse zygotes. We also observed a single homologous recombination (HR-driven gene replacement event when this ZFN pair was co-injected with a targeting vector. Our experiments demonstrate the feasibility of achieving both gene ablation through NHEJ and gene replacement by HR by using the OPEN ZFN technology directly in mouse zygotes.

  2. FISH comets show that the salvage enzyme TK1 contributes to gene-specific DNA repair

    Directory of Open Access Journals (Sweden)

    Stephen eDownes

    2014-08-01

    Full Text Available Thymidine kinase 1 (TK1 is a salvage enzyme that phosphorylates thymidine, imported from surrounding fluids, to create dTMP, which is further phosphorylated to the DNA precursor dTTP. TK1 deficiency has for a long time been known to cause increased cellular sensitivity to DNA damage. We have examined preferential strand break repair of DNA domains in TK1+ and TK1- clones of the Raji cell line, by the Comet-FISH technique, in bulk DNA and in the actively transcribed tumour suppressor (TP53 and human telomerase reverse transcriptase (hTERT gene regions, over 1 hour after 5Gy γ-irradiation. Results showed that repair of the TP53 and hTERT gene regions was more efficient in TK1+ compared to TK1- cells, while levels of genomic DNA repair were consistant between the two cell-lines. The targeted gene-specific repair in TK+ cells occurred rapidly, mainly over the first 15 minute repair-period. Therefore, TK1 is needed for preferential repair of actively transcribed regions, through a previously unsuspected mechanism. In principle, TK1 could exert its protective effects through supply of a supplementary dTTP pool for accurate repair of damaged genes; but Raji TK1+ cells in thymidine free media still show preferential repair of transcribed regions. TK1 therefore does not exert its protective effects through dTTP pools, but through another unidentified mechanism, which affects sensitivity to and mutagenicity by DNA damaging agents.

  3. FISH comets show that the salvage enzyme TK1 contributes to gene-specific DNA repair

    Science.gov (United States)

    McAllister, Katherine A.; Yasseen, Akeel A.; McKerr, George; Downes, C. S.; McKelvey-Martin, Valerie J.

    2014-01-01

    Thymidine kinase 1 (TK1) is a salvage enzyme that phosphorylates thymidine, imported from surrounding fluids, to create dTMP, which is further phosphorylated to the DNA precursor dTTP. TK1 deficiency has for a long time been known to cause increased cellular sensitivity to DNA damage. We have examined preferential strand break repair of DNA domains in TK1+ and TK1- clones of the Raji cell line, by the Comet-FISH technique, in bulk DNA and in the actively transcribed tumor suppressor (TP53) and human telomerase reverse transcriptase (hTERT) gene regions, over 1 h after 5Gy γ-irradiation. Results showed that repair of the TP53 and hTERT gene regions was more efficient in TK1+ compared to TK1- cells, a trend also reflected to a lesser degree in genomic DNA repair between the cell-lines. The targeted gene-specific repair in TK+ cells occurred rapidly, mainly over the first 15 min repair-period. Therefore, TK1 is needed for preferential repair of actively transcribed regions, through a previously unsuspected mechanism. In principle, TK1 could exert its protective effects through supply of a supplementary dTTP pool for accurate repair of damaged genes; but Raji TK1+ cells in thymidine free media still show preferential repair of transcribed regions. TK1 therefore does not exert its protective effects through dTTP pools, but through another unidentified mechanism, which affects sensitivity to and mutagenicity by DNA damaging agents. PMID:25152750

  4. GC-biased gene conversion impacts ribosomal DNA evolution in vertebrates, angiosperms, and other eukaryotes.

    Science.gov (United States)

    Escobar, Juan S; Glémin, Sylvain; Galtier, Nicolas

    2011-09-01

    Ribosomal DNA (rDNA) is one of the most conserved genes in eukaryotes. The multiples copies of rDNA in the genome evolve in a concerted manner, through unequal crossing over and/or gene conversion, two mechanisms related to homologous recombination. Recombination increases local GC content in several organisms through a process known as GC-biased gene conversion (gBGC). gBGC has been well characterized in mammals, birds, and grasses, but its phylogenetic distribution across the tree of life is poorly understood. Here, we test the hypothesis that recombination affects the evolution of base composition in 18S rDNA and examine the reliability of this thoroughly studied molecule as a marker of gBGC in eukaryotes. Phylogenetic analyses of 18S rDNA in vertebrates and angiosperms reveal significant heterogeneity in the evolution of base composition across both groups. Mammals, birds, and grasses experience increases in the GC content of the 18S rDNA, consistent with previous genome-wide analyses. In addition, we observe increased GC contents in Ostariophysi ray-finned fishes and commelinid monocots (i.e., the clade including grasses), suggesting that the genomes of these two groups have been affected by gBGC. Polymorphism analyses in rDNA confirm that gBGC, not mutation bias, is the most plausible explanation for these patterns. We also find that helix and loop sites of the secondary structure of ribosomal RNA do not evolve at the same pace: loops evolve faster than helices, whereas helices are GC richer than loops. We extend analyses to major lineages of eukaryotes and suggest that gBGC might have also affected base composition in Giardia (Diplomonadina), nudibranch gastropods (Mollusca), and Asterozoa (Echinodermata).

