Study of the repeatability of histone genes in the ploidy series of wheat and Aegilops

International Nuclear Information System (INIS)

Vakhitov, V.A.; Kulikov, A.M.

1986-01-01

The hDNA content and number of histone genes in the genomes of different wheat and Aegilops species have been determined by molecular hybridization of DNA with 125 I-histone DNA of Drosophila (L-repeat) on nitrocellulose filters. It has been demonstrated that the proportion of hDNA in the total DNA of diploid and polyploid wheat species is (1.3-7.7) x 10 -3 % (57-850 genes), and in the ploidy series of Aegilops species (2.0-8.0) x 10 -3 % (89-780 genes). The repeatability of the histone genes generally increases at each ploidy level in the species with higher DNA content. At the same time, it has been demonstrated that the DNA content is not the only factor determining repeatability of the histone genes, as some diploid and allopolyploid species have similar number of these genes. It has been concluded that genetic mechanisms are involved in the regulation of the number of histone genes

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover.

Science.gov (United States)

Richardson, Gavin D

2016-05-23

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4',6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types.

Atypical ploidy cycles, Spo11, and the evolution of meiosis.

Science.gov (United States)

Bloomfield, Gareth

2016-06-01

The Spo11 protein induces DNA double strand breaks before the first division of meiosis, enabling the formation of the chiasmata that physically link homologous chromosomes as they align. Spo11 is an ancient and well conserved protein, related in sequence and structure to a DNA topoisomerase subunit found in Archaea as well as a subset of eukaryotes. However the origins of its meiotic function are unclear. This review examines some apparent exceptions to the rule that Spo11 activity is specific to, and required for meiosis. Spo11 appears to function in the context of unusual forms of ploidy reduction in some protists and fungi. One lineage of amoebae, the dictyostelids, is thought to undergo meiosis during its sexual cycle despite having lost Spo11 entirely. Further experimental characterisation of these and other non-canonical ploidy cycling mechanisms may cast light of the evolution of meiosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression

KAUST Repository

Nagymihály, Marianna

2017-04-13

The formation of symbiotic nodule cells in Medicago truncatula is driven by successive endoreduplication cycles and transcriptional reprogramming in different temporal waves including the activation of more than 600 cysteine-rich NCR genes expressed only in nodules. We show here that the transcriptional waves correlate with growing ploidy levels and have investigated how the epigenome changes during endoreduplication cycles. Differential DNA methylation was found in only a small subset of symbiotic nodule-specific genes, including more than half of the NCR genes, whereas in most genes DNA methylation was unaffected by the ploidy levels and was independent of the genes\\' active or repressed state. On the other hand, expression of nodule-specific genes correlated with ploidy-dependent opening of the chromatin as well as, in a subset of tested genes, with reduced H3K27me3 levels combined with enhanced H3K9ac levels. Our results suggest that endoreduplication-dependent epigenetic changes contribute to transcriptional reprogramming in the differentiation of symbiotic cells.

Reference cells and ploidy in the comet assay

Directory of Open Access Journals (Sweden)

Gunnar eBrunborg

2015-02-01

Full Text Available In the comet assay, single cells are analyzed with respect to their level of DNA damage. Discrimination of the individual cell or cell type based on DNA content, with concomitant scoring of the DNA damage, is useful since this may allow analysis of mixtures of cells. Different cells can then be characterized based on their ploidy, cell cycle stage, or genome size. We here describe two applications of such a cell type-specific comet assay: (i Testicular cell suspensions, analyzed on the basis of their ploidy during spermatogenesis; and (ii reference cells in the form of fish erythrocytes which can be included as internal standards to correct for inter-assay variations. With standard fluorochromes used in the comet assay, the total staining signal from each cell – whether damaged or undamaged – was found to be associated with the cell’s DNA content. Analysis of the fluorescence intensity of single cells is straightforward since these data are available in scoring systems based on image analysis. The analysis of testicular cell suspensions provides information on cell type specific composition, susceptibility to genotoxicants, and DNA repair. Internal reference cells, either untreated or carrying defined numbers of lesions induced by ionizing radiation, are useful for investigation of experimental factors that can cause variation in comet assay results, and for routine inclusion in experiments to facilitate standardization of methods and comparison of comet assay data obtained in different experiments or in different laboratories. They can also be used - in combination with a reference curve - to quantify the DNA lesions induced by a certain treatment. Fish cells of a range of genome sizes, both greater and smaller than human, are suitable for this purpose and they are inexpensive.

Evaluation of Tumor Heterogeneity of Prostate Carcinoma by Flow- and Image DNA Cytometry and Histopathological Grading

Directory of Open Access Journals (Sweden)

Naining Wang

2000-01-01

Full Text Available Background. Heterogeneity of prostate carcinoma is one of the reasons for pretreatment underestimation of tumor aggressiveness. We studied tumor heterogeneity and the probability of finding the highest tumor grade and DNA aneuploidy with relation to the number of biopsies. Material and methods. Specimens simulating core biopsies from five randomly selected tumor areas from each of 16 Böcking’s grade II and 23 grade III prostate carcinomas were analyzed for tumor grade and DNA ploidy by flow‐ and fluorescence image cytometry (FCM, FICM. Cell cycle composition was measured by FCM. Results. By determination of ploidy and cell cycle composition, morphologically defined tumors can further be subdivided. Heterogeneity of tumor grade and DNA ploidy (FCM was 54% and 50%. Coexistence of diploid tumor cells in aneuploid specimens represents another form of tumor heterogeneity. The proportion of diploid tumor cells decreased significantly with tumor grade and with increase in the fraction of proliferating cell of the aneuploid tumor part. The probability of estimating the highest tumor grade or aneuploidy increased from 40% for one biopsy to 95% for 5 biopsies studied. By combining the tumor grade with DNA ploidy, the probability of detecting a highly aggressive tumor increased from 40% to 70% and 90% for one and two biopsies, respectively. Conclusion. Specimens of the size of core biopsies can be used for evaluation of DNA ploidy and cell cycle composition. Underestimation of aggressiveness of prostate carcinoma due to tumor heterogeneity is minimized by simultaneous study of the tumor grade and DNA ploidy more than by increasing the number of biopsies. The biological significance of coexistent diploid tumor cell in aneuploid lesions remains to be evaluated.

Ploidy levels among species in the 'Oxalis tuberosa alliance' as inferred by flow cytometry.

Science.gov (United States)

Emshwiller, Eve

2002-06-01

The 'Oxalis tuberosa alliance' is a group of Andean Oxalis species allied to the Andean tuber crop O. tuberosa Molina (Oxalidaceae), commonly known as 'oca'. As part of a larger project studying the origins of polyploidy and domestication of cultivated oca, flow cytometry was used to survey DNA ploidy levels among Bolivian and Peruvian accessions of alliance members. In addition, this study provided a first assessment of C-values in the alliance by estimating nuclear DNA contents of these accessions using chicken erythrocytes as internal standard. Ten Bolivian accessions of cultivated O. tuberosa were confirmed to be octoploid, with a mean nuclear DNA content of approx. 3.6 pg/2C. Two Peruvian wild Oxalis species, O. phaeotricha and O. picchensis, were inferred to be tetraploid (both with approx. 1.67 pg/2C), the latter being one of the putative progenitors of O. tuberosa identified by chloroplast-expressed glutamine synthetase data in prior work. The remaining accessions (from 78 populations provisionally identified as 35 species) were DNA diploid, with nuclear DNA contents varying from 0.79 to 1.34 pg/2C.

Visual selection and maintenance of the cell lines with high plant regeneration ability and low ploidy level in Dianthus acicularis by monitoring with flow cytometry analysis.

Science.gov (United States)

Shiba, Tomonori; Mii, Masahiro

2005-12-01

Efficient plant regeneration system from cell suspension cultures was established in D. acicularis (2n=90) by monitoring ploidy level and visual selection of the cultures. The ploidy level of the cell cultures closely related to the shoot regeneration ability. The cell lines comprising original ploidy levels (2C+4C cells corresponding to DNA contents of G1 and G2 cells of diploid plant, respectively) showed high regeneration ability, whereas those containing the cells with 8C or higher DNA C-values showed low or no regeneration ability. The highly regenerable cell lines thus selected consisted of compact cell clumps with yellowish color and relatively moderate growth, suggesting that it is possible to select visually the highly regenerable cell lines with the original ploidy level. All the regenerated plantlets from the highly regenerable cell cultures exhibited normal phenotypes and no variations in ploidy level were observed by flow cytometry (FCM) analysis.

FISHtrees 3.0: Tumor Phylogenetics Using a Ploidy Probe.

Science.gov (United States)

Gertz, E Michael; Chowdhury, Salim Akhter; Lee, Woei-Jyh; Wangsa, Darawalee; Heselmeyer-Haddad, Kerstin; Ried, Thomas; Schwartz, Russell; Schäffer, Alejandro A

2016-01-01

Advances in fluorescence in situ hybridization (FISH) make it feasible to detect multiple copy-number changes in hundreds of cells of solid tumors. Studies using FISH, sequencing, and other technologies have revealed substantial intra-tumor heterogeneity. The evolution of subclones in tumors may be modeled by phylogenies. Tumors often harbor aneuploid or polyploid cell populations. Using a FISH probe to estimate changes in ploidy can guide the creation of trees that model changes in ploidy and individual gene copy-number variations. We present FISHtrees 3.0, which implements a ploidy-based tree building method based on mixed integer linear programming (MILP). The ploidy-based modeling in FISHtrees includes a new formulation of the problem of merging trees for changes of a single gene into trees modeling changes in multiple genes and the ploidy. When multiple samples are collected from each patient, varying over time or tumor regions, it is useful to evaluate similarities in tumor progression among the samples. Therefore, we further implemented in FISHtrees 3.0 a new method to build consensus graphs for multiple samples. We validate FISHtrees 3.0 on a simulated data and on FISH data from paired cases of cervical primary and metastatic tumors and on paired breast ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). Tests on simulated data show improved accuracy of the ploidy-based approach relative to prior ploidyless methods. Tests on real data further demonstrate novel insights these methods offer into tumor progression processes. Trees for DCIS samples are significantly less complex than trees for paired IDC samples. Consensus graphs show substantial divergence among most paired samples from both sets. Low consensus between DCIS and IDC trees may help explain the difficulty in finding biomarkers that predict which DCIS cases are at most risk to progress to IDC. The FISHtrees software is available at ftp://ftp.ncbi.nih.gov/pub/FISHtrees.

Condensin HEAT subunits required for DNA repair, kinetochore/centromere function and ploidy maintenance in fission yeast.

Directory of Open Access Journals (Sweden)

Xingya Xu

Full Text Available Condensin, a central player in eukaryotic chromosomal dynamics, contains five evolutionarily-conserved subunits. Two SMC (structural maintenance of chromosomes subunits contain ATPase, hinge, and coiled-coil domains. One non-SMC subunit is similar to bacterial kleisin, and two other non-SMC subunits contain HEAT (similar to armadillo repeats. Here we report isolation and characterization of 21 fission yeast (Schizosaccharomyces pombe mutants for three non-SMC subunits, created using error-prone mutagenesis that resulted in single-amino acid substitutions. Beside condensation, segregation, and DNA repair defects, similar to those observed in previously isolated SMC and cnd2 mutants, novel phenotypes were observed for mutants of HEAT-repeats containing Cnd1 and Cnd3 subunits. cnd3-L269P is hypersensitive to the microtubule poison, thiabendazole, revealing defects in kinetochore/centromere and spindle assembly checkpoints. Three cnd1 and three cnd3 mutants increased cell size and doubled DNA content, thereby eliminating the haploid state. Five of these mutations reside in helix B of HEAT repeats. Two non-SMC condensin subunits, Cnd1 and Cnd3, are thus implicated in ploidy maintenance.

Assessment of ploidy stability of the somatic embryogenesis process in Quercus suber L. using flow cytometry

Czech Academy of Sciences Publication Activity Database

Loureiro, J.; Pinto, G.; Lopes, T.; Doležel, Jaroslav; Santos, C.

2005-01-01

Roč. 221, - (2005), s. 815-822 ISSN 0032-0935 Institutional research plan: CEZ:AV0Z50380511 Keywords : Flow cytometry * ploidy stability * nuclear DNA content Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.108, year: 2005

Ploidy, cytokinetics, and histology features of aggressive versus less aggressive uterine cervical squamous cell carcinomas

International Nuclear Information System (INIS)

Johnson, T.S.; Peters, L.J.; Adelson, M.; Williamson, K.D.; Sneige, N.; Katz, R.L.; Freedman, R.S.

1985-01-01

The authors are investigating the interrelationships of flow cytometric measured ploidy, S-fraction with histology features of uterine cervical squamous cell cancers in an attempt to identify aggressive, high risk tumors and less aggressive tumors. Experimentally, pre-radiotherapy biopsy specimens are being studied using flow ploidy and cell-cycle analysis and microscopic scoring for histology features. The results to date for some 200 patients indicate that there are identifyable aggressive tumors, at high risk for 2 yr local control within each stage of disease and differentiation category (WD, MD, PD). These aggressive tumors usually have high degree DNA abnormalities (triploid or greater), high proliferative activity (%S≥20) compared to the less aggressive tumors characterized by diploid/near diploid DNA content, low to moderate %S (2-19, mean 12). Expression of high S-fraction appears to reflect high growth activity or growth potential and characterizes the aggressive tumors

Ploidy Levels among Species in the ‘Oxalis tuberosa Alliance’ as Inferred by Flow Cytometry

Science.gov (United States)

EMSHWILLER, EVE

2002-01-01

The ‘Oxalis tuberosa alliance’ is a group of Andean Oxalis species allied to the Andean tuber crop O. tuberosa Molina (Oxalidaceae), commonly known as ‘oca’. As part of a larger project studying the origins of polyploidy and domestication of cultivated oca, flow cytometry was used to survey DNA ploidy levels among Bolivian and Peruvian accessions of alliance members. In addition, this study provided a first assessment of C‐values in the alliance by estimating nuclear DNA contents of these accessions using chicken erythrocytes as internal standard. Ten Bolivian accessions of cultivated O. tuberosa were confirmed to be octoploid, with a mean nuclear DNA content of approx. 3·6 pg/2C. Two Peruvian wild Oxalis species, O. phaeotricha and O. picchensis, were inferred to be tetraploid (both with approx. 1·67 pg/2C), the latter being one of the putative progenitors of O. tuberosa identified by chloroplast‐expressed glutamine synthetase data in prior work. The remaining accessions (from 78 populations provisionally identified as 35 species) were DNA diploid, with nuclear DNA contents varying from 0·79 to 1·34 pg/2C. PMID:12102530

Nuclear grade and DNA ploidy in stage IV breast cancer with only visceral metastases at initial diagnosis.

Science.gov (United States)

De Lena, M; Barletta, A; Marzullo, F; Rabinovich, M; Leone, B; Vallejo, C; Machiavelli, M; Romero, A; Perez, J; Lacava, J; Cuevas, M A; Rodriguez, R; Schittulli, F; Paradisco, A

1996-01-01

The presence of early metastases to distant sites in breast cancer patients is an infrequent event whose mechanisms are still not clear. The aim of this study was to evaluate the biologic and clinical role of DNA ploidy and cell nuclear grade of primary tumors in the metastatic process of a series of stage IV previously untreated breast cancer patients with only visceral metastases. DNA flow cytometry analysis on paraffin-embedded material and cell nuclear grading of primary tumors was performed on a series of 50 breast cancer patients with only visceral metastases at the time of initial diagnosis. Aneuploidy was found in 28/46 (61%) of evaluable cases and was independent of site of involvement, clinical response, time of progression and overall survival of patients. Of the 46 cases evaluable for nuclear grade, 5 (11%), 16 (35%) and 25 (54%) were classified as G1 (well-differentiated) G2 and G3, respectively. Nuclear grade also was unrelated to response to therapy and overall survival, whereas time to progression was significantly longer in G1-2 than G3 tumors with the logrank test (P < 0.03) and multivariate analysis. Our results seem to stress the difficulty to individualize different prognostic subsets from a series of breast cancer patients with only visceral metastases at initial diagnosis according to DNA flow cytometry and nuclear grade.

A method for the determination of bacterial spore DNA content based on isotopic labelling, spore germination and diphenylamine assay; ploidy of spores of several Bacillus species

International Nuclear Information System (INIS)

Hauser, P.M.; Karamata, D.

1992-01-01

A reliable method for measuring the spore DNA content, based on radioactive DNA labelling, spore germination in absence of DNA replication and diphenylamine assay, was developed. The accuracy of the method, within 10 - 15%, is adequate for determining the number of chromosomes per spore, provided that the genome size is known. B subtilis spores were shown to be invariably monogenomic, while those of larger bacilli Bacillus megaterium, Bacillus cereus and Bacillus thuringiensis, often, if not invariably, contain two genomes. Attempts to modify the spore DNA content of B subtilis by altering the richness of the sporulation medium, the sporulation conditions (liquid or solid medium), or by mutation, were apparently unsuccessful. An increase of spore size with medium richness, not accompanied by an increase in DNA content, was observed. The implication of the apparently species-specific spore ploidy and the influence of the sporulation conditions on spore size and shape are discussed

The ploidy races of Atriplex confertifolia (chenopodiaceae)

Science.gov (United States)

Stewart C. Sanderson

2011-01-01

Previous accounts of polyploidy in the North American salt desert shrub Atriplex confertifolia (shadscale) have dealt with the distribution of polyploidy and the morphological and secondary chemical differences between races. The present study amplifies these studies and reveals additional ploidy-flavonoid races, with ploidy levels known to extend from 2x to 12x, and...

Ploidy level variability in South American fescues (festuca L., poaceae): use of flow cytometry in up to 5 1/2-year-old caryopses and herbarium specimens.

Science.gov (United States)

Smarda, P; Stancík, D

2006-01-01

Ploidy levels and chromosome numbers for 24 species of Festuca L. from 29 sites in Bolivia, Colombia, Ecuador and Venezuela are given. The ploidy level of 22 species is reported for the first time. A higher proportion of tetraploids in northern South America and the high frequency of polyploids in the whole continent are documented. In combination with chromosome counts, ploidy level was determined using flow cytometry in 4- to 5 1/2 -year-old herbarium specimens and mature caryopses. Flow cytometric determination from seeds was more reliable than determination from herbarium specimens. In herbarium specimens, the youngest, fresh green leaves, still hidden in sheaths, seem to be most suitable for cytometric determination. In old, brownish leaves, or poorly preserved herbarium specimens, the degradation of DNA signal in flow histograms was documented. DNA content measured in seeds was always higher than that measured in herbarium specimens, which may be caused by the presence of different cytosolic compounds. Differences of about 15% in relative DNA content of F. sodiroana and F. vaginalis was found in simultaneous measurements in seeds.

Study of quantitative genetics of gum arabic production complicated by variability in ploidy level of Acacia senegal (L.) Willd

DEFF Research Database (Denmark)

Diallo, Adja Madjiguene; Nielsen, Lene Rostgaard; Hansen, Jon Kehlet

2015-01-01

Gum arabic is an important international commodity produced by trees of Acacia senegal across Sahelian Africa, but documented results of breeding activities are limited. The objective of this study was to provide reliable estimates of quantitative genetic parameters in order to shed light on the ...... stress the importance of testing ploidy levels of selected material and use of genetic markers to qualify the assumptions in the quantitative genetic analysis....... that progenies consisted of both diploid and polyploid trees, and growth, gum yield, and gum quality varied substantially among ploidy level, populations, and progenies. Analysis of molecular variance and estimates of outcrossing rate supported that trees within open-pollinated families of diploids were half...... sibs, while the open-pollinated families of polyploids showed low variation within families. The difference in sibling relationship observed between ploidy levels complicated estimation of genetic parameters. However, based on the diploid trees, we conclude that heritability in gum arabic production...

Analysis of Different Ploidy and Parent–Offspring Genomic DNA Methylation in the Loach Misgurnus anguillicaudatus

Directory of Open Access Journals (Sweden)

He Zhou

2016-08-01

Full Text Available In this study, we selected natural polyploidy loach (diploid, triploid and tetraploid and hybrid F1 generation obverse cross (4 × 2 and inverse cross (2 × 4 by diploids and tetraploids as the research model. The MSAP (methylation-sensitive amplified polymorphism reaction system was established by our laboratory to explore methylation levels and pattern diversification features at the whole genome level of the polyploidy loach. The results showed that the total methylation and full methylation rates decreased on increased ploidy individuals; moreover, the hemimethylation rate showed no consistent pattern. Compared with diploid loach, the methylation patterns of tetraploid sites changed 68.17%, and the methylation patterns of triploid sites changed 73.05%. The proportion of hypermethylation genes is significantly higher than the proportion of demethylation genes. The methylation level of reciprocal cross F1 generation is lower than the male diploid and higher than the female tetraploid. The hemimethylation and total methylation rate of the cross hybrid F1 generation is significantly higher than the orthogonal F1 generation (p < 0.01. After readjusting, the methylation pattern of genome DNA of reciprocal hybrids changed 69.59% and 72.83%, respectively.

Analysis of Different Ploidy and Parent–Offspring Genomic DNA Methylation in the Loach Misgurnus anguillicaudatus

Science.gov (United States)

Zhou, He; Ma, Tian-Yu; Zhang, Rui; Xu, Qi-Zheng; Shen, Fu; Qin, Yan-Jie; Xu, Wen; Wang, Yuan; Li, Ya-Juan

2016-01-01

In this study, we selected natural polyploidy loach (diploid, triploid and tetraploid) and hybrid F1 generation obverse cross (4 × 2) and inverse cross (2 × 4) by diploids and tetraploids as the research model. The MSAP (methylation-sensitive amplified polymorphism) reaction system was established by our laboratory to explore methylation levels and pattern diversification features at the whole genome level of the polyploidy loach. The results showed that the total methylation and full methylation rates decreased on increased ploidy individuals; moreover, the hemimethylation rate showed no consistent pattern. Compared with diploid loach, the methylation patterns of tetraploid sites changed 68.17%, and the methylation patterns of triploid sites changed 73.05%. The proportion of hypermethylation genes is significantly higher than the proportion of demethylation genes. The methylation level of reciprocal cross F1 generation is lower than the male diploid and higher than the female tetraploid. The hemimethylation and total methylation rate of the cross hybrid F1 generation is significantly higher than the orthogonal F1 generation (p < 0.01). After readjusting, the methylation pattern of genome DNA of reciprocal hybrids changed 69.59% and 72.83%, respectively. PMID:27556458

Nuclear ploidy of neonatal rat livers: effects of two hepatic carcinogens (mirex and dimethylnitrosamine)

International Nuclear Information System (INIS)

Carlson, J.; Abraham, R.

1985-01-01

The effect of two hepatic carcinogens, dimethylnitrosamine (DMN) (genotoxic) and mirex (epigenetic), on polyploidization in 12-d-old neonatal rats was investigated by Coulter counteranalysis and [ 3 H] thymidine uptake in isolated hepatic nuclear classes. DMN disturbed the normal ploidy development in the neonatal liver and the proportion of nuclei in the ploidy classes by inducing the premature formation of a significant population of tetraploids with a concommitant reduction in diploids. A great proportion of the replicative activity was present in tetraploid nuclei as measured by the incorporation of [ 3 H] thymidine. The labeling index and number of mitoses were also increased. In contrast to DMN, mirex had no influence on polyploidization. The neonatal rats used in these studies thus offer an opportunity to investigate in vivo the mode of action of genotoxic versus epigenetic compounds with reference to their effect on DNA

Radiation induced reproductive death as a function of mammalian cell ploidy

International Nuclear Information System (INIS)

Philbrick, D.A.

1976-09-01

Mammalian cells containing different multiples of the diploid chromosome set were created through drug induction and cell fusion. In all cell strains used the chromosome number was determined from metaphase spreads, as well as from DNA content and cell size. The survival of cells as a function of radiation dose was determined for cell lines with differing chromosome complements at 37 0 C, 4 0 C, in hypertonic media, while frozen, and with increasing levels of incorporated IUdR. Survival of frozen diploid and hypotetraploid Chinese hamster cells was determined following varying numbers of decays of incorporated 3 HTdR and 125 IUdR. The percent of reproductively viable cells following irradiation is a function of the cell ploidy, i.e., the number of haploid sets of chromosomes contained in the cell genome. At 37 0 C and in hypertonic media, the Chinese hamster cells of progressively higher ploidies are increasingly sensitive to irradiation. As the number of chromosomes per unit cell volume increases the radiosensitivity increases. Both trends suggest interaction between chromosomes as an important cause of cell death

Image cytometry: nuclear and chromosomal DNA quantification.

Science.gov (United States)

Carvalho, Carlos Roberto; Clarindo, Wellington Ronildo; Abreu, Isabella Santiago

2011-01-01

Image cytometry (ICM) associates microscopy, digital image and software technologies, and has been particularly useful in spatial and densitometric cytological analyses, such as DNA ploidy and DNA content measurements. Basically, ICM integrates methodologies of optical microscopy calibration, standard density filters, digital CCD camera, and image analysis softwares for quantitative applications. Apart from all system calibration and setup, cytological protocols must provide good slide preparations for efficient and reliable ICM analysis. In this chapter, procedures for ICM applications employed in our laboratory are described. Protocols shown here for human DNA ploidy determination and quantification of nuclear and chromosomal DNA content in plants could be used as described, or adapted for other studies.

Flow cytometry determination of ploidy level in winged bean ...

African Journals Online (AJOL)

Ploidy determination and mutation breeding of crop plants are inseparable twins given that mutation breeding is hinged majorly on polyploidization of crop's chromosome number. The present research was aimed at determining the ploidy level of 20 accessions of winged bean (Psophoscarpus tetragonolobus) using known ...

Simultaneous use of multiplex ligation-dependent probe amplification assay and flow cytometric DNA ploidy analysis in patients with acute leukemia.

Science.gov (United States)

Reyes-Núñez, Virginia; Galo-Hooker, Evelyn; Pérez-Romano, Beatriz; Duque, Ricardo E; Ruiz-Arguelles, Alejandro; Garcés-Eisele, Javier

2018-01-01

The aim of this work was to simultaneously use multiplex ligation-dependent probe amplification (MLPA) assay and flow cytometric DNA ploidy analysis (FPA) to detect aneuploidy in patients with newly diagnosed acute leukemia. MLPA assay and propidium iodide FPA were used to test samples from 53 consecutive patients with newly diagnosed acute leukemia referred to our laboratory for immunophenotyping. Results were compared by nonparametric statistics. The combined use of both methods significantly increased the rate of detection of aneuploidy as compared to that obtained by each method alone. The limitations of one method are somehow countervailed by the other and vice versa. MPLA and FPA yield different yet complementary information concerning aneuploidy in acute leukemia. The simultaneous use of both methods might be recommended in the clinical setting. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

Analysis of Cellular DNA Content by Flow Cytometry.

Science.gov (United States)

Darzynkiewicz, Zbigniew; Huang, Xuan; Zhao, Hong

2017-11-01

Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA-content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin-embedded tissues. Available algorithms are listed for deconvolution of DNA-content-frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

Induced mutagenesis in wheat at various ploidy levels

International Nuclear Information System (INIS)

Valeva, S.A.

1975-01-01

Different wheat species with 2x=14,28 and 42 were treated with ethylene imine(EI) and gamma-rays, and the M 1 damage and M 2 mutation frequency recorded. The resistance towards mutagenic treatment, in general, increased with ploidy level, but in each ploidy group the cultivated varieties were tolerant than the wild and primitive forms of the same species or ploidy level. It was also observed that the manifestation of mutagenic damage is expressed differently for different parameters taken for recording the extent of injury. There was no direct correlation between the sensitivity expressed through M 1 , injury and the mutation frequency recorded in M 2 , although in hexploid bread wheat a more sensitive variety (Bezostava-1) was also more mutable than the other variety of the same species (Beltskaya-32). (author)

DNA-index and stereological estimation of nuclear volume in primary and metastatic malignant melanomas

DEFF Research Database (Denmark)

Sørensen, Flemming Brandt; Kristensen, I B; Grymer, F

1990-01-01

The aim of this study was to investigate the relationship between physical nuclear volume and ploidy level in malignant melanomas, and to analyse the heterogeneity of these two parameters among primary and corresponding secondary tumours. Unbiased stereological estimates of nuclear volume can...

Radiation induced reproductive death as a function of mammalian cell ploidy

Energy Technology Data Exchange (ETDEWEB)

Philbrick, D.A.

1976-09-01

Mammalian cells containing different multiples of the diploid chromosome set were created through drug induction and cell fusion. In all cell strains used the chromosome number was determined from metaphase spreads, as well as from DNA content and cell size. The survival of cells as a function of radiation dose was determined for cell lines with differing chromosome complements at 37/sup 0/C, 4/sup 0/C, in hypertonic media, while frozen, and with increasing levels of incorporated IUdR. Survival of frozen diploid and hypotetraploid Chinese hamster cells was determined following varying numbers of decays of incorporated /sup 3/HTdR and /sup 125/IUdR. The percent of reproductively viable cells following irradiation is a function of the cell ploidy, i.e., the number of haploid sets of chromosomes contained in the cell genome. At 37/sup 0/C and in hypertonic media, the Chinese hamster cells of progressively higher ploidies are increasingly sensitive to irradiation. As the number of chromosomes per unit cell volume increases the radiosensitivity increases. Both trends suggest interaction between chromosomes as an important cause of cell death.

The study of triploid progenies crossed between different ploidy ...

African Journals Online (AJOL)

The study of triploid progenies crossed between different ploidy grapes. L Sun, G Zhang, A Yan, H Xu. Abstract. The cross between different ploidy grape was one of the effective ways to obtain new seedless cultivars, in this study, through testing the changes of the ovule weight and observing its anatomical structure, the ...

Does Ploidy Level Directly Control Cell Size? Counterevidence from Arabidopsis Genetics

OpenAIRE

Tsukaya, Hirokazu

2013-01-01

Ploidy level affects cell size in many organisms, and ploidy-dependent cell enlargement has been used to breed many useful organisms. However, how polyploidy affects cell size remains unknown. Previous studies have explored changes in transcriptome data caused by polyploidy, but have not been successful. The most naïve theory explaining ploidy-dependent cell enlargement is that increases in gene copy number increase the amount of protein, which in turn increases the cell volume. This hypothes...

Chromosome numbers and DNA content in some species of Mecardonia (Gratiolae, Plantaginaceae)

Science.gov (United States)

Sosa, María M.; Angulo, María B.; Greppi, Julián A.; Bugallo, Verónica

2016-01-01

Abstract Cytogenetic characterization and determination of DNA content by flow cytometry of five species of Mecardonia Ruiz et Pavon, 1798 (Gratiolae, Plantaginaceae) was performed. This is the first study of nuclear DNA content carried out in the genus. Mitotic analysis revealed a base chromosome number x = 11 for all entities and different ploidy levels, ranging from diploid (2n = 2x = 22) to hexaploid (2n = 6x = 66). The results include the first report of the chromosome numbers for Mecardonia flagellaris (Chamisso & Schlechtendal, 1827) (2n = 22), Mecardonia grandiflora (Bentham) Pennell, 1946 (2n = 22), Mecardonia kamogawae Greppi & Hagiwara, 2011 (2n = 66), and Mecardonia sp. (2n = 44). The three ploidy levels here reported suggest that polyploidy is common in Mecardonia and appear to be an important factor in the evolution of this genus. The 2C- and 1Cx-values were also estimated in all the species. The 2C-values ranged from 1.91 to 5.29 pg. The 1Cx-values ranged from 0.88 to 1.03 pg. The general tendency indicated a decrease in the 1Cx-value with increasing ploidy level. The significance of the results is discussed in relation to taxonomy of the genus. PMID:28123693

Organization and variation analysis of 5S rDNA in different ploidy-level hybrids of red crucian carp × topmouth culter.

Science.gov (United States)

He, Weiguo; Qin, Qinbo; Liu, Shaojun; Li, Tangluo; Wang, Jing; Xiao, Jun; Xie, Lihua; Zhang, Chun; Liu, Yun

2012-01-01

Through distant crossing, diploid, triploid and tetraploid hybrids of red crucian carp (Carassius auratus red var., RCC♀, Cyprininae, 2n = 100) × topmouth culter (Erythroculter ilishaeformis Bleeker, TC♂, Cultrinae, 2n = 48) were successfully produced. Diploid hybrids possessed 74 chromosomes with one set from RCC and one set from TC; triploid hybrids harbored 124 chromosomes with two sets from RCC and one set from TC; tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from TC. The 5S rDNA of the three different ploidy-level hybrids and their parents were sequenced and analyzed. There were three monomeric 5S rDNA classes (designated class I: 203 bp; class II: 340 bp; and class III: 477 bp) in RCC and two monomeric 5S rDNA classes (designated class IV: 188 bp, and class V: 286 bp) in TC. In the hybrid offspring, diploid hybrids inherited three 5S rDNA classes from their female parent (RCC) and only class IV from their male parent (TC). Triploid hybrids inherited class II and class III from their female parent (RCC) and class IV from their male parent (TC). Tetraploid hybrids gained class II and class III from their female parent (RCC), and generated a new 5S rDNA sequence (designated class I-N). The specific paternal 5S rDNA sequence of class V was not found in the hybrid offspring. Sequence analysis of 5S rDNA revealed the influence of hybridization and polyploidization on the organization and variation of 5S rDNA in fish. This is the first report on the coexistence in vertebrates of viable diploid, triploid and tetraploid hybrids produced by crossing parents with different chromosome numbers, and these new hybrids are novel specimens for studying the genomic variation in the first generation of interspecific hybrids, which has significance for evolution and fish genetics.

Evaluation of Prognostic Factors Following Flow-Cytometric DNA Analysis after Cytokeratin Labelling: I. Breast Cancer

Directory of Open Access Journals (Sweden)

Pauline Wimberger

2002-01-01

Full Text Available In gynecologic oncology valid prognostic factors are necessary to estimate the course of disease and to define biologically similar subgroups for analysis of therapeutic efficacy. The presented study is a prospective study concerning prognostic significance of DNA ploidy and S‐phase fraction in breast cancer following enrichment of tumor cells by cytokeratin labelling. Epithelial cells were labeled by FITC‐conjugated cytokeratin antibody (CK 5, 6, 8, and CK 17 prior to flow cytometric cell cycle analysis in 327 fresh specimens of primary breast cancer. Univariate analysis in breast cancer detected the prognostic significance of DNA‐ploidy, S‐phase fraction and CV (coefficient of variation of G0G1‐peak of tumor cells for clinical outcome, especially for nodal‐negative patients. Multivariate analysis could not confirm prognostic evidence of DNA‐ploidy and S‐phase fraction. In conclusion, in breast cancer no clinical significance for determination of DNA‐parameters was found.

A Hidden Markov Model Approach for Simultaneously Estimating Local Ancestry and Admixture Time Using Next Generation Sequence Data in Samples of Arbitrary Ploidy.

Science.gov (United States)

Corbett-Detig, Russell; Nielsen, Rasmus

2017-01-01

Admixture-the mixing of genomes from divergent populations-is increasingly appreciated as a central process in evolution. To characterize and quantify patterns of admixture across the genome, a number of methods have been developed for local ancestry inference. However, existing approaches have a number of shortcomings. First, all local ancestry inference methods require some prior assumption about the expected ancestry tract lengths. Second, existing methods generally require genotypes, which is not feasible to obtain for many next-generation sequencing projects. Third, many methods assume samples are diploid, however a wide variety of sequencing applications will fail to meet this assumption. To address these issues, we introduce a novel hidden Markov model for estimating local ancestry that models the read pileup data, rather than genotypes, is generalized to arbitrary ploidy, and can estimate the time since admixture during local ancestry inference. We demonstrate that our method can simultaneously estimate the time since admixture and local ancestry with good accuracy, and that it performs well on samples of high ploidy-i.e. 100 or more chromosomes. As this method is very general, we expect it will be useful for local ancestry inference in a wider variety of populations than what previously has been possible. We then applied our method to pooled sequencing data derived from populations of Drosophila melanogaster on an ancestry cline on the east coast of North America. We find that regions of local recombination rates are negatively correlated with the proportion of African ancestry, suggesting that selection against foreign ancestry is the least efficient in low recombination regions. Finally we show that clinal outlier loci are enriched for genes associated with gene regulatory functions, consistent with a role of regulatory evolution in ecological adaptation of admixed D. melanogaster populations. Our results illustrate the potential of local ancestry

DNA-content in isolated nuclei of postembryonic stages of progeny from normal and irradiated males of Tetranychus urticae (Acari Tetranychidoe)

International Nuclear Information System (INIS)

Tempelaar, M.J.

1980-01-01

The lC DNA-content of isolated nuclei of postembryonic stages of Tetranychus urticae stained with the classic and a modified Schiff's reagent was cytophotometrically estimated as 0.1 pg, a low value in animals. For many tissues of this arrhenotokus species the ploidy ratio between males and females is 1:2, indicating the absence of sex-related differences in ploidy. In addition, DNA measurements were performed to evaluate irradiation-experiments, starting with X-irradiation of mature sperm in males with doses known from previous work to induce chromosomal fragments that are subject to loss and missegregation in the embryonic mitotic stages of the female progeny despite the presumed holokinetic nature of the chromosomes. The DNA-content of the nuclei of the surviving postembryonic preadult stages did not indicate the occurrence of nuclei with in-between male/female values, ruling out loss and missegregation of fragments as important factors in postembryonic lethality. Abnormally low DNA-values in some adult females could be attributed to development of embryos before oviposition caused by radiation-induced effects. (orig.) [de

Change in ploidy status from hyperdiploid to near-tetraploid in multiple myeloma associated with bortezomib/lenalidomide resistance.

Science.gov (United States)

Pavlistova, Lenka; Zemanova, Zuzana; Sarova, Iveta; Lhotska, Halka; Berkova, Adela; Spicka, Ivan; Michalova, Kyra

2014-01-01

Ploidy is an important prognostic factor in the risk stratification of multiple myeloma (MM) patients. Patients with MM can be divided into two groups according to the modal number of chromosomes: nonhyperdiploid (NH-MM) and hyperdiploid (H-MM), which has a more favorable outcome. The two ploidy groups represent two different oncogenetic pathways determined at the premalignant stage. The ploidy subtype also persists during the course of the disease, even during progression after the therapy, with only very rare cases of ploidy conversion. The clinical significance of ploidy conversion and its relation to drug resistance have been previously discussed. Here, we describe a female MM patient with a rare change in her ploidy status from H-MM to NH-MM, detected by cytogenetic and molecular cytogenetic examinations of consecutive bone marrow aspirates. We hypothesize that ploidy conversion (from H-MM to NH-MM) is associated with disease progression and acquired resistance to bortezomib/lenalidomide therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

OpenAIRE

Baars, Destiny L.; Takle, Kendra A.; Heier, Jonathon; Pelegri, Francisco

2016-01-01

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole...

Histogram score contributes for reliability of DNA content estimatives in Brachiaria spp Notas do histograma contribuem para a confiabilidade das estimativas do conteúdo de DNA de Brachiaria spp

Directory of Open Access Journals (Sweden)

Ana Luiza de Oliveira Timbó

2012-12-01

Full Text Available Flow cytometry allows to estimate the DNA content of a large number of plants quickly. However, inadequate protocols can compromise the reliability of these estimates leading to variations in the values of DNA content the same species. The objective of this study was to propose an efficient protocol to estimate the DNA content of Brachiaria spp. genotypes with different ploidy levels using flow cytometry. We evaluated four genotypes (B. ruziziensis diploid and artificially tetraploidized; a tetraploid B. brizantha and a natural triploid hybrid, three buffer solutions (MgSO4, Galbraith and Tris-HCl and three species as internal reference standards (Raphanus sativus, Solanum lycopersicum e Pisum sativum. The variables measured were: histogram score (1-5, coefficient of variation and estimation of DNA content. The best combination for the analysis of Brachiaria spp. DNA content was the use of MgSO4 buffer with R. sativus as a internal reference standard. Genome sizes expressed in picograms of DNA are presented for all genotypes and the importance of the histogram score on the results reliability of DNA content analyses were discussed.A citometria de fluxo permite estimar o conteúdo de DNA de um grande número de plantas rapidamente. No entanto, protocolos inadequados podem comprometer a confiabilidade dessas estimativas, levando a variações nos valores de conteúdo de DNA para uma mesma espécie. Neste trabalho, objetivou-se propor um protocolo eficiente para a estimativa do conteúdo de DNA de genótipos de Brachiaria spp. com diferentes níveis de ploidia, utilizando a citometria de fluxo. Foram avaliados quatro genótipos (B. ruziziensis, diploide e tetraploidizada artificialmente; B. brizantha tetraploide e um híbrido natural triploide, 3 soluções tampões (MgSO4, Galbraith e Tris-HCl e três espécies como padrões de referência interno (Raphanus sativus, Solanum lycopersicum e Pisum sativum. As variáveis mensuradas foram: nota do

Effect of phosphorus availability on the selection of species with different ploidy levels and genome sizes in a long-term grassland fertilization experiment.

Science.gov (United States)

Šmarda, Petr; Hejcman, Michal; Březinová, Alexandra; Horová, Lucie; Steigerová, Helena; Zedek, František; Bureš, Petr; Hejcmanová, Pavla; Schellberg, Jürgen

2013-11-01

Polyploidy and increased genome size are hypothesized to increase organismal nutrient demands, namely of phosphorus (P), which is an essential and abundant component of nucleic acids. Therefore, polyploids and plants with larger genomes are expected to be selectively disadvantaged in P-limited environments. However, this hypothesis has yet to be experimentally tested. We measured the somatic DNA content and ploidy level in 74 vascular plant species in a long-term fertilization experiment. The differences between the fertilizer treatments regarding the DNA content and ploidy level of the established species were tested using phylogeny-based statistics. The percentage and biomass of polyploid species clearly increased with soil P in particular fertilizer treatments, and a similar but weaker trend was observed for the DNA content. These increases were associated with the dominance of competitive life strategy (particularly advantageous in the P-treated plots) in polyploids and the enhanced competitive ability of dominant polyploid grasses at high soil P concentrations, indicating their increased P limitation. Our results verify the hypothesized effect of P availability on the selection of polyploids and plants with increased genome sizes, although the relative contribution of increased P demands vs increased competitiveness as causes of the observed pattern requires further evaluation. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Ploidy and liquid-holding recovery of yeasts sensitive to radiation and nitrous acid

International Nuclear Information System (INIS)

Arman, I.P.; Dutova, T.A.

1975-01-01

NA-Inactivation of isogeneic yeasts Sacch. cerevisiae from the collection of LIYaF, normal and sensitive to radiation and Na (xrs1-5 mutation), was studied as a function of the ploidy of the genome and the conditions of incubation after the influence of the mutagen. Normal cells of highly homozygous PG strains exhibit a protective effect of ploidy: the haploid is the most sensitive to the inactivating action of NA, and with increasing number of chromosome sets the resistance increases substantially. With a different polyploid series of yeasts of the same origin, where di-, tri-, and tetraploids are homozygous for the mutation xrs1-5, this effect is absent. Moreover, the doubling of the genome leads to a sharp increase in the sensitivity of the cells to NA, while the shape of the dose-versus-effect curves becomes exponential, in contrast to sigmoid for the initial control strains. This fact of the ''reverse effect of ploidy'' is evidence of an impairment of the reapir of dominant lethals - the basic cause of death of diploid and polyploid cells - in the xrs1-5 strains. Exposure of yeasts in buffer for 24 h (LHR conditions) after the influence of NA modifies the level of inactivation, depending on the genotype and ploidy of the cells. In yeast strains of a different origin (from Berkeley, United States) with a normal sensitivity to NA (n, 2n, 3n), the survival under LHR conditions is practically unchanged for 24 h. The haploids of highly homozygous strains (LIYaF) - normal and xrs1-5 mutant - also do not recover. However, diploidizationand a further increase in the number of genomes leads to the fact that the death under LH conditions increases sharply in normal highly homozygous yeasts (PG) - by at least an order of magnitude in 24 h of incubation in buffer; in this case the titer of the cells remains constant, while the loss of viability is proportional to the time. A significant effect of recovery is detected in homozygotes for the xrs1 mutation (2n, 3n, 4n). It

Agronomic Trait Variations and Ploidy Differentiation of Kiwiberries in Northwest China: Implication for Breeding

Directory of Open Access Journals (Sweden)

Ying Zhang

2017-05-01

Full Text Available Polyploid plants often have higher biomass and superior crop qualities. Breeders therefore search for crop germplasm with higher ploidy levels; however, whether higher ploidy levels are associated with better performance remains unclear. Actinidia arguta and related species, whose commercialized fruit are referred to as kiwiberries, harbor a series of ploidy races in nature, offering an opportunity to determine the link between ploidy levels and agronomic traits. In the present study, we determined the ploidy levels of A. arguta var. arguta, A. arguta var. giraldii, and A. melanandra in 16 natural populations using flow cytometry, and examined 31 trait variations in fruits, leaves and flowers by field observations, microscopic examination and laboratory analyses. Our results showed that octaploid and decaploid A. arguta var. giraldii had larger dimension of leaves than tetraploid A. arguta var. arguta and A. melanandra, but their fruits were significantly smaller. In addition, A. arguta var. giraldii (8x and 10x had higher contents of nutrients such as ascorbic acid and amino acids; however, some important agronomic traits, including the content of total sugar and total acid, were significantly lower in the octaploids and decaploids. Moreover, octaploids and decaploids did not result in greater ecological adaptability for the challenging environments and climates. In conclusion, the differentiation of ecological adaptability and traits among natural kiwiberries' cytotypes suggested that higher ploidy levels are not inevitably advantageous in plants. The findings of A. arguta and related taxa in geographical distribution and agronomic trait variations will facilitate their germplasm domestication.

Nuclear DNA content of the pigeon orchid (Dendrobium crumenatum Sw. with the analysis of flow cytometry

Directory of Open Access Journals (Sweden)

Upatham Meesawat

2008-05-01

Full Text Available Nuclear DNA content for the adult plants grown in a greenhouse and in vitro young plantlets of the pigeon orchid (Dendrobium crumenatum Sw. was analyzed using flow cytometry. The resulting 2C DNA values ranged from 2.30±0.14 pgto 2.43±0.06 pg. However, nuclear DNA ploidy levels of long-term in vitro plantlets were found to be triploid and tetraploid.These ploidy levels were confirmed by chromosome counting. Tetraploid individuals (2n = 4x = 76 had approximately two times DNA content than diploid (2n = 2x = 38 individuals. This variation may be due to prolonged cultivation and thepresence of exogenous plant growth regulators.

Counting DNA: estimating the complexity of a test tube of DNA.

Science.gov (United States)

Faulhammer, D; Lipton, R J; Landweber, L F

1999-10-01

We consider the problem of estimation of the 'complexity' of a test tube of DNA. The complexity of a test tube is the number of different kinds of strands of DNA in the test tube. It is quite easy to estimate the number of total strands in a test tube, especially if the strands are all the same length. Estimation of the complexity is much less clear. We propose a simple kind of DNA computation that can estimate the complexity.

Prognostic significance of DNA aneuploidy in diffuse malignant mesothelioma

Energy Technology Data Exchange (ETDEWEB)

Isobe, Hiroshi; Sridhar, K.S.; Doria, R. [Univ. of Miami School of Medicine, FL (United States)] [and others

1995-01-01

DNA ploidy of pepsin digested preparations of 48 paraffin-embedded specimens from 19 patients with histologically confirmed malignant mesothelioma was determined by laser flow cytometry. Eight of the 19 tumors (42%) were diploid and 11 (58%) were aneuploid. Of the aneuploid tumors, only one showed multiploidy. The median survival time of the patients with diploid tumors was 19, 16, and 14 months from the onset of symptoms, diagnosis, and treatment, respectively. The median survival in patients with aneuploid tumors was 8, 7, and 7 months from the onset of first symptoms, diagnosis, and treatment. Thus, patients with diploid tumors lived longer than patients with aneuploid tumors. These results suggest that DNA ploidy analysis may be of prognostic value in malignant mesothelioma. 31 refs., 2 figs., 3 tabs.

Determination of chromosomal ploidy in Agave ssp. | Lingling ...

African Journals Online (AJOL)

Chromosome observation is necessary to elucidate the structure, function and organization of Agave plants' genes and genomes. However, few researches about chromosome observation of Agave ssp. were done, not only because their chromosome numbers are large, but also because their ploidies are complicated.

Focus on the Musa collection: Ploidy levels revealed

Czech Academy of Sciences Publication Activity Database

Kubaláková, Marie; Doležel, Jaroslav; Van den Houwe, I.; Roux, N.; Swennen, R.

2005-01-01

Roč. 14, - (2005), s. 34-36 ISSN 1023-0076 R&D Projects: GA AV ČR IAA6038204 Grant - others:IAEA res. contract No. 12230 Institutional research plan: CEZ:AV0Z50380511 Keywords : Musa * ploidy * flow cytometry Subject RIV: EB - Genetics ; Molecular Biology

Increasing genetic gain by reducing ploidy in potato

Science.gov (United States)

While potato cultivars in major world production regions are tetraploid, wild and cultivated potatoes in the crop’s center of origin range from diploid to hexaploid. Landrace potato varieties cannot be distinguished based on ploidy. Contrary to popular belief, tetraploidy does not appear to be neces...

Ploidy of Bovine Nuclear Transfer Blastocysts Blastomere Donors

DEFF Research Database (Denmark)

Booth, P J; VIUFF, D; THOMSEN, P D

2000-01-01

The higher rate of embryonic loss in nuclear transfer compared to in vitro produced embryos may be due to chromosome abnormalities that occur during preimplantation in vitro devel- opment. Because little is known about ploidy errors in nuclear transfer embryos, this was ex- amined using embryos...... cultured until day 7 at which time blastocyst nuclei were extracted and chromosome abnormalities were evaluated by fluorescent in situ hybridization using two probes that bind to the subcentromeric regions on chromosomes 6 and 7. In 16 nuclear transfer blastocysts generated from 5 donor embryos, 53.8 6 20...... comprised mainly triploid (8.2 6 10.3 [0–26.3]: SD [range]) and tetraploid (10.6 6 19.9 [0–54.9]) nuclei with other ploidy com- binations accounting for only 0.9 6 2.1 [0–2.1]% of deviant nuclei. The proportion of com- pletely normal nuclear transfer embryos was no less than those produced by in vitro...

Development of novel EST-SSR markers for ploidy identification based on de novo transcriptome assembly for Misgurnus anguillicaudatus.

Science.gov (United States)

Feng, Bing; Yi, Soojin V; Zhang, Manman; Zhou, Xiaoyun

2018-01-01

The co-existence of several ploidy types in natural populations makes the cyprinid loach Misgurnus anguillicaudatus an exciting model system to study the genetic and phenotypic consequences of ploidy variations. A first step in such effort is to identify the specific ploidy of an individual. Currently popular methods of karyotyping via cytological preparation or flow cytometry require a large amount of tissue (such as blood) samples, which can be damaging or fatal to the fishes. Here, we developed novel microsatellite markers (SSR markers) from M. anguillicaudatus and show that they can effectively discriminate ploidy using samples collected in a minimally invasive way. Specifically, we generated whole genome transcriptomes from multiple M. anguillicaudatus using the Illumina paired-end sequencing. Approximately 150 million raw reads were assembled into 76,544 non-redundant unigenes. A total of 8,194 potential SSR markers were identified. We selected 98 pairs with more than five tandem repeats for further assays. Out of 45 putative EST-SSR markers that successfully amplified and harbored polymorphism in diploids, 11 markers displayed high variability in tetraploids. We further demonstrate that a set of five EST-SSR markers selected from these are sufficient to distinguish ploidy levels, by first validating them on 69 reference specimens with known ploidy levels and then subsequently using fresh-collected 96 ploidy-unknown specimens. The results from EST-SSR markers are highly concordant with those from independent flow cytometry analysis. The novel EST-SSR markers developed here should facilitate genetic studies of polyploidy in the emerging model system M. anguillicaudatus.

Host ploidy, parasitism and immune defence in a coevolutionary snail-trematode system.

Science.gov (United States)

Osnas, E E; Lively, C M

2006-01-01

We studied the role of host ploidy and parasite exposure on immune defence allocation in a snail-trematode system (Potamopyrgus antipodarum-Microphallus sp.). In the field, haemocyte (the defence cell) concentration was lowest in deep-water habitats where infection is relatively low and highest in shallow-water habitats where infection is common. Because the frequency of asexual triploid snails is positively correlated with depth, we also experimentally studied the role of ploidy by exposing both diploid sexual and triploid asexual snails to Microphallus eggs. We found that triploid snails had lower haemocyte concentrations than did diploids in both parasite-addition and parasite-free treatments. We also found that both triploids and diploids increased their numbers of large granular haemocytes at similar rates after parasite exposure. Because triploid P. antipodarum have been shown to be more resistant to allopatric parasites than diploids, the current results suggest that the increased resistance of triploids is because of intrinsic genetic properties rather than to greater allocation to defence cells. This finding is consistent with recent theory on the advantages of increased ploidy for hosts combating coevolving parasites.

Nuclear DNA-Content in Mesenchymal Lesions in Dogs: Its Value as Marker of Malignancy and Extent of Genomic Instability

International Nuclear Information System (INIS)

Boerkamp, Kim M.; Rutteman, Gerard R.; Kik, Marja J. L.; Kirpensteijn, Jolle; Schulze, Christoph; Grinwis, Guy C. M.

2012-01-01

DNA-aneuploidy may reflect the malignant nature of mesenchymal proliferations and herald gross genomic instability as a mechanistic factor in tumor genesis. DNA-ploidy and -index were determined by flow cytometry in canine inflammatory or neoplastic mesenchymal tissues and related to clinico-pathological features, biological behavior and p53 gene mutational status. Half of all sarcomas were aneuploid. Benign mesenchymal neoplasms were rarely aneuploid and inflammatory lesions not at all. The aneuploidy rate was comparable to that reported for human sarcomas with significant variation amongst subtypes. DNA-ploidy status in canines lacked a relation with histological grade of malignancy, in contrast to human sarcomas. While aneuploidy was related to the development of metastases in soft tissue sarcomas it was not in osteosarcomas. No relation amongst sarcomas was found between ploidy status and presence of P53 gene mutations. Heterogeneity of the DNA index between primary and metastatic sarcoma sites was present in half of the cases examined. Hypoploidy is more common in canine sarcomas and hyperploid cases have less deviation of the DNA index than human sarcomas. The variation in the presence and extent of aneuploidy amongst sarcoma subtypes indicates variation in genomic instability. This study strengthens the concept of interspecies variation in the evolution of gross chromosomal aberrations during cancer development

Nuclear DNA-Content in Mesenchymal Lesions in Dogs: Its Value as Marker of Malignancy and Extent of Genomic Instability

Science.gov (United States)

Boerkamp, Kim M.; Rutteman, Gerard R.; Kik, Marja J. L.; Kirpensteijn, Jolle; Schulze, Christoph; Grinwis, Guy C. M.

2012-01-01

DNA-aneuploidy may reflect the malignant nature of mesenchymal proliferations and herald gross genomic instability as a mechanistic factor in tumor genesis. DNA-ploidy and -index were determined by flow cytometry in canine inflammatory or neoplastic mesenchymal tissues and related to clinico-pathological features, biological behavior and p53 gene mutational status. Half of all sarcomas were aneuploid. Benign mesenchymal neoplasms were rarely aneuploid and inflammatory lesions not at all. The aneuploidy rate was comparable to that reported for human sarcomas with significant variation amongst subtypes. DNA-ploidy status in canines lacked a relation with histological grade of malignancy, in contrast to human sarcomas. While aneuploidy was related to the development of metastases in soft tissue sarcomas it was not in osteosarcomas. No relation amongst sarcomas was found between ploidy status and presence of P53 gene mutations. Heterogeneity of the DNA index between primary and metastatic sarcoma sites was present in half of the cases examined. Hypoploidy is more common in canine sarcomas and hyperploid cases have less deviation of the DNA index than human sarcomas. The variation in the presence and extent of aneuploidy amongst sarcoma subtypes indicates variation in genomic instability. This study strengthens the concept of interspecies variation in the evolution of gross chromosomal aberrations during cancer development. PMID:24213507

Nuclear DNA-Content in Mesenchymal Lesions in Dogs: Its Value as Marker of Malignancy and Extent of Genomic Instability

Energy Technology Data Exchange (ETDEWEB)

Boerkamp, Kim M., E-mail: K.M.Boerkamp@uu.nl; Rutteman, Gerard R. [Department of Clinical Science of Companion Animals, Faculty of Veterinary Medicine, UU, Yalelaan 104, 3584 CM, Utrecht (Netherlands); Kik, Marja J. L. [Department of Pathobiology, Faculty of Veterinary Medicine, UU, Yalelaan 1, 3508 TD, Utrecht (Netherlands); Kirpensteijn, Jolle [Department of Clinical Science of Companion Animals, Faculty of Veterinary Medicine, UU, Yalelaan 104, 3584 CM, Utrecht (Netherlands); Schulze, Christoph; Grinwis, Guy C. M. [Department of Pathobiology, Faculty of Veterinary Medicine, UU, Yalelaan 1, 3508 TD, Utrecht (Netherlands)

2012-12-03

DNA-aneuploidy may reflect the malignant nature of mesenchymal proliferations and herald gross genomic instability as a mechanistic factor in tumor genesis. DNA-ploidy and -index were determined by flow cytometry in canine inflammatory or neoplastic mesenchymal tissues and related to clinico-pathological features, biological behavior and p53 gene mutational status. Half of all sarcomas were aneuploid. Benign mesenchymal neoplasms were rarely aneuploid and inflammatory lesions not at all. The aneuploidy rate was comparable to that reported for human sarcomas with significant variation amongst subtypes. DNA-ploidy status in canines lacked a relation with histological grade of malignancy, in contrast to human sarcomas. While aneuploidy was related to the development of metastases in soft tissue sarcomas it was not in osteosarcomas. No relation amongst sarcomas was found between ploidy status and presence of P53 gene mutations. Heterogeneity of the DNA index between primary and metastatic sarcoma sites was present in half of the cases examined. Hypoploidy is more common in canine sarcomas and hyperploid cases have less deviation of the DNA index than human sarcomas. The variation in the presence and extent of aneuploidy amongst sarcoma subtypes indicates variation in genomic instability. This study strengthens the concept of interspecies variation in the evolution of gross chromosomal aberrations during cancer development.

Nuclear DNA-Content in Mesenchymal Lesions in Dogs: Its Value as Marker of Malignancy and Extent of Genomic Instability

Directory of Open Access Journals (Sweden)

Christoph Schulze

2012-12-01

Full Text Available DNA-aneuploidy may reflect the malignant nature of mesenchymal proliferations and herald gross genomic instability as a mechanistic factor in tumor genesis. DNA-ploidy and -index were determined by flow cytometry in canine inflammatory or neoplastic mesenchymal tissues and related to clinico-pathological features, biological behavior and p53 gene mutational status. Half of all sarcomas were aneuploid. Benign mesenchymal neoplasms were rarely aneuploid and inflammatory lesions not at all. The aneuploidy rate was comparable to that reported for human sarcomas with significant variation amongst subtypes. DNA-ploidy status in canines lacked a relation with histological grade of malignancy, in contrast to human sarcomas. While aneuploidy was related to the development of metastases in soft tissue sarcomas it was not in osteosarcomas. No relation amongst sarcomas was found between ploidy status and presence of P53 gene mutations. Heterogeneity of the DNA index between primary and metastatic sarcoma sites was present in half of the cases examined. Hypoploidy is more common in canine sarcomas and hyperploid cases have less deviation of the DNA index than human sarcomas. The variation in the presence and extent of aneuploidy amongst sarcoma subtypes indicates variation in genomic instability. This study strengthens the concept of interspecies variation in the evolution of gross chromosomal aberrations during cancer development.

Comparison of DNA aneuploidy, chromosome 1 abnormalities, MYCN amplification and CD44 expression as prognostic factors in neuroblastoma.

Science.gov (United States)

Christiansen, H; Sahin, K; Berthold, F; Hero, B; Terpe, H J; Lampert, F

1995-01-01

A comparison of the prognostic impact of five molecular variables in a large series was made, including tests of their nonrandom association and multivariate analysis. Molecular data were available for 377 patients and MYCN amplification, cytogenetic chromosome 1p deletion, loss of chromosome 1p heterozygosity, DNA ploidy and CD44 expression were investigated. Their interdependence and influence on event-free survival was tested uni- and multivariately using Pearson's chi 2-test, Kaplan-Meier estimates, log rank tests and the Cox's regression model. MYCN amplification was present in 18% (58/322) of cases and predicted poorer prognosis in localised (P < 0.001), metastatic (P = 0.002) and even 4S (P = 0.040) disease. CD44 expression was found in 86% (127/148) of cases, and was a marker for favourable outcome in patients with neuroblastoma stages 1-3 (P = 0.003) and 4 (P = 0.017). Chromosome 1p deletion was cytogenetically detected in 51% (28/55), and indicated reduced event-free survival in localised neuroblastoma (P = 0.020). DNA ploidy and loss of heterozygosity on chromosome 1p were of less prognostic value. Most factors of prognostic significance were associated with each other. By multivariate analysis, MYCN was selected as the only relevant factor. Risk estimation of high discriminating power is, therefore, possible for patients with localised and metastatic neuroblastoma using stage and MYCN.

Quantification of ploidy in proteobacteria revealed the existence of monoploid, (mero-oligoploid and polyploid species.

Directory of Open Access Journals (Sweden)

Vito Pecoraro

Full Text Available Bacteria are generally assumed to be monoploid (haploid. This assumption is mainly based on generalization of the results obtained with the most intensely studied model bacterium, Escherichia coli (a gamma-proteobacterium, which is monoploid during very slow growth. However, several species of proteobacteria are oligo- or polyploid, respectively. To get a better overview of the distribution of ploidy levels, genome copy numbers were quantified in four species of three different groups of proteobacteria. A recently developed Real Time PCR approach, which had been used to determine the ploidy levels of halophilic archaea, was optimized for the quantification of genome copy numbers of bacteria. Slow-growing (doubling time 103 minutes and fast-growing (doubling time 25 minutes E. coli cultures were used as a positive control. The copy numbers of the origin and terminus region of the chromosome were determined and the results were in excellent agreement with published data. The approach was also used to determine the ploidy levels of Caulobacter crescentus (an alpha-proteobacterium and Wolinella succinogenes (an epsilon-proteobacterium, both of which are monoploid. In contrast, Pseudomonas putida (a gamma-proteobacterium contains 20 genome copies and is thus polyploid. A survey of the proteobacteria with experimentally-determined genome copy numbers revealed that only three to four of 11 species are monoploid and thus monoploidy is not typical for proteobacteria. The ploidy level is not conserved within the groups of proteobacteria, and there are no obvious correlations between the ploidy levels with other parameters like genome size, optimal growth temperature or mode of life.

