Showing papers on "Nuclear DNA published in 1994"
TL;DR: A simple, rapid, and selective procedure for extraction of the degraded, low-molecular-weight DNA from apoptotic cells, and the ladder pattern was detected from DNA extracted from 1-2 x 10(6) HL-60 cells, by flow cytometric criteria, as well as from blood and bone marrow samples from leukemic patients undergoing chemotherapy.
661 citations
TL;DR: The utility of exon-primed, intron-crossing amplifications is tested by analyzing the variability of actin intron sequences from humpback, blue, and bowhead whales and comparing the results with mitochondrial DNA (mtDNA) haplotype data.
Abstract: Powerful analyses of population structure require information from multiple genetic loci. To help develop a molecular toolbox for obtaining this information, we have designed universal oligonucleotide primers that span conserved intron-exon junctions in a wide variety of animal phyla. We test the utility of exon-primed, intron-crossing amplifications by analyzing the variability of actin intron sequences from humpback, blue, and bowhead whales and comparing the results with mitochondrial DNA (mtDNA) haplotype data. Humpback actin introns fall into two major clades that exist in different frequencies in different oceanic populations. It is surprising that Hawaii and California populations, which are very distinct in mtDNAs, are similar in actin intron alleles. This discrepancy between mtDNA and nuclear DNA results may be due either to differences in genetic drift in mitochondrial and nuclear genes or to preferential movement of males, which do not transmit mtDNA to offspring, between separate breeding grounds. Opposing mtDNA and nuclear DNA results can help clarify otherwise hidden patterns of structure in natural populations.
435 citations
TL;DR: The quantitative approach may allow one to estimate oxidative DNA damage in an animal or individual exposed to prooxidant conditions associated with lifestyle, genetic predisposition, degenerative diseases, or environmental toxins by estimating the rate of oxidative DNA damaged within the body or in a population of cells.
Abstract: High-performance liquid chromatography with electrochemical detection is a highly sensitive and selective method for detecting oxo8dG and oxo8Gua, biomarkers of oxidative DNA damage. When employed together with the DNA isolation and monoclonal antibody-based immunoaffinity purification methods described, oxo8dG and oxo8Gua, in DNA and urine can be readily detected and quantitated, offering a powerful approach for assessing oxidative DNA damage in vivo. Application of the technique to the detection of oxo8dG from DNA permits quantitation of the steadystate levels of this oxidatively modified deoxynucleoside and overcomes the detection problems associated with the extremely low levels present in DNA. In addition, the selectivity gained by this detection method eliminates the problem of separating the signal for oxo8dG from those of normal deoxynucleosides. The quantitation of oxo8dG and oxo8Gua, in biological fluids is noninvasive and complements the measurement of oxo8dG in DNA by estimating the rate of oxidative DNA damage occurring within the body or in a population of cells. This analytical approach may allow one to estimate oxidative DNA damage in animal or individual exposed to prooxidant conditions associated with lifestyle, genetic predisposition, degenerative diseases, or environmental toxins. Furthermore, these assays may allow one to assess the potentially beneficial effects of intervention strategies that protect DNA from such damage.
319 citations
TL;DR: The results of the study showed that the size of nuclear genome of Musa is smaller than previously estimated, and it was found that nuclear DNA content of M. balbisiana (genome BB) is significantly lower than that ofM.
Abstract: Cell nuclei were isolated from leaf tissues of wild banana (Musa balbisiana, M. acuminata ssp.banksii andM. acuminata ssp.errans) and of the two vegetative clones of diploid cultivar “Pisang Mas”. Relative fluorescence intensity was measured on propidium iodide-stained nuclei by flow cytometry. Nuclei isolated fromGlycine max with known nuclear genome size were used as internal standard to determine nuclear DNA content ofMusa in absolute units. The results of the study showed that the size of nuclear genome ofMusa is smaller than previously estimated. In general, it is smaller in comparison with many other angiosperms. Furthermore, it was found that nuclear DNA content ofM. balbisiana (genome BB) is significantly lower than that ofM. acuminata subspecies and cultivars (genome AA). This finding should permit estimation of genome composition in triploidMusa clones with expected hybrid composition. Flow cytometry is proposed as a useful technique with potential applications in taxonomy, breeding and biotechnology ofMusa.
250 citations
TL;DR: The hypothesis that oxidative molecular damage is a causal factor in senescence is supported, and the 8OHdG levels were found to be inversely associated with the life expectancy of houseflies.