  5. Gene Analysis of Free Fetal DNA in Maternal Plasma

    Institute of Scientific and Technical Information of China (English)

    CHEN; Hanping(

    2001-01-01

    [1]王凤水 王宇.基因扩增产物的固相杂交--酶联显色方法的建立.中国生物化学与分子生物学报 1998 14(2):192[2]Lin Z Xiang F C.Whole genome amplification from a single cell:implication for genetic analysis.Proc Natl Acad Sci USA 1992 89:5847[3]Lo Y M D Waiscoat J S Pater P et al.Prenatal sex determination by DNA amplification from maternal blood.Lancet 1989 2:1363[4]Lo Y M D Pater P Baigent C N et al.Prenatal sex determination from maternal peripheral blood using the polymerase chain reaction.Hum Genet 1993 90:483[5]Adindfi M Gillmer M D G.Non-invasive approaches to the prenatal diagnosis of selected inherited disorders using fetal nucleated cells eventually present in the maternal circulation.Prenat Diag 1992 12:1[6]Bianchi D W Mahr A Zickwolf G K et al.Detection of fetal with 47 XY +21 karyotype in maternal peripheral blood.Hum Genet 1992 90:368

  6. DNA transposon activity is associated with increased mutation rates in genes of rice and other grasses.

    Science.gov (United States)

    Wicker, Thomas; Yu, Yeisoo; Haberer, Georg; Mayer, Klaus F X; Marri, Pradeep Reddy; Rounsley, Steve; Chen, Mingsheng; Zuccolo, Andrea; Panaud, Olivier; Wing, Rod A; Roffler, Stefan

    2016-09-07

    DNA (class 2) transposons are mobile genetic elements which move within their 'host' genome through excising and re-inserting elsewhere. Although the rice genome contains tens of thousands of such elements, their actual role in evolution is still unclear. Analysing over 650 transposon polymorphisms in the rice species Oryza sativa and Oryza glaberrima, we find that DNA repair following transposon excisions is associated with an increased number of mutations in the sequences neighbouring the transposon. Indeed, the 3,000 bp flanking the excised transposons can contain over 10 times more mutations than the genome-wide average. Since DNA transposons preferably insert near genes, this is correlated with increases in mutation rates in coding sequences and regulatory regions. Most importantly, we find this phenomenon also in maize, wheat and barley. Thus, these findings suggest that DNA transposon activity is a major evolutionary force in grasses which provide the basis of most food consumed by humankind.

  7. Optimizing hyaluronidase dose and plasmid DNA delivery greatly improves gene electrotransfer efficiency in rat skeletal muscle

    DEFF Research Database (Denmark)

    Åkerström, Thorbjörn; Vedel, Kenneth; Needham Andersen, Josefine

    2015-01-01

    Transfection of rat skeletal muscle in vivo is a widely used research model. However, gene electrotransfer protocols have been developed for mice and yield variable results in rats. We investigated whether changes in hyaluronidase pre-treatment and plasmid DNA delivery can improve transfection...... efficiency in rat skeletal muscle. We found that pre-treating the muscle with a hyaluronidase dose suitable for rats (0.56. U/g b.w.) prior to plasmid DNA injection increased transfection efficiency by >200% whereas timing of the pre-treatment did not affect efficiency. Uniformly distributing plasmid DNA...... delivery across the muscle by increasing the number of plasmid DNA injections further enhanced transfection efficiency whereas increasing plasmid dose from 0.2 to 1.6. μg/g b.w. or vehicle volume had no effect. The optimized protocol resulted in ~80% (CI95%: 79-84%) transfected muscle fibers...

  8. DNA Nanotechnology for Precise Control over Drug Delivery and Gene Therapy.

    Science.gov (United States)

    Angell, Chava; Xie, Sibai; Zhang, Liangfang; Chen, Yi

    2016-03-02

    Nanomedicine has been growing exponentially due to its enhanced drug targeting and reduced drug toxicity. It uses the interactions where nanotechnological components and biological systems communicate with each other to facilitate the delivery performance. At this scale, the physiochemical properties of delivery systems strongly affect their capacities. Among current delivery systems, DNA nanotechnology shows many advantages because of its unprecedented engineering abilities. Through molecular recognition, DNA nanotechnology can be used to construct a variety of nanostructures with precisely controllable size, shape, and surface chemistry, which can be appreciated in the delivery process. In this review, different approaches that are currently used for the construction of DNA nanostructures are reported. Further, the utilization of these DNA nanostructures with the well-defined parameters for the precise control in drug delivery and gene therapy is discussed.

  9. Plasma cell differentiation is coupled to division-dependent DNA hypomethylation and gene regulation.

    Science.gov (United States)

    Barwick, Benjamin G; Scharer, Christopher D; Bally, Alexander P R; Boss, Jeremy M

    2016-10-01

    The epigenetic processes that regulate antibody-secreting plasma cells are not well understood. Here, analysis of plasma cell differentiation revealed DNA hypomethylation of 10% of CpG loci that were overrepresented at enhancers. Inhibition of DNA methylation enhanced plasma cell commitment in a cell-division-dependent manner. Analysis of B cells differentiating in vivo stratified by cell division revealed a fivefold increase in mRNA transcription coupled to DNA hypomethylation. Demethylation occurred first at binding motifs for the transcription factors NF-κB and AP-1 and later at those for the transcription factors IRF and Oct-2 and was coincident with activation and differentiation gene-expression programs in a cell-division-dependent manner. These data provide mechanistic insight into cell-division-coupled transcriptional and epigenetic reprogramming and suggest that DNA hypomethylation reflects the cis-regulatory history of plasma cell differentiation.

  10. Individual variation of adipose gene expression and identification of covariated genes by cDNA microarrays

    NARCIS (Netherlands)

    Boeuf, S.; Keijer, J.; Franssen-Hal, van N.L.W.; Klaus, S.

    2002-01-01

    Gene expression profiling through the application of microarrays provides comprehensive assessment of gene expression levels in a given tissue or cell population, as well as information on changes of gene expression in altered physiological or pathological situations. Microarrays are particularly su

  11. Species-level para- and polyphyly in DNA barcode gene trees

    DEFF Research Database (Denmark)

    Mutanen, Marko; Kivelä, Sami M.; Vos, Rutger A.

    2016-01-01

    between species and gene genealogies, as indicated by situations where conspecific individuals do not form a monophyletic cluster in a gene tree. In two previous reviews, non-monophyly has been reported as being common in mitochondrial DNA gene trees. We developed a novel web service "Monophylizer...... to their nearest neighbors or exceptionally high levels of intraspecific variability. Cases of polyphyly in COI trees arising as a result of deep intraspecific divergence are negligible, as the detected cases reflected misidentifications or methodological errors. Taking into consideration variation in sampling...