High-resolution DNA content analysis of microbiopsy samples in oral lichen planus.

Science.gov (United States)

Pentenero, M; Monticone, M; Marino, R; Aiello, C; Marchitto, G; Malacarne, D; Giaretti, W; Gandolfo, S; Castagnola, P

2017-04-01

DNA aneuploidy has been reported to be a predictor of poor prognosis in both premalignant and malignant lesions. In oral lichen planus (OLP), this hypothesis remains to be proved. This study aimed to determine the rate of occurrence of DNA aneuploidy in patients with OLP by high-resolution DNA flow cytometry. Patients with OLP were consecutively enrolled. Tissue samples were subdivided for formalin fixation and routine histological assessment and for immediate storage at -20°C for later DNA ploidy analysis, which was performed by DAPI staining of the extracted nuclei and excitation with a UV lamp. The DNA aneuploid sublines were characterized by the DNA Index. A DNA aneuploid status was observed in two of 77 patients with OLP (2.6%). When considering the clinical aspect of the OLP lesions, both DNA aneuploid cases had a reticular clinical aspect. DNA aneuploidy is an uncommon event in OLP and less frequent compared to other non-dysplastic and non-OLP oral potentially malignant disorders. The extremely low rate of DNA aneuploidy could represent an occasional finding or reflect the low rate of malignant transformation observed in patients with OLP even if the real prognostic value of DNA ploidy analysis in patients with OLP remains to be confirmed. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Ploidy and genome composition of Musa germplasm at the ...

African Journals Online (AJOL)

GRACE

2006-07-03

Jul 3, 2006 ... Musa spp (bananas and plantains) constitute a hybrid-polyploid complex and are classified according to different genome compositions such as AA, BB, AB, AAA, AAB, ABB, AAAA, ABBB, AAAB and. AABB. Knowledge of ploidy and exact genome compositions of the parental material is essential for.

Clonal heterogeneity of small-cell anaplastic carcinoma of the lung demonstrated by flow-cytometric DNA analysis

DEFF Research Database (Denmark)

Vindeløv, L L; Hansen, H H; Christensen, I J

1980-01-01

Flow-cytometric DNA analysis yields information on ploidy and proliferative characteristics of a cell population. The analysis was implemented on small-cell anaplastic carcinoma of the lung using a rapid detergent technique for the preparation of fine-needle aspirates for DNA determination and a ...

Ploidy levels and reproductive behaviour in invasive Hieracium pilosella in Patagonia

Czech Academy of Sciences Publication Activity Database

Krahulec, František; Krahulcová, Anna

2011-01-01

Roč. 11, - (2011), s. 25-31 ISSN 1619-0033 R&D Projects: GA ČR GA206/08/0890 Institutional research plan: CEZ:AV0Z60050516 Keywords : Patagonia * ploidy levels * hybridization Subject RIV: EF - Botanics

The effect of sub-lethal doses on the ploidy level in rats hepatocytes with aging

International Nuclear Information System (INIS)

Ekhtiar, A. M.

2004-11-01

It has been shown that the polyploidization levels in rat's hepatocytes increased with aging. The high LET ionizing radiation also induce cell polyploidization by two different means: cells and nuclei fusion, and mitosis restriction after DNA replication. The purpose of the present study was to determine the kinetic of rat's hepatocytes polyploidization with ageing, and the late effects of low doses of gamma irradiation on polyploidization. To this end, three groups of rats were used. Each group composed of 175 four weeks old animals. The first was served as a control, the second and the third groups were irradiated with 4 and 2 Gy respectively, of gamma irradiation at the age of one month. Of each group, 7-8 animals were monthly scarified (for two years), and their liver tissues were used to obtain cell suspensions which were further fixed in gradual series concentrations of ethanol. After staining with Propidum Iodide 'PI' (10 6 cells per ml of PI used at 10 - 5 M final concentration), the cells were analyzed on a FACS Vantage Flow Cytometer (Becton Dickinson). In the control, the results showed: 1) A decrease of cell fraction that contained normal diploid until steady level. 2) Biphasic changes of fraction tetraploidy cells (increase until age of 4 month followed by decrease). 3) The fraction of octaploidy cells appeared at age of 3-4 month and increased continuously with the aging. In accompanied to life-span reductions of 4 Gy irradiated animals, the DNA contents were similar to those in control groups in addition to some quantities variation due to a programmed cell death (Apoptosis) induced by irradiation and regenerations. These variations persisted till the age of 7 month, in additional to reduce the spin-life of irradiated animals. The irradiation with 2 Gy induced some quantities variation in comparison with nonirradiated group, appeared in the reduction of rate conversion from one ploidy class to another, and in shift with 2-3 months of the second pike

Geographic Variation in Festuca rubra L. Ploidy Levels and Systemic Fungal Endophyte Frequencies.

Directory of Open Access Journals (Sweden)

Serdar Dirihan

Full Text Available Polyploidy and symbiotic Epichloë fungal endophytes are common and heritable characteristics that can facilitate environmental range expansion in grasses. Here we examined geographic patterns of polyploidy and the frequency of fungal endophyte colonized plants in 29 Festuca rubra L. populations from eight geographic sites across latitudes from Spain to northernmost Finland and Greenland. Ploidy seemed to be positively and negatively correlated with latitude and productivity, respectively. However, the correlations were nonlinear; 84% of the plants were hexaploids (2n = 6x = 42, and the positive correlation between ploidy level and latitude is the result of only four populations skewing the data. In the southernmost end of the gradient 86% of the plants were tetraploids (2n = 4x = 28, whereas in the northernmost end of the gradient one population had only octoploid plants (2n = 8x = 56. Endophytes were detected in 22 out of the 29 populations. Endophyte frequencies varied among geographic sites, and populations and habitats within geographic sites irrespective of ploidy, latitude or productivity. The highest overall endophyte frequencies were found in the southernmost end of the gradient, Spain, where 69% of plants harbored endophytes. In northern Finland, endophytes were detected in 30% of grasses but endophyte frequencies varied among populations from 0% to 75%, being higher in meadows compared to riverbanks. The endophytes were detected in 36%, 30% and 27% of the plants in Faroe Islands, Iceland and Switzerland, respectively. Practically all examined plants collected from southern Finland and Greenland were endophyte-free, whereas in other geographic sites endophyte frequencies were highly variable among populations. Common to all populations with high endophyte frequencies is heavy vertebrate grazing. We propose that the detected endophyte frequencies and ploidy levels mirror past distribution history of F. rubra after the last glaciation

Botanical indices of ploidy levels in some African accessions of ...

African Journals Online (AJOL)

Twenty-nine accessions of Oryza punctata Kotschy ex Steud, from local and other African habitats were studied to establish the attributes that can delineate the two ploidy levels based on agro-botanical, foliar epidermal and nodal anatomical characteristics. The diploid plants of O. punctata are early-maturing annuals with a ...

Induction of ploidy level increments in an asporogenous industrial strain of the yeast Saccaromyces cerevisiae by UV irradiation

International Nuclear Information System (INIS)

Sasaki, Takashi

1992-01-01

Cells of an asporogenous industrial strain of the yeast Saccaromyces cerevisiae were irradiated with UV light by using a method that was developed previously. This treatment gave rise to large-cell clones among the surviving cells, from which colonies consisting of cells with a normal morphology and a prototropic property were obtained. The large-cell trait of these was stably inheritable, with the cell volumes being about twice that of the parent for 7 years on a slant agar medium at 4C with repeated transfers. The cellular DNA content of these clones, in comparison to those of two authentic haploid strains, was determined by chemical analysis. The ratio of the DNA contents showed that the parent and its large-cell derivatives were a diploid and tetraploids, respectively. No abnormality was found in the chromosomal DNA patterns of the large-cell clones, at least as determined by agarose gel electrophoresis with a CHEF-DR II pulsed-field electrophoresis system. These findings led to the conclusion that the UV light method is applicable for inducing ploidy level increments in the widely used yeast species S. cerevisiae

Comparative Population Dynamics of Two Closely Related Species Differing in Ploidy Level

Czech Academy of Sciences Publication Activity Database

Černá, L.; Münzbergová, Zuzana

2013-01-01

Roč. 8, č. 10 (2013), no.-e75563 E-ISSN 1932-6203 Institutional support: RVO:67985939 Keywords : ploidy level * clonal growth * life-table response experiments Subject RIV: EF - Botanics Impact factor: 3.534, year: 2013

Diversity among Cynodon accessions and taxa based on DNA amplification fingerprinting.

Science.gov (United States)

Assefa, S; Taliaferro, C M; Anderson, M P; de los Reyes, B G; Edwards, R M

1999-06-01

The genus Cynodon (Gramineae), comprised of 9 species, is geographically widely distributed and genetically diverse. Information on the amounts of molecular genetic variation among and within Cynodon taxa is needed to enhance understanding of phylogenetic relations and facilitate germplasm management and breeding improvement efforts. Genetic relatedness among 62 Cynodon accessions, representing eight species, was assessed using DNA amplification fingerprinting (DAF). Ten 8-mer oligonucleotides were used to amplify specific Cynodon genomic sequences. The DNA amplification products of individual accessions were scored for presence (1) or absence (0) of bands. Similarity matrices were developed and the accessions were grouped by cluster (UPGMA) and principal coordinate analysis. Analyses were conducted within ploidy level (2x = 18 and 4x = 36) and over ploidy levels. Each primer revealed polymorphic loci among accessions within species. Of 539 loci (bands) scored, 496 (92%) were polymorphic. Cynodon arcuatus was clearly separated from other species by numerous monomorphic bands. The strongest species similarities were between C. aethiopicus and C. arcuatus, C. transvaalensis and C. plectostachyus, and C. incompletus and C. nlemfuensis. Intraspecific variation was least for C. aethiopicus, C. arcuatus, and C. transvaalensis, and greatest for C. dactylon. Accessions of like taxonomic classification were generally clustered, except the cosmopolitan C. dactylon var. dactylon and C. dactylon var. afganicus. Within taxa, accessions differing in chromosome number clustered in all instances indicating the 2x and 4x forms to be closely related. Little, if any, relationship was found between relatedness as indicated by the DAF profiles and previous estimates of hybridization potential between the different taxa.

Environmental DNA (eDNA sampling improves occurrence and detection estimates of invasive burmese pythons.

Directory of Open Access Journals (Sweden)

Margaret E Hunter

Full Text Available Environmental DNA (eDNA methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR for the Burmese python (Python molurus bivittatus, Northern African python (P. sebae, boa constrictor (Boa constrictor, and the green (Eunectes murinus and yellow anaconda (E. notaeus. Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive

Environmental DNA (eDNA) sampling improves occurrence and detection estimates of invasive burmese pythons.

Science.gov (United States)

Hunter, Margaret E; Oyler-McCance, Sara J; Dorazio, Robert M; Fike, Jennifer A; Smith, Brian J; Hunter, Charles T; Reed, Robert N; Hart, Kristen M

2015-01-01

Environmental DNA (eDNA) methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR) for the Burmese python (Python molurus bivittatus), Northern African python (P. sebae), boa constrictor (Boa constrictor), and the green (Eunectes murinus) and yellow anaconda (E. notaeus). Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive constrictors

DNA flow cytometric analysis in variable types of hydropic placentas

Directory of Open Access Journals (Sweden)

Fatemeh Atabaki pasdar

2015-05-01

Full Text Available Background: Differential diagnosis between complete hydatidiform mole, partial hydatidiform mole and hydropic abortion, known as hydropic placentas is still a challenge for pathologists but it is very important for patient management. Objective: We analyzed the nuclear DNA content of various types of hydropic placentas by flowcytometry. Materials and Methods: DNA ploidy analysis was performed in 20 non-molar (hydropic and non-hydropic spontaneous abortions and 20 molar (complete and partial moles, formalin-fixed, paraffin-embedded tissue samples by flow cytometry. The criteria for selection were based on the histopathologic diagnosis. Results: Of 10 cases histologically diagnosed as complete hydatiform mole, 9 cases yielded diploid histograms, and 1 case was tetraploid. Of 10 partial hydatidiform moles, 8 were triploid and 2 were diploid. All of 20 cases diagnosed as spontaneous abortions (hydropic and non-hydropic yielded diploid histograms. Conclusion: These findings signify the importance of the combined use of conventional histology and ploidy analysis in the differential diagnosis of complete hydatidiform mole, partial hydatidiform mole and hydropic abortion.

Determination of ploidy level and isolation of genes encoding acetyl-CoA carboxylase in Japanese Foxtail (Alopecurus japonicus.

Directory of Open Access Journals (Sweden)

Hongle Xu

Full Text Available Ploidy level is important in biodiversity studies and in developing strategies for isolating important plant genes. Many herbicide-resistant weed species are polyploids, but our understanding of these polyploid weeds is limited. Japanese foxtail, a noxious agricultural grass weed, has evolved herbicide resistance. However, most studies on this weed have ignored the fact that there are multiple copies of target genes. This may complicate the study of resistance mechanisms. Japanese foxtail was found to be a tetraploid by flow cytometer and chromosome counting, two commonly used methods in the determination of ploidy levels. We found that there are two copies of the gene encoding plastidic acetyl-CoA carboxylase (ACCase in Japanese foxtail and all the homologous genes are expressed. Additionally, no difference in ploidy levels or ACCase gene copy numbers was observed between an ACCase-inhibiting herbicide-resistant and a herbicide-sensitive population in this study.

Relook TURBT in superficial bladder cancer: its importance and its correlation with the tumor ploidy.

Science.gov (United States)

Dwivedi, Udai S; Kumar, Abhay; Das, Suren K; Trivedi, Sameer; Kumar, Mohan; Sunder, Shyam; Singh, Pratap B

2009-01-01

To evaluate various prognostic factor predictors of residual growth in Relook transurethral resection of bladder tumor (TURBT) in superficial bladder cancer. Also, to evaluate the role of Relook TURBT along with the ploidy for prediction of recurrence and stage progression in these patients. Fifty patients with superficial bladder cancer underwent TURBT after complete evaluation. Ploidy of the tumor specimen was evaluated by flow cytometry. After 4 to 6 weeks of initial TURBT, these patients underwent Relook TURBT. Final treatment was given after the results of the histological evaluation of these specimens. Patients who underwent bladder sparing treatment were followed-up. Of the patients, 28.5% had residual tumor in Relook TURBT. Growth was found to be at the same site in 66.7% and at a different site 33.3%; 75% had single while 25% had multiple residual growth. Residual malignant tissue had a statistically significant correlation with size of the tumor (>3 cm), appearance (solid tumor), number (>3), grade (high), and multiple previous resections. Overall, the up-migration of stage and grade leads to change in treatment in 41.6%; 5 underwent radical cystectomy and 1 opted for radiotherapy; in 2 patients, intravesical BCG was given. In follow-up of mean 11.5 months, 16.6% had recurrence. Presence of residual growth in Relook TURBT along with number, size, morphology, and multiple previous resections were found to have significant correlation with the recurrence in these patients. Ploidy and grade of the tumor were not found to have correlation. Multiple, more than 3 cm, solid high grade tumor with > 3 previous resections were predictors of presence of residual tumor in Relook TURBT. Presence of residual growth is a significant risk factor for recurrence. Ploidy was not found to be significantly correlated with recurrence.

Ploidy mosaicism and allele-specific gene expression differences in the allopolyploid Squalius alburnoides

Directory of Open Access Journals (Sweden)

Matos Isa

2011-12-01

Full Text Available Abstract Background Squalius alburnoides is an Iberian cyprinid fish resulting from an interspecific hybridisation between Squalius pyrenaicus females (P genome and males of an unknown Anaecypris hispanica-like species (A genome. S. alburnoides is an allopolyploid hybridogenetic complex, which makes it a likely candidate for ploidy mosaicism occurrence, and is also an interesting model to address questions about gene expression regulation and genomic interactions. Indeed, it was previously suggested that in S. alburnoides triploids (PAA composition silencing of one of the three alleles (mainly of the P allele occurs. However, not a whole haplome is inactivated but a more or less random inactivation of alleles varying between individuals and even between organs of the same fish was seen. In this work we intended to correlate expression differences between individuals and/or between organs to the occurrence of mosaicism, evaluating if mosaics could explain previous observations and its impact on the assessment of gene expression patterns. Results To achieve our goal, we developed flow cytometry and cell sorting protocols for this system generating more homogenous cellular and transcriptional samples. With this set-up we detected 10% ploidy mosaicism within the S. alburnoides complex, and determined the allelic expression profiles of ubiquitously expressed genes (rpl8; gapdh and β-actin in cells from liver and kidney of mosaic and non-mosaic individuals coming from different rivers over a wide geographic range. Conclusions Ploidy mosaicism occurs sporadically within the S. alburnoides complex, but in a frequency significantly higher than reported for other organisms. Moreover, we could exclude the influence of this phenomenon on the detection of variable allelic expression profiles of ubiquitously expressed genes (rpl8; gapdh and β-actin in cells from liver and kidney of triploid individuals. Finally, we determined that the expression patterns

DNA index determination with Automated Cellular Imaging System (ACIS in Barrett's esophagus: Comparison with CAS 200

Directory of Open Access Journals (Sweden)

Klein Michael

2005-08-01

Full Text Available Abstract Background For solid tumors, image cytometry has been shown to be more sensitive for diagnosing DNA content abnormalities (aneuploidy than flow cytometry. Image cytometry has often been performed using the semi-automated CAS 200 system. Recently, an Automated Cellular Imaging System (ACIS was introduced to determine DNA content (DNA index, but it has not been validated. Methods Using the CAS 200 system and ACIS, we compared the DNA index (DI obtained from the same archived formalin-fixed and paraffin embedded tissue samples from Barrett's esophagus related lesions, including samples with specialized intestinal metaplasia without dysplasia, low-grade dysplasia, high-grade dysplasia and adenocarcinoma. Results Although there was a very good correlation between the DI values determined by ACIS and CAS 200, the former was 25% more sensitive in detecting aneuploidy. ACIS yielded a mean DI value 18% higher than that obtained by CAS 200 (p t test. In addition, the average time required to perform a DNA ploidy analysis was shorter with the ACIS (30–40 min than with the CAS 200 (40–70 min. Results obtained by ACIS gave excellent inter-and intra-observer variability (coefficient of correlation >0.9 for both, p Conclusion Compared with the CAS 200, the ACIS is a more sensitive and less time consuming technique for determining DNA ploidy. Results obtained by ACIS are also highly reproducible.

Flow cytometric DNA ploidy analysis of ovarian granulosa cell tumors

NARCIS (Netherlands)

D. Chadha; C.J. Cornelisse; A. Schabert (A.)

1990-01-01

textabstractAbstract The nuclear DNA content of 50 ovarian tumors initially diagnosed as granulosa cell tumors was measured by flow cytometry using paraffin-embedded archival material. The follow-up period of the patients ranged from 4 months to 19 years. Thirty-eight tumors were diploid or

Surveys of environmental DNA (eDNA): a new approach to estimate occurrence in Vulnerable manatee populations

Science.gov (United States)

Hunter, Margaret; Meigs-Friend, Gaia; Ferrante, Jason; Takoukam Kamla, Aristide; Dorazio, Robert; Keith Diagne, Lucy; Luna, Fabia; Lanyon, Janet M.; Reid, James P.

2018-01-01

Environmental DNA (eDNA) detection is a technique used to non-invasively detect cryptic, low density, or logistically difficult-to-study species, such as imperiled manatees. For eDNA measurement, genetic material shed into the environment is concentrated from water samples and analyzed for the presence of target species. Cytochrome bquantitative PCR and droplet digital PCR eDNA assays were developed for the 3 Vulnerable manatee species: African, Amazonian, and both subspecies of the West Indian (Florida and Antillean) manatee. Environmental DNA assays can help to delineate manatee habitat ranges, high use areas, and seasonal population changes. To validate the assay, water was analyzed from Florida’s east coast containing a high-density manatee population and produced 31564 DNA molecules l-1on average and high occurrence (ψ) and detection (p) estimates (ψ = 0.84 [0.40-0.99]; p = 0.99 [0.95-1.00]; limit of detection 3 copies µl-1). Similar occupancy estimates were produced in the Florida Panhandle (ψ = 0.79 [0.54-0.97]) and Cuba (ψ = 0.89 [0.54-1.00]), while occupancy estimates in Cameroon were lower (ψ = 0.49 [0.09-0.95]). The eDNA-derived detection estimates were higher than those generated using aerial survey data on the west coast of Florida and may be effective for population monitoring. Subsequent eDNA studies could be particularly useful in locations where manatees are (1) difficult to identify visually (e.g. the Amazon River and Africa), (2) are present in patchy distributions or are on the verge of extinction (e.g. Jamaica, Haiti), and (3) where repatriation efforts are proposed (e.g. Brazil, Guadeloupe). Extension of these eDNA techniques could be applied to other imperiled marine mammal populations such as African and Asian dugongs.

Diversity and endemism in deglaciated areas: ploidy, relative genome size and niche differentiation in the Galium pusillum complex (Rubiaceae) in Northern and Central Europe

Science.gov (United States)

Kolář, Filip; Lučanová, Magdalena; Vít, Petr; Urfus, Tomáš; Chrtek, Jindřich; Fér, Tomáš; Ehrendorfer, Friedrich; Suda, Jan

2013-01-01

Background and Aims Plants endemic to areas covered by ice sheets during the last glaciation represent paradigmatic examples of rapid speciation in changing environments, yet very few systems outside the harsh arctic zone have been comprehensively investigated so far. The Galium pusillum aggregate (Rubiaceae) is a challenging species complex that exhibits a marked differentiation in boreal parts of Northern Europe. As a first step towards understanding its evolutionary history in deglaciated regions, this study assesses cytological variation and ecological preferences of the northern endemics and compares the results with corresponding data for species occurring in neighbouring unglaciated parts of Central and Western Europe. Methods DNA flow cytometry was used together with confirmatory chromosome counts to determine ploidy levels and relative genome sizes in 1158 individuals from 181 populations. A formalized analysis of habitat preferences was applied to explore niche differentiation among species and ploidy levels. Key Results The G. pusillum complex evolved at diploid and tetraploid levels in Northern Europe, in contrast to the high-polyploid evolution of most other northern endemics. A high level of eco-geographic segregation was observed between different species (particularly along gradients of soil pH and competition) which is unusual for plants in deglaciated areas and most probably contributes to maintaining species integrity. Relative monoploid DNA contents of the species from previously glaciated regions were significantly lower than those of their counterparts from mostly unglaciated Central Europe, suggesting independent evolutionary histories. Conclusions The aggregate of G. pusillum in Northern Europe represents an exceptional case with a geographically vicariant and ecologically distinct diploid/tetraploid species endemic to formerly glaciated areas. The high level of interspecific differentiation substantially widens our perception of the

An Estimate of the Total DNA in the Biosphere.

Science.gov (United States)

Landenmark, Hanna K E; Forgan, Duncan H; Cockell, Charles S

2015-06-01

Modern whole-organism genome analysis, in combination with biomass estimates, allows us to estimate a lower bound on the total information content in the biosphere: 5.3 × 1031 (±3.6 × 1031) megabases (Mb) of DNA. Given conservative estimates regarding DNA transcription rates, this information content suggests biosphere processing speeds exceeding yottaNOPS values (1024 Nucleotide Operations Per Second). Although prokaryotes evolved at least 3 billion years before plants and animals, we find that the information content of prokaryotes is similar to plants and animals at the present day. This information-based approach offers a new way to quantify anthropogenic and natural processes in the biosphere and its information diversity over time.

Evaluation of the Environmental DNA Method for Estimating Distribution and Biomass of Submerged Aquatic Plants.

Science.gov (United States)

Matsuhashi, Saeko; Doi, Hideyuki; Fujiwara, Ayaka; Watanabe, Sonoko; Minamoto, Toshifumi

2016-01-01

The environmental DNA (eDNA) method has increasingly been recognized as a powerful tool for monitoring aquatic animal species; however, its application for monitoring aquatic plants is limited. To evaluate eDNA analysis for estimating the distribution of aquatic plants, we compared its estimated distributions with eDNA analysis, visual observation, and past distribution records for the submerged species Hydrilla verticillata. Moreover, we conducted aquarium experiments using H. verticillata and Egeria densa and analyzed the relationships between eDNA concentrations and plant biomass to investigate the potential for biomass estimation. The occurrences estimated by eDNA analysis closely corresponded to past distribution records, and eDNA detections were more frequent than visual observations, indicating that the method is potentially more sensitive. The results of the aquarium experiments showed a positive relationship between plant biomass and eDNA concentration; however, the relationship was not always significant. The eDNA concentration peaked within three days of the start of the experiment in most cases, suggesting that plants do not release constant amounts of DNA. These results showed that eDNA analysis can be used for distribution surveys, and has the potential to estimate the biomass of aquatic plants.

An analytical framework for estimating aquatic species density from environmental DNA

Science.gov (United States)

Chambert, Thierry; Pilliod, David S.; Goldberg, Caren S.; Doi, Hideyuki; Takahara, Teruhiko

2018-01-01

Environmental DNA (eDNA) analysis of water samples is on the brink of becoming a standard monitoring method for aquatic species. This method has improved detection rates over conventional survey methods and thus has demonstrated effectiveness for estimation of site occupancy and species distribution. The frontier of eDNA applications, however, is to infer species density. Building upon previous studies, we present and assess a modeling approach that aims at inferring animal density from eDNA. The modeling combines eDNA and animal count data from a subset of sites to estimate species density (and associated uncertainties) at other sites where only eDNA data are available. As a proof of concept, we first perform a cross-validation study using experimental data on carp in mesocosms. In these data, fish densities are known without error, which allows us to test the performance of the method with known data. We then evaluate the model using field data from a study on a stream salamander species to assess the potential of this method to work in natural settings, where density can never be known with absolute certainty. Two alternative distributions (Normal and Negative Binomial) to model variability in eDNA concentration data are assessed. Assessment based on the proof of concept data (carp) revealed that the Negative Binomial model provided much more accurate estimates than the model based on a Normal distribution, likely because eDNA data tend to be overdispersed. Greater imprecision was found when we applied the method to the field data, but the Negative Binomial model still provided useful density estimates. We call for further model development in this direction, as well as further research targeted at sampling design optimization. It will be important to assess these approaches on a broad range of study systems.

Evolution in African tropical trees displaying ploidy-habitat association: The genus Afzelia (Leguminosae).

Science.gov (United States)

Donkpegan, Armel S L; Doucet, Jean-Louis; Migliore, Jérémy; Duminil, Jérôme; Dainou, Kasso; Piñeiro, Rosalía; Wieringa, Jan J; Champluvier, Dominique; Hardy, Olivier J

2017-02-01

Polyploidy has rarely been documented in rain forest trees but it has recently been found in African species of the genus Afzelia (Leguminosae), which is composed of four tetraploid rain forest species and two diploid dry forest species. The genus Afzelia thus provides an opportunity to examine how and when polyploidy and habitat shift occurred in Africa, and whether they are associated. In this study, we combined three plastid markers (psbA, trnL, ndhF), two nuclear markers (ribosomal ITS and the single-copy PEPC E7 gene), plastomes (obtained by High Throughput Sequencing) and morphological traits, with an extensive taxonomic and geographic sampling to explore the evolutionary history of Afzelia. Both nuclear DNA and morphological vegetative characters separated diploid from tetraploid lineages. Although the two African diploid species were well differentiated genetically and morphologically, the relationships among the tetraploid species were not resolved. In contrast to the nuclear markers, plastid markers revealed that one of the diploid species forms a well-supported clade with the tetraploids, suggesting historical hybridisation, possibly in relation with genome duplication (polyploidization) and habitat shift from dry to rain forests. Molecular dating based on fossil-anchored gene phylogenies indicates that extant Afzelia started diverging c. 14.5 or 20Ma while extant tetraploid species started diverging c. 7.0 or 9.4Ma according to plastid and nuclear DNA, respectively. Additional studies of tropical polyploid plants are needed to assess whether the ploidy-habitat association observed in African Afzelia would reflect a role of polyploidization in niche divergence in the tropics. Copyright © 2016 Elsevier Inc. All rights reserved.

Flow cytometric and morphological analyses of Pinus pinaster somatic embryogenesis.

Science.gov (United States)

Marum, Liliana; Loureiro, João; Rodriguez, Eleazar; Santos, Conceição; Oliveira, M Margarida; Miguel, Célia

2009-09-25

An approach combining morphological profiling and flow cytometric analysis was used to assess genetic stability during the several steps of somatic embryogenesis in Pinus pinaster. Embryogenic cell lines of P. pinaster were established from immature zygotic embryos excised from seeds obtained from open-pollinated trees. During the maturation stage, phenotype of somatic embryos was characterized as being either normal or abnormal. Based upon the prevalent morphological traits, different types of abnormal embryos underwent further classification and quantification. Nuclear DNA content of maritime pine using the zygotic embryos was estimated to be 57.04 pg/2C, using propidium iodide flow cytometry. According to the same methodology, no significant differences (P< or =0.01) in DNA ploidy were detected among the most frequently observed abnormal phenotypes, embryogenic cell lines, zygotic and normal somatic embryos, and somatic embryogenesis-derived plantlets. Although the differences in DNA ploidy level do not exclude the occurrence of a low level of aneuploidy, the results obtained point to the absence of major changes in ploidy level during the somatic embryogenesis process of this economically important species. Therefore, our primary goal of true-to-typeness was assured at this level.

Ploidy Variation in Hybrids from Interploid 3x X 2x Crosses in Musa

Directory of Open Access Journals (Sweden)

Osuji, JO.

1997-01-01

Full Text Available Hybrids were obtained after in vitro germination of embryos from interploid crosses between triploid 'French' plantain cultivars (Musa spp. AAB group 'Ntanga 2' and 'Bobby Tannap' with diploid banana (Ivlusa acuminata subsp. burmannicoidesj 'Calcutta 4'. Cross-pollinated bunches were harvested at full maturity and ripened with acetylene in a room for 4 days. Seeds were extracted from peeled ripe fruits by squashing. Embryos from the seeds were excised aseptically after 2 days and germinated in vitro. Seedlings were subsequently planted in early evaluation trials after acclimatising in the greenhouse. Chromosome counts were carried out on root tips of mature and maiden suckers to determine ploidy levels using a modified squashing technique. Counts showed that two of the hybrids were aneuploids (trisomies with somatic chromosome number of 2n = 2x + 1 = 23, one hybrid was diploid while the other two were tetraploids. Tetraploids are the most promising hybrids for the genetic improvement of plantains. Diploids are valuable material for further improvement of the plantain genome at this ploidy level. Trisomies provide means for further characterisation of the Musa genome and physical gene mapping in plantain and banana.

Feulgen-DNA response and chromatin condensation in Malpighian tubules of Melipona rufiventris and Melipona quadrifasciata (Hymenoptera, Apoidea).

Science.gov (United States)

Mampumbu, André Roberto; Mello, Maria Luiza S

2008-08-01

Melipona quadrifasciata and Melipona rufiventris are stingless bee species which present low and high heterochromatin content, respectively, on their mitotic chromosomes as assessed visually after a C-banding assay. However, these species do not show differences in the C-banding responses of their Malpighian tubule interphase nuclei. In the present study, the Feulgen-DNA response, which could inform on differences in DNA depurination due to differences in chromatin condensation, was compared in the cell nuclei of the Malpighian tubules of these species. It was hypothesized that differences in acid hydrolysis kinetics patterns, as assessed by Feulgen reaction and studied microspectrophotometrically, could discriminate M. quadrifasciata and M. rufiventris interphase nuclei not distinguishable with the C-banding method. Feulgen-DNA values corresponding to more than one ploidy class were found in both species; these values at the hydrolysis time corresponding to the maximal DNA depurination for each ploidy degree were higher in M. quadrifasciata, reflecting a higher DNA content in the Malpighian tubule cell nuclei of this species compared to those of M. rufiventris at the same larval instar. The maximal Feulgen-DNA values of M. quadrifasciata after short (50 min) and long (90 min) hydrolysis times were found to be closer to each other, while those of M. rufiventris occurred sharply at the long hydrolysis time, indicating that DNA depurination in M. quadrifasciata occurred faster. This result is probably related to the involvement of differences in chromatin condensation; it agrees with the idea that M. rufiventris contains more heterochromatin than M. quadrifasciata, which is supported by the analysis of results obtained with the image analysis parameter average absorption ratio. The depurination kinetics studied here with the Feulgen reaction were revealed to be more pertinent than the C-banding technique in establishing differences in levels of chromatin condensation

Karyotype analysis, DNA content and molecular screening in Lippia alba (Verbenaceae

Directory of Open Access Journals (Sweden)

Patrícia M.O. Pierre

2011-09-01

Full Text Available Cytogenetic analyses, of pollen viability, nuclear DNA content and RAPD markers were employed to study three chemotypes of Lippia alba (Mill. (Verbenaceae in order to understand the genetic variation among them. Different ploidy levels and mixoploid individuals were observed. This work comprises the first report of different chromosome numbers (cytotypes in L. alba. The chromosome numbers of La2-carvone and La3-linalool chemotypes suggested that they are polyploids. Flow cytometric analysis showed an increase of nuclear DNA content that was not directly proportional to ploidy level variation. A cluster analysis based on RAPD markers revealed that La3-linalool shares genetic markers with La1-citral and La2-carvone. The analysis showed that the majority of genetic variation of La3-linalool could be a consequence of ixoploidy. ur data indicates that sexual reproduction aong those three chemotypes is unlikely and suggests the beginning of reproductive isolation. The results demonstrated that chromosome analysis, nuclear DNA content estimation and RAPD markers constitute excellent tools for detecting genetic variation among L. alba chemotypes.Análises citogenéticas, de viabilidade do pólen, do conteúdo de DNA nuclear e marcadores RAPD foram empregadas no estudo de três quimiotipos de Lippia alba (Mill. (Verbenaceae visando contribuir para o entendimento da variação genética entre os mesmos. Diferentes níveis de ploidia e indivíduos mixoploides foram observados. Este trabalho compreende o primeiro relato de diferentes números cromossômicos (citótipos em L. alba. Os números cromossômicos dos quimiotipos La2-carvona e La3-linalol sugere que eles seja poliploides. A análise da citometria de fluxo mostrou um aumento do conteúdo de DNA nuclear que não foi diretamente proporcional à variação no nível de ploidia. A análise de agrupamento baseada nos marcadores RAPD demonstrou que La3-linalol compartilha marcadores genéticos com La1

Mating system and ploidy influence levels of inbreeding depression in Clarkia (Onagraceae).

Science.gov (United States)

Barringer, Brian C; Geber, Monica A

2008-05-01

Inbreeding depression is the reduction in offspring fitness associated with inbreeding and is thought to be one of the primary forces selecting against the evolution of self-fertilization. Studies suggest that most inbreeding depression is caused by the expression of recessive deleterious alleles in homozygotes whose frequency increases as a result of self-fertilization or mating among relatives. This process leads to the selective elimination of deleterious alleles such that highly selfing species may show remarkably little inbreeding depression. Genome duplication (polyploidy) has also been hypothesized to influence levels of inbreeding depression, with polyploids expected to exhibit less inbreeding depression than diploids. We studied levels of inbreeding depression in allotetraploid and diploid species of Clarkia (Onagraceae) that vary in mating system (each cytotype was represented by an outcrossing and a selfing species). The outcrossing species exhibited more inbreeding depression than the selfing species for most fitness components and for two different measures of cumulative fitness. In contrast, though inbreeding depression was generally lower for the polyploid species than for the diploid species, the difference was statistically significant only for flower number and one of the two measures of cumulative fitness. Further, we detected no significant interaction between mating system and ploidy in determining inbreeding depression. In sum, our results suggest that a taxon's current mating system is more important than ploidy in influencing levels of inbreeding depression in natural populations of these annual plants.

Sequential steps in DNA replication are inhibited to ensure reduction of ploidy in meiosis

Science.gov (United States)

Hua, Hui; Namdar, Mandana; Ganier, Olivier; Gregan, Juraj; Méchali, Marcel; Kearsey, Stephen E.

2013-01-01

Meiosis involves two successive rounds of chromosome segregation without an intervening S phase. Exit from meiosis I is distinct from mitotic exit, in that replication origins are not licensed by Mcm2-7 chromatin binding, but spindle disassembly occurs during a transient interphase-like state before meiosis II. The absence of licensing is assumed to explain the block to DNA replication, but this has not been formally tested. Here we attempt to subvert this block by expressing the licensing control factors Cdc18 and Cdt1 during the interval between meiotic nuclear divisions. Surprisingly, this leads only to a partial round of DNA replication, even when these factors are overexpressed and effect clear Mcm2-7 chromatin binding. Combining Cdc18 and Cdt1 expression with modulation of cyclin-dependent kinase activity, activation of Dbf4-dependent kinase, or deletion of the Spd1 inhibitor of ribonucleotide reductase has little additional effect on the extent of DNA replication. Single-molecule analysis indicates this partial round of replication results from inefficient progression of replication forks, and thus both initiation and elongation replication steps may be inhibited in late meiosis. In addition, DNA replication or damage during the meiosis I–II interval fails to arrest meiotic progress, suggesting absence of checkpoint regulation of meiosis II entry. PMID:23303250

Estimating HPV DNA Deposition Between Sexual Partners Using HPV Concordance, Y Chromosome DNA Detection, and Self-reported Sexual Behaviors.

Science.gov (United States)

Malagón, Talía; Burchell, Ann N; El-Zein, Mariam; Guénoun, Julie; Tellier, Pierre-Paul; Coutlée, François; Franco, Eduardo L

2017-12-05

Detection of human papillomavirus (HPV) DNA in genital samples may not always represent true infections but may be depositions from infected sexual partners. We examined whether sexual risk factors and a biomarker (Y chromosome DNA) were associated with genital HPV partner concordance and estimated the fraction of HPV detections potentially attributable to partner deposition. The HITCH study enrolled young women attending a university or college in Montréal, Canada, and their male partners, from 2005 to 2010. We tested baseline genital samples for Y chromosome DNA and HPV DNA using polymerase chain reaction. Type-specific HPV concordance was 42.4% in partnerships where at least one partner was HPV DNA positive. Y chromosome DNA predicted type-specific HPV concordance in univariate analyses, but in multivariable models the independent predictors of concordance were days since last vaginal sex (26.5% higher concordance 0-1 vs 8-14 days after last vaginal sex) and condom use (22.6% higher concordance in never vs always users). We estimated that 14.1% (95% confidence interval [CI], 6.3-21.9%) of HPV DNA detections in genital samples were attributable to vaginal sex in the past week. A substantial proportion of HPV DNA detections may be depositions due to recent unprotected vaginal sex. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Overcoming the species hybridization barrier by ploidy manipulation in the genus Oryza.

Science.gov (United States)

Tonosaki, Kaoru; Sekine, Daisuke; Ohnishi, Takayuki; Ono, Akemi; Furuumi, Hiroyasu; Kurata, Nori; Kinoshita, Tetsu

2018-02-01

In most eudicot and monocot species, interspecific and interploidy crosses generally display abnormalities in the endosperm that are the major cause of a post-zygotic hybridization barrier. In some eudicot species, however, this type of hybridization barrier can be overcome by the manipulation of ploidy levels of one parental species, suggesting that the molecular mechanisms underlying the species hybridization barrier can be circumvented by genome dosage. We previously demonstrated that endosperm barriers in interspecific and interploidy crosses in the genus Oryza involve overlapping but different mechanisms. This result contrasts with those in the genus Arabidopsis, which shows similar outcomes in both interploidy and interspecific crosses. Therefore, we postulated that an exploration of pathways for overcoming the species hybridization barrier in Oryza endosperm, by manipulating the ploidy levels in one parental species, might provide novel insights into molecular mechanisms. We showed that fertile hybrid seeds could be produced by an interspecific cross of female tetraploid Oryza sativa and male diploid Oryza longistaminata. Although the rate of nuclear divisions did not return to normal levels in the hybrid endosperm, the timing of cellularization, nucellus degeneration and the accumulation of storage products were close to normal levels. In addition, the expression patterns of the imprinted gene MADS87 and YUCCA11 were changed when the species barrier was overcome. These results suggest that the regulatory machinery for developmental transitions and imprinted gene expression are likely to play a central role in overcoming species hybridization barriers by genome dosage in the genus Oryza. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Nuclear/Nucleolar morphometry and DNA image cytometry as a combined diagnostic tool in pathology of prostatic carcinoma

Energy Technology Data Exchange (ETDEWEB)

Kavantzas, N.; Agapitos, E.; Lazaris, A. C.; Pavlopulos, P.M.; Sofikitis, N.; Davaris, P. [National University of Athens, Dept. of Pathology, Medical School, Athens (Greece)

2001-12-01

Paraffin tissue sections from 50 patients with prostate adenocarcinoma were used to study nuclear and nucleolar morphometric features by image analysis. The results were compared to DNA ploidy and Gleason grade. In the examined histological samples nuclear and nucleolar areas were positively interrelated. It was also noticed that the higher the percentage of nucleolated nuclei, the bigger the nuclear and nucleolar areas. The morphometric characteristics did not differ significantly among the four grades of the examined specimens. In well-differentiated carcinomas the DNA index was lower than in the rest at a statistically significant level. Hypodiploid carcinomas were found to possess significantly bigger nuclear areas than any other DNA index group. Morphonuclear evidence of anaplasia and DNA aneuploidy may be used as diagnostic tools in prostate cancer in addition to Gleason grade.

Nuclear/Nucleolar morphometry and DNA image cytometry as a combined diagnostic tool in pathology of prostatic carcinoma

International Nuclear Information System (INIS)

Kavantzas, N.; Agapitos, E.; Lazaris, A. C.; Pavlopulos, P.M.; Sofikitis, N.; Davaris, P.

2001-01-01

Paraffin tissue sections from 50 patients with prostate adenocarcinoma were used to study nuclear and nucleolar morphometric features by image analysis. The results were compared to DNA ploidy and Gleason grade. In the examined histological samples nuclear and nucleolar areas were positively interrelated. It was also noticed that the higher the percentage of nucleolated nuclei, the bigger the nuclear and nucleolar areas. The morphometric characteristics did not differ significantly among the four grades of the examined specimens. In well-differentiated carcinomas the DNA index was lower than in the rest at a statistically significant level. Hypodiploid carcinomas were found to possess significantly bigger nuclear areas than any other DNA index group. Morphonuclear evidence of anaplasia and DNA aneuploidy may be used as diagnostic tools in prostate cancer in addition to Gleason grade

Plant DNA barcodes can accurately estimate species richness in poorly known floras.

Science.gov (United States)

Costion, Craig; Ford, Andrew; Cross, Hugh; Crayn, Darren; Harrington, Mark; Lowe, Andrew

2011-01-01

Widespread uptake of DNA barcoding technology for vascular plants has been slow due to the relatively poor resolution of species discrimination (∼70%) and low sequencing and amplification success of one of the two official barcoding loci, matK. Studies to date have mostly focused on finding a solution to these intrinsic limitations of the markers, rather than posing questions that can maximize the utility of DNA barcodes for plants with the current technology. Here we test the ability of plant DNA barcodes using the two official barcoding loci, rbcLa and matK, plus an alternative barcoding locus, trnH-psbA, to estimate the species diversity of trees in a tropical rainforest plot. Species discrimination accuracy was similar to findings from previous studies but species richness estimation accuracy proved higher, up to 89%. All combinations which included the trnH-psbA locus performed better at both species discrimination and richness estimation than matK, which showed little enhanced species discriminatory power when concatenated with rbcLa. The utility of the trnH-psbA locus is limited however, by the occurrence of intraspecific variation observed in some angiosperm families to occur as an inversion that obscures the monophyly of species. We demonstrate for the first time, using a case study, the potential of plant DNA barcodes for the rapid estimation of species richness in taxonomically poorly known areas or cryptic populations revealing a powerful new tool for rapid biodiversity assessment. The combination of the rbcLa and trnH-psbA loci performed better for this purpose than any two-locus combination that included matK. We show that although DNA barcodes fail to discriminate all species of plants, new perspectives and methods on biodiversity value and quantification may overshadow some of these shortcomings by applying barcode data in new ways.

Estimates of DNA strand breakage in bottlenose dolphin (Tursiops truncatus leukocytes measured with the Comet and DNA diffusion assays

Directory of Open Access Journals (Sweden)

Adriana Díaz

2009-01-01

Full Text Available The analysis of DNA damage by mean of Comet or single cell gel electrophoresis (SCGE assay has been commonly used to assess genotoxic impact in aquatic animals being able to detect exposure to low concentrations of contaminants in a wide range of species. The aims of this work were 1 to evaluate the usefulness of the Comet to detect DNA strand breakage in dolphin leukocytes, 2 to use the DNA diffusion assay to determine the amount of DNA strand breakage associated with apoptosis or necrosis, and 3 to determine the proportion of DNA strand breakage that was unrelated to apoptosis and necrosis. Significant intra-individual variation was observed in all of the estimates of DNA damage. DNA strand breakage was overestimated because a considerable amount (~29% of the DNA damage was derived from apoptosis and necrosis. The remaining DNA damage in dolphin leukocytes was caused by factors unrelated to apoptosis and necrosis. These results indicate that the DNA diffusion assay is a complementary tool that can be used together with the Comet assay to assess DNA damage in bottlenose dolphins.

Ploidy race distributions since the Last Glacial Maximum in the North American desert shrub, Larrea tridentata

Science.gov (United States)

Hunter, K.L.; Betancourt, J.L.; Riddle, B.R.; Van Devender, T. R.; Cole, K.L.; Geoffrey, Spaulding W.

2000-01-01

1 A classic biogeographic pattern is the alignment of diploid, tetraploid and hexaploid races of creosote bush (Larrea tridentata) across the Chihuahuan, Sonoran and Mohave Deserts of western North America. We used statistically robust differences in guard cell size of modern plants and fossil leaves from packrat middens to map current and past distributions of these ploidy races since the Last Glacial Maximum (LGM). 2 Glacial/early Holocene (26-10 14C kyr BP or thousands of radiocarbon years before present) populations included diploids along the lower Rio Grande of west Texas, 650 km removed from sympatric diploids and tetraploids in the lower Colorado River Basin of south-eastern California/south-western Arizona. Diploids migrated slowly from lower Rio Grande refugia with expansion into the northern Chihuahuan Desert sites forestalled until after ???4.0 14C kyr BP. Tetraploids expanded from the lower Colorado River Basin into the northern limits of the Sonoran Desert in central Arizona by 6.4 14C kyr BP. Hexaploids appeared by 8.5 14C kyr BP in the lower Colorado River Basin, reaching their northernmost limits (???37??N) in the Mohave Desert between 5.6 and 3.9 14C kyr BP. 3 Modern diploid isolates may have resulted from both vicariant and dispersal events. In central Baja California and the lower Colorado River Basin, modern diploids probably originated from relict populations near glacial refugia. Founder events in the middle and late Holocene established diploid outposts on isolated limestone outcrops in areas of central and southern Arizona dominated by tetraploid populations. 4 Geographic alignment of the three ploidy races along the modern gradient of increasingly drier and hotter summers is clearly a postglacial phenomenon, but evolution of both higher ploidy races must have happened before the Holocene. The exact timing and mechanism of polyploidy evolution in creosote bush remains a matter of conjecture. ?? 2001 Blackwell Science Ltd.

Transgene-induced gene silencing is not affected by a change in ploidy level.

Directory of Open Access Journals (Sweden)

Daniela Pignatta

Full Text Available BACKGROUND: Whole genome duplication, which results in polyploidy, is a common feature of plant populations and a recurring event in the evolution of flowering plants. Polyploidy can result in changes to gene expression and epigenetic instability. Several epigenetic phenomena, occurring at the transcriptional or post-transcriptional level, have been documented in allopolyploids (polyploids derived from species hybrids of Arabidopsis thaliana, yet findings in autopolyploids (polyploids derived from the duplication of the genome of a single species are limited. Here, we tested the hypothesis that an increase in ploidy enhances transgene-induced post-transcriptional gene silencing using autopolyploids of A. thaliana. METHODOLOGY/PRINCIPAL FINDINGS: Diploid and tetraploid individuals of four independent homozygous transgenic lines of A. thaliana transformed with chalcone synthase (CHS inverted repeat (hairpin constructs were generated. For each line diploids and tetraploids were compared for efficiency in post-transcriptional silencing of the endogenous CHS gene. The four lines differed substantially in their silencing efficiency. Yet, diploid and tetraploid plants derived from these plants and containing therefore identical transgene insertions showed no difference in the efficiency silencing CHS as assayed by visual scoring, anthocyanin assays and quantification of CHS mRNA. CONCLUSIONS/SIGNIFICANCE: Our results in A. thaliana indicated that there is no effect of ploidy level on transgene-induced post-transcriptional gene silencing. Our findings that post-transcriptional mechanisms were equally effective in diploids and tetraploids supports the use of transgene-driven post-transcriptional gene silencing as a useful mechanism to modify gene expression in polyploid species.

Plant DNA barcodes can accurately estimate species richness in poorly known floras.

Directory of Open Access Journals (Sweden)

Craig Costion

Full Text Available BACKGROUND: Widespread uptake of DNA barcoding technology for vascular plants has been slow due to the relatively poor resolution of species discrimination (∼70% and low sequencing and amplification success of one of the two official barcoding loci, matK. Studies to date have mostly focused on finding a solution to these intrinsic limitations of the markers, rather than posing questions that can maximize the utility of DNA barcodes for plants with the current technology. METHODOLOGY/PRINCIPAL FINDINGS: Here we test the ability of plant DNA barcodes using the two official barcoding loci, rbcLa and matK, plus an alternative barcoding locus, trnH-psbA, to estimate the species diversity of trees in a tropical rainforest plot. Species discrimination accuracy was similar to findings from previous studies but species richness estimation accuracy proved higher, up to 89%. All combinations which included the trnH-psbA locus performed better at both species discrimination and richness estimation than matK, which showed little enhanced species discriminatory power when concatenated with rbcLa. The utility of the trnH-psbA locus is limited however, by the occurrence of intraspecific variation observed in some angiosperm families to occur as an inversion that obscures the monophyly of species. CONCLUSIONS/SIGNIFICANCE: We demonstrate for the first time, using a case study, the potential of plant DNA barcodes for the rapid estimation of species richness in taxonomically poorly known areas or cryptic populations revealing a powerful new tool for rapid biodiversity assessment. The combination of the rbcLa and trnH-psbA loci performed better for this purpose than any two-locus combination that included matK. We show that although DNA barcodes fail to discriminate all species of plants, new perspectives and methods on biodiversity value and quantification may overshadow some of these shortcomings by applying barcode data in new ways.

The agamic complex of Pilosella (Asteraceae) in Bulgaria and in the southwestern Romania: variation in ploidy level and breeding systems

Czech Academy of Sciences Publication Activity Database

Krahulcová, Anna; Vladimirov, V.; Krahulec, František; Bräutigam, S.

2009-01-01

Roč. 15, č. 3 (2009), s. 377-384 ISSN 1310-7771 R&D Projects: GA ČR GA206/07/0059; GA ČR GA206/08/0890 Institutional research plan: CEZ:AV0Z60050516 Keywords : Pilosella * ploidy level * breeding system Subject RIV: EF - Botanics

Determination of Ploidy Level and Nuclear DNA Content in the Droseraceae by Flow Cytometry

Czech Academy of Sciences Publication Activity Database

Hoshi, Y.; Azumatani, M.; Suyama, T.; Adamec, Lubomír

2017-01-01

Roč. 82, č. 3 (2017), s. 321-327 ISSN 0011-4545 Institutional support: RVO:67985939 Keywords : nuclear DNA content * genome size * Droseraceae Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Plant sciences, botany Impact factor: 0.913, year: 2016

Estimation of absorbed dose in cell nuclei due to DNA-bound /sup 3/H

Energy Technology Data Exchange (ETDEWEB)

Saito, M; Ishida, M R; Streffer, C; Molls, M

1985-04-01

The average absorbed dose due to DNA-bound /sup 3/H in a cell nucleus was estimated by a Monte Carlo simulation for a model nucleus which was assumed to be spheroidal. The volume of the cell nucleus was the major dose-determining factor for cell nuclei which have the same DNA content and the same specific activity of DNA. This result was applied to estimating the accumulated dose in the cell nuclei of organs of young mice born from mother mice which ingested /sup 3/H-thymidine with drinking water during pregnancy. The values of dose-modifying factors for the accumulated dose due to DNA-bound /sup 3/H compared to the dose due to an assumed homogenous distribution of /sup 3/H in organ were found to be between about 2 and 6 for the various organs.

Extensive analysis of native and non-native Centaurea solstitialis L. populations across the world shows no traces of polyploidization

Directory of Open Access Journals (Sweden)

Ramona-Elena Irimia

2017-08-01

Full Text Available Centaurea solstitialis L. (yellow starthistle, Asteraceae is a Eurasian native plant introduced as an exotic into North and South America, and Australia, where it is regarded as a noxious invasive. Changes in ploidy level have been found to be responsible for numerous plant biological invasions, as they are involved in trait shifts critical to invasive success, like increased growth rate and biomass, longer life-span, or polycarpy. C. solstitialis had been reported to be diploid (2n = 2x = 16 chromosomes, however, actual data are scarce and sometimes contradictory. We determined for the first time the absolute nuclear DNA content by flow cytometry and estimated ploidy level in 52 natural populations of C. solstitialis across its native and non-native ranges, around the world. All the C. solstitialis populations screened were found to be homogeneously diploid (average 2C value of 1.72 pg, SD = ±0.06 pg, with no significant variation in DNA content between invasive and non-invasive genotypes. We did not find any meaningful difference among the extensive number of native and non-native C. solstitialis populations sampled around the globe, indicating that the species invasive success is not due to changes in genome size or ploidy level.

Extensive analysis of native and non-native Centaurea solstitialis L. populations across the world shows no traces of polyploidization.

Science.gov (United States)

Irimia, Ramona-Elena; Montesinos, Daniel; Eren, Özkan; Lortie, Christopher J; French, Kristine; Cavieres, Lohengrin A; Sotes, Gastón J; Hierro, José L; Jorge, Andreia; Loureiro, João

2017-01-01

Centaurea solstitialis L. (yellow starthistle, Asteraceae) is a Eurasian native plant introduced as an exotic into North and South America, and Australia, where it is regarded as a noxious invasive. Changes in ploidy level have been found to be responsible for numerous plant biological invasions, as they are involved in trait shifts critical to invasive success, like increased growth rate and biomass, longer life-span, or polycarpy. C . solstitialis had been reported to be diploid (2 n  = 2 x  = 16 chromosomes), however, actual data are scarce and sometimes contradictory. We determined for the first time the absolute nuclear DNA content by flow cytometry and estimated ploidy level in 52 natural populations of C . solstitialis across its native and non-native ranges, around the world. All the C. solstitialis populations screened were found to be homogeneously diploid (average 2C value of 1.72 pg, SD = ±0.06 pg), with no significant variation in DNA content between invasive and non-invasive genotypes. We did not find any meaningful difference among the extensive number of native and non-native C . solstitialis populations sampled around the globe, indicating that the species invasive success is not due to changes in genome size or ploidy level.

Making Epidermal Bladder Cells Bigger: Developmental- and Salinity-Induced Endopolyploidy in a Model Halophyte.

Science.gov (United States)

Barkla, Bronwyn J; Rhodes, Timothy; Tran, Kieu-Nga T; Wijesinghege, Chathura; Larkin, John C; Dassanayake, Maheshi

2018-06-01

Endopolyploidy occurs when DNA replication takes place without subsequent mitotic nuclear division, resulting in cell-specific ploidy levels within tissues. In plants, endopolyploidy plays an important role in sustaining growth and development, but only a few studies have demonstrated a role in abiotic stress response. In this study, we investigated the function of ploidy level and nuclear and cell size in leaf expansion throughout development and tracked cell type-specific ploidy in the halophyte Mesembryanthemum crystallinum In addition to developmental endopolyploidy, we examined the effects of salinity stress on ploidy level. We focused specifically on epidermal bladder cells (EBC), which are modified balloon-like trichomes, due to their large size and role in salt accumulation. Our results demonstrate that ploidy increases as the leaves expand in a similar manner for each leaf type, and ploidy levels up to 512C were recorded for nuclei in EBC of leaves of adult plants. Salt treatment led to a significant increase in ploidy levels in the EBC, and these cells showed spatially related differences in their ploidy and nuclear and cell size depending on the positions on the leaf and stem surface. Transcriptome analysis highlighted salinity-induced changes in genes involved in DNA replication, cell cycle, endoreduplication, and trichome development in EBC. The increase in cell size and ploidy observed in M. crystallinum under salinity stress may contribute to salt tolerance by increasing the storage capacity for sodium sequestration brought about by higher metabolic activity driving rapid cell enlargement in the leaf tissue and EBC. © 2018 American Society of Plant Biologists. All rights reserved.

DNA-based population density estimation of black bear at northern ...

African Journals Online (AJOL)

The analysis of deoxyribonucleic acid (DNA) microsatellites from hair samples obtained by the non-invasive method of traps was used to estimate the population density of black bears (Ursus americanus eremicus) in a mountain located at the county of Lampazos, Nuevo Leon, Mexico. The genotyping of bears was ...

Variation in ploidy level and phenology can result in large and unexpected differences in demography and climatic sensitivity between closely related ferns.

NARCIS (Netherlands)

Groot, de G.A.; Zuidema, P.A.; Groot, H.; During, H.J.

2012-01-01

• Premise of the study: Current environmental changes may affect the dynamics and viability of plant populations. This environmental sensitivity may differ between species of different ploidy level because polyploidization can influence life history traits. We compared the demography and climatic

Towards resolving the Knautia arvensis agg. (Dipsacaceae) puzzle: primary and secondary contact zones and ploidy segregation at landscape and microgeographic scales

Czech Academy of Sciences Publication Activity Database

Kolář, F.; Štech, M.; Trávníček, Pavel; Rauchová, Jana; Urfus, Tomáš; Vít, Petr; Kubešová, Magdalena; Suda, Jan

2009-01-01

Roč. 103, č. 6 (2009), s. 963-974 ISSN 0305-7364 R&D Projects: GA AV ČR(CZ) KJB601110627 Institutional research plan: CEZ:AV0Z60050516 Keywords : Knautia arvensis * polyploidy * ploidy mixture Subject RIV: EF - Botanics Impact factor: 3.501, year: 2009

DNA replication and the repair of DNA strand breaks in nuclei of Physarum polycephalum. Terminal report, August 1, 1978-March 31, 1980

International Nuclear Information System (INIS)

Brewer, E.N.; Evans, T.E.

1980-01-01

Nuclei isolated from Physarum are able to replicate approximately 15% of the total genome in a manner which is qualitatively similar to the DNA replication process occurring in the intact organism. Such nuclei, however, are defective in the joining of Okazaki intermediates in vitro. Two DNA polymerase species, isolated from nuclei or intact plasmodia of this organism, can be separated by sucrose density gradient centrifugation. Total DNA polymerase activity is low in nuclei isolated during mitosis. A heat-stable glycoprotein material present in aqueous nuclear extracts stimulates DNA synthesis in well-washed nuclei. A sub-nuclear preparation active in DNA synthesis in vitro has been obtained from isolated nuclei of Physarum. Radiation-induced DNA double-strand breaks are rejoined in intact plasmodia and isolated nuclei of Physarum in a cell cycle-dependent manner. This phenomenon does not appear to be due to an intrinsic difference in nuclear DNA endonuclease activity at different times of the mitotic cycle. DNA strand breaks and repair induced by the carcinogen 4-nitroquinoline-1-oxide is similar in several respects to that resulting from exposure of the organism to ionizing radiation. Temperature sensitive strains of Physarum have been constructed and preliminary genetical and biochemical characterizations have been carried out. Two of the strains appear to be conditionally defective in DNA metabolism. An isogenic ploidal series of amoebae has been prepared and characterized as to uv and ionizing radiation sensitivity (in terms of cell survival). There is a direct relationship between ploidy and resistance to uv whereas ploidal change does not appear to affect the response to ionizing radiation

The effects of strain and ploidy on the physiological responses of rainbow trout (Oncorhynchus mykiss) to pH 9.5 exposure.

Science.gov (United States)

Thompson, William A; Rodela, Tamara M; Richards, Jeffrey G

2015-05-01

We characterized the physiological effects of exposure to pH9.5 on one domesticated and four wild strains of diploid and triploid juvenile rainbow trout (Oncorhynchus mykiss) over two consecutive years. In the first year, 35-70% of the individuals from the wild strains showed a loss of equilibrium (LOE) at 12 h exposure to pH9.5, with all fish from wild strains experiencing a LOE by 48 h. In contrast, trout strains and ploidies. Plasma chloride decreased at 24h exposure in all trout strains and ploidies, but recovered by 72 h. No change was observed in plasma sodium. Overall, our data suggest that the domesticated strain of trout is more tolerant of pH9.5 than the wild strains, but these differences in tolerance cannot be explained by our sub-lethal assessment of ammonia balance or ion regulation. Copyright © 2015 Elsevier Inc. All rights reserved.

Hydrodynamic characterization and molecular weight estimation of ultrasonically sheared DNA

International Nuclear Information System (INIS)

Casal, J. I.; Garces, F.; Garcia-Sacristan, A.

