Showing papers on "Nuclear DNA published in 2002"

Changes in Mitochondrial DNA as a Marker of Nucleoside Toxicity in HIV-Infected Patients

Abstract: Background Nucleoside analogues can induce toxic effects on mitochondria by inhibiting the human DNA polymerase γ. The toxic effects can range from increased serum lactate levels to potentially fatal lactic acidosis. We studied changes in mitochondrial DNA relative to nuclear DNA in the peripheral-blood cells of patients with symptomatic, nucleoside-induced hyperlactatemia. Methods Total DNA was extracted from blood cells. A nuclear gene and a mitochondrial gene were quantified by real-time polymerase chain reaction. Three groups were studied: 24 controls not infected with the human immunodeficiency virus (HIV), 47 HIV-infected asymptomatic patients who had never been treated with antiretroviral drugs, and 8 HIV-infected patients who were receiving antiretroviral drugs and had symptomatic hyperlactatemia. The patients in the last group were studied longitudinally before, during, and after antiretroviral therapy. Results Symptomatic hyperlactatemia was associated with marked reductions in the ratios of mit...

Nature of DNA Damage in Ejaculated Human Spermatozoa and the Possible Involvement of Apoptosis

Abstract: Numerous studies have shown the presence of DNA strand breaks in human ejaculated spermatozoa. The nature of this nuclear anomaly and its relationship to patient etiology is however poorly understood. The aim of this study was to investigate the relationship between nuclear DNA damage, assessed using the TUNEL assay and a number of key apoptotic markers, including Fas, Bcl-x, and p53, in ejaculated human spermatozoa from men with normal and abnormal semen parameters. We also determined the nature of the DNA damage by examining the percentage of ejaculated spermatozoa exhibiting DNA damage using the comet assay and by challenging sperm chromatin to attack by micrococcal nuclease S7 and DNase I. We show that TUNEL positivity and apoptotic markers do not always exist in unison; however, semen samples that had a low sperm concentration and poor morphology were more likely to show high levels of TUNEL positivity and Fas and p53 expression. In addition, the DNA damage in ejaculated human sperm is represented by both single- and double-stranded DNA breaks, and access to the DNA is restricted by the compacted nature of ejaculated spermatozoa. This DNA protection is poorer in men with abnormal semen parameters. We propose that the presence of DNA damage is not directly linked to an apoptotic process occurring in spermatozoa and arises due to problems in the nuclear remodeling process. Subsequently, the presence of apoptotic proteins in ejaculated spermatozoa may be linked to defects in cytoplasmic remodeling during the later stages of spermatogenesis.

Mitochondrial DNA repair and aging.

Abstract: The mitochondrial electron transport chain plays an important role in energy production in aerobic organisms and is also a significant source of reactive oxygen species that damage DNA, RNA and proteins in the cell. Oxidative damage to the mitochondrial DNA is implicated in various degenerative diseases, cancer and aging. The importance of mitochondrial ROS in age-related degenerative diseases is further strengthened by studies using animal models, Caenorhabditis elegans, Drosophila and yeast. Research in the last several years shows that mitochondrial DNA is more susceptible to various carcinogens and ROS when compared to nuclear DNA. DNA damage in mammalian mitochondria is repaired by base excision repair (BER). Studies have shown that mitochondria contain all the enzymes required for BER. Mitochondrial DNA damage, if not repaired, leads to disruption of electron transport chain and production of more ROS. This vicious cycle of ROS production and mtDNA damage ultimately leads to energy depletion in the cell and apoptosis.

Early Detection of DNA Strand Breaks in the Brain After Transient Focal Ischemia: Implications for the Role of DNA Damage in Apoptosis and Neuronal Cell Death