  12. Conditions for gene disruption by homologous recombination of exogenous DNA into the Sulfolobus solfataricus genome

    Directory of Open Access Journals (Sweden)

    Sonja-Verena Albers

    2008-01-01

    Full Text Available The construction of directed gene deletion mutants is an essential tool in molecular biology that allows functional studies on the role of genes in their natural environment. For hyperthermophilic archaea, it has been difficult to obtain a reliable system to construct such mutants. However, during the past years, systems have been developed for Thermococcus kodakarensis and two Sulfolobus species, S. acidocaldarius and derivatives of S. solfataricus 98/2. Here we describe an optimization of the method for integration of exogenous DNA into S. solfataricus PBL 2025, an S. solfataricus 98/2 derivative, based on lactose auxotrophy that now allows for routine gene inactivation.

  13. In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma.

    Directory of Open Access Journals (Sweden)

    Bekim Sadikovic

    Full Text Available Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.

  14. Genomic survey, gene expression analysis and structural modeling suggest diverse roles of DNA methyltransferases in legumes.

    Directory of Open Access Journals (Sweden)

    Rohini Garg

    Full Text Available DNA methylation plays a crucial role in development through inheritable gene silencing. Plants possess three types of DNA methyltransferases (MTases, namely Methyltransferase (MET, Chromomethylase (CMT and Domains Rearranged Methyltransferase (DRM, which maintain methylation at CG, CHG and CHH sites. DNA MTases have not been studied in legumes so far. Here, we report the identification and analysis of putative DNA MTases in five legumes, including chickpea, soybean, pigeonpea, Medicago and Lotus. MTases in legumes could be classified in known MET, CMT, DRM and DNA nucleotide methyltransferases (DNMT2 subfamilies based on their domain organization. First three MTases represent DNA MTases, whereas DNMT2 represents a transfer RNA (tRNA MTase. Structural comparison of all the MTases in plants with known MTases in mammalian and plant systems have been reported to assign structural features in context of biological functions of these proteins. The structure analysis clearly specified regions crucial for protein-protein interactions and regions important for nucleosome binding in various domains of CMT and MET proteins. In addition, structural model of DRM suggested that circular permutation of motifs does not have any effect on overall structure of DNA methyltransferase domain. These results provide valuable insights into role of various domains in molecular recognition and should facilitate mechanistic understanding of their function in mediating specific methylation patterns. Further, the comprehensive gene expression analyses of MTases in legumes provided evidence of their role in various developmental processes throughout the plant life cycle and response to various abiotic stresses. Overall, our study will be very helpful in establishing the specific functions of DNA MTases in legumes.

  15. Genomic survey, gene expression analysis and structural modeling suggest diverse roles of DNA methyltransferases in legumes.

    Science.gov (United States)

    Garg, Rohini; Kumari, Romika; Tiwari, Sneha; Goyal, Shweta

    2014-01-01

    DNA methylation plays a crucial role in development through inheritable gene silencing. Plants possess three types of DNA methyltransferases (MTases), namely Methyltransferase (MET), Chromomethylase (CMT) and Domains Rearranged Methyltransferase (DRM), which maintain methylation at CG, CHG and CHH sites. DNA MTases have not been studied in legumes so far. Here, we report the identification and analysis of putative DNA MTases in five legumes, including chickpea, soybean, pigeonpea, Medicago and Lotus. MTases in legumes could be classified in known MET, CMT, DRM and DNA nucleotide methyltransferases (DNMT2) subfamilies based on their domain organization. First three MTases represent DNA MTases, whereas DNMT2 represents a transfer RNA (tRNA) MTase. Structural comparison of all the MTases in plants with known MTases in mammalian and plant systems have been reported to assign structural features in context of biological functions of these proteins. The structure analysis clearly specified regions crucial for protein-protein interactions and regions important for nucleosome binding in various domains of CMT and MET proteins. In addition, structural model of DRM suggested that circular permutation of motifs does not have any effect on overall structure of DNA methyltransferase domain. These results provide valuable insights into role of various domains in molecular recognition and should facilitate mechanistic understanding of their function in mediating specific methylation patterns. Further, the comprehensive gene expression analyses of MTases in legumes provided evidence of their role in various developmental processes throughout the plant life cycle and response to various abiotic stresses. Overall, our study will be very helpful in establishing the specific functions of DNA MTases in legumes.

  16. DNA methylation of extracellular matrix remodeling genes in children exposed to arsenic.

    Science.gov (United States)

    Gonzalez-Cortes, Tania; Recio-Vega, Rogelio; Lantz, Robert Clark; Chau, Binh T

    2017-08-15

    Several novel mechanistic findings regarding to arsenic's pathogenesis has been reported and some of them suggest that the etiology of some arsenic induced diseases are due in part to heritable changes to the genome via epigenetic processes such as DNA methylation, histone maintenance, and mRNA expression. Recently, we reported that arsenic exposure during in utero and early life was associated with impairment in the lung function and abnormal receptor for advanced glycation endproducts (RAGE), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) sputum levels. Based on our results and the reported arsenic impacts on DNA methylation, we designed this study in our cohort of children exposed in utero and early childhood to arsenic with the aim to associate DNA methylation of MMP9, TIMP1 and RAGE genes with its protein sputum levels and with urinary and toenail arsenic levels. The results disclosed hypermethylation in MMP9 promotor region in the most exposed children; and an increase in the RAGE sputum levels among children with the mid methylation level; there were also positive associations between MMP9 DNA methylation with arsenic toenail concentrations; RAGE DNA methylation with iAs, and %DMA; and finally between TIMP1 DNA methylation with the first arsenic methylation. A negative correlation between MMP9 sputum levels with its DNA methylation was registered. In conclusion, arsenic levels were positive associated with the DNA methylation of extracellular matrix remodeling genes;, which in turn could modifies the biological process in which they are involved causing or predisposing to lung diseases. Copyright © 2017. Published by Elsevier Inc.