1981-01-01

The sedimentation coefficients and intrinsic viscosities of ultrasonically sheared calf thymus DNA have been determined. The molecular weight estimation according to this parameters have been compared with the ones obtained from the electrophoretic migration rates based on the calibration proposed using the known molecular weight restriction fragments of X-ENA. (Author) 35 refs

Ploidia e fertilidade de pólen em progênies de citros Ploidy and pollen fertility in citrus hybrids

Directory of Open Access Journals (Sweden)

Eduardo Cesar Brugnara

2008-01-01

Full Text Available Este trabalho foi desenvolvido para estimar a fertilidade do pólen e determinar o nível de ploidia em progênies dos cruzamentos da tangerineira 'Montenegrina' (C. deliciosa Ten. com a tangerineira 'King' (C. nobilis Lour. e com a laranjeira 'Caipira' (C. sinensis (L. Osb.. As plantas, de pés-francos e com idade entre 11 e 12 anos, são mantidas na Estação Experimental Agronômica da Universidade Federal do Rio Grande do Sul, em Eldorado do Sul. Avaliaram-se o nível de ploidia, através da contagem dos cromossomos em células em meiose, e a fertilidade do pólen, por coloração do pólen com carmim propiônico, em 2005 e 2006. Todos os híbridos avaliados são diplóides e a fertilidade de pólen variou de zero a 98%. C27 - híbrido de 'Montenegrina' x 'Caipira' - revelou-se praticamente estéril, e a fertilidade de C21, do mesmo cruzamento, é de 10%. A menor fertilidade observada no cruzamento da 'Montenegrina' x 'King' foi de 42%.This work was performed to estimate pollen fertility and determinate the ploidy level of 11 to 12 years old progenies of crosses of 'Montenegrina' mandarin (Citrus deliciosa Ten. with 'King' mandarin (C. nobilis Lour. and with 'Caipira' sweet orange (C. sinensis (L. Osb. maintained at Estação Experimental Agronômica of UFRGS, in Eldorado do Sul, Brazil. The ploidy level was determined by chromosome countings in meiosis cells and pollen fertility evaluated by staining pollen grains with propionic carmine. All evaluated hybrids are diploid and pollen fertility varied from zero to 98%. C27, a hybrid 'Montenegrina' x 'Caipira', was found is sterile, and C21, from the same cross, showed 10 % fertility. The lowest observed fertility in the progeny 'Montenegrina' x 'King' was 42 %.

A rapid assessment method to estimate the distribution of juvenile Chinook Salmon in tributary habitats using eDNA and occupancy estimation

Science.gov (United States)

Matter, A.; Falke, Jeffrey A.; López, J. Andres; Savereide, James W.

2018-01-01

Identification and protection of water bodies used by anadromous species are critical in light of increasing threats to fish populations, yet often challenging given budgetary and logistical limitations. Noninvasive, rapid‐assessment, sampling techniques may reduce costs and effort while increasing species detection efficiencies. We used an intrinsic potential (IP) habitat model to identify high‐quality rearing habitats for Chinook Salmon Oncorhynchus tshawytscha and select sites to sample throughout the Chena River basin, Alaska, for juvenile occupancy using an environmental DNA (eDNA) approach. Water samples were collected from 75 tributary sites in 2014 and 2015. The presence of Chinook Salmon DNA in water samples was assessed using a species‐specific quantitative PCR (qPCR) assay. The IP model predicted over 900 stream kilometers in the basin to support high‐quality (IP ≥ 0.75) rearing habitat. Occupancy estimation based on eDNA samples indicated that 80% and 56% of previously unsampled sites classified as high or low IP (IP Salmon DNA from three replicate water samples was high (p = 0.76) but varied with drainage area (km2). A power analysis indicated high power to detect proportional changes in occupancy based on parameter values estimated from eDNA occupancy models, although power curves were not symmetrical around zero, indicating greater power to detect positive than negative proportional changes in occupancy. Overall, the combination of IP habitat modeling and occupancy estimation provided a useful, rapid‐assessment method to predict and subsequently quantify the distribution of juvenile salmon in previously unsampled tributary habitats. Additionally, these methods are flexible and can be modified for application to other species and in other locations, which may contribute towards improved population monitoring and management.

Neotropical bats: estimating species diversity with DNA barcodes.

Directory of Open Access Journals (Sweden)

Elizabeth L Clare

Full Text Available DNA barcoding using the cytochrome c oxidase subunit 1 gene (COI is frequently employed as an efficient method of species identification in animal life and may also be used to estimate species richness, particularly in understudied faunas. Despite numerous past demonstrations of the efficiency of this technique, few studies have attempted to employ DNA barcoding methodologies on a large geographic scale, particularly within tropical regions. In this study we survey current and potential species diversity using DNA barcodes with a collection of more than 9000 individuals from 163 species of Neotropical bats (order Chiroptera. This represents one of the largest surveys to employ this strategy on any animal group and is certainly the largest to date for land vertebrates. Our analysis documents the utility of this tool over great geographic distances and across extraordinarily diverse habitats. Among the 163 included species 98.8% possessed distinct sets of COI haplotypes making them easily recognizable at this locus. We detected only a single case of shared haplotypes. Intraspecific diversity in the region was high among currently recognized species (mean of 1.38%, range 0-11.79% with respect to birds, though comparable to other bat assemblages. In 44 of 163 cases, well-supported, distinct intraspecific lineages were identified which may suggest the presence of cryptic species though mean and maximum intraspecific divergence were not good predictors of their presence. In all cases, intraspecific lineages require additional investigation using complementary molecular techniques and additional characters such as morphology and acoustic data. Our analysis provides strong support for the continued assembly of DNA barcoding libraries and ongoing taxonomic investigation of bats.

A Ploidy Difference Represents an Impassable Barrier for Hybridisation in Animals. Is There an Exception among Botiid Loaches (Teleostei: Botiidae?

Directory of Open Access Journals (Sweden)

Jörg Bohlen

Full Text Available One of the most efficient mechanisms to keep animal lineages separate is a difference in ploidy level (number of whole genome copies, since hybrid offspring from parents with different ploidy level are functionally sterile. In the freshwater fish family Botiidae, ploidy difference has been held responsible for the separation of its two subfamilies, the evolutionary tetraploid Botiinae and the diploid Leptobotiinae. Diploid and tetraploid species coexist in the upper Yangtze, the Pearl River and the Red River basins in China. Interestingly, the species 'Botia' zebra from the Pearl River basin combines a number of morphological characters that otherwise are found in the diploid genus Leptobotia with morphological characters of the tetraploid genus Sinibotia, therefore the aim of the present study is to test weather 'B.' zebra is the result of a hybridisation event between species from different subfamilies with different ploidy level. A closer morphological examination indeed demonstrates a high similarity of 'B.' zebra to two co-occurring species, the diploid Leptobotia guilinensis and the tetraploid Sinibotia pulchra. These two species thus could have been the potential parental species in case of a hybrid origin of 'B.' zebra. The morphologic analysis further reveals that 'B.' zebra bears even the diagnostic characters of the genera Leptobotia (Leptobotiinae and Sinibotia (Botiinae. In contrast, a comparison of six allozyme loci between 'B.' zebra, L. guilinensis and S. pulchra showed only similarities between 'B.' zebra and S. pulchra, not between 'B.' zebra and L. guilinensis. Six specimens of 'B.' zebra that were cytogenetically analysed were tetraploid with 4n = 100. The composition of the karyotype (18% metacentric, 18% submetacentric, 36% subtelocentric and 28% acrocentric chromosomes differs from those of L. guilinensis (12%, 24%, 20% and 44% and S. pulchra (20%, 26%, 28% and 26%, and cannot be obtained by any combination of genomes from

Local climate and cultivation, but not ploidy, predict functional trait variation in Bouteloua gracilis (Poaceae)

Science.gov (United States)

Butterfield, Bradley J.; Wood, Troy E.

2015-01-01

Efforts to improve the diversity of seed 18 resources for important restoration species has become a high priority for land managers in many parts of the world. Relationships between functional trait values and the environment from which seed sources are collected can provide important insights into patterns of local adaptation and guidelines for seed transfer. However, little is known about which functional traits exhibit genetic differentiation across populations of restoration species and thus may contribute to local adaptation. Here, we report the results of a common garden experiment aimed at assessing genetic (including ploidy level) and environmental regulation of several functional traits among populations of Bouteloua gracilis, a dominant C4 grass and the most highly utilized restoration species across much of the Colorado Plateau. We found that leaf size and specific leaf area (SLA) varied significantly among populations, and were strongly correlated with the source population environment from which seeds were collected. However, variation in ploidy level had no significant effect on functional traits. Leaves of plants grown from commercial seed releases were significantly larger and had lower SLA than those from natural populations, a result that is concordant with the overall relation between climate and these two functional traits. We suggest that the patterns of functional trait variation shown here may extend to other grass species in the western USA, and may serve as useful proxies for more extensive genecology research. Furthermore, we argue that care should be taken to develop commercial seed lines with functional trait values that match those of natural populations occupying climates similar to target restoration sites.

Changes of ploidy and sexuality status of "Carassius auratus" populations in the drainage area of the River Dyje (Czech Republic)

Czech Academy of Sciences Publication Activity Database

Lusková, Věra; Halačka, Karel; Vetešník, Lukáš; Lusk, Stanislav

2004-01-01

Roč. 4, č. 2 (2004), s. 165-171 ISSN 1642-3593. [International Symposium on the Ecology of Fluvial Fishes /9./. Lodz, 23.06.2003-26.06.2003] R&D Projects: GA ČR GA206/00/0668; GA AV ČR IBS5045111 Institutional research plan: CEZ:AV0Z6093917 Keywords : Carassius auratus * goldfish * ploidy status Subject RIV: EH - Ecology, Behaviour

Flow Cytometric DNA Analysis Using Cytokeratin Labeling for Identification of Tumor Cells in Carcinomas of the Breast and the Female Genital Tract

Directory of Open Access Journals (Sweden)

Rainer Kimmig

2001-01-01

Full Text Available Flow cytometric assessment of DNA‐ploidy and S‐phase fraction in malignant tumors is compromised by the heterogeneity of cell subpopulations derived from the malignant and surrounding connective tissue, e.g., tumor, stromal and inflammatory cells. To evaluate the effect on quality of DNA cell cycle analysis and determination of DNA ploidy, cytokeratin labeling of epithelial cells was used for tumor cell enrichment in breast, ovarian, cervical and endometrial cancer prior to DNA analysis. In a prospective study, tumor cell subpopulations of 620 malignant tumors were labeled by a FITC‐conjugated cytokeratin antibody (CK 5, 6, CK18 and CK 5, 6, 8 and CK 17, respectively prior to flow cytometric cell cycle analysis. Compared to total cell analysis, detection rate of DNA‐aneuploid tumors following cytokeratin labeling was increased from 62% to 76.5% in breast cancer, from 68% to 77% in ovarian cancer, from 60% to 80% in cervical cancer and from 30% to 53% in endometrial cancer. Predominantly in DNA‐diploid tumors, a significantly improved detection of S‐phase fraction of the tumor cells was shown due to the elimination of contaminating nonproliferating “normal cells”. S‐phase fraction following tumor cell enrichment was increased by 10% (mean following cytokeratin staining in ovarian and endometrial cancer, by 30% in breast cancer and even by 70% in cervical cancer compared to total cell analysis. Thus, diagnostic accuracy of DNA‐analysis was enhanced by cytokeratin labeling of tumor cells for all tumor entities investigated.

Estimating DNA coverage and abundance in metagenomes using a gamma approximation

Energy Technology Data Exchange (ETDEWEB)

Hooper, Sean D; Dalevi, Daniel; Pati, Amrita; Mavromatis, Konstantinos; Ivanova, Natalia N; Kyrpides, Nikos C

2010-01-01

Shotgun sequencing generates large numbers of short DNA reads from either an isolated organism or, in the case of metagenomics projects, from the aggregate genome of a microbial community. These reads are then assembled based on overlapping sequences into larger, contiguous sequences (contigs). The feasibility of assembly and the coverage achieved (reads per nucleotide or distinct sequence of nucleotides) depend on several factors: the number of reads sequenced, the read length and the relative abundances of their source genomes in the microbial community. A low coverage suggests that most of the genomic DNA in the sample has not been sequenced, but it is often difficult to estimate either the extent of the uncaptured diversity or the amount of additional sequencing that would be most efficacious. In this work, we regard a metagenome as a population of DNA fragments (bins), each of which may be covered by one or more reads. We employ a gamma distribution to model this bin population due to its flexibility and ease of use. When a gamma approximation can be found that adequately fits the data, we may estimate the number of bins that were not sequenced and that could potentially be revealed by additional sequencing. We evaluated the performance of this model using simulated metagenomes and demonstrate its applicability on three recent metagenomic datasets.

Forensic individual age estimation with DNA: From initial approaches to methylation tests.

Science.gov (United States)

Freire-Aradas, A; Phillips, C; Lareu, M V

2017-07-01

Individual age estimation is a key factor in forensic science analysis that can provide very useful information applicable to criminal, legal, and anthropological investigations. Forensic age inference was initially based on morphological inspection or radiography and only later began to adopt molecular approaches. However, a lack of accuracy or technical problems hampered the introduction of these DNA-based methodologies in casework analysis. A turning point occurred when the epigenetic signature of DNA methylation was observed to gradually change during an individual´s lifespan. In the last four years, the number of publications reporting DNA methylation age-correlated changes has gradually risen and the forensic community now has a range of age methylation tests applicable to forensic casework. Most forensic age predictor models have been developed based on blood DNA samples, but additional tissues are now also being explored. This review assesses the most widely adopted genes harboring methylation sites, detection technologies, statistical age-predictive analyses, and potential causes of variation in age estimates. Despite the need for further work to improve predictive accuracy and establishing a broader range of tissues for which tests can analyze the most appropriate methylation sites, several forensic age predictors have now been reported that provide consistency in their prediction accuracies (predictive error of ±4 years); this makes them compelling tools with the potential to contribute key information to help guide criminal investigations. Copyright © 2017 Central Police University.

Prolonged decay of molecular rate estimates for metazoan mitochondrial DNA

Directory of Open Access Journals (Sweden)

Martyna Molak

2015-03-01

Full Text Available Evolutionary timescales can be estimated from genetic data using the molecular clock, often calibrated by fossil or geological evidence. However, estimates of molecular rates in mitochondrial DNA appear to scale negatively with the age of the clock calibration. Although such a pattern has been observed in a limited range of data sets, it has not been studied on a large scale in metazoans. In addition, there is uncertainty over the temporal extent of the time-dependent pattern in rate estimates. Here we present a meta-analysis of 239 rate estimates from metazoans, representing a range of timescales and taxonomic groups. We found evidence of time-dependent rates in both coding and non-coding mitochondrial markers, in every group of animals that we studied. The negative relationship between the estimated rate and time persisted across a much wider range of calibration times than previously suggested. This indicates that, over long time frames, purifying selection gives way to mutational saturation as the main driver of time-dependent biases in rate estimates. The results of our study stress the importance of accounting for time-dependent biases in estimating mitochondrial rates regardless of the timescale over which they are inferred.

Age Estimation with DNA: From Forensic DNA Fingerprinting to Forensic (Epi)Genomics: A Mini-Review.

Science.gov (United States)

Parson, Walther

2018-01-23

Forensic genetics developed from protein-based techniques a quarter of a century ago and became famous as "DNA fingerprinting," this being based on restriction fragment length polymorphisms (RFLPs) of high-molecular-weight DNA. The amplification of much smaller short tandem repeat (STR) sequences using the polymerase chain reaction soon replaced RFLP analysis and advanced to become the gold standard in genetic identification. Meanwhile, STR multiplexes have been developed and made commercially available which simultaneously amplify up to 30 STR loci from as little as 15 cells or fewer. The enormous information content that comes with the large variety of observed STR genotypes allows for genetic individualisation (with the exception of identical twins). Carefully selected core STR loci form the basis of intelligence-led DNA databases that provide investigative leads by linking unsolved crime scenes and criminals through their matched STR profiles. Nevertheless, the success of modern DNA fingerprinting depends on the availability of reference material from suspects. In order to provide new investigative leads in cases where such reference samples are absent, forensic scientists started to explore the prediction of phenotypic traits from the DNA of the evidentiary sample. This paradigm change now uses DNA and epigenetic markers to forecast characteristics that are useful to triage further investigative work. So far, the best investigated externally visible characteristics are eye, hair and skin colour, as well as geographic ancestry and age. Information on the chronological age of a stain donor (or any sample donor) is elemental for forensic investigations in a number of aspects and has, therefore, been explored by researchers in some detail. Among different methodological approaches tested to date, the methylation-sensitive analysis of carefully selected DNA markers (CpG sites) has brought the most promising results by providing prediction accuracies of ±3-4 years

Tanzanian malignant lymphomas: WHO classification, presentation, ploidy, proliferation and HIV/EBV association

Directory of Open Access Journals (Sweden)

Castro Juan

2010-07-01

Full Text Available Abstract Background In Tanzania, the International Working Formulation [WF] rather than the WHO Classification is still being used in diagnosing malignant lymphomas (ML and the biological characterization including the HIV/EBV association is sketchy, thus restraining comparison, prognostication and application of established therapeutic protocols. Methods Archival, diagnostic ML biopsies (N = 336, available sera (N = 35 screened by ELISA for HIV antibodies and corresponding clinical/histological reports at Muhimbili National Hospital (MNH in Tanzania between 1996 and 2006 were retrieved and evaluated. A fraction (N = 174 were analyzed by histopathology and immunohistochemistry (IHC. Selected biopsies were characterized by flow-cytometry (FC for DNA ploidy (N = 60 and some by in-situ hybridization (ISH for EBV-encoded RNA (EBER, N = 37. Results A third (38.8%, 109/281 of the ML patients with available clinical information had extranodal disease presentation. A total of 158 out of 174 biopsies selected for immunophenotyping were confirmed to be ML which were mostly (84. 8%, 134/158 non-Hodgkin lymphoma (NHL. Most (83.6%, 112/134 of NHL were B-cell lymphomas (BCL (CD20+, of which 50.9%, (57/112 were diffuse large B-cell (DLBCL. Out of the 158 confirmed MLs, 22 (13.9% were T-cell [CD3+] lymphomas (TCL and 24 (15.2% were Hodgkin lymphomas (HL [CD30+]. Furthermore, out of the 60 FC analyzed ML cases, 27 (M:F ratio 2:1 were DLBCL, a slight majority (55.6%, 15/27 with activated B-cell like (ABC and 45% (12/27 with germinal center B-cell like (GCB immunophenotype. Overall, 40% (24/60 ML were aneuploid mostly (63.0%, 17/27 the DLBCL and TCL (54.5%, 6/11. DNA index (DI of FC-analyzed ML ranged from 1.103-2.407 (median = 1.51 and most (75.0% aneuploid cases showed high (>40% cell proliferation by Ki-67 reactivity. The majority (51.4%, 19/37 of EBER ISH analyzed lymphoma biopsies were positive. Of the serologically tested MLs, 40.0% (14/35 were HIV positive

Tanzanian malignant lymphomas: WHO classification, presentation, ploidy, proliferation and HIV/EBV association

Science.gov (United States)

2010-01-01

Background In Tanzania, the International Working Formulation [WF] rather than the WHO Classification is still being used in diagnosing malignant lymphomas (ML) and the biological characterization including the HIV/EBV association is sketchy, thus restraining comparison, prognostication and application of established therapeutic protocols. Methods Archival, diagnostic ML biopsies (N = 336), available sera (N = 35) screened by ELISA for HIV antibodies and corresponding clinical/histological reports at Muhimbili National Hospital (MNH) in Tanzania between 1996 and 2006 were retrieved and evaluated. A fraction (N = 174) were analyzed by histopathology and immunohistochemistry (IHC). Selected biopsies were characterized by flow-cytometry (FC) for DNA ploidy (N = 60) and some by in-situ hybridization (ISH) for EBV-encoded RNA (EBER, N = 37). Results A third (38.8%, 109/281) of the ML patients with available clinical information had extranodal disease presentation. A total of 158 out of 174 biopsies selected for immunophenotyping were confirmed to be ML which were mostly (84. 8%, 134/158) non-Hodgkin lymphoma (NHL). Most (83.6%, 112/134) of NHL were B-cell lymphomas (BCL) (CD20+), of which 50.9%, (57/112) were diffuse large B-cell (DLBCL). Out of the 158 confirmed MLs, 22 (13.9%) were T-cell [CD3+] lymphomas (TCL) and 24 (15.2%) were Hodgkin lymphomas (HL) [CD30+]. Furthermore, out of the 60 FC analyzed ML cases, 27 (M:F ratio 2:1) were DLBCL, a slight majority (55.6%, 15/27) with activated B-cell like (ABC) and 45% (12/27) with germinal center B-cell like (GCB) immunophenotype. Overall, 40% (24/60) ML were aneuploid mostly (63.0%, 17/27) the DLBCL and TCL (54.5%, 6/11). DNA index (DI) of FC-analyzed ML ranged from 1.103-2.407 (median = 1.51) and most (75.0%) aneuploid cases showed high (>40%) cell proliferation by Ki-67 reactivity. The majority (51.4%, 19/37) of EBER ISH analyzed lymphoma biopsies were positive. Of the serologically tested MLs, 40.0% (14/35) were HIV

Frequent occurrence of mitochondrial DNA mutations in Barrett's metaplasia without the presence of dysplasia.

Directory of Open Access Journals (Sweden)

Soong Lee

Full Text Available BACKGROUND: Barrett's esophagus (BE is one of the most common premalignant lesions and can progress to esophageal adenocarcinoma (EA. The numerous molecular events may play a role in the neoplastic transformation of Barrett's mucosa such as the change of DNA ploidy, p53 mutation and alteration of adhesion molecules. However, the molecular mechanism of the progression of BE to EA remains unclear and most studies of mitochondrial DNA (mtDNA mutations in BE have performed on BE with the presence of dysplasia. METHODS/FINDINGS: Thus, the current study is to investigate new molecular events (Barrett's esophageal tissue-specific-mtDNA alterations/instabilities in mitochondrial genome and causative factors for their alterations using the corresponding adjacent normal mucosal tissue (NT and tissue (BT from 34 patients having Barrett's metaplasia without the presence of dysplasia. Eighteen patients (53% exhibited mtDNA mutations which were not found in adjacent NT. mtDNA copy number was about 3 times higher in BT than in adjacent NT. The activity of the mitochondrial respiratory chain enzyme complexes in tissues from Barrett's metaplasia without the presence of dysplasia was impaired. Reactive oxygen species (ROS level in BT was significantly higher than those in corresponding samples. CONCLUSION/SIGNIFICANCE: High ROS level in BT may contribute to the development of mtDNA mutations, which may play a crucial role in disease progression and tumorigenesis in BE.

Multiple data sources improve DNA-based mark-recapture population estimates of grizzly bears.

Science.gov (United States)

Boulanger, John; Kendall, Katherine C; Stetz, Jeffrey B; Roon, David A; Waits, Lisette P; Paetkau, David

2008-04-01

A fundamental challenge to estimating population size with mark-recapture methods is heterogeneous capture probabilities and subsequent bias of population estimates. Confronting this problem usually requires substantial sampling effort that can be difficult to achieve for some species, such as carnivores. We developed a methodology that uses two data sources to deal with heterogeneity and applied this to DNA mark-recapture data from grizzly bears (Ursus arctos). We improved population estimates by incorporating additional DNA "captures" of grizzly bears obtained by collecting hair from unbaited bear rub trees concurrently with baited, grid-based, hair snag sampling. We consider a Lincoln-Petersen estimator with hair snag captures as the initial session and rub tree captures as the recapture session and develop an estimator in program MARK that treats hair snag and rub tree samples as successive sessions. Using empirical data from a large-scale project in the greater Glacier National Park, Montana, USA, area and simulation modeling we evaluate these methods and compare the results to hair-snag-only estimates. Empirical results indicate that, compared with hair-snag-only data, the joint hair-snag-rub-tree methods produce similar but more precise estimates if capture and recapture rates are reasonably high for both methods. Simulation results suggest that estimators are potentially affected by correlation of capture probabilities between sample types in the presence of heterogeneity. Overall, closed population Huggins-Pledger estimators showed the highest precision and were most robust to sparse data, heterogeneity, and capture probability correlation among sampling types. Results also indicate that these estimators can be used when a segment of the population has zero capture probability for one of the methods. We propose that this general methodology may be useful for other species in which mark-recapture data are available from multiple sources.

Variation and geographical distribution of ploidy levels in Pennisetum section Brevivalvula (Poaceae) in Burkina Faso, Benin and southern Niger

OpenAIRE

Renno, Jean-François; Schmelzer, G.; De Jong, J.H.

1995-01-01

#Pennisetum$ sect. #Brevivalvula$ is a species complex characterized by polyploidy and apoximis. Ploidy level was assessed by DAPI-flow cytometry for 304 plants of the section, originating from Burkina Faso, Benin and southern Niger. The results were confirmed for 54 plants based on chromosome counts. The samples show four euploidy levels (with x = 9) distributed among five species : #P. hordeoides$ (2n = 36, 54), #P. pedicellatum$ (2n = 36, 45, 54), #P. polystachion$ (2n = 18, 36, 45, 54), #...

Switchgrass genomic diversity, ploidy, and evolution: novel insights from a network-based SNP discovery protocol.

Directory of Open Access Journals (Sweden)

Fei Lu

Full Text Available Switchgrass (Panicum virgatum L. is a perennial grass that has been designated as an herbaceous model biofuel crop for the United States of America. To facilitate accelerated breeding programs of switchgrass, we developed both an association panel and linkage populations for genome-wide association study (GWAS and genomic selection (GS. All of the 840 individuals were then genotyped using genotyping by sequencing (GBS, generating 350 GB of sequence in total. As a highly heterozygous polyploid (tetraploid and octoploid species lacking a reference genome, switchgrass is highly intractable with earlier methodologies of single nucleotide polymorphism (SNP discovery. To access the genetic diversity of species like switchgrass, we developed a SNP discovery pipeline based on a network approach called the Universal Network-Enabled Analysis Kit (UNEAK. Complexities that hinder single nucleotide polymorphism discovery, such as repeats, paralogs, and sequencing errors, are easily resolved with UNEAK. Here, 1.2 million putative SNPs were discovered in a diverse collection of primarily upland, northern-adapted switchgrass populations. Further analysis of this data set revealed the fundamentally diploid nature of tetraploid switchgrass. Taking advantage of the high conservation of genome structure between switchgrass and foxtail millet (Setaria italica (L. P. Beauv., two parent-specific, synteny-based, ultra high-density linkage maps containing a total of 88,217 SNPs were constructed. Also, our results showed clear patterns of isolation-by-distance and isolation-by-ploidy in natural populations of switchgrass. Phylogenetic analysis supported a general south-to-north migration path of switchgrass. In addition, this analysis suggested that upland tetraploid arose from upland octoploid. All together, this study provides unparalleled insights into the diversity, genomic complexity, population structure, phylogeny, phylogeography, ploidy, and evolutionary dynamics

Estimating abundance of Sitka black-tailed deer using DNA from fecal pellets

Science.gov (United States)

Todd J. Brinkman; David K. Person; F. Stuart Chapin; Winston Smith; Kris J. Hundertmark

2011-01-01

Densely vegetated environments have hindered collection of basic population parameters on forest-dwelling ungulates. Our objective was to develop a mark-recapture technique that used DNA from fecal pellets to overcome constraints associated with estimating abundance of ungulates in landscapes where direct observation is difficult. We tested our technique on Sitka black...

Effects of sampling conditions on DNA-based estimates of American black bear abundance

Science.gov (United States)

Laufenberg, Jared S.; Van Manen, Frank T.; Clark, Joseph D.

2013-01-01

DNA-based capture-mark-recapture techniques are commonly used to estimate American black bear (Ursus americanus) population abundance (N). Although the technique is well established, many questions remain regarding study design. In particular, relationships among N, capture probability of heterogeneity mixtures A and B (pA and pB, respectively, or p, collectively), the proportion of each mixture (π), number of capture occasions (k), and probability of obtaining reliable estimates of N are not fully understood. We investigated these relationships using 1) an empirical dataset of DNA samples for which true N was unknown and 2) simulated datasets with known properties that represented a broader array of sampling conditions. For the empirical data analysis, we used the full closed population with heterogeneity data type in Program MARK to estimate N for a black bear population in Great Smoky Mountains National Park, Tennessee. We systematically reduced the number of those samples used in the analysis to evaluate the effect that changes in capture probabilities may have on parameter estimates. Model-averaged N for females and males were 161 (95% CI = 114–272) and 100 (95% CI = 74–167), respectively (pooled N = 261, 95% CI = 192–419), and the average weekly p was 0.09 for females and 0.12 for males. When we reduced the number of samples of the empirical data, support for heterogeneity models decreased. For the simulation analysis, we generated capture data with individual heterogeneity covering a range of sampling conditions commonly encountered in DNA-based capture-mark-recapture studies and examined the relationships between those conditions and accuracy (i.e., probability of obtaining an estimated N that is within 20% of true N), coverage (i.e., probability that 95% confidence interval includes true N), and precision (i.e., probability of obtaining a coefficient of variation ≤20%) of estimates using logistic regression. The capture probability

Aurora-A overexpression and aneuploidy predict poor outcome in serous ovarian carcinoma.

Science.gov (United States)

Lassus, Heini; Staff, Synnöve; Leminen, Arto; Isola, Jorma; Butzow, Ralf

2011-01-01

Aurora-A is a potential oncogene and therapeutic target in ovarian carcinoma. It is involved in mitotic events and overexpression leads to centrosome amplification and chromosomal instability. The objective of this study was to evaluate the clinical significance of Aurora-A and DNA ploidy in serous ovarian carcinoma. Serous ovarian carcinomas were analysed for Aurora-A protein by immunohistochemistry (n=592), Aurora-A copy number by CISH (n=169), Aurora-A mRNA by real-time PCR (n=158) and DNA ploidy by flowcytometry (n=440). Overexpression of Aurora-A was found in 27% of the tumors, cytoplasmic overexpression in 11% and nuclear in 17%. The cytoplasmic and nuclear overexpression were nearly mutually exclusive. Both cytoplasmic and nuclear overexpression were associated with shorter survival, high grade, high proliferation index and aberrant p53. Interestingly, only cytoplasmic expression was associated with aneuploidy and expression of phosphorylated Aurora-A. DNA ploidy was associated with poor patient outcome as well as aggressive clinicopathological parameters. In multivariate analysis, Aurora-A overexpression appeared as an independent prognostic factor for disease-free survival, together with grade, stage and ploidy. Aurora-A protein expression is strongly linked with poor patient outcome and aggressive disease characteristics, which makes Aurora-A a promising biomarker and a potential therapeutic target in ovarian carcinoma. Cytoplasmic and nuclear Aurora-A protein may have different functions. DNA aneuploidy is a strong predictor of poor prognosis in serous ovarian carcinoma. Copyright © 2010 Elsevier Inc. All rights reserved.

New method for estimating clustering of DNA lesions induced by physical/chemical mutagens using fluorescence anisotropy.

Science.gov (United States)

Akamatsu, Ken; Shikazono, Naoya; Saito, Takeshi

2017-11-01

We have developed a new method for estimating the localization of DNA damage such as apurinic/apyrimidinic sites (APs) on DNA using fluorescence anisotropy. This method is aimed at characterizing clustered DNA damage produced by DNA-damaging agents such as ionizing radiation and genotoxic chemicals. A fluorescent probe with an aminooxy group (AlexaFluor488) was used to label APs. We prepared a pUC19 plasmid with APs by heating under acidic conditions as a model for damaged DNA, and subsequently labeled the APs. We found that the observed fluorescence anisotropy (r obs ) decreases as averaged AP density (λ AP : number of APs per base pair) increases due to homo-FRET, and that the APs were randomly distributed. We applied this method to three DNA-damaging agents, 60 Co γ-rays, methyl methanesulfonate (MMS), and neocarzinostatin (NCS). We found that r obs -λ AP relationships differed significantly between MMS and NCS. At low AP density (λ AP  < 0.001), the APs induced by MMS seemed to not be closely distributed, whereas those induced by NCS were remarkably clustered. In contrast, the AP clustering induced by 60 Co γ-rays was similar to, but potentially more likely to occur than, random distribution. This simple method can be used to estimate mutagenicity of ionizing radiation and genotoxic chemicals. Copyright © 2017 Elsevier Inc. All rights reserved.

Joint Estimation of Contamination, Error and Demography for Nuclear DNA from Ancient Humans

Science.gov (United States)

Slatkin, Montgomery

2016-01-01

When sequencing an ancient DNA sample from a hominin fossil, DNA from present-day humans involved in excavation and extraction will be sequenced along with the endogenous material. This type of contamination is problematic for downstream analyses as it will introduce a bias towards the population of the contaminating individual(s). Quantifying the extent of contamination is a crucial step as it allows researchers to account for possible biases that may arise in downstream genetic analyses. Here, we present an MCMC algorithm to co-estimate the contamination rate, sequencing error rate and demographic parameters—including drift times and admixture rates—for an ancient nuclear genome obtained from human remains, when the putative contaminating DNA comes from present-day humans. We assume we have a large panel representing the putative contaminant population (e.g. European, East Asian or African). The method is implemented in a C++ program called ‘Demographic Inference with Contamination and Error’ (DICE). We applied it to simulations and genome data from ancient Neanderthals and modern humans. With reasonable levels of genome sequence coverage (>3X), we find we can recover accurate estimates of all these parameters, even when the contamination rate is as high as 50%. PMID:27049965

Mitochondrial DNA single nucleotide polymorphism associated with weight estimated breeding values in Nelore cattle (Bos indicus

Directory of Open Access Journals (Sweden)

Fernando Henrique Biase

2007-01-01

Full Text Available We sampled 119 Nelore cattle (Bos indicus, 69 harboring B. indicus mtDNA plus 50 carrying Bos taurus mtDNA, to estimate the frequencies of putative mtDNA single nucleotide polymorphisms (SNPs and investigate their association with Nelore weight and scrotal circumference estimated breeding values (EBVs. The PCR restriction fragment length polymorphism (PCR-RFLP method was used to detect polymorphisms in the mitochondrial asparagine, cysteine, glycine, leucine and proline transporter RNA (tRNA genes (tRNAasn, tRNAcys, tRNAgly, tRNAleu and tRNApro. The 50 cattle carrying B. taurus mtDNA were monomorphic for all the tRNA gene SNPs analyzed, suggesting that they are specific to mtDNA from B. indicus cattle. No tRNAcys or tRNAgly polymorphisms were detected in any of the cattle but we did detect polymorphic SNPs in the tRNAasn, tRNAleu and tRNApro genes in the cattle harboring B. indicus mtDNA, with the same allele observed in the B. taurus sequence being present in the following percentage of cattle harboring B. indicus mtDNA: 72.46% for tRNAasn, 95.23% for tRNAleu and 90.62% for tRNApro. Analyses of variance using the tRNAasn SNP as the independent variable and EBVs as the dependent variable showed that the G -> T SNP was significantly associated (p < 0.05 with maternal EBVs for weight at 120 and 210 days (p < 0.05 and animal's EBVs for weight at 210, 365 and 455 days. There was no association of the tRNAasn SNP with the scrotal circumference EBVs. These results confirm that mtDNA can affect weight and that mtDNA polymorphisms can be a source of genetic variation for quantitative traits.

The effect of ploidy and temporal changes in the biochemical profile of gibel carp (Carassius gibelio): a cyprinid fish species with dual reproductive strategies

Czech Academy of Sciences Publication Activity Database

Vetešník, Lukáš; Halačka, Karel; Šimková, A.

2013-01-01

Roč. 39, č. 2 (2013), s. 171-180 ISSN 0920-1742 R&D Projects: GA ČR GP524/09/P620; GA ČR GBP505/12/G112 Institutional support: RVO:68081766 Keywords : Biochemical profile of blood * Gibel carp * Ploidy * Temporal variability Subject RIV: EG - Zoology Impact factor: 1.676, year: 2013

Asymmetric epigenetic modification and elimination of rDNA sequences by polyploidization in wheat.

Science.gov (United States)

Guo, Xiang; Han, Fangpu

2014-11-01

rRNA genes consist of long tandem repeats clustered on chromosomes, and their products are important functional components of the ribosome. In common wheat (Triticum aestivum), rDNA loci from the A and D genomes were largely lost during the evolutionary process. This biased DNA elimination may be related to asymmetric transcription and epigenetic modifications caused by the polyploid formation. Here, we observed both sets of parental nucleolus organizing regions (NORs) were expressed after hybridization, but asymmetric silencing of one parental NOR was immediately induced by chromosome doubling, and reversing the ploidy status could not reactivate silenced NORs. Furthermore, increased CHG and CHH DNA methylation on promoters was accompanied by asymmetric silencing of NORs. Enrichment of H3K27me3 and H3K9me2 modifications was also observed to be a direct response to increased DNA methylation and transcriptional inactivation of NOR loci. Both A and D genome NOR loci with these modifications started to disappear in the S4 generation and were completely eliminated by the S7 generation in synthetic tetraploid wheat. Our results indicated that asymmetric epigenetic modification and elimination of rDNA sequences between different donor genomes may lead to stable allopolyploid wheat with increased differentiation and diversity. © 2014 American Society of Plant Biologists. All rights reserved.

The use of caspase inhibitors in pulsed-field gel electrophoresis may improve the estimation of radiation-induced DNA repair and apoptosis

International Nuclear Information System (INIS)

Balart, Josep; Pueyo, Gemma; Llobet, Lara I de; Baro, Marta; Sole, Xavi; Marin, Susanna; Casanovas, Oriol; Mesia, Ricard; Capella, Gabriel

2011-01-01

Radiation-induced DNA double-strand break (DSB) repair can be tested by using pulsed-field gel electrophoresis (PFGE) in agarose-encapsulated cells. However, previous studies have reported that this assay is impaired by the spontaneous DNA breakage in this medium. We investigated the mechanisms of this fragmentation with the principal aim of eliminating it in order to improve the estimation of radiation-induced DNA repair. Samples from cancer cell cultures or xenografted tumours were encapsulated in agarose plugs. The cell plugs were then irradiated, incubated to allow them to repair, and evaluated by PFGE, caspase-3, and histone H2AX activation (γH2AX). In addition, apoptosis inhibition was evaluated through chemical caspase inhibitors. We confirmed that spontaneous DNA fragmentation was associated with the process of encapsulation, regardless of whether cells were irradiated or not. This DNA fragmentation was also correlated to apoptosis activation in a fraction of the cells encapsulated in agarose, while non-apoptotic cell fraction could rejoin DNA fragments as was measured by γH2AX decrease and PFGE data. We were able to eliminate interference of apoptosis by applying specific caspase inhibitors, and improve the estimation of DNA repair, and apoptosis itself. The estimation of radiation-induced DNA repair by PFGE may be improved by the use of apoptosis inhibitors. The ability to simultaneously determine DNA repair and apoptosis, which are involved in cell fate, provides new insights for using the PFGE methodology as functional assay

Adaptive response to DNA-damaging agents in natural Saccharomyces cerevisiae populations from "Evolution Canyon", Mt. Carmel, Israel.

Directory of Open Access Journals (Sweden)

Gabriel A Lidzbarsky

2009-06-01

Full Text Available Natural populations of most organisms, especially unicellular microorganisms, are constantly exposed to harsh environmental factors which affect their growth. UV radiation is one of the most important physical parameters which influences yeast growth in nature. Here we used 46 natural strains of Saccharomyces cerevisiae isolated from several natural populations at the "Evolution Canyon" microsite (Nahal Oren, Mt. Carmel, Israel. The opposing slopes of this canyon share the same geology, soil, and macroclimate, but they differ in microclimatic conditions. The interslope differences in solar radiation (200%-800% more on the "African" slope caused the development of two distinct biomes. The south-facing slope is sunnier and has xeric, savannoid "African" environment while the north-facing slope is represented by temperate, "European" forested environment. Here we studied the phenotypic response of the S. cerevisiae strains to UVA and UVC radiations and to methyl methanesulfonate (MMS in order to evaluate the interslope effect on the strains' ability to withstand DNA-damaging agents.We exposed our strains to the different DNA-damaging agents and measured survival by counting colony forming units. The strains from the "African" slope were more resilient to both UVA and MMS than the strains from the "European" slope. In contrast, we found that there was almost no difference between strains (with similar ploidy from the opposite slopes, in their sensitivity to UVC radiation. These results suggest that the "African" strains are more adapted to higher solar radiation than the "European" strains. We also found that the tetraploids strains were more tolerant to all DNA-damaging agents than their neighboring diploid strains, which suggest that high ploidy level might be a mechanism of adaptation to high solar radiation.Our results and the results of parallel studies with several other organisms, suggest that natural selection appears to select, at a

Effect of low dose tritium on mouse lymphocyte DNA estimated by comet assay

International Nuclear Information System (INIS)

Ichimasa, Yusuke; Otsuka, Kensuke; Maruyama, Satoko; Tauchi, Hiroshi; Ichimasa, Michiko; Uda, Tatsuhiko

2003-01-01

This paper deals with low dose effect of HTO on mouse lymphocytes DNA (in vitro irradiation) estimated by the comet assay using ICR male mouse of 20 to 23 weeks old. Lymphocytes were isolated by centrifugation of whole blood sample on Ficoll-Paque solution and embedded in agarose gel just after mixed with HTO. After lymphocytes were exposed to 17-50 mGy of HTO, the agarose gel slides were washed to remove HTO and cell lysis treatment on the slides was conducted before electrophoresis. The individual comets on stained slides after electrophoresis were analyzed using imaging software. No significant DNA damages were observed. (author)

Autoregressive-model-based missing value estimation for DNA microarray time series data.

Science.gov (United States)

Choong, Miew Keen; Charbit, Maurice; Yan, Hong

2009-01-01

Missing value estimation is important in DNA microarray data analysis. A number of algorithms have been developed to solve this problem, but they have several limitations. Most existing algorithms are not able to deal with the situation where a particular time point (column) of the data is missing entirely. In this paper, we present an autoregressive-model-based missing value estimation method (ARLSimpute) that takes into account the dynamic property of microarray temporal data and the local similarity structures in the data. ARLSimpute is especially effective for the situation where a particular time point contains many missing values or where the entire time point is missing. Experiment results suggest that our proposed algorithm is an accurate missing value estimator in comparison with other imputation methods on simulated as well as real microarray time series datasets.

Karyological and flow cytometric evidence of triploid specimens in Bufo viridis (Amphibia Anura

Directory of Open Access Journals (Sweden)

D Cavallo

2010-01-01

Full Text Available Karyological and flow cytometric (FCM analyses were performed on a group of 14 green toads of the Bufo viridis species from seven Eurasian populations. Both approaches gave concordant results concerning the DNA ploidy level. All the populations examined were represented exclusively by diploid or tetraploid specimens, except one, where triploids were found. Results evidenced an interpopulation variability in DNA content against the same ploidy level, as well as an unusually high number of triploids in a particular reproductive place. The origin of polyploidy and the presence and persistence of a high number of triploids in a particular population are discussed.

Ploidy plasticity: a rapid and reversible strategy for adaptation to stress.

Science.gov (United States)

Berman, Judith

2016-05-01

Organisms must be able to grow in a broad range of conditions found in their normal growth environment and for a species to survive, at least some cells in a population must adapt rapidly to extreme stress conditions that kill the majority of cells.Candida albicans, the most prevalent fungal pathogen of humans resides as a commensal in a broad range of niches within the human host. Growth conditions in these niches are highly variable and stresses such exposure to antifungal drugs can inhibit population growth abruptly. One of the mechanisms C. albicans uses to adapt rapidly to severe stresses is aneuploidy-a change in the total number of chromosomes such that one or more chromosomes are present in excess or are missing. Aneuploidy is quite common in wild isolates of fungi and other eukaryotic microbes. Aneuploidy can be achieved by chromosome nondisjunction during a simple mitosis, and in stress conditions it begins to appear after two mitotic divisions via a tetraploid intermediate. Aneuploidy usually resolves to euploidy (a balanced number of chromosomes), but not necessarily to diploidy. Aneuploidy of a specific chromosome can confer new phenotypes by virtue of the copy number of specific genes on that chromosome relative to the copies of other genes. Thus, it is not aneuploidy per se, but the relative copy number of specific genes that confers many tested aneuploidy-associated phenotypes. Aneuploidy almost always carries a fitness cost, as cells express most proteins encoded by genes on the aneuploid chromosome in proportion to the number of DNA copies of the gene. This is thought to be due to imbalances in the stoichiometry of different components of large complexes. Despite this, fitness is a relative function-and if stress is severe and population growth has slowed considerably, then even small growth advantages of some aneuploidies can provide a selective advantage. Thus, aneuploidy appears to provide a transient solution to severe and sudden stress

DNA analysis in three populations of African spinach (Basella spp.)

International Nuclear Information System (INIS)

Grasso, G.; Van Duren, M.; Lee, K.S.; Morpurgo, R.

1997-01-01

African spinach (Basella spp.) is an important vegetable in West Africa, and was introduced by early colonialists. Its alien origin is supported by its narrow genetic variability. Flowcytometry and RAPD polymorphism were used to investigate genetic variation in three populations of Basella - 'Congo native', 'Cong domesticated', and an introduced cultivar, 'Sri Lanka' from Sri Lanka. Normal spinach (Spinacia oleracea) cv. 'Prince F 1 Hybrid' was used to test sensitivity and to verify detection of genetic variation. Nuclei were isolated from young leaves of Basella, stained with DAPI and ethidium bromide, and ploidy level and total DNA content were determined by using a flowcytometer. The two sexually propagated populations, 'Cong domesticated' and 'Sri Lanka' showed very low amount of genetic variation as revealed by RAPD analysis; the third population 'Congo native' showed a limited amount of polymorphism. (author). 8 refs, 1 fig., 2 tabs

Calibration of denaturing agarose gels for molecular weight estimation of DNA: size determination of the single-stranded genomes of parvoviruses

Energy Technology Data Exchange (ETDEWEB)

Snyder, C.E. (Oak Ridge National Lab., TN); Schmoyer, R.L.; Bates, R.C.; Mitra, S.

1982-01-01

Vertical slab gel electrophoresis of DNA with CH/sub 3/HgOH-containing agarose produces sharp bands whose mobilities are suitable for size estimation of single-stranded DNA containing 600 to 20,000 bases. The relationship of electrophoretic mobility to size of DNA over this range is a smooth, S-shaped function, and an empirical model was developed to express the relationship. The model involves terms in squared and reciprocal mobilities, and produced excellent fit of known standard markers to measured mobilities. It was used to estimate the sizes of six parvovirus DNAs: Kilham rat virus (KRV), H-1, LuIII, and minute virus of mice (MVM) DNAs had molecular weights of 1.66 to 1.70 x 10/sup 6/, while the molecular weight of bovine parvovirus (BPV) DNA was 1.84 x 10/sup 6/ and that of adenoassociated virus (AAV) DNA was 1.52 x 10/sup 6/.

Meta-analysis of the predictive value of DNA aneuploidy in malignant transformation of oral potentially malignant disorders.

Science.gov (United States)

Alaizari, Nader A; Sperandio, Marcelo; Odell, Edward W; Peruzzo, Daiane; Al-Maweri, Sadeq A

2018-02-01

DNA aneuploidy is an imbalance of chromosomal DNA content that has been highlighted as a predictor of biological behavior and risk of malignant transformation. To date, DNA aneuploidy in oral potentially malignant diseases (OPMD) has been shown to correlate strongly with severe dysplasia and high-risk lesions that appeared non-dysplastic can be identified by ploidy analysis. Nevertheless, the prognostic value of DNA aneuploidy in predicting malignant transformation of OPMD remains to be validated. The aim of this meta-analysis was to assess the role of DNA aneuploidy in predicting malignant transformation in OPMD. The questions addressed were (i) Is DNA aneuploidy a useful marker to predict malignant transformation in OPMD? (ii) Is DNA diploidy a useful negative marker of malignant transformation in OPMD? These questions were addressed using the PECO method. Five studies assessing aneuploidy as a risk marker of malignant change were pooled into the meta-analysis. Aneuploidy was found to be associated with a 3.12-fold increased risk to progress into cancer (RR=3.12, 95% CI 1.86-5.24). Based on the five studies meta-analyzed, "no malignant progression" was more likely to occur in DNA diploid OPMD by 82% when compared to aneuploidy (RR=0.18, 95% CI 0.08-0.41). In conclusion, aneuploidy is a useful marker of malignant transformation in OPMD, although a diploid result should be interpreted with caution. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Environmental DNA method for estimating salamander distribution in headwater streams, and a comparison of water sampling methods.

Science.gov (United States)

Katano, Izumi; Harada, Ken; Doi, Hideyuki; Souma, Rio; Minamoto, Toshifumi

2017-01-01

Environmental DNA (eDNA) has recently been used for detecting the distribution of macroorganisms in various aquatic habitats. In this study, we applied an eDNA method to estimate the distribution of the Japanese clawed salamander, Onychodactylus japonicus, in headwater streams. Additionally, we compared the detection of eDNA and hand-capturing methods used for determining the distribution of O. japonicus. For eDNA detection, we designed a qPCR primer/probe set for O. japonicus using the 12S rRNA region. We detected the eDNA of O. japonicus at all sites (with the exception of one), where we also observed them by hand-capturing. Additionally, we detected eDNA at two sites where we were unable to observe individuals using the hand-capturing method. Moreover, we found that eDNA concentrations and detection rates of the two water sampling areas (stream surface and under stones) were not significantly different, although the eDNA concentration in the water under stones was more varied than that on the surface. We, therefore, conclude that eDNA methods could be used to determine the distribution of macroorganisms inhabiting headwater systems by using samples collected from the surface of the water.

An Accurate Estimate of the Free Energy and Phase Diagram of All-DNA Bulk Fluids

Directory of Open Access Journals (Sweden)

Emanuele Locatelli

2018-04-01

Full Text Available We present a numerical study in which large-scale bulk simulations of self-assembled DNA constructs have been carried out with a realistic coarse-grained model. The investigation aims at obtaining a precise, albeit numerically demanding, estimate of the free energy for such systems. We then, in turn, use these accurate results to validate a recently proposed theoretical approach that builds on a liquid-state theory, the Wertheim theory, to compute the phase diagram of all-DNA fluids. This hybrid theoretical/numerical approach, based on the lowest-order virial expansion and on a nearest-neighbor DNA model, can provide, in an undemanding way, a parameter-free thermodynamic description of DNA associating fluids that is in semi-quantitative agreement with experiments. We show that the predictions of the scheme are as accurate as those obtained with more sophisticated methods. We also demonstrate the flexibility of the approach by incorporating non-trivial additional contributions that go beyond the nearest-neighbor model to compute the DNA hybridization free energy.

An MCMC Algorithm for Target Estimation in Real-Time DNA Microarrays

Directory of Open Access Journals (Sweden)

Vikalo Haris

2010-01-01

Full Text Available DNA microarrays detect the presence and quantify the amounts of nucleic acid molecules of interest. They rely on a chemical attraction between the target molecules and their Watson-Crick complements, which serve as biological sensing elements (probes. The attraction between these biomolecules leads to binding, in which probes capture target analytes. Recently developed real-time DNA microarrays are capable of observing kinetics of the binding process. They collect noisy measurements of the amount of captured molecules at discrete points in time. Molecular binding is a random process which, in this paper, is modeled by a stochastic differential equation. The target analyte quantification is posed as a parameter estimation problem, and solved using a Markov Chain Monte Carlo technique. In simulation studies where we test the robustness with respect to the measurement noise, the proposed technique significantly outperforms previously proposed methods. Moreover, the proposed approach is tested and verified on experimental data.

The merits of DNA content and cell kinetic parameters for the assessment of intrinsic cellular radiosensitivity to photon and high-LET neutron irradiation

International Nuclear Information System (INIS)

Theron, C.S.; Serafin, A.; Bohm, L.; Slabbert, J.P.

1997-01-01

Differences of the intrinsic cellular radiosensitivity between tumours make the selection of patients for specific radiation schedules very difficult. The reasons for these variations are still unclear, but are thought to be due to genomic and cellular characteristics. Radiosensitivities vary between cell cycle stages, with S-phase cells being most radioresistant and G2/M phase cells most radiosensitive. It is also well established that most tumour cells have an abnormal ploidy. DNA content and cellular proliferation kinetics therefore could influence the intrinsic radiosensitivity. This prompted us to assess the merits of these parameters as predictors of radiation response. (authors)

Hybridization between two cryptic filamentous brown seaweeds along the shore: analysing pre- and postzygotic barriers in populations of individuals with varying ploidy levels.

Science.gov (United States)

Montecinos, Alejandro E; Guillemin, Marie-Laure; Couceiro, Lucia; Peters, Akira F; Stoeckel, Solenn; Valero, Myriam

2017-07-01

We aimed to study the importance of hybridization between two cryptic species of the genus Ectocarpus, a group of filamentous algae with haploid-diploid life cycles that include the principal genetic model organism for the brown algae. In haploid-diploid species, the genetic structure of the two phases of the life cycle can be analysed separately in natural populations. Such life cycles provide a unique opportunity to estimate the frequency of hybrid genotypes in diploid sporophytes and meiotic recombinant genotypes in haploid gametophytes allowing the effects of reproductive barriers preventing fertilization or preventing meiosis to be untangle. The level of hybridization between E. siliculosus and E. crouaniorum was quantified along the European coast. Clonal cultures (568 diploid, 336 haploid) isolated from field samples were genotyped using cytoplasmic and nuclear markers to estimate the frequency of hybrid genotypes in diploids and recombinant haploids. We identified admixed individuals using microsatellite loci, classical assignment methods and a newly developed Bayesian method (XPloidAssignment), which allows the analysis of populations that exhibit variations in ploidy level. Over all populations, the level of hybridization was estimated at 8.7%. Hybrids were exclusively observed in sympatric populations. More than 98% of hybrids were diploids (40% of which showed signs of aneuploidy) with a high frequency of rare alleles. The near absence of haploid recombinant hybrids demonstrates that the reproductive barriers are mostly postzygotic and suggests that abnormal chromosome segregation during meiosis following hybridization of species with different genome sizes could be a major cause of interspecific incompatibility in this system. © 2017 John Wiley & Sons Ltd.

Differential immunoadsorption coupled with rate nephelometry for estimation of DNA-binding immunoglobulins

International Nuclear Information System (INIS)

DeBari, V.A.; Nicotra, J.; Blaney, J.F.; Schultz, E.F.; Needle, M.A.

1984-01-01

The authors describe a technique for estimating the mass of anti-DNA antibodies by immunonephelometry of serum immunoglobulins (IgG, IgA, IgM) before and after adsorption onto DNA bound to agarose-polylysine columns. Sixteen patients with systemic lupus erythematosus and 16 age- and sex-matched controls were studied. Precision was determined for high-value (in 10 patients) and low-value (in nine controls) ranges for each of the immunoglobulins. They found anti-DNA antibody concentrations (mean +/- SD) in systemic lupus erythematosus of 1.981 +/- 1.015 g/L for IgG 0.257 +/- 0.215 g/L for IgA and 0.282 +/- 0.234 g/L for IgM. Sensitivity and linearity are such that fivefold dilutions of patients' serum with either a buffered albumin solution or control serum yielded values close to the expected values for IgG. Similarly diluted sera gave inordinately high values in the radiometric binding assay. Neither parametric (linear regression) nor nonparametric correlation methods (Spearman's rank and Kendall's tau) show a significant correlation between patients' data obtained by the present technique and that by a radiometric binding assay, although combined data from patients and controls demonstrate a significant nonparametric correlation

Hydrodynamic characterization and molecular weight estimation of ultrasonically sheared DNA; Caracterizacion hidrodinamica y estimacion de pesos moleculares de DNA degradado por ultrasonidos

Energy Technology Data Exchange (ETDEWEB)

Casal, J I; Garces, F; Garcia-Sacristan, A

1981-07-01

The sedimentation coefficients and intrinsic viscosities of ultrasonically sheared calf thymus DNA have been determined. The molecular weight estimation according to this parameters have been compared with the ones obtained from the electrophoretic migration rates based on the calibration proposed using the known molecular weight restriction fragments of X-ENA. (Author) 35 refs.

DNA analysis in three populations of African spinach (Basella spp.)

Energy Technology Data Exchange (ETDEWEB)

Grasso, G; Van Duren, M; Lee, K S; Morpurgo, R [Agriculture and Biotechnology Lab., International Atomic Energy Agency, Seiberdorf (Austria)

1997-07-01

African spinach (Basella spp.) is an important vegetable in West Africa, and was introduced by early colonialists. Its alien origin is supported by its narrow genetic variability. Flowcytometry and RAPD polymorphism were used to investigate genetic variation in three populations of Basella - `Congo native`, `Cong domesticated`, and an introduced cultivar, `Sri Lanka` from Sri Lanka. Normal spinach (Spinacia oleracea) cv. `Prince F{sub 1} Hybrid` was used to test sensitivity and to verify detection of genetic variation. Nuclei were isolated from young leaves of Basella, stained with DAPI and ethidium bromide, and ploidy level and total DNA content were determined by using a flowcytometer. The two sexually propagated populations, `Cong domesticated` and `Sri Lanka` showed very low amount of genetic variation as revealed by RAPD analysis; the third population `Congo native` showed a limited amount of polymorphism. (author). 8 refs, 1 fig., 2 tabs.

Estimation of the radiation-induced DNA double-strand breaks number by considering cell cycle and absorbed dose per cell nucleus.

Science.gov (United States)

Mori, Ryosuke; Matsuya, Yusuke; Yoshii, Yuji; Date, Hiroyuki

2018-05-01

DNA double-strand breaks (DSBs) are thought to be the main cause of cell death after irradiation. In this study, we estimated the probability distribution of the number of DSBs per cell nucleus by considering the DNA amount in a cell nucleus (which depends on the cell cycle) and the statistical variation in the energy imparted to the cell nucleus by X-ray irradiation. The probability estimation of DSB induction was made following these procedures: (i) making use of the Chinese Hamster Ovary (CHO)-K1 cell line as the target example, the amounts of DNA per nucleus in the logarithmic and the plateau phases of the growth curve were measured by flow cytometry with propidium iodide (PI) dyeing; (ii) the probability distribution of the DSB number per cell nucleus for each phase after irradiation with 1.0 Gy of 200 kVp X-rays was measured by means of γ-H2AX immunofluorescent staining; (iii) the distribution of the cell-specific energy deposition via secondary electrons produced by the incident X-rays was calculated by WLTrack (in-house Monte Carlo code); (iv) according to a mathematical model for estimating the DSB number per nucleus, we deduced the induction probability density of DSBs based on the measured DNA amount (depending on the cell cycle) and the calculated dose per nucleus. The model exhibited DSB induction probabilities in good agreement with the experimental results for the two phases, suggesting that the DNA amount (depending on the cell cycle) and the statistical variation in the local energy deposition are essential for estimating the DSB induction probability after X-ray exposure.

PACE: Probabilistic Assessment for Contributor Estimation- A machine learning-based assessment of the number of contributors in DNA mixtures.

Science.gov (United States)

Marciano, Michael A; Adelman, Jonathan D

2017-03-01

The deconvolution of DNA mixtures remains one of the most critical challenges in the field of forensic DNA analysis. In addition, of all the data features required to perform such deconvolution, the number of contributors in the sample is widely considered the most important, and, if incorrectly chosen, the most likely to negatively influence the mixture interpretation of a DNA profile. Unfortunately, most current approaches to mixture deconvolution require the assumption that the number of contributors is known by the analyst, an assumption that can prove to be especially faulty when faced with increasingly complex mixtures of 3 or more contributors. In this study, we propose a probabilistic approach for estimating the number of contributors in a DNA mixture that leverages the strengths of machine learning. To assess this approach, we compare classification performances of six machine learning algorithms and evaluate the model from the top-performing algorithm against the current state of the art in the field of contributor number classification. Overall results show over 98% accuracy in identifying the number of contributors in a DNA mixture of up to 4 contributors. Comparative results showed 3-person mixtures had a classification accuracy improvement of over 6% compared to the current best-in-field methodology, and that 4-person mixtures had a classification accuracy improvement of over 20%. The Probabilistic Assessment for Contributor Estimation (PACE) also accomplishes classification of mixtures of up to 4 contributors in less than 1s using a standard laptop or desktop computer. Considering the high classification accuracy rates, as well as the significant time commitment required by the current state of the art model versus seconds required by a machine learning-derived model, the approach described herein provides a promising means of estimating the number of contributors and, subsequently, will lead to improved DNA mixture interpretation. Copyright © 2016

Prepubertal goat oocytes from large follicles result in similar blastocyst production and embryo ploidy than those from adult goats.

Science.gov (United States)

Romaguera, R; Moll, X; Morató, R; Roura, M; Palomo, M J; Catalá, M G; Jiménez-Macedo, A R; Hammami, S; Izquierdo, D; Mogas, T; Paramio, M T

2011-07-01

Developmental competence of oocytes from prepubertal females is lower than those from adult females. Oocyte development competence is positively related to follicular diameter. Most of the follicles of prepubertal goat ovaries are smaller than 3 mm. The aim of this study was to compare oocytes of two follicle sizes (goats with oocytes from adult goats in relation to their in vitro production and quality of blastocysts. Oocytes from prepubertal goats were obtained from slaughterhouse ovaries and selected according to the follicle diameter whereas oocytes from adult goats were recovered in vivo by LOPU technique without prior selection of follicle size. COCs were IVM for 27 h, IVF at the conventional conditions with fresh semen and presumptive zygotes were cultured in SOF medium for 8 days. Blastocysts obtained were vitrified and after warming their blastocoele re-expansion and the ploidy by FISH technique were assessed. We found significant differences between blastocysts yield of oocytes recovered from follicles smaller than 3 mm of prepubertal goats compared to those from adult goats (5.45% vs 20. 83%, respectively) however, these differences disappear if oocytes were recovered form large follicles (18.07%). A total of 28 blastocysts were analysed and 96.43% showed mixoploidy. Age did not affect the number of embryos with abnormal ploidy or blastocyst re-expansion after warming. Furthermore, the percentage of diploid blastomeres per embryo was similar in the 3 groups studied, adult, prepubertal from follicles ≥ 3 mm and goats 45 days old were not different to the blastocysts produced from adult goats, both in terms of quantity and quality. Copyright © 2011 Elsevier Inc. All rights reserved.