Abstract: Using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL), we investigated the evolution of DNA strand breaks, a marker of DNA damage, in rat brain after 1 h of middle cerebral artery occlusion and various durations of reperfusion. DNA single-strand breaks (SSBs) detected by PANT were present in neurons after as little as 1 min of reperfusion. Numbers of neurons containing an SSB increased progressively in the ischemic core but decreased in the ischemic penumbra after 1 h of reperfusion. DNA double-strand breaks (DSBs) detected by TUNEL were first seen in neurons after 1 h of reperfusion, and their numbers then increased progressively in the ischemic core, with a regional distribution similar to that of SSBs. However, the number of SSB-containing cells was greater than that of DSB-containing cells at all time points tested. SSB-containing cells detected within the first hour of reperfusion were exclusively neuronal and exhibited normal nuclear morphology. At 16-72 h of reperfusion, many SSB- and DSB-containing cells, including both neurons and astrocytes, showed morphological changes consistent with apoptosis. Gel electrophoresis of DNA isolated from the ischemic core showed DNA fragmentation at 24 h, when both SSBs and DSBs were present, but not at 1 h, when few DSBs were detected. These results suggest that damage to nuclear DNA is an early event after neuronal ischemia and that the accumulation of unrepaired DNA SSBs may contribute to delayed ischemic neuronal death, perhaps by triggering apoptosis.

Mechanisms and rates of genome expansion and contraction in flowering plants

Abstract: Plant genomes are exceptional for their great variation in genome size, an outcome derived primarily from their frequent polyploid origins and from the amplification of retrotransposons. Although most studies of plant genome size variation have focused on developmental or physiological effects of nuclear DNA content that might influence plant fitness, more recent studies have begun to investigate possible mechanisms for plant genome expansion and contraction. Analyses of 'relatively neutral' genome components, like transposable elements, have been particularly fruitful, largely due to the enormous growth in genomic sequence information from many different plant species. Current data suggest that unequal recombination can slow the growth in genome size caused by retrotransposon amplification, but that illegitimate recombination and other deletion processes may be primarily responsible for the removal of non-essential DNA from small genome plants.

Pattern of Organization of Human Mitochondrial Pseudogenes in the Nuclear Genome

Abstract: Mitochondrial pseudogenes in the human nuclear genome have been previously described, mostly as a source of artifacts during the analysis of the mitochondrial genome. With the availability of the complete human genome sequence, we performed a comprehensive analysis of mtDNA insertions into the nucleus. We found 612 independent integrations that are evenly distributed among all chromosomes as well as within each individual chromosome. The identified pseudogenes account for a content of at least 0.016% of the human nuclear DNA. Up to 30% of a chromosome's mtDNA pseudogene content is composed of fragments that encompass two or more adjacent mitochondrial genes, and we found no correlation between the abundance of mitochondrial transcripts and the multiplicity of integrations. These observations indicate that the migrations of mitochondrial DNA sequences to the nucleus were predominantly DNA mediated. Phylogenetic analysis of the mtDNA pseudogenes and mtDNA sequences of primates indicate a continuous transfer into the nucleus. Because of the limited window of opportunity for mtDNA transfer to the germline, sperm mtDNA, which is released from degenerating mitochondria after fertilization, could be an important source of nuclear mtDNA pseudogenes.

ZEN1 Is a Key Enzyme in the Degradation of Nuclear DNA during Programmed Cell Death of Tracheary Elements

Abstract: Tracheary elements (TEs) have a unique cell death program in which the rapid collapse of the vacuole triggers the beginning of nuclear degradation. Although various nucleases are known to function in nuclear DNA degradation in animal apoptosis, it is unclear what hydrolase is involved in nuclear degradation in plants. In this study, we demonstrated that an S1-type nuclease, Zinnia endonuclease 1 (ZEN1), functions directly in nuclear DNA degradation during programmed cell death (PCD) of TEs. In-gel DNase assay demonstrated the presence of a 24-kD Ca2+/Mg2+-dependent nuclease and a 40-kD Zn2+-dependent nuclease as well as ZEN1 in 60-h-cultured cells that included differentiating TEs. Such cell extracts possessed the ability to degrade the nuclear DNA isolated from Zinnia elegans cells in the presence of Zn2+, and its activity was suppressed by an anti-ZEN1 antibody, indicating that ZEN1 is a central DNase responsible for nuclear DNA degradation. The introduction of the antisense ZEN1 gene into Zinnia cells cultured for 40 h specifically suppressed the degradation of nuclear DNA in TEs undergoing PCD but did not affect vacuole collapse. Based on these results, a common mechanism between animal and plant PCD is discussed.