  17. Immunogenicity of DNA and Recombinant Sendai Virus Vaccines Expressing the HIV-1 gag Gene

    Institute of Scientific and Technical Information of China (English)

    Xia FENG; Shuang-qing YU; Tsugumine Shu; Tetsuro Matano; Mamoru Hasegawa; Xiao-li WANG; Hong-tao MA; Hong-xia LI; Yi ZENG

    2008-01-01

    Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendal virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1(+)-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens.

  18. Associations between DNA methylation and schizophrenia-related intermediate phenotypes - a gene set enrichment analysis.

    Science.gov (United States)

    Hass, Johanna; Walton, Esther; Wright, Carrie; Beyer, Andreas; Scholz, Markus; Turner, Jessica; Liu, Jingyu; Smolka, Michael N; Roessner, Veit; Sponheim, Scott R; Gollub, Randy L; Calhoun, Vince D; Ehrlich, Stefan

    2015-06-03

    Multiple genetic approaches have identified microRNAs as key effectors in psychiatric disorders as they post-transcriptionally regulate expression of thousands of target genes. However, their role in specific psychiatric diseases remains poorly understood. In addition, epigenetic mechanisms such as DNA methylation, which affect the expression of both microRNAs and coding genes, are critical for our understanding of molecular mechanisms in schizophrenia. Using clinical, imaging, genetic, and epigenetic data of 103 patients with schizophrenia and 111 healthy controls of the Mind Clinical Imaging Consortium (MCIC) study of schizophrenia, we conducted gene set enrichment analysis to identify markers for schizophrenia-associated intermediate phenotypes. Genes were ranked based on the correlation between DNA methylation patterns and each phenotype, and then searched for enrichment in 221 predicted microRNA target gene sets. We found the predicted hsa-miR-219a-5p target gene set to be significantly enriched for genes (EPHA4, PKNOX1, ESR1, among others) whose methylation status is correlated with hippocampal volume independent of disease status. Our results were strengthened by significant associations between hsa-miR-219a-5p target gene methylation patterns and hippocampus-related neuropsychological variables. IPA pathway analysis of the respective predicted hsa-miR-219a-5p target genes revealed associated network functions in behavior and developmental disorders. Altered methylation patterns of predicted hsa-miR-219a-5p target genes are associated with a structural aberration of the brain that has been proposed as a possible biomarker for schizophrenia. The (dys)regulation of microRNA target genes by epigenetic mechanisms may confer additional risk for developing psychiatric symptoms. Further study is needed to understand possible interactions between microRNAs and epigenetic changes and their impact on risk for brain-based disorders such as schizophrenia.

  19. A broadly applicable method to characterize large DNA viruses and adenoviruses based on the DNA polymerase gene

    Directory of Open Access Journals (Sweden)

    Montgomery Roy D

    2006-04-01

    Full Text Available Abstract Background Many viral pathogens are poorly characterized, are difficult to culture or reagents are lacking for confirmatory diagnoses. We have developed and tested a robust assay for detecting and characterizing large DNA viruses and adenoviruses. The assay is based on the use of degenerate PCR to target a gene common to these viruses, the DNA polymerase, and sequencing the products. Results We evaluated our method by applying it to fowl adenovirus isolates, catfish herpesvirus isolates, and largemouth bass ranavirus (iridovirus from cell culture and lymphocystis disease virus (iridovirus and avian poxvirus from tissue. All viruses with the exception of avian poxvirus produced the expected product. After optimization of extraction procedures, and after designing and applying an additional primer we were able to produce polymerase gene product from the avian poxvirus genome. The sequence data that we obtained demonstrated the simplicity and potential of the method for routine use in characterizing large DNA viruses. The adenovirus samples were demonstrated to represent 2 types of fowl adenovirus, fowl adenovirus 1 and an uncharacterized avian adenovirus most similar to fowl adenovirus 9. The herpesvirus isolate from blue catfish was shown to be similar to channel catfish virus (Ictalurid herpesvirus 1. The case isolate of largemouth bass ranavirus was shown to exactly match the type specimen and both were similar to tiger frog virus and frog virus 3. The lymphocystis disease virus isolate from largemouth bass was shown to be related but distinct from the two previously characterized lymphocystis disease virus isolates suggesting that it may represent a distinct lymphocystis disease virus species. Conclusion The method developed is rapid and broadly applicable to cell culture isolates and infected tissues. Targeting a specific gene for in the large DNA viruses and adenoviruses provide a common reference for grouping the newly identified

  20. Informational gene phylogenies do not support a fourth domain of life for nucleocytoplasmic large DNA viruses.

    Directory of Open Access Journals (Sweden)

    Tom A Williams

    Full Text Available Mimivirus is a nucleocytoplasmic large DNA virus (NCLDV with a genome size (1.2 Mb and coding capacity ( 1000 genes comparable to that of some cellular organisms. Unlike other viruses, Mimivirus and its NCLDV relatives encode homologs of broadly conserved informational genes found in Bacteria, Archaea, and Eukaryotes, raising the possibility that they could be placed on the tree of life. A recent phylogenetic analysis of these genes showed the NCLDVs emerging as a monophyletic group branching between Eukaryotes and Archaea. These trees were interpreted as evidence for an independent "fourth domain" of life that may have contributed DNA processing genes to the ancestral eukaryote. However, the analysis of ancient evolutionary events is challenging, and tree reconstruction is susceptible to bias resulting from non-phylogenetic signals in the data. These include compositional heterogeneity and homoplasy, which can lead to the spurious grouping of compositionally-similar or fast-evolving sequences. Here, we show that these informational gene alignments contain both significant compositional heterogeneity and homoplasy, which were not adequately modelled in the original analysis. When we use more realistic evolutionary models that better fit the data, the resulting trees are unable to reject a simple null hypothesis in which these informational genes, like many other NCLDV genes, were acquired by horizontal transfer from eukaryotic hosts. Our results suggest that a fourth domain is not required to explain the available sequence data.