Anatomy and morphology character of five Indonesian banana cultivars (Musa spp. of different ploidy level

Directory of Open Access Journals (Sweden)

ISSIREP SUMARDI

2010-04-01

Full Text Available Sumardi I, Wulandari M (2011 Anatomy and morphology character of five Indonesian banana cultivars (Musa spp. of different ploidy level. Biodiversitas 12: 167-175. In Indonesia there are many cultivars of banana, and some of them produce edible fruits. Beside their morphology, the character which necessary as a tool for classification is anatomical character. The aim of this research were to describe the anatomical character and morphology of fives Indonesian banana cultivars based on their level of ploidy. The cultivars were collected from Banana Germplasm Plantation, Yogyakarta District, Indonesia. The samples of roots, rhizome, and leaf were collected from five banana cultivars i.e.: Musa acuminata cv Penjalin, M.balbisiana cv Kluthuk warangan, M.acuminata cv Ambon warangan, M.paradisiaca cv Raja nangka , and M. paradisiaca cv Kluthuk susu. For anatomy observation samples were prepared using paraffin method, stained with 1% safranin in 70% ethanol. To observe the structure of stomata and epidermis surface, slide were prepared using modification of whole mount method. Slides were observed using Olympus BHB microscope completed with Olympus camera BM-10A. Stem and leaf morphology character of diploid level (AA and BB genome is different with triploid level (AAA, AAB, and ABB genome. Anatomy and morphology character of root and rhizome of banana in diploid level (AA and BB genome and triploid level (AAA, AAB, and ABB genome is quite similar. Distribution of stomata is found in leaf and pseudostem. Stomata is found in adaxial and abaxial epidermis layer. The size of guard cells in triploid cultivars was longer than that diploid cultivars. The root composse of epidermis layer, cortex and cylinder vascular of five cultivar’s root show anomalous structure. Rhizome consist of peripheric and centre zone. Anatomically, this was no differences in the rizome structur among five banana cultivars. The row of vascular bundles act as demarcation area

DNA fragmentation and nuclear phenotype in tendons exposed to low-intensity infrared laser

Science.gov (United States)

de Paoli, Flavia; Ramos Cerqueira, Larissa; Martins Ramos, Mayara; Campos, Vera M.; Ferreira-Machado, Samara C.; Geller, Mauro; de Souza da Fonseca, Adenilson

2015-03-01

Clinical protocols are recommended in device guidelines outlined for treating many diseases on empirical basis. However, effects of low-intensity infrared lasers at fluences used in clinical protocols on DNA are controversial. Excitation of endogenous chromophores in tissues and free radicals generation could be described as a consequence of laser used. DNA lesions induced by free radicals cause changes in DNA structure, chromatin organization, ploidy degrees and cell death. In this work, we investigated whether low-intensity infrared laser therapy could alter the fibroblasts nuclei characteristics and induce DNA fragmentation. Tendons of Wistar rats were exposed to low-intensity infrared laser (830 nm), at different fluences (1, 5 and 10 J/cm2), in continuous wave (power output of 10mW, power density of 79.6 mW/cm2). Different frequencies were analyzed for the higher fluence (10 J/cm2), at pulsed emission mode (2.5, 250 and 2500 Hz), with the laser source at surface of skin. Geometric, densitometric and textural parameters obtained for Feulgen-stained nuclei by image analysis were used to define nuclear phenotypes. Significant differences were observed on the nuclear phenotype of tendons after exposure to laser, as well as, high cell death percentages was observed for all fluences and frequencies analyzed here, exception 1 J/cm2 fluence. Our results indicate that low-intensity infrared laser can alter geometric, densitometric and textural parameters in tendon fibroblasts nuclei. Laser can also induce DNA fragmentation, chromatin lost and consequently cell death, using fluences, frequencies and emission modes took out from clinical protocols.

Estimating black bear density using DNA data from hair snares

Science.gov (United States)

Gardner, B.; Royle, J. Andrew; Wegan, M.T.; Rainbolt, R.E.; Curtis, P.D.

2010-01-01

DNA-based mark-recapture has become a methodological cornerstone of research focused on bear species. The objective of such studies is often to estimate population size; however, doing so is frequently complicated by movement of individual bears. Movement affects the probability of detection and the assumption of closure of the population required in most models. To mitigate the bias caused by movement of individuals, population size and density estimates are often adjusted using ad hoc methods, including buffering the minimum polygon of the trapping array. We used a hierarchical, spatial capturerecapture model that contains explicit components for the spatial-point process that governs the distribution of individuals and their exposure to (via movement), and detection by, traps. We modeled detection probability as a function of each individual's distance to the trap and an indicator variable for previous capture to account for possible behavioral responses. We applied our model to a 2006 hair-snare study of a black bear (Ursus americanus) population in northern New York, USA. Based on the microsatellite marker analysis of collected hair samples, 47 individuals were identified. We estimated mean density at 0.20 bears/km2. A positive estimate of the indicator variable suggests that bears are attracted to baited sites; therefore, including a trap-dependence covariate is important when using bait to attract individuals. Bayesian analysis of the model was implemented in WinBUGS, and we provide the model specification. The model can be applied to any spatially organized trapping array (hair snares, camera traps, mist nests, etc.) to estimate density and can also account for heterogeneity and covariate information at the trap or individual level. ?? The Wildlife Society.

Genome size variation and incidence of polyploidy in Scrophulariaceae sensu lato from the Iberian Peninsula.

Science.gov (United States)

Castro, Mariana; Castro, Sílvia; Loureiro, João

2012-01-01

In the last decade, genomic studies using DNA markers have strongly influenced the current phylogeny of angiosperms. Genome size and ploidy level have contributed to this discussion, being considered important characters in biosystematics, ecology and population biology. Despite the recent increase in studies related to genome size evolution and polyploidy incidence, only a few are available for Scrophulariaceae. In this context, we assessed the value of genome size, mostly as a taxonomic marker, and the role of polyploidy as a process of genesis and maintenance of plant diversity in Scrophulariaceae sensu lato in the Iberian Peninsula. Large-scale analyses of genome size and ploidy-level variation across the Iberian Peninsula were performed using flow cytometry. One hundred and sixty-two populations of 59 distinct taxa were analysed. A bibliographic review on chromosome counts was also performed. From the 59 sampled taxa, 51 represent first estimates of genome size. The majority of the Scrophulariaceae species presented very small to small genome sizes (2C ≤ 7.0 pg). Furthermore, in most of the analysed genera it was possible to use this character to separate several taxa, independently if these genera were homoploid or heteroploid. Also, some genome-related phenomena were detected, such as intraspecific variation of genome size in some genera and the possible occurrence of dysploidy in Verbascum spp. With respect to polyploidy, despite a few new DNA ploidy levels having been detected in Veronica, no multiple cytotypes have been found in any taxa. This work contributed with important basic scientific knowledge on genome size and polyploid incidence in the Scrophulariaceae, providing important background information for subsequent studies, with several perspectives for future studies being opened.

Quantitative investigation of reproduction of gonosomal condensed chromatin during trophoblast cell polyploidization and endoreduplication in the east-european field vole Microtus rossiaemeridionalis

Directory of Open Access Journals (Sweden)

Bogdanova Margarita S

2003-04-01

Full Text Available Abstract Simultaneous determinations of DNA content in cell nuclei and condensed chromatin bodies formed by heterochromatized regions of sex chromosomes (gonosomal chromatin bodies, GCB have been performed in two trophoblast cell populations of the East-European field vole Microtus rossiaemeridionalis: in the proliferative population of trophoblast cells of the junctional zone of placenta and in the secondary giant trophoblast cells. One or two GCBs have been observed in trophoblast cell nuclei of all embryos studied (perhaps both male and female. In the proliferative trophoblast cell population characterized by low ploidy levels (2–16c and in the highly polyploid population of secondary giant trophoblast cells (32–256c the total DNA content in GCB increased proportionally to the ploidy level. In individual GCBs the DNA content also rose proportionally to the ploidy level in nuclei both with one and with two GCBs in both trophoblast cell populations. Some increase in percentage of nuclei with 2–3 GCBs was shown in nuclei of the placenta junctional zone; this may be accounted for by genome multiplication via uncompleted mitoses. In nuclei of the secondary giant trophoblast cells (16–256c the number of GCBs did not exceed 2, and the fraction of nuclei with two GCBs did not increase, which suggests the polytene nature of sex chromosomes in these cells. In all classes of ploidy the DNA content in trophoblast cell nuclei with the single GCB was lower than in nuclei with two and more GCBs. This can indicate that the single GCB in many cases does not derive from fusion of two GCBs. The measurements in individual GCBs suggest that different heterochromatized regions of the X- and Y-chromosome may contribute in GCB formation.

Salix transect of Europe: variation in ploidy and genome size in willow-associated common nettle, Urtica dioica L. sens. lat., from Greece to arctic Norway

OpenAIRE

Quentin Cronk; Oriane Hidalgo; Jaume Pellicer; Diana Percy; Ilia Leitch

2016-01-01

Abstract Background The common stinging nettle, Urtica dioica L. sensu lato, is an invertebrate "superhost", its clonal patches maintaining large populations of insects and molluscs. It is extremely widespread in Europe and highly variable, and two ploidy levels (diploid and tetraploid) are known. However, geographical patterns in cytotype variation require further study. New information We assembled a collection of nettles in conjunction with a transect of Europe from the Aegean to Arctic No...

Spontaneous unscheduled DNA synthesis in human lymphocytes

International Nuclear Information System (INIS)

Forell, B.; Myers, L.S. Jr.; Norman, A.

1979-01-01

The rate of spontaneous unscheduled DNA synthesis in human lymphocytes was estimated from measurements of tritiated thymidine incorporation into double-stranded DNA (ds-DNA) during incubation of cells in vitro. The contribution of scheduled DNA synthesis to the observed incorporation was reduced by inhibiting replication with hydroxyurea and by separating freshly replicated single-stranded DNA (ss-DNA) from repaired ds-DNA by column chromatography. The residual contribution of scheduled DNA synthesis was estimated by observing effects on thymidine incorporation of: (a) increasing the rate of production of apurinic sites, and alternatively, (b) increasing the number of cells in S-phase. Corrections based on estimates of endogenous pool size were also made. The rate of spontaneous unscheduled DNA synthesis is estimated to be 490 +- 120 thymidine molecules incorporated per cell per hour. These results compare favorably with estimates made from rates of depurination and depyrimidination of DNA, measured in molecular systems if we assume thymidine is incorporated by a short patch mechanism which incorporates an average of four bases per lesion

The aryl hydrocarbon receptor (AHR) transcription factor regulates megakaryocytic polyploidization.

Science.gov (United States)

Lindsey, Stephan; Papoutsakis, Eleftherios T

2011-02-01

We propose that the aryl hydrocarbon receptor (AHR) is a novel transcriptional regulator of megakaryopoietic polyploidization. Functional evidence was obtained that AHR impacts in vivo megakaryocytic differentiation and maturation; compared to wild-type mice, AHR-null mice had lower platelet counts, fewer numbers of newly synthesized platelets, increased bleeding times and lower-ploidy megakaryocytes (Mks). AHR mRNA increased 3·6-fold during ex vivo megakaryocytic differentiation, but reduced or remained constant during parallel isogenic granulocytic or erythroid differentiation. We interrogated the role of AHR in megakaryopoiesis using a validated Mk model of megakaryopoiesis, the human megakaryoblastic leukaemia CHRF cell line. Upon CHRF Mk differentiation, AHR mRNA and protein levels increased, AHR protein shifted from the cytoplasm to the nucleus and AHR binding to its consensus DNA binding sequence increased. Protein and mRNA levels of the AHR transcriptional target HES1 also increased. Mk differentiation of CHRF cells where AHR or HES1 was knocked-down using RNAi resulted in lower ploidy distributions and cells that were incapable of reaching ploidy classes ≥16n. AHR knockdown also resulted in increased DNA synthesis of lower ploidy cells, without impacting apoptosis. Together, these data support a role for AHR in Mk polyploidization and in vivo platelet function, and warrant further detailed investigations. © 2011 Blackwell Publishing Ltd.

Use of spatial capture-recapture modeling and DNA data to estimate densities of elusive animals

Science.gov (United States)

Kery, Marc; Gardner, Beth; Stoeckle, Tabea; Weber, Darius; Royle, J. Andrew

2011-01-01

Assessment of abundance, survival, recruitment rates, and density (i.e., population assessment) is especially challenging for elusive species most in need of protection (e.g., rare carnivores). Individual identification methods, such as DNA sampling, provide ways of studying such species efficiently and noninvasively. Additionally, statistical methods that correct for undetected animals and account for locations where animals are captured are available to efficiently estimate density and other demographic parameters. We collected hair samples of European wildcat (Felis silvestris) from cheek-rub lure sticks, extracted DNA from the samples, and identified each animals' genotype. To estimate the density of wildcats, we used Bayesian inference in a spatial capture-recapture model. We used WinBUGS to fit a model that accounted for differences in detection probability among individuals and seasons and between two lure arrays. We detected 21 individual wildcats (including possible hybrids) 47 times. Wildcat density was estimated at 0.29/km2 (SE 0.06), and 95% of the activity of wildcats was estimated to occur within 1.83 km from their home-range center. Lures located systematically were associated with a greater number of detections than lures placed in a cell on the basis of expert opinion. Detection probability of individual cats was greatest in late March. Our model is a generalized linear mixed model; hence, it can be easily extended, for instance, to incorporate trap- and individual-level covariates. We believe that the combined use of noninvasive sampling techniques and spatial capture-recapture models will improve population assessments, especially for rare and elusive animals.

Environmental DNA (eDNA): A tool for quantifying the abundant but elusive round goby (Neogobius melanostomus)

Science.gov (United States)

Nevers, Meredith; Byappanahalli, Muruleedhara; Morris, Charles C.; Shively, Dawn; Przybyla-Kelly, Katarzyna; Spoljaric, Ashley M.; Dickey, Joshua; Roseman, Edward

2018-01-01

Environmental DNA (eDNA) is revolutionizing biodiversity monitoring, occupancy estimates, and real-time detections of invasive species. In the Great Lakes, the round goby (Neogobius melanostomus), an invasive benthic fish from the Black Sea, has spread to encompass all five lakes and many tributaries, outcompeting or consuming native species; however, estimates of round goby abundance are confounded by behavior and habitat preference, which impact reliable methods for estimating their population. By integrating eDNA into round goby monitoring, improved estimates of biomass may be obtainable. We conducted mesocosm experiments to estimate rates of goby DNA shedding and decay. Further, we compared eDNA with several methods of traditional field sampling to compare its use as an alternative/complementary monitoring method. Environmental DNA decay was comparable to other fish species, and first-order decay was lower at 12°C (k = 0.043) than at 19°C (k = 0.058). Round goby eDNA was routinely detected in known invaded sites of Lake Michigan and its tributaries (range log10 4.8–6.2 CN/L), but not upstream of an artificial fish barrier. Traditional techniques (mark-recapture, seining, trapping) in Lakes Michigan and Huron resulted in fewer, more variable detections than eDNA, but trapping and eDNA were correlated (Pearson R = 0.87). Additional field testing will help correlate round goby abundance with eDNA, providing insight on its role as a prey fish and its impact on food webs.

Applying of centrifugal chromatography on DEAE cellulose and viscosity measurement to estimate damage caused by gamma irradiation in lymphocyte DNA

International Nuclear Information System (INIS)

Olinski, R.

1977-01-01

DNA isolated from limphocytes of pig blood was irradiated by γ radiation in the range of 0.5-50 Krads. Changes caused by irradiation (single and double breaks) were determined by using viscosity measurement and centrifugal chromatography on DEAE cellulose. Study of DNA chromatograms showed possibility to apply centrifugal chromatography on DEAE cellulose to estimate changes caused by irradiation. (author)

[The polyploidization characteristics of the hepatocytes of the mouse-like hamster Calomyscus mystax].

Science.gov (United States)

Anatskaia, O V; Malikov, V G; Meĭer, M N; Kudriavtsev, B N

1995-01-01

A cytophotometric measurement of DNA content in hepatocytes of maturing mouse-like hamsters was made. Cells belonging to ordinary mammalian ploidy classes 2c, 2c x 2, 4c, and 4c x 2 made about 90% of the hepatocyte population. The share of binucleated cells wa high (about 80%), the majority of these cells being 2c X 2 hepatocytes. Binucleated cells with tetraploid and diploid nuclei occur in almost every animal. An average hepatocyte ploidy level in mouse-like hamster is 4.6c. The main peculiarity of parenchymal liver cell populations is that up 5% of hepatocytes contain 3--11 nuclei of different ploidy classes. Multinucleated cells increase in number from 1.5% to 4% within the period from one year (the age of maturation) to two years. Later on their percentage does not change. It is found that in binucleated and multinucleated hepatocytes DNA synthesis can proceed asynchronously. Asynchrony in DNA synthesis elevates as the number of nuclei increases. Among the 2c x 2 and 2c x 3 cells an uneven distribution of 3H-thymidine label can occur, respectively, in 5 and in 50% cases, whereas all the cells with more than 3 nuclei display an uneven an uneven 3H-thymidin label distribution. The formation of multinucleated cells is supposed to be associated with asynchrony in DNA-synthesis in binucleated cells and with the restitution of mitosis.

Estimation of pre-meiotic DNA synthesis period in the dog spermatozoa

International Nuclear Information System (INIS)

Ghosal, S.K.; Bandyopadhyay, T.; De, S.; Beauregard, L.J.

1976-01-01

About 10 μCi of 3 H-thymidine was injected into each of 4 arbitarary sites in each testis of 6 dogs. Biopsies were taken at 4-hour intervals coverning a period from 20.0 to 22.2 days post-injection. Kinetics of labelled spermatocytes was followed employing Kodak NTB-3 emulsion to conventionally prepared air-dried slides. The technique for calculating pre-meiosis DNA synthesis duration is same as that for estimating S period in mitotic cells. Current investigation suggests that the mean duration of pre-meiotic S period of Canine spermatocytes is 20.4 hrs as compared to 29 and 40 hrs in spermatocytes of mouse and golden hamster respectively. (author)

Abnormal meiosis in an intersectional allotriploid of Populus L. and segregation of ploidy levels in 2x × 3x progeny.

Directory of Open Access Journals (Sweden)

Jun Wang

Full Text Available Triploid plants are usually highly aborted owing to unbalanced meiotic chromosome segregation, but limited viable gametes can participate in the transition to different ploidy levels. In this study, numerous meiotic abnormalities were found with high frequency in an intersectional allotriploid poplar (Populus alba × P. berolinensis 'Yinzhong', including univalents, precocious chromosome migration, lagging chromosomes, chromosome bridges, micronuclei, and precocious cytokinesis, indicating high genetic imbalance in this allotriploid. Some micronuclei trigger mini-spindle formation in metaphase II and participate in cytokinesis to form polyads with microcytes. Unbalanced chromosome segregation and chromosome elimination resulted in the formation of microspores with aneuploid chromosome sets. Fusion of sister nuclei occurs in microsporocytes with precocious cytokinesis, which could form second meiotic division restitution (SDR-type gametes. However, SDR-type gametes likely contain incomplete chromosome sets due to unbalanced segregation of homologous chromosomes during the first meiotic division in triploids. Misorientation of spindles during the second meiotic division, such as fused and tripolar spindles with low frequency, could result in the formation of first meiotic division restitution (FDR-type unreduced gametes, which most likely contain three complete chromosome sets. Although 'Yinzhong' yields 88.7% stainable pollen grains with wide diameter variation from 23.9 to 61.3 μm, the pollen viability is poor (2.78% ± 0.38. A cross of 'Yinzhong' pollen with a diploid female clone produced progeny with extensive segregation of ploidy levels, including 29 diploids, 18 triploids, 4 tetraploids, and 48 aneuploids, suggesting the formation of viable aneuploidy and unreduced pollen in 'Yinzhong'. Individuals with different chromosome compositions are potential to analyze chromosomal function and to integrate the chromosomal dosage variation into

Polyploidisation and geographic differentiation drive diversification in a European High Mountain Plant Group (Doronicum clusii Aggregate, Asteraceae).

Science.gov (United States)

Pachschwöll, Clemens; Escobar García, Pedro; Winkler, Manuela; Schneeweiss, Gerald M; Schönswetter, Peter

2015-01-01

Range shifts (especially during the Pleistocene), polyploidisation and hybridization are major factors affecting high-mountain biodiversity. A good system to study their role in the European high mountains is the Doronicum clusii aggregate (Asteraceae), whose four taxa (D. clusii s.s., D. stiriacum, D. glaciale subsp. glaciale and D. glaciale subsp. calcareum) are differentiated geographically, ecologically (basiphilous versus silicicolous) and/or via their ploidy levels (diploid versus tetraploid). Here, we use DNA sequences (three plastid and one nuclear spacer) and AFLP fingerprinting data generated for 58 populations to infer phylogenetic relationships, origin of polyploids-whose ploidy level was confirmed by chromosomally calibrated DNA ploidy level estimates-and phylogeographic history. Taxonomic conclusions were informed, among others, by a Gaussian clustering method for species delimitation using dominant multilocus data. Based on molecular data we identified three lineages: (i) silicicolous diploid D. clusii s.s. in the Alps, (ii) silicicolous tetraploid D. stiriacum in the eastern Alps (outside the range of D. clusii s.s.) and the Carpathians and (iii) the basiphilous diploids D. glaciale subsp. glaciale (eastern Alps) and D. glaciale subsp. calcareum (northeastern Alps); each taxon was identified as distinct by the Gaussian clustering, but the separation of D. glaciale subsp. calcareum and D. glaciale subsp. glaciale was not stable, supporting their taxonomic treatment as subspecies. Carpathian and Alpine populations of D. stiriacum were genetically differentiated suggesting phases of vicariance, probably during the Pleistocene. The origin (autopolyploid versus allopolyploid) of D. stiriacum remained unclear. Doronicum glaciale subsp. calcareum was genetically and morphologically weakly separated from D. glaciale subsp. glaciale but exhibited significantly higher genetic diversity and rarity. This suggests that the more widespread D. glaciale subsp

Polyploidisation and geographic differentiation drive diversification in a European High Mountain Plant Group (Doronicum clusii Aggregate, Asteraceae.

Directory of Open Access Journals (Sweden)

Clemens Pachschwöll

Full Text Available Range shifts (especially during the Pleistocene, polyploidisation and hybridization are major factors affecting high-mountain biodiversity. A good system to study their role in the European high mountains is the Doronicum clusii aggregate (Asteraceae, whose four taxa (D. clusii s.s., D. stiriacum, D. glaciale subsp. glaciale and D. glaciale subsp. calcareum are differentiated geographically, ecologically (basiphilous versus silicicolous and/or via their ploidy levels (diploid versus tetraploid. Here, we use DNA sequences (three plastid and one nuclear spacer and AFLP fingerprinting data generated for 58 populations to infer phylogenetic relationships, origin of polyploids-whose ploidy level was confirmed by chromosomally calibrated DNA ploidy level estimates-and phylogeographic history. Taxonomic conclusions were informed, among others, by a Gaussian clustering method for species delimitation using dominant multilocus data. Based on molecular data we identified three lineages: (i silicicolous diploid D. clusii s.s. in the Alps, (ii silicicolous tetraploid D. stiriacum in the eastern Alps (outside the range of D. clusii s.s. and the Carpathians and (iii the basiphilous diploids D. glaciale subsp. glaciale (eastern Alps and D. glaciale subsp. calcareum (northeastern Alps; each taxon was identified as distinct by the Gaussian clustering, but the separation of D. glaciale subsp. calcareum and D. glaciale subsp. glaciale was not stable, supporting their taxonomic treatment as subspecies. Carpathian and Alpine populations of D. stiriacum were genetically differentiated suggesting phases of vicariance, probably during the Pleistocene. The origin (autopolyploid versus allopolyploid of D. stiriacum remained unclear. Doronicum glaciale subsp. calcareum was genetically and morphologically weakly separated from D. glaciale subsp. glaciale but exhibited significantly higher genetic diversity and rarity. This suggests that the more widespread D. glaciale

DNA:DNA hybridization studies on the pink-pigmented facultative methylotrophs.

Science.gov (United States)

Hood, D W; Dow, C S; Green, P N

1987-03-01

The genomic relatedness among 36 strains of pink-pigmented facultatively methylotrophic bacteria (PPFMs) was estimated by determination of DNA base composition and by DNA:DNA hybridization studies. A reproducible hybridization system was developed for the rapid analysis of multiple DNA samples. Results indicated that the PPFMs comprise four major and several minor homology groups, and that they should remain grouped in a single genus, Methylobacterium.

Phylogenetic relationships of the Gomphales based on nuc-25S-rDNA, mit-12S-rDNA, and mit-atp6-DNA combined sequences

Science.gov (United States)

Admir J. Giachini; Kentaro Hosaka; Eduardo Nouhra; Joseph Spatafora; James M. Trappe

2010-01-01

Phylogenetic relationships among Geastrales, Gomphales, Hysterangiales, and Phallales were estimated via combined sequences: nuclear large subunit ribosomal DNA (nuc-25S-rDNA), mitochondrial small subunit ribosomal DNA (mit-12S-rDNA), and mitochondrial atp6 DNA (mit-atp6-DNA). Eighty-one taxa comprising 19 genera and 58 species...

Polymorphism discovery and allele frequency estimation using high-throughput DNA sequencing of target-enriched pooled DNA samples

Directory of Open Access Journals (Sweden)

Mullen Michael P

2012-01-01

Full Text Available Abstract Background The central role of the somatotrophic axis in animal post-natal growth, development and fertility is well established. Therefore, the identification of genetic variants affecting quantitative traits within this axis is an attractive goal. However, large sample numbers are a pre-requisite for the identification of genetic variants underlying complex traits and although technologies are improving rapidly, high-throughput sequencing of large numbers of complete individual genomes remains prohibitively expensive. Therefore using a pooled DNA approach coupled with target enrichment and high-throughput sequencing, the aim of this study was to identify polymorphisms and estimate allele frequency differences across 83 candidate genes of the somatotrophic axis, in 150 Holstein-Friesian dairy bulls divided into two groups divergent for genetic merit for fertility. Results In total, 4,135 SNPs and 893 indels were identified during the resequencing of the 83 candidate genes. Nineteen percent (n = 952 of variants were located within 5' and 3' UTRs. Seventy-two percent (n = 3,612 were intronic and 9% (n = 464 were exonic, including 65 indels and 236 SNPs resulting in non-synonymous substitutions (NSS. Significant (P ® MassARRAY. No significant differences (P > 0.1 were observed between the two methods for any of the 43 SNPs across both pools (i.e., 86 tests in total. Conclusions The results of the current study support previous findings of the use of DNA sample pooling and high-throughput sequencing as a viable strategy for polymorphism discovery and allele frequency estimation. Using this approach we have characterised the genetic variation within genes of the somatotrophic axis and related pathways, central to mammalian post-natal growth and development and subsequent lactogenesis and fertility. We have identified a large number of variants segregating at significantly different frequencies between cattle groups divergent for calving

Assessment of somaclonal variation in somatic embryo-derived plants of yacon [Smallanthus sonchifolius (Poepp. and Endl.) H. Robinson] using inter simple sequence repeat analysis and flow cytometry

Czech Academy of Sciences Publication Activity Database

Viehmannová, I.; Bortlová, Z.; Vítámvás, J.; Čepková, P.; Eliášová, Kateřina; Svobodová, E.; Trávníčková, M.

2014-01-01

Roč. 17, č. 2 (2014) ISSN 0717-3458 Institutional support: RVO:61389030 Keywords : DNA polymorphism * Molecular markers * Ploidy level Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.681, year: 2014

Glycogen content in hepatocytes is related with their size in normal rat liver but not in cirrhotic one.

Science.gov (United States)

Bezborodkina, Natalia N; Chestnova, Anna Yu; Vorobev, Mikhail L; Kudryavtsev, Boris N

2016-04-01

Hepatocytes differ from one another by the degree of the ploidy, size, position in the liver lobule, and level of the DNA-synthetic processes. It is believed, that the cell size exerts substantial influence on the metabolism of the hepatocytes and the glycogen content in them. The aim of the present study was to test this hypothesis. Dry weight of hepatocytes, their ploidy and glycogen content were determined in the normal and the cirrhotic rat liver. Liver cirrhosis in rats was produced by chronic inhalation of CCl4 vapours in the course of 6 months. A combined cytophotometric method was used. Dry weight of the cell, its glycogen and DNA content were successively measured on a mapped preparation. Hepatocytes of each ploidy class in the normal and the cirrhotic rat liver accumulated glycogen at the same rate. In the normal liver, there was a distinct correlation between the size of hepatocytes and glycogen content in them. This correlation was observed in each ploidy class, and was especially pronounced in the class of mononucleate tetraploid hepatocytes. In the cirrhotic liver, there was no correlation between the size of the cells and their glycogen content. The impairment of liver lobular structure probably explains the observed lack of correlation between hepatocyte size and their glycogen content in the cirrhotic liver. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Polyploidy creates higher diversity among Cynodon accessions as assessed by molecular markers.

Science.gov (United States)

Gulsen, Osman; Sever-Mutlu, Songul; Mutlu, Nedim; Tuna, Metin; Karaguzel, Osman; Shearman, Robert C; Riordan, Terrance P; Heng-Moss, Tiffany M

2009-05-01

Developing a better understanding of associations among ploidy level, geographic distribution, and genetic diversity of Cynodon accessions could be beneficial to bermudagrass breeding programs, and would enhance our understanding of the evolutionary biology of this warm season grass species. This study was initiated to: (1) determine ploidy analysis of Cynodon accessions collected from Turkey, (2) investigate associations between ploidy level and diversity, (3) determine whether geographic and ploidy distribution are related to nuclear genome variation, and (4) correlate among four nuclear molecular marker systems for Cynodon accessions' genetic analyses. One hundred and eighty-two Cynodon accessions collected in Turkey from an area south of the Taurus Mountains along the Mediterranean cost and ten known genotypes were genotyped using sequence related amplified polymorphism (SRAP), peroxidase gene polymorphism (POGP), inter-simple sequence repeat (ISSR), and random amplified polymorphic DNA (RAPD). The diploids, triploids, tetraploids, pentaploids, and hexaploids revealed by flow cytometry had a linear present band frequency of 0.36, 0.47, 0.49, 0.52, and 0.54, respectively. Regression analysis explained that quadratic relationship between ploidy level and band frequency was the most explanatory (r = 0.62, P Cynodon accessions' genetic structure can aid to enhance breeding programs and broaden genetic base of commercial cultivars.

Role of Tumor Suppressor P53 in Megakaryopoiesis and Platelet Function

Science.gov (United States)

Apostolidis, Pani A.; Woulfe, Donna S.; Chavez, Massiel; Miller, William M.; Papoutsakis, Eleftherios T.

2011-01-01

The pathobiological role of p53 has been widely studied, however its role in normophysiology is relatively unexplored. We previously showed that p53 knock-down increased ploidy in megakaryocytic cultures. This study aims to examine the effect of p53 loss on in vivo megakaryopoiesis, platelet production and function, and to investigate the basis for greater ploidy in p53−/− megakaryocytic cultures. Here, we used flow cytometry to analyze ploidy, DNA synthesis and apoptosis in murine cultured and bone marrow megakaryocytes following thrombopoietin administration and to analyze fibrinogen binding to platelets in vitro. Culture of p53−/− marrow cells for 6 days with thrombopoietin gave rise to 1.7-fold more megakaryocytes, 26.1±3.6% of which reached ploidy classes ≥64N compared to 8.2±0.9% of p53+/+ megakaryocytes. This was due to 30% greater DNA synthesis in p53−/− megakaryocytes and 31% greater apoptosis in p53+/+ megakaryocytes by day 4 of culture. Although the bone marrow and spleen steady-state megakaryocytic content and ploidy were similar in p53+/+ and p53−/− mice, thrombopoietin administration resulted in increased megakaryocytic polyploidization in p53−/− mice. Although their platelet counts were normal, p53−/− mice exhibited significantly longer bleeding times and p53−/− platelets were less sensitive than p53+/+ platelets to agonist-induced fibrinogen binding and P-selectin secretion. In summary, our in vivo and ex-vivo studies indicate that p53 loss leads to increased polyploidization during megakaryopoiesis. Our findings also suggest for the first time a direct link between p53 loss and the development of fully functional platelets resulting in hemostatic deficiencies. PMID:22024107

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae

Directory of Open Access Journals (Sweden)

Laura Carolina Valencia

2011-01-01

Full Text Available The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM and 4-nitroquinoline-1-oxide (4NQO was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 !g/mL yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV < 10%. The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies.

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae).

Science.gov (United States)

Valencia, Laura Carolina; García, Adriana; Ramírez-Pinilla, Martha Patricia; Fuentes, Jorge Luis

2011-10-01

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies.

Levels of Intra-specific AFLP Diversity in Tuber-Bearing Potato Species with Different Breeding Systems and Ploidy Levels

Directory of Open Access Journals (Sweden)

Glenn J. Bryan

2017-09-01

Full Text Available DNA-based marker analysis of plant genebank material has become a useful tool in the evaluation of levels of genetic diversity and for the informed use and maintenance of germplasm. In this study, we quantify levels of amplified fragment length polymorphism (AFLP in representative accessions of wild and cultivated potato species of differing geographic origin, ploidy, and breeding system. We generated 449 polymorphic AFLP fragments in 619 plants, representing multiple plants (16–23 from 17 accessions of 14 potato taxa as well as single plants sampled from available accessions (from 3 to 56 of the same 14 taxa. Intra-accession diversities were compared to those of a synthetic ‘taxon-wide’ population comprising a single individual from a variable number of available accessions of each sampled taxon. Results confirm the expected considerably lower levels of polymorphism within accessions of self-compatible as compared to self-incompatible taxa. We observed broadly similar levels of ‘taxon-wide’ polymorphism among self-compatible and self-incompatible species, with self-compatible taxa showing only slightly lower rates of polymorphism. The most diverse accessions were the two cultivated potato accessions examined, the least diverse being the Mexican allohexaploids Solanum demissum and S. iopetalum. Generally allopolyploid self-compatible accessions exhibited lower levels of diversity. Some purported self-incompatible accessions showed relatively low levels of marker diversity, similar to the more diverse self-compatible material surveyed. Our data indicate that for self-compatible species a single plant is highly representative of a genebank accession. The situation for self-incompatible taxa is less clear, and sampling strategies used will depend on the type of investigation. These results have important implications for those seeking novel trait variation (e.g., disease resistance in gene banks as well as for the selection of individuals

Real-Time PCR Quantification of Chloroplast DNA Supports DNA Barcoding of Plant Species.

Science.gov (United States)

Kikkawa, Hitomi S; Tsuge, Kouichiro; Sugita, Ritsuko

2016-03-01

Species identification from extracted DNA is sometimes needed for botanical samples. DNA quantification is required for an accurate and effective examination. If a quantitative assay provides unreliable estimates, a higher quantity of DNA than the estimated amount may be used in additional analyses to avoid failure to analyze samples from which extracting DNA is difficult. Compared with conventional methods, real-time quantitative PCR (qPCR) requires a low amount of DNA and enables quantification of dilute DNA solutions accurately. The aim of this study was to develop a qPCR assay for quantification of chloroplast DNA from taxonomically diverse plant species. An absolute quantification method was developed using primers targeting the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene using SYBR Green I-based qPCR. The calibration curve was generated using the PCR amplicon as the template. DNA extracts from representatives of 13 plant families common in Japan. This demonstrates that qPCR analysis is an effective method for quantification of DNA from plant samples. The results of qPCR assist in the decision-making will determine the success or failure of DNA analysis, indicating the possibility of optimization of the procedure for downstream reactions.

Quantitative estimation of the extent of alkylation of DNA following treatment of mammalian cells with non-radioactive alkylating agents

Energy Technology Data Exchange (ETDEWEB)

Snyder, R.D. (Univ. of Tennessee, Oak Ridge); Regan, J.D.

1981-01-01

Alkaline sucrose sedimentation has been used to quantitate phosphotriester formation following treatment of human cells with the monofunctional alkylating agents methyl and ethyl methanesulfonate. These persistent alkaline-labile lesions are not repaired during short-term culture conditions and thus serve as a useful and precise index of the total alkylation of the DNA.Estimates of alkylation by this procedure compare favorably with direct estimates by use of labeled alkylating agents.

Iron may induce both DNA synthesis and repair in rat hepatocytes stimulated by EGF/pyruvate

Energy Technology Data Exchange (ETDEWEB)

Chenoufi, N.; Loreal, O.; Cariou, S.; Hubert, N.; Lescoat, G. [Univ. Hospital Pontchaillou, Unite de Recherches Hepatologiques, INSERM U 49, Rennes (France); Drenou, B. [Univ. Hospital Pontchaillou, Lab. d`Hematologie et d`Immunologie, Rennes (France); Leroyer, P.; Brissot, P. [Univ. Hospital Pontchaillou, Clinique des Maladies du Foie, Rennes (France)

1997-03-01

Background/Aims: Hepatocellular carcinoma develops frequently in the course of genetic hemochromatosis, and a role of iron overload in hepatic carcinogenesis is strongly suggested. Methods: The aim of our study was to investigate the effect of iron exposure on DNA synthesis of adult rat hepatocytes maintained in primary culture stimulated or not by EGF/pyruvate and exposed to iron-citrate complex. Results: In EGF/pyruvate-stimulated cultures, the level of [{sup 3}H] methyl thymidine incorporation was strongly increased as compared to unstimulated cultures. The addition of iron to stimulated cultures increased [{sup 3}H] methyl thymidine incorporation. The mitotic index was also significantly higher at 72 h. However,the number of cells found in the cell layer was not significantly different from iron-citrate free culture. By flow cytometry, no difference in cell ploidy was found between iron-treated and untreated EGF/pyruvate-stimulated cultures. A significant increase in LDH leakage reflecting a toxic effect of iron was found in the cell medium 48 h after cell seeding. In addition, [{sup 3}H] methyl thymidine incorporation in the presence of hydroxyurea was increased in iron-treated compared to untreated cultures. Conclusions: Our results show that DNA synthesis is increased in the presence of iron in rat hepatocyte cultures stimulated by EGF/pyruvate, and they suggest that DNA synthesis is likely to be related both to cell proliferation and to DNA repair. These observations may allow better understanding of the role of iron overload in the development of hepatocellular carcinoma. (au) 61 refs.

OligoHeatMap (OHM): an online tool to estimate and display hybridizations of oligonucleotides onto DNA sequences.

Science.gov (United States)

Croce, Olivier; Chevenet, François; Christen, Richard

2008-07-01

The efficiency of molecular methods involving DNA/DNA hybridizations depends on the accurate prediction of the melting temperature (T(m)) of the duplex. Many softwares are available for T(m) calculations, but difficulties arise when one wishes to check if a given oligomer (PCR primer or probe) hybridizes well or not on more than a single sequence. Moreover, the presence of mismatches within the duplex is not sufficient to estimate specificity as it does not always significantly decrease the T(m). OHM (OligoHeatMap) is an online tool able to provide estimates of T(m) for a set of oligomers and a set of aligned sequences, not only as text files of complete results but also in a graphical way: T(m) values are translated into colors and displayed as a heat map image, either stand alone or to be used by softwares such as TreeDyn to be included in a phylogenetic tree. OHM is freely available at http://bioinfo.unice.fr/ohm/, with links to the full source code and online help.

An immunosurveillance mechanism controls cancer cell ploidy.

Science.gov (United States)

Senovilla, Laura; Vitale, Ilio; Martins, Isabelle; Tailler, Maximilien; Pailleret, Claire; Michaud, Mickaël; Galluzzi, Lorenzo; Adjemian, Sandy; Kepp, Oliver; Niso-Santano, Mireia; Shen, Shensi; Mariño, Guillermo; Criollo, Alfredo; Boilève, Alice; Job, Bastien; Ladoire, Sylvain; Ghiringhelli, François; Sistigu, Antonella; Yamazaki, Takahiro; Rello-Varona, Santiago; Locher, Clara; Poirier-Colame, Vichnou; Talbot, Monique; Valent, Alexander; Berardinelli, Francesco; Antoccia, Antonio; Ciccosanti, Fabiola; Fimia, Gian Maria; Piacentini, Mauro; Fueyo, Antonio; Messina, Nicole L; Li, Ming; Chan, Christopher J; Sigl, Verena; Pourcher, Guillaume; Ruckenstuhl, Christoph; Carmona-Gutierrez, Didac; Lazar, Vladimir; Penninger, Josef M; Madeo, Frank; López-Otín, Carlos; Smyth, Mark J; Zitvogel, Laurence; Castedo, Maria; Kroemer, Guido

2012-09-28

Cancer cells accommodate multiple genetic and epigenetic alterations that initially activate intrinsic (cell-autonomous) and extrinsic (immune-mediated) oncosuppressive mechanisms. Only once these barriers to oncogenesis have been overcome can malignant growth proceed unrestrained. Tetraploidization can contribute to oncogenesis because hyperploid cells are genomically unstable. We report that hyperploid cancer cells become immunogenic because of a constitutive endoplasmic reticulum stress response resulting in the aberrant cell surface exposure of calreticulin. Hyperploid, calreticulin-exposing cancer cells readily proliferated in immunodeficient mice and conserved their increased DNA content. In contrast, hyperploid cells injected into immunocompetent mice generated tumors only after a delay, and such tumors exhibited reduced DNA content, endoplasmic reticulum stress, and calreticulin exposure. Our results unveil an immunosurveillance system that imposes immunoselection against hyperploidy in carcinogen- and oncogene-induced cancers.

Correlation of DNA content and nucleomorphometric features with World Health Organization grading of meningiomas.

Science.gov (United States)

Grunewald, J P; Röhl, F W; Kirches, E; Dietzmann, K

1998-02-01

Many studies dealing with extracranial cancer showed a strong correlation of DNA ploidy to a poor clinical outcome, recurrence, or malignancy. In brain tumors, analysis of DNA content did not always provided significant diagnostic information. In this study, DNA density and karyometric parameters of 50 meningiomas (26 Grade I, 10 Grade II, 14 Grade III) were quantitatively evaluated by digital cell image analyses of Feulgen-stained nuclei. In particular, the densitometric parameter SEXT, which describes nuclear DNA content, as well as the morphometric values LENG (a computer-assisted measurement of nuclear circumference), AREA (a computer-assisted measurement of nuclear area), FCON (a parameter that describes nuclear roundness), and CONC (a describing nuclear contour), evaluated with the software IMAGE C, were correlated to World Health Organization (WHO) grading using univariate and multivariate methods. AREA and LENG values showed significant differences between tumors of Grades I and III. FCON values were unable to distinguish WHO Grade III from Grade I/II but were useful in clearly separating Grade II from Grade I tumors. CONC values detected differences between WHO Grades II and I/III tumors but not between the latter. SEXT values clearly distinguished Grade III from Grade I/II tumors. The 1c, 2c, 2.5c, and 5c exceeding rates showed no predictive values. Only the 6c exceeding rate showed a significant difference between Grades I and III. These results outline the characteristic features of the atypical (Grade II) meningiomas, which make them a recognizable tumor entity distinct from benign and anaplastic meningiomas. The combination of DNA densitometric and morphometric findings seems to be a powerful addition to the histopathologic classification of meningiomas, as suggested by the WHO.

Estimates of population genetic diversity in brown bullhead catfish by DNA fingerprinting

Energy Technology Data Exchange (ETDEWEB)

Roth, A.C.; Wessendarp, T.K.; Gordon, D.A.; Smith, M.K. [Environmental Protection Agency, Cincinnati, OH (United States); Lattier, D.L. [Oak Ridge Inst. for Science and Education, Cincinnati, OH (United States); Hertzberg, V.; Leonard, A. [Univ. of Cincinnati, OH (United States). Dept. of Environmental Health

1994-12-31

Estimates of population genetic diversity may be a sensitive indicator of environmental impact, since limiting the effective breeding population by any means will result in loss of some variant genotypes, as has been demonstrated by allozyme analysis. DNA fingerprinting techniques are also coming into use for population analyses, and the authors chose to apply fingerprinting analysis three populations of brown bullhead catfish collected in Northern Ohio. DNA was isolated from the red blood cells of individual fish. Purified DNAs were digested with EcoR1 restriction enzyme; the digests were then sized on a 1% agarose gel, transferred to nylon membranes and probed with a radiolabeled M13 probe using the Westneat hybridization protocol (Southern blotting). This method effects fragments containing VNTR (variable number of tandem repeat) sequences complementary to the M13, which are highly variable among individual catfish. Hybridized bands were visualized by a Molecular Dynamics phosphorimager and recorded and analyzed with its proprietary Imagequant image analysis program, Excel and SAS. A total of 10 variable bands were identified and their presence or absence scored in each individual. These data were analyzed to determine between and within-population similarity indices as well as population heterozygosity and genetic diversity measures.

Gastric lymphomas in Turkey. Analysis of prognostic factors with special emphasis on flow cytometric DNA content.

Science.gov (United States)

Aydin, Z D; Barista, I; Canpinar, H; Sungur, A; Tekuzman, G

2000-07-01

In contrast to DNA ploidy, to the authors' knowledge the prognostic significance of S-phase fraction (SPF) in gastric lymphomas has not been determined. In the current study, the prognostic significance of various parameters including SPF and DNA aneuploidy were analyzed and some distinct epidemiologic and biologic features of gastric lymphomas in Turkey were found. A series of 78 gastric lymphoma patients followed at Hacettepe University is reported. DNA flow cytometry was performed for 34 patients. The influence of various parameters on survival was investigated with the log rank test. The Cox proportional hazards model was fitted to identify independent prognostic factors. The median age of the patients was 50 years. There was no correlation between patient age and tumor grade. DNA content analysis revealed 4 of the 34 cases to be aneuploid with DNA index values < 1.0. The mean SPF was 33.5%. In the univariate analysis, surgical resection of the tumor, modified Ann Arbor stage, performance status, response to first-line chemotherapy, lactate dehydrogenase (LDH) level, and SPF were important prognostic factors for disease free survival (DFS). The same parameters, excluding LDH level, were important for determining overall survival (OS). In the multivariate analysis, surgical resection of the tumor, disease stage, performance status, and age were found to be important prognostic factors for OS. To the authors' knowledge the current study is the first to demonstrate the prognostic significance of SPF in gastric lymphomas. The distinguishing features of Turkish gastric lymphoma patients are 1) DNA indices of aneuploid cases that all are < 1.0, which is a unique feature; 2) a lower percentage of aneuploid cases; 3) a higher SPF; 4) a younger age distribution; and 5) lack of an age-grade correlation. The authors conclude that gastric lymphomas in Turkey have distinct biologic and epidemiologic characteristics. Copyright 2000 American Cancer Society.

Validity of the tritiated thymidine method for estimating bacterial growth rates: measurement of isotope dilution during DNA synthesis

International Nuclear Information System (INIS)

Pollard, P.C.; Moriarty, D.J.W.

1984-01-01

The rate of tritiated thymidine incorporation into DNA was used to estimate bacterial growth rates in aquatic environments. To be accurate, the calculation of growth rates has to include a factor for the dilution of isotope before incorporation. The validity of an isotope dilution analysis to determine this factor was verified in experiments reported here with cultures of a marine bacterium growing in a chemostat. Growth rates calculated from data on chemostat dilution rates and cell density agreed well with rates calculated by tritiated thymidine incorporation into DNA and isotope dilution analysis. With sufficiently high concentrations of exogenous thymidine, de novo synthesis of deoxythymidine monophosphate was inhibited, thereby preventing the endogenous dilution of isoope. The thymidine technique was also shown to be useful for measuring growth rates of mixed suspensions of bacteria growing anaerobically. Thymidine was incorporated into the DNA of a range of marine pseudomonads that were investigated. Three species did not take up thymidine. The common marine cyanobacterium Synechococcus species did not incorporate thymidine into DNA

Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Science.gov (United States)

Avanzi, Mauro P; Chen, Amanda; He, Wu; Mitchell, W Beau

2012-11-01

Large-scale in vitro production of platelets (PLTs) from cord blood stem cells is one goal of stem cell research. One step toward this goal will be to produce polyploid megakaryocytes capable of releasing high numbers of PLTs. Megakaryocyte polyploidization requires distinct cytoskeletal and cellular mechanisms, including actin polymerization, myosin activation, microtubule formation, and increased DNA production. In this study we variably combined inhibition of these principal mechanisms of cytokinesis with the goal of driving polyploidization in megakaryocytes. Megakaryocytes were derived from umbilical cord blood and cultured with reagents that inhibit distinct mechanisms of cytokinesis: Rho-Rock inhibitor (RRI), Src inhibitor (SI), nicotinamide (NIC), aurora B inhibitor (ABI), and myosin light chain kinase inhibitor (MLCKI). Combinations of reagents were used to determine their interactions and to maximize megakaryocyte ploidy. Treatment with RRI, NIC, SI, and ABI, but not with MLCKI, increased the final ploidy and RRI was the most effective single reagent. RRI and MLCKI, both inhibitors of MLC activation, resulted in opposite ploidy outcomes. Combinations of reagents also increased ploidy and the use of NIC, SI, and ABI was as effective as RRI alone. Addition of MLCKI to NIC, SI, and ABI reached the highest level of polyploidization. Megakaryocyte polyploidization results from modulation of a combination of distinct cytokinesis pathways. Reagents targeting distinct cytoskeletal pathways produced additive effects in final megakaryocyte ploidy. The RRI, however, showed no additive effect but produced a high final ploidy due to overlapping inhibition of multiple cytokinesis pathways. © 2012 American Association of Blood Banks.

Application of DNA barcoding in biodiversity studies of shallow-water octocorals: molecular proxies agree with morphological estimates of species richness in Palau

Science.gov (United States)

McFadden, C. S.; Brown, A. S.; Brayton, C.; Hunt, C. B.; van Ofwegen, L. P.

2014-06-01

The application of DNA barcoding to anthozoan cnidarians has been hindered by their slow rates of mitochondrial gene evolution and the failure to identify alternative molecular markers that distinguish species reliably. Among octocorals, however, multilocus barcodes can distinguish up to 70 % of morphospecies, thereby facilitating the identification of species that are ecologically important but still very poorly known taxonomically. We tested the ability of these imperfect DNA barcodes to estimate species richness in a biodiversity survey of the shallow-water octocoral fauna of Palau using multilocus ( COI, mtMutS, 28S rDNA) sequences obtained from 305 specimens representing 38 genera of octocorals. Numbers and identities of species were estimated independently (1) by a taxonomic expert using morphological criteria and (2) by assigning sequences to molecular operational taxonomic units (MOTUs) using predefined genetic distance thresholds. Estimated numbers of MOTUs ranged from 73 to 128 depending on the barcode and distance threshold applied, bracketing the estimated number of 118 morphospecies. Concordance between morphospecies identifications and MOTUs ranged from 71 to 75 % and differed little among barcodes. For the speciose and ecologically dominant genus Sinularia, however, we were able to identify 95 % of specimens correctly simply by comparing mtMutS sequences and in situ photographs of colonies to an existing vouchered database. Because we lack a clear understanding of species boundaries in most of these taxa, numbers of morphospecies and MOTUs are both estimates of the true species diversity, and we cannot currently determine which is more accurate. Our results suggest, however, that the two methods provide comparable estimates of species richness for shallow-water Indo-Pacific octocorals. Use of molecular barcodes in biodiversity surveys will facilitate comparisons of species richness and composition among localities and over time, data that do not

Apparent polyploidization after gamma irradiation: pitfalls in the use of quantitative polymerase chain reaction (qPCR) for the estimation of mitochondrial and nuclear DNA gene copy numbers.

Science.gov (United States)

Kam, Winnie W Y; Lake, Vanessa; Banos, Connie; Davies, Justin; Banati, Richard

2013-05-30

Quantitative polymerase chain reaction (qPCR) has been widely used to quantify changes in gene copy numbers after radiation exposure. Here, we show that gamma irradiation ranging from 10 to 100 Gy of cells and cell-free DNA samples significantly affects the measured qPCR yield, due to radiation-induced fragmentation of the DNA template and, therefore, introduces errors into the estimation of gene copy numbers. The radiation-induced DNA fragmentation and, thus, measured qPCR yield varies with temperature not only in living cells, but also in isolated DNA irradiated under cell-free conditions. In summary, the variability in measured qPCR yield from irradiated samples introduces a significant error into the estimation of both mitochondrial and nuclear gene copy numbers and may give spurious evidence for polyploidization.

Estimation of isolation times of the island species in the Drosophila simulans complex from multilocus DNA sequence data.

Directory of Open Access Journals (Sweden)

Shannon R McDermott

2008-06-01

Full Text Available The Drosophila simulans species complex continues to serve as an important model system for the study of new species formation. The complex is comprised of the cosmopolitan species, D. simulans, and two island endemics, D. mauritiana and D. sechellia. A substantial amount of effort has gone into reconstructing the natural history of the complex, in part to infer the context in which functional divergence among the species has arisen. In this regard, a key parameter to be estimated is the initial isolation time (t of each island species. Loci in regions of low recombination have lower divergence within the complex than do other loci, yet divergence from D. melanogaster is similar for both classes. This might reflect gene flow of the low-recombination loci subsequent to initial isolation, but it might also reflect differential effects of changing population size on the two recombination classes of loci when the low-recombination loci are subject to genetic hitchhiking or pseudohitchhikingNew DNA sequence variation data for 17 loci corroborate the prior observation from 13 loci that DNA sequence divergence is reduced in genes of low recombination. Two models are presented to estimate t and other relevant parameters (substitution rate correction factors in lineages leading to the island species and, in the case of the 4-parameter model, the ratio of ancestral to extant effective population size from the multilocus DNA sequence data.In general, it appears that both island species were isolated at about the same time, here estimated at approximately 250,000 years ago. It also appears that the difference in divergence patterns of genes in regions of low and higher recombination can be reconciled by allowing a modestly larger effective population size for the ancestral population than for extant D. simulans.

DNA damage and carcinogenesis

International Nuclear Information System (INIS)

Stelow, R.B.

1980-01-01

Although cancer may arise as a result of many different types of molecular changes, there is little reason to doubt that changes to DNA are one of the more important ones in cancer initiation. Although DNA repair mechanisms seem able to eliminate a very large fraction of deleterious changes to DNA, we not only have little insight into the molecular mechanisms involved in such repair, but have a negligible amount of information to permit us to estimate the shape of dose response relations at low doses. The case of skin cancer is a special one, in that the average population is exposed to sufficient solar uv so that the effects of small increments in uv dose may be estimated. An approximate 85% reduction in DNA repair increases skin cancer incidence 10 4 fold

Application of DNA-DNA colony hybridization to the detection of catabolic genotypes in environmental samples

International Nuclear Information System (INIS)

Sayler, G.S.; Shields, M.S.; Tedford, E.T.; Breen, A.; Hooper, S.W.; Sirotkin, K.M.; Davis, J.W.

1985-01-01

The application of preexisting DNA hybridization techniques was investigated for potential in determining populations of specific gene sequences in environmental samples. Cross-hybridizations among two degradative plasmids, TOL and NAH, and two cloning vehicles, pLAFR1 and RSF1010, were determined. The detection limits for the TOL plasmid against a nonhomologous plasmid-bearing bacterial background was ascertained. The colony hybridization technique allowed detection of one colony containing TOL plasmid among 10(6) Escherichia coli colonies of nonhomologous DNA. Comparisons between population estimates derived from growth on selective substrates and from hybridizations were examined. Findings indicated that standard sole carbon source enumeration procedures for degradative populations lead to overestimations due to nonspecific growth of other bacteria on the microcontaminant carbon sources present in the media. Population estimates based on the selective growth of a microcosm population on two aromatic substrates (toluene and naphthalene) and estimates derived from DNA-DNA colony hybridizations, using the TOL or NAH plasmid as a probe, corresponded with estimates of substrate mineralization rates and past exposure to environmental contaminants. The applications of such techniques are hoped to eventually allow enumeration of any specific gene sequences in the environment, including both anabolic and catabolic genes. In addition, this procedure should prove useful in monitoring recombinant DNA clones released into environmental situations

Genetic diversity among Korean bermudagrass (Cynodon spp.) ecotypes characterized by morphological, cytological and molecular approaches.

Science.gov (United States)

Kang, Si-Yong; Lee, Geung-Joo; Lim, Ki Byung; Lee, Hye Jung; Park, In Sook; Chung, Sung Jin; Kim, Jin-Baek; Kim, Dong Sub; Rhee, Hye Kyung

2008-04-30

The genus Cynodon comprises ten species. The objective of this study was to evaluate the genetic diversity of Korean bermudagrasses at the morphological, cytological and molecular levels. Morphological parameters, the nuclear DNA content and ploidy levels were observed in 43 bermudagrass ecotypes. AFLP markers were evaluated to define the genetic diversity, and chromosome counts were made to confirm the inferred cytotypes. Nuclear DNA contents were in the ranges 1.42-1.56, 1.94-2.19, 2.54, and 2.77-2.85 pg/2C for the triploid, tetraploid, pentaploid, and hexaploid accessions, respectively. The inferred cytotypes were triploid (2n = 3x = 27), tetraploid (2n = 4x = 36), pentaploid (2n = 5x = 45), and hexaploid (2n = 6x = 54), but the majority of the collections were tetraploid (81%). Mitotic chromosome counts verified the corresponding ploidy levels. The fast growing fine-textured ecotypes had lower ploidy levels, while the pentaploids and hexaploids were coarse types. The genetic similarity ranged from 0.42 to 0.94 with an average of 0.64. UPGMA cluster analysis and principle coordinate analysis separated the ecotypes into 6 distinct groups. The genetic similarity suggests natural hybridization between the different cytotypes, which could be useful resources for future breeding and genetic studies.

Variabilita ve fenologii a ploidních hladinách původních a invazních populací kypreje vrbice (Lythrum salicaria) v širším geografickém měřítku

Czech Academy of Sciences Publication Activity Database

Bastlová, D.; Květ, Jan; Kubátová, B.; Trávníček, Pavel; Čurn, V.; Suda, Jan

2008-01-01

Roč. 43, č. 23 (2008), s. 103-112 ISSN 1212-3323 Institutional research plan: CEZ:AV0Z60870520; CEZ:AV0Z60050516 Keywords : cytotype * cytogeography * distribution * DNA ploidy level * Flow cytometry * invasion * Lythrum salicaria Subject RIV: EF - Botanics

Polyploidy induction of Trigonella foenum-greaum L

African Journals Online (AJOL)

Hp

2011-08-10

Aug 10, 2011 ... colchicine allowed generation of diploid and mixoploid plants with a mixoploidy ... Ahmad F, Acharya SN, Mir Z, Mir PS (2000). Localization and ... Koutoulis A, Roy AT, Price A, Sherriff L, Leggett G (2005). DNA ploidy level of ...

Application of DNA fingerprints for cell-line individualization.

OpenAIRE

Gilbert, D A; Reid, Y A; Gail, M H; Pee, D; White, C; Hay, R J; O'Brien, S J

1990-01-01

DNA fingerprints of 46 human cell lines were derived using minisatellite probes for hypervariable genetic loci. The incidence of 121 HaeIII DNA fragments among 33 cell lines derived from unrelated individuals was used to estimate allelic and genotypic frequencies for each fragment and for composite individual DNA fingerprints. We present a quantitative estimate of the extent of genetic difference between individuals, an estimate based on the percentage of restriction fragments at which they d...

FBI's DNA analysis program

Science.gov (United States)

Brown, John R.

1994-03-01

Forensic DNA profiling technology is a significant law enforcement tool due to its superior discriminating power. Applying the principles of population genetics to the DNA profile obtained in violent crime investigations results in low frequency of occurrence estimates for the DNA profile. These estimates often range from a frequency of occurrence of 1 in 50 unrelated individuals to 1 in a million unrelated individuals or even smaller. It is this power to discriminate among individuals in the population that has propelled forensic DNA technology to the forefront of forensic testing in violent crime cases. Not only is the technology extremely powerful in including or excluding a criminal suspect as the perpetrator, but it also gives rise to the potential of identifying criminal suspects in cases where the investigators of unknown suspect cases have exhausted all other available leads.

Accuracy Rates of Sex Estimation by Forensic Anthropologists through Comparison with DNA Typing Results in Forensic Casework.

Science.gov (United States)

Thomas, Richard M; Parks, Connie L; Richard, Adam H

2016-09-01

A common task in forensic anthropology involves the estimation of the biological sex of a decedent by exploiting the sexual dimorphism between males and females. Estimation methods are often based on analysis of skeletal collections of known sex and most include a research-based accuracy rate. However, the accuracy rates of sex estimation methods in actual forensic casework have rarely been studied. This article uses sex determinations based on DNA results from 360 forensic cases to develop accuracy rates for sex estimations conducted by forensic anthropologists. The overall rate of correct sex estimation from these cases is 94.7% with increasing accuracy rates as more skeletal material is available for analysis and as the education level and certification of the examiner increases. Nine of 19 incorrect assessments resulted from cases in which one skeletal element was available, suggesting that the use of an "undetermined" result may be more appropriate for these cases. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Accurate Estimation of the Standard Binding Free Energy of Netropsin with DNA

Directory of Open Access Journals (Sweden)

Hong Zhang

2018-01-01

Full Text Available DNA is the target of chemical compounds (drugs, pollutants, photosensitizers, etc., which bind through non-covalent interactions. Depending on their structure and their chemical properties, DNA binders can associate to the minor or to the major groove of double-stranded DNA. They can also intercalate between two adjacent base pairs, or even replace one or two base pairs within the DNA double helix. The subsequent biological effects are strongly dependent on the architecture of the binding motif. Discriminating between the different binding patterns is of paramount importance to predict and rationalize the effect of a given compound on DNA. The structural characterization of DNA complexes remains, however, cumbersome at the experimental level. In this contribution, we employed all-atom molecular dynamics simulations to determine the standard binding free energy of DNA with netropsin, a well-characterized antiviral and antimicrobial drug, which associates to the minor groove of double-stranded DNA. To overcome the sampling limitations of classical molecular dynamics simulations, which cannot capture the large change in configurational entropy that accompanies binding, we resort to a series of potentials of mean force calculations involving a set of geometrical restraints acting on collective variables.

Comparative analysis of intermuscular bones in fish of different ploidies.