Abstract: The objective of this study was to explore the relationship between oxidative molecular damage and the aging process by determining whether such damage is associated with the rate of aging, using the adult housefly as the experimental organism. Because the somatic tissues in the housefly consist of long-lived postmitotic cells, it provides an excellent model system for studying cumulative age-related cellular alterations. Rate of aging in the housefly was manipulated by varying the rate of metabolism (physical activity). The concentration of 8-hydroxydeoxyguanosine (80HdG) was used as an indicator of DNA oxidation. Exposure of live flies to x-rays and hyperoxia elevated the level of 8OHdG. The level of 8OHdG in mitochondrial as well as total DNA increased with the age of flies. Mitochondrial DNA was 3 times more susceptible to age-related oxidative damage than nuclear DNA. A decrease in the level of physical activity of the flies was found to prolong the life-span and corresponding reduce the level of 8OHdG in both mitochondrial and total DNA. Under all conditions examined, mitochondrial DNA exhibited a higher level of oxidative damage than total DNA. The 8OHdG levels were found to be inversely associated with the life expectancy of houseflies. The pattern of age-associated accrural of 8OHdG was virtually identical to that of protein carbonyl content. Altoghether, results of this study support the hypothesis that oxidative molecular damage is a causal factor in senescence.
184 citations
TL;DR: A novel source of nuclear DNA information from random amplified polymorphisms (RAPD) and a wide‐range mitochondrial DNA information (cytochrome b, cytochrome oxidase, and 12s rRNA sequence, RFLP from 4‐base and 6‐base recognition endonucleases) are used to reconstruct the population phylogeny of the western Canary Island lizard, Gallotia galloti.
Abstract: A novel source of nuclear DNA information from random amplified polymorphisms (RAPD) and a wide-range mitochondrial DNA information (cytochrome b, cytochrome oxidase, and 12s rRNA sequence, RFLP from 4-base and 6-base recognition endonucleases) are used to reconstruct the population phylogeny of the western Canary Island lizard, Gallotia galloti, which, for geological reasons, has been subject to dispersal but not vicariance. Interpretation of DNA phylogenies in terms of colonization sequence indicates that G. galloti arose in Tenerife and dispersed westward in two independent pathways: north from north Tenerife to La Palma, and south from south Tenerife to Gomera to Hierro. The direction and timing of colonization by DNA divergence is entirely compatible with geological time and sequence of island origin.
148 citations
TL;DR: Pairwise comparisons between individual RFLP loci showed that the majority of alleles at these loci were in gametic equilibrium, as is expected in a random-mating population of wheat pathogen S. nodorum.
Abstract: Anonymous nuclear DNA markers were used to characterize the genetic structure of field populations of the wheat pathogen Stagonospora nodorum. High levels of genetic and genotypic diversity were found when DNA probes that hybridized to single restriction fragment length polymorphism (RFLP) loci and to a dispersed, repetitive DNA sequence were used. The populations surveyed contained a high number of different genotypes distributed on a small scale. In the majority of cases, separate lesions on the same leaf were caused by different genotypes of S. nodorum. Pairwise comparisons between individual RFLP loci showed that the majority of alleles at these loci were in gametic equilibrium, as is expected in a random-mating population [...]
148 citations
TL;DR: This work compared six sets of nuclear and mitochondrial noncoding DNA sequences of primates and obtained the estimates of the evolutionary rate of insertion and deletion, and the maximum-parsimony principle was applied to locate insertions and deletions on a given phylogenetic tree.
Abstract: Insertions and deletions are responsible for gaps in aligned nucleotide sequences, but they have been usually ignored when the number of nucleotide substitutions was estimated. We compared six sets of nuclear and mitochondrial noncoding DNA sequences of primates and obtained the estimates of the evolutionary rate of insertion and deletion. The maximum-parsimony principle was applied to locate insertions and deletions on a given phylogenetic tree. Deletions were about twice as frequent as insertions for nuclear DNA, and single-nucleotide insertions and deletions were the most frequent in all events. The rate of insertion and deletion was found to be rather constant among branches of the phylogenetic tree, and the rate (approximately 2.0/kb/Myr) for mitochondrial DNA was found to be much higher than that (approximately 0.2/kb/Myr) for nuclear DNA. The rates of nucleotide substitution were about 10 times higher than the rate of insertion and deletion for both nuclear and mitochondrial DNA.
115 citations
TL;DR: A linear method for the search of eukaryotic nuclear tRNA genes in DNA databases is described, which relies on the recognition of two intragenic control regions known as A and B boxes, a transcription termination signal, and on the evaluation of the spacing between these elements.
Abstract: A linear method for the search of eukaryotic nuclear tRNA genes in DNA databases is described. Based on a modified version of the general weight matrix procedure, our algorithm relies on the recognition of two intragenic control regions known as A and B boxes, a transcription termination signal, and on the evaluation of the spacing between these elements. The scanning of the eukaryotic nuclear DNA database using this search algorithm correctly identified 933 of the 940 known tRNA genes (0.74% of false negatives). Thirty new potential tRNA genes were identified, and the transcriptional activity of two of them was directly verified by in vitro transcription. The total false positive rate of the algorithm was 0.014%. Structurally unusual tRNA genes, like those coding for selenocysteine tRNAs, could also be recognized using a set of rules concerning their specific properties, and one human gene coding for such tRNA was identified. Some of the newly identified tRNA genes were found in rather uncommon genomic positions: 2 in centromeric regions and 3 within introns. Furthermore, the presence of extragenically located B boxes in tRNA genes from various organisms could be detected through a specific subroutine of the standard search program.
109 citations
TL;DR: Nuclear DNA helicase II has been purified to near-homogeneity by exploiting its high affinity to poly[(rI).(rC)]-agarose, and may function in both DNA and RNA unwinding.
Abstract: Nuclear DNA helicase II (NDH II) has been purified to near-homogeneity by exploiting its high affinity to poly[(rI).(rC)]-agarose. The purified enzyme was obtained as two catalytically active forms of 130- and 100-kDa molecular mass, respectively. After treatment with cyanogen bromide, the separated polypeptides displayed very similar digestion patterns. Thus, the 100-kDa form most likely is a proteolytic product of the 130-kDa polypeptide. For DNA unwinding, NDH II could use any of the four rNTPs or dNTPs with Km values between 20 and 100 microM. DNA unwinding was stimulated up to 20-fold by substrates that contained single-stranded 3'-tails. NDH II-catalyzed DNA unwinding was strongly inhibited by RNA, but was little affected by DNA. The strongest RNA inhibitor, poly[(rI).(rC)], was also the strongest effector of the NTPase activity of NDH II. The binding constant for poly[(rI).(rC)] binding was about 2 x 10(7) M-1; the minimal binding site size was determined as 16 nucleotides. In agreement with its high affinity to RNA, NDH II unwound double-stranded RNA. RNA unwinding required the presence of a nucleoside triphosphate and a divalent cation (Mg2+). Thus, like the prototypic replicative helicase large T antigen of simian virus 40, NDH II may function in both DNA and RNA unwinding.
105 citations
TL;DR: A relatively small and stable nuclear genome typifies the Prunus species investigated, consistent with their low, basic chromosome number (x = 8).
Abstract: Using laser flow cytometry, nuclear DNA amounts were estimated for 12 Prunus species, representing three subgenera (Prunophora (Prunus), Amygdalus, and Cerasus (Lithocerasus)), two interspecific hybrids, four cultivars, and a synthetic polyploid series of peach consisting of haploids, diploids, triploids, and tetraploids (periclinal cytochimeras). Peach nuclear DNA content ranged from 0.30 pg for the haploid nuclei to 1.23 pg for the tetraploid nuclei. The diploid genome of peach is relatively small and was estimated to be 0.60 ± 0.03 pg (or 5.8 × 10 8 nucleotide base pairs). The polyploid series represented the expected arithmetic progression, as genome size positively correlated with ploidy level (i.e., DNA content was proportional to chromosome number). The DNA content for the 12 diploid species and two interspecific diploid hybrids ranged from 0.57 to 0.79 pg. Genome size estimates were verified independently by Southern blot analysis, using restriction fragment length polymorphism clones as gene-copy equivalents. Thus, a relatively small and stable nuclear genome typifies the Prunus species investigated, consistent with their low, basic chromosome number (x = 8).
TL;DR: The nuclear DNA content estimates obtained by flow cytometry for a group of twelve Eucalyptus species and five fast-growing hybrids that includes those most widely planted throughout the world are reported, showing the closer the species were in phylogenetic relationship the more similar were their nuclearDNA content values.
Abstract: This paper reports the nuclear DNA content estimates obtained by flow cytometry for a group of twelve Eucalyptus species and five fast-growing hybrids that includes those most widely planted throughout the world. Estimates of nuclear (2C) DNA content for the species surveyed ranged from 0.77 pg/2C for Eucalyptuscitriodora Hook. (subgenus Corymbia) to 1.47 pg/2C for Eucalyptussaligna Smith (subgenus Symphyomyrtus). This range corresponds to a haploid genome size range of 370–700 megabase pairs. The average physical equivalent of a 1 cM distance could be as low as 200 kilobase pairs in Eucalyptus, an attractive feature for positional cloning efforts in woody plants. The closer the species were in phylogenetic relationship the more similar were their nuclear DNA content values. All the interspecific hybrids surveyed displayed a nuclear DNA content in the expected intermediate range between the respective parental species, with the exception of one originating from Rio Claro, Brazil, whose exact parentage is ...
TL;DR: Southern hybridization provided evidence for at least one family of highly repetitive DNA and, furthermore, revealed different amounts of repetitive DNA in the four genotypes, but these sequences cannot account for the 100% increase in the nuclear DNA amount in mutant P24, relative to wild type.
Abstract: A wild-type (WT) strain of the moss Physcomitrella patens (Hedw.) B.S.G., two mutants derived from it (PC22 and P24), and a somatic hybrid, PC22(+)P24, were analysed. Staining of metaphases revealed 54±2 chromosomes in the somatic hybrid and 27 chromosomes in the wild type and the two mutants. Using flow cytometry (FCM), DNA contents were calculated to be 0.6 pg (WT, PC22), 1.2 pg (P24), and 1.6 pg (PC22(+)P24) per nucleus, respectively. Southern hybridization provided evidence for at least one family of highly repetitive DNA and, furthermore, revealed different amounts of repetitive DNA in the four genotypes. However, these sequences cannot account for the 100% increase in the nuclear DNA amount in mutant P24, relative to wild type. In FCM analyses every moss geno-type generated just one single peak of fluorescence, indicating an arrest in the cell cycle during the daytime. Thermal denaturation of wild-type DNA revealed a G+C content of 34.6% for total DNA and 38.6% for plastid DNA. A cDNA library of 1.2 × 106 independent clones was established, from which sequences homologous to cab and rbcS, respectively, were isolated. These genes show significant homologies to those of higher plants, and, likewise, comprise multigene families. No restriction fragment length polymorphisms could be detected between the four moss genotypes using these cDNA probes.
TL;DR: The nuclear DNA content of 53 accessions from 24 Ipomoea (Convolvulaceae) species, including four sweetpotato cultivars, was determined by flow cytometry of DAPI-stained nuclei, and a strong correlation between DNA values determined by Feulgen mi- crospectrophotometry and flow cytometer has been shown.
Abstract: The nuclear DNA content of 53 accessions from 24 Ipomoea (Convolvulaceae) species, including four sweetpotato cultivars, was determined by flow cytometry of DAPI-stained nuclei. Ploidy level and DNA content were significantly correlated within the genus, but more highly so within species that contained multiple cytotypes. DNA content of cultivated Z. batatas (L.) Lam. (4.8 to 5.3 pg/2C nucleus) and feral tetraploid I. batatas (3.0 to 3.5 pg/2C nucleus) was estimated from the known DNA content of chicken erythrocytes (2.33 pg), which were used as an internal standard. Tetraploid forms of Z. cordato-triloba Dennstedt also were identified. Ploidy analysis using flow cytometry is rapid and suitable for large-scale experiments such as studying the genetic structure of populations of Z. batatas and related species. Chemical name used: 4´,6-diamidino-2-phenylindole (DAPI). Nuclear DNA content can be used to estimate ploidy level, provided that there has been sufficient cytological investigation to base the study on known chromosome numbers and ploidy ranges. The quantitation of DNA content often has been based on mi- crospectrophotometry of Feulgen-stained nuclei in squashed tis- sues (Bennett and Smith, 1976; Bennett et al., 1982). This method requires careful standardization of tissue samples and fixation, reaction, and spectrophotometer conditions (Price, 1988). More recently, flow cytometry of nuclei stained with DNA fluoro- chromes has been recognized as a fast, accurate alternative to microspectrophotometry (Dolezel, 1991; Galbraith, 1989). A strong correlation between DNA values determined by Feulgen mi- crospectrophotometry and flow cytometry has been shown (Michelson et al., 1991). The flow cytometer uses a laser beam that is focused on a small sample stream containing suspended nuclei. The nuclei can be stained with DNA fluorochromes such as ethidium bromide or propidium iodide (intercalating dyes), DAPI (specific for A-T base pairs), and mithramycin (specific for G-C base pairs). Computer-assisted measurements of fluorescence emission per fluorescent particle allow the generation of a histo- gram reflecting the distribution of fluorescence intensity among a fixed number of particles. Several thousand particles can be measured within minutes by flow cytometry, which has a capacity of at least two orders of magnitude greater than that of mi- crospectrophotometry. The genus Ipomoea (Convolvulaceae) is thought to contain >500 species with known ploidy levels of 2x,4x, and 6x and a basic chromosome number of x = 15 (Jones, 1964, 1968). The high basic chromosome number and small chromosome size in species of this genus make cytological studies laborious and time consuming. Morphological plasticity within the genus has led to considerable
TL;DR: The chapter focuses on the use of 6-diamidino-2-phenylindole (DAPI) for flow cytometry (FCM), which proved to be a specific, highly fluorescent stain, very well suited for FCM of DNA in whole cells, nuclei, and chromosomes.
Abstract: Publisher Summary Protocols are available for high-resolution analysis of nuclear DNA that enable to discriminate somatic cells from male and female donors and to characterize precisely the ploidy state of tumors This chapter discusses protocols developed for use in cells obtained from cell suspensions and solid tissues. The procedures include fixation with 70% ethanol. Thus, they are applicable in specimens that have to be stored before measurement. The chapter focuses on the use of 6-diamidino-2-phenylindole (DAPI) for flow cytometry (FCM). DAPI proved to be a specific, highly fluorescent stain, very well suited for FCM of DNA in whole cells, nuclei, and chromosomes. The protocols are recommended for accurate and reproducible measurements of the nuclear DNA, especially for the assessment of DNA content variations in cell populations affected by chemical or physical mutagens and for the reliable detection and characterization of aneuploid cell clones in tumors.
TL;DR: A homolog of cdc2 from Aspergillus nidulans that can complement the Schizosaccharomyces pombe cDC2-33 mutation is cloned and sequenced and designated nimXcdc2 as it is not allelic to any of the other nim genes (nimA to nimW) of A. ndulans.
Abstract: We have cloned and sequenced a homolog of cdc2 from Aspergillus nidulans that can complement the Schizosaccharomyces pombe cdc2-33 mutation. The gene was deleted and is required for continued nuclear DNA replication but not for mitochondrial DNA replication. Three different temperature-sensitive alleles were generated by reverse genetics. All of the mutations generate the nim phenotype of A. nidulans. The new gene was designated nimXcdc2 as it is not allelic to any of the other nim genes (nimA to nimW) of A. nidulans. Reciprocal shift experiments place an essential function for nimXcdc2 in G1 and G2. Antipeptide antibodies were generated that detect NIMXcdc2, and antisera were also generated to detect NIMEcyclinB. The two p34cdc2 protein species previously detected in A. nidulans, p34 and p37, both precipitate using NIMXcdc2 C-terminus-specific antibodies but only p34 co-precipitates with NIMEcyclinB. Dephosphorylation of denatured p34 converts it to the p37 form, showing p37 to be the non-phosphorylated form of NIMXcdc2. The phosphorylation of p34 is therefore associated with its interaction with NIMEcyclinB.
TL;DR: The maintenance of the spotted appearance of the replication clusters through all stages of the three subsequent cell cycles may be an indication in favour of the hypothesis that such clusters represent structurally stable replicon complexes held together by the nuclear matrix and the chromosome scaffold.
Abstract: Nuclear DNA replication was monitored 'in situ' in pea nuclei with the bromodeoxyuridine antibody technique. The labelling appeared to be restricted to a number of finely distinct spots. The labelling was followed through three subsequent cell cycles in meristematic and differentiating pea root cells. The results show that the spots as seen just after the labelling persist distinctly over the mitotic chromosomes as well as in the nuclei of the following cell cycles up to 44 hours after the pulse. Moreover, they are also present in the nuclei of differentiating cells. The spots over the mitotic chromosomes in specific cases give rise to a dynamic banding. Nuclei of the second and third cycle show absence of labelling in specific zones, owing to the segregation of the labelled strands of chromosomal DNA. The maintenance of the spotted appearance of the replication clusters through all stages of the three subsequent cell cycles may be an indication in favour of the hypothesis that such clusters represent structurally stable replicon complexes held together by the nuclear matrix and the chromosome scaffold.
TL;DR: Results suggest that hydroxy radical is produced through a Fenton reaction by Cu2+ and H2O2 released from the glycated Cu,Zn-SOD, and participates in nuclear DNA cleavage, which may partly explain the deterioration of organs under diabetic conditions.
Abstract: Human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) undergoes site-specific and random fragmentation by non-enzymic glycosylation (glycation). Released Cu2+ from the glycated Cu,Zn-SOD probably facilitates a Fenton reaction to convert H2O2 into hydroxy radical, which then participates in the non-specific fragmentation [Ookawara et al. (1992) J. Biol. Chem. 267, 18505-18510]. In the present study, we investigated the effects of glycated Cu,Zn-SOD on cloned DNA fragments and nuclear DNA and analysed the formation of 8-hydroxydeoxyguanosine (8-OH-dG). Incubation of cloned DNA fragments with Cu,Zn-SOD and reducing sugars resulted in cleavage of the DNA. The extent of the cleavage corresponded to the reducing capacity of the sugar. Metal-chelating reagents, EDTA and bathocuproine, and an H2O2 scavenger, catalase, inhibited the DNA cleavage. Hydroxy radical scavengers and aminoguanidine, an inhibitor of glycation, also inhibited the reaction. Moreover, the glycation of Cu,Zn-SOD caused the substantial formation of 8-OH-dG in DNA. When isolated nuclei were incubated with CuCl2 plus H2O2, nuclear DNA cleavage was observed. Incubation of isolated nuclei with Cu,Zn-SOD that had been pre-incubated with glucose also resulted in nuclear DNA cleavage. These results suggest that hydroxy radical is produced through a Fenton reaction by Cu2+ and H2O2 released from the glycated Cu,Zn-SOD, and participates in nuclear DNA cleavage. This mechanism may partly explain the deterioration of organs under diabetic conditions.
TL;DR: This article focuses on the most dramatic cell‐type‐specific DNA organization, that found in spermatozoa, in which the entire genome is reorganized into an inactive state that is more highly condensed than mitotic chromosomes.
Abstract: The tertiary structure of the DNA that makes up the eukaryotic genome is remarkably plastic, taking many different forms in response to the different needs of the cell. During the cell cycle of one cell, the DNA is replicated, reorganized into mitotic chromosomes, and decondensed into interphase chromatin. Within one cell at any given point in time, the chromatin is divided into hetero- and euchromatin reflecting active and inactive states of the DNA. This organization varies within one organism since different parts of the genome are active in different cell types. This article focuses on the most dramatic cell-type-specific DNA organization, that found in spermatozoa, in which the entire genome is reorganized into an inactive state that is more highly condensed than mitotic chromosomes. This unique example of eukaryotic DNA organization offers some interesting clues to the still unanswered questions about the role that the three-dimensional packaging of DNA plays in its function.
TL;DR: Competition between FIAUTP and dTTP and the relative ease of accumulation of FIAUMP in mitochondrial DNA by DNA pol-gamma in vitro together may relate to clinical FIAU toxicity.
Abstract: Fialuridine [FIAU, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5- iodouridine] was used in clinical trials for chronic hepatitis B virus infection and was extremely toxic. Evidence suggested targets of FIAU toxicity included mitochondria, but toxic mechanisms were unclear. Since FIAU is a thymidine analog, we reasoned that triphosphorylated FIAU (FIAUTP) could be incorporated into mitochondrial DNA by DNA pol-gamma and into genomic DNA by DNA polymerases alpha, beta, delta, and epsilon. All five purified mammalian DNA polymerases incorporated FIAUMP into the nascent DNA chain during in vitro DNA synthesis. When FIAUTP was substituted for dTTP, oligonucleotide products were generated efficiently by DNA pol-gamma and were similar to those generated in the presence of the four normal dNTPs. In contrast, oligonucleotide products generated by the four nuclear DNA polymerases in the presence of FIAUTP were significantly reduced in length relative to those generated in the presence of dTTP. In parallel kinetic assays, FIAUTP competitively inhibited the accumulation of radiolabeled dTTP into DNA by DNA pol-gamma. The Ki with DNA pol-gamma was 0.04 microM, the lowest Ki among the mammalian DNA polymerases. Competition between FIAUTP and dTTP and the relative ease of accumulation of FIAUMP in mitochondrial DNA by DNA pol-gamma in vitro together may relate to clinical FIAU toxicity.
TL;DR: The quantitative assessment of 8-OHdG by ISB assay, with fmol sensitivity and direct analysis using unhydrolyzed DNA, should prove a highly valuable alternative to currently used approaches to detecting 8- OHdG in enzymatic DNA hydrolysates.
Abstract: The modified DNA base 8-hydroxyguanine has been implicated in spontaneous mutagenesis, carcinogenesis and cellular aging. Polyclonal antibodies specific for the 8-hydroxy-2'-deoxyguanosine moiety in oxidized DNA were used for sensitive detection and quantitation of this biomarker of oxidative damage to cellular DNA. The analysis was performed with immunoslot blot assay (ISB) of oxidized DNA modified in vitro with methylene blue plus light and upon H2O2 treatment of cultured human cells. The level of 8-OHdG in DNA exposed to 90 and 120 min light in the presence of 100 microM methylene blue showed 15.96 +/- 2.4 and 22.65 +/- 3.65 pmol/micrograms DNA compared to 0.107 +/- 0.024 pmol/micrograms in commercial calf thymus DNA control. Inherent damage, due to cellular endogenous oxidation of DNA, increased significantly upon inhibition of catalase activity in human cells with 10 mM azide. The damage increased further on exposure of azide-treated cells to H2O2. The amounts of 8-OHdG following treatment of cells with 10 and 100 microM H2O2 were determined to be 205 +/- 42 and 333 +/- 17.5 pmol/micrograms DNA respectively. Very low but quantifiable antibody binding was seen with the 'control unoxidized' human nuclear DNA. This DNA, obtained under controlled conditions to restrict the induction of 8-OHdG during isolation, provides a background level of 0.022 +/- 0.005 pmol 8-OHdG/micrograms DNA. The quantitative assessment of 8-OHdG by ISB assay, with fmol sensitivity and direct analysis using unhydrolyzed DNA, should prove a highly valuable alternative to currently used approaches to detecting 8-OHdG in enzymatic DNA hydrolysates.
TL;DR: As previously demonstrated, chromium-DNA adducts in total nuclear DNA decreased within the first 24 h, but thereafter persisted at a similar level, whereas the adduct levels in the 1SF and bulk chromatin fractions did not change up to 48 h after treatment.
Abstract: The distributions of chromium-DNA adducts and DNA-protein crosslinks induced by treatment of intact CHO cells with carcinogenic chromium were examined in distinct chromatin subfractions: a chromatin subfraction released by digestion of isolated nuclei with micrococcal nuclease (1SF, 14% of total nuclear DNA), bulk chromatin (74% of total DNA) and a nuclear matrix fraction (12% of total DNA). The identity of the matrix fraction was confirmed by hybridization of DNA from each subfraction with a cDNA probe prepared from total mRNA isolated from CHO cells, which showed that the 1SF and nuclear matrix fractions were 2.3- and 3.8-fold enriched in actively transcribed genes respectively, compared to total unfractionated DNA. Immediately following treatment of cells with 150 microM sodium chromate for 2 h the binding of chromium to each chromatin fraction was found to be non-uniform. Compared with total unfractionated nuclei, the nuclear matrix fractions were enriched in chromatin-bound chromium (3.4-fold), whereas the bulk chromatin fraction was relatively depleted (0.5-fold). Approximately 13% of nuclear chromium was associated with the detergent-soluble lipid component of nuclei. A similar distribution of chromatin-bound chromium was also apparent 24 h after the chromate treatment. Immediately after the 2 h chromate treatment, chromium-DNA adducts were detected in all the chromatin subfractions. Total nuclear and bulk chromatin DNA contained similar levels of this type of damage. The 1SF fraction was depleted approximately 3-fold in this type of damage compared with total nuclear DNA. In contrast, the nuclear matrix was markedly enriched in chromium-DNA adducts (approximately 4-fold compared with total nuclear DNA) at this time. As previously demonstrated, chromium-DNA adducts in total nuclear DNA decreased within the first 24 h, but thereafter persisted at a similar level. Chromium-DNA adducts in nuclear matrix DNA also reached maximum levels at the end of the 2 h treatment and decreased to 68% and 39% of this level by 24 and 48 h after treatment respectively. In contrast, the adduct levels in the 1SF and bulk chromatin fractions did not change up to 48 h after treatment. Chromium-induced DNA-protein crosslinks, which were stable to 8 M urea and 2% SDS, occurred almost exclusively in the nuclear matrix fraction. The crosslinks in this fraction reached a maximum level at the end of the 2 h treatment, but returned to control levels 24 h later.(ABSTRACT TRUNCATED AT 400 WORDS)
TL;DR: It is concluded that rapid DNA loss in the first and subsequent generations was not only associated with the substantial increase in the seed set, but it also helped in the establishment and stabilization of the tetraploid.
Abstract: The 2C and 4C nuclear DNA amounts were estimated in eight diploid species, belonging to three diverse genera (Vicia, Tephrosia, and Phlox) and their corresponding colchitetraploids. In P. drummondi...
TL;DR: The data suggest that mtDNA is inherited uniparentally in P. infestans with no evidence for segregation, elimination or recombination of types.
TL;DR: The cloning and characterization of a cDNA clone AtTopII encoding the first reported TOPII from higher plants is reported, which shows strong homologies to TOPII sequences reported from other eukaryotes, particularly in the regions that are highly conserved among different species.
Abstract: Topoisomerase II (TOPII) is an important enzyme involved in DNA replication and chromosome condensation. The level of TOPII expression has been correlated with the proliferative state of eukaryotic cells. Here we report the cloning and characterization of a cDNA clone AtTopII encoding the first reported TOPII from higher plants. AtTopII is 4603 base pairs (bp) in length and encodes an open reading frame of 1473 amino acid residues. One interesting feature of AtTopII is the presence of a 110 bp direct repeat in the last one-third of the cDNA. Analysis of the genomic sequence within this region by PCR revealed that this duplication includes a small intron of 89 bp. Conservation of sequences within this repeated intron suggests that this in-frame duplication may be a relatively recent event. The deduced amino acid sequence of AtTopII shows strong homologies to TOPII sequences reported from other eukaryotes, particularly in the regions that are highly conserved among different species. Southern blot analysis with Arabidopsis DNA indicates that AtTopII is a single-copy gene while Northern blots detected a 5.0 kb transcript, the level of which is substantially higher in young seedlings than in mature plants. Using a polyclonal antiserum raised against the C-terminal one-third of AtTOPII, we found that the protein is localized in the nucleus and its level is correlated with the proliferative state of the particular tissue.
TL;DR: Evidence is found that each allele can be inherited from either sex, that there are no barriers to fertilization between gametes of different genotypes, and that most larvae have genotypes compatible with Mendelian inheritance of the locus.
Abstract: We examined the inheritance of theMytilus edulis CaM-1 Intron 3 locus, a non-coding DNA locus with two potentially neutral length-variants. The polymerase chain reaction (PCR) was used to determine theCaM-1 genotype for 799 larvae obtained from 11 laboratory crosses. Larvae were typed singly only 8 to 24 h after fertilization. We find evidence that each allele can be inherited from either sex, that there are no barriers to fertilization between gametes of different genotypes, and that most larvae have genotypes compatible with Mendelian inheritance of the locus. Deviations from expected genotype frequencies were found in some crosses; we suggest that a contributing factor is aneuploidy in early larvae, but a major cause is more likely to be selection at a locus linked toCaM-1, occurring under the artificial laboratory culture conditions. This study demonstrates the feasibility of applying molecular genetic techniques to early larval stages of marine bivalves, and presents a new non-destructive biopsy method for DNA analysis from living adult mussels.
TL;DR: The results have confirmed and extended the possibility of sex identification in species of birds characterized by sexual monomorphism by evaluating the small DNA content difference which exists between males and females.
Abstract: A high interspecific karyotype variability has been evidenced in birds especially in Falconiformes and Strigiformes. Avian cytogenetic analysis, conventionally used for this study, presents several difficulties. We used flow cytometric analysis in order to obtain further information on the DNA patterns of different species of birds belonging to the above-mentioned orders. Our study was performed on blood samples while chicken erythrocytes and human lymphocytes, with known cytometric DNA content, were used as reference cells. The blood samples of the birds under study were stained, simultaneously to the reference cell, with a lysis-staining buffer containing propidium iodide. The nuclear DNA content of the bird samples was calculated as DNA index in relation to reference cells, and was expressed as nuclear DNA mass in picograms (pg) with respect to the standard value of 7.0 pg per human lymphocyte nucleus. The results obtained showed an interspecific variability of DNA content and evidenced the usefulness of FCM analysis as a rapid and easy tool for studying the DNA pattern of different species of birds. Moreover, our results have confirmed and extended the possibility of sex identification in species of birds characterized by sexual monomorphism by evaluating the small DNA content difference which exists between males and females. © 1994 Wiley-Liss, Inc.
TL;DR: The main objective of this study was to estimate the nuclear DNA content in ten rice species by flow cytometry, which allowed rapid and reliable determination of the ploidy level of anther culture-derived plants.
Abstract: Accurate estimations of nuclear DNA content of rice are important because this crop is being used in many types of molecular studies. The main objective of this study was to estimate the nuclear DNA content in ten rice species by flow cytometry. Most of the values obtained were lower than those obtained in earlier studies using Feulgen microdensitometry. Significant differences in genome size among rice species were found. Oryza glaberrima had the smallest genome (0.73-0.76 pg/2C), while O. minuta and O. latifolia had the largest values (ca. 2.33 pg/2C). Tetraploid species had more nuclear DNA than diploid ones. The AA genome had less DNA (0.86-0.96 pg/2C) than the CC (1.14-1.17 pg/2C) and EE (1.99 pg/2C) genomes. The AA genome of O. saliva was larger than the AgA g genome of O. glaberrima and the A1A 1 genome of O. longistaminata, which had mean values of 0.73-0.76 and 0.78 pg/2C, respectively. Indica (IR36) and japonica (Yukihikari) cultivars of O. saliva also showed significant differences. Three different nuclear DNA levels (1.93, 1.85, and 1.31 pg/2C) were found among five O. ridleyi plants analyzed. Flow cytometry also allowed rapid and reliable determination of the ploidy level of anther culture-derived plants.
TL;DR: While the rate of O6-EtGua elimination from DNA was decelerated it was not abolished, suggesting the possible involvement of additional repair systems that might be co-regulated with AT.
Abstract: Inter-individual and cell-cell variability of repair of O6-alkylguanines (O6-AlkGua) in nuclear DNA was studied at the single-cell level in peripheral lymphocytes from healthy donors and in leukaemic cells isolated from patients with chronic lymphatic leukaemia (CLL) or acute myeloid leukaemia (AML). Cells were pulse exposed to N-ethyl- or N-(n-)butyl-N-nitrosourea in vitro, and O6-AlkGua residues in DNA were quantified using an anti-(O6-AlkGua) monoclonal antibody and electronically intensified fluorescence. The kinetics of O6-AlkGua elimination revealed considerable inter-individual differences in O6-ethylguanine (O6-EtGua) half-life (t1/2) values in DNA, ranging from 1.5 to 4.5 h (five AML patients), from 0.8 to 2.8 h (five CLL patients) and from 1.2 to 7.3 h (five healthy donors). The elimination from DNA of equimolar amounts of O6-butylguanine was generally 3-5 times slower in comparison with O6-EtGua. The t1/2 values of individual samples varied in parallel for both DNA alkylation products. Upon preincubation with O6-benzylguanine, the activity of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AT) in both lymphocytes and leukaemic blasts was reduced to < or = 1%. However, while the rate of O6-EtGua elimination from DNA was decelerated it was not abolished, suggesting the possible involvement of additional repair systems that might be co-regulated with AT. Within individual samples, no major cell subpopulations were observed whose repair kinetics would differ significantly from the remaining cells.