  1. TRAV gene usage in pig T-cell receptor alpha cDNA.

    Science.gov (United States)

    Yamamoto, Ryuji; Uenishi, Hirohide; Hatsuse, Hiromi; Sato, Eimei; Awata, Takashi; Yasue, Hiroshi; Takagaki, Yohtaroh

    2005-05-01

    Pig (Sus scrofa) TRA clones were isolated from cDNA libraries of total RNA from two different sources, the thymus of a 1-month-old LW strain pig and the peripheral blood lymphocytes of a 5-month-old Clawn strain pig. Among 103 complete TRA cDNA clones from both sources, 33 different TRAV genes were identified. By comparing their sequence identities against one another, these pig TRAV genes were grouped into 20 subgroups, including 13 subgroups, each containing only a single member. All of these pig subgroups gave corresponding human and mouse functional counterparts, suggesting their functional commonality. An exception was the Va01 gene segment, which lacked a functional human counterpart. The present report provides groundwork for studies on pig TRA expression.

  2. Distinct DNA-based epigenetic switches trigger transcriptional activation of silent genes in human dermal fibroblasts.

    Science.gov (United States)

    Pandian, Ganesh N; Taniguchi, Junichi; Junetha, Syed; Sato, Shinsuke; Han, Le; Saha, Abhijit; AnandhaKumar, Chandran; Bando, Toshikazu; Nagase, Hiroki; Vaijayanthi, Thangavel; Taylor, Rhys D; Sugiyama, Hiroshi

    2014-01-24

    The influential role of the epigenome in orchestrating genome-wide transcriptional activation instigates the demand for the artificial genetic switches with distinct DNA sequence recognition. Recently, we developed a novel class of epigenetically active small molecules called SAHA-PIPs by conjugating selective DNA binding pyrrole-imidazole polyamides (PIPs) with the histone deacetylase inhibitor SAHA. Screening studies revealed that certain SAHA-PIPs trigger targeted transcriptional activation of pluripotency and germ cell genes in mouse and human fibroblasts, respectively. Through microarray studies and functional analysis, here we demonstrate for the first time the remarkable ability of thirty-two different SAHA-PIPs to trigger the transcriptional activation of exclusive clusters of genes and noncoding RNAs. QRT-PCR validated the microarray data, and some SAHA-PIPs activated therapeutically significant genes like KSR2. Based on the aforementioned results, we propose the potential use of SAHA-PIPs as reagents capable of targeted transcriptional activation.

  3. On the origin and evolutionary consequences of gene body DNA methylation.

    Science.gov (United States)

    Bewick, Adam J; Ji, Lexiang; Niederhuth, Chad E; Willing, Eva-Maria; Hofmeister, Brigitte T; Shi, Xiuling; Wang, Li; Lu, Zefu; Rohr, Nicholas A; Hartwig, Benjamin; Kiefer, Christiane; Deal, Roger B; Schmutz, Jeremy; Grimwood, Jane; Stroud, Hume; Jacobsen, Steven E; Schneeberger, Korbinian; Zhang, Xiaoyu; Schmitz, Robert J

    2016-08-01

    In plants, CG DNA methylation is prevalent in the transcribed regions of many constitutively expressed genes (gene body methylation; gbM), but the origin and function of gbM remain unknown. Here we report the discovery that Eutrema salsugineum has lost gbM from its genome, to our knowledge the first instance for an angiosperm. Of all known DNA methyltransferases, only CHROMOMETHYLASE 3 (CMT3) is missing from E. salsugineum Identification of an additional angiosperm, Conringia planisiliqua, which independently lost CMT3 and gbM, supports that CMT3 is required for the establishment of gbM. Detailed analyses of gene expression, the histone variant H2A.Z, and various histone modifications in E. salsugineum and in Arabidopsis thaliana epigenetic recombinant inbred lines found no evidence in support of any role for gbM in regulating transcription or affecting the composition and modification of chromatin over evolutionary timescales.

  4. RNA-DNA differences are rarer in proto-oncogenes than in tumor suppressor genes.

    Science.gov (United States)

    Gao, Feng; Lin, Yan; Zhang, Randy Ren

    2012-01-01

    It has long been assumed that DNA sequences and corresponding RNA transcripts are almost identical; a recent discovery, however, revealed widespread RNA-DNA differences (RDDs), which represent a largely unexplored aspect of human genome variation. It has been speculated that RDDs can affect disease susceptibility and manifestations; however, almost nothing is known about how RDDs are related to disease. Here, we show that RDDs are rarer in proto-oncogenes than in tumor suppressor genes; the number of RDDs in coding exons, but not in 3'UTR and 5'UTR, is significantly lower in the former than the latter, and this trend is especially pronounced in non-synonymous RDDs, i.e., those cause amino acid changes. A potential mechanism is that, unlike proto-oncogenes, the requirement of tumor suppressor genes to have both alleles affected to cause tumor 'buffers' these genes to tolerate more RDDs.

  5. Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins.

    Science.gov (United States)

    Maeder, Morgan L; Angstman, James F; Richardson, Marcy E; Linder, Samantha J; Cascio, Vincent M; Tsai, Shengdar Q; Ho, Quan H; Sander, Jeffry D; Reyon, Deepak; Bernstein, Bradley E; Costello, Joseph F; Wilkinson, Miles F; Joung, J Keith

    2013-12-01

    Genome-wide studies have defined cell type-specific patterns of DNA methylation that are important for regulating gene expression in both normal development and disease. However, determining the functional significance of specific methylation events remains challenging, owing to the lack of methods for removing such modifications in a targeted manner. Here we describe an approach for efficient targeted demethylation of specific CpGs in human cells using fusions of engineered transcription activator-like effector (TALE) repeat arrays and the TET1 hydroxylase catalytic domain. Using these TALE-TET1 fusions, we demonstrate that modification of critical methylated promoter CpG positions can lead to substantial increases in the expression of endogenous human genes. Our results delineate a strategy for understanding the functional significance of specific CpG methylation marks in the context of endogenous gene loci and validate programmable DNA demethylation reagents with potential utility for research and therapeutic applications.