Science.gov (United States)

Li, Ling; Zhong, Zezhou; Zeng, Ming; Liu, Shaojun; Zhou, Yi; Xiao, Jun; Wang, Jun; Liu, Yun

2013-04-01

We documented the number, morphology, and distribution of intermuscular bones in five fishes of different ploidy: Carassius auratus (Abbr.WCC, 2n=100), Carassius auratus variety PengZe (Abbr.PZCC, 3n=150), improved triploid crucian carp (Abbr.ITCC, 3n=150), improved red crucian carp (Carassius auratus red var., Abbr.IRCC, ♀, 2n=100), and improved allotetraploids (Abbr.G×AT, ♂, 4n=200). The number of intermuscular bones in WCC, PZCC, and G×AT ranged from 78 to 83 ([Formula: see text]=81), 80 to 86 ([Formula: see text]=84), and 77 to 84 ([Formula: see text]=82), respectively. The numbers in ITCC and IRCC were significantly lower, ranging from 77 to 82 ([Formula: see text]=79) and 58 to 77 ([Formula: see text]=71), respectively. The average number of intermuscular bones in each sarcomere, ranked in order from highest to lowest, was 0.721 (WCC), 0.673 (PZCC), 0.653 (G×AT), 0.633 (ITCC), and 0.608 (IRCC). There was no difference between ITCC and G×AT or between G×AT and PZCC. However, the average number of intermuscular bones in the sarcomeres of ITCC, WCC, and PZCC differed significantly, as did that of IRCC and the four other kinds of fish. The intermuscular bone of these five fishes was divided into seven shape categories, non-forked (), one-end-unequal-bi-fork (), one-end-equal-bi-fork (Y), one-end-multi-fork, two-end-bi-fork, two-end-multi-fork, and tree-branch types. Generally, the morphological complexity was higher in the anterior intermuscular bones than in the posterior body. The number of intermuscular bones was similar but not equal between the left and right sides of the body. ITCC had significantly fewer intermuscular bones than either WCC or PZCC, making it of greater commercial value. Additionally, IRCC and ITCC had fewer intermuscular bones than WCC. Our observations are significant in both fish bone developmental biology and genetic breeding.

Estimation and quantification of human DNA in dental calculus: A pilot study.

Science.gov (United States)

Singh, Udita; Goel, Saurabh

2017-01-01

Identification using DNA has proved its accuracy multiple times in the field of forensic investigations. Investigators usually rely on either teeth or bone as the DNA reservoirs. However, there are instances where the skeletal or dental remains are not available or not preserved properly. Moreover, due to religious beliefs, the family members of the dead do not allow the investigating team to damage the remains for the sole purpose of identification. To investigate the presence of human DNA in dental calculus and to quantify the amount, if present. This prospective single-blinded pilot study included twenty subjects selected from the patients visiting a dental college. The samples of dental calculus were collected from the thickest portion of calculus deposited on the lingual surfaces of mandibular incisors. These samples were decontaminated and subjected to gel electrophoresis for DNA extraction. DNA was found in 85% cases. The amount of DNA varied from 21 to 37 μg/ml of dental calculus. Dental calculus is a rich reservoir of human DNA.

Cr(VI) induces DNA damage, cell cycle arrest and polyploidization: a flow cytometric and comet assay study in Pisum sativum.

Science.gov (United States)

Rodriguez, Eleazar; Azevedo, Raquel; Fernandes, Pedro; Santos, Conceição

2011-07-18

Chromium(VI) is recognized as the most toxic valency of Cr, but its genotoxicity and cytostaticity in plants is still poorly studied. In order to analyze Cr(VI) cyto- and gentotoxicity, Pisum sativum L. plants were grown in soil and watered with solutions with different concentrations of Cr up to 2000 mg/L. After 28 days of exposure, leaves showed no significant variations in either cell cycle dynamics or ploidy level. As for DNA damage, flow cytometric (FCM) histograms showed significant differences in full peak coefficient of variation (FPCV) values, suggesting clastogenicity. This is paralleled by the Comet assay results, showing an increase in DNA damage for 1000 and 2000 mg/L. In roots, exposure to 2000 mg/L resulted in cell cycle arrest at the G(2)/M checkpoint. It was also verified that under the same conditions 40% of the individuals analyzed suffered polyploidization having both 2C and 4C levels. DNA damage analysis by the Comet assay and FCM revealed dose-dependent increases in DNA damage and FPCV. Through this, we have unequivocally demonstrated for the first time in plants that Cr exposure can result in DNA damage, cell cycle arrest, and polyploidization. Moreover, we critically compare the validity of the Comet assay and FCM in evaluating cytogenetic toxicity tests in plants and demonstrate that the data provided by both techniques complement each other and present high correlation levels. In conclusion, the data presented provides new insight on Cr effects in plants in general and supports the use of the parameters tested in this study as reliable endpoints for this metal toxicity in plants. © 2011 American Chemical Society

Building a Phylogenetic Tree of the Human and Ape Superfamily Using DNA-DNA Hybridization Data

Science.gov (United States)

Maier, Caroline Alexander

2004-01-01

The study describes the process of DNA-DNA hybridization and the history of its use by Sibley and Alquist in simple, straightforward, and interesting language that students easily understand to create their own phylogenetic tree of the hominoid superfamily. They calibrate the DNA clock and use it to estimate the divergence dates of the various…

Estimation of a Killer Whale (Orcinus orca Population's Diet Using Sequencing Analysis of DNA from Feces.

Directory of Open Access Journals (Sweden)

Michael J Ford

Full Text Available Estimating diet composition is important for understanding interactions between predators and prey and thus illuminating ecosystem function. The diet of many species, however, is difficult to observe directly. Genetic analysis of fecal material collected in the field is therefore a useful tool for gaining insight into wild animal diets. In this study, we used high-throughput DNA sequencing to quantitatively estimate the diet composition of an endangered population of wild killer whales (Orcinus orca in their summer range in the Salish Sea. We combined 175 fecal samples collected between May and September from five years between 2006 and 2011 into 13 sample groups. Two known DNA composition control groups were also created. Each group was sequenced at a ~330bp segment of the 16s gene in the mitochondrial genome using an Illumina MiSeq sequencing system. After several quality controls steps, 4,987,107 individual sequences were aligned to a custom sequence database containing 19 potential fish prey species and the most likely species of each fecal-derived sequence was determined. Based on these alignments, salmonids made up >98.6% of the total sequences and thus of the inferred diet. Of the six salmonid species, Chinook salmon made up 79.5% of the sequences, followed by coho salmon (15%. Over all years, a clear pattern emerged with Chinook salmon dominating the estimated diet early in the summer, and coho salmon contributing an average of >40% of the diet in late summer. Sockeye salmon appeared to be occasionally important, at >18% in some sample groups. Non-salmonids were rarely observed. Our results are consistent with earlier results based on surface prey remains, and confirm the importance of Chinook salmon in this population's summer diet.

Isolation of mouse mesenchymal stem cells with normal ploidy from bone marrows by reducing oxidative stress in combination with extracellular matrix

Directory of Open Access Journals (Sweden)

Wang Fang

2011-07-01

Full Text Available Abstract Background Isolation of mouse MSCs (mMSCs with normal ploidy from bone marrow remains challenging. mMSCs isolated under 20% O2 are frequently contaminated by overgrown hematopoietic cells, and could also be especially vulnerable to oxidative damage, resulting in chromosomal instability. Culture under low oxygen or extracellular matrix (ECM improves proliferation of MSCs in several species. We tested the hypothesis that culture under low oxygen in combination with ECM prepared from mouse embryonic fibroblast (MEF-ECM could be used to purify proliferative mMSCs, and to reduce oxidative damage and maintain their chromosomal stability. Results Optimization of culture conditions under 20% O2 resulted in immortalization of mMSCs, showing extensive chromosome abnormalities, consistent with previous studies. In contrast, culture under low oxygen (2% O2 improved proliferation of mMSCs and reduced oxidative damage, such that mMSCs were purified simply by plating at low density under 2% O2. MEF-ECM reduced oxidative damage and enhanced proliferation of mMSCs. However, these isolated mMSCs still exhibited high frequency of chromosome abnormalities, suggesting that low oxygen or in combination with MEF-ECM was insufficient to fully protect mMSCs from oxidative damage. Notably, antioxidants (alpha -phenyl-t-butyl nitrone (PBN and N-acetylcysteine (NAC further reduced DNA damage and chromosomal abnormalities, and increased proliferation of mMSCs. mMSCs isolated by the combination method were successfully used to generate induced pluripotent stem (iPS cells by ectopic expression of Oct4, Sox2, Klf4 and c-Myc. Conclusions We have developed a technique that allows to reduce the number of karyotypic abnormalities for isolation of primary mMSCs and for limited culture period by combination of low oxygen, MEF-ECM, antioxidants and low density plating strategy. The effectiveness of the new combination method is demonstrated by successful generation of i

Reef-associated crustacean fauna: biodiversity estimates using semi-quantitative sampling and DNA barcoding

Science.gov (United States)

Plaisance, L.; Knowlton, N.; Paulay, G.; Meyer, C.

2009-12-01

The cryptofauna associated with coral reefs accounts for a major part of the biodiversity in these ecosystems but has been largely overlooked in biodiversity estimates because the organisms are hard to collect and identify. We combine a semi-quantitative sampling design and a DNA barcoding approach to provide metrics for the diversity of reef-associated crustacean. Twenty-two similar-sized dead heads of Pocillopora were sampled at 10 m depth from five central Pacific Ocean localities (four atolls in the Northern Line Islands and in Moorea, French Polynesia). All crustaceans were removed, and partial cytochrome oxidase subunit I was sequenced from 403 individuals, yielding 135 distinct taxa using a species-level criterion of 5% similarity. Most crustacean species were rare; 44% of the OTUs were represented by a single individual, and an additional 33% were represented by several specimens found only in one of the five localities. The Northern Line Islands and Moorea shared only 11 OTUs. Total numbers estimated by species richness statistics (Chao1 and ACE) suggest at least 90 species of crustaceans in Moorea and 150 in the Northern Line Islands for this habitat type. However, rarefaction curves for each region failed to approach an asymptote, and Chao1 and ACE estimators did not stabilize after sampling eight heads in Moorea, so even these diversity figures are underestimates. Nevertheless, even this modest sampling effort from a very limited habitat resulted in surprisingly high species numbers.

Quantification of damage in DNA recovered from highly degraded samples – a case study on DNA in faeces

Directory of Open Access Journals (Sweden)

Eveson J Paige

2006-08-01

Full Text Available Abstract Background Poorly preserved biological tissues have become an important source of DNA for a wide range of zoological studies. Measuring the quality of DNA obtained from these samples is often desired; however, there are no widely used techniques available for quantifying damage in highly degraded DNA samples. We present a general method that can be used to determine the frequency of polymerase blocking DNA damage in specific gene-regions in such samples. The approach uses quantitative PCR to measure the amount of DNA present at several fragment sizes within a sample. According to a model of random degradation the amount of available template will decline exponentially with increasing fragment size in damaged samples, and the frequency of DNA damage (λ can be estimated by determining the rate of decline. Results The method is illustrated through the analysis of DNA extracted from sea lion faecal samples. Faeces contain a complex mixture of DNA from several sources and different components are expected to be differentially degraded. We estimated the frequency of DNA damage in both predator and prey DNA within individual faecal samples. The distribution of fragment lengths for each target fit well with the assumption of a random degradation process and, in keeping with our expectations, the estimated frequency of damage was always less in predator DNA than in prey DNA within the same sample (mean λpredator = 0.0106 per nucleotide; mean λprey = 0.0176 per nucleotide. This study is the first to explicitly define the amount of template damage in any DNA extracted from faeces and the first to quantify the amount of predator and prey DNA present within individual faecal samples. Conclusion We present an approach for characterizing mixed, highly degraded PCR templates such as those often encountered in ecological studies using non-invasive samples as a source of DNA, wildlife forensics investigations and ancient DNA research. This method will

Application of DNA fingerprints for cell-line individualization.

Science.gov (United States)

Gilbert, D A; Reid, Y A; Gail, M H; Pee, D; White, C; Hay, R J; O'Brien, S J

1990-09-01

DNA fingerprints of 46 human cell lines were derived using minisatellite probes for hypervariable genetic loci. The incidence of 121 HaeIII DNA fragments among 33 cell lines derived from unrelated individuals was used to estimate allelic and genotypic frequencies for each fragment and for composite individual DNA fingerprints. We present a quantitative estimate of the extent of genetic difference between individuals, an estimate based on the percentage of restriction fragments at which they differ. The average percent difference (APD) among pairwise combinations from the population of 33 unrelated cell lines was 76.9%, compared with the APD in band sharing among cell lines derived from the same individual (less than or equal to 1.2%). Included in this survey were nine additional cell lines previously implicated as HeLa cell derivatives, and these lines were clearly confirmed as such by DNA fingerprints (APD less than or equal to 0.6%). On the basis of fragment frequencies in the tested cell line population, a simple genetic model was developed to estimate the frequencies of each DNA fingerprint in the population. The median incidence was 2.9 X 10(-17), and the range was 2.4 X 10(-21) to 6.6 X 10(-15). This value approximates the probability that a second cell line selected at random from unrelated individuals will match a given DNA fingerprint. Related calculations address the chance that any two DNA fingerprints would be identical among a large group of cell lines. This estimate is still very slight; for example, the chance of two or more common DNA fingerprints among 1 million distinct individuals is less than .001. The procedure provides a straightforward, easily interpreted, and statistically robust method for identification and individualization of human cells.

Dynamic formation of asexual diploid and polyploid lineages: multilocus analysis of Cobitis reveals the mechanisms maintaining the diversity of clones.

Directory of Open Access Journals (Sweden)

Karel Janko

Full Text Available Given the hybrid genomic constitutions and increased ploidy of many asexual animals, the identification of processes governing the origin and maintenance of clonal diversity provides useful information about the evolutionary consequences of interspecific hybridization, asexuality and polyploidy. In order to understand the processes driving observed diversity of biotypes and clones in the Cobitis taenia hybrid complex, we performed fine-scale genetic analysis of Central European hybrid zone between two sexual species using microsatellite genotyping and mtDNA sequencing. We found that the hybrid zone is populated by an assemblage of clonally (gynogenetically reproducing di-, tri- and tetraploid hybrid lineages and that successful clones, which are able of spatial expansion, recruit from two ploidy levels, i.e. diploid and triploid. We further compared the distribution of observed estimates of clonal ages to theoretical distributions simulated under various assumptions and showed that new clones are most likely continuously recruited from ancestral populations. This suggests that the clonal diversity is maintained by dynamic equilibrium between origination and extinction of clonal lineages. On the other hand, an interclonal selection is implied by nonrandom spatial distribution of individual clones with respect to the coexisting sexual species. Importantly, there was no evidence for sexually reproducing hybrids or clonally reproducing non-hybrid forms. Together with previous successful laboratory synthesis of clonal Cobitis hybrids, our data thus provide the most compelling evidence that 1 the origin of asexuality is causally linked to interspecific hybridization; 2 successful establishment of clones is not restricted to one specific ploidy level and 3 the initiation of clonality and polyploidy may be dynamic and continuous in asexual complexes.

Chromosome numbers and reproductive systems of selected representatives of Pilosella from the Krkonoše Mts (the Sudetes Mts). Part 3

Czech Academy of Sciences Publication Activity Database

Krahulcová, Anna; Krahulec, František; Bräutigam, S.; Chrtek, Jindřich

2013-01-01

Roč. 85, č. 2 (2013), s. 179-192 ISSN 0032-7786 R&D Projects: GA ČR GA206/08/0890; GA ČR GAP506/10/1363 Institutional support: RVO:67985939 Keywords : DNA ploidy level * hybridization * Pilosella Subject RIV: EF - Botanics Impact factor: 2.778, year: 2013

PDA: Pooled DNA analyzer

Directory of Open Access Journals (Sweden)

Lin Chin-Yu

2006-04-01

Full Text Available Abstract Background Association mapping using abundant single nucleotide polymorphisms is a powerful tool for identifying disease susceptibility genes for complex traits and exploring possible genetic diversity. Genotyping large numbers of SNPs individually is performed routinely but is cost prohibitive for large-scale genetic studies. DNA pooling is a reliable and cost-saving alternative genotyping method. However, no software has been developed for complete pooled-DNA analyses, including data standardization, allele frequency estimation, and single/multipoint DNA pooling association tests. This motivated the development of the software, 'PDA' (Pooled DNA Analyzer, to analyze pooled DNA data. Results We develop the software, PDA, for the analysis of pooled-DNA data. PDA is originally implemented with the MATLAB® language, but it can also be executed on a Windows system without installing the MATLAB®. PDA provides estimates of the coefficient of preferential amplification and allele frequency. PDA considers an extended single-point association test, which can compare allele frequencies between two DNA pools constructed under different experimental conditions. Moreover, PDA also provides novel chromosome-wide multipoint association tests based on p-value combinations and a sliding-window concept. This new multipoint testing procedure overcomes a computational bottleneck of conventional haplotype-oriented multipoint methods in DNA pooling analyses and can handle data sets having a large pool size and/or large numbers of polymorphic markers. All of the PDA functions are illustrated in the four bona fide examples. Conclusion PDA is simple to operate and does not require that users have a strong statistical background. The software is available at http://www.ibms.sinica.edu.tw/%7Ecsjfann/first%20flow/pda.htm.

DNA level and stereologic estimates of nuclear volume in squamous cell carcinomas of the uterine cervix. A comparative study with analysis of prognostic impact

DEFF Research Database (Denmark)

Sørensen, Flemming Brandt; Bichel, P; Jakobsen, A

1992-01-01

Grading of malignancy in squamous cell carcinomas of the uterine cervix is based on qualitative, morphologic examination and suffers from poor reproducibility. Using modern stereology, unbiased estimates of the three-dimensional, volume-weighted mean nuclear volume (nuclear vv), were obtained...... in pretreatment biopsies from 51 patients treated for cervical cancer in clinical Stages I through III (mean age of 56 years, follow-up period greater than 5 years). In addition, conventional, two-dimensional morphometric estimates of nuclear and mitotic features were obtained. DNA indices (DI) were estimated...

Scaffolded DNA origami of a DNA tetrahedron molecular container.

Science.gov (United States)

Ke, Yonggang; Sharma, Jaswinder; Liu, Minghui; Jahn, Kasper; Liu, Yan; Yan, Hao

2009-06-01

We describe a strategy of scaffolded DNA origami to design and construct 3D molecular cages of tetrahedron geometry with inside volume closed by triangular faces. Each edge of the triangular face is approximately 54 nm in dimension. The estimated total external volume and the internal cavity of the triangular pyramid are about 1.8 x 10(-23) and 1.5 x 10(-23) m(3), respectively. Correct formation of the tetrahedron DNA cage was verified by gel electrophoresis, atomic force microscopy, transmission electron microscopy, and dynamic light scattering techniques.

Scaffolded DNA Origami of a DNA Tetrahedron Molecular Container

DEFF Research Database (Denmark)

Ke, Yongang; Sharma, Jaswinder; Liu, Minghui

2009-01-01

We describe a strategy of scaffolded DNA origami to design and construct 3D molecular cages of tetrahedron geometry with inside volume closed by triangular faces. Each edge of the triangular face is ∼54 nm in dimension. The estimated total external volume and the internal cavity of the triangular...... pyramid are about 1.8 × 10-23 and 1.5 × 10-23 m3, respectively. Correct formation of the tetrahedron DNA cage was verified by gel electrophoresis, atomic force microscopy, transmission electron microscopy, and dynamic light scattering techniques....

DNA level and stereologic estimates of nuclear volume in squamous cell carcinomas of the uterine cervix. A comparative study with analysis of prognostic impact

DEFF Research Database (Denmark)

Sørensen, Flemming Brandt; Bichel, P; Jakobsen, A

1992-01-01

Grading of malignancy in squamous cell carcinomas of the uterine cervix is based on qualitative, morphologic examination and suffers from poor reproducibility. Using modern stereology, unbiased estimates of the three-dimensional, volume-weighted mean nuclear volume (nuclear vv), were obtained...... in pretreatment biopsies from 51 patients treated for cervical cancer in clinical Stages I through III (mean age of 56 years, follow-up period greater than 5 years). In addition, conventional, two-dimensional morphometric estimates of nuclear and mitotic features were obtained. DNA indices (DI) were estimated...... carcinoma of the uterine cervix....

The practical analysis of food: the development of Sakalar quantification table of DNA (SQT-DNA).

Science.gov (United States)

Sakalar, Ergün

2013-11-15

Practical and highly sensitive Sakalar quantification table of DNA (SQT-DNA) has been developed for the detection% of species-specific DNA amount in food products. Cycle threshold (Ct) data were obtained from multiple curves of real-time qPCR. The statistical analysis was done to estimate the concentration of standard dilutions. Amplicon concentrations versus each Ct value were assessed by the predictions of targets at known concentrations. SQT-DNA was prepared by using the percentage versus each Ct values. The applicability of SQT-DNA to commercial foods was proved by using sausages containing varying ratios of beef, chicken, and soybean. The results showed that SQT-DNA can be used to directly quantify food DNA by a single PCR without the need to construct a standart curve in parallel with the samples every time the experiment is performed, and also quantification by SQT-DNA is as reliable as standard curve quantification for a wide range of DNA concentrations. Copyright © 2013 Elsevier Ltd. All rights reserved.

Estimates of Continental Ancestry Vary Widely among Individuals with the Same mtDNA Haplogroup

Science.gov (United States)

Emery, Leslie S.; Magnaye, Kevin M.; Bigham, Abigail W.; Akey, Joshua M.; Bamshad, Michael J.

2015-01-01

The association between a geographical region and an mtDNA haplogroup(s) has provided the basis for using mtDNA haplogroups to infer an individual’s place of origin and genetic ancestry. Although it is well known that ancestry inferences using mtDNA haplogroups and those using genome-wide markers are frequently discrepant, little empirical information exists on the magnitude and scope of such discrepancies between multiple mtDNA haplogroups and worldwide populations. We compared genetic-ancestry inferences made by mtDNA-haplogroup membership to those made by autosomal SNPs in ∼940 samples of the Human Genome Diversity Panel and recently admixed populations from the 1000 Genomes Project. Continental-ancestry proportions often varied widely among individuals sharing the same mtDNA haplogroup. For only half of mtDNA haplogroups did the highest average continental-ancestry proportion match the highest continental-ancestry proportion of a majority of individuals with that haplogroup. Prediction of an individual’s mtDNA haplogroup from his or her continental-ancestry proportions was often incorrect. Collectively, these results indicate that for most individuals in the worldwide populations sampled, mtDNA-haplogroup membership provides limited information about either continental ancestry or continental region of origin. PMID:25620206

Ploidy-Dependent Unreductional Meiotic Cell Division in Polyploid Wheat

Science.gov (United States)

Meiosis includes one round of DNA replication and two successive nuclear divisions, i.e. meiosis I (reductional) and meiosis II (equational). This specialized cell division reduces chromosomes in half and generates haploid gametes in sexual reproduction of eukaryotes. It ensures faithful transmiss...

DNA methylation results depend on DNA integrity – role of post mortem interval

Directory of Open Access Journals (Sweden)

Mathias eRhein

2015-05-01

Full Text Available Major questions of neurological and psychiatric mechanisms involve the brain functions on a molecular level and cannot be easily addressed due to limitations in access to tissue samples. Post mortem studies are able to partly bridge the gap between brain tissue research retrieved from animal trials and the information derived from peripheral analysis (e.g. measurements in blood cells in patients. Here, we wanted to know how fast DNA degradation is progressing under controlled conditions in order to define thresholds for tissue quality to be used in respective trials. Our focus was on the applicability of partly degraded samples for bisulfite sequencing and the determination of simple means to define cut-off values.After opening the brain cavity, we kept two consecutive pig skulls at ambient temperature (19-21°C and removed cortex tissue up to a post mortem interval (PMI of 120h. We calculated the percentage of degradation on DNA gel electrophoresis of brain DNA to estimate quality and relate this estimation spectrum to the quality of human post-mortem control samples. Functional DNA quality was investigated by bisulfite sequencing of two functionally relevant genes for either the serotonin receptor 5 (SLC6A4 or aldehyde dehydrogenase 2 (ALDH2.Testing our approach in a heterogeneous collective of human blood and brain samples, we demonstrate integrity of measurement quality below the threshold of 72h PMI.While sequencing technically worked for all timepoints irrespective of conceivable DNA degradation, there is a good correlation between variance of methylation to degradation levels documented in the gel (R2=0.4311, p=0.0392 for advancing post mortem intervals (PMI. This otherwise elusive phenomenon is an important prerequisite for the interpretation and evaluation of samples prior to in-depth processing via an affordable and easy assay to estimate identical sample quality and thereby comparable methylation measurements.

Sedimentation properties of DNA-membrane complexes and yield of DNA breaks at irradiation of mammalian cells

International Nuclear Information System (INIS)

Erzgraber, G.; Kozubek, S.; Lapidus, I.L.

1985-01-01

The dependence of the relative sedimentation velocity of DNA-membrane complexes on the dose of irradiation and time of incubation of Chinese Hamster cells is analysed. It is concluded that the initial part of the curve provides the information on the occurrence of single strand breaks in DNA; the position of the local maximum allows us to calculate the yield of DNA double strand breaks. The reparation decay constant can be estimated as well

Reproduction at the extremes: pseudovivipary, hybridization and genetic mosaicism in Posidonia australis (Posidoniaceae).

Science.gov (United States)

Sinclair, Elizabeth A; Statton, John; Hovey, Renae; Anthony, Janet M; Dixon, Kingsley W; Kendrick, Gary A

2016-02-01

Organisms occupying the edges of natural geographical ranges usually survive at the extreme limits of their innate physiological tolerances. Extreme and prolonged fluctuations in environmental conditions, often associated with climate change and exacerbated at species' geographical range edges, are known to trigger alternative responses in reproduction. This study reports the first observations of adventitious inflorescence-derived plantlet formation in the marine angiosperm Posidonia australis, growing at the northern range edge (upper thermal and salinity tolerance) in Shark Bay, Western Australia. These novel plantlets are described and a combination of microsatellite DNA markers and flow cytometry is used to determine their origin. Polymorphic microsatellite DNA markers were used to generate multilocus genotypes to determine the origin of the adventitious inflorescence-derived plantlets. Ploidy and genome size were estimated using flow cytometry. All adventitious plantlets were genetically identical to the maternal plant and were therefore the product of a novel pseudoviviparous reproductive event. It was found that 87 % of the multilocus genotypes contained three alleles in at least one locus. Ploidy was identical in all sampled plants. The genome size (2 C value) for samples from Shark Bay and from a separate site much further south was not significantly different, implying they are the same ploidy level and ruling out a complete genome duplication (polyploidy). Survival at range edges often sees the development of novel responses in the struggle for survival and reproduction. This study documents a physiological response at the trailing edge, whereby reproductive strategy can adapt to fluctuating conditions and suggests that the lower-than-usual water temperature triggered unfertilized inflorescences to 'switch' to growing plantlets that were adventitious clones of their maternal parent. This may have important long-term implications as both genetic and

Assessing environmental DNA detection in controlled lentic systems.

Science.gov (United States)

Moyer, Gregory R; Díaz-Ferguson, Edgardo; Hill, Jeffrey E; Shea, Colin

2014-01-01

Little consideration has been given to environmental DNA (eDNA) sampling strategies for rare species. The certainty of species detection relies on understanding false positive and false negative error rates. We used artificial ponds together with logistic regression models to assess the detection of African jewelfish eDNA at varying fish densities (0, 0.32, 1.75, and 5.25 fish/m3). Our objectives were to determine the most effective water stratum for eDNA detection, estimate true and false positive eDNA detection rates, and assess the number of water samples necessary to minimize the risk of false negatives. There were 28 eDNA detections in 324, 1-L, water samples collected from four experimental ponds. The best-approximating model indicated that the per-L-sample probability of eDNA detection was 4.86 times more likely for every 2.53 fish/m3 (1 SD) increase in fish density and 1.67 times less likely for every 1.02 C (1 SD) increase in water temperature. The best section of the water column to detect eDNA was the surface and to a lesser extent the bottom. Although no false positives were detected, the estimated likely number of false positives in samples from ponds that contained fish averaged 3.62. At high densities of African jewelfish, 3-5 L of water provided a >95% probability for the presence/absence of its eDNA. Conversely, at moderate and low densities, the number of water samples necessary to achieve a >95% probability of eDNA detection approximated 42-73 and >100 L, respectively. Potential biases associated with incomplete detection of eDNA could be alleviated via formal estimation of eDNA detection probabilities under an occupancy modeling framework; alternatively, the filtration of hundreds of liters of water may be required to achieve a high (e.g., 95%) level of certainty that African jewelfish eDNA will be detected at low densities (i.e., <0.32 fish/m3 or 1.75 g/m3).

Cdc14 phosphatase directs centrosome re-duplication at the meiosis I to meiosis II transition in budding yeast [version 2; referees: 3 approved, 1 approved with reservations

Directory of Open Access Journals (Sweden)

Colette Fox

2017-02-01

Full Text Available Background Gametes are generated through a specialized cell division called meiosis, in which ploidy is reduced by half because two consecutive rounds of chromosome segregation, meiosis I and meiosis II, occur without intervening DNA replication. This contrasts with the mitotic cell cycle where DNA replication and chromosome segregation alternate to maintain the same ploidy. At the end of mitosis, cyclin-dependent kinases (CDKs are inactivated. This low CDK state in late mitosis/G1 allows for critical preparatory events for DNA replication and centrosome/spindle pole body (SPB duplication. However, their execution is inhibited until S phase, where further preparatory events are also prevented. This “licensing” ensures that both the chromosomes and the centrosomes/SPBs replicate exactly once per cell cycle, thereby maintaining constant ploidy. Crucially, between meiosis I and meiosis II, centrosomes/SPBs must be re-licensed, but DNA re-replication must be avoided. In budding yeast, the Cdc14 protein phosphatase triggers CDK down regulation to promote exit from mitosis. Cdc14 also regulates the meiosis I to meiosis II transition, though its mode of action has remained unclear. Methods Fluorescence and electron microscopy was combined with proteomics to probe SPB duplication in cells with inactive or hyperactive Cdc14. Results We demonstrate that Cdc14 ensures two successive nuclear divisions by re-licensing SPBs at the meiosis I to meiosis II transition. We show that Cdc14 is asymmetrically enriched on a single SPB during anaphase I and provide evidence that this enrichment promotes SPB re-duplication. Cells with impaired Cdc14 activity fail to promote extension of the SPB half-bridge, the initial step in morphogenesis of a new SPB. Conversely, cells with hyper-active Cdc14 duplicate SPBs, but fail to induce their separation. Conclusion Our findings implicate reversal of key CDK-dependent phosphorylations in the differential licensing of

Risk assessment of DNA-reactive carcinogens in food

International Nuclear Information System (INIS)

Jeffrey, A.M.; Williams, G.M.

2005-01-01

Risk assessment of DNA-reactive carcinogens in food requires knowledge of the extent of DNA damage in the target organ which results from the competition between DNA adduct formation and repair. Estimates of DNA adduct levels can be made by direct measurement or indirectly as a consequence of their presence, for example, by tumor formation in animal models or exposed populations epidemiologically. Food-borne DNA-reactive carcinogens are present from a variety of sources. They are generally not intrinsically DNA-reactive but require bioactivation to DNA-reactive metabolites a process which may be modulated by the compound itself or the presence of other xenobiotics. A single DNA reactant may form several distinct DNA adducts each undergoing different rates of repair. Some DNA reactants may be photochemically activated or produce reactive oxygen species and thus indirect oxidative DNA damage. The levels of DNA adducts arising from exposures influenced by variations in the doses, the frequency with which an individual is exposed, and rates of DNA repair for specific adducts. Each adduct has a characteristic efficiency with which it induces mutations. Based on experience with the well-studied DNA-reactive food carcinogen aflatoxin B 1 (AFB 1 ), a limit of 20 ppb or ∼30 μg/day has been set and is considered a tolerable daily intake (TDI). Since AFB 1 is considered a potent carcinogen, doses of 32 P-postlabeling or the use of surrogates such as hemoglobin adducts, together with approaches to evaluate the results. A discussion of approaches to estimating possible threshold effects for DNA-reactive carcinogens is made

DNA-based hair sampling to identify road crossings and estimate population size of black bears in Great Dismal Swamp National Wildlife Refuge, Virginia

OpenAIRE

Wills, Johnny

2008-01-01

The planned widening of U.S. Highway 17 along the east boundary of Great Dismal Swamp National Wildlife Refuge (GDSNWR) and a lack of knowledge about the refugeâ s bear population created the need to identify potential sites for wildlife crossings and estimate the size of the refugeâ s bear population. I collected black bear hair in order to collect DNA samples to estimate population size, density, and sex ratio, and determine road crossing locations for black bears (Ursus americanus) in G...

Mediterranean species of Caulerpa are polyploid with smaller genomes in the invasive ones.

Directory of Open Access Journals (Sweden)

Elena Varela-Álvarez

Full Text Available Caulerpa species are marine green algae, which often act as invasive species with rapid clonal proliferation when growing outside their native biogeographical borders. Despite many publications on the genetics and ecology of Caulerpa species, their life history and ploidy levels are still to be resolved and are the subject of large controversy. While some authors claimed that the thallus found in nature has a haplodiplobiontic life cycle with heteromorphic alternation of generations, other authors claimed a diploid or haploid life cycle with only one generation involved. DAPI-staining with image analysis and microspectrophotometry were used to estimate relative nuclear DNA contents in three species of Caulerpa from the Mediterranean, at individual, population and species levels. Results show that ploidy levels and genome size vary in these three Caulerpa species, with a reduction in genome size for the invasive ones. Caulerpa species in the Mediterranean are polyploids in different life history phases; all sampled C. taxifolia and C. racemosa var. cylindracea were in haplophasic phase, but in C. prolifera, the native species, individuals were found in both diplophasic and haplophasic phases. Different levels of endopolyploidy were found in both C. prolifera and C. racemosa var. cylindracea. Life history is elucidated for the Mediterranean C. prolifera and it is hypothesized that haplophasic dominance in C. racemosa var. cylindracea and C. taxifolia is a beneficial trait for their invasive strategies.

Condensin II mutation causes T-cell lymphoma through tissue-specific genome instability

Science.gov (United States)

Woodward, Jessica; Taylor, Gillian C.; Soares, Dinesh C.; Boyle, Shelagh; Sie, Daoud; Read, David; Chathoth, Keerthi; Vukovic, Milica; Tarrats, Nuria; Jamieson, David; Campbell, Kirsteen J.; Blyth, Karen; Acosta, Juan Carlos; Ylstra, Bauke; Arends, Mark J.; Kranc, Kamil R.; Jackson, Andrew P.; Bickmore, Wendy A.

2016-01-01

Chromosomal instability is a hallmark of cancer, but mitotic regulators are rarely mutated in tumors. Mutations in the condensin complexes, which restructure chromosomes to facilitate segregation during mitosis, are significantly enriched in cancer genomes, but experimental evidence implicating condensin dysfunction in tumorigenesis is lacking. We report that mice inheriting missense mutations in a condensin II subunit (Caph2nes) develop T-cell lymphoma. Before tumors develop, we found that the same Caph2 mutation impairs ploidy maintenance to a different extent in different hematopoietic cell types, with ploidy most severely perturbed at the CD4+CD8+ T-cell stage from which tumors initiate. Premalignant CD4+CD8+ T cells show persistent catenations during chromosome segregation, triggering DNA damage in diploid daughter cells and elevated ploidy. Genome sequencing revealed that Caph2 single-mutant tumors are near diploid but carry deletions spanning tumor suppressor genes, whereas P53 inactivation allowed Caph2 mutant cells with whole-chromosome gains and structural rearrangements to form highly aggressive disease. Together, our data challenge the view that mitotic chromosome formation is an invariant process during development and provide evidence that defective mitotic chromosome structure can promote tumorigenesis. PMID:27737961

Use of DNA markers in forest tree improvement research

Science.gov (United States)

D.B. Neale; M.E. Devey; K.D. Jermstad; M.R. Ahuja; M.C. Alosi; K.A. Marshall

1992-01-01

DNA markers are rapidly being developed for forest trees. The most important markers are restriction fragment length polymorphisms (RFLPs), polymerase chain reaction- (PCR) based markers such as random amplified polymorphic DNA (RAPD), and fingerprinting markers. DNA markers can supplement isozyme markers for monitoring tree improvement activities such as; estimating...

Comments on mutagenesis risk estimation

International Nuclear Information System (INIS)

Russell, W.L.

1976-01-01

Several hypotheses and concepts have tended to oversimplify the problem of mutagenesis and can be misleading when used for genetic risk estimation. These include: the hypothesis that radiation-induced mutation frequency depends primarily on the DNA content per haploid genome, the extension of this concept to chemical mutagenesis, the view that, since DNA is DNA, mutational effects can be expected to be qualitatively similar in all organisms, the REC unit, and the view that mutation rates from chronic irradiation can be theoretically and accurately predicted from acute irradiation data. Therefore, direct determination of frequencies of transmitted mutations in mammals continues to be important for risk estimation, and the specific-locus method in mice is shown to be not as expensive as is commonly supposed for many of the chemical testing requirements

Capturing a DNA duplex under near-physiological conditions

Science.gov (United States)

Zhang, Huijuan; Xu, Wei; Liu, Xiaogang; Stellacci, Francesco; Thong, John T. L.

2010-10-01

We report in situ trapping of a thiolated DNA duplex with eight base pairs into a polymer-protected gold nanogap device under near-physiological conditions. The double-stranded DNA was captured by electrophoresis and covalently attached to the nanogap electrodes through sulfur-gold bonding interaction. The immobilization of the DNA duplex was confirmed by direct electrical measurements under near-physiological conditions. The conductance of the DNA duplex was estimated to be 0.09 μS. We also demonstrate the control of DNA dehybridization by heating the device to temperatures above the melting point of the DNA.

Quantifying DNA melting transitions using single-molecule force spectroscopy

International Nuclear Information System (INIS)

Calderon, Christopher P; Chen, W-H; Harris, Nolan C; Kiang, C-H; Lin, K-J

2009-01-01

We stretched a DNA molecule using an atomic force microscope (AFM) and quantified the mechanical properties associated with B and S forms of double-stranded DNA (dsDNA), molten DNA, and single-stranded DNA. We also fit overdamped diffusion models to the AFM time series and used these models to extract additional kinetic information about the system. Our analysis provides additional evidence supporting the view that S-DNA is a stable intermediate encountered during dsDNA melting by mechanical force. In addition, we demonstrated that the estimated diffusion models can detect dynamical signatures of conformational degrees of freedom not directly observed in experiments.

Quantifying DNA melting transitions using single-molecule force spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Calderon, Christopher P [Department of Computational and Applied Mathematics, Rice University, Houston, TX (United States); Chen, W-H; Harris, Nolan C; Kiang, C-H [Department of Physics and Astronomy, Rice University, Houston, TX (United States); Lin, K-J [Department of Chemistry, National Chung Hsing University, Taichung, Taiwan (China)], E-mail: chkiang@rice.edu

2009-01-21

We stretched a DNA molecule using an atomic force microscope (AFM) and quantified the mechanical properties associated with B and S forms of double-stranded DNA (dsDNA), molten DNA, and single-stranded DNA. We also fit overdamped diffusion models to the AFM time series and used these models to extract additional kinetic information about the system. Our analysis provides additional evidence supporting the view that S-DNA is a stable intermediate encountered during dsDNA melting by mechanical force. In addition, we demonstrated that the estimated diffusion models can detect dynamical signatures of conformational degrees of freedom not directly observed in experiments.

Environmental DNA (eDNA) Detection Probability Is Influenced by Seasonal Activity of Organisms.

Science.gov (United States)

de Souza, Lesley S; Godwin, James C; Renshaw, Mark A; Larson, Eric

2016-01-01

Environmental DNA (eDNA) holds great promise for conservation applications like the monitoring of invasive or imperiled species, yet this emerging technique requires ongoing testing in order to determine the contexts over which it is effective. For example, little research to date has evaluated how seasonality of organism behavior or activity may influence detection probability of eDNA. We applied eDNA to survey for two highly imperiled species endemic to the upper Black Warrior River basin in Alabama, US: the Black Warrior Waterdog (Necturus alabamensis) and the Flattened Musk Turtle (Sternotherus depressus). Importantly, these species have contrasting patterns of seasonal activity, with N. alabamensis more active in the cool season (October-April) and S. depressus more active in the warm season (May-September). We surveyed sites historically occupied by these species across cool and warm seasons over two years with replicated eDNA water samples, which were analyzed in the laboratory using species-specific quantitative PCR (qPCR) assays. We then used occupancy estimation with detection probability modeling to evaluate both the effects of landscape attributes on organism presence and season of sampling on detection probability of eDNA. Importantly, we found that season strongly affected eDNA detection probability for both species, with N. alabamensis having higher eDNA detection probabilities during the cool season and S. depressus have higher eDNA detection probabilities during the warm season. These results illustrate the influence of organismal behavior or activity on eDNA detection in the environment and identify an important role for basic natural history in designing eDNA monitoring programs.

Effects of ionizing radiations on DNA replication in cultured mammalian cells

International Nuclear Information System (INIS)

Makino, F.; Okada, S.

1975-01-01

The dose-response curve of [ 3 H] thymidine incorporation into the acid-insoluble fraction of cultured mammalian cells, grown in the presence of 10 -4 M cold thymidine, is different from that of incorporation in the absence of cold thymidine. For quantitative estimation of net DNA synthesis in nonirradiated and irradiated cells, two methods were used: isolation of newly synthesized BUdR-labeled DNA by CsCl gradient centrifugation and a fluorometric estimation of DNA content in the synchronized population. Both methods showed that the depression of [ 3 H]thymidine incorporation in the presence of cold thymidine reflected a depression of net DNA synthesis. Radiosensitive steps in DNA synthesis were examined by the use of alkaline sucrose gradient centrifugation. The rate of replication along the DNA strands was inhibited to a lesser extent than that of over-all DNA synthesis. The labeling patterns of DNA exposed to [ 3 H]thymidine for 20 min indicated that ionizing radiation preferentially interfered with the formation of small-size 3 H-labeled DNA pieces. These results suggest that the initiation of DNA replication is more radiosensitive than the elongation of DNA strands whose replication has already been initiated. (U.S.)

Radiosensitivity of mesothelioma cell lines

International Nuclear Information System (INIS)

Haekkinen, A.M.; Laasonen, A.; Linnainmaa, K.; Mattson, K.; Pyrhoenen, S.

1996-01-01

The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters α and β of the linear quadratic model (LQ-model) and mean inactivation dose (D MID ) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean α value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The α/β ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.)

Radiosensitivity of mesothelioma cell lines

Energy Technology Data Exchange (ETDEWEB)

Haekkinen, A.M. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland); Laasonen, A. [Dept. of Pathology, Central Hospital of Etelae-Pohjanmaa, Seinaejoki (Finland); Linnainmaa, K. [Dept. of Industrial Hygiene and Toxicology, Inst. of Occupational Health, Helsinki (Finland); Mattson, K. [Dept. Pulmonary Medicine, Univ. Central Hospital, Helsinki (Finland); Pyrhoenen, S. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland)

1996-10-01

The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters {alpha} and {beta} of the linear quadratic model (LQ-model) and mean inactivation dose (D{sub MID}) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean {alpha} value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The {alpha}/{beta} ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.).

Bacterial diversity in a soil sample from Uranium mining waste pile as estimated via a culture-independent 16S rDNA approach

International Nuclear Information System (INIS)

Satchanska, G.; Golovinsky, E.; Selenska-Pobell, S.

2004-01-01

Bacterial diversity was studied in a soil sample collected from a uranium mining waste pile situated near the town of Johanngeorgenstadt, Germany. As estimated by ICP-MS analysis the studied sample was highly contaminated with Fe, Al, Mn, Zn, As, Pb and U. The 16S rDNA retrieval, applied in this study, demonstrated that more than the half of the clones of the constructed 16S rDNA library were represented by individual RFLP profiles. This indicates that the composition of the bacterial community in the sample was very complex. However, several 16S rDNA RFLP groups were found to be predominant and they were subjected to a sequence analysis. The most predominant group, which represented about 13% of the clones of the 16S rDNA library, was affiliated with the Holophaga/Acidobacterium phylum. Significant was also the number of the proteobacterial sequences which were distributed in one predominant α-proteobacterial cluster representing 11% of the total number of clones and in two equal-sized β- and γ-proteobacterial clusters representing each 6% of the clones. Two smaller groups representing both 2% of the clones were affiliated with Nitrospira and with the novel division WS3. Three of the analysed sequences were evaluated as a novel, not yet described lineage and one as a putative chimera. (authors)

Cross-linking and relaxation of supercoiled DNA by psoralen and light

International Nuclear Information System (INIS)

Yoakum, G.H.; Cole, R.S.

1978-01-01

Photoreaction of 4,5',8-trimethylpsoralen with superhelical ColE1 and ColE1amp DNA was studied. Changes in mobilities in agarose gels, formation of interstrand cross-links, and DNA strand breaks were determined. Psoralen and light treatment removed negative superhelical turns, and extensive treatments failed to produce positive superhelical turns in covalently closed plasmid DNA. The rate of relaxation of superhelical turns by psoralen photobinding appeared to be directly proportional to the number of superhelical turns remaining. A unique reaction mechanism is presented to explain these results. By this interpretation the initial rate of psoralen photobinding to superhelical DNA was estimated to be 3 times that for linear DNA, and the ratio of cross-linking to monofunctional adducts appears to be dependent on the superhelical conformation of the DNA. The estimated ratio of psoralen molecules bound to DNA strand breaks was 1.7 . 10 4 :1, and 70% of this breakage is caused by the light alone. (Auth.)

Carcinogen-induced damage to DNA

International Nuclear Information System (INIS)

Strauss, B.; Altamirano, M.; Bose, K.; Sklar, R.; Tatsumi, K.

1979-01-01

Human cells respond to carcinogen-induced damage in their DNA in at least two ways. The first response, excision repair, proceeds by at least three variations, depending on the nature of the damage. Nucleotide excision results in relatively large repair patches but few free DNA breaks, since the endonuclease step is limiting. Apurinic repair is characterized by the appearance of numerous breaks in the DNA and by short repair patches. The pathways behave as though they function independently. Lymphoic cells derived from a xeroderma pigmentosum complementation group C patient are deficient in their ability to perform nucleotide excision and also to excise 6 methoxyguanine adducts, but they are apurinic repair competent. Organisms may bypass damage in their DNA. Lymphoblastoid cells, including those derived from xeroderma pigmentosum treated with 3 H-anti-BPDE, can replicate their DNA at low doses of carcinogen. Unexcised 3 H is found in the light or parental strand of the resulting hybrid DNA when replication occurs in medium with BrdUrd. This observation indicates a bypass reaction occurring by a mechanism involving branch migration at DNA growing points. Branch migration in DNA preparations have been observed, but the evidence is that most occurs in BrdUrd-containing DNA during cell lysis. The measurement of the bifilarly substituted DNA resulting from branch migration is a convenient method of estimating the proportion of new synthesis remaining in the vicinity of the DNA growing point. Treatment with carcinogens or caffeine results in accumulation of DNA growing points accompanied by the synthesis of shortened pieces of daughter DNA

DNA Duplex Length and Salt Concentration Dependence of Enthalpy−Entropy Compensation Parameters for DNA Melting

KAUST Repository

Starikov, E. B.

2009-08-20

Systematical differential calorimetry experiments on DNA oligomers with different lengths and placed in water solutions with various added salt concentrations may, in principle, unravel important information about the structure and dynamics of the DNA and their water-counterion surrounding. With this in mind, to reinterpret the most recent results of calorimetric experiments on DNA oligomers of such a kind, the recent enthalpy-entropy compensation theory has been used. It is demonstrated that the application of the latter could enable direct estimation of thermodynamic parameters of the microphase transitions connected to the changes in DNA dynamical regimes versus the length of the biopolymers and the ionic strengths of their water solutions, and this calls for much more systematical experimental and theoretical studies in this field. © 2009 American Chemical Society.

Genome size evolution in Ontario ferns (Polypodiidae): evolutionary correlations with cell size, spore size, and habitat type and an absence of genome downsizing.

Science.gov (United States)

Henry, Thomas A; Bainard, Jillian D; Newmaster, Steven G

2014-10-01

Genome size is known to correlate with a number of traits in angiosperms, but less is known about the phenotypic correlates of genome size in ferns. We explored genome size variation in relation to a suite of morphological and ecological traits in ferns. Thirty-six fern taxa were collected from wild populations in Ontario, Canada. 2C DNA content was measured using flow cytometry. We tested for genome downsizing following polyploidy using a phylogenetic comparative analysis to explore the correlation between 1Cx DNA content and ploidy. There was no compelling evidence for the occurrence of widespread genome downsizing during the evolution of Ontario ferns. The relationship between genome size and 11 morphological and ecological traits was explored using a phylogenetic principal component regression analysis. Genome size was found to be significantly associated with cell size, spore size, spore type, and habitat type. These results are timely as past and recent studies have found conflicting support for the association between ploidy/genome size and spore size in fern polyploid complexes; this study represents the first comparative analysis of the trend across a broad taxonomic group of ferns.

Prognostic value of flow cytometry in surgically treated primary gastric lymphoma Valor pronóstico de la citometría de flujo en el linfoma gástrico

Directory of Open Access Journals (Sweden)

F. Fernández

2006-11-01

Full Text Available Aim: to investigate whether flow cytometry could help to define the optimal therapeutic strategy of primary gastric lymphomas. Material and method: retrospective study of 46 patients having primary gastric lymphoma -according to Dawson criteria- in Ann Arbor stage I E and II E, who were surgically treated. From selected paraffin-embedded tissue blocks of the tumor, DNA content was studied by flow cytometry (FC. Other pathological tumor features were analysed by hematoxiline-eosine and Giemsa stains as well as immunohistochemical study; any possible influence on postoperative survival was investigated through statistical analysis. Results: the DNA ploidy pattern was diploid in 40 cases (87% and aneuploid (hyperdiploid in 6 (13%. Postoperative survival probability (PSP was 62.7% at 5 years. Statistical analysis showed significant prognostic value for Ann Arbor classification -with higher PSP for stage I E (p = 0.009- and FC parameters: diploid tumors had higher PSP than aneuploid tumors. Also tumors having S-phase (p = 0.044 or G2-M phase values (p = 0.023 under the respective mean values had higher PSP. No influence on PSP was found for wall invasion, Helicobacter pylori infection, Isaacson's histologic type or resection margin involvement. No significant relationship was appreciated between Isaacson's histologic type and DNA ploidy patterns. Conclusion: FC could be useful in assessing gastric lymphoma prognosis.

Cooperative DNA Recognition Modulated by an Interplay between Protein-Protein Interactions and DNA-Mediated Allostery.

Directory of Open Access Journals (Sweden)

Felipe Merino

2015-06-01

Full Text Available Highly specific transcriptional regulation depends on the cooperative association of transcription factors into enhanceosomes. Usually, their DNA-binding cooperativity originates from either direct interactions or DNA-mediated allostery. Here, we performed unbiased molecular simulations followed by simulations of protein-DNA unbinding and free energy profiling to study the cooperative DNA recognition by OCT4 and SOX2, key components of enhanceosomes in pluripotent cells. We found that SOX2 influences the orientation and dynamics of the DNA-bound configuration of OCT4. In addition SOX2 modifies the unbinding free energy profiles of both DNA-binding domains of OCT4, the POU specific and POU homeodomain, despite interacting directly only with the first. Thus, we demonstrate that the OCT4-SOX2 cooperativity is modulated by an interplay between protein-protein interactions and DNA-mediated allostery. Further, we estimated the change in OCT4-DNA binding free energy due to the cooperativity with SOX2, observed a good agreement with experimental measurements, and found that SOX2 affects the relative DNA-binding strength of the two OCT4 domains. Based on these findings, we propose that available interaction partners in different biological contexts modulate the DNA exploration routes of multi-domain transcription factors such as OCT4. We consider the OCT4-SOX2 cooperativity as a paradigm of how specificity of transcriptional regulation is achieved through concerted modulation of protein-DNA recognition by different types of interactions.

Targeted and Untargeted Lipidomics of Emiliania huxleyi Viral Infection and Life Cycle Phases Highlights Molecular Biomarkers of Infection, Susceptibility, and Ploidy

Directory of Open Access Journals (Sweden)

Jonathan Eliott Hunter

2015-10-01

Full Text Available Marine viruses that infect phytoplankton strongly influence the ecology and evolution of their hosts. Emiliania huxleyi is characterized by a biphasic life cycle composed of a diploid (2N and haploid (1N phase; diploid cells are susceptible to infection by specific coccolithoviruses, yet haploid cells are resistant. Glycosphingolipids (GSLs play a role during infection, but their molecular distribution in haploid cells is unknown. We present mass spectrometric analyses of lipids from cultures of uninfected diploid, infected diploid, and uninfected haploid E. huxleyi. Known viral GSLs were present in the infected diploid cultures as expected, but surprisingly, trace amounts of viral GSLs were also detected in the uninfected haploid cells. Sialic-acid GSLs have been linked to viral susceptibility in diploid cells, but were found to be absent in the haploid cultures, suggesting a mechanism of haploid resistance to infection. Additional untargeted high-resolution mass spectrometry data processed via multivariate analysis unveiled a number of novel biomarkers of infected, non-infected, and haploid cells. These data expand our understanding on the dynamics of lipid metabolism during E. huxleyi host/virus interactions and highlight potential novel biomarkers for infection, susceptibility, and ploidy.

Cell proliferation and DNA dependent DNA polymerase estimation in acute lymphoblastic leukaemia during treatment with prednisone and vincristine

International Nuclear Information System (INIS)

Lange Wantzin, G.

1979-01-01

The presence of DNA polymerase and primer-template DNA in lymphoblast nuclei by measuring the in vitro incorporation of 3 H-thymidine-5'-triphosphate ( 3 H-TTP) was studied in 10 patients with acute lymphoblastic leukemia. Protein synthesis and various other cytokinetic parameters were also studied. After prednisone (P) administration a marked decrease in 3 H-TTP labelling index ( 3 H-TTP LI) was apparent together with an inhibition of 3 H-leucine incorporation ( 3 H-LEU LI) into lymphoblasts. A moderate decrease in 3 H-TDR labelling index ( 3 H-TDR LI) and a later decrease in mitotic index (MI) were seen. Single cell DNA measurements showed a depletion of 3 H-TDR labelled lymphoblasts in early part of S-phase apparent at 24 h lasting up to 54 h after P administration. Vincristine given as a flash injection later in the study period caused an immediate rise of the MI, at the same time the P induced decline in 3 H-TTP LI, 3 H-TDR LI and 3 H-LEU LI were continued in most patients. P is thought to damage the cells both in and outside the cell cycle. In the cell cycle the effect of P is an arresting effect in G 1 . (author)

Mutant DNA quantification by digital PCR can be confounded by heating during DNA fragmentation.

Science.gov (United States)

Kang, Qing; Parkin, Brian; Giraldez, Maria D; Tewari, Muneesh

2016-04-01

Digital PCR (dPCR) is gaining popularity as a DNA mutation quantification method for clinical specimens. Fragmentation prior to dPCR is required for non-fragmented genomic DNA samples; however, the effect of fragmentation on DNA analysis has not been well-studied. Here we evaluated three fragmentation methods for their effects on dPCR point mutation assay performance. Wild-type (WT) human genomic DNA was fragmented by heating, restriction digestion, or acoustic shearing using a Covaris focused-ultrasonicator. dPCR was then used to determine the limit of blank (LoB) by quantifying observed WT and mutant allele counts of the proto-oncogenes KRAS and BRAF in the WT DNA sample. DNA fragmentation by heating to 95°C, while the simplest and least expensive method, produced a high background mutation frequency for certain KRAS mutations relative to the other methods. This was due to heat-induced mutations, specifically affecting dPCR assays designed to interrogate guanine to adenine (G>A) mutations. Moreover, heat-induced fragmentation overestimated gene copy number, potentially due to denaturation and partition of single-stranded DNA into different droplets. Covaris acoustic shearing and restriction enzyme digestion showed similar LoBs and gene copy number estimates to one another. It should be noted that moderate heating, commonly used in genomic DNA extraction protocols, did not significantly increase observed KRAS mutation counts.

Extraction of ultrashort DNA molecules from herbarium specimens.

Science.gov (United States)

Gutaker, Rafal M; Reiter, Ella; Furtwängler, Anja; Schuenemann, Verena J; Burbano, Hernán A

2017-02-01

DNA extracted from herbarium specimens is highly fragmented; therefore, it is crucial to use extraction protocols that retrieve short DNA molecules. Improvements in extraction and DNA library preparation protocols for animal remains have allowed efficient retrieval of molecules shorter than 50 bp. Here, we applied these improvements to DNA extraction protocols for herbarium specimens and evaluated extraction performance by shotgun sequencing, which allows an accurate estimation of the distribution of DNA fragment lengths. Extraction with N-phenacylthiazolium bromide (PTB) buffer decreased median fragment length by 35% when compared with cetyl-trimethyl ammonium bromide (CTAB); modifying the binding conditions of DNA to silica allowed for an additional decrease of 10%. We did not observe a further decrease in length for single-stranded DNA (ssDNA) versus double-stranded DNA (dsDNA) library preparation methods. Our protocol enables the retrieval of ultrashort molecules from herbarium specimens, which will help to unlock the genetic information stored in herbaria.

Ligation bias in Illumina next-generation DNA libraries

DEFF Research Database (Denmark)

Seguin-Orlando, Andaine; Schubert, Mikkel; Clary, Joel

2013-01-01

Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products,...... for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries.......Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by......-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate...

DNA adducts as molecular dosimeters

International Nuclear Information System (INIS)

Lucier, G.W.

1990-01-01

There is compelling evidence that DNA adducts play an important role in the actions of many pulmonary carcinogens. During the last ten years sensitive methods (antibodies and 32 P-postlabeling) have been developed that permit detection of DNA adducts in tissues of animals or humans exposed to low levels of some genotoxic carcinogens. This capability has led to approaches designed to more reliably estimate the shape of the dose-response curve in the low dose region for a few carcinogens. Moreover, dosimetry comparisions can, in some cases, be made between animals and humans which help in judging the adequacy of animal models for human risk assessments. There are several points that need to be considered in the evaluation of DNA adducts as a molecular dosimeter. For example, DNA adduct formation is only one of many events that are needed for tumor development and some potent carcinogens do not form DNA adducts; i.e., TCDD. Other issues that need to be considered are DNA adduct heterogeneity, DNA repair, relationship of DNA adducts to somatic mutation and cell specificity in DNA adduct formation and persistence. Molecular epidemiology studies often require quantitation of adducts in cells such as lymphocytes which may or may not be reliable surrogates for adduct concentrations in target issues. In summary, accurate quantitation of low levels of DNA adducts may provide data useful in species to species extrapolation of risk including the development of more meaningful human monitoring programs

The More the Merrier: Recent Hybridization and Polyploidy in Cardamine

Czech Academy of Sciences Publication Activity Database

Mandáková, T.; Kovařík, Aleš; Zozomová-Lihová, J.; Shimuzi-Inatsugi, R.; Shimizu, K.K.; Mummenhoff, K.; Marhold, K.; Lysák, M. A.

2013-01-01

Roč. 25, č. 9 (2013), s. 3280 ISSN 1040-4651 R&D Projects: GA ČR(CZ) GA13-10057S; GA MŠk(CZ) ED1.1.00/02.0068 Grant - others:GA ČR(CZ) GAP501/10/1014 Institutional support: RVO:68081707 Keywords : CHROMOSOME-NUMBER REDUCTION * MIXED-PLOIDY POPULATIONS * NUCLEAR RIBOSOMAL DNA Subject RIV: BO - Biophysics Impact factor: 9.575, year: 2013

Molecular pathology and age estimation.

Science.gov (United States)

Meissner, Christoph; Ritz-Timme, Stefanie

2010-12-15

Over the course of our lifetime a stochastic process leads to gradual alterations of biomolecules on the molecular level, a process that is called ageing. Important changes are observed on the DNA-level as well as on the protein level and are the cause and/or consequence of our 'molecular clock', influenced by genetic as well as environmental parameters. These alterations on the molecular level may aid in forensic medicine to estimate the age of a living person, a dead body or even skeletal remains for identification purposes. Four such important alterations have become the focus of molecular age estimation in the forensic community over the last two decades. The age-dependent accumulation of the 4977bp deletion of mitochondrial DNA and the attrition of telomeres along with ageing are two important processes at the DNA-level. Among a variety of protein alterations, the racemisation of aspartic acid and advanced glycation endproducs have already been tested for forensic applications. At the moment the racemisation of aspartic acid represents the pinnacle of molecular age estimation for three reasons: an excellent standardization of sampling and methods, an evaluation of different variables in many published studies and highest accuracy of results. The three other mentioned alterations often lack standardized procedures, published data are sparse and often have the character of pilot studies. Nevertheless it is important to evaluate molecular methods for their suitability in forensic age estimation, because supplementary methods will help to extend and refine accuracy and reliability of such estimates. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

GEOGRAPHIC DISTRIBUTION OF MOLECULAR VARIANCE WITHIN THE BLUE MARLIN (MAKAIRA NIGRICANS): A HIERARCHICAL ANALYSIS OF ALLOZYME, SINGLE-COPY NUCLEAR DNA, AND MITOCHONDRIAL DNA MARKERS.

Science.gov (United States)

Buonaccorsi, Vincent P; Reece, Kimberly S; Morgan, Lee W; Graves, John E

1999-04-01

This study presents a comparative hierarchical analysis of variance applied to three classes of molecular markers within the blue marlin (Makaira nigricans). Results are reported from analyses of four polymorphic allozyme loci, four polymorphic anonymously chosen single-copy nuclear DNA (scnDNA) loci, and previously reported restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA). Samples were collected within and among the Atlantic and Pacific Oceans over a period of several years. Although moderate levels of genetic variation were detected at both polymorphic allozyme (H = 0.30) and scnDNA loci (H = 0.37), mtDNA markers were much more diverse (h = 0.85). Allele frequencies were significantly different between Atlantic and Pacific Ocean samples at three of four allozyme loci and three of four scnDNA loci. Estimates of allozyme genetic differentiation (θ O ) ranged from 0.00 to 0.15, with a mean of 0.08. The θ O values for scnDNA loci were similar to those of allozymes, ranging from 0.00 to 0.12 with a mean of 0.09. MtDNA RFLP divergence between oceans (θ O = 0.39) was significantly greater than divergence detected at nuclear loci (95% nuclear confidence interval = 0.04-0.11). The fourfold smaller effective population size of mtDNA and male-mediated gene flow may account for the difference observed between nuclear and mitochondrial divergence estimates. © 1999 The Society for the Study of Evolution.