Divergence of mitochondrial dna is not corroborated by nuclear dna, morphology, or behavior in drosophila simulans

Abstract: We ask whether the observed mitochondrial DNA (mtDNA) population subdivision of Drosophila simulans is indicative of organismal structure or of specific processes acting on the mitochondrial genome. Factors either intrinsic or extrinsic to the host genome may influence the evolutionary dynamics of mtDNA. Potential intrinsic factors include adaptation of the mitochondrial genome and of nucleomitochondrial gene complexes specific to the local environment. An extrinsic force that has been shown to influence mtDNA evolution in invertebrates is the bacterial endosymbiont Wolbachia. Evidence presented in this study suggests that mtDNA is not a good indicator of organismal subdivision in D. simulans. Furthermore, there is no evidence to suggest that Wolbachia causes any reduction in nuclear gene flow in this species. The observed differentiation in mtDNA is not corroborated by data from NADH: ubiquinone reductase 75kD subunit precursor or the Alcohol dehydrogenase-related loci, from the shape or size of the male genital arch, or from assortative premating behavior. We discuss these results in relation to a mitochondrial genetic species concept and the potential for Wolbachia-induced incompatibility to be a mechanism of speciation in insects. We conclude with an iterated appeal to include phylogenetic and statistical tests of neutrality as a supplement to phylogenetic and population genetic analyses when using mtDNA as an evolutionary marker.

Maintenance of mitochondrial DNA integrity: repair and degradation.

Abstract: Mitochondria have their own genome, which is essential for proper oxidative phosphorylation and hence for a large part of ATP production in a cell. Although mitochondrial DNA-less (rho0) cells can survive under certain conditions, the integrity of the mitochondrial genome is critical for the survival of multicellular organisms. Mitochondrial DNA (mtDNA) is damaged more than nuclear DNA because mitochondria produce a large amount of reactive oxygen species and tend to accumulate toxic xenobiotics. Therefore, there is keen interest in mechanisms that maintain the integrity of mtDNA. DNA repair may play an important role. The repair of mtDNA has been investigated less intensely than nuclear DNA repair because, for a long time, it was thought that mitochondria lacked DNA repair systems. In fact, DNA damage can be repaired in mitochondria. Base-excision repair in mitochondria is well established. The enzymes responsible for mtDNA repair have been identified and are encoded by the same genes as their nuclear counterparts. Mitochondrion-targeting sequences are generated through alternative splicing of mRNAs, alternative use of transcription initiation sites, or alternative use of translation initiation sites. In addition to DNA repair, the degradation of damaged mtDNA may be tolerated because there are multiple copies of mtDNA molecules in a cell.

Discrimination of DNA and RNA in cells by a vital fluorescent probe: Lifetime imaging of SYTO13 in healthy and apoptotic cells

Abstract: Background Of the few vital DNA and RNA probes, the SYTO dyes are the most specific for nucleic acids. However, they show no spectral contrast upon DNA or RNA binding. We show that fluorescence lifetime imaging using two-photon excitation of SYTO13 allows differential and simultaneous imaging of DNA and RNA in living cells, as well as sequential and repetitive assessment of staining patterns. Methods Two-photon imaging of SYTO13 is combined with lifetime contrast, using time-gated detection. We focus on distinguishing DNA and RNA in healthy and apoptotic Chinese hamster ovary cells. Results In healthy cells, SYTO13 has a fluorescence lifetime of 3.4 ± 0.2 ns when associated with nuclear DNA. Bound to RNA, its lifetime is 4.1 ± 0.1 ns. After induction of apoptosis, clusters of SYTO13 with fluorescence lifetime of 3.4 ± 0.2 ns become apparent in the cytoplasm. They are identified as mitochondrial DNA on the basis of colocalization experiments with the DNA-specific dye, DRAQ5, and the mitochondrial-specific dye, CMXRos. Upon progression of apoptosis, the lifetime of SYTO13 attached to DNA shortens significantly, which is indicative of changes in the molecular environment of the dye. Conclusions We have characterized SYTO13 as a vital lifetime probe, allowing repetitive and differential imaging of DNA and RNA. Cytometry 47:226–235, 2002. © 2002 Wiley-Liss, Inc.

Attenuation of the formation of DNA-repair foci containing RAD51 in Fanconi anaemia

Abstract: The role of the Fanconi anaemia genes in DNA repair was examined by a quantitative analysis of nuclear DNA repair foci in FA primary fibroblasts after ionising irradiation using antibodies directed against RAD51, MRE11 and BRCA1 for visualisation. IR induced foci detected with anti-RAD51, but not those detected with anti-MRE11, are reduced in fibroblasts of all eight FA complementation groups in comparison to control cells. Correction of FA-A, FA-C and FA-G cells by retroviral cDNA transfer specifically corrected the RAD51-foci response but did not affect formation of foci containing BRCA1 or MRE11. Since all FA cells, except FA-D1, lack the monoubiquitinated FANCD2-L protein, this isoform is likely to be involved in the formation of nuclear foci containing RAD51 in diploid FA cells. FA-D1 cells show the same attenuation in RAD51 foci formation, suggesting that the unknown FANCD1 protein is similarly involved in RAD51 foci formation, either independently or as a subsequent step in the FANCD2 pathway. These findings indicate that Fanconi anaemia cells have an impairment in the RAD51-dependent homologous recombination pathway for DNA repair, explaining their chromosomal instability and extreme sensitivity to DNA cross-linking agents.

H2O2 induces translocation of APE/Ref-1 to mitochondria in the Raji B-cell line

Abstract: Reactive oxygen species (ROS) are generated as by-products of respiration and are used as signal transducing intermediates in out-in signaling pathways ROS are also generated during inflammatory responses and it has been shown that hydrogen peroxide may trigger activation of B-lymphocytes, similar to cross-linking of surface immunoglobulins On the other hand, both exogenous and endogenous generated ROS are a major source of nuclear and mitochondrial DNA (mtDNA) damage The base excision repair (BER) enzyme APE/Ref-1 normally repairs small nuclear DNA lesion such as oxidized or alkylated bases It is not clear though whether DNA repair mechanisms able to abolish oxidative damage from nuclear DNA are present into mitochondria too Here we show by confocal microscopy and Western blot analysis that in the B-lymphocyte Raji cell line a fraction of APE/Ref-1 rapidly re-localizes into mitochondria following H(2)O(2) activation Targeting of APE/Ref-1 to mitochondria is not associated with cytochrome-c loss or apoptosis induction These findings indicate that the APE/Ref-1 translocates to mitochondria in response to oxidative stress and thereby it might exert a protective function

The Complete Mitochondrial Sequence of Tarsius bancanus: Evidence for an Extensive Nucleotide Compositional Plasticity of Primate Mitochondrial DNA

Abstract: Inconsistencies between phylogenetic interpretations obtained from independent sources of molecular data occasionally hamper the recovery of the true evolutionary history of certain taxa. One prominent example concerns the primate infraordinal relationships. Phylogenetic analyses based on nuclear DNA sequences traditionally represent Tarsius as a sister group to anthropoids. In contrast, mitochondrial DNA (mtDNA) data only marginally support this affiliation or even exclude Tarsius from primates. Two possible scenarios might cause this conflict: a period of adaptive molecular evolution or a shift in the nucleotide composition of higher primate mtDNAs through directional mutation pressure. To test these options, the entire mt genome of Tarsius bancanus was sequenced and compared with mtDNA of representatives of all major primate groups and mammals. Phylogenetic reconstructions at both the amino acid (AA) and DNA level of the protein-coding genes led to faulty tree topologies depending on the algorithms used for reconstruction. We propose that these artifactual affiliations rather reflect the nucleotide compositional similarity than phylogenetic relatedness and favor the directional mutation pressure hypothesis because: (1) the overall nucleotide composition changes dramatically on the lineage leading to higher primates at both silent and nonsilent sites, and (2) a highly significant correlation exists between codon usage and the nucleotide composition at the third, silent codon position. Comparisons of mt genes with mt pseudogenes that presumably transferred to the nucleus before the directional mutation pressure took place indicate that the ancestral DNA composition is retained in the relatively fossilized mtDNA-like sequences, and that the directed acceleration of the substitution rate in higher primates is restricted to mtDNA.

The mitochondrial protein frataxin prevents nuclear damage

Abstract: The mitochondrial protein frataxin helps maintain appropriate iron levels in the mitochondria of yeast and humans. A deficiency of this protein in humans causes Friedreich's ataxia, while its complete absence in yeast (Delta yfh1 mutant) results in loss of mitochondrial DNA, apparently due to radicals generated by excess iron. We found that the absence of frataxin in yeast also leads to nuclear damage, as evidenced by inducibility of a nuclear DNA damage reporter, increased chromosomal instability including recombination and mutation, and greater sensitivity to DNA-damaging agents, as well as slow growth. Addition of a human frataxin mutant did not prevent nuclear damage, although it partially complemented the Delta yfh1 mutant in preventing mitochondrial DNA loss. The effects in Delta yfh1 mutants result from reactive oxygen species (ROS), since (i) Delta yfh1 cells produce more hydrogen peroxide, (ii) the effects are alleviated by a radical scavenger and (iii) the glutathione peroxidase gene prevents an increase in mutation rates. Thus, the frataxin protein is concluded to have a protective role for the nucleus as well as the mitochondria.

SV40 large T-antigen disturbs the formation of nuclear DNA-repair foci containing MRE11.

Abstract: The accumulation of DNA repair proteins at the sites of DNA damage can be visualized in mutagenized cells at the single cell level as discrete nuclear foci by immunofluorescent staining. Formation of nuclear foci in irradiated human fibroblasts, as detected by antibodies directed against the DNA repair protein MRE11, is significantly disturbed by the presence of the viral oncogene, SV40 large T-antigen. The attenuation of foci formation was found in both T-antigen immortalized cells and in cells transiently expressing T-antigen, indicating that it is not attributable to secondary mutations but to T-antigen expression itself. ATM-mediated nibrin phosphorylation was not altered, thus the disturbance of MRE11 foci formation by T-antigen is independent of this event. The decrease in MRE11 foci was particularly pronounced in T-antigen immortalized cells from the Fanconi anaemia complementation group FA-D2. FA-D2 cells produce essentially no MRE11 DNA repair foci after ionizing irradiation and have a significantly increased cellular radiosensitivity at low radiation doses. The gene mutated in FA-D2 cells, FANCD2, codes for a protein which also locates to nuclear foci and may, therefore, be involved in MRE11 foci formation, at least in T-antigen immortalized cells. This finding possibly links Fanconi anaemia proteins to the frequently reported increased sensitivity of Fanconi anaemia cells to transformation by SV40. From a practical stand point these findings are particularly relevant to the many studies on DNA repair which exploit the advantages of SV40 immortalized cell lines. The interference of T-antigen with DNA repair processes, as demonstrated here, should be borne in mind when interpreting such studies.

Programmed cell death during rice leaf senescence is nonapoptotic.

Abstract: Summary • The cellular events associated with programmed cell death during leaf senescence in rice (Oryza sativa) plants are reported here. • The cytological sequence of senescence-related changes in rice leaves was studied by transmission electron microscopy, in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay and DNA ladder assay. • Cell death in senescing mesophyll cells was marked by depletion of cytoplasm in a tightly controlled manner. However, no apparent morphological feature associated with apoptosis was observed. Nuclear DNA fragmentation was detectable as early as during leaf unfolding and the subsequent developmental and senescent stages. The occurrence of DNA fragmentation correlated well with the size-shift of chromosomal DNA on agarose gel after electrophoresis. However, DNA fragmentation was not accompanied by generation of oligonucleosomal DNA fragments. • These features of cell death occurring during leaf senescence in monocot rice are quite different from features characteristic of apoptosis in animals. The implications of these results for cellular events associated with rice leaf senescence are discussed.

J-binding protein increases the level and retention of the unusual base J in trypanosome DNA

Abstract: Summary The nuclear DNA of Trypanosoma brucei and other kinetoplastid flagellates contains the unusual base β-d-glucosyl-hydroxymethyluracil, called J, replacing part of the thymine in repetitive sequences We have described a 100 kDa protein that specifically binds to J in duplex DNA We have now disrupted the genes for this J-binding protein (JBP) in T brucei The disruption does not affect growth, gene expression or the stability of some repetitive DNA sequences Unexpectedly, however, the JBP KO trypanosomes contain only about 5% of the wild-type level of J in their DNA Excess J, randomly introduced into T brucei DNA by growing the cells in the presence of the J precursor 5-hydroxymethyldeoxyuridine, is lost by simple dilution as the KO trypanosomes multiply, showing that JBP does not protect J against removal In contrast, cells containing JBP lose excess J only sluggishly We conclude that JBP is able to activate the thymine modification enzymes to introduce additional J in regions of DNA already containing a basal level of J We propose that JBP is a novel DNA modification maintenance protein

Determining Ploidy Level and Nuclear DNA Content in Rubus by Flow Cytometry

Abstract: Nuclear DNA flow cytometry was used to differentiate ploidy level and determine nuclear DNA content in Rubus. Nuclei suspensions were prepared from leaf discs of young leaves following published protocols with modifica- tions. DNA was stained with propidium iodide. Measurement of fluorescence of 40 genotypes, whose published ploidy ranged from diploid to dodecaploid, indicated that fluorescence increased with an increase in chromosome number. Ploidy level accounted for 99% of the variation in fluorescence intensity (r 2 = 0.99) and variation among ploidy levels was much higher than within ploidy levels. This protocol was used successfully for genotypes representing eight different Rubus subgenera. Rubus ursinus Cham. and Schldl., a native blackberry species in the Pacific Northwest, which has been reported to have 6x, 8x, 9x, 10x, 11x, and 12x forms, was extensively tested. Genotypes of R. ursinus were predominantly 12x, but 6x, 7x, 8x, 9x, 11x, and 13x forms were found as well. Attempts to confirm the 13x estimates with manual counts were unsuccessful. Ploidy level of 103 genotypes in the USDA-ARS breeding program was determined by flow cytometry. Flow cytometry confirmed that genotypes from crosses among 7x and 4x parents had chromosome numbers that must be the result of nonreduced gametes. This technique was effective in differentiating chromosome numbers differing by 1x, but was not able to differentiate aneuploids. Nuclear DNA contents of 21 diploid Rubus species from five subgenera were determined by flow cytometry. Idaeobatus, Chamaebatus, and Anaplobatus were significantly lower in DNA content than those of Rubus and Cylactis. In the Rubus subgenus, R. hispidus and R. canadensis had the lowest DNA content and R. sanctus had the highest DNA content, 0.59 and 0.75 pg, respectively. Idaeobatus had greater variation in DNA content among diploid species than the Rubus subgenus, with the highest being from R. ellipticus (0.69 pg) and lowest from R. illecebrosus (0.47 pg).

A chloroplast-resident DNA methyltransferase is responsible for hypermethylation of chloroplast genes in Chlamydomonas maternal gametes.

Abstract: Chloroplast DNA of the green alga Chlamydomonas reinhardtii is maternally inherited. Methylation mapping directly revealed that, before mating, chloroplast DNA of maternal (mating type plus; mt+) gametes is heavily methylated whereas that of paternal (mating type minus; mt−) gametes is not. Indirect immunofluorescence analyses with anti-5-methylcytosine mAbs visually showed methylation to occur exclusively in chloroplast DNA of mt+ gametes, and not in mt− gametes or nuclear DNA of either mt. To clarify the relationship between methylation and maternal inheritance of chloroplast DNA, we have isolated and characterized a cDNA encoding a DNA methyltransferase. The deduced protein, CrMET1, consists of 1,344 aa and contains a conserved catalytic domain at the C terminal and a nonconserved N-terminal region. The predicted N-terminal region has an arginine-rich domain, suggesting CrMET1 is transferred to chloroplasts. This finding could be directly shown by green fluorescent protein epifluorescence microscopy analyses. CrMET1 transcripts were found to be absent in both mt+ and mt− vegetative cells. Upon gametogenesis, however, transcript levels clearly increased in mt+ but not mt− cells. These experiments suggest that the CrMET1 protein is located in chloroplasts and that it specifically methylates cytosine residues of chloroplast DNA in mt+ gametes. This conclusion was further strengthened by the observation that, during gametogenesis, CrMET1 is expressed in a mt− mutant, mat-1, whose chloroplast DNA is heavily methylated in gametes and paternally inherited. The results provide evidence that cytosine methylation plays a critical role in maternal inheritance of chloroplast genes in C. reinhardtii.

Variation in chromosome numbers and nuclear DNA contents in genetic resources of Lactuca L. species (Asteraceae).

Abstract: Fifty accessions of 25 Lactuca species,L. serriola ×L. sativa and Mycelismuralis were analyzed for chromosome number and relative DNA amountvariation. In the majority of Lactuca species studiedchromosome counts, as earlier reported (n = 8, 9, 17), were verified; however,for L. dregeana andL. homblei (probablyL. schweinfurthii orL. longespicata) the chromosome number(n = 9) was determined for the first time. Relative nuclear DNA content,estimated by using flow cytometry (DAPI staining), showed that 2C DNA contentranged from 2.02 pg in L.capensis to 17.96 pg inL. canadensis. Statistical and clusteranalysis of data based on relative nuclear DNA contents correspond fairly wellwith recently accepted taxonomic classification of the genusLactuca. However, the position of certain species as wellas clarification of taxonomic determinations of someLactuca accessions needs further examination.

Suppressive role of endogenous regucalcin in the enhancement of deoxyribonucleic acid synthesis activity in the nucleus of regenerating rat liver.

Abstract: The role of endogenous regucalcin in the regulation of deoxyribonucleic acid (DNA) synthesis activity in the nuclei of regenerating rat liver after partial hepatectomy was investigated. The addition of regucalcin (0.25 and 0.5 μM) in the reaction mixture caused a significant decrease in the nuclear DNA synthesis activity of normal rat liver. This decrease was also seen in the presence of Ca2+-chelator EGTA (0.4 mM), indicating that the effect of regucalcin is not related to nuclear Ca2+. Nuclear DNA activity was significantly increased in the presence of anti-regucalcin monoclonal antibody (10–50 ng/ml) in the reaction mixture. The effect was completely abolished by the addition of regucalcin (0.5 μM). Nuclear DNA synthesis activity was significantly increased at 24, 48, and 72 h after partial heptectomy. The effect of anti-regucalcin monoclonal antibody (25 ng/ml) in increasing nuclear DNA synthesis activity was significantly enhanced at 24 and 48 h after partial hepatectomy. The presence of staurospone (10−6 M), trifluoperazine (2 × 10−5 M), or PD98059 (10−5 M) in the reaction mixture caused a significant decrease in DNA synthesis activity in the nuclei obtained at 24 after partial hepateactomy. The effect of these inhibitors in the presence of anti-regucalcin monoclonal antibody (25 ng/ml) was greater than that in the absence of the antibody. The present study suggests that endogenous regucalcin plays a suppressive role in the enhancement of nuclear DNA synthesis activity in regenerating liver with cell proliferation after partial hepatectomy in rats. J. Cell. Biochem. 85: 516–522, 2002. © 2002 Wiley-Liss, Inc.

The clinical value of sperm nuclear DNA assessment.

Abstract: Traditional semen analysis is essential for the diagnosis of male infertility. A number of studies over the past decade have reported that a significant contributing factor to male fertility that is not revealed as part of semen analysis is sperm DNA, specifically its composition and organization. Exogenous and endogenous factors can cause damage to sperm DNA. For example, topoisomerase II activity, which is necessary for sperm DNA packaging, can adversely influence the competence of sperm DNA if the activity of the enzyme is abnormal. Germ cell apoptosis can be induced by oxygen radicals produced from environmental (for example cigarette smoke) or testicular (for example localized ischaemia) sources. Several assays have been developed that are useful for assessing sperm DNA composition and organization. To date, each of these assays has revealed that when sperm DNA has been damaged or packaged improperly there is a concomitant and often significant decline in male fertility.

Single-copy nuclear DNA sequences obtained from noninvasively collected primate feces.

Abstract: Noninvasively collected primate feces have been shown to provide a useful source of mitochondrial DNA for sequencing and nuclear microsatellite DNA for size analysis. In this study, single-copy nuclear DNA sequences were obtained from noninvasively collected fecal samples of two species of wild tamarins, Saguinus fuscicollis and S. mystax, in the context of a project on the functional utility of color vision. Noninvasive genotyping of the X-linked opsin gene is important for future studies of selection and adaptation at this locus in a number of primate species. The wide range of techniques that can now be applied successfully to DNA extracted from feces introduces a broad spectrum of potential genetic studies that can be undertaken on primates, without the need for intrusive or invasive methods.