  6. Biomphalaria glabrata transcriptome: cDNA microarray profiling identifies resistant- and susceptible-specific gene expression in haemocytes from snail strains exposed to Schistosoma mansoni

    Directory of Open Access Journals (Sweden)

    Rollinson David

    2008-12-01

    Full Text Available Abstract Background Biomphalaria glabrata is an intermediate snail host for Schistosoma mansoni, one of the important schistosomes infecting man. B. glabrata/S. mansoni provides a useful model system for investigating the intimate interactions between host and parasite. Examining differential gene expression between S. mansoni-exposed schistosome-resistant and susceptible snail lines will identify genes and pathways that may be involved in snail defences. Results We have developed a 2053 element cDNA microarray for B. glabrata containing clones from ORESTES (Open Reading frame ESTs libraries, suppression subtractive hybridization (SSH libraries and clones identified in previous expression studies. Snail haemocyte RNA, extracted from parasite-challenged resistant and susceptible snails, 2 to 24 h post-exposure to S. mansoni, was hybridized to the custom made cDNA microarray and 98 differentially expressed genes or gene clusters were identified, 94 resistant-associated and 4 susceptible-associated. Quantitative PCR analysis verified the cDNA microarray results for representative transcripts. Differentially expressed genes were annotated and clustered using gene ontology (GO terminology and Kyoto Encyclopaedia of Genes and Genomes (KEGG pathway analysis. 61% of the identified differentially expressed genes have no known function including the 4 susceptible strain-specific transcripts. Resistant strain-specific expression of genes implicated in innate immunity of invertebrates was identified, including hydrolytic enzymes such as cathepsin L, a cysteine proteinase involved in lysis of phagocytosed particles; metabolic enzymes such as ornithine decarboxylase, the rate-limiting enzyme in the production of polyamines, important in inflammation and infection processes, as well as scavenging damaging free radicals produced during production of reactive oxygen species; stress response genes such as HSP70; proteins involved in signalling, such as importin 7

  7. Focal DNA copy number changes in neuroblastoma target MYCN regulated genes.

    Directory of Open Access Journals (Sweden)

    Candy Kumps

    Full Text Available Neuroblastoma is an embryonic tumor arising from immature sympathetic nervous system cells. Recurrent genomic alterations include MYCN and ALK amplification as well as recurrent patterns of gains and losses of whole or large partial chromosome segments. A recent whole genome sequencing effort yielded no frequently recurring mutations in genes other than those affecting ALK. However, the study further stresses the importance of DNA copy number alterations in this disease, in particular for genes implicated in neuritogenesis. Here we provide additional evidence for the importance of focal DNA copy number gains and losses, which are predominantly observed in MYCN amplified tumors. A focal 5 kb gain encompassing the MYCN regulated miR-17~92 cluster as sole gene was detected in a neuroblastoma cell line and further analyses of the array CGH data set demonstrated enrichment for other MYCN target genes in focal gains and amplifications. Next we applied an integrated genomics analysis to prioritize MYCN down regulated genes mediated by MYCN driven miRNAs within regions of focal heterozygous or homozygous deletion. We identified RGS5, a negative regulator of G-protein signaling implicated in vascular normalization, invasion and metastasis, targeted by a focal homozygous deletion, as a new MYCN target gene, down regulated through MYCN activated miRNAs. In addition, we expand the miR-17~92 regulatory network controlling TGFß signaling in neuroblastoma with the ring finger protein 11 encoding gene RNF11, which was previously shown to be targeted by the miR-17~92 member miR-19b. Taken together, our data indicate that focal DNA copy number imbalances in neuroblastoma (1 target genes that are implicated in MYCN signaling, possibly selected to reinforce MYCN oncogene addiction and (2 serve as a resource for identifying new molecular targets for treatment.

  8. DNA vaccines

    Science.gov (United States)

    Gregersen, Jens-Peter

    2001-12-01

    Immunization by genes encoding immunogens, rather than with the immunogen itself, has opened up new possibilities for vaccine research and development and offers chances for new applications and indications for future vaccines. The underlying mechanisms of antigen processing, immune presentation and regulation of immune responses raise high expectations for new and more effective prophylactic or therapeutic vaccines, particularly for vaccines against chronic or persistent infectious diseases and tumors. Our current knowledge and experience of DNA vaccination is summarized and critically reviewed with particular attention to basic immunological mechanisms, the construction of plasmids, screening for protective immunogens to be encoded by these plasmids, modes of application, pharmacokinetics, safety and immunotoxicological aspects. DNA vaccines have the potential to accelerate the research phase of new vaccines and to improve the chances of success, since finding new immunogens with the desired properties is at least technically less demanding than for conventional vaccines. However, on the way to innovative vaccine products, several hurdles have to be overcome. The efficacy of DNA vaccines in humans appears to be much less than indicated by early studies in mice. Open questions remain concerning the persistence and distribution of inoculated plasmid DNA in vivo, its potential to express antigens inappropriately, or the potentially deleterious ability to insert genes into the host cell's genome. Furthermore, the possibility of inducing immunotolerance or autoimmune diseases also needs to be investigated more thoroughly, in order to arrive at a well-founded consensus, which justifies the widespread application of DNA vaccines in a healthy population.