DNA level and stereologic estimates of nuclear volume in squamous cell carcinomas of the uterine cervix. A comparative study with analysis of prognostic impact

DEFF Research Database (Denmark)

Sørensen, Flemming Brandt; Bichel, P; Jakobsen, A

1992-01-01

Grading of malignancy in squamous cell carcinomas of the uterine cervix is based on qualitative, morphologic examination and suffers from poor reproducibility. Using modern stereology, unbiased estimates of the three-dimensional, volume-weighted mean nuclear volume (nuclear vv), were obtained...... in pretreatment biopsies from 51 patients treated for cervical cancer in clinical Stages I through III (mean age of 56 years, follow-up period greater than 5 years). In addition, conventional, two-dimensional morphometric estimates of nuclear and mitotic features were obtained. DNA indices (DI) were estimated...... of nuclear vv were only of marginal prognostic significance (2P = 0.07). However, Cox multivariate regression analysis showed independent prognostic value of patient age and nuclear vv along with clinical stage and DI. All other investigated variables were rejected from the model. A prognostic index...

Comparison of protocols for genomic DNA extraction from 'velame ...

African Journals Online (AJOL)

usuario

2013-07-24

Jul 24, 2013 ... involving C. linearifolius, we compared the efficiency of six protocols for genomic DNA extraction previously ... phytic, with diverse aspect and floristics, average rainfall between ..... The variation observed for DNA concentrations estimated with .... performed with protocol 1 (data not shown), or still, bands.

Cell proliferation and DNA dependent DNA polymerase estimation in acute lymphoblastic leukaemia during treatment with prednisone and vincristine

Energy Technology Data Exchange (ETDEWEB)

Lange Wantzin, G [Rigshospitalet, Copenhagen (Denmark)

1979-01-01

The presence of DNA polymerase and primer-template DNA in lymphoblast nuclei by measuring the in vitro incorporation of /sup 3/H-thymidine-5'-triphosphate (/sup 3/H-TTP) was studied in 10 patients with acute lymphoblastic leukemia. Protein synthesis and various other cytokinetic parameters were also studied. After prednisone (P) administration a marked decrease in /sup 3/H-TTP labelling index (/sup 3/H-TTP LI) was apparent together with an inhibition of /sup 3/H-leucine incorporation (/sup 3/H-LEU LI) into lymphoblasts. A moderate decrease in /sup 3/H-TDR labelling index (/sup 3/H-TDR LI) and a later decrease in mitotic index (MI) were seen. Single cell DNA measurements showed a depletion of /sup 3/H-TDR labelled lymphoblasts in early part of S-phase apparent at 24 h lasting up to 54 h after P administration. Vincristine given as a flash injection later in the study period caused an immediate rise of the MI, at the same time the P induced decline in /sup 3/H-TTP LI, /sup 3/H-TDR LI and /sup 3/H-LEU LI were continued in most patients. P is thought to damage the cells both in and outside the cell cycle. In the cell cycle the effect of P is an arresting effect in G/sub 1/.

Evaluating the efficacy of DNA differential extraction methods for sexual assault evidence.

Science.gov (United States)

Klein, Sonja B; Buoncristiani, Martin R

2017-07-01

Analysis of sexual assault evidence, often a mixture of spermatozoa and victim epithelial cells, represents a significant portion of a forensic DNA laboratory's case load. Successful genotyping of sperm DNA from these mixed cell samples, particularly with low amounts of sperm, depends on maximizing sperm DNA recovery and minimizing non-sperm DNA carryover. For evaluating the efficacy of the differential extraction, we present a method which uses a Separation Potential Ratio (SPRED) to consider both sperm DNA recovery and non-sperm DNA removal as variables for determining separation efficiency. In addition, we describe how the ratio of male-to-female DNA in the sperm fraction may be estimated by using the SPRED of the differential extraction method in conjunction with the estimated ratio of male-to-female DNA initially present on the mixed swab. This approach may be useful for evaluating or modifying differential extraction methods, as we demonstrate by comparing experimental results obtained from the traditional differential extraction and the Erase Sperm Isolation Kit (PTC © ) procedures. Copyright © 2017 Elsevier B.V. All rights reserved.

Distribution of DTHS3 satellite DNA across 12 bivalve species Eva ...

Indian Academy of Sciences (India)

Windows User

In this work, characterization of DTHS3 satellite DNA was further expanded within the Class. Bivalvia. Monomer variants of DTHS3 satDNA were compared in 12 bivalve species belonging to two different Subclasses, Heterodonta and Pteriomorphia. This satDNA, whose age is estimated to a minimum of 516 Ma, ...

Evaluation of cell number and DNA content in mouse embryos cultivated with uranium; Evaluacion del numero de celulas y el contenido de DNA en embriones murinos cultivados con uranio

Energy Technology Data Exchange (ETDEWEB)

Kundt, Mirian S; Cabrini, Romulo L [Comision Nacional de Energia Atomica, General San Martin (Argentina). Dept. de Radiobiologia

2000-07-01

The evaluation of the degree of development, the number of cells and the DNA content, were used to evaluate the embryotoxicity of uranium. Embryos at a one cell stage were cultured with uranyl nitrate hexahydrate (UN) at a final concentration of uranium (U) of 26, 52 and 104 {mu}gU/ml. At 24 hs of culture, the embryos at the 2 cell stage, were put in new wells with the same concentrations of U as the previous day, until the end of the period of incubation at 72 hs. At 72 hs of culture, 87% of the original one cell embryos were at morula stage, and in those cultivated with uranium, the percentage decreased significantly to 77; 63.24 and 40.79% respectively for the different U concentrations. Those embryos that exhibited a normal morphology, were selected and fixed on slides. The number of cells per embryo was evaluated in Giemsa stained preparations. The DNA content was evaluated cytophotometrically in Feulgen stained nuclei. The number of cells decreased significantly from 20,3 {+-} 5.6 in the control to 19 {+-} 6; 14 {+-} 3 and 13.9 {+-} 5.6 for the different concentrations. All the embryos evaluated showed one easy recognizable polar body, which was used a haploid indicator (n). The content of DNA was measured in a total of 20 control embryos and 16 embryos cultivated with UN. In control embryos, 92,7% of the nuclei presented a normal ploidy from 2n to 4n, 2,9% nuclei were hypoploid and 4,4% were hyperploid. The percentage of hypoploid nuclei rose in a dose-dependent fashion to 3.45; 44.45 and 50.34% respectively for the embryos cultured at the different U concentrations. The results indicate that U is embryotoxic, that its effects are dose dependent at the concentrations used in this study and that even those embryos that show a normal morphology, can be genetically affected. We show that the model employed is extremely sensitive. It is possible to use the preimplantation embryos, as a model to test the effect of possibly mutagenic agents of the nuclear industry

K-ras2 Activation and Genome Instability Increase Proliferation and Size of FAP Adenomas

Directory of Open Access Journals (Sweden)

Anna Rapallo

1999-01-01

Full Text Available The possible role of K‐ras2 mutations and aneuploidy toward increase of proliferation and adenoma size in Familial Adenomatous Polyposis (FAP adenomas is not known. The present study addresses these issues by investigating 147 colorectal adenomas obtained from four FAP patients. The majority of adenomas had size lower than or equal to 10 mm (86%, low grade dysplasia (63%, and were preferentially located in the right colon (60%. Normal mucosa samples were obtained from 19 healthy donors. Three synchronous adenocarcinomas were also investigated. K‐ras2 mutation spectrum was analysed by PCR and Sequence Specific Oligonucleotide (SSO hybridization, while flow cytometry (FCM was used for evaluating degree of DNA ploidy and S‐phase fraction. Overall, incidences of K‐ras2 mutations, DNA aneuploidy and high S‐phase values (>7.2% were 6.6%, 5.4% and 10.5%, respectively. In particular, among the adenomas with size lower than 5 mm, K‐ras2 mutation and DNA aneuploidy frequencies were only slightly above 1%. Statistically significant correlations were found between K‐ras2 and size, DNA ploidy and size and K‐ras2 and S‐phase (p. In particular, among the wild type K‐ras2 adenomas, high S‐phase values were detected in 8% of the cases versus 57% among the K‐ras2 mutated adenomas (p=0.0005. The present series of FAP adenomas indicates that K‐ras2 activation and gross genomic changes play a role toward a proliferative gain and tumour growth in size.

Improving cluster-based missing value estimation of DNA microarray data.

Science.gov (United States)

Brás, Lígia P; Menezes, José C

2007-06-01

We present a modification of the weighted K-nearest neighbours imputation method (KNNimpute) for missing values (MVs) estimation in microarray data based on the reuse of estimated data. The method was called iterative KNN imputation (IKNNimpute) as the estimation is performed iteratively using the recently estimated values. The estimation efficiency of IKNNimpute was assessed under different conditions (data type, fraction and structure of missing data) by the normalized root mean squared error (NRMSE) and the correlation coefficients between estimated and true values, and compared with that of other cluster-based estimation methods (KNNimpute and sequential KNN). We further investigated the influence of imputation on the detection of differentially expressed genes using SAM by examining the differentially expressed genes that are lost after MV estimation. The performance measures give consistent results, indicating that the iterative procedure of IKNNimpute can enhance the prediction ability of cluster-based methods in the presence of high missing rates, in non-time series experiments and in data sets comprising both time series and non-time series data, because the information of the genes having MVs is used more efficiently and the iterative procedure allows refining the MV estimates. More importantly, IKNN has a smaller detrimental effect on the detection of differentially expressed genes.

Serum Removal from Culture Induces Growth Arrest, Ploidy Alteration, Decrease in Infectivity and Differential Expression of Crucial Genes in Leishmania infantum Promastigotes.

Directory of Open Access Journals (Sweden)

Pedro J Alcolea

Full Text Available Leishmania infantum is one of the species responsible for visceral leishmaniasis. This species is distributed basically in the Mediterranean basin. A recent outbreak in humans has been reported in Spain. Axenic cultures are performed for most procedures with Leishmania spp. promastigotes. This model is stable and reproducible and mimics the conditions of the gut of the sand fly host, which is the natural environment of promastigote development. Culture media are undefined because they contain mammalian serum, which is a rich source of complex lipids and proteins. Serum deprivation slows down the growth kinetics and therefore, yield in biomass. In fact, we have confirmed that the growth rate decreases, as well as infectivity. Ploidy is also affected. Regarding the transcriptome, a high-throughput approach has revealed a low differential expression rate but important differentially regulated genes. The most remarkable profiles are: up-regulation of the GINS Psf3, the fatty acyl-CoA synthase (FAS1, the glyoxylase I (GLO1, the hydrophilic surface protein B (HASPB, the methylmalonyl-CoA epimerase (MMCE and an amastin gene; and down-regulation of the gPEPCK and the arginase. Implications for metabolic adaptations, differentiation and infectivity are discussed herein.

Serum Removal from Culture Induces Growth Arrest, Ploidy Alteration, Decrease in Infectivity and Differential Expression of Crucial Genes in Leishmania infantum Promastigotes.

Science.gov (United States)

Alcolea, Pedro J; Alonso, Ana; Moreno-Izquierdo, Miguel A; Degayón, María A; Moreno, Inmaculada; Larraga, Vicente

2016-01-01

Leishmania infantum is one of the species responsible for visceral leishmaniasis. This species is distributed basically in the Mediterranean basin. A recent outbreak in humans has been reported in Spain. Axenic cultures are performed for most procedures with Leishmania spp. promastigotes. This model is stable and reproducible and mimics the conditions of the gut of the sand fly host, which is the natural environment of promastigote development. Culture media are undefined because they contain mammalian serum, which is a rich source of complex lipids and proteins. Serum deprivation slows down the growth kinetics and therefore, yield in biomass. In fact, we have confirmed that the growth rate decreases, as well as infectivity. Ploidy is also affected. Regarding the transcriptome, a high-throughput approach has revealed a low differential expression rate but important differentially regulated genes. The most remarkable profiles are: up-regulation of the GINS Psf3, the fatty acyl-CoA synthase (FAS1), the glyoxylase I (GLO1), the hydrophilic surface protein B (HASPB), the methylmalonyl-CoA epimerase (MMCE) and an amastin gene; and down-regulation of the gPEPCK and the arginase. Implications for metabolic adaptations, differentiation and infectivity are discussed herein.

The effect of ancient DNA damage on inferences of demographic histories

DEFF Research Database (Denmark)

Axelsson, Erik; Willerslev, Eske; Gilbert, Marcus Thomas Pius

2008-01-01

The field of ancient DNA (aDNA) is casting new light on many evolutionary questions. However, problems associated with the postmortem instability of DNA may complicate the interpretation of aDNA data. For example, in population genetic studies, the inclusion of damaged DNA may inflate estimates o...... for a change in effective population size in this data set vanishes once the effects of putative damage are removed. Our results suggest that population genetic analyses of aDNA sequences, which do not accurately account for damage, should be interpreted with great caution....

Temporal patterns of damage and decay kinetics of DNA retrieved from plant herbarium specimens.

Science.gov (United States)

Weiß, Clemens L; Schuenemann, Verena J; Devos, Jane; Shirsekar, Gautam; Reiter, Ella; Gould, Billie A; Stinchcombe, John R; Krause, Johannes; Burbano, Hernán A

2016-06-01

Herbaria archive a record of changes of worldwide plant biodiversity harbouring millions of specimens that contain DNA suitable for genome sequencing. To profit from this resource, it is fundamental to understand in detail the process of DNA degradation in herbarium specimens. We investigated patterns of DNA fragmentation and nucleotide misincorporation by analysing 86 herbarium samples spanning the last 300 years using Illumina shotgun sequencing. We found an exponential decay relationship between DNA fragmentation and time, and estimated a per nucleotide fragmentation rate of 1.66 × 10(-4) per year, which is six times faster than the rate estimated for ancient bones. Additionally, we found that strand breaks occur specially before purines, and that depurination-driven DNA breakage occurs constantly through time and can to a great extent explain decreasing fragment length over time. Similar to what has been found analysing ancient DNA from bones, we found a strong correlation between the deamination-driven accumulation of cytosine to thymine substitutions and time, which reinforces the importance of substitution patterns to authenticate the ancient/historical nature of DNA fragments. Accurate estimations of DNA degradation through time will allow informed decisions about laboratory and computational procedures to take advantage of the vast collection of worldwide herbarium specimens.

Kinetic mechanism of DNA polymerase I (Klenow)

International Nuclear Information System (INIS)

Kuchta, R.D.; Mizrahi, V.; Benkovic, P.A.; Johnson, K.A.; Benkovic, S.J.

1987-01-01

The minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment of DNA polymerase I (KF) from Escherichia coli has been determined with short DNA oligomers of defined sequence, labeled with [ 32 P]-nucleotides. A key feature of this scheme is a minimal two-step sequence that interconverts the ternary KF-DNA/sub n/-dNTP and KF-DNA/sub n+1/-PP/sub i/ complexes. The rate is not limited by the actual polymerization but by a separate step, possibly important in ensuring fidelity. Evidence for this sequence is supplied by the observation of biphasic kinetics in single-turnover pyrophosphorolysis experiments (the microscopic reverse of polymerization). Data analysis then provides an estimate of the internal equilibrium constant. The dissociations of DNA, dNTP, and PP/sub i/ from the various binary and ternary complexes were measured by partitioning (isotope-trapping) experiments. The rate constant for DNA dissociation from KF is sequence dependent and is rate limiting during nonprocessive DNA synthesis. The combination of single-turnover (both directions) and isotope-trapping experiments provides sufficient information to permit a quantitative evaluation of the kinetic scheme for specific DNA sequences

Multifragment alleles in DNA fingerprints of the parrot, Amazona ventralis

Science.gov (United States)

Brock, M.K.; White, B.N.

1991-01-01

Human DNA probes that identify variable numbers of tandem repeat loci are being used to generate DNA fingerprints in many animal and plant species. In most species the majority of the sc rable autoradiographic bands of the DNA fingerprint represent alleles from numerous unlinked loci. This study was initiated to use DNA fingerprints to determine the amount of band-sharing among captive Hispaniolan parrots (Amazona ventralis) with known genetic relationships. This would form the data base to examine DNA fingerprints of the closely related and endangered Puerto Rican parrot (A. vittata) and to estimate the degree of inbreeding in the relic population. We found by segregation analysis of the bands scored in the DNA fingerprints of the Hispaniolan parrots that there may be as few as two to five loci identified by the human 33.15 probe. Furthermore, at one locus we identified seven alleles, one of which is represented by as many as 19 cosegregating bands. It is unknown how common multiband alleles might be in natural populations, and their existence will cause problems in the assessment of relatedness by band-sharing analysis. We believe, therefore, that a pedigree analysis should be included in all DNA fingerprinting studies, where possible, in order to estimate the number of loci identified by a minisatellite DNA probe and to examine the nature of their alleles.

Feasibility of measuring radiation-induced DNA double strand breaks and their repair by pulsed field gel electrophoresis in freshly isolated cells from the mouse RIF-1 tumor

International Nuclear Information System (INIS)

Waarde, Maria A.W.H. van; Assen, Annette J. van; Konings, Antonius W.T.; Kampinga, Harm H.

1996-01-01

Purpose: To examine the technical feasibility of pulsed field gel electrophoresis (PFGE) as a predictive assay for the radio responsiveness of tumors. Induction and repair of DNA double strand breaks (DSBs) in a freshly prepared cell suspension from a RIF-1 tumor (irradiated ex vivo) was compared with DSB induction and repair in exponentially growing RIF-1 cells in culture (irradiated in vitro). Methods and Materials: A murine RIF-1 tumor grown in vivo was digested, and cells were exposed to x-rays (ex vivo) at doses of 1 to 75 Gy. DNA damage was measured using CHEF (clamped homogeneous electric fields) electrophoresis. Repair kinetics were studied at 37 deg. C for 4 h after irradiation. Radiosensitivity was determined by clonogenic assay, and cell cycle distributions by flow cytometry. For comparison, a trypsinized suspension of exponentially growing RIF-1 cells in vitro was run parallel with each ex vivo experiment. Results: Induction of DSBs, expressed as % DNA extracted from the plug, was similar in the in vitro and ex vivo irradiated cells. Compared to repair rates in in vitro cultured RIF-1 cells, repair kinetics in a freshly prepared cell suspension from the tumor were decreased, unrelated to differences in radiosensitivity. Differences in repair could not be explained by endogenous DNA degradation, nor by influences of enzymes used for digestion of the tumor. A lower plating efficiency and differences in ploidy (as revealed by flow cytometry) were the only reproducible differences between in vivo and in vitro grown cells that may explain the differences in repair kinetics. Conclusions: The current results do not support the idea that PFGE is a technique robust enough to be a predictive assay for the radiosensitivity of tumor cells

Nature of vegetative cycle in Phytophthora parasitica Dastur and palmivora (BUTLER) BUTLER

International Nuclear Information System (INIS)

Le Grand-Pernot, Francoise; Pellegrin, F.

1976-01-01

Up to now, neither cytological nor genetical studies have clearly demonstrated the 'ploidy' degree of somatic nuclei in different species of Phytophthora. Two complementary studies, using biological effect of γ rays upon zoospores, and microspectrophotometric techniques measuring relative amount of DNA by nucleus in zoospores or in hyphae, allowed to demonstrate diploid nature of the vegetative phase in P. palmivora (BUTLER) BUTLER and P. parasitica DASTUR cycles [fr

Sensitive Fluorescent Sensor for Recognition of HIV-1 dsDNA by Using Glucose Oxidase and Triplex DNA

Directory of Open Access Journals (Sweden)

Yubin Li

2018-01-01

Full Text Available A sensitive fluorescent sensor for sequence-specific recognition of double-stranded DNA (dsDNA was developed on the surface of silver-coated glass slide (SCGS. Oligonucleotide-1 (Oligo-1 was designed to assemble on the surface of SCGS and act as capture DNA, and oligonucleotide-2 (Oligo-2 was designed as signal DNA. Upon addition of target HIV-1 dsDNA (Oligo-3•Oligo-4, signal DNA could bind on the surface of silver-coated glass because of the formation of C•GoC in parallel triplex DNA structure. Biotin-labeled glucose oxidase (biotin-GOx could bind to signal DNA through the specific interaction of biotin-streptavidin, thereby GOx was attached to the surface of SCGS, which was dependent on the concentration of target HIV-1 dsDNA. GOx could catalyze the oxidation of glucose and yield H2O2, and the HPPA can be oxidized into a fluorescent product in the presence of HRP. Therefore, the concentration of target HIV-1 dsDNA could be estimated with fluorescence intensity. Under the optimum conditions, the fluorescence intensity was proportional to the concentration of target HIV-1 dsDNA over the range of 10 pM to 1000 pM, the detection limit was 3 pM. Moreover, the sensor had good sequence selectivity and practicability and might be applied for the diagnosis of HIV disease in the future.

Alkaline elution of DNA from mammalian cells on cellulose triacetate filters

International Nuclear Information System (INIS)

Moss, A.J. Jr.; Nagle, W.A.; Henle, K.J.; Prior, R.M.

1984-01-01

The alkaline elution technique is widely used for the estimation of cellular DNA damage because of its sensitivity in the biologically relevant dose range. The authors have extended the original studies and provide additional characterization of the cellulose triacetate alkaline elution method. This filter material permits the elution of approximately 80 percent of cellular DNA from untreated V79 cells. the total radioactivity in the system was compartmentalized with respect to 1) lysing solution, 2) washing solution, 3) elution fractions, and 4) membrane retained activity. In these studies [/sup 3/H]-thymidine labeled untreated internal control cells were co-eluted with X-irradiated [/sup 14/C]-thymidine labeled cells. For the estimation of DNA damage, elution profiles for treated cells were directly compared with untreated internal control cells. The quantity of DNA eluting in excess of the labeled internal control per fraction is directly proportional to the extent of DNA damage in the treated sample. Using the technique the necessity of an irradiated internal control is eliminated

Evaluation of cell number and DNA content in mouse embryos cultivated with uranium

International Nuclear Information System (INIS)

Kundt, Mirian S.; Cabrini, Romulo L.

2000-01-01

The evaluation of the degree of development, the number of cells and the DNA content, were used to evaluate the embryotoxicity of uranium. Embryos at a one cell stage were cultured with uranyl nitrate hexahydrate (UN) at a final concentration of uranium (U) of 26, 52 and 104 μgU/ml. At 24 hs of culture, the embryos at the 2 cell stage, were put in new wells with the same concentrations of U as the previous day, until the end of the period of incubation at 72 hs. At 72 hs of culture, 87% of the original one cell embryos were at morula stage, and in those cultivated with uranium, the percentage decreased significantly to 77; 63.24 and 40.79% respectively for the different U concentrations. Those embryos that exhibited a normal morphology, were selected and fixed on slides. The number of cells per embryo was evaluated in Giemsa stained preparations. The DNA content was evaluated cytophotometrically in Feulgen stained nuclei. The number of cells decreased significantly from 20,3 ± 5.6 in the control to 19 ± 6; 14 ± 3 and 13.9 ± 5.6 for the different concentrations. All the embryos evaluated showed one easy recognizable polar body, which was used a haploid indicator (n). The content of DNA was measured in a total of 20 control embryos and 16 embryos cultivated with UN. In control embryos, 92,7% of the nuclei presented a normal ploidy from 2n to 4n, 2,9% nuclei were hypoploid and 4,4% were hyperploid. The percentage of hypoploid nuclei rose in a dose-dependent fashion to 3.45; 44.45 and 50.34% respectively for the embryos cultured at the different U concentrations. The results indicate that U is embryotoxic, that its effects are dose dependent at the concentrations used in this study and that even those embryos that show a normal morphology, can be genetically affected. We show that the model employed is extremely sensitive. It is possible to use the preimplantation embryos, as a model to test the effect of possibly mutagenic agents of the nuclear industry. (author)

Evaluation of five DNA extraction methods for purification of DNA from atherosclerotic tissue and estimation of prevalence of Chlamydia pneumoniae in tissue from a Danish population undergoing vascular repair

Directory of Open Access Journals (Sweden)

Lindholt Jes S

2003-09-01

Full Text Available Abstract Background To date PCR detection of Chlamydia pneumoniae DNA in atherosclerotic lesions from Danish patients has been unsuccessful. To establish whether non-detection was caused by a suboptimal DNA extraction method, we tested five different DNA extraction methods for purification of DNA from atherosclerotic tissue. Results The five different DNA extraction methods were tested on homogenate of atherosclerotic tissue spiked with C. pneumoniae DNA or EB, on pure C. pneumoniae DNA samples and on whole C. pneumoniae EB. Recovery of DNA was measured with a C. pneumoniae-specific quantitative real-time PCR. A DNA extraction method based on DNA-binding to spin columns with a silica-gel membrane (DNeasy Tissue kit showed the highest recovery rate for the tissue samples and pure DNA samples. However, an automated extraction method based on magnetic glass particles (MagNA Pure performed best on intact EB and atherosclerotic tissue spiked with EB. The DNeasy Tissue kit and MagNA Pure methods and the highly sensitive real-time PCR were subsequently used on 78 atherosclerotic tissue samples from Danish patients undergoing vascular repair. None of the samples were positive for C. pneumoniae DNA. The atherosclerotic samples were tested for inhibition by spiking with two different, known amounts of C. pneumoniae DNA and no samples showed inhibition. Conclusion As a highly sensitive PCR method and an optimised DNA extraction method were used, non-detection in atherosclerotic tissue from the Danish population was probably not caused by use of inappropriate methods. However, more samples may need to be analysed per patient to be completely certain on this. Possible methodological and epidemiological reasons for non-detection of C. pneumoniae DNA in atherosclerotic tissue from the Danish population are discussed. Further testing of DNA extraction methods is needed as this study has shown considerable intra- and inter-method variation in DNA recovery.

Cell type specific DNA methylation in cord blood: A 450K-reference data set and cell count-based validation of estimated cell type composition.

Science.gov (United States)

Gervin, Kristina; Page, Christian Magnus; Aass, Hans Christian D; Jansen, Michelle A; Fjeldstad, Heidi Elisabeth; Andreassen, Bettina Kulle; Duijts, Liesbeth; van Meurs, Joyce B; van Zelm, Menno C; Jaddoe, Vincent W; Nordeng, Hedvig; Knudsen, Gunn Peggy; Magnus, Per; Nystad, Wenche; Staff, Anne Cathrine; Felix, Janine F; Lyle, Robert

2016-09-01

Epigenome-wide association studies of prenatal exposure to different environmental factors are becoming increasingly common. These studies are usually performed in umbilical cord blood. Since blood comprises multiple cell types with specific DNA methylation patterns, confounding caused by cellular heterogeneity is a major concern. This can be adjusted for using reference data consisting of DNA methylation signatures in cell types isolated from blood. However, the most commonly used reference data set is based on blood samples from adult males and is not representative of the cell type composition in neonatal cord blood. The aim of this study was to generate a reference data set from cord blood to enable correct adjustment of the cell type composition in samples collected at birth. The purity of the isolated cell types was very high for all samples (>97.1%), and clustering analyses showed distinct grouping of the cell types according to hematopoietic lineage. We explored whether this cord blood and the adult peripheral blood reference data sets impact the estimation of cell type composition in cord blood samples from an independent birth cohort (MoBa, n = 1092). This revealed significant differences for all cell types. Importantly, comparison of the cell type estimates against matched cell counts both in the cord blood reference samples (n = 11) and in another independent birth cohort (Generation R, n = 195), demonstrated moderate to high correlation of the data. This is the first cord blood reference data set with a comprehensive examination of the downstream application of the data through validation of estimated cell types against matched cell counts.

Towards quantitative viromics for both double-stranded and single-stranded DNA viruses

Directory of Open Access Journals (Sweden)

Simon Roux

2016-12-01

Full Text Available Background Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA viral genomes captured in quantitative viral metagenomes (viromes. This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation. Methods Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses. Results Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5% of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA viruses and bacteriophages from the Microviridae family, can be among the most abundant viral genomes in a sample. Discussion Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.

Distribution and diversity of cytotypes in Dianthus broteri as evidenced by genome size variations.

Science.gov (United States)

Balao, Francisco; Casimiro-Soriguer, Ramón; Talavera, María; Herrera, Javier; Talavera, Salvador

2009-10-01

Studying the spatial distribution of cytotypes and genome size in plants can provide valuable information about the evolution of polyploid complexes. Here, the spatial distribution of cytological races and the amount of DNA in Dianthus broteri, an Iberian carnation with several ploidy levels, is investigated. Sample chromosome counts and flow cytometry (using propidium iodide) were used to determine overall genome size (2C value) and ploidy level in 244 individuals of 25 populations. Both fresh and dried samples were investigated. Differences in 2C and 1Cx values among ploidy levels within biogeographical provinces were tested using ANOVA. Geographical correlations of genome size were also explored. Extensive variation in chromosomes numbers (2n = 2x = 30, 2n = 4x = 60, 2n = 6x = 90 and 2n = 12x =180) was detected, and the dodecaploid cytotype is reported for the first time in this genus. As regards cytotype distribution, six populations were diploid, 11 were tetraploid, three were hexaploid and five were dodecaploid. Except for one diploid population containing some triploid plants (2n = 45), the remaining populations showed a single cytotype. Diploids appeared in two disjunct areas (south-east and south-west), and so did tetraploids (although with a considerably wider geographic range). Dehydrated leaf samples provided reliable measurements of DNA content. Genome size varied significantly among some cytotypes, and also extensively within diploid (up to 1.17-fold) and tetraploid (1.22-fold) populations. Nevertheless, variations were not straightforwardly congruent with ecology and geographical distribution. Dianthus broteri shows the highest diversity of cytotypes known to date in the genus Dianthus. Moreover, some cytotypes present remarkable internal genome size variation. The evolution of the complex is discussed in terms of autopolyploidy, with primary and secondary contact zones.

Polyploidisation and Geographic Differentiation Drive Diversification in a European High Mountain Plant Group (Doronicum clusii Aggregate, Asteraceae)

Science.gov (United States)

Pachschwöll, Clemens; Escobar García, Pedro; Winkler, Manuela; Schneeweiss, Gerald M.; Schönswetter, Peter

2015-01-01

Range shifts (especially during the Pleistocene), polyploidisation and hybridization are major factors affecting high-mountain biodiversity. A good system to study their role in the European high mountains is the Doronicum clusii aggregate (Asteraceae), whose four taxa (D. clusii s.s., D. stiriacum, D. glaciale subsp. glaciale and D. glaciale subsp. calcareum) are differentiated geographically, ecologically (basiphilous versus silicicolous) and/or via their ploidy levels (diploid versus tetraploid). Here, we use DNA sequences (three plastid and one nuclear spacer) and AFLP fingerprinting data generated for 58 populations to infer phylogenetic relationships, origin of polyploids—whose ploidy level was confirmed by chromosomally calibrated DNA ploidy level estimates—and phylogeographic history. Taxonomic conclusions were informed, among others, by a Gaussian clustering method for species delimitation using dominant multilocus data. Based on molecular data we identified three lineages: (i) silicicolous diploid D. clusii s.s. in the Alps, (ii) silicicolous tetraploid D. stiriacum in the eastern Alps (outside the range of D. clusii s.s.) and the Carpathians and (iii) the basiphilous diploids D. glaciale subsp. glaciale (eastern Alps) and D. glaciale subsp. calcareum (northeastern Alps); each taxon was identified as distinct by the Gaussian clustering, but the separation of D. glaciale subsp. calcareum and D. glaciale subsp. glaciale was not stable, supporting their taxonomic treatment as subspecies. Carpathian and Alpine populations of D. stiriacum were genetically differentiated suggesting phases of vicariance, probably during the Pleistocene. The origin (autopolyploid versus allopolyploid) of D. stiriacum remained unclear. Doronicum glaciale subsp. calcareum was genetically and morphologically weakly separated from D. glaciale subsp. glaciale but exhibited significantly higher genetic diversity and rarity. This suggests that the more widespread D. glaciale subsp

Assessment of mitochondrial functions in Daphnia pulex clones using high-resolution respirometry.

Science.gov (United States)

Kake-Guena, Sandrine A; Touisse, Kamal; Vergilino, Roland; Dufresne, France; Blier, Pierre U; Lemieux, Hélène

2015-06-01

The objectives of our study were to adapt a method to measure mitochondrial function in intact mitochondria from the small crustacean Daphnia pulex and to validate if this method was sensitive enough to characterize mitochondrial metabolism in clones of the pulex complex differing in ploidy levels, mitochondrial DNA haplotypes, and geographic origins. Daphnia clones belonging to the Daphnia pulex complex represent a powerful model to delineate the link between mitochondrial DNA evolution and mitochondrial phenotypes, as single genotypes with divergent mtDNA can be grown under various experimental conditions. Our study included two diploid clones from temperate environments and two triploid clones from subarctic environments. The whole animal permeabilization and measurement of respiration with high-resolution respirometry enabled the measurement of the functional capacity of specific mitochondrial complexes in four clones. When expressing the activity as ratios, our method detected significant interclonal variations. In the triploid subarctic clone from Kuujjurapik, a higher proportion of the maximal physiological oxidative phosphorylation (OXPHOS) capacity of mitochondria was supported by complex II, and a lower proportion by complex I. The triploid subarctic clone from Churchill (Manitoba) showed the lowest proportion of the maximal OXPHOS supported by complex II. Additional studies are required to determine if these differences in mitochondrial functions are related to differences in mitochondrial haplotypes or ploidy level and if they might be associated with fitness divergences and therefore selective value. © 2015 Wiley Periodicals, Inc.

DNA damage in plant herbarium tissue.

Science.gov (United States)

Staats, Martijn; Cuenca, Argelia; Richardson, James E; Vrielink-van Ginkel, Ria; Petersen, Gitte; Seberg, Ole; Bakker, Freek T

2011-01-01

Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of amplifiable template molecules, but may also lead to the generation of erroneous sequence information. A qualitative and quantitative assessment of DNA post-mortem damage is essential to determine the accuracy of molecular data from herbarium specimens. In this study we present an assessment of DNA damage as miscoding lesions in herbarium specimens using 454-sequencing of amplicons derived from plastid, mitochondrial, and nuclear DNA. In addition, we assess DNA degradation as a result of strand breaks and other types of polymerase non-bypassable damage by quantitative real-time PCR. Comparing four pairs of fresh and herbarium specimens of the same individuals we quantitatively assess post-mortem DNA damage, directly after specimen preparation, as well as after long-term herbarium storage. After specimen preparation we estimate the proportion of gene copy numbers of plastid, mitochondrial, and nuclear DNA to be 2.4-3.8% of fresh control DNA and 1.0-1.3% after long-term herbarium storage, indicating that nearly all DNA damage occurs on specimen preparation. In addition, there is no evidence of preferential degradation of organelle versus nuclear genomes. Increased levels of C→T/G→A transitions were observed in old herbarium plastid DNA, representing 21.8% of observed miscoding lesions. We interpret this type of post-mortem DNA damage-derived modification to have arisen from the hydrolytic deamination of cytosine during long-term herbarium storage. Our results suggest that reliable sequence data can be obtained from herbarium specimens.

Nondetectability of restriction fragments and independence of DNA fragment sizes within and between loci in RFLP typing of DNA

Energy Technology Data Exchange (ETDEWEB)

Chakraborty, R.; Zhong, Y.; Jin, L. (Univ. of Texas Health Science Center, Houston, TX (United States)); Budowle, B. (FBI Academy, Quantico, VA (United States))

1994-08-01

The authors provide experimental evidence showing that, during the restriction-enzyme digestion of DNA samples, some of the HaeIII-digested DNA fragments are small enough to prevent their reliable sizing on a Southern gel. As a result of such nondetectability of DNA fragments, individuals who show a single-band DNA profile at a VNTR locus may not necessarily be true homozygotes. In a population database, when the presence of such nondetectable alleles is ignored, they show that a pseudodependence of alleles within as well as across loci may occur. Using a known statistical method, under the hypothesis of independence of alleles within loci, they derive an efficient estimate of null allele frequency, which may be subsequently used for testing allelic independence within and across loci. The estimates of null allele frequencies, thus derived, are shown to agree with direct experimental data on the frequencies of HaeIII-null alleles. Incorporation of null alleles into the analysis of the forensic VNTR database suggests that the assumptions of allelic independence within and between loci are appropriate. In contrast, a failure to incorporate the occurrence of null alleles would provide a wrong inference regarding the independence of alleles within and between loci. 47 refs., 2 figs., 4 tabs.

Effect of cell phone-like electromagnetic radiation on primary human thyroid cells.

Science.gov (United States)

Silva, Veronica; Hilly, Ohad; Strenov, Yulia; Tzabari, Cochava; Hauptman, Yirmi; Feinmesser, Raphael

2016-01-01

To evaluate the potential carcinogenic effects of radiofrequency energy (RFE) emitted by cell phones on human thyroid primary cells. Primary thyroid cell culture was prepared from normal thyroid tissue obtained from patients who underwent surgery at our department. Subconfluent thyroid cells were irradiated under different conditions inside a cell incubator using a device that simulates cell phone-RFE. Proliferation of control and irradiated cells was assessed by the immunohistochemical staining of antigen Kiel clone-67 (Ki-67) and tumor suppressor p53 (p53) expression. DNA ploidy and the stress biomarkers heat shock protein 70 (HSP70) and reactive oxygen species (ROS) was evaluated by fluorescence-activated cell sorting (FACS). Our cells highly expressed thyroglobulin (Tg) and sodium-iodide symporter (NIS) confirming the origin of the tissue. None of the irradiation conditions evaluated here had an effect neither on the proliferation marker Ki-67 nor on p53 expression. DNA ploidy was also not affected by RFE, as well as the expression of the biomarkers HSP70 and ROS. Our conditions of RFE exposure seem to have no potential carcinogenic effect on human thyroid cells. Moreover, common biomarkers usually associated to environmental stress also remained unchanged. We failed to find an association between cell phone-RFE and thyroid cancer. Additional studies are recommended.

Risk assessment of DNA-reactive carcinogens in food.

Science.gov (United States)

Jeffrey, A M; Williams, G M

2005-09-01

Risk assessment of DNA-reactive carcinogens in food requires knowledge of the extent of DNA damage in the target organ which results from the competition between DNA adduct formation and repair. Estimates of DNA adduct levels can be made by direct measurement or indirectly as a consequence of their presence, for example, by tumor formation in animal models or exposed populations epidemiologically. Food-borne DNA-reactive carcinogens are present from a variety of sources. They are generally not intrinsically DNA-reactive but require bioactivation to DNA-reactive metabolites a process which may be modulated by the compound itself or the presence of other xenobiotics. A single DNA reactant may form several distinct DNA adducts each undergoing different rates of repair. Some DNA reactants may be photochemically activated or produce reactive oxygen species and thus indirect oxidative DNA damage. The levels of DNA adducts arising from exposures influenced by variations in the doses, the frequency with which an individual is exposed, and rates of DNA repair for specific adducts. Each adduct has a characteristic efficiency with which it induces mutations. Based on experience with the well-studied DNA-reactive food carcinogen aflatoxin B(1) (AFB(1)), a limit of 20 ppb or approximately 30 microg/day has been set and is considered a tolerable daily intake (TDI). Since AFB(1) is considered a potent carcinogen, doses of carcinogens is made.

DNA Methylation Biomarkers: Cancer and Beyond

Directory of Open Access Journals (Sweden)

Thomas Mikeska

2014-09-01

Full Text Available Biomarkers are naturally-occurring characteristics by which a particular pathological process or disease can be identified or monitored. They can reflect past environmental exposures, predict disease onset or course, or determine a patient’s response to therapy. Epigenetic changes are such characteristics, with most epigenetic biomarkers discovered to date based on the epigenetic mark of DNA methylation. Many tissue types are suitable for the discovery of DNA methylation biomarkers including cell-based samples such as blood and tumor material and cell-free DNA samples such as plasma. DNA methylation biomarkers with diagnostic, prognostic and predictive power are already in clinical trials or in a clinical setting for cancer. Outside cancer, strong evidence that complex disease originates in early life is opening up exciting new avenues for the detection of DNA methylation biomarkers for adverse early life environment and for estimation of future disease risk. However, there are a number of limitations to overcome before such biomarkers reach the clinic. Nevertheless, DNA methylation biomarkers have great potential to contribute to personalized medicine throughout life. We review the current state of play for DNA methylation biomarkers, discuss the barriers that must be crossed on the way to implementation in a clinical setting, and predict their future use for human disease.

Genome size estimation: a new methodology

Science.gov (United States)

Álvarez-Borrego, Josué; Gallardo-Escárate, Crisitian; Kober, Vitaly; López-Bonilla, Oscar

2007-03-01

Recently, within the cytogenetic analysis, the evolutionary relations implied in the content of nuclear DNA in plants and animals have received a great attention. The first detailed measurements of the nuclear DNA content were made in the early 40's, several years before Watson and Crick proposed the molecular structure of the DNA. In the following years Hewson Swift developed the concept of "C-value" in reference to the haploid phase of DNA in plants. Later Mirsky and Ris carried out the first systematic study of genomic size in animals, including representatives of the five super classes of vertebrates as well as of some invertebrates. From these preliminary results it became evident that the DNA content varies enormously between the species and that this variation does not bear relation to the intuitive notion from the complexity of the organism. Later, this observation was reaffirmed in the following years as the studies increased on genomic size, thus denominating to this characteristic of the organisms like the "Paradox of the C-value". Few years later along with the no-codification discovery of DNA the paradox was solved, nevertheless, numerous questions remain until nowadays unfinished, taking to denominate this type of studies like the "C-value enigma". In this study, we reported a new method for genome size estimation by quantification of fluorescence fading. We measured the fluorescence intensity each 1600 milliseconds in DAPI-stained nuclei. The estimation of the area under the graph (integral fading) during fading period was related with the genome size.

Structural DNA Nanotechnology: Artificial Nanostructures for Biomedical Research.

Science.gov (United States)

Ke, Yonggang; Castro, Carlos; Choi, Jong Hyun

2018-04-04

Structural DNA nanotechnology utilizes synthetic or biologic DNA as designer molecules for the self-assembly of artificial nanostructures. The field is founded upon the specific interactions between DNA molecules, known as Watson-Crick base pairing. After decades of active pursuit, DNA has demonstrated unprecedented versatility in constructing artificial nanostructures with significant complexity and programmability. The nanostructures could be either static, with well-controlled physicochemical properties, or dynamic, with the ability to reconfigure upon external stimuli. Researchers have devoted considerable effort to exploring the usability of DNA nanostructures in biomedical research. We review the basic design methods for fabricating both static and dynamic DNA nanostructures, along with their biomedical applications in fields such as biosensing, bioimaging, and drug delivery. Expected final online publication date for the Annual Review of Biomedical Engineering Volume 20 is June 4, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Better estimation of protein-DNA interaction parameters improve prediction of functional sites

Directory of Open Access Journals (Sweden)

O'Flanagan Ruadhan A

2008-12-01

Full Text Available Abstract Background Characterizing transcription factor binding motifs is a common bioinformatics task. For transcription factors with variable binding sites, we need to get many suboptimal binding sites in our training dataset to get accurate estimates of free energy penalties for deviating from the consensus DNA sequence. One procedure to do that involves a modified SELEX (Systematic Evolution of Ligands by Exponential Enrichment method designed to produce many such sequences. Results We analyzed low stringency SELEX data for E. coli Catabolic Activator Protein (CAP, and we show here that appropriate quantitative analysis improves our ability to predict in vitro affinity. To obtain large number of sequences required for this analysis we used a SELEX SAGE protocol developed by Roulet et al. The sequences obtained from here were subjected to bioinformatic analysis. The resulting bioinformatic model characterizes the sequence specificity of the protein more accurately than those sequence specificities predicted from previous analysis just by using a few known binding sites available in the literature. The consequences of this increase in accuracy for prediction of in vivo binding sites (and especially functional ones in the E. coli genome are also discussed. We measured the dissociation constants of several putative CAP binding sites by EMSA (Electrophoretic Mobility Shift Assay and compared the affinities to the bioinformatics scores provided by methods like the weight matrix method and QPMEME (Quadratic Programming Method of Energy Matrix Estimation trained on known binding sites as well as on the new sites from SELEX SAGE data. We also checked predicted genome sites for conservation in the related species S. typhimurium. We found that bioinformatics scores based on SELEX SAGE data does better in terms of prediction of physical binding energies as well as in detecting functional sites. Conclusion We think that training binding site detection

Calibration and LOD/LOQ estimation of a chemiluminescent hybridization assay for residual DNA in recombinant protein drugs expressed in E. coli using a four-parameter logistic model.

Science.gov (United States)

Lee, K R; Dipaolo, B; Ji, X

2000-06-01

Calibration is the process of fitting a model based on reference data points (x, y), then using the model to estimate an unknown x based on a new measured response, y. In DNA assay, x is the concentration, and y is the measured signal volume. A four-parameter logistic model was used frequently for calibration of immunoassay when the response is optical density for enzyme-linked immunosorbent assay (ELISA) or adjusted radioactivity count for radioimmunoassay (RIA). Here, it is shown that the same model or a linearized version of the curve are equally useful for the calibration of a chemiluminescent hybridization assay for residual DNA in recombinant protein drugs and calculation of performance measures of the assay.

DNA conformation on surfaces measured by fluorescence self-interference.

Science.gov (United States)

Moiseev, Lev; Unlü, M Selim; Swan, Anna K; Goldberg, Bennett B; Cantor, Charles R

2006-02-21

The conformation of DNA molecules tethered to the surface of a microarray may significantly affect the efficiency of hybridization. Although a number of methods have been applied to determine the structure of the DNA layer, they are not very sensitive to variations in the shape of DNA molecules. Here we describe the application of an interferometric technique called spectral self-interference fluorescence microscopy to the precise measurement of the average location of a fluorescent label in a DNA layer relative to the surface and thus determine specific information on the conformation of the surface-bound DNA molecules. Using spectral self-interference fluorescence microscopy, we have estimated the shape of coiled single-stranded DNA, the average tilt of double-stranded DNA of different lengths, and the amount of hybridization. The data provide important proofs of concept for the capabilities of novel optical surface analytical methods of the molecular disposition of DNA on surfaces. The determination of DNA conformations on surfaces and hybridization behavior provide information required to move DNA interfacial applications forward and thus impact emerging clinical and biotechnological fields.

Fabrication of DNA/Hydroxyapatite nanocomposites by simulated body fluid for gene delivery

Energy Technology Data Exchange (ETDEWEB)

Takeshita, Takayuki; Okamoto, Masami [Advanced Polymeric Nanostructured Materials Engineering, Graduate School of Engineering, Toyota Technological Institute, 2-12-1 Hisakata, Tempaku, Nagoya 468-8511 (Japan)

2015-05-22

The hydroxyapatite (HA) formation on the surface of DNA molecules in simulated body fluid (SBF) was examined. The osteoconductivity is estimated using SBF having ion concentrations approximately equal to those of human blood plasma. After immersion for 4 weeks in SBF at 36.5 °C, the HA crystallites possessing 1-14 micrometer in diameter grew on the surface of DNA molecules. The leaf flake-like and spherical shapes morphologies were observed through scanning electron microscopy analysis. Original peaks of both of DNA and HA were characterized by fourier transform infrared spectroscopy. The Ca/P ratio (1.1-1.5) in HA was estimated by energy dispersive X-ray analysis. After biomineralization, the calculated weight ratio of DNA/HA was 18/82 by thermogravimetry/differential thermal analysis. The molecular orbital computer simulation has been used to probe the interaction of DNA with two charge-balancing ions, CaOH{sup +} and CaH{sub 2}PO{sub 4}{sup +}. The adsorption enthalpy of the two ions on DNA having negative value was the evidence for the interface in mineralization of HA in SBF.

Relationship between DNA replication and DNA repair in human lymphocytes proliferating in vitro in the presence and in absence of mutagen

International Nuclear Information System (INIS)

Szyfter, K.; Wielgosz, M.Sz.; Kujawski, M.; Jaloszynski, P.; Zajaczek, S.

1995-01-01

The effects of mutagens on DNA replication and DNA repair were studied in peripheral blood lymphocytes (PBL) obtained from 21 healthy subjects, 2 samples from healthy heterozygote of ''Xeroderma pigmentosum'' (XP) and 2 samples from patient with clinically recognised XP. Inter-individual variations were found in DNA replication and in the level of spontaneous DNA repair measured under standard culture condition. Exposure of human PBL proliferating in vitro to B(a)P was followed by a partial inhibition of replicative DNA synthesis in all subjects and by an induction of DNA repair in healthy subjects. In XP patients DNA repair synthesis remained at the level attributed to spontaneous DNA repair. The response to mutagen varied individually. Results were analysed statistically. It was established that the studied indices of DNA synthesis correlate well with each other. The highest correlation was found between the levels of spontaneous and B(a)P-induced DNA repair. It is concluded that the level of spontaneous DNA repair is predictive for an estimation of cells ability to repair DNA damage. Inter-individual variations in the inhibition of DNA replication and in DNA repair synthesis are also dependent on the type of mutagen as shown by effects of other mutagens. Different effects of mutagen exposure on the inhibition of DNA replicative synthesis and induction of DNA repair can be explained by genetically controlled differences in the activity of enzymes responsible for mutagen processing and lesion removal. (author). 37 refs, 2 figs, 2 tabs

Correction of the lack of commutability between plasmid DNA and genomic DNA for quantification of genetically modified organisms using pBSTopas as a model.

Science.gov (United States)

Zhang, Li; Wu, Yuhua; Wu, Gang; Cao, Yinglong; Lu, Changming

2014-10-01

Plasmid calibrators are increasingly applied for polymerase chain reaction (PCR) analysis of genetically modified organisms (GMOs). To evaluate the commutability between plasmid DNA (pDNA) and genomic DNA (gDNA) as calibrators, a plasmid molecule, pBSTopas, was constructed, harboring a Topas 19/2 event-specific sequence and a partial sequence of the rapeseed reference gene CruA. Assays of the pDNA showed similar limits of detection (five copies for Topas 19/2 and CruA) and quantification (40 copies for Topas 19/2 and 20 for CruA) as those for the gDNA. Comparisons of plasmid and genomic standard curves indicated that the slopes, intercepts, and PCR efficiency for pBSTopas were significantly different from CRM Topas 19/2 gDNA for quantitative analysis of GMOs. Three correction methods were used to calibrate the quantitative analysis of control samples using pDNA as calibrators: model a, or coefficient value a (Cva); model b, or coefficient value b (Cvb); and the novel model c or coefficient formula (Cf). Cva and Cvb gave similar estimated values for the control samples, and the quantitative bias of the low concentration sample exceeded the acceptable range within ±25% in two of the four repeats. Using Cfs to normalize the Ct values of test samples, the estimated values were very close to the reference values (bias -13.27 to 13.05%). In the validation of control samples, model c was more appropriate than Cva or Cvb. The application of Cf allowed pBSTopas to substitute for Topas 19/2 gDNA as a calibrator to accurately quantify the GMO.

Interaction of sulforaphane with DNA and RNA.

Directory of Open Access Journals (Sweden)

Farzaneh Abassi Joozdani

Full Text Available Sulforaphane (SFN is an isothiocyanate found in cruciferous vegetables with anti-inflammatory, anti-oxidant and anti-cancer activities. However, the antioxidant and anticancer mechanism of sulforaphane is not well understood. In the present research, we reported binding modes, binding constants and stability of SFN-DNA and -RNA complexes by Fourier transform infrared (FTIR and UV-Visible spectroscopic methods. Spectroscopic evidence showed DNA intercalation with some degree of groove binding. SFN binds minor and major grooves of DNA and backbone phosphate (PO2, while RNA binding is through G, U, A bases with some degree of SFN-phosphate (PO2 interaction. Overall binding constants were estimated to be K(SFN-DNA=3.01 (± 0.035×10(4 M(-1 and K(SFN-RNA= 6.63 (±0.042×10(3 M(-1. At high SFN concentration (SFN/RNA = 1/1, DNA conformation changed from B to A occurred, while RNA remained in A-family structure.

The use of PCR techniques to detect genetic variations in Cassava (Manihot esculenta L. Crantz): minisatellite and RAPD analysis

International Nuclear Information System (INIS)

Pawlicki, N.; Sangwan, R.S.; Sangwan-Norreel, B.; Koffi Konan, N.

1998-01-01

Cassava is an important tuber crop grown in the tropical and subtropical regions. Recently, we developed protocols for efficient somatic embryogenesis using zygotic embryos and nodal axillary meristems in order to reduce the genotype effect. Thereafter flow cytophotometry and randomly amplified polymorphic DNA (RAPD) markers were used to assess the ploidy level and the genetic fidelity of cassava plants regenerated by somatic embryogenesis. No change in the ploidy level of the regenerated plants was observed in comparison with the control plants. In the same way, monomorphic profiles of RAPD were obtained for the different cassava plants regenerated by somatic embryogenesis. The genetic analysis of calli showed only a few differences. Using two pairs of heterologous micro satellite primers developed in a wild African grass, a monomorphic pattern was also detected. Moreover, cultivars of different origins were also analysed using these PCR techniques. Our data from RAPD and materialistic analyses suggested that these techniques can be efficiently used to detect genetic variations in cassava. (author)

Sequenza: allele-specific copy number and mutation profiles from tumor sequencing data

DEFF Research Database (Denmark)

Favero, Francesco; Joshi, Tejal; Marquard, Andrea Marion

2015-01-01

: Comparison between Sequenza/exome and SNP/ASCAT revealed strong correlation in cellularity (Pearson’s r = 0.90) and ploidy estimates (r = 0.42, or r = 0.94 after manual inspecting alternative solutions). This performance was noticeably superior to previously published algorithms. In addition, in artificial...

Single-cell analysis of ploidy and centrosomes underscores the peculiarity of normal hepatocytes.

Directory of Open Access Journals (Sweden)

Francesca Faggioli

Full Text Available Polyploidization is the most well recognized feature of the liver. Yet, a quantitative and behavioral analysis of centrosomes and DNA content in normal hepatocytes has been limited by the technical challenges of methods available. By using a novel approach employing FISH for chromosomes 18, X and Y we provide, for the first time, a detailed analysis of DNA copies during physiological development in the liver at single cell level. We demonstrate that aneuploidy and unbalanced DNA content in binucleated hepatocytes are common features in normal adult liver. Despite the common belief that hepatocytes contain 1, 2 or no more than 4 centrosomes, our double staining for centrosome associated proteins reveals extranumerary centrosomes in a high percentage of cells as early as 15 days of age. We show that in murine liver the period between 15 days and 1.5 months marks the transition from a prevalence of mononucleated cells to up to 75% of binucleated cells. Our data demonstrate that this timing correlates with a switch in centrosomes number. At 15 days the expected 1 or 2 centrosomes converge with several hepatocytes that contain 3 centrosomes; at 1.5 months the percentage of cells with 3 centrosomes decreases concomitantly with the increase of cells with more than 4 centrosomes. Our analysis shows that the extranumerary centrosomes emerge in concomitance with the process of binucleation and polyploidization and maintain α-tubulin nucleation activity. Finally, by integrating interphase FISH and immunofluorescent approaches, we detected an imbalance between centrosome number and DNA content in liver cells that deviates from the equilibrium expected in normal cells. We speculate that these unique features are relevant to the peculiar biological function of liver cells which are continuously challenged by stress, a condition that could predispose to genomic instability.

Population genetic data for six non-combined DNA index system ...

African Journals Online (AJOL)

The respective values for the combined power of exclusion in these populations were 0.94 and 0.99. The allele frequency data generated can be used for estimating DNA profile frequencies for the studied populations residing in South Africa. Key words: Allele frequencies, MiniSTR, DNA typing, population data, South Africa ...

Configurational entropy change of netropsin and distamycin upon DNA minor-groove binding.

Science.gov (United States)

Dolenc, Jozica; Baron, Riccardo; Oostenbrink, Chris; Koller, Joze; van Gunsteren, Wilfred F

2006-08-15

Binding of a small molecule to a macromolecular target reduces its conformational freedom, resulting in a negative entropy change that opposes the binding. The goal of this study is to estimate the configurational entropy change of two minor-groove-binding ligands, netropsin and distamycin, upon binding to the DNA duplex d(CGCGAAAAACGCG).d(CGCGTTTTTCGCG). Configurational entropy upper bounds based on 10-ns molecular dynamics simulations of netropsin and distamycin in solution and in complex with DNA in solution were estimated using the covariance matrix of atom-positional fluctuations. The results suggest that netropsin and distamycin lose a significant amount of configurational entropy upon binding to the DNA minor groove. The estimated changes in configurational entropy for netropsin and distamycin are -127 J K(-1) mol(-1) and -104 J K(-1) mol(-1), respectively. Estimates of the configurational entropy contributions of parts of the ligands are presented, showing that the loss of configurational entropy is comparatively more pronounced for the flexible tails than for the relatively rigid central body.

DNA repair in human bronchial epithelial cells

International Nuclear Information System (INIS)

Fornace, A.J. Jr.; Lechner, J.F.; Grafstrom, R.C.; Harris, C.C.

1982-01-01

The purpose of this investigation was to compare the response of human cell types (bronchial epithelial cells and fibroblasts and skin fibroblasts) to various DNA damaging agents. Repair of DNA single strand breaks (SSB) induced by 5 krads of X-ray was similar for all cell types; approximately 90% of the DNA SSB were rejoined within one hour. During excision repair of DNA damage from u.v.-radiation, the frequencies of DNA SSB as estimated by the alkaline elution technique, were similar in all cell types. Repair replication as measured by BND cellulose chromatography was also similar in epithelial and fibroblastic cells after u.v.-irradiation. Similar levels of SSB were also observed in epithelial and fibroblastic cells after exposure to chemical carcinogens: 7,12-dimethylbenz[a]anthracene; benzo[a]pyrene diol epoxide (BPDE); or N-methyl-N-nitro-N-nitrosoguanidine. Significant repair replication of BPDE-induced DNA damage was detected in both bronchial epithelial and fibroblastic cells, although the level in fibroblasts was approximately 40% of that in epithelial cells. The pulmonary carcinogen asbestos did not damage DNA. DNA-protein crosslinks induced by formaldehyde were rapidly removed in bronchial cells. Further, epithelial and fibroblastic cells, which were incubated with formaldehyde and the polymerase inhibitor combination of cytosine arabinoside and hydroxyurea, accumulated DNA SSB at approximately equal frequencies. These results should provide a useful background for further investigations of the response of human bronchial cells to various DNA damaging agents

Nuclear and mitochondrial DNA quantification of various forensic materials.

Science.gov (United States)

Andréasson, H; Nilsson, M; Budowle, B; Lundberg, H; Allen, M

2006-12-01

Due to the different types and quality of forensic evidence materials, their DNA content can vary substantially, and particularly low quantities can impact the results in an identification analysis. In this study, the quantity of mitochondrial and nuclear DNA was determined in a variety of materials using a previously described real-time PCR method. DNA quantification in the roots and distal sections of plucked and shed head hairs revealed large variations in DNA content particularly between the root and the shaft of plucked hairs. Also large intra- and inter-individual variations were found among hairs. In addition, DNA content was estimated in samples collected from fingerprints and accessories. The quantification of DNA on various items also displayed large variations, with some materials containing large amounts of nuclear DNA while no detectable nuclear DNA and only limited amounts of mitochondrial DNA were seen in others. Using this sensitive real-time PCR quantification assay, a better understanding was obtained regarding DNA content and variation in commonly analysed forensic evidence materials and this may guide the forensic scientist as to the best molecular biology approach for analysing various forensic evidence materials.

DNA replication and the repair of DNA strand breaks in nuclei of Physarum polycephalum. Progress report, September 1, 1977--July 31, 1978

International Nuclear Information System (INIS)

Brewer, E.N.; Nygaard, O.F.; Kuncio, G.

1978-01-01

Isolated nuclei and intact plasmodia of Physarum contain a heat-stable stimulator of nuclear DNA replication. This substance has been purified extensively and found to contain both protein and carbohydrate. The molecular weight, estimated by gel filtration, is ca. 30,000 d. The purified material does not exhibit DNA polymerase or DNase activity, and does not stimulate DNA polymerase activity per se. In the presence of the stimulatory factor, DNA chain elongation occurs at an elevated rate, and continues for a longer time than in its absence, but G 2 nuclei are not stimulated to initiate DNA synthesis. Double-strand breaks in nuclear DNA of irradiated plasmodia are repaired in vitro to a greater extent following nuclear isolation during G 2 , and the DNA of unirradiated plasmodia is less susceptible to double-strand breakage during cell-free nuclear incubation, than is the DNA of S-phase nuclei. This correlation suggests a common basis for both observations, for example an increase in deoxyribonuclease activity or a decrease in DNA ligase activity during the S period. This, in turn, may account for the cell cycle-dependent sensitivity of this organism, in terms of mitotic delay, to ionizing radiation

Modeling the Sensitivity of Field Surveys for Detection of Environmental DNA (eDNA.

Directory of Open Access Journals (Sweden)

Martin T Schultz

Full Text Available The environmental DNA (eDNA method is the practice of collecting environmental samples and analyzing them for the presence of a genetic marker specific to a target species. Little is known about the sensitivity of the eDNA method. Sensitivity is the probability that the target marker will be detected if it is present in the water body. Methods and tools are needed to assess the sensitivity of sampling protocols, design eDNA surveys, and interpret survey results. In this study, the sensitivity of the eDNA method is modeled as a function of ambient target marker concentration. The model accounts for five steps of sample collection and analysis, including: 1 collection of a filtered water sample from the source; 2 extraction of DNA from the filter and isolation in a purified elution; 3 removal of aliquots from the elution for use in the polymerase chain reaction (PCR assay; 4 PCR; and 5 genetic sequencing. The model is applicable to any target species. For demonstration purposes, the model is parameterized for bighead carp (Hypophthalmichthys nobilis and silver carp (H. molitrix assuming sampling protocols used in the Chicago Area Waterway System (CAWS. Simulation results show that eDNA surveys have a high false negative rate at low concentrations of the genetic marker. This is attributed to processing of water samples and division of the extraction elution in preparation for the PCR assay. Increases in field survey sensitivity can be achieved by increasing sample volume, sample number, and PCR replicates. Increasing sample volume yields the greatest increase in sensitivity. It is recommended that investigators estimate and communicate the sensitivity of eDNA surveys to help facilitate interpretation of eDNA survey results. In the absence of such information, it is difficult to evaluate the results of surveys in which no water samples test positive for the target marker. It is also recommended that invasive species managers articulate concentration

Nuclear DNA content variation in life history phases of the Bonnemasoniaceae (Rhodophyta).

Science.gov (United States)

Salvador Soler, Noemi; Gómez Garreta, Amelia; Ribera Siguan, Ma Antonia; Kapraun, Donald F

2014-01-01

Nuclear DNA content in gametophytes and sporophytes or the prostrate phases of the following species of Bonnemaisoniaceae (Asparagopsis armata, Asparagopsis taxiformis, Bonnemaisonia asparagoides, Bonnemaisonia clavata and Bonnemaisonia hamifera) were estimated by image analysis and static microspectrophotometry using the DNA-localizing fluorochrome DAPI (4', 6-diamidino-2-phenylindole, dilactate) and the chicken erythrocytes standard. These estimates expand on the Kew database of DNA nuclear content. DNA content values for 1C nuclei in the gametophytes (spermatia and vegetative cells) range from 0.5 pg to 0.8 pg, and for 2C nuclei in the sporophytes or the prostrate phases range from 1.15-1.7 pg. Although only the 2C and 4C values were observed in the sporophyte or the prostrate phase, in the vegetative cells of the gametophyte the values oscillated from 1C to 4C, showing the possible start of endopolyploidy. The results confirm the alternation of nuclear phases in these Bonnemaisoniaceae species, in those that have tetrasporogenesis, as well as those that have somatic meiosis. The availability of a consensus phylogenetic tree for Bonnemaisoniaceae has opened the way to determine evolutionary trends in DNA contents. Both the estimated genome sizes and the published chromosome numbers for Bonnemaisoniaceae suggest a narrow range of values consistent with the conservation of an ancestral genome.

Nuclear DNA Content Variation in Life History Phases of the Bonnemasoniaceae (Rhodophyta)

Science.gov (United States)

Salvador Soler, Noemi; Gómez Garreta, Amelia; Ribera Siguan, Mª Antonia; Kapraun, Donald F.

2014-01-01

Nuclear DNA content in gametophytes and sporophytes or the prostrate phases of the following species of Bonnemaisoniaceae (Asparagopsis armata, Asparagopsis taxiformis, Bonnemaisonia asparagoides, Bonnemaisonia clavata and Bonnemaisonia hamifera) were estimated by image analysis and static microspectrophotometry using the DNA-localizing fluorochrome DAPI (4′, 6-diamidino-2-phenylindole, dilactate) and the chicken erythrocytes standard. These estimates expand on the Kew database of DNA nuclear content. DNA content values for 1C nuclei in the gametophytes (spermatia and vegetative cells) range from 0.5 pg to 0.8 pg, and for 2C nuclei in the sporophytes or the prostrate phases range from 1.15–1.7 pg. Although only the 2C and 4C values were observed in the sporophyte or the prostrate phase, in the vegetative cells of the gametophyte the values oscillated from 1C to 4C, showing the possible start of endopolyploidy. The results confirm the alternation of nuclear phases in these Bonnemaisoniaceae species, in those that have tetrasporogenesis, as well as those that have somatic meiosis. The availability of a consensus phylogenetic tree for Bonnemaisoniaceae has opened the way to determine evolutionary trends in DNA contents. Both the estimated genome sizes and the published chromosome numbers for Bonnemaisoniaceae suggest a narrow range of values consistent with the conservation of an ancestral genome. PMID:24465835

A proposal of a novel DNA modification mechanism induced by irradiation

International Nuclear Information System (INIS)

Oka, Toshitaka

2016-01-01

This article depicts a proposal of a novel DNA modification mechanism induced by irradiation, and is written as an award work from Japanese Society of Radiation Chemistry. The mechanism of DNA modification induced by K-shell photoabsorption of nitrogen and oxygen atoms was investigated by electron paramagnetic resonance and x-ray absorption near edge structure measurements of calf thymus DNA film. The EPR intensities for DNA film were twofold times larger than those estimated based on the photoabsorption cross section. This suggests that the DNA film itself forms unpaired electron species through the excitation of enhanced electron recapturing, known as the postcollision interaction process. (author)

An "escape clock" for estimating the turnover of SIV DNA in resting CD4⁺ T cells.

Directory of Open Access Journals (Sweden)

Jeanette Reece

Full Text Available Persistence of HIV DNA presents a major barrier to the complete control of HIV infection under current therapies. Most studies suggest that cells with latently integrated HIV decay very slowly under therapy. However, it is much more difficult to study the turnover and persistence of HIV DNA during active infection. We have developed an "escape clock" approach for measuring the turnover of HIV DNA in resting CD4+ T cells. This approach studies the replacement of wild-type (WT SIV DNA present in early infection by CTL escape mutant (EM strains during later infection. Using a strain-specific real time PCR assay, we quantified the relative amounts of WT and EM strains in plasma SIV RNA and cellular SIV DNA. Thus we can track the formation and turnover of SIV DNA in sorted resting CD4+ T cells. We studied serial plasma and PBMC samples from 20 SIV-infected Mane-A*10 positive pigtail macaques that have a signature Gag CTL escape mutation. In animals with low viral load, WT virus laid down early in infection is extremely stable, and the decay of this WT species is very slow, consistent with findings in subjects on anti-retroviral medications. However, during active, high level infection, most SIV DNA in resting cells was turning over rapidly, suggesting a large pool of short-lived DNA produced by recent infection events. Our results suggest that, in order to reduce the formation of a stable population of SIV DNA, it will be important either to intervene very early or intervene during active replication.

A model for the induction of DNA damages and their evolution into cell clonogenic inactivation

International Nuclear Information System (INIS)

Yamaguchi, Hiroshi; Ohara, Hiroshi; Waker, A.J.

2006-01-01

The dependence of the initial production of DNA damages on radiation quality was examined by using a proposed new model on the basis of target theory. For the estimation of DNA damage-production by different radiation qualities, five possible modes of radiation action, including both direct and indirect effects, were assumed inside a target the molecular structure of which was defined to consist of 10 base-pairs of DNA surrounded by water molecules. The induction of DNA damage was modeled on the basis of comparisons between the primary ionization mean free path and the distance between pairs of ionized atoms, such distance being characteristic on the mode of radiation action. The OH radicals per average energy to produce an ion pair on the nanosecond time scale was estimated and used for indirect action. Assuming a relation between estimated yields of DNA damages and experimental inactivation cross sections for AT-cells, the present model enabled the quantitative reproduction of experimental results for AT-cell killing under aerobic or hypoxic conditions. The results suggest a higher order organization of DNA in a way that there will be at least two types of water environment, one filling half the space surrounding DNA with a depth of 3.7-4.3 nm and the other filling all space with a depth 4.6-4.9 nm. (author)

Forensic DNA methylation profiling from evidence material for investigative leads

Science.gov (United States)

Lee, Hwan Young; Lee, Soong Deok; Shin, Kyoung-Jin

2016-01-01

DNA methylation is emerging as an attractive marker providing investigative leads to solve crimes in forensic genetics. The identification of body fluids that utilizes tissue-specific DNA methylation can contribute to solving crimes by predicting activity related to the evidence material. The age estimation based on DNA methylation is expected to reduce the number of potential suspects, when the DNA profile from the evidence does not match with any known person, including those stored in the forensic database. Moreover, the variation in DNA implicates environmental exposure, such as cigarette smoking and alcohol consumption, thereby suggesting the possibility to be used as a marker for predicting the lifestyle of potential suspect. In this review, we describe recent advances in our understanding of DNA methylation variations and the utility of DNA methylation as a forensic marker for advanced investigative leads from evidence materials. [BMB Reports 2016; 49(7): 359-369] PMID:27099236

DNA measurements after radiation-induced tissue structure of thyroid gland of rats Análise do conteúdo de DNA (ploidia em tecidos de glândula tireóide de ratos após indução por radiação

Directory of Open Access Journals (Sweden)

Roberto Souza Camargo

2005-06-01

Full Text Available INTRODUCTION: Thyroid gland exposures to radiation induce nuclear chromosomal alteration. Objective: To evaluate the DNA content of thyroid gland submitted to radiation. MATERIALS AND METHODS: We radiated 75 rats while 25 were not radiated to be used as control group. Exposure was conducted by the use of Cobalt-60 radioactive source in the right anterior cervical region in a field of 3-30cm, comprising the second and the sixth tracheal rings with 600-centigray (cGY doses. The DNA content (ploidy was obtained with Feulgen-thionin stain and was quantified with CAS 200 quantitative measurement equipment. RESULTS: Diploid pattern was obtained in 88 cases (95.7%, independently of time of exposure: on the other hand, aneuploidy was observed in four cases (4.3% only in the group sacrificed to the 33 days. Eight cases were excluded due to technical reasons. CONCLUSION: The early aneuploid pattern found in our study certainly corroborated that radiation affects thyroid gland with important consequences in terms of disorders.INTRODUÇÃO: A exposição da tireóide à irradiação está associada à alteração do componente cromossômico nessa glândula. OBJETIVO: Avaliar o conteúdo de DNA (ploidia em glândula tireóide submetida à radiação. MATERIAL E MÉTODOS: Foram irradiadas tireóides de 75 ratos; 25 foram usados como grupo controle e não sofreram irradiação. A exposição à irradiação foi realizada com o uso de Cobalt-60 na região cervical anterior direita com espaço de 3-30cm, com anel traqueal de 2º e 6º e dose de 600 centigrays (cGY. O conteúdo de DNA (ploidia foi obtido com o método de Feulgen-Thionin e quantificado com o aparelho CAS 200 de citometria estática. RESULTADO: Dos 92 casos, 88 (95,7% foram diplóides, independente do tempo de exposição; aneuploidia foi observada em quatro casos (4,3%, somente no grupo sacrificado aos 33 dias. Oito casos foram excluídos por problemas técnicos. CONCLUSÃO: A irradiação afeta

Amplification of DNA mixtures--Missing data approach

DEFF Research Database (Denmark)

Tvedebrink, Torben; Eriksen, Poul Svante; Mogensen, Helle Smidt

2008-01-01

This paper presents a model for the interpretation of results of STR typing of DNA mixtures based on a multivariate normal distribution of peak areas. From previous analyses of controlled experiments with mixed DNA samples, we exploit the linear relationship between peak heights and peak areas...... DNA samples, it is only possible to observe the cumulative peak heights and areas. Complying with this latent structure, we use the EM-algorithm to impute the missing variables based on a compound symmetry model. That is the measurements are subject to intra- and inter-loci correlations not depending...... on the actual alleles of the DNA profiles. Due to factorization of the likelihood, properties of the normal distribution and use of auxiliary variables, an ordinary implementation of the EM-algorithm solves the missing data problem. We estimate the parameters in the model based on a training data set. In order...

Opportunities for measuring DNA synthesis time by quantitative autoradiography

International Nuclear Information System (INIS)

Vasileva, D.

1980-01-01

DNA sysntesis time (Tsub(s)) in cells of the canine erythropoiesis and myelopoiesis pools was determined by quantitative autoradiography according to Doermer. In contrast to mitosis labelling for Tsub(s) estimation as so far applied, this technique uses well-differentiated cells. After blocking endogeneous DNA synthesis with 5-fluorodeoxyuridine, its further course becomes dependent on exogeneous supply of thymidine, in the form of 14 C-thymidine. From incroporation of the latter into the individual cell within a definite time span (3-7 min) and taking into account its total amount, Tsub(s) may be calculated. The data thus obtained were found to agree with Tsub(s) values as estimated from the labelled mitosis curve

Estimation of serum concentration of parvovirus B19 DNA by PCR in patients with chronic anemia

DEFF Research Database (Denmark)

Hornsleth, A.; Carlsen, K. M.; Christensen, Laurids Siig

1994-01-01

Parvovirus B19 DNA was detected in serum samples from 10 out of 42 patients with chronic anaemia, the majority of whom suffered from aplastic anaemia, haemolytic anaemia, pure red cell anaemia or myelodysplastic syndrome. Nested PCR methods with sensitivities of 0.005-0.05 fg DNA were developed. ...

Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

Science.gov (United States)

McCutchen-Maloney, Sandra L.

2002-01-01

Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

Effects of humic acid on DNA quantification with Quantifiler® Human DNA Quantification kit and short tandem repeat amplification efficiency.

Science.gov (United States)

Seo, Seung Bum; Lee, Hye Young; Zhang, Ai Hua; Kim, Hye Yeon; Shin, Dong Hoon; Lee, Soong Deok

2012-11-01

Correct DNA quantification is an essential part to obtain reliable STR typing results. Forensic DNA analysts often use commercial kits for DNA quantification; among them, real-time-based DNA quantification kits are most frequently used. Incorrect DNA quantification due to the presence of PCR inhibitors may affect experiment results. In this study, we examined the alteration degree of DNA quantification results estimated in DNA samples containing a PCR inhibitor by using a Quantifiler® Human DNA Quantification kit. For experiments, we prepared approximately 0.25 ng/μl DNA samples containing various concentrations of humic acid (HA). The quantification results were 0.194-0.303 ng/μl at 0-1.6 ng/μl HA (final concentration in the Quantifiler reaction) and 0.003-0.168 ng/μl at 2.4-4.0 ng/μl HA. Most DNA quantity was undetermined when HA concentration was higher than 4.8 ng/μl HA. The C (T) values of an internal PCR control (IPC) were 28.0-31.0, 36.5-37.1, and undetermined at 0-1.6, 2.4, and 3.2 ng/μl HA. These results indicate that underestimated DNA quantification results may be obtained in the DNA sample with high C (T) values of IPC. Thus, researchers should carefully interpret the DNA quantification results. We additionally examined the effects of HA on the STR amplification by using an Identifiler® kit and a MiniFiler™ kit. Based on the results of this study, it is thought that a better understanding of various effects of HA would help researchers recognize and manipulate samples containing HA.

DNA repair, human cancer and assessment of radiation hazards

International Nuclear Information System (INIS)

Paterson, M.C.; Myers, D.K.

1979-09-01

Cancers, like genetic defects, are thought to be caused primarily by changes in DNA. Part of the evidence in support of this hypothesis derives from the study of certain rare hereditary disorders in man associated with high risk of cancer. Cells derived from patients suffering from at least one of these disorders, ataxia telangiectasia, appear to be defective in their ability to repair the damage caused by radiation and/or certain other environmental agents. Studies of the consequences of DNA repair suggest that currently accepted estimates of the carcinogenic hazards of low level radiation are substantially correct. There would appear to be some margin of safety involved in these risk estimates for the majority of the population, but any major reduction in the currently accepted risk estimates appears inadvisable in view of the existence of potentially radiosensitive subgroups forming a minority in the general population. (author)

Ecological niche modelling and nDNA sequencing support a new, morphologically cryptic beetle species unveiled by DNA barcoding.

Science.gov (United States)

Hawlitschek, Oliver; Porch, Nick; Hendrich, Lars; Balke, Michael

2011-02-09

DNA sequencing techniques used to estimate biodiversity, such as DNA barcoding, may reveal cryptic species. However, disagreements between barcoding and morphological data have already led to controversy. Species delimitation should therefore not be based on mtDNA alone. Here, we explore the use of nDNA and bioclimatic modelling in a new species of aquatic beetle revealed by mtDNA sequence data. The aquatic beetle fauna of Australia is characterised by high degrees of endemism, including local radiations such as the genus Antiporus. Antiporus femoralis was previously considered to exist in two disjunct, but morphologically indistinguishable populations in south-western and south-eastern Australia. We constructed a phylogeny of Antiporus and detected a deep split between these populations. Diagnostic characters from the highly variable nuclear protein encoding arginine kinase gene confirmed the presence of two isolated populations. We then used ecological niche modelling to examine the climatic niche characteristics of the two populations. All results support the status of the two populations as distinct species. We describe the south-western species as Antiporus occidentalis sp.n. In addition to nDNA sequence data and extended use of mitochondrial sequences, ecological niche modelling has great potential for delineating morphologically cryptic species.

Antigen-antibody reactions of UV-irradiated phage DNA

International Nuclear Information System (INIS)

Fink, A.

1976-01-01

The observation of others could be confirmed that UV-irradiated DNA is a better immunogen than unirradiated DNA. The author's immune sera contained a high amount of antibodies with a specific action against photoproducts in the DNA. The thymine dimer was identified as relevant photoproduct and thus as antigenic determinant. In comparison, the amount of unspecific antibodies reacting with denaturated DNA was low and varied between sera. Thymin-dimer antibodies showed a high specificity without cross-reaction with other pyrimidine dimers such as anti CC and anti CT; they belong to the class of IgG molecules. UV-irradiated dinucleotide dTpT is sufficient to induce the formation of antibodies reacting with the cis-syn thymine dimers in UV-irradiated DNA. Antibody binding is proportional to the UV doses applied to the DNA. When using completely denaturated DNA, there is a linear increase changing into a plateau at higher doses. The extent of antigen-antibody binding is strongly dependent on the degree of denaturation of the DNA. With increasing denaturation, the antibody binding of the DNA increases. The antigen-antibody reaction can thus be used to estimate the degree of denaturation of the DNA. There were no signs of an influence of the degree of denaturation of the DNA on the quantum yield of thymine dimers. The different amounts of antibodies is therefore due to the masking of thymine dimers in native DNA. When irradiating intact phage particles, there was no sign of an influence of the phages' protein covers on the antibody binding capacity of DNA compared with DNA irradiated in vitro. (orig.) [de

Salix transect of Europe: variation in ploidy and genome size in willow-associated common nettle, Urtica dioica L. sens. lat., from Greece to arctic Norway.

Science.gov (United States)

Cronk, Quentin; Hidalgo, Oriane; Pellicer, Jaume; Percy, Diana; Leitch, Ilia J

2016-01-01

The common stinging nettle, Urtica dioica L. sensu lato, is an invertebrate "superhost", its clonal patches maintaining large populations of insects and molluscs. It is extremely widespread in Europe and highly variable, and two ploidy levels (diploid and tetraploid) are known. However, geographical patterns in cytotype variation require further study. We assembled a collection of nettles in conjunction with a transect of Europe from the Aegean to Arctic Norway (primarily conducted to examine the diversity of Salix and Salix -associated insects). Using flow cytometry to measure genome size, our sample of 29 plants reveals 5 diploids and 24 tetraploids. Two diploids were found in SE Europe (Bulgaria and Romania) and three diploids in S. Finland. More detailed cytotype surveys in these regions are suggested. The tetraploid genome size (2C value) varied between accessions from 2.36 to 2.59 pg. The diploids varied from 1.31 to 1.35 pg per 2C nucleus, equivalent to a haploid genome size of c. 650 Mbp. Within the tetraploids, we find that the most northerly samples (from N. Finland and arctic Norway) have a generally higher genome size. This is possibly indicative of a distinct population in this region.

DNA synthesis in vitro in human fibroblast preparations

Energy Technology Data Exchange (ETDEWEB)

Kaufmann, W.K.

1983-01-01

When confluent cultures of human fibroblasts were ultraviolet irradiated and either permeabilized or lysed, three types of DNA synthesis were subsequently observed during incubation in vitro: (A) a low level of DNA replication, which ceased after 15-30 min incubation at 37/sup 0/C; (B) radiation-dependent reparative gap-filling, which also ceased after 15 min at 37/sup 0/C; and (C) radiation-independent DNA synthesis, which was not semiconservative and proceeded at a linear rate for 1 hr at 37/sup 0/C. Normal and xeroderma pigmentosum fibroblasts displayed different rates of radiation-dependent reparative gap-filling after lysis but similar rates of radiation-independent DNA synthesis. The rates of DNA replication and radiation-independent DNA synthesis were less in the permeable cell system than in the lysed cell system, whereas radiation-dependent reparative gap-filling was the same in both. Preparations of permeable and lysed cells activated radiation-dependent reparative gap-filling at about 15% of the rate estimated for intact cells. No radiation-dependent DNA strand breaks, as assayed by alkaline elution, were observed in the lysed cell preparation. Some radiation-dependent breaks were observed in the permeable cell preparation, but radiation-dependent DNA breakage was less than that seen in intact cells. This inability to incise DNA at damaged sites could account for the low rate of activation of reparative gap-filling in vitro. DNA strand breaks were produced in fibroblast preparations nonspecifically during lysis or permeabilization and incubation in vitro, and this breakage of DNA probably was responsible for the radiation-independent DNA synthesis.

Asynchronous Magnetic Bead Rotation (AMBR Microviscometer for Label-Free DNA Analysis

Directory of Open Access Journals (Sweden)

Yunzi Li

2014-03-01

Full Text Available We have developed a label-free viscosity-based DNA detection system, using paramagnetic beads as an asynchronous magnetic bead rotation (AMBR microviscometer. We have demonstrated experimentally that the bead rotation period is linearly proportional to the viscosity of a DNA solution surrounding the paramagnetic bead, as expected theoretically. Simple optical measurement of asynchronous microbead motion determines solution viscosity precisely in microscale volumes, thus allowing an estimate of DNA concentration or average fragment length. The response of the AMBR microviscometer yields reproducible measurement of DNA solutions, enzymatic digestion reactions, and PCR systems at template concentrations across a 5000-fold range. The results demonstrate the feasibility of viscosity-based DNA detection using AMBR in microscale aqueous volumes.

Supercoiled circular DNA of an insect granulosis virus.

Science.gov (United States)

Tweeten, K A; Bulla, L A; Consigli, R A

1977-08-01

The DNA of the granulosis virus of the Indian meal moth, Plodia interpunctella, was characterized by physical chemical and electron microscopic techniques. Twenty-five percent of the DNA extracted from purified virus was isolated as supercoiled circular molecules. The remaining 75% consisted of relaxed circular molecules. These molecular forms were indicated by the production of two radioactive bands during sedimentation of (3)H-labeled granulosis virus DNA in alkaline sucrose gradients or in equilibrium density gradients of neutral cesium chloride/propidium iodide. Electron microscopic visualization of the DNA that banded at the higher density in the latter gradients revealed supercoiled structures whereas that of DNA that banded at the lower density demonstrated relaxed circular molecules. The superhelical molecules were converted to relaxed circles by treatment with pancreatic DNase. The molecular weight of the viral DNA was calculated to be 81 x 10(6) by sedimentation in neutral sucrose and 78 x 10(6) by sedimentation in alkaline sucrose. The molecular weight estimated from length measurements in electron micrographs was 76 x 10(6). The buoyant density of the granulosis virus DNA was 1.703 g/cm(3) and that of its insect host DNA was 1.697 g/cm(3). Equilibrium sedimentation in cesium chloride and thermal denaturation indicated G + C contents of 44% and 39% for the viral and host DNA, respectively.

Comparative analysis of protocols for DNA extraction from soybean caterpillars.

Science.gov (United States)

Palma, J; Valmorbida, I; da Costa, I F D; Guedes, J V C

2016-04-07

Genomic DNA extraction is crucial for molecular research, including diagnostic and genome characterization of different organisms. The aim of this study was to comparatively analyze protocols of DNA extraction based on cell lysis by sarcosyl, cetyltrimethylammonium bromide, and sodium dodecyl sulfate, and to determine the most efficient method applicable to soybean caterpillars. DNA was extracted from specimens of Chrysodeixis includens and Spodoptera eridania using the aforementioned three methods. DNA quantification was performed using spectrophotometry and high molecular weight DNA ladders. The purity of the extracted DNA was determined by calculating the A260/A280 ratio. Cost and time for each DNA extraction method were estimated and analyzed statistically. The amount of DNA extracted by these three methods was sufficient for PCR amplification. The sarcosyl method yielded DNA of higher purity, because it generated a clearer pellet without viscosity, and yielded high quality amplification products of the COI gene I. The sarcosyl method showed lower cost per extraction and did not differ from the other methods with respect to preparation times. Cell lysis by sarcosyl represents the best method for DNA extraction in terms of yield, quality, and cost effectiveness.

DNA content analysis allows discrimination between Trypanosoma cruzi and Trypanosoma rangeli.

Science.gov (United States)

Naves, Lucila Langoni; da Silva, Marcos Vinícius; Fajardo, Emanuella Francisco; da Silva, Raíssa Bernardes; De Vito, Fernanda Bernadelli; Rodrigues, Virmondes; Lages-Silva, Eliane; Ramírez, Luis Eduardo; Pedrosa, André Luiz

2017-01-01

Trypanosoma cruzi, a human protozoan parasite, is the causative agent of Chagas disease. Currently the species is divided into six taxonomic groups. The genome of the CL Brener clone has been estimated to be 106.4-110.7 Mb, and DNA content analyses revealed that it is a diploid hybrid clone. Trypanosoma rangeli is a hemoflagellate that has the same reservoirs and vectors as T. cruzi; however, it is non-pathogenic to vertebrate hosts. The haploid genome of T. rangeli was previously estimated to be 24 Mb. The parasitic strains of T. rangeli are divided into KP1(+) and KP1(-). Thus, the objective of this study was to investigate the DNA content in different strains of T. cruzi and T. rangeli by flow cytometry. All T. cruzi and T. rangeli strains yielded cell cycle profiles with clearly identifiable G1-0 (2n) and G2-M (4n) peaks. T. cruzi and T. rangeli genome sizes were estimated using the clone CL Brener and the Leishmania major CC1 as reference cell lines because their genome sequences have been previously determined. The DNA content of T. cruzi strains ranged from 87,41 to 108,16 Mb, and the DNA content of T. rangeli strains ranged from 63,25 Mb to 68,66 Mb. No differences in DNA content were observed between KP1(+) and KP1(-) T. rangeli strains. Cultures containing mixtures of the epimastigote forms of T. cruzi and T. rangeli strains resulted in cell cycle profiles with distinct G1 peaks for strains of each species. These results demonstrate that DNA content analysis by flow cytometry is a reliable technique for discrimination between T. cruzi and T. rangeli isolated from different hosts.

Force-dependent melting of supercoiled DNA at thermophilic temperatures.

Science.gov (United States)

Galburt, E A; Tomko, E J; Stump, W T; Ruiz Manzano, A

2014-01-01

Local DNA opening plays an important role in DNA metabolism as the double-helix must be melted before the information contained within may be accessed. Cells finely tune the torsional state of their genomes to strike a balance between stability and accessibility. For example, while mesophilic life forms maintain negatively superhelical genomes, thermophilic life forms use unique mechanisms to maintain relaxed or even positively supercoiled genomes. Here, we use a single-molecule magnetic tweezers approach to quantify the force-dependent equilibrium between DNA melting and supercoiling at high temperatures populated by Thermophiles. We show that negatively supercoiled DNA denatures at 0.5 pN lower tension at thermophilic vs. mesophilic temperatures. This work demonstrates the ability to monitor DNA supercoiling at high temperature and opens the possibility to perform magnetic tweezers assays on thermophilic systems. The data allow for an estimation of the relative energies of base-pairing and DNA bending as a function of temperature and support speculation as to different general mechanisms of DNA opening in different environments. Lastly, our results imply that average in vivo DNA tensions range between 0.3 and 1.1 pN. Copyright © 2014 Elsevier B.V. All rights reserved.

[Correlation between PMI and DNA degradation of costicartilage and dental pulp cells in human being].

Science.gov (United States)

Long, Ren; Wang, Wei-ping; Xiong, Ping

2005-08-01

To probe the correlation between the postmortem interval (PMI) and the DNA degradation of costicartilage and dental pulp cells in human being after death, and to seek a new method for estimating PMI. The image cytometry was used to measure the DNA degradation under different ambient temperatures (30-35 degrees C, 15-20 degrees C) in 0-15 days after death. The average DNA content of two kinds of tissue was degradated with the prolongation of PMI. But there was a plateau period of 0-4 days for dental pulp cells of human being in 15-20 degrees C. There was a high negative correlativity PPMI. PMI could be estimated accurately according to the DNA degradation of costicartilage and dental pulp cells in human being after death.

Comparison of methods for quantification of global DNA methylation in human cells and tissues.

Directory of Open Access Journals (Sweden)

Sofia Lisanti

Full Text Available DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the "gold standard" of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases.

DNA amount of X and B chromosomes in the grasshoppers Eyprepocnemis plorans and Locusta migratoria.

Science.gov (United States)

Ruiz-Ruano, F J; Ruiz-Estévez, M; Rodríguez-Pérez, J; López-Pino, J L; Cabrero, J; Camacho, J P M

2011-01-01

We analyzed the DNA amount in X and B chromosomes of 2 XX/X0 grasshopper species (Eyprepocnemis plorans and Locusta migratoria), by means of Feulgen image analysis densitometry (FIAD), using previous estimates in L. migratoria as standard (5.89 pg). We first analyzed spermatids of 0B males and found a bimodal distribution of integrated optical densities (IODs), suggesting that one peak corresponded to +X and the other to -X spermatids. The difference between the 2 peaks corresponded to the X chromosome DNA amount, which was 1.28 pg in E. plorans and 0.80 pg in L. migratoria. In addition, the +X peak in E. plorans gave an estimate of the C-value in this species (10.39 pg). We next analyzed diplotene cells from 1B males in E. plorans and +B males in L. migratoria (a species where Bs are mitotically unstable and no integer B number can be defined for an individual) and measured B chromosome IOD relative to X chromosome IOD, within the same cell, taking advantage of the similar degree of condensation for both positively heteropycnotic chromosomes at this meiotic stage. From this proportion, we estimated the DNA amount for 3 different B chromosome variants found in individuals from 3 E. plorans Spanish populations (0.54 pg for B1 from Saladares, 0.51 pg for B2 from Salobreña and 0.64 for B24 from Torrox). Likewise, we estimated the DNA amount of the B chromosome in L. migratoria to be 0.15 pg. To automate measurements, we wrote a GPL3 licensed Python program (pyFIA). We discuss the utility of the present approach for estimating X and B chromosome DNA amount in a variety of situations, and the meaning of the DNA amount estimates for X and B chromosomes in these 2 species. Copyright © 2011 S. Karger AG, Basel.

Geant4-DNA coupling and validation in the GATE Monte Carlo platform for DNA molecules irradiation in a calculation grid environment

International Nuclear Information System (INIS)

Pham, Quang Trung

2014-01-01

The Monte Carlo simulation methods are successfully being used in various areas of medical physics but also at different scales, for example, from the radiation therapy treatment planning systems to the prediction of the effects of radiation in cancer cells. The Monte Carlo simulation platform GATE based on the Geant4 tool-kit offers features dedicated to simulations in medical physics (nuclear medicine and radiotherapy). For radiobiology applications, the Geant4-DNA physical models are implemented to track particles till very low energy (eV) and are adapted for estimation of micro-dosimetric quantities. In order to implement a multi-scale Monte Carlo platform, we first validated the physical models of Geant4-DNA, and integrated them into GATE. Finally, we validated this implementation in the context of radiation therapy and proton therapy. In order to validate the Geant4-DNA physical models, dose point kernels for monoenergetic electrons (10 keV to 100 keV) were simulated using the physical models of Geant4-DNA and were compared to those simulated with Geant4 Standard physical models and another Monte Carlo code EGSnrc. The range and the stopping powers of electrons (7.4 eV to 1 MeV) and protons (1 keV to 100 MeV) calculated with GATE/Geant4-DNA were then compared with literature. We proposed to simulate with the GATE platform the impact of clinical and preclinical beams on cellular DNA. We modeled a clinical proton beam of 193.1 MeV, 6 MeV clinical electron beam and a X-ray irradiator beam. The beams models were validated by comparing absorbed dose computed and measured in liquid water. Then, the beams were used to calculate the frequency of energy deposits in DNA represented by different geometries. First, the DNA molecule was represented by small cylinders: 2 nm x 2 nm (∼10 bp), 5 nm x 10 nm (nucleosome) and 25 nm x 25 nm (chromatin fiber). All these cylinders were placed randomly in a sphere of liquid water (500 nm radius). Then we reconstructed the DNA

Quantification of transplant-derived circulating cell-free DNA in absence of a donor genotype.

Science.gov (United States)

Sharon, Eilon; Shi, Hao; Kharbanda, Sandhya; Koh, Winston; Martin, Lance R; Khush, Kiran K; Valantine, Hannah; Pritchard, Jonathan K; De Vlaminck, Iwijn

2017-08-01

Quantification of cell-free DNA (cfDNA) in circulating blood derived from a transplanted organ is a powerful approach to monitoring post-transplant injury. Genome transplant dynamics (GTD) quantifies donor-derived cfDNA (dd-cfDNA) by taking advantage of single-nucleotide polymorphisms (SNPs) distributed across the genome to discriminate donor and recipient DNA molecules. In its current implementation, GTD requires genotyping of both the transplant recipient and donor. However, in practice, donor genotype information is often unavailable. Here, we address this issue by developing an algorithm that estimates dd-cfDNA levels in the absence of a donor genotype. Our algorithm predicts heart and lung allograft rejection with an accuracy that is similar to conventional GTD. We furthermore refined the algorithm to handle closely related recipients and donors, a scenario that is common in bone marrow and kidney transplantation. We show that it is possible to estimate dd-cfDNA in bone marrow transplant patients that are unrelated or that are siblings of the donors, using a hidden Markov model (HMM) of identity-by-descent (IBD) states along the genome. Last, we demonstrate that comparing dd-cfDNA to the proportion of donor DNA in white blood cells can differentiate between relapse and the onset of graft-versus-host disease (GVHD). These methods alleviate some of the barriers to the implementation of GTD, which will further widen its clinical application.

The re-entrant cholesteric phase of DNA

Science.gov (United States)

Yevdokimov, Yu. M.; Skuridin, S. G.; Salyanov, V. I.; Semenov, S. V.; Shtykova, E. V.; Dadinova, L. A.; Kompanets, O. N.; Kats, E. I.

2017-07-01

The character of packing of double-stranded DNA molecules in particles of liquid-crystal dispersions formed as a result of the phase exclusion of DNA molecules from aqueous salt polyethylene glycol solutions has been estimated by comparing the circular dichroism (CD) spectra of these dispersions recorded at different osmotic pressures and temperatures. It is shown that the first cycle of heating of dispersion particles with hexagonally packed double-stranded DNA molecules leads to the occurrence of abnormal optical activity of these particles, which manifests itself in the form of a strong negative CD band, characteristic of DNA cholesterics. Moreover, subsequent cooling is accompanied by a further increase in the abnormal optical activity, which indicates the existence of the "hexagonal → cholesteric packing" phase transition, controlled by both the osmotic pressure of the solution and its temperature. The result obtained can be described in terms of "quasi-nematic" layers composed of orientationally ordered DNA molecules in the structure of dispersion particles. There are two possible ways of packing for these layers, which determine their hexagonal or cholesteric spatial structure. The second heating → cooling cycle confirms these results and is indicative of possible differences in the packing of double-stranded DNA molecules in the hexagonal phase, which depend on the osmotic pressure of the solution.

Construction and characterization of normalized cDNA libraries by 454 pyrosequencing and estimation of DNA methylation levels in three distantly related termite species.

Directory of Open Access Journals (Sweden)

Yoshinobu Hayashi

Full Text Available In termites, division of labor among castes, categories of individuals that perform specialized tasks, increases colony-level productivity and is the key to their ecological success. Although molecular studies on caste polymorphism have been performed in termites, we are far from a comprehensive understanding of the molecular basis of this phenomenon. To facilitate future molecular studies, we aimed to construct expressed sequence tag (EST libraries covering wide ranges of gene repertoires in three representative termite species, Hodotermopsis sjostedti, Reticulitermes speratus and Nasutitermes takasagoensis. We generated normalized cDNA libraries from whole bodies, except for guts containing microbes, of almost all castes, sexes and developmental stages and sequenced them with the 454 GS FLX titanium system. We obtained >1.2 million quality-filtered reads yielding >400 million bases for each of the three species. Isotigs, which are analogous to individual transcripts, and singletons were produced by assembling the reads and annotated using public databases. Genes related to juvenile hormone, which plays crucial roles in caste differentiation of termites, were identified from the EST libraries by BLAST search. To explore the potential for DNA methylation, which plays an important role in caste differentiation of honeybees, tBLASTn searches for DNA methyltransferases (dnmt1, dnmt2 and dnmt3 and methyl-CpG binding domain (mbd were performed against the EST libraries. All four of these genes were found in the H. sjostedti library, while all except dnmt3 were found in R. speratus and N. takasagoensis. The ratio of the observed to the expected CpG content (CpG O/E, which is a proxy for DNA methylation level, was calculated for the coding sequences predicted from the isotigs and singletons. In all of the three species, the majority of coding sequences showed depletion of CpG O/E (less than 1, and the distributions of CpG O/E were bimodal, suggesting

Construction and characterization of normalized cDNA libraries by 454 pyrosequencing and estimation of DNA methylation levels in three distantly related termite species.

Science.gov (United States)

Hayashi, Yoshinobu; Shigenobu, Shuji; Watanabe, Dai; Toga, Kouhei; Saiki, Ryota; Shimada, Keisuke; Bourguignon, Thomas; Lo, Nathan; Hojo, Masaru; Maekawa, Kiyoto; Miura, Toru

2013-01-01

In termites, division of labor among castes, categories of individuals that perform specialized tasks, increases colony-level productivity and is the key to their ecological success. Although molecular studies on caste polymorphism have been performed in termites, we are far from a comprehensive understanding of the molecular basis of this phenomenon. To facilitate future molecular studies, we aimed to construct expressed sequence tag (EST) libraries covering wide ranges of gene repertoires in three representative termite species, Hodotermopsis sjostedti, Reticulitermes speratus and Nasutitermes takasagoensis. We generated normalized cDNA libraries from whole bodies, except for guts containing microbes, of almost all castes, sexes and developmental stages and sequenced them with the 454 GS FLX titanium system. We obtained >1.2 million quality-filtered reads yielding >400 million bases for each of the three species. Isotigs, which are analogous to individual transcripts, and singletons were produced by assembling the reads and annotated using public databases. Genes related to juvenile hormone, which plays crucial roles in caste differentiation of termites, were identified from the EST libraries by BLAST search. To explore the potential for DNA methylation, which plays an important role in caste differentiation of honeybees, tBLASTn searches for DNA methyltransferases (dnmt1, dnmt2 and dnmt3) and methyl-CpG binding domain (mbd) were performed against the EST libraries. All four of these genes were found in the H. sjostedti library, while all except dnmt3 were found in R. speratus and N. takasagoensis. The ratio of the observed to the expected CpG content (CpG O/E), which is a proxy for DNA methylation level, was calculated for the coding sequences predicted from the isotigs and singletons. In all of the three species, the majority of coding sequences showed depletion of CpG O/E (less than 1), and the distributions of CpG O/E were bimodal, suggesting the presence of

Prenatal exclusion of Norrie disease with flanking DNA markers.

Science.gov (United States)

Gal, A; Uhlhaas, S; Glaser, D; Grimm, T

1988-10-01

Three polymorphic DNA markers linked to the locus of Norrie disease were used for indirect genotype analysis in a ten-wk-old fetus at risk for the disease. When haplotypes of the family members and the estimated recombination frequency between Norrie gene and each of the DNA marker loci DXS7, DXS84, and DXS146 were taken into account, the risk that the fetus had inherited the mutation was about 1%.

Mitochondrial DNA variation, but not nuclear DNA, sharply divides morphologically identical chameleons along an ancient geographic barrier.

Directory of Open Access Journals (Sweden)

Dan Bar Yaacov

Full Text Available The Levant is an important migration bridge, harboring border-zones between Afrotropical and palearctic species. Accordingly, Chameleo chameleon, a common species throughout the Mediterranean basin, is morphologically divided in the southern Levant (Israel into two subspecies, Chamaeleo chamaeleon recticrista (CCR and C. c. musae (CCM. CCR mostly inhabits the Mediterranean climate (northern Israel, while CCM inhabits the sands of the north-western Negev Desert (southern Israel. AFLP analysis of 94 geographically well dispersed specimens indicated moderate genetic differentiation (PhiPT = 0.097, consistent with the classical division into the two subspecies, CCR and CCM. In contrast, sequence analysis of a 637 bp coding mitochondrial DNA (mtDNA fragment revealed two distinct phylogenetic clusters which were not consistent with the morphological division: one mtDNA cluster consisted of CCR specimens collected in regions northern of the Jezreel Valley and another mtDNA cluster harboring specimens pertaining to both the CCR and CCM subspecies but collected southern of the Jezreel Valley. AMOVA indicated clear mtDNA differentiation between specimens collected northern and southern to the Jezreel Valley (PhiPT = 0.79, which was further supported by a very low coalescent-based estimate of effective migration rates. Whole chameleon mtDNA sequencing (∼17,400 bp generated from 11 well dispersed geographic locations revealed 325 mutations sharply differentiating the two mtDNA clusters, suggesting a long allopatric history further supported by BEAST. This separation correlated temporally with the existence of an at least 1 million year old marine barrier at the Jezreel Valley exactly where the mtDNA clusters meet. We discuss possible involvement of gender-dependent life history differences in maintaining such mtDNA genetic differentiation and suggest that it reflects (ancient local adaptation to mitochondrial-related traits.

Mitochondrial DNA evolution in the genus Equus.

Science.gov (United States)

George, M; Ryder, O A

1986-11-01

Employing mitochondrial DNA (mtDNA) restriction-endonuclease maps as the basis of comparison, we have investigated the evolutionary affinities of the seven species generally recognized as the genus Equus. Individual species' cleavage maps contained an average of 60 cleavage sites for 16 enzymes, of which 29 were invariant for all species. Based on an average divergence rate of 2%/Myr, the variation between species supports a divergence of extant lineages from a common ancestor approximately 3.9 Myr before the present. Comparisons of cleavage maps between Equus przewalskii (Mongolian wild horse) and E. caballus (domestic horse) yielded estimates of nucleotide sequence divergence ranging from 0.27% to 0.41%. This range was due to intraspecific variation, which was noted only for E. caballus. For pairwise comparisons within this family, estimates of sequence divergence ranged from 0% (E. hemionus onager vs. E. h. kulan) to 7.8% (E. przewalskii vs. E. h. onager). Trees constructed according to the parsimony principle, on the basis of 31 phylogenetically informative restriction sites, indicate that the three extant zebra species represent a monophyletic group with E. grevyi and E. burchelli antiquorum diverging most recently. The phylogenetic relationships of E. africanus and E. hemionus remain enigmatic on the basis of the mtDNA analysis, although a recent divergence is unsupported.

DNA expressions - A formal notation for DNA

NARCIS (Netherlands)

Vliet, Rudy van

2015-01-01

We describe a formal notation for DNA molecules that may contain nicks and gaps. The resulting DNA expressions denote formal DNA molecules. Different DNA expressions may denote the same molecule. Such DNA expressions are called equivalent. We examine which DNA expressions are minimal, which

Environmental DNA reflects spatial and temporal jellyfish distribution.

Directory of Open Access Journals (Sweden)

Toshifumi Minamoto

Full Text Available Recent development of environmental DNA (eDNA analysis allows us to survey underwater macro-organisms easily and cost effectively; however, there have been no reports on eDNA detection or quantification for jellyfish. Here we present the first report on an eDNA analysis of marine jellyfish using Japanese sea nettle (Chrysaora pacifica as a model species by combining a tank experiment with spatial and temporal distribution surveys. We performed a tank experiment monitoring eDNA concentrations over a range of time intervals after the introduction of jellyfish, and quantified the eDNA concentrations by quantitative real-time PCR. The eDNA concentrations peaked twice, at 1 and 8 h after the beginning of the experiment, and became stable within 48 h. The estimated release rates of the eDNA in jellyfish were higher than the rates previously reported in fishes. A spatial survey was conducted in June 2014 in Maizuru Bay, Kyoto, in which eDNA was collected from surface water and sea floor water samples at 47 sites while jellyfish near surface water were counted on board by eye. The distribution of eDNA in the bay corresponded with the distribution of jellyfish inferred by visual observation, and the eDNA concentration in the bay was ~13 times higher on the sea floor than on the surface. The temporal survey was conducted from March to November 2014, in which jellyfish were counted by eye every morning while eDNA was collected from surface and sea floor water at three sampling points along a pier once a month. The temporal fluctuation pattern of the eDNA concentrations and the numbers of observed individuals were well correlated. We conclude that an eDNA approach is applicable for jellyfish species in the ocean.

Human papillomavirus types 16 and 18 in adenocarcinoma of the uterine cervix

International Nuclear Information System (INIS)

Leminen, A.; Paavonen, J.; Vesterinen, E.; Wahlstroem, T.R.; Rantala, I.; Lehtinen, M.

1991-01-01

Many reports have shown a link between human papillomavirus (HPV) and cervical squamous neoplasia. However, the association of HPV with cervical adenocarcinoma has been studied less extensively. The authors evaluated the presence of HPV-DNA in 106 patients with adenocarcinoma of the uterine cervix by in situ hybridization, using 35 S-labeled probes for HPV 16 DNA and HPV 18 DNA. The overall prevalence of HPV-DNA was 18% (19 of 106). HPV 16 was present in 2 (2%) cases, HPV 18 was observed in 15 (14%) cases, and both HPV 16 and HPV 18 were found in 2 (2%) cases. There was a correlation between HPV-DNA positivity and tumor stage (P less than 0.01) and tumor size (P less than 0.05), but there was no relationship between HPV-DNA positivity and tumor differentiation, proliferation (S-phase fraction), ploidy, lymph node metastases, or five-year survival rate. These results suggest that HPV 18 DNA is associated with cervical adenocarcinoma but the presence of HPV 18 has no influence on overall survival

Inhibition of radiation-induced DNA strand breaks by hoechst 33258: OH-radical scavenging and DNA radical quenching

International Nuclear Information System (INIS)

Adhikary, A.; Bothe, E.; Von Sonntag, C.; Adhikary, A.

1997-01-01

The minor-groove-binding dye Hoechst 33258 has been found to protect pBR322 DNA in aqueous solution against radiation-induced single-strand breaks (ssb). This protective effect has been assumed to be largely due to the scavenging of the strand-break-generating OH radicals by Hoechst. From D 37 values for ssb at different Hoechst concentrations the value of the OH radical scavenging constant of DNA-bound Hoechst has been estimated at k Ho/DNA = 2.7 * 10 11 dm 3 mol -1 . This unexpectedly high value has led us to study the reactions of OH radicals with Hoechst in the absence and in the presence of double-stranded calf thymus DNA (ds DNA) by pulse radiolysis, and the formation of radiation-induced ssb by low angle laser light scattering. The D 37 /D 37 0 values at different Hoechst concentrations agree with the values obtained by Martin and al. and demonstrate the protection. However, this protection cannot be explained on the basis of OH radical scavenging alone using the above rate constants. There must, in addition, be some quenching of DNA radicals. Hoechst radicals are formed in the later ms time range, i.e a long time after the disappearance of the OH radicals. This delayed Hoechst radical formation has been assigned to a a reaction of DNA radicals with Hoechst, thereby inhibiting strand breakage. In confirmation, pulse radiolysis of aqueous solution of nucleotides in the presence of Hoechst yields a similar delayed Hoechst radical formation. The data indicate that in DNA the cross-section of this quenching has a diameter of 3 to 4 base pairs per Hoechst molecule. (N.C.)

DNA content variation and its significance in the evolution of the genus Micrasterias (Desmidiales, Streptophyta.

Directory of Open Access Journals (Sweden)

Aloisie Poulíčková

Full Text Available It is now clear that whole genome duplications have occurred in all eukaryotic evolutionary lineages, and that the vast majority of flowering plants have experienced polyploidisation in their evolutionary history. However, study of genome size variation in microalgae lags behind that of higher plants and seaweeds. In this study, we have addressed the question whether microalgal phylogeny is associated with DNA content variation in order to evaluate the evolutionary significance of polyploidy in the model genus Micrasterias. We applied flow-cytometric techniques of DNA quantification to microalgae and mapped the estimated DNA content along the phylogenetic tree. Correlations between DNA content and cell morphometric parameters were also tested using geometric morphometrics. In total, DNA content was successfully determined for 34 strains of the genus Micrasterias. The estimated absolute 2C nuclear DNA amount ranged from 2.1 to 64.7 pg; intraspecific variation being 17.4-30.7 pg in M. truncata and 32.0-64.7 pg in M. rotata. There were significant differences between DNA contents of related species. We found strong correlation between the absolute nuclear DNA content and chromosome numbers and significant positive correlation between the DNA content and both cell size and number of terminal lobes. Moreover, the results showed the importance of cell/life cycle studies for interpretation of DNA content measurements in microalgae.

A statistical mixture model for estimating the proportion of unreduced pollen grains in perennial ryegrass (Lolium perenne L.) via the size of pollen grains

NARCIS (Netherlands)

Jansen, R.C.; Nijs, A.P.M. den

1993-01-01

The size of pollen grains is commonly used to indicate the ploidy level of pollen grains. In this paper observations of the diameter of pollen grains are evaluated from one diploid accession of perennial ryegrass (Lolium perenne L.), which was expected to produce diploid (unreduced) pollen grains in

DNA barcoding of odonates from the Upper Plata basin: Database creation and genetic diversity estimation.

Directory of Open Access Journals (Sweden)

Ricardo Koroiva

Full Text Available We present a DNA barcoding study of Neotropical odonates from the Upper Plata basin, Brazil. A total of 38 species were collected in a transition region of "Cerrado" and Atlantic Forest, both regarded as biological hotspots, and 130 cytochrome c oxidase subunit I (COI barcodes were generated for the collected specimens. The distinct gap between intraspecific (0-2% and interspecific variation (15% and above in COI, and resulting separation of Barcode Index Numbers (BIN, allowed for successful identification of specimens in 94% of cases. The 6% fail rate was due to a shared BIN between two separate nominal species. DNA barcoding, based on COI, thus seems to be a reliable and efficient tool for identifying Neotropical odonate specimens down to the species level. These results underscore the utility of DNA barcoding to aid specimen identification in diverse biological hotspots, areas that require urgent action regarding taxonomic surveys and biodiversity conservation.

The correlation between cell-free DNA and tumour burden was estimated by PET/CT in patients with advanced NSCLC

DEFF Research Database (Denmark)

Nygaard, A D; Holdgaard, Paw; Spindler, K-L G

2014-01-01

Background:Cell-free DNA (cfDNA) circulating in the blood holds a possible prognostic value in malignant diseases. Under malignant conditions, the level of cfDNA increases but the biological mechanism remains to be fully understood. We aimed to examine the correlation between cfDNA and total tumour...... burden defined by positron emission tomography (PET) parameters.Methods:Patients with advanced non-small cell lung cancer (NSCLC) were enrolled into a prospective biomarker trial. Before treatment, plasma was extracted and the level of cfDNA was determined by qPCR. An (18)F-fluorodeoxyglucose ((18)F...... analysis. MTV>the median was associated with a significantly shorter OS (P=0.02). There was no significant difference in OS according to TLG (P=0.08).Conclusion:Cell-free DNA may not be a simple measure of tumour burden, but seems to reflect more complex mechanisms of tumour biology, making it attractive...

A model capturing novel strand symmetries in bacterial DNA

International Nuclear Information System (INIS)

Sobottka, Marcelo; Hart, Andrew G.

2011-01-01

Highlights: → We propose a simple stochastic model to construct primitive DNA sequences. → The model provide an explanation for Chargaff's second parity rule in primitive DNA sequences. → The model is also used to predict a novel type of strand symmetry in primitive DNA sequences. → We extend the results for bacterial DNA sequences and compare distributional properties intrinsic to the model to statistical estimates from 1049 bacterial genomes. → We find out statistical evidences that the novel type of strand symmetry holds for bacterial DNA sequences. -- Abstract: Chargaff's second parity rule for short oligonucleotides states that the frequency of any short nucleotide sequence on a strand is approximately equal to the frequency of its reverse complement on the same strand. Recent studies have shown that, with the exception of organellar DNA, this parity rule generally holds for double-stranded DNA genomes and fails to hold for single-stranded genomes. While Chargaff's first parity rule is fully explained by the Watson-Crick pairing in the DNA double helix, a definitive explanation for the second parity rule has not yet been determined. In this work, we propose a model based on a hidden Markov process for approximating the distributional structure of primitive DNA sequences. Then, we use the model to provide another possible theoretical explanation for Chargaff's second parity rule, and to predict novel distributional aspects of bacterial DNA sequences.

The role of DNA base excision repair in brain homeostasis and disease

DEFF Research Database (Denmark)

Akbari, Mansour; Morevati, Marya; Croteau, Deborah

2015-01-01

Chemical modification and spontaneous loss of nucleotide bases from DNA are estimated to occur at the rate of thousands per human cell per day. DNA base excision repair (BER) is a critical mechanism for repairing such lesions in nuclear and mitochondrial DNA. Defective expression or function of p...... energy homeostasis, mitochondrial function and cellular bioenergetics, with especially strong influence on neurological function. Further studies in this area could lead to novel approaches to prevent and treat human neurodegenerative disease....

Novel nuclei isolation buffer for flow cytometric genome size estimation of Zingiberaceae: a comparison with common isolation buffers.

Science.gov (United States)

Sadhu, Abhishek; Bhadra, Sreetama; Bandyopadhyay, Maumita

2016-11-01

Cytological parameters such as chromosome numbers and genome sizes of plants are used routinely for studying evolutionary aspects of polyploid plants. Members of Zingiberaceae show a wide range of inter- and intrageneric variation in their reproductive habits and ploidy levels. Conventional cytological study in this group of plants is severely hampered by the presence of diverse secondary metabolites, which also affect their genome size estimation using flow cytometry. None of the several nuclei isolation buffers used in flow cytometry could be used very successfully for members of Zingiberaceae to isolate good quality nuclei from both shoot and root tissues. The competency of eight nuclei isolation buffers was compared with a newly formulated buffer, MB01, in six different genera of Zingiberaceae based on the fluorescence intensity of propidium iodide-stained nuclei using flow cytometric parameters, namely coefficient of variation of the G 0 /G 1 peak, debris factor and nuclei yield factor. Isolated nuclei were studied using fluorescence microscopy and bio-scanning electron microscopy to analyse stain-nuclei interaction and nuclei topology, respectively. Genome contents of 21 species belonging to these six genera were determined using MB01. Flow cytometric parameters showed significant differences among the analysed buffers. MB01 exhibited the best combination of analysed parameters; photomicrographs obtained from fluorescence and electron microscopy supported the superiority of MB01 buffer over other buffers. Among the 21 species studied, nuclear DNA contents of 14 species are reported for the first time. Results of the present study substantiate the enhanced efficacy of MB01, compared to other buffers tested, in the generation of acceptable cytograms from all species of Zingiberaceae studied. Our study facilitates new ways of sample preparation for further flow cytometric analysis of genome size of other members belonging to this highly complex polyploid family

Understanding environmental DNA detection probabilities: A case study using a stream-dwelling char Salvelinus fontinalis

Science.gov (United States)

Wilcox, Taylor M; Mckelvey, Kevin S.; Young, Michael K.; Sepulveda, Adam; Shepard, Bradley B.; Jane, Stephen F; Whiteley, Andrew R.; Lowe, Winsor H.; Schwartz, Michael K.

2016-01-01

Environmental DNA sampling (eDNA) has emerged as a powerful tool for detecting aquatic animals. Previous research suggests that eDNA methods are substantially more sensitive than traditional sampling. However, the factors influencing eDNA detection and the resulting sampling costs are still not well understood. Here we use multiple experiments to derive independent estimates of eDNA production rates and downstream persistence from brook trout (Salvelinus fontinalis) in streams. We use these estimates to parameterize models comparing the false negative detection rates of eDNA sampling and traditional backpack electrofishing. We find that using the protocols in this study eDNA had reasonable detection probabilities at extremely low animal densities (e.g., probability of detection 0.18 at densities of one fish per stream kilometer) and very high detection probabilities at population-level densities (e.g., probability of detection > 0.99 at densities of ≥ 3 fish per 100 m). This is substantially more sensitive than traditional electrofishing for determining the presence of brook trout and may translate into important cost savings when animals are rare. Our findings are consistent with a growing body of literature showing that eDNA sampling is a powerful tool for the detection of aquatic species, particularly those that are rare and difficult to sample using traditional methods.

Radiation-induced DNA damage in halogenated pyrimidine incorporated cells and its correlation with radiosensitivity

International Nuclear Information System (INIS)

Watanabe, R.; Nikjoo, H.

2003-01-01

Cells with DNA containing 5-halogenated pyrimidines in place of thymidine show significant reductions of slope (Do) and shoulder (Dq) of their radiation survival curves. Similar radiosensitization has also been observed in the yield of DNA strand breaks. The purpose of this study is to obtain an insight into the mechanism of cell lethality by examining the relationship between the spectrum of DNA damage and the cell survival. In this study we estimated the enhancement of strand breaks due to incorporation of halogenated pyrimidine, the complexity of DNA damage and the probability of the initial DNA damage leading to cell inactivation. Monte Carlo track structure methods were used to model and simulate the induction of strand breakage by X-rays. The increase of DNA strand break was estimated by assuming the excess strand break was caused by the highly reactive uracil radicals at the halouracil substituted sites. The assumption of the enhancement mechanism of strand breaks was examined and verified by comparison with experimental data for induction of SSB and DSB. The calculated DNA damage spectrum shows the increase in complexity of strand breaks is due to incorporation of halogenated pyrimidines. The increase in the yield of DSB and cell lethality show similar trend at various degrees of halogenated pyrimidine substitution. We asked the question whether this agreement supports the hypothesis that DSB is responsible for cell lethality? The estimated number of lethal damage from the cell survival using a linear-quadratic model is much less than the initial yield of DSB. This work examines the correlation of cell lethality as a function of frequencies of complex form of double strand breaks

A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis

Science.gov (United States)

Down, Thomas A.; Rakyan, Vardhman K.; Turner, Daniel J.; Flicek, Paul; Li, Heng; Kulesha, Eugene; Gräf, Stefan; Johnson, Nathan; Herrero, Javier; Tomazou, Eleni M.; Thorne, Natalie P.; Bäckdahl, Liselotte; Herberth, Marlis; Howe, Kevin L.; Jackson, David K.; Miretti, Marcos M.; Marioni, John C.; Birney, Ewan; Hubbard, Tim J. P.; Durbin, Richard; Tavaré, Simon; Beck, Stephan

2009-01-01

DNA methylation is an indispensible epigenetic modification of mammalian genomes. Consequently there is great interest in strategies for genome-wide/whole-genome DNA methylation analysis, and immunoprecipitation-based methods have proven to be a powerful option. Such methods are rapidly shifting the bottleneck from data generation to data analysis, necessitating the development of better analytical tools. Until now, a major analytical difficulty associated with immunoprecipitation-based DNA methylation profiling has been the inability to estimate absolute methylation levels. Here we report the development of a novel cross-platform algorithm – Bayesian Tool for Methylation Analysis (Batman) – for analyzing Methylated DNA Immunoprecipitation (MeDIP) profiles generated using arrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). The latter is an approach we have developed to elucidate the first high-resolution whole-genome DNA methylation profile (DNA methylome) of any mammalian genome. MeDIP-seq/MeDIP-chip combined with Batman represent robust, quantitative, and cost-effective functional genomic strategies for elucidating the function of DNA methylation. PMID:18612301

Regulating DNA Self-assembly by DNA-Surface Interactions.

Science.gov (United States)

Liu, Longfei; Li, Yulin; Wang, Yong; Zheng, Jianwei; Mao, Chengde

2017-12-14

DNA self-assembly provides a powerful approach for preparation of nanostructures. It is often studied in bulk solution and involves only DNA-DNA interactions. When confined to surfaces, DNA-surface interactions become an additional, important factor to DNA self-assembly. However, the way in which DNA-surface interactions influence DNA self-assembly is not well studied. In this study, we showed that weak DNA-DNA interactions could be stabilized by DNA-surface interactions to allow large DNA nanostructures to form. In addition, the assembly can be conducted isothermally at room temperature in as little as 5 seconds. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Relation between sedimentation behaviour of DNA-membrane complexes and DNA single- and double-strand breaks after irradiation with gamma-rays, pulse neutrons and 12C ions

International Nuclear Information System (INIS)

Erzgraber, G.; Lapidus, I.L.

1985-01-01

The experimental data on sedimentation behaviour of DNA-membrane complexes at radiation of the Chinese hamster cells (V79-4) in a wide dose range of 127 Cs γ-rays, pulse neutrons (reactor IBR-2, Laboratory of Neutron Physics, JINR, Dubna) are accelerated 12 C ions (cyclotron U-200, Laboratory of Nuclear Reactions, JINR, Dubna) are presented An assumption on the role of DNA single- and double-strend breaks in changing the sedimentation properties of DNA-membrane complexes has been confirmed by the experiments with radiation of different quality. The possibility of estimating induction and repair of DNA breaks on the basis of dependence of the relative sedimentation velocity of complexes on the irradiation does is discussed

Quantification of transplant-derived circulating cell-free DNA in absence of a donor genotype.

Directory of Open Access Journals (Sweden)

Eilon Sharon

2017-08-01

Full Text Available Quantification of cell-free DNA (cfDNA in circulating blood derived from a transplanted organ is a powerful approach to monitoring post-transplant injury. Genome transplant dynamics (GTD quantifies donor-derived cfDNA (dd-cfDNA by taking advantage of single-nucleotide polymorphisms (SNPs distributed across the genome to discriminate donor and recipient DNA molecules. In its current implementation, GTD requires genotyping of both the transplant recipient and donor. However, in practice, donor genotype information is often unavailable. Here, we address this issue by developing an algorithm that estimates dd-cfDNA levels in the absence of a donor genotype. Our algorithm predicts heart and lung allograft rejection with an accuracy that is similar to conventional GTD. We furthermore refined the algorithm to handle closely related recipients and donors, a scenario that is common in bone marrow and kidney transplantation. We show that it is possible to estimate dd-cfDNA in bone marrow transplant patients that are unrelated or that are siblings of the donors, using a hidden Markov model (HMM of identity-by-descent (IBD states along the genome. Last, we demonstrate that comparing dd-cfDNA to the proportion of donor DNA in white blood cells can differentiate between relapse and the onset of graft-versus-host disease (GVHD. These methods alleviate some of the barriers to the implementation of GTD, which will further widen its clinical application.