  9. GENE TRANSFER ON Betta imbellis THROUGH TRANSFECTION METHOD WITH DIFFERENT DNA CONCENTRATION

    Directory of Open Access Journals (Sweden)

    Eni Kusrini

    2016-12-01

    Full Text Available Big size betta (Giant have a high economic value compared to normal size betta, and over expression of growth hormone gene can produce giant fish.  As an initial step of giant transgenic betta productions, this study was conducted in order to obtain DNA plasmid concentration which provide higher hatching and survival rate of betta larvae.  Construction of PhGH pCcBA gene contains growth hormone gene of Siamese catfish (PhGH and it is controlled by the CCBA promoter. Betta imbellis broodstocks were spawned naturally, and embryos were collected 1-2 minutes after spawning time. One hundred embryos were dipped in 2 mL of transfectan X-treme gene which containp CcBA-PhGH construction genes (50 µg/mL, on room temperature for about 30 minutes. Treatments on this study were different transfectant : DNA plasmid ratiosnamely:A (0,75 µL: 0,25 µL; B (0,75 µL : 0,50 µL; C (0,75 µL: 0,75 µL, D as Control 1(without transfectant, 0,25 µL DNA; E.as Control 2(0,75 µL transfectant, without DNA, and Fas control 3 (without transfectant and without DNA. Every treatments was repeated three times.  Transfection embryos were hatched on a container (1L Volume. Study results showed that hatching rate and larvae survival rate  (4 days after hatching on treatment A were the same with the control, but slightly higher than B and C treatments. PCR analysis with DNA template showing that PhGH gene were found on embryos and larvae (pooled sample of treatment A, B and C. Furthermore, RT-PCR analysis showing the existence of mRNA PhGH expression on embryos and larvae (pooled sample. Therefore, embryo transfection with transfectant ratio 0,75 µL and  DNA 0,25 µLshowing the best results.

  10. Cell-specific DNA methylation patterns of retina-specific genes.

    Directory of Open Access Journals (Sweden)

    Shannath L Merbs

    Full Text Available Many studies have demonstrated that epigenetic mechanisms are important in the regulation of gene expression during embryogenesis, gametogenesis, and other forms of tissue-specific gene regulation. We sought to explore the possible role of epigenetics, specifically DNA methylation, in the establishment and maintenance of cell type-restricted gene expression in the retina. To assess the relationship between DNA methylation status and expression level of retinal genes, bisulfite sequence analysis of the 1000 bp region around the transcription start sites (TSS of representative rod and cone photoreceptor-specific genes and gene expression analysis were performed in the WERI and Y79 human retinoblastoma cell lines. Next, the homologous genes in mouse were bisulfite sequenced in the retina and in non-expressing tissues. Finally, bisulfite sequencing was performed on isolated photoreceptor and non-photoreceptor retinal cells isolated by laser capture microdissection. Differential methylation of rhodopsin (RHO, retinal binding protein 3 (RBP3, IRBP cone opsin, short-wave-sensitive (OPN1SW, cone opsin, middle-wave-sensitive (OPN1MW, and cone opsin, long-wave-sensitive (OPN1LW was found in the retinoblastoma cell lines that inversely correlated with gene expression levels. Similarly, we found tissue-specific hypomethylation of the promoter region of Rho and Rbp3 in mouse retina as compared to non-expressing tissues, and also observed hypomethylation of retinal-expressed microRNAs. The Rho and Rbp3 promoter regions were unmethylated in expressing photoreceptor cells and methylated in non-expressing, non-photoreceptor cells from the inner nuclear layer. A third regional hypomethylation pattern of photoreceptor-specific genes was seen in a subpopulation of non-expressing photoreceptors (Rho in cones from the Nrl -/- mouse and Opn1sw in rods. These results demonstrate that a number of photoreceptor-specific genes have cell-specific differential DNA

  11. Effects of aspirin on metastasis-associated gene expression detected by cDNA microarray

    Institute of Scientific and Technical Information of China (English)

    Xue-qin GAO; Jin-xiang HAN; Hai-yan HUANG; Shi YAN; Chang-zheng SONG; Hai-nan HUANG

    2004-01-01

    AIM: To investigate the effect of aspirin on the metastasis-associated gene expression in 3AO ovarian cancer cells.METHODS: 3AO cells were treated with aspirin at the concentration of 1.2 mmol/L for 16 and 48 h, respectively.The total RNA was extracted with Trizol reagents and reverse transcribed with Superscript II and hybridized with cDNA microarray (containing oncogenes, tumor suppressor genes, signal transduction pathway molecules, adhesive molecules, growth factors and ESTs) fabricated in our lab. After normalization, the ratio of gene expression of aspirin treated to untreated 3AO cells being either 2 fold up higher or 0.5 fold down (lower) were defined as differential expression. Semi-quantitative RT-PCR was used to validate the microarray results. RESULTS: Among the 447 metastasis-associated genes, 4 genes were up-regulated and 14 genes were down-regulated in 3AO cells treated with aspirin for 16 h compared with untreated cells. While 24 genes were up-regulated and 10 genes were down-regulated in cells treated with aspirin for 48 h. Several up or down-regulated gene expression changes continued from 16 h to 48 h. CONCLUSION: Aspirin might exert its anti-metastasis effects on ovarian cancer by affecting metastasis-associated gene expression.

  12. Interruptions in gene expression drive highly expressed operons to the leading strand of DNA replication.

    Science.gov (United States)

    Price, Morgan N; Alm, Eric J; Arkin, Adam P

    2005-01-01

    In bacteria, most genes are on the leading strand of replication, a phenomenon attributed to collisions between the DNA and RNA polymerases. In Escherichia coli, these collisions slow the movement of the replication fork through actively transcribed genes only if they are coded on the lagging strand. For genes on both strands, however, these collisions sever nascent transcripts and interrupt gene expression. Based on these observations, we propose a new theory to explain strand bias: genes whose expression is important for fitness are selected to the leading strand because this reduces the duration of these interruptions. Our theory predicts that multi-gene operons, which are subject to longer interruptions, should be more strongly selected to the leading strand than singleton transcripts. We show that this is true even after controlling for the tendency for essential genes, which are strongly biased to the leading strand, to occur in operons. Our theory also predicts that other factors that are associated with strand bias should have stronger effects for genes that are in operons. We find that expression level and phylogenetic ubiquity are correlated with strand bias for both essential and non-essential genes, but only for genes in operons.

  13. The impact of endurance exercise on global and AMPK gene-specific DNA methylation

    Energy Technology Data Exchange (ETDEWEB)

    King-Himmelreich, Tanya S.; Schramm, Stefanie; Wolters, Miriam C.; Schmetzer, Julia; Möser, Christine V.; Knothe, Claudia [pharmazentrum frankfurt/ZAFES, Institut für Klinische Pharmakologie, Klinikum der Goethe-Universität Frankfurt, Theodor Stern Kai 7, 60590, Frankfurt am Main (Germany); Resch, Eduard [Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project Group for Translational Medicine & Pharmacology (TMP), 60596, Frankfurt/Main (Germany); Peil, Johannes [Sports Clinic, Bad Nauheim, MCI GmbH, In der Aue 30-32, 61231, Bad Nauheim (Germany); Geisslinger, Gerd [pharmazentrum frankfurt/ZAFES, Institut für Klinische Pharmakologie, Klinikum der Goethe-Universität Frankfurt, Theodor Stern Kai 7, 60590, Frankfurt am Main (Germany); Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project Group for Translational Medicine & Pharmacology (TMP), 60596, Frankfurt/Main (Germany); Niederberger, Ellen, E-mail: e.niederberger@em.uni-frankfurt.de [pharmazentrum frankfurt/ZAFES, Institut für Klinische Pharmakologie, Klinikum der Goethe-Universität Frankfurt, Theodor Stern Kai 7, 60590, Frankfurt am Main (Germany)

    2016-05-27

    Alterations in gene expression as a consequence of physical exercise are frequently described. The mechanism of these regulations might depend on epigenetic changes in global or gene-specific DNA methylation levels. The AMP-activated protein kinase (AMPK) plays a key role in maintenance of energy homeostasis and is activated by increases in the AMP/ATP ratio as occurring in skeletal muscles after sporting activity. To analyze whether exercise has an impact on the methylation status of the AMPK promoter, we determined the AMPK methylation status in human blood samples from patients before and after sporting activity in the context of rehabilitation as well as in skeletal muscles of trained and untrained mice. Further, we examined long interspersed nuclear element 1 (LINE-1) as indicator of global DNA methylation changes. Our results revealed that light sporting activity in mice and humans does not alter global DNA methylation but has an effect on methylation of specific CpG sites in the AMPKα2 gene. These regulations were associated with a reduced AMPKα2 mRNA and protein expression in muscle tissue, pointing at a contribution of the methylation status to AMPK expression. Taken together, these results suggest that exercise influences AMPKα2 gene methylation in human blood and eminently in the skeletal muscle of mice and therefore might repress AMPKα2 gene expression. -- Highlights: •AMPK gene methylation increases after moderate endurance exercise in humans and mice. •AMPKα mRNA and protein decrease after moderate endurance exercise in mice. •Global DNA methylation is not affected under the same conditions.

  14. Obesity is associated with depot-specific alterations in adipocyte DNA methylation and gene expression.

    Science.gov (United States)

    Sonne, Si Brask; Yadav, Rachita; Yin, Guangliang; Dalgaard, Marlene Danner; Myrmel, Lene Secher; Gupta, Ramneek; Wang, Jun; Madsen, Lise; Kajimura, Shingo; Kristiansen, Karsten

    2017-04-03

    The present study aimed to identify genes exhibiting concomitant obesity-dependent changes in DNA methylation and gene expression in adipose tissues in the mouse using diet-induced obese (DIO) C57BL/6J and genetically obese ob/ob mice as models. Mature adipocytes were isolated from epididymal and inguinal adipose tissues of ob/ob and DIO C57BL/6J mice. DNA methylation was analyzed by MeDIP-sequencing and gene expression by microarray analysis. The majority of differentially methylated regions (DMRs) were hypomethylated in obese mice. Global methylation of long interspersed elements indicated that hypomethylation did not reflect methyl donor deficiency. In both DIO and ob/ob mice, we observed more obesity-associated methylation changes in epididymal than in inguinal adipocytes. Assignment of DMRs to promoter, exon, intron and intergenic regions demonstrated that DIO-induced changes in DNA methylation in C57BL/6J mice occurred primarily in exons, whereas inguinal adipocytes of ob/ob mice exhibited a higher enrichment of DMRs in promoter regions than in other regions of the genome, suggesting an influence of leptin on DNA methylation in inguinal adipocytes. We observed altered methylation and expression of 9 genes in epididymal adipocytes, including the known obesity-associated genes, Ehd2 and Kctd15, and a novel candidate gene, Irf8, possibly involved in immune type 1/type2 balance. The use of 2 obesity models enabled us to dissociate changes associated with high fat feeding from those associated with obesity per se. This information will be of value in future studies on the mechanisms governing the development of obesity and changes in adipocyte function associated with obesity.

  15. Polymorphisms of the DNA repair genes XRCC1 and XRCC3 in a Brazilian population

    Directory of Open Access Journals (Sweden)

    Duarte Márcia Cristina

    2005-01-01

    Full Text Available In several DNA repair genes, polymorphisms may result in reduced repair capacity, which has been implicated as a risk factor for various types of cancer. The frequency of the polymorphic alleles varies among populations, suggesting an ethnic distribution of genotypes. We genotyped 300 healthy Southeastern Brazilian individuals (262 of European ancestry and 38 of African ancestry for polymorphisms of codons 194 and 399 of the XRCC1 base excision repair pathway gene and of codon 241 of the XRCC3 homologous recombination repair pathway gene. The allele frequencies were 0.07 for the Arg194Trp and 0.33 for the Arg399Gln codons of the XRCC1 gene and 0.35 for the Thr241Met codon of the XRCC3 gene. The genotypic frequencies were within Hardy-Weinberg