From Gene Trees to a Dated Allopolyploid Network: Insights from the Angiosperm Genus Viola (Violaceae)

Science.gov (United States)

Marcussen, Thomas; Heier, Lise; Brysting, Anne K.; Oxelman, Bengt; Jakobsen, Kjetill S.

2015-01-01

Allopolyploidization accounts for a significant fraction of speciation events in many eukaryotic lineages. However, existing phylogenetic and dating methods require tree-like topologies and are unable to handle the network-like phylogenetic relationships of lineages containing allopolyploids. No explicit framework has so far been established for evaluating competing network topologies, and few attempts have been made to date phylogenetic networks. We used a four-step approach to generate a dated polyploid species network for the cosmopolitan angiosperm genus Viola L. (Violaceae Batch.). The genus contains ca 600 species and both recent (neo-) and more ancient (meso-) polyploid lineages distributed over 16 sections. First, we obtained DNA sequences of three low-copy nuclear genes and one chloroplast region, from 42 species representing all 16 sections. Second, we obtained fossil-calibrated chronograms for each nuclear gene marker. Third, we determined the most parsimonious multilabeled genome tree and its corresponding network, resolved at the section (not the species) level. Reconstructing the “correct” network for a set of polyploids depends on recovering all homoeologs, i.e., all subgenomes, in these polyploids. Assuming the presence of Viola subgenome lineages that were not detected by the nuclear gene phylogenies (“ghost subgenome lineages”) significantly reduced the number of inferred polyploidization events. We identified the most parsimonious network topology from a set of five competing scenarios differing in the interpretation of homoeolog extinctions and lineage sorting, based on (i) fewest possible ghost subgenome lineages, (ii) fewest possible polyploidization events, and (iii) least possible deviation from expected ploidy as inferred from available chromosome counts of the involved polyploid taxa. Finally, we estimated the homoploid and polyploid speciation times of the most parsimonious network. Homoploid speciation times were estimated by

Endogenous DNA Damage and Risk of Testicular Germ Cell Tumors

Energy Technology Data Exchange (ETDEWEB)

Cook, M B; Sigurdson, A J; Jones, I M; Thomas, C B; Graubard, B I; Korde, L; Greene, M H; McGlynn, K A

2008-01-18

Testicular germ cell tumors (TGCT) are comprised of two histologic groups, seminomas and nonseminomas. We postulated that the possible divergent pathogeneses of these histologies may be partially explained by variable endogenous DNA damage. To assess our hypothesis, we conducted a case-case analysis of seminomas and nonseminomas using the alkaline comet assay to quantify single-strand DNA breaks and alkali-labile sites. The Familial Testicular Cancer study and the U.S. Radiologic Technologists cohort provided 112 TGCT cases (51 seminomas & 61 nonseminomas). A lymphoblastoid cell line was cultured for each patient and the alkaline comet assay was used to determine four parameters: tail DNA, tail length, comet distributed moment (CDM) and Olive tail moment (OTM). Odds ratios (OR) and 95% confidence intervals (95%CI) were estimated using logistic regression. Values for tail length, tail DNA, CDM and OTM were modeled as categorical variables using the 50th and 75th percentiles of the seminoma group. Tail DNA was significantly associated with nonseminoma compared to seminoma (OR{sub 50th percentile} = 3.31, 95%CI: 1.00, 10.98; OR{sub 75th percentile} = 3.71, 95%CI: 1.04, 13.20; p for trend=0.039). OTM exhibited similar, albeit statistically non-significant, risk estimates (OR{sub 50th percentile} = 2.27, 95%CI: 0.75, 6.87; OR{sub 75th percentile} = 2.40, 95%CI: 0.75, 7.71; p for trend=0.12) whereas tail length and CDM showed no association. In conclusion, the results for tail DNA and OTM indicate that endogenous DNA damage levels are higher in patients who develop nonseminoma compared with seminoma. This may partly explain the more aggressive biology and younger age-of-onset of this histologic subgroup compared with the relatively less aggressive, later-onset seminoma.

Coulomb and CH-π interactions in (6-4) photolyase-DNA complex dominate DNA binding and repair abilities.

Science.gov (United States)

Terai, Yuma; Sato, Ryuma; Yumiba, Takahiro; Harada, Ryuhei; Shimizu, Kohei; Toga, Tatsuya; Ishikawa-Fujiwara, Tomoko; Todo, Takeshi; Iwai, Shigenori; Shigeta, Yasuteru; Yamamoto, Junpei

2018-05-14

(6-4) Photolyases ((6-4)PLs) are flavoenzymes that repair the carcinogenic UV-induced DNA damage, pyrimidine(6-4)pyrimidone photoproducts ((6-4)PPs), in a light-dependent manner. Although the reaction mechanism of DNA photorepair by (6-4)PLs has been intensively investigated, the molecular mechanism of the lesion recognition remains obscure. We show that a well-conserved arginine residue in Xenopus laevis (6-4)PL (Xl64) participates in DNA binding, through Coulomb and CH-π interactions. Fragment molecular orbital calculations estimated attractive interaction energies of -80-100 kcal mol-1 for the Coulomb interaction and -6 kcal mol-1 for the CH-π interaction, and the loss of either of them significantly reduced the affinity for (6-4)PP-containing oligonucleotides, as well as the quantum yield of DNA photorepair. From experimental and theoretical observations, we formulated a DNA binding model of (6-4)PLs. Based on the binding model, we mutated this Arg in Xl64 to His, which is well conserved among the animal cryptochromes (CRYs), and found that the CRY-type mutant exhibited reduced affinity for the (6-4)PP-containing oligonucleotides, implying the possible molecular origin of the functional diversity of the photolyase/cryptochrome superfamily.

Changing numbers of spawning cutthroat trout in tributary streams of Yellowstone Lake and estimates of grizzly bears visiting streams from DNA

Science.gov (United States)

Haroldson, M.A.; Gunther, K.A.; Reinhart, Daniel P.; Podruzny, S.R.; Cegelski, C.; Waits, L.; Wyman, T.C.; Smith, J.

2005-01-01

Spawning Yellowstone cutthroat trout (Oncorhynchus clarki) provide a source of highly digestible energy for grizzly bears (Ursus arctos) that visit tributary streams to Yellowstone Lake during the spring and early summer. During 1985–87, research documented grizzly bears fishing on 61% of the 124 tributary streams to the lake. Using track measurements, it was estimated that a minimum of 44 grizzly bears fished those streams annually. During 1994, non-native lake trout (Salvelinus namaycush) were discovered in Yellowstone Lake. Lake trout are efficient predators and have the potential to reduce the native cutthroat population and negatively impact terrestrial predators that use cutthroat trout as a food resource. In 1997, we began sampling a subset of streams (n = 25) from areas of Yellowstone Lake surveyed during the previous study to determine if changes in spawner numbers or bear use had occurred. Comparisons of peak numbers and duration suggested a considerable decline between study periods in streams in the West Thumb area of the lake. The apparent decline may be due to predation by lake trout. Indices of bear use also declined on West Thumb area streams. We used DNA from hair collected near spawning streams to estimate the minimum number of bears visiting the vicinity of spawning streams. Seventy-four individual bears were identified from 429 hair samples. The annual number of individuals detected ranged from 15 in 1997 to 33 in 2000. Seventy percent of genotypes identified were represented by more than 1 sample, but only 31% of bears were documented more than 1 year of the study. Sixty-two (84%) bears were only documented in 1 segment of the lake, whereas 12 (16%) were found in 2–3 lake segments. Twenty-seven bears were identified from hair collected at multiple streams. One bear was identified on 6 streams in 2 segments of the lake and during 3 years of the study. We used encounter histories derived from DNA and the Jolly-Seber procedure in Program MARK

Mitochondrial DNA diversity of present-day Aboriginal Australians and implications for human evolution in Oceania.

Science.gov (United States)

Nagle, Nano; Ballantyne, Kaye N; van Oven, Mannis; Tyler-Smith, Chris; Xue, Yali; Wilcox, Stephen; Wilcox, Leah; Turkalov, Rust; van Oorschot, Roland A H; van Holst Pellekaan, Sheila; Schurr, Theodore G; McAllister, Peter; Williams, Lesley; Kayser, Manfred; Mitchell, R John

2017-03-01

Aboriginal Australians are one of the more poorly studied populations from the standpoint of human evolution and genetic diversity. Thus, to investigate their genetic diversity, the possible date of their ancestors' arrival and their relationships with neighboring populations, we analyzed mitochondrial DNA (mtDNA) diversity in a large sample of Aboriginal Australians. Selected mtDNA single-nucleotide polymorphisms and the hypervariable segment haplotypes were analyzed in 594 Aboriginal Australians drawn from locations across the continent, chiefly from regions not previously sampled. Most (~78%) samples could be assigned to mtDNA haplogroups indigenous to Australia. The indigenous haplogroups were all ancient (with estimated ages >40 000 years) and geographically widespread across the continent. The most common haplogroup was P (44%) followed by S (23%) and M42a (9%). There was some geographic structure at the haplotype level. The estimated ages of the indigenous haplogroups range from 39 000 to 55 000 years, dates that fit well with the estimated date of colonization of Australia based on archeological evidence (~47 000 years ago). The distribution of mtDNA haplogroups in Australia and New Guinea supports the hypothesis that the ancestors of Aboriginal Australians entered Sahul through at least two entry points. The mtDNA data give no support to the hypothesis of secondary gene flow into Australia during the Holocene, but instead suggest long-term isolation of the continent.

Oxidative damage to DNA and lipids as biomarkers of exposure to air pollution

DEFF Research Database (Denmark)

Møller, Peter; Loft, Steffen

2010-01-01

BACKGROUND: Air pollution is thought to exert health effects through oxidative stress, which causes damage to DNA and lipids. OBJECTIVE: We determined whether levels of oxidatively damaged DNA and lipid peroxidation products in cells or bodily fluids from humans are useful biomarkers...... of biologically effective dose in studies of the health effects of exposure to particulate matter (PM) from combustion processes. DATA SOURCES: We identified publications that reported estimated associations between environmental exposure to PM and oxidative damage to DNA and lipids in PubMed and EMBASE. We also...... identified publications from reference lists and articles cited in the Web of Science. DATA EXTRACTION: For each study, we obtained information on the estimated effect size to calculate the standardized mean difference (unitless) and determined the potential for errors in exposure assessment and analysis...

Structure of DNA damaged by UV and psoralen

International Nuclear Information System (INIS)

Sung-hou Kim; Tomic, M.T.; Wemmer, D.E.; Pearlman, D.; Holbrook, S.

1988-01-01

The authors have used NMR methods to determine a three-dimensional model of an 8 base-pair DNA fragment cross-linked with psoralen. The duplex form of the self-complementary deoxyribonucleotide d-GGGTACCC, contains a psoralen cross-linkable site at the center of the duplex. The cross-link was formed by UV irradiation of a mixture of the purified DNA octamer and 4'-(aminomethyl)-4,5',8-trimethylpsoralen (AMT). Structural information was obtained using one and two-dimensional NMR techniques. Two-dimensional NOE experiments were used to assign the spectrum and estimate distances for many pairs of protons in the cross-linked DNA. Structural parameters obtained are qualitatively consistent with a previously proposed model for kinked and unwound cross-linked B-form DNA derived from crystallography and molecular modeling. The NMR derived model has a 53 degree bend into the major groove occuring primarily at the site of drug addition, and a 56 degree unwinding spanning the 8 base pair duplex. (author)

DNA-DNA hybridization determined in micro-wells using covalent attachment of DNA

DEFF Research Database (Denmark)

Christensen, H.; Angen, Øystein; Mutters, R.

2000-01-01

The present study was aimed at reducing the time and labour used to perform DNA-DNA hybridizations for classification of bacteria at the species level. A micro-well-format DNA hybridization method was developed and validated. DNA extractions were performed by a small-scale method and DNA...... was sheared mechanically into fragments of between 400 and 700 bases. The hybridization conditions were calibrated according to DNA similarities obtained by the spectrophotometric method using strains within the family Pasteurellaceae, Optimal conditions were obtained with 300 ng DNA added per well and bound...... by covalent attachment to NucleoLink. Hybridization was performed with 500 ng DNA, 5% (w/w) of which was labelled with photo-activatable biotin (competitive hybridization) for 2.5 h at 65 degrees C in 2 x SSC followed by stringent washing with 2 x SSC at the same temperature. The criteria for acceptance...

The persistence of human DNA in soil following surface decomposition.

Science.gov (United States)

Emmons, Alexandra L; DeBruyn, Jennifer M; Mundorff, Amy Z; Cobaugh, Kelly L; Cabana, Graciela S

2017-09-01

Though recent decades have seen a marked increase in research concerning the impact of human decomposition on the grave soil environment, the fate of human DNA in grave soil has been relatively understudied. With the purpose of supplementing the growing body of literature in forensic soil taphonomy, this study assessed the relative persistence of human DNA in soil over the course of decomposition. Endpoint PCR was used to assess the presence or absence of human nuclear and mitochondrial DNA, while qPCR was used to evaluate the quantity of human DNA recovered from the soil beneath four cadavers at the University of Tennessee's Anthropology Research Facility (ARF). Human nuclear DNA from the soil was largely unrecoverable, while human mitochondrial DNA was detectable in the soil throughout all decomposition stages. Mitochondrial DNA copy abundances were not significantly different between decomposition stages and were not significantly correlated to soil edaphic parameters tested. There was, however, a significant positive correlation between mitochondrial DNA copy abundances and the human associated bacteria, Bacteroides, as estimated by 16S rRNA gene abundances. These results show that human mitochondrial DNA can persist in grave soil and be consistently detected throughout decomposition. Copyright © 2017 The Chartered Society of Forensic Sciences. Published by Elsevier B.V. All rights reserved.

Algorithm for post-clustering curation of DNA amplicon data yields reliable biodiversity estimates

DEFF Research Database (Denmark)

Froslev, Tobias Guldberg; Kjoller, Rasmus; Bruun, Hans Henrik

2017-01-01

by high-throughput sequencing of amplified marker genes. LULU identifies errors by combining sequence similarity and co-occurrence patterns. To validate the LULU method, we use a unique data set of high quality survey data of vascular plants paired with plant ITS2 metabarcoding data of DNA extracted from...

Mitochondrial DNA levels in Huntington disease leukocytes and dermal fibroblasts.

Science.gov (United States)

Jędrak, Paulina; Krygier, Magdalena; Tońska, Katarzyna; Drozd, Małgorzata; Kaliszewska, Magdalena; Bartnik, Ewa; Sołtan, Witold; Sitek, Emilia J; Stanisławska-Sachadyn, Anna; Limon, Janusz; Sławek, Jarosław; Węgrzyn, Grzegorz; Barańska, Sylwia

2017-08-01

Huntington disease (HD) is an inherited neurodegenerative disorder caused by mutations in the huntingtin gene. Involvement of mitochondrial dysfunctions in, and especially influence of the level of mitochondrial DNA (mtDNA) on, development of this disease is unclear. Here, samples of blood from 84 HD patients and 79 controls, and dermal fibroblasts from 10 HD patients and 9 controls were analysed for mtDNA levels. Although the type of mitochondrial haplogroup had no influence on the mtDNA level, and there was no correlation between mtDNA level in leukocytes in HD patients and various parameters of HD severity, some considerable differences between HD patients and controls were identified. The average mtDNA/nDNA relative copy number was significantly higher in leukocytes, but lower in fibroblasts, of symptomatic HD patients relative to the control group. Moreover, HD women displayed higher mtDNA levels in leukocytes than HD men. Because this is the largest population analysed to date, these results might contribute to explanation of discrepancies between previously published studies concerning levels of mtDNA in cells of HD patients. We suggest that the size of the investigated population and type of cells from which DNA is isolated could significantly affect results of mtDNA copy number estimation in HD. Hence, these parameters should be taken into consideration in studies on mtDNA in HD, and perhaps also in other diseases where mitochondrial dysfunction occurs.

Quantification of DNA in simple eukaryotic cells using Fourier transform infrared spectroscopy.

Science.gov (United States)

Whelan, Donna R; Bambery, Keith R; Puskar, Ljiljana; McNaughton, Don; Wood, Bayden R

2013-10-01

A technique capable of detecting and monitoring nucleic acid concentration offers potential in diagnosing cancer and further developing an understanding of the biochemistry of disease. The application of Fourier transform infrared (FTIR) spectroscopy has previously been hindered by the supposed non-Beer-Lambert absorption behavior of DNA in intact cells making elucidation of the DNA bands difficult. We use known composition DNA/hemoglobin standards to successfully estimate the DNA content in avian erythrocyte nuclei (44.2%) and intact erythrocytes (12.8%). Furthermore we demonstrate that the absorption of cellular DNA does follow the Beer-Lambert Law and highlights the role of conformation and hydration in FTIR spectroscopy of biological samples. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Worldwide F(ST) estimates relative to five continental-scale populations.

Science.gov (United States)

Steele, Christopher D; Court, Denise Syndercombe; Balding, David J

2014-11-01

We estimate the population genetics parameter FST (also referred to as the fixation index) from short tandem repeat (STR) allele frequencies, comparing many worldwide human subpopulations at approximately the national level with continental-scale populations. FST is commonly used to measure population differentiation, and is important in forensic DNA analysis to account for remote shared ancestry between a suspect and an alternative source of the DNA. We estimate FST comparing subpopulations with a hypothetical ancestral population, which is the approach most widely used in population genetics, and also compare a subpopulation with a sampled reference population, which is more appropriate for forensic applications. Both estimation methods are likelihood-based, in which FST is related to the variance of the multinomial-Dirichlet distribution for allele counts. Overall, we find low FST values, with posterior 97.5 percentiles estimates, and are also about half the magnitude of STR-based estimates from population genetics surveys that focus on distinct ethnic groups rather than a general population. Our findings support the use of FST up to 3% in forensic calculations, which corresponds to some current practice.

A colorimetric platform for sensitively differentiating telomere DNA with different lengths, monitoring G-quadruplex and dsDNA based on silver nanoclusters and unmodified gold nanoparticles

Science.gov (United States)

Qu, Fei; Chen, Zeqiu; You, Jinmao; Song, Cuihua

2018-05-01

Human telomere DNA plays a vital role in genome integrity control and carcinogenesis as an indication for extensive cell proliferation. Herein, silver nanoclusters (Ag NCs) templated by polymer and unmodified gold nanoparticles (Au NPs) are designed as a new colorimetric platform for sensitively differentiating telomere DNA with different lengths, monitoring G-quadruplex and dsDNA. Ag NCs can produce the aggregation of Au NPs, so the color of Au NPs changes to blue and the absorption peak moves to 700 nm. While the telomere DNA can protect Au NPs from aggregation, the color turns to red again and the absorption band blue shift. Benefiting from the obvious color change, we can differentiate the length of telomere DNA by naked eyes. As the length of telomere DNA is longer, the variation of color becomes more noticeable. The detection limits of telomere DNA containing 10, 22, 40, 64 bases are estimated to be 1.41, 1.21, 0.23 and 0.22 nM, respectively. On the other hand, when telomere DNA forms G-quadruplex in the presence of K+, or dsDNA with complementary sequence, both G-quadruplex and dsDNA can protect Au NPs better than the unfolded telomere DNA. Hence, a new colorimetric platform for monitoring structure conversion of DNA is established by Ag NCs-Au NPs system, and to prove this type of application, a selective K+ sensor is developed.

Single Molecule Study of DNA Organization and Recombination

Science.gov (United States)

Xiao, Botao

We have studied five projects related to DNA organization and recombination using mainly single molecule force-spectroscopy and statistical tools. First, HU is one of the most abundant DNA-organizing proteins in bacterial chromosomes and participates in gene regulation. We report experiments that study the dependence of DNA condensation by HU on force, salt and HU concentration. A first important result is that at physiological salt levels, HU only bends DNA, resolving a previous paradox of why a chromosome-compacting protein should have a DNA-stiffening function. A second major result is quantitative demonstration of strong dependencies of HU-DNA dissociation on both salt concentration and force. Second, we have used a thermodynamic Maxwell relation to count proteins driven off large DNAs by tension, an effect important to understanding DNA organization. Our results compare well with estimates of numbers of proteins HU and Fis in previous studies. We have also shown that a semi-flexible polymer model describes our HU experimental data well. The force-dependent binding suggests mechano-chemical mechanisms for gene regulation. Third, the elusive role of protein H1 in chromatin has been clarified with purified H1 and Xenopus extracts. We find that H1 compacts DNA by both bending and looping. Addition of H1 enhances chromatin formation and maintains the plasticity of the chromatin. Fourth, the topology and mechanics of DNA twisting are critical to DNA organization and recombination. We have systematically measured DNA extension as a function of linking number density from 0.08 to -2 with holding forces from 0.2 to 2.4 pN. Unlike previous proposals, the DNA extension decreases with negative linking number. Finally, DNA recombination is a dynamic process starting from enzyme-DNA binding. We report that the Int-DBD domain of lambda integrase binds to DNA without compaction at low Int-DBD concentration. High concentration of Int-DBD loops DNA below a threshold force

Detection of environmental carcinogens-DNA

International Nuclear Information System (INIS)

Pfohl-Leszkowicz, A.; Guillemaut, G.; Rether, B.; Masfaraud, J.F.; Haguenoer, J.M.

1995-01-01

It has been estimated that majority of human cancer is due to environmental factors including pollutants in air, soil, water and food, work places exposure and personal habits such as smoking. After penetration in organism, xenobiotics could be directly excreted or are bio transformed by oxidation or reduction in more hydrophilic compounds which could be conjugate and then eliminated in urine. But in some case, the biotransformation leads to electrophilic compounds which interact with macromolecules such as DNA, forming addition products named adduct. The 32 P post-labelling method, inspired by recent developments in the methodology for sequencing nucleic acids, is an extremely sensitive method for assessing and quantifying DNA adducts and is applicable to structurally diverse classes of chemicals. In the first study, we have analysed hepatic DNA from fish living in the River Rhone downstream and upstream from a polychlorinated biphenyl incineration plant. Our results suggest that fish are exposed to genotoxic chemicals. In another study, leave DNA from healthy and declining hop were analysed. The total adduct level is 3 time higher in declining hop. A comparison between DNA adducts from several vegetal cells cultured in presence of heptachlor and DNA adduct in declining hop, confirmed the implication of heptachlor. In these examples, our data indicate the usefulness of the 32 P-post labelling method to assess the contamination of the environment by genotoxic pollutants. Epidemiological data suggested that increasing exposure to airborne PAH contributes to increase risk cancer in this population. Exposure-dependent adducts were detected in while blood cells in coke oven workers. The adduct levels is function of the level of pollutant. In the last example we have analysed lung tissue from patient with cancer. We observed many adducts in peritumoral tissue, while few adducts could be detected in tumoral tissues. (author)

Robust k-mer frequency estimation using gapped k-mers.

Science.gov (United States)

Ghandi, Mahmoud; Mohammad-Noori, Morteza; Beer, Michael A

2014-08-01

Oligomers of fixed length, k, commonly known as k-mers, are often used as fundamental elements in the description of DNA sequence features of diverse biological function, or as intermediate elements in the constuction of more complex descriptors of sequence features such as position weight matrices. k-mers are very useful as general sequence features because they constitute a complete and unbiased feature set, and do not require parameterization based on incomplete knowledge of biological mechanisms. However, a fundamental limitation in the use of k-mers as sequence features is that as k is increased, larger spatial correlations in DNA sequence elements can be described, but the frequency of observing any specific k-mer becomes very small, and rapidly approaches a sparse matrix of binary counts. Thus any statistical learning approach using k-mers will be susceptible to noisy estimation of k-mer frequencies once k becomes large. Because all molecular DNA interactions have limited spatial extent, gapped k-mers often carry the relevant biological signal. Here we use gapped k-mer counts to more robustly estimate the ungapped k-mer frequencies, by deriving an equation for the minimum norm estimate of k-mer frequencies given an observed set of gapped k-mer frequencies. We demonstrate that this approach provides a more accurate estimate of the k-mer frequencies in real biological sequences using a sample of CTCF binding sites in the human genome.

Extraction and phylogenetic survey of extracellular and intracellular DNA in marine sediments

DEFF Research Database (Denmark)

Torti, Andrea

indeed inflate richness estimates of sediments microbial communities, and point to a role of bioturbation in shaping the prokaryotic diversity of the eDNA pool at the investigated site. Analysis of 18S RNA gene sequences revealed a diverse collection of eukaryotic taxa throughout the sediment column......DNA, and validated for minimal cell lysis during the eDNA extraction process. The optimized method was applied to investigate and compare the bacterial, archaeal, and eukaryotic diversity within iDNA and eDNA pools, in the context of differing geochemical and lithological zones in the Holocene sediment column...... of Aarhus Bay (Demark). Using high-throughput sequencing technologies, I first explored whether, and to what extent, prokaryotic eDNA parallels the phylogenetic composition of the local microbiome. Phylogenetic analyses revealed that, in near-surface sediments influenced by faunal activities, 50% of all...

Regulation of the demographic structure in isomorphic biphasic life cycles at the spatial fine scale.

Directory of Open Access Journals (Sweden)

Vasco Manuel Nobre de Carvalho da Silva Vieira

Full Text Available Isomorphic biphasic algal life cycles often occur in the environment at ploidy abundance ratios (Haploid:Diploid different from 1. Its spatial variability occurs within populations related to intertidal height and hydrodynamic stress, possibly reflecting the niche partitioning driven by their diverging adaptation to the environment argued necessary for their prevalence (evolutionary stability. Demographic models based in matrix algebra were developed to investigate which vital rates may efficiently generate an H:D variability at a fine spatial resolution. It was also taken into account time variation and type of life strategy. Ploidy dissimilarities in fecundity rates set an H:D spatial structure miss-fitting the ploidy fitness ratio. The same happened with ploidy dissimilarities in ramet growth whenever reproductive output dominated the population demography. Only through ploidy dissimilarities in looping rates (stasis, breakage and clonal growth did the life cycle respond to a spatially heterogeneous environment efficiently creating a niche partition. Marginal locations were more sensitive than central locations. Related results have been obtained experimentally and numerically for widely different life cycles from the plant and animal kingdoms. Spore dispersal smoothed the effects of ploidy dissimilarities in fertility and enhanced the effects of ploidy dissimilarities looping rates. Ploidy dissimilarities in spore dispersal could also create the necessary niche partition, both over the space and time dimensions, even in spatial homogeneous environments and without the need for conditional differentiation of the ramets. Fine scale spatial variability may be the key for the prevalence of isomorphic biphasic life cycles, which has been neglected so far.

Sequence-dependent DNA deformability studied using molecular dynamics simulations.

Science.gov (United States)

Fujii, Satoshi; Kono, Hidetoshi; Takenaka, Shigeori; Go, Nobuhiro; Sarai, Akinori

2007-01-01

Proteins recognize specific DNA sequences not only through direct contact between amino acids and bases, but also indirectly based on the sequence-dependent conformation and deformability of the DNA (indirect readout). We used molecular dynamics simulations to analyze the sequence-dependent DNA conformations of all 136 possible tetrameric sequences sandwiched between CGCG sequences. The deformability of dimeric steps obtained by the simulations is consistent with that by the crystal structures. The simulation results further showed that the conformation and deformability of the tetramers can highly depend on the flanking base pairs. The conformations of xATx tetramers show the most rigidity and are not affected by the flanking base pairs and the xYRx show by contrast the greatest flexibility and change their conformations depending on the base pairs at both ends, suggesting tetramers with the same central dimer can show different deformabilities. These results suggest that analysis of dimeric steps alone may overlook some conformational features of DNA and provide insight into the mechanism of indirect readout during protein-DNA recognition. Moreover, the sequence dependence of DNA conformation and deformability may be used to estimate the contribution of indirect readout to the specificity of protein-DNA recognition as well as nucleosome positioning and large-scale behavior of nucleic acids.

Beyond DNA repair: DNA-PK function in cancer

OpenAIRE

Goodwin, Jonathan F.; Knudsen, Karen E.

2014-01-01

The DNA-dependent protein kinase (DNA-PK) is a pivotal component of the DNA repair machinery that governs the response to DNA damage, serving to maintain genome integrity. However, the DNA-PK kinase component was initially isolated with transcriptional complexes, and recent findings have illuminated the impact of DNA-PK-mediated transcriptional regulation on tumor progression and therapeutic response. DNA-PK expression has also been correlated with poor outcome in selected tumor types, furthe...

DNA replication stress restricts ribosomal DNA copy number.

Science.gov (United States)

Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

2017-09-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

DNA replication stress restricts ribosomal DNA copy number

Science.gov (United States)

Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

2017-01-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

DNA replication stress restricts ribosomal DNA copy number.

Directory of Open Access Journals (Sweden)

Devika Salim

2017-09-01

Full Text Available Ribosomal RNAs (rRNAs in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

DNA damage and plasma homocysteine levels are associated with ...

African Journals Online (AJOL)

STORAGESEVER

2010-01-18

Jan 18, 2010 ... (Fluitest Glu, Biocon Solutions Pte Ltd, Singapore). Cholesterol, ... migration in the comet tail was taken as an estimate of DNA damage and is ..... fever, and dietary energy intake on weight gain in rural Bangladeshi children.

DNA Origami-Graphene Hybrid Nanopore for DNA Detection.

Science.gov (United States)

Barati Farimani, Amir; Dibaeinia, Payam; Aluru, Narayana R

2017-01-11

DNA origami nanostructures can be used to functionalize solid-state nanopores for single molecule studies. In this study, we characterized a nanopore in a DNA origami-graphene heterostructure for DNA detection. The DNA origami nanopore is functionalized with a specific nucleotide type at the edge of the pore. Using extensive molecular dynamics (MD) simulations, we computed and analyzed the ionic conductivity of nanopores in heterostructures carpeted with one or two layers of DNA origami on graphene. We demonstrate that a nanopore in DNA origami-graphene gives rise to distinguishable dwell times for the four DNA base types, whereas for a nanopore in bare graphene, the dwell time is almost the same for all types of bases. The specific interactions (hydrogen bonds) between DNA origami and the translocating DNA strand yield different residence times and ionic currents. We also conclude that the speed of DNA translocation decreases due to the friction between the dangling bases at the pore mouth and the sequencing DNA strands.

Bone marrow cellularity in normal and polycythemic mice estimated by DNA incorporation of /sup 3/H-TdR

Energy Technology Data Exchange (ETDEWEB)

Blackwell, L.H.; Ledney, G.D.

1982-07-01

Nucleated bone marrow cell numbers in normal and polycythemic mice were determined using /sup 3/H-thymidine (/sup 3/H-TdR). The cellularities were estimated by extrapolating the exponential disappearance of labeled cells after a single injection of /sup 3/H-TdR to the time of injection. Dermestid beetles (Anthrenus piceus) were used to prepare tissue-free skeletons labeled with /sup 3/H-TdR. The correlation between tritium activity in bone marrow DNA and tritium derived from the combusted skeleton was determined. The total skeletal cellularity determined by isotope dilution analysis in both normal and polycythemic mice was 2.6 x 10(8) cells/mouse or 17.6 x 10(9) cells/kg body weight. Although the red cell component of the marrow was reduced in the polycythemic mouse, the total numbers of nucleated cells in both types of animals were similar. The differential distribution of cells in the polycythemic animal showed a twofold increase in granulocytic cells, which may explain the identical nucleated cell count in normal and in polycythemic mice.

Multiple origins of polyploidy in the phylogeny of southern African barbs (Cyprinidae) as inferred from mtDNA markers.

Science.gov (United States)

Tsigenopoulos, C S; Ráb, P; Naran, D; Berrebi, P

2002-06-01

The cyprinid genus Barbus, with more than 800 nominal species, is an apparently polyphyletic assemblage to which a number of unrelated species, groups and/or assemblages have been assigned. It includes species that exhibit three different ploidy levels: diploid, tetraploid and hexaploid. Several lineages of the family Cyprinidae constitute a major component of the African freshwater ichthyofauna, having about 500 species, and fishes assigned to the genus 'Barbus' have the most species on the continent. We used complete sequences of the mitochondrial cytochrome b gene in order to infer phylogenetic relationships between diploid, tetraploid and hexaploid species of 'Barbus' occurring in southern Africa, the only region where representatives of all of the three ploidy levels occur. The results indicate that most of the lineages are incorrectly classified in the genus 'Barbus'. The southern African tetraploids probably originated from southern African diploids. They constitute a monophyletic group distinct from tetraploids occurring in the Euro-Mediterranean region (Barbus sensu stricto). The 'small' African diploid species seem to be paraphyletic, while the 'large' African hexaploid barbs species are of a single, recent origin and form a monophyletic group. The evidence of multiple, independent origins of polyploidy occurring in the African cyprinine cyprinids thus provides a significant contribution to the knowledge on the systematic diversity of these fishes, and warrants a thorough taxonomic reorganization of the genus.

Melanesian mtDNA complexity.

Directory of Open Access Journals (Sweden)

Jonathan S Friedlaender

Full Text Available Melanesian populations are known for their diversity, but it has been hard to grasp the pattern of the variation or its underlying dynamic. Using 1,223 mitochondrial DNA (mtDNA sequences from hypervariable regions 1 and 2 (HVR1 and HVR2 from 32 populations, we found the among-group variation is structured by island, island size, and also by language affiliation. The more isolated inland Papuan-speaking groups on the largest islands have the greatest distinctions, while shore dwelling populations are considerably less diverse (at the same time, within-group haplotype diversity is less in the most isolated groups. Persistent differences between shore and inland groups in effective population sizes and marital migration rates probably cause these differences. We also add 16 whole sequences to the Melanesian mtDNA phylogenies. We identify the likely origins of a number of the haplogroups and ancient branches in specific islands, point to some ancient mtDNA connections between Near Oceania and Australia, and show additional Holocene connections between Island Southeast Asia/Taiwan and Island Melanesia with branches of haplogroup E. Coalescence estimates based on synonymous transitions in the coding region suggest an initial settlement and expansion in the region at approximately 30-50,000 years before present (YBP, and a second important expansion from Island Southeast Asia/Taiwan during the interval approximately 3,500-8,000 YBP. However, there are some important variance components in molecular dating that have been overlooked, and the specific nature of ancestral (maternal Austronesian influence in this region remains unresolved.

Current developments in forensic interpretation of mixed DNA samples (Review)

Science.gov (United States)

HU, NA; CONG, BIN; LI, SHUJIN; MA, CHUNLING; FU, LIHONG; ZHANG, XIAOJING

2014-01-01

A number of recent improvements have provided contemporary forensic investigations with a variety of tools to improve the analysis of mixed DNA samples in criminal investigations, producing notable improvements in the analysis of complex trace samples in cases of sexual assult and homicide. Mixed DNA contains DNA from two or more contributors, compounding DNA analysis by combining DNA from one or more major contributors with small amounts of DNA from potentially numerous minor contributors. These samples are characterized by a high probability of drop-out or drop-in combined with elevated stutter, significantly increasing analysis complexity. At some loci, minor contributor alleles may be completely obscured due to amplification bias or over-amplification, creating the illusion of additional contributors. Thus, estimating the number of contributors and separating contributor genotypes at a given locus is significantly more difficult in mixed DNA samples, requiring the application of specialized protocols that have only recently been widely commercialized and standardized. Over the last decade, the accuracy and repeatability of mixed DNA analyses available to conventional forensic laboratories has greatly advanced in terms of laboratory technology, mathematical models and biostatistical software, generating more accurate, rapid and readily available data for legal proceedings and criminal cases. PMID:24748965

LET-effects in DNA

International Nuclear Information System (INIS)

Kraft, G.; Taucher-Scholz, G.; Heilmann, J.

1995-01-01

The use of heavy particles in radiobiological experiments provides a fundamental tool to study the influence of different ionization densities and to prove the physical basis of models and theories. The knowledge of the interaction of high LET radiation (LET: linear energy transfer [keV/microm]) with biological matter is of great importance for the application of neutrons, protons and heavier ions in radiotherapy. It is also essential in radioprotection to estimate the risk in case of exposure to high LET radiation. In this contribution, an introductory view on the physical properties of ions is given and the cellular response to high LET radiation is summarized. Then the measurements of strand break induction of DNA in solution and in intracellular DNA are reported and compared to cell survival. The possibility of changes in the quality of the lesions is discussed and finally the present status of model calculations in comparison to the experiments is given

The Genomic Pattern of tDNA Operon Expression in E. coli.

Directory of Open Access Journals (Sweden)

2005-06-01

Full Text Available In fast-growing microorganisms, a tRNA concentration profile enriched in major isoacceptors selects for the biased usage of cognate codons. This optimizes translational rate for the least mass invested in the translational apparatus. Such translational streamlining is thought to be growth-regulated, but its genetic basis is poorly understood. First, we found in reanalysis of the E. coli tRNA profile that the degree to which it is translationally streamlined is nearly invariant with growth rate. Then, using least squares multiple regression, we partitioned tRNA isoacceptor pools to predicted tDNA operons from the E. coli K12 genome. Co-expression of tDNAs in operons explains the tRNA profile significantly better than tDNA gene dosage alone. Also, operon expression increases significantly with proximity to the origin of replication, oriC, at all growth rates. Genome location explains about 15% of expression variation in a form, at a given growth rate, that is consistent with replication-dependent gene concentration effects. Yet the change in the tRNA profile with growth rate is less than would be expected from such effects. We estimated per-copy expression rates for all tDNA operons that were consistent with independent estimates for rDNA operons. We also found that tDNA operon location, and the location dependence of expression, were significantly different in the leading and lagging strands. The operonic organization and genomic location of tDNA operons are significant factors influencing their expression. Nonrandom patterns of location and strandedness shown by tDNA operons in E. coli suggest that their genomic architecture may be under selection to satisfy physiological demand for tRNA expression at high growth rates.

Simple Laboratory methods to measure cell proliferation using DNA synthesis property

Directory of Open Access Journals (Sweden)

Madhavan H N

2007-01-01

Full Text Available This is a mini-review on the techniques to measure proliferation of cells by estimation of DNA synthesis. This is not an exhaustive review of literature, but a bird’s eye view of a few selected articles which may provide the technical details to the readers.The nucleus of a cell occupies about 10-30% of the cells space, depends on the type of genetic material (DNA -DeoxyriboNucleic Acid. DNA is a long, double-stranded, helical molecule which carries the genetic information. Duplication of the DNA takes place by the phenomena of replication. One copy of double-stranded DNA molecule forms two double-stranded DNA molecules. DNA replication is the fundamental process used in all living organisms as it is the basis for biological inheritance. This process is known also as Mitosis in somatic cells. In Mitosis, the duplication process results in two genetically identical "daughter" cells from a single "parent" cell. The resulting double-stranded DNA molecules are identical; proof reading and error-checking mechanisms exist to ensure near perfect pair. Mitosis is divided into six phases: prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis.

Application of DNA markers against illegal logging as a new tool for the Forest Guard Service

OpenAIRE

Nowakowska, Justyna A.

2011-01-01

DNA markers are currently the most precise tool for forest tree species identification and can be used for comparative analyses of plant material. Molecular diagnosis of evidence and reference material is based on comparing the structure of DNA markers duplicated in the PCR reaction and estimation of the DNA profiles obtained in studied wood samples. For this purpose, the microsatellite DNA markers are the most suitable tool because of their high polymorphism and accurate detection of structu...

Shifts in microbial populations in Rusitec fermenters as affected by the type of diet and impact of the method for estimating microbial growth (15N v. microbial DNA).

Science.gov (United States)

Mateos, I; Ranilla, M J; Saro, C; Carro, M D

2017-11-01

Rusitec fermenters are in vitro systems widely used to study ruminal fermentation, but little is known about the microbial populations establishing in them. This study was designed to assess the time evolution of microbial populations in fermenters fed medium- (MC; 50% alfalfa hay : concentrate) and high-concentrate diets (HC; 15 : 85 barley straw : concentrate). Samples from solid (SOL) and liquid (LIQ) content of fermenters were taken immediately before feeding on days 3, 8 and 14 of incubation for quantitative polymerase chain reaction and automated ribosomal intergenic spacer analysis analyses. In SOL, total bacterial DNA concentration and relative abundance of Ruminococcus flavefaciens remained unchanged over the incubation period, but protozoal DNA concentration and abundance of Fibrobacter succinogenes, Ruminococcus albus and fungi decreased and abundance of methanogenic archaea increased. In LIQ, total bacterial DNA concentration increased with time, whereas concentration of protozoal DNA and abundance of methanogens and fungi decreased. Diet×time interactions were observed for bacterial and protozoal DNA and relative abundance of F. succinogenes and R. albus in SOL, as well as for protozoal DNA in LIQ. Bacterial diversity in SOL increased with time, but no changes were observed in LIQ. The incubated diet influenced all microbial populations, with the exception of total bacteria and fungi abundance in LIQ. Bacterial diversity was higher in MC-fed than in HC-fed fermenters in SOL, but no differences were detected in LIQ. Values of pH, daily production of volatile fatty acids and CH4 and isobutyrate proportions remained stable over the incubation period, but other fermentation parameters varied with time. The relationships among microbial populations and fermentation parameters were in well agreement with those previously reported in in vivo studies. Using 15N as a microbial marker or quantifying total microbial DNA for estimating microbial protein synthesis

Evaluating droplet digital PCR for the quantification of human genomic DNA: converting copies per nanoliter to nanograms nuclear DNA per microliter.

Science.gov (United States)

Duewer, David L; Kline, Margaret C; Romsos, Erica L; Toman, Blaza

2018-05-01

The highly multiplexed polymerase chain reaction (PCR) assays used for forensic human identification perform best when used with an accurately determined quantity of input DNA. To help ensure the reliable performance of these assays, we are developing a certified reference material (CRM) for calibrating human genomic DNA working standards. To enable sharing information over time and place, CRMs must provide accurate and stable values that are metrologically traceable to a common reference. We have shown that droplet digital PCR (ddPCR) limiting dilution end-point measurements of the concentration of DNA copies per volume of sample can be traceably linked to the International System of Units (SI). Unlike values assigned using conventional relationships between ultraviolet absorbance and DNA mass concentration, entity-based ddPCR measurements are expected to be stable over time. However, the forensic community expects DNA quantity to be stated in terms of mass concentration rather than entity concentration. The transformation can be accomplished given SI-traceable values and uncertainties for the number of nucleotide bases per human haploid genome equivalent (HHGE) and the average molar mass of a nucleotide monomer in the DNA polymer. This report presents the considerations required to establish the metrological traceability of ddPCR-based mass concentration estimates of human nuclear DNA. Graphical abstract The roots of metrological traceability for human nuclear DNA mass concentration results. Values for the factors in blue must be established experimentally. Values for the factors in red have been established from authoritative source materials. HHGE stands for "haploid human genome equivalent"; there are two HHGE per diploid human genome.

Factors influencing detection of eDNA from a stream-dwelling amphibian

Science.gov (United States)

Pilliod, David S.; Goldberg, Caren S.; Arkle, Robert S.; Waits, Lisette P.

2013-01-01

Environmental DNA (eDNA) methods for detecting and estimating abundance of aquatic species are emerging rapidly, but little is known about how processes such as secretion rate, environmental degradation, and time since colonization or extirpation from a given site affect eDNA measurements. Using stream-dwelling salamanders and quantitative PCR (qPCR) analysis, we conducted three experiments to assess eDNA: (i) production rate; (ii) persistence time under different temperature and light conditions; and (iii) detectability and concentration through time following experimental introduction and removal of salamanders into previously unoccupied streams. We found that 44–50 g individuals held in aquaria produced 77 ng eDNA/h for 2 h, after which production either slowed considerably or began to equilibrate with degradation. eDNA in both full-sun and shaded treatments degraded exponentially to 2) and when samples were collected within 5 m of the animals. Concentrations of eDNA detected were very low and increased steadily from 6–24 h after introduction, reaching 0.0022 ng/L. Within 1 h of removing salamanders from the stream, eDNA was no longer detectable. These results suggest that eDNA detectability and concentration depend on production rates of individuals, environmental conditions, density of animals, and their residence time.

At the forefront: evidence of the applicability of using environmental DNA to quantify the abundance of fish populations in natural lentic waters with additional sampling considerations

Science.gov (United States)

Klobucar, Stephen L.; Rodgers, Torrey W.; Budy, Phaedra

2017-01-01

Environmental DNA (eDNA) sampling has proven to be a valuable tool for detecting species in aquatic ecosystems. Within this rapidly evolving field, a promising application is the ability to obtain quantitative estimates of relative species abundance based on eDNA concentration rather than traditionally labor-intensive methods. We investigated the relationship between eDNA concentration and Arctic char (Salvelinus alpinus) abundance in five well-studied natural lakes; additionally, we examined the effects of different temporal (e.g., season) and spatial (e.g., depth) scales on eDNA concentration. Concentrations of eDNA were linearly correlated with char population estimates ( = 0.78) and exponentially correlated with char densities ( = 0.96 by area; 0.82 by volume). Across lakes, eDNA concentrations were greater and more homogeneous in the water column during mixis; however, when stratified, eDNA concentrations were greater in the hypolimnion. Overall, our findings demonstrate that eDNA techniques can produce effective estimates of relative fish abundance in natural lakes. These findings can guide future studies to improve and expand eDNA methods while informing research and management using rapid and minimally invasive sampling.

Electron microscope autoradiography of isolated DNA molecules

International Nuclear Information System (INIS)

Delain, Etienne; Bouteille, Michel

1980-01-01

Autoradiographs of 3 H-thymidine-labelled DNA molecules were observed with an electron microscope. After ten months of exposure significant labelling was obtained with tritiated T7 DNA molecules which had a specific activity of 630,000 cpm/μg. Although isolated DNA molecules were not stretched out to such an extent that they could be rigorously compared to straight 'hot lines', the resolution was estimated and found to be similar to that obtained by autoradiography on thin plastic sections. The H.D. value was of the order of 1600A. From the known specific activity of the macromolecules, it was possible to compare the expected number of disintegrations from the samples to the number of grains obtained on the autoradiograms. This enabled us to calculate 1/ The absolute autoradiographic efficiency and 2/ The per cent ratio of thymidine residues labelled with tritium. These results throw some light on the resolution and sensitivity of electron microscope autoradiography of shadowed isolated macromolecules as compared to thin plastic sections

Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication

Energy Technology Data Exchange (ETDEWEB)

Haruta, Mayumi [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Shimada, Midori, E-mail: midorism@med.nagoya-cu.ac.jp [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Nishiyama, Atsuya; Johmura, Yoshikazu [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Le Tallec, Benoît; Debatisse, Michelle [Institut Curie, Centre de Recherche, 26 rue d’Ulm, CNRS UMR 3244, 75248 ParisCedex 05 (France); Nakanishi, Makoto, E-mail: mkt-naka@med.nagoya-cu.ac.jp [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan)

2016-01-22

The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. - Highlights: • DNMT1 depletion results in an abnormal DNA replication program. • Aberrant DNA replication is independent of the DNA damage checkpoint in DNMT1cKO. • DNMT1 catalytic activity and RFT domain are required for proper DNA replication. • DNMT1 catalytic activity and RFT domain are required for cell proliferation.

Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication

International Nuclear Information System (INIS)

Haruta, Mayumi; Shimada, Midori; Nishiyama, Atsuya; Johmura, Yoshikazu; Le Tallec, Benoît; Debatisse, Michelle; Nakanishi, Makoto

2016-01-01

The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. - Highlights: • DNMT1 depletion results in an abnormal DNA replication program. • Aberrant DNA replication is independent of the DNA damage checkpoint in DNMT1cKO. • DNMT1 catalytic activity and RFT domain are required for proper DNA replication. • DNMT1 catalytic activity and RFT domain are required for cell proliferation.

Quantitative measurement of ultraviolet-induced damage in cellular DNA by an enzyme immunodot assay

International Nuclear Information System (INIS)

Wakizaka, A.; Nishizawa, Y.; Aiba, N.; Okuhara, E.; Takahashi, S.

1989-01-01

A simple enzyme immunoassay procedure was developed for the quantitative determination of 254-nm uv-induced DNA damage in cells. With the use of specific antibodies to uv-irradiated DNA and horseradish peroxidase-conjugated antibody to rabbit IgG, the extent of damaged DNA in uv-irradiated rat spleen mononuclear cells was quantitatively measurable. Through the use of this method, the amount of damaged DNA present in 2 X 10(5) cells irradiated at a dose of 75 J/m2 was estimated to be 7 ng equivalents of the standard uv-irradiated DNA. In addition, when the cells, irradiated at 750 J/m2, were incubated for 1 h, the antigenic activity of DNA decreased by 40%, suggesting that a repair of the damaged sites in DNA had proceeded to some extent in the cells

DNA stable-isotope probing (DNA-SIP).

Science.gov (United States)

Dunford, Eric A; Neufeld, Josh D

2010-08-02

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

Electrostatic force microscopy: imaging DNA and protein polarizations one by one

International Nuclear Information System (INIS)

Mikamo-Satoh, Eriko; Yamada, Fumihiko; Takagi, Akihiko; Matsumoto, Takuya; Kawai, Tomoji

2009-01-01

We present electrostatic force microscopy images of double-stranded DNA and transcription complex on an insulating mica substrate obtained with molecular resolution using a frequency-mode noncontact atomic force microscope. The electrostatic potential images show that both DNA and transcription complexes are polarized with an upward dipole moment. Potential differences of these molecules from the mica substrate enabled us to estimate dipole moments of isolated DNA and transcription complex in zero external field to be 0.027 D/base and 0.16 D/molecule, respectively. Scanning capacitance microscopy demonstrates characteristic contrast inversion between DNA and transcription complex images, indicating the difference in electric polarizability of these molecules. These findings indicate that the electrostatic properties of individual biological molecules can be imaged on an insulator substrate while retaining complex formation.

The dynamic interplay between DNA topoisomerases and DNA topology.

Science.gov (United States)

Seol, Yeonee; Neuman, Keir C

2016-11-01

Topological properties of DNA influence its structure and biochemical interactions. Within the cell, DNA topology is constantly in flux. Transcription and other essential processes, including DNA replication and repair, not only alter the topology of the genome but also introduce additional complications associated with DNA knotting and catenation. These topological perturbations are counteracted by the action of topoisomerases, a specialized class of highly conserved and essential enzymes that actively regulate the topological state of the genome. This dynamic interplay among DNA topology, DNA processing enzymes, and DNA topoisomerases is a pervasive factor that influences DNA metabolism in vivo. Building on the extensive structural and biochemical characterization over the past four decades that has established the fundamental mechanistic basis of topoisomerase activity, scientists have begun to explore the unique roles played by DNA topology in modulating and influencing the activity of topoisomerases. In this review we survey established and emerging DNA topology-dependent protein-DNA interactions with a focus on in vitro measurements of the dynamic interplay between DNA topology and topoisomerase activity.

Ancient DNA

DEFF Research Database (Denmark)

Willerslev, Eske; Cooper, Alan

2004-01-01

ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair......ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair...

Separation/extraction, detection, and interpretation of DNA mixtures in forensic science (review).

Science.gov (United States)

Tao, Ruiyang; Wang, Shouyu; Zhang, Jiashuo; Zhang, Jingyi; Yang, Zihao; Sheng, Xiang; Hou, Yiping; Zhang, Suhua; Li, Chengtao

2018-05-25

Interpreting mixed DNA samples containing material from multiple contributors has long been considered a major challenge in forensic casework, especially when encountering low-template DNA (LT-DNA) or high-order mixtures that may involve missing alleles (dropout) and unrelated alleles (drop-in), among others. In the last decades, extraordinary progress has been made in the analysis of mixed DNA samples, which has led to increasing attention to this research field. The advent of new methods for the separation and extraction of DNA from mixtures, novel or jointly applied genetic markers for detection and reliable interpretation approaches for estimating the weight of evidence, as well as the powerful massively parallel sequencing (MPS) technology, has greatly extended the range of mixed samples that can be correctly analyzed. Here, we summarized the investigative approaches and progress in the field of forensic DNA mixture analysis, hoping to provide some assistance to forensic practitioners and to promote further development involving this issue.

Evaluation of DNA damage using microwave dielectric absorption spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Hirayama, Makoto; Matuo, Youichrou; Izumi, Yoshinobu [Research Institute of Nuclear Engineering, University of Fukui, Fukui (Japan); Sunagawa, Takeyoshi [Fukui University of Technology, Fukui (Japan)

2016-12-15

Evaluation of deoxyribonucleic acid (DNA)-strand break is important to elucidate the biological effect of ionizing radiations. The conventional methods for DNA-strand break evaluation have been achieved by Agarose gel electrophoresis and others using an electrical property of DNAs. Such kinds of DNA-strand break evaluation systems can estimate DNA-strand break, according to a molecular weight of DNAs. However, the conventional method needs pre-treatment of the sample and a relatively long period for analysis. They do not have enough sensitivity to detect the strand break products in the low-dose region. The sample is water, methanol and plasmid DNA solution. The plasmid DNA pUC118 was multiplied by using Escherichia coli JM109 competent cells. The resonance frequency and Q-value were measured by means of microwave dielectric absorption spectroscopy. When a sample is located at a center of the electric field, resonance curve of the frequency that existed as a standing wave is disturbed. As a result, the perturbation effect to perform a resonance with different frequency is adopted. The resonance frequency shifted to higher frequency with an increase in a concentration of methanol as the model of the biological material, and the Q-value decreased. The absorption peak in microwave power spectrum of the double-strand break plasmid DNA shifted from the non-damaged plasmid DNA. Moreover, the sharpness of absorption peak changed resulting in change in Q-value. We confirmed that a resonance frequency shifted to higher frequency with an increase in concentration of the plasmid DNA. We developed a new technique for an evaluation of DNA damage. In this paper, we report the evaluation method of DNA damage using microwave dielectric absorption spectroscopy.

Evaluation of DNA damage using microwave dielectric absorption spectroscopy

International Nuclear Information System (INIS)

Hirayama, Makoto; Matuo, Youichrou; Izumi, Yoshinobu; Sunagawa, Takeyoshi

2016-01-01

Evaluation of deoxyribonucleic acid (DNA)-strand break is important to elucidate the biological effect of ionizing radiations. The conventional methods for DNA-strand break evaluation have been achieved by Agarose gel electrophoresis and others using an electrical property of DNAs. Such kinds of DNA-strand break evaluation systems can estimate DNA-strand break, according to a molecular weight of DNAs. However, the conventional method needs pre-treatment of the sample and a relatively long period for analysis. They do not have enough sensitivity to detect the strand break products in the low-dose region. The sample is water, methanol and plasmid DNA solution. The plasmid DNA pUC118 was multiplied by using Escherichia coli JM109 competent cells. The resonance frequency and Q-value were measured by means of microwave dielectric absorption spectroscopy. When a sample is located at a center of the electric field, resonance curve of the frequency that existed as a standing wave is disturbed. As a result, the perturbation effect to perform a resonance with different frequency is adopted. The resonance frequency shifted to higher frequency with an increase in a concentration of methanol as the model of the biological material, and the Q-value decreased. The absorption peak in microwave power spectrum of the double-strand break plasmid DNA shifted from the non-damaged plasmid DNA. Moreover, the sharpness of absorption peak changed resulting in change in Q-value. We confirmed that a resonance frequency shifted to higher frequency with an increase in concentration of the plasmid DNA. We developed a new technique for an evaluation of DNA damage. In this paper, we report the evaluation method of DNA damage using microwave dielectric absorption spectroscopy

Quantification of DNA cleavage specificity in Hi-C experiments.

Science.gov (United States)

Meluzzi, Dario; Arya, Gaurav

2016-01-08

Hi-C experiments produce large numbers of DNA sequence read pairs that are typically analyzed to deduce genomewide interactions between arbitrary loci. A key step in these experiments is the cleavage of cross-linked chromatin with a restriction endonuclease. Although this cleavage should happen specifically at the enzyme's recognition sequence, an unknown proportion of cleavage events may involve other sequences, owing to the enzyme's star activity or to random DNA breakage. A quantitative estimation of these non-specific cleavages may enable simulating realistic Hi-C read pairs for validation of downstream analyses, monitoring the reproducibility of experimental conditions and investigating biophysical properties that correlate with DNA cleavage patterns. Here we describe a computational method for analyzing Hi-C read pairs to estimate the fractions of cleavages at different possible targets. The method relies on expressing an observed local target distribution downstream of aligned reads as a linear combination of known conditional local target distributions. We validated this method using Hi-C read pairs obtained by computer simulation. Application of the method to experimental Hi-C datasets from murine cells revealed interesting similarities and differences in patterns of cleavage across the various experiments considered. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Mitochondrial DNA Copy Number in Sleep Duration Discordant Monozygotic Twins.

Science.gov (United States)

Wrede, Joanna E; Mengel-From, Jonas; Buchwald, Dedra; Vitiello, Michael V; Bamshad, Michael; Noonan, Carolyn; Christiansen, Lene; Christensen, Kaare; Watson, Nathaniel F

2015-10-01

Mitochondrial DNA (mtDNA) copy number is an important component of mitochondrial function and varies with age, disease, and environmental factors. We aimed to determine whether mtDNA copy number varies with habitual differences in sleep duration within pairs of monozygotic twins. Academic clinical research center. 15 sleep duration discordant monozygotic twin pairs (30 twins, 80% female; mean age 42.1 years [SD 15.0]). Sleep duration was phenotyped with wrist actigraphy. Each twin pair included a "normal" (7-9 h/24) and "short" (sleeping twin. Fasting peripheral blood leukocyte DNA was assessed for mtDNA copy number via the n-fold difference between qPCR measured mtDNA and nuclear DNA creating an mtDNA measure without absolute units. We used generalized estimating equation linear regression models accounting for the correlated data structure to assess within-pair effects of sleep duration on mtDNA copy number. Mean within-pair sleep duration difference per 24 hours was 94.3 minutes (SD 62.6 min). We found reduced sleep duration (β = 0.06; 95% CI 0.004, 0.12; P sleep efficiency (β = 0.51; 95% CI 0.06, 0.95; P sleep duration was associated with a decrease in mtDNA copy number of 0.06. Likewise, a 1% decrease in actigraphy-defined sleep efficiency was associated with a decrease in mtDNA copy number of 0.51. Reduced sleep duration and sleep efficiency were associated with reduced mitochondrial DNA copy number in sleep duration discordant monozygotic twins offering a potential mechanism whereby short sleep impairs health and longevity through mitochondrial stress. © 2015 Associated Professional Sleep Societies, LLC.

DNA accumulation on ventilation system filters in university buildings in Singapore.

Science.gov (United States)

Luhung, Irvan; Wu, Yan; Xu, Siyu; Yamamoto, Naomichi; Chang, Victor Wei-Chung; Nazaroff, William W

2017-01-01

Biological particles deposit on air handling system filters as they process air. This study reports and interprets abundance and diversity information regarding biomass accumulation on ordinarily used filters acquired from several locations in a university environment. DNA-based analysis was applied both to quantify (via DNA fluorometry and qPCR) and to characterize (via high-throughput sequencing) the microbial material on filters, which mainly processed recirculated indoor air. Results were interpreted in relation to building occupancy and ventilation system operational parameters. Based on accumulated biomass, average DNA concentrations per AHU filter surface area across nine indoor locations after twelve weeks of filter use were in the respective ranges 1.1 to 41 ng per cm2 for total DNA, 0.02 to 3.3 ng per cm2 for bacterial DNA and 0.2 to 2.0 ng DNA per cm2 for fungal DNA. The most abundant genera detected on the AHU filter samples were Clostridium, Streptophyta, Bacillus, Acinetobacter and Ktedonobacter for bacteria and Aspergillus, Cladosporium, Nigrospora, Rigidoporus and Lentinus for fungi. Conditional indoor airborne DNA concentrations (median (range)) were estimated to be 13 (2.6-107) pg/m3 for total DNA, 0.4 (0.05-8.4) pg/m3 for bacterial DNA and 2.3 (1.0-5.1) pg/m3 for fungal DNA. Conditional airborne concentrations and the relative abundances of selected groups of genera correlate well with occupancy level. Bacterial DNA was found to be more responsive than fungal DNA to differences in occupancy level and indoor environmental conditions.

Direct qPCR quantification using the Quantifiler(®) Trio DNA quantification kit.

Science.gov (United States)

Liu, Jason Yingjie

2014-11-01

The effectiveness of a direct quantification assay is essential to the adoption of the combined direct quantification/direct STR workflow. In this paper, the feasibility of using the Quantifiler(®) Trio DNA quantification kit for the direct quantification of forensic casework samples was investigated. Both low-level touch DNA samples and blood samples were collected on PE swabs and quantified directly. The increased sensitivity of the Quantifiler(®) Trio kit enabled the detection of less than 10pg of DNA in unprocessed touch samples and also minimizes the stochastic effect experienced by different targets in the same sample. The DNA quantity information obtained from a direct quantification assay using the Quantifiler(®) Trio kit can also be used to accurately estimate the optimal input DNA quantity for a direct STR amplification reaction. The correlation between the direct quantification results (Quantifiler(®) Trio kit) and the direct STR results (GlobalFiler™ PCR amplification kit(*)) for low-level touch DNA samples indicates that direct quantification using the Quantifiler(®) Trio DNA quantification kit is more reliable than the Quantifiler(®) Duo DNA quantification kit for predicting the STR results of unprocessed touch DNA samples containing less than 10pg of DNA. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Repulsive DNA-DNA interactions accelerate viral DNA packaging in phage Phi29.

Science.gov (United States)

Keller, Nicholas; delToro, Damian; Grimes, Shelley; Jardine, Paul J; Smith, Douglas E

2014-06-20

We use optical tweezers to study the effect of attractive versus repulsive DNA-DNA interactions on motor-driven viral packaging. Screening of repulsive interactions accelerates packaging, but induction of attractive interactions by spermidine(3+) causes heterogeneous dynamics. Acceleration is observed in a fraction of complexes, but most exhibit slowing and stalling, suggesting that attractive interactions promote nonequilibrium DNA conformations that impede the motor. Thus, repulsive interactions facilitate packaging despite increasing the energy of the theoretical optimum spooled DNA conformation.

Quantification bias caused by plasmid DNA conformation in quantitative real-time PCR assay.

Science.gov (United States)

Lin, Chih-Hui; Chen, Yu-Chieh; Pan, Tzu-Ming

2011-01-01

Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification.