Showing papers on "Nuclear DNA published in 2001"

Oxidative stress, DNA damage and the Y chromosome.

Abstract: Recent advances in understanding of male infertility have implicated two major causative factors, oxidative stress and Y chromosome deletions. A major cause of oxidative stress appears to be the high rate of reactive oxygen species generation associated with the retention of excess residual cytoplasm in the sperm midpiece. Other possible causes include the redox cycling of xenobiotics, and antioxidant depletion or apoptosis. Oxidative stress induces peroxidative damage in the sperm plasma membrane and DNA damage in both the mitochondrial and nuclear genomes. Nuclear DNA damage in the germ line of the father may be associated with pathology in the offspring, including childhood cancer and infertility. Gene deletions on the non-recombining region of the Y chromosome account for the infertility observed in about 15% of patients with azoospermia and 5-10% of subjects with severe oligozoospermia. The Y chromosome is particularly susceptible to gene deletions because of the inability of the haploid genome to deploy recombination repair in retrieving lost genetic information. Aberrant recombination, defective chromatin packaging, abortive apoptosis and oxidative stress may all be involved in the aetiology of DNA damage in the germ line. The factors responsible for Y chromosome deletions in spermatozoa remain unresolved but may be one facet of a central reproductive problem: controlling the amount of oxidative stress experienced by germ cells during their differentiation and maturation in the male reproductive tract.

Quantitative polymerase chain reaction analysis of DNA from noninvasive samples for accurate microsatellite genotyping of wild chimpanzees (Pan troglodytes verus)

Abstract: Noninvasive samples are useful for molecular genetic analyses of wild animal populations. However, the low DNA content of such samples makes DNA amplification difficult, and there is the potential for erroneous results when one of two alleles at heterozygous microsatellite loci fails to be amplified. In this study we describe an assay designed to measure the amount of amplifiable nuclear DNA in low DNA concentration extracts from noninvasive samples. We describe the range of DNA amounts obtained from chimpanzee faeces and shed hair samples and formulate a new efficient approach for accurate microsatellite genotyping. Prescreening of extracts for DNA quantity is recommended for sorting of samples for likely success and reliability. Repetition of results remains extensive for analysis of microsatellite amplifications beginning from low starting amounts of DNA, but is reduced for those with higher DNA content.

Mitochondrial DNA in human malignancy.

Abstract: Alterations in expression of mitochondrial DNA (mtDNA)-encoded polypeptides required for oxidative phosphorylation and cellular ATP generation may be a general characteristic of cancer cells. Mitochondrial DNA has been proposed to be involved in carcinogenesis because of high susceptibility to mutations and limited repair mechanisms in comparison to nuclear DNA. Since mtDNA lacks introns, it has been suggested that most mutations will occur in coding sequences and subsequent accumulation of mutations may lead to tumor formation. The mitochondrial genome is dependent upon the nuclear genome for transcription, translation, replication and repair, but precise mechanisms for how the two genomes interact and integrate with each other are poorly understood. In solid tumors, elevated expression of mtDNA-encoded subunits of the mitochondrial electron respiratory chain may reflect mitochondrial adaptation to perturbations in cellular energy requirements. In this paper, we review mitochondrial genomic aberrations reported in solid tumors of the breast, colon, stomach, liver, kidney, bladder, head/neck and lung as well as for hematologic diseases such as leukemia, myelodysplastic syndrome and lymphoma. We include data for elevated expression of mtDNA-encoded electron respiratory chain subunits in breast, colon and liver cancers and also the mutations reported in cancers of the colon, stomach, bladder, head/neck and lung. Finally, we examine the role of reactive oxygen species (ROS) generated by mitochondria in the process of carcinogenesis.

The PET1-CMS Mitochondrial Mutation in Sunflower Is Associated with Premature Programmed Cell Death and Cytochrome c Release

Abstract: In mammals, mitochondria have been shown to play a key intermediary role in apoptosis, a morphologically distinct form of programmed cell death (PCD), for example, through the release of cytochrome c, which activates a proteolytic enzyme cascade, resulting in specific nuclear DNA degradation and cell death. In plants, PCD is a feature of normal development, including the penultimate stage of anther development, leading to dehiscence and pollen release. However, there is little evidence that plant mitochondria are involved in PCD. In a wide range of plant species, anther and/or pollen development is disrupted in a class of mutants termed CMS (for cytoplasmic male sterility), which is associated with mutations in the mitochondrial genome. On the basis of the manifestation of a number of morphological and biochemical markers of apoptosis, we have shown that the PET1-CMS cytoplasm in sunflower causes premature PCD of the tapetal cells, which then extends to other anther tissues. These features included cell condensation, oligonucleosomal cleavage of nuclear DNA, separation of chromatin into delineated masses, and initial persistence of mitochondria. In addition, immunocytochemical analysis revealed that cytochrome c was released partially from the mitochondria into the cytosol of tapetal cells before the gross morphological changes associated with PCD. The decrease in cytochrome c content in mitochondria isolated from male sterile florets preceded a decrease in the integrity of the outer mitochondrial membrane and respiratory control ratio. Our data suggest that plant mitochondria, like mammalian mitochondria, play a key role in the induction of PCD. The tissue-specific nature of the CMS phenotype is discussed with regard to cellular respiratory demand and PCD during normal anther development.

Mitochondrial DNA Instability and Peri-Implantation Lethality Associated with Targeted Disruption of Nuclear Respiratory Factor 1 in Mice

Abstract: In vitro studies have implicated nuclear respiratory factor 1 (NRF-1) in the transcriptional expression of nuclear genes required for mitochondrial respiratory function, as well as for other fundamental cellular activities. We investigated here the in vivo function of NRF-1 in mammals by disrupting the gene in mice. A portion of the NRF-1 gene that encodes the nuclear localization signal and the DNA-binding and dimerization domains was replaced through homologous recombination by a β-galactosidase–neomycin cassette. In the mutant allele, β-galactosidase expression is under the control of the NRF-1 promoter. Embryos homozygous for NRF-1 disruption die between embryonic days 3.5 and 6.5. β-Galactosidase staining was observed in growing oocytes and in 2.5- and 3.5-day-old embryos, demonstrating that the NRF-1 gene is expressed during oogenesis and during early stages of embryogenesis. Moreover, the embryonic expression of NRF-1 did not result from maternal carryover. While most isolated wild-type and NRF-1+/− blastocysts can develop further in vitro, the NRF-1−/− blastocysts lack this ability despite their normal morphology. Interestingly, a fraction of the blastocysts from heterozygous matings had reduced staining intensity with rhodamine 123 and NRF-1−/− blastocysts had markedly reduced levels of mitochondrial DNA (mtDNA). The depletion of mtDNA did not coincide with nuclear DNA fragmentation, indicating that mtDNA loss was not associated with increased apoptosis. These results are consistent with a specific requirement for NRF-1 in the maintenance of mtDNA and respiratory chain function during early embryogenesis.

Detection of mitochondrial DNA mutations in pancreatic cancer offers a "mass"-ive advantage over detection of nuclear DNA mutations.

Abstract: We sequenced the complete 16.5-kb mitochondrial genome (mtDNA) in 15 pancreatic cancer cell lines and xenografts. Homoplasmic mtDNA somatic mutations and novel variants were identified in nearly all samples. Southern blot analysis and direct sequencing of mutation sites showed that the intracellular mass of mtDNA was greatly (6-8-fold) increased in pancreatic cancer cells in relation to corresponding normal cells; this property accounted for and greatly facilitated the identification of these mutations among the dense desmoplastic host reaction characteristic of primary pancreatic cancers. Structural characteristics and mathematical modeling of the evolution of mtDNA mutations suggested that many of the mutations identified might represent a random evolution of homoplasmic variants, rather than necessarily being a product of selective pressures. Complete sequencing of the nuclear MnSOD gene, which protects cells from the mitogenic and toxic effects of oxygen radicals, did not reveal any mutations. Nevertheless, the nearly ubiquitous prevalence and high copy number of mtDNA mutations suggest that they be considered of promising clinical utility in diagnostic applications.

Incongruence of Mitochondrial and Nuclear Gene Trees in the Carabid Beetles Ohomopterus

Abstract: We studied the molecular phylogeny of the carabid subgenus Ohomopterus (genus Carabus), using two mitochondrial (mt) DNA regions (16SrRNA and NADH dehydrogenase subunit 5) and three nuclear DNA regions (wingless, phosphoenolpyruvate carboxykinase, and an anonymous locus). We revisited the previously reported incongruence between the distribution of mtDNA markers and morphologically defined species (Su et al., 1996; J. Mol. Evol. 43:662-671), which those authors attributed to "type switching", a concerted change in many morphological characters that results in the repeated evolution of a particular morphological type. Our mtDNA gene tree obtained from 44 individuals representing all 15 currently recognized species of Ohomopterus revealed that haplotypes isolated from individuals of a single "species" were frequently separated into distant clades, confirming the previous report. The three nuclear markers generally conformed better-with the morphologically defined species than did the mitochondrial markers. The phylogenetic signal in mtDNA and nuclear DNA data differed strongly, and these two partitions were significantly incongruent with each other according to the incongruence length difference test of Farris et al. (1994; Cladistics 10:315-320), although the three nuclear partitions were not homogeneous either. Our results did not support the type-switching hypothesis that had been proposed to fit the morphological data to the mitochondrial gene tree: The incongruence of the mtDNA tree with other nuclear markers indicates that the mtDNA-based tree does not reflect species history any better than the morphological data do. Incongruence of gene trees in Ohomopterus may have been promoted by the complex processes of geographic isolation and hybridization in the Japanese Archipelago that have led to occasional gene flow and recombination between separated entities. The occurrence of reticulate patterns in this group is intriguing, because species of Ohomopterus exhibit extremely divergent genitalic structures that represent a highly efficient reproductive isolation mechanism.

A reliable assessment of 8-oxo-2-deoxyguanosine levels in nuclear and mitochondrial DNA using the sodium iodide method to isolate DNA.

Abstract: A major controversy in the area of DNA biochemistry concerns the actual in vivo levels of oxidative damage in DNA. We show here that 8-oxo-2-deoxyguanosine (oxo8dG) generation during DNA isolation is eliminated using the sodium iodide (NaI) isolation method and that the level of oxo8dG in nuclear DNA (nDNA) is almost one-hundredth of the level obtained using the classical phenol method. We found using NaI that the ratio of oxo8dG/10(5 )deoxyguanosine (dG) in nDNA isolated from mouse tissues ranged from 0.032 +/- 0.002 for liver to 0.015 +/- 0.003 for brain. We observed a significant increase (10-fold) in oxo8dG in nDNA isolated from liver tissue after 2 Gy of gamma-irradiation when NaI was used to isolate DNA. The turnover of oxo8dG in nDNA was rapid, e.g. disappearance of oxo8dG in the mouse liver in vivo after gamma-irradiation had a half-life of 11 min. The levels of oxo8dG in mitochondrial DNA isolated from liver, heart and brain were 6-, 16- and 23-fold higher than nDNA from these tissues. Thus, our results showed that the steady-state levels of oxo8dG in mouse tissues range from 180 to 360 lesions in the nuclear genome and from one to two lesions in 100 mitochondrial genomes.

Mutations in the complex I NDUFS2 gene of patients with cardiomyopathy and encephalomyopathy.

Abstract: Human complex I is built up and regulated by genes encoded by the mitochondrial DNA (mtDNA) as well as the nuclear DNA (nDNA). In recent years, attention mainly focused on the relation between complex I deficiency and mtDNA mutations. However, a high percentage of consanguinity and an autosomal-recessive mode of inheritance observed within our patient group as well as the absence of common mtDNA mutations make a nuclear genetic cause likely. The NDUFS2 protein is part of complex I of many pro- and eukaryotes. The nuclear gene coding for this protein is therefore an important candidate for mutational detection studies in enzymatic complex I deficient patients. Screening of patient NDUFS2 cDNA by reverse transcriptase-polymerase chain reaction (RT-PCR) in combination with direct DNA sequencing revealed three missense mutations resulting in the substitution of conserved amino acids in three families.

Poxviruses and the origin of the eukaryotic nucleus.

Abstract: A number of molecular forms of DNA polymerases have been reported to be involved in eukaryotic nuclear DNA replication, with contributions from α-, δ-, and e-polymerases. It has been reported that δ-polymerase possessed a central role in DNA replication in archaea, whose ancestry are thought to be closely related to the ancestor of eukaryotes. Indeed, in vitro experiment shown here suggests that δ-polymerase has the potential ability to start DNA synthesis immediately after RNA primer synthesis. Therefore, the question arises, where did the α-polymerase come from? Phylogenetic analysis based on the nucleotide sequence of several conserved regions reveals that two poxviruses, vaccinia and variola viruses, have polymerases similar to eukaryotic α-polymerase rather than δ-polymerase, while adenovirus, herpes family viruses, and archaeotes have eukaryotic δ-like polymerases, suggesting that the eukaryotic α-polymerase gene is derived from a poxvirus-like organism, which had some eukaryote-like characteristics. Furthermore, the poxvirus's proliferation independent from the host-cell nucleus suggests the possibility that this virus could infect non-nucleated cells, such as ancestral eukaryotes. I wish to propose here a new hypothesis for the origin of the eukaryotic nucleus, posing symbiotic contact of an orthopoxvirus ancestor with an archaebacterium, whose genome already had a δ-like polymerase gene.

Genome size in 21 Artemisia L. species (Asteraceae, Anthemideae): Systematic, evolutionary, and ecological implications

Abstract: Genome size was estimated by flow cytometry in 24 populations belonging to 22 Artemisia taxa (21 species, 1 with two subspecies), which represent the distinct subgenera, life forms, basic chromosome numbers, and ploidy lev- els in the genus. 2C nuclear DNA content values range from 3.5 to 25.65 pg, which represents a more than sevenfold variation. DNA content per haploid genome ranges from 1.75 to 5.76 pg. DNA amount is very well correlated with karyotype length and ploidy level. Some variations in genome size have systematic and evolutionary implications, whereas others are linked to ecological selection pressures.

Infrequent Genetic Exchange and Recombination in the Mitochondrial Genome of Candida albicans

Abstract: Previous analyses of diploid nuclear genotypes have concluded that recombination has occurred in populations of the yeast Candida albicans. To address the possibilities of clonality and recombination in an effectively haploid genome, we sequenced seven regions of mitochondrial DNA (mtDNA) in 45 strains of C. albicans from human immunodeficiency virus-positive patients in Toronto, Canada, and 3 standard reference isolates of C. albicans, CA, CAI4, and WO-1. Among a total of 2,553 nucleotides in the seven regions, 62 polymorphic nucleotide sites and seven indels defined nine distinct mtDNA haplotypes among the 48 strains. Five of these haplotypes occurred in more than one strain, indicating clonal proliferation of mtDNA. Phylogenetic analysis of mtDNA haplotypes resulted in one most-parsimonious tree. Most of the nucleotide sites undergoing parallel change in this tree were clustered in blocks that corresponded to sequenced regions. Because of the existence of these blocks, the apparent homoplasy can be attributed to infrequent, past genetic exchange and recombination between individuals and cannot be attributed to parallel mutation. Among strains sharing the same mtDNA haplotypes, multilocus nuclear genotypes were more similar than expected from a random comparison of nuclear DNA genotypes, suggesting that clonal proliferation of the mitochondrial genome was accompanied by clonal proliferation of the nuclear genome.

Mitochondrial defects in neurodegenerative disease.

Abstract: Over the past 12 years, a wide variety of neurodegenerative diseases has been linked to mutations in mitochondrial genes located in either the mitochondrial DNA (mtDNA) or the nuclear DNA (nDNA). These disorders encompass an array of unorthodox inheritance patterns and a plethora of symptoms ranging from lethal neonatal multi-symptom disorders to later onset myopathies, cardiomyopathies, movement disorders, and dementias. The bases for the genetic and phenotypic variability of mitochondrial diseases lie in the multiplicity of the mitochondria genes dispersed across the human genome and the variety of cellular pathways and functions in which the mitochondria play a central role.

Apoptosis. DNA destroyers.

Abstract: Proteins with quite mundane functions in healthy cells often behave very differently during cell suicide. One protein normally involved in copying mitochondrial DNA actually degrades nuclear DNA in dying cells.

Antisense-mediated decrease in DNA ligase III expression results in reduced mitochondrial DNA integrity.

Abstract: The human DNA ligase III gene encodes both nuclear and mitochondrial proteins. Abundant evidence supports the conclusion that the nuclear DNA ligase III protein plays an essential role in both base excision repair and homologous recombination. However, the role of DNA ligase III protein in mitochondrial genome dynamics has been obscure. Human tumor-derived HT1080 cells were transfected with an antisense DNA ligase III expression vector and clones with diminished levels of DNA ligase III activity identified. Mitochondrial protein extracts prepared from these clones had decreased levels of DNA ligase III relative to extracts from cells transfected with a control vector. Analysis of these clones revealed that the DNA ligase III antisense mRNA-expressing cells had reduced mtDNA content compared to control cells. In addition, the residual mtDNA present in these cells had numerous single-strand nicks that were not detected in mtDNA from control cells. Cells expressing antisense ligase III also had diminished capacity to restore their mtDNA to pre-irradiation levels following exposure to gamma-irradiation. An antisense-mediated reduction in cellular DNA ligase IV had no effect on the copy number or integrity of mtDNA. This observation, coupled with other evidence, suggests that DNA ligase IV is not present in the mitochondria and does not play a role in maintaining mtDNA integrity. We conclude that DNA ligase III is essential for the proper maintenance of mtDNA in cultured mammalian somatic cells.

Genome Size and Pollen Viability as Taxonomic Criteria: Application to the Genus Hosta

Abstract: Genome size (C values) and pollen viability staining were applied as new criteria to investigate the taxonomy of the genus Hosta Tratt. (Hostaceae). Nearly all species of the genus Hosta have the same basic chromosome number (2n = 2x = 60). However, the nuclear DNA contents, as measured by flow cytometry with propidium iodide, could be demonstrated to range between 17.2 to 26.6 pg. This implies that the largest genome contains roughly 1010 more base pairs than the smallest. Therefore, nuclear DNA content is a very relevant taxonomic trait that can be measured simply by flow cytometry. In addition, differences in overall DNA composition were demonstrated by comparing to DAPI fluorescence. In general, genome size data confirmed the division into three subgenera. The geographical distribution of genome sizes indicates the migration pattern of Hosta throughout East Asia. The species belonging to the mainly Korean subgenus Bryocles, with a low nuclear DNA content (17.2 - 19.3 pg), can now largely be distinguished from the mainly Japanese species of the subgenus Giboshi (21.3 - 26.5 pg). The exception is H. longissima, that with only 19.6 pg provides a nice example of a decrease in DNA content. On the mainland, as well as on Honshu, species with increased and decreased DNA content have evolved independently. The usefulness of pollen viability to detect hybrids in Hosta was demonstrated in a large series of artificial crosses between bona fide species. Consequently, pollen viability was measured in all available Hosta described as species. Several had low pollen viability and were concluded to be hybrids. Morphology and DNA content confirmed this in most cases. The resulting 23 species approximate the number of Hosta species that follows from the combined studies by Fujita (197618) on the Japanese species and Chung (1991 a11) on the Korean species.

Relative susceptibilities of mitochondrial and nuclear DNA to damage induced by hydrogen peroxide in two mouse germ cell lines.

Abstract: The objective of this study was to determine the relative susceptibilities to the damaging effects of hydrogen peroxide of DNA in the mitochondrial and nuclear compartments of two murine germ cell lines. We used a quantitative polymerase chain reaction assay (QPCR) to measure gene- and mitochondrial-specific DNA damage and examined for the presence of alkali-labile sites using alkaline gel electrophoresis. No DNA damage was observed in a nuclear gene (β-globin) in response to hydrogen peroxide treatment. In addition, no increase in alkali-labile sites was observed. However, mitochondrial DNA suffered extensive damage which increased in a dose-dependent manner. These results demonstrate that the nuclear DNA in these germ cell lines is relatively resistant to peroxide-mediated DNA damage, and that mitochondrial DNA is a sensitive biomarker for oxidative stress in these cells.

Eggs yield nuclear DNA from egg-laying female cowbirds, their embryos and offspring

Abstract: Here we report methods forextracting maternal DNA from avian eggshells oroffspring DNA from eggshells and embryos. Thesemethods offer alternative techniques forobtaining DNA from oviparous organisms. UsingDNA extracted from eggshells, we obtainmicrosatellite genotypes of the brood parasiticbrown-headed cowbird (Molothrus ater)female that laid the eggs and/or her hatchedoffspring. Using DNA extracted from embryos, weobtain microsatellite genotypes of offspring.We demonstrate that separate extractionsperformed on the embryo and shell from a singleegg can provide DNA from the embryo and itsmother, respectively. This single-egg approachfor obtaining both maternal and embryonic DNAsimplifies paternity analyses because allelesunique to the embryo can be considered paternalin origin. Finally we report two newmicrosatellite loci and primer sequences forbrown-headed cowbirds.

Mitochondrial DNA in aging and degenerative disease

Abstract: The mitochondrial DNA encodes only a few gene products compared to the nuclear DNA. These products, however, play a decisive role in determining cell function. Should this DNA mutate spontaneously or be damaged by free radicals the functionality of the gene products will be compromised. A number of mitochondrial genetic diseases have been identified. Some of these are quite serious and involve the central nervous system as well as muscle, heart, liver and kidney. Aging has been characterized by a gradual increase in base deletions in this DNA. This increase in deletion mutation has been suggested to be the cumulative result of exposure to free radicals.

Nuclear DNA Contents of Phalaenopsis sp. and Doritis pulcherrima

Abstract: ADDITIONAL INDEX WORDS. Orchidaceae, endoreduplication, flow cytometry, genome size ABSTRACT. Nuclear DNA contents were estimated by flow cytometry in 18 Phalaenopsis Blume species and Doritis pulcherrima Lindl. DNA amounts differed 6.07-fold, from 2.74 pg/diploid nuclear DNA content (2C) in P. sanderiana Rchb.f. to 16.61 pg/2C in P. parishii Rchb.f. Nuclear DNA contents of P. aphrodite Rchb.f. clones, W01-38 (2n = 2x = 38), W01-41 (2n = 3x = 57), and W01-22 (2n = 4x = 76), displayed a linear relationship with their chromosome numbers, indicating the accuracy of flow cytometry. Our results also suggest that the 2C-values of the Phalaenopsis sp. correlate with their chromosome sizes. The comparative analyses of DNA contents may provide information to molecular geneticists and systematists for genome analysis in Phalaenopsis. Endoreduplication was found in various tissues of P. equestris at different levels. The highest degree of endoreduplication in P. equestris was detected in leaves.

DNA repair inhibition and cancer therapy.

Abstract: The DNA repair process in mammalian cells is a multi-pathway mechanism that protects cells from the plethora of DNA damaging agents that are known to attack nuclear DNA. Moreover, the majority of current anticancer therapies (e.g. ionising radiation and chemotoxic therapies) rely on this ability to create DNA lesions, leading to apoptosis/cell death. A cells natural ability to repair such DNA damage is a major cause of resistance to these existing antitumour agents. It seems logical, therefore, that by modulating these repair mechanisms, greater killing effect to anticancer agents would occur. Experimental data support this, either through knockout studies or by the use of pharmacological inhibitors which target some of the key regulatory proteins involved in the DNA repair process. Several of these key DNA repair proteins which are actively under investigation as novel sites for intervention in cancer biology are discussed.

Evolutionary genetics - Clonal inheritance of avian mitochondrial DNA

Abstract: We have taken a new approach to test the commonly accepted, but recently questioned, principle of clonal inheritance of vertebrate mitochondrial DNA (mtDNA) by relating its inheritance to a female-specific marker of nuclear DNA. Whereas this is impossible in organisms with male heterogamy (such as mammals), we show here that genealogies of mtDNA and the female-specific W chromosome of a bird species are completely concordant. Our results indicate that inheritance of mtDNA is free of detectable recombination effects over an evolutionary timescale.

Cryptic speciation in Pelobates fuscus (Anura, Pelobatidae): evidence from DNA flow cytometry

Abstract: The amount of nuclear DNA in 173 specimens of Pelobates f. fuscus from 34 localities in Russia, Ukraine, Moldavia and Latvia was determined by DNA e ow cytometry. Two distinct groups with different genome sizes were identie ed. The ranges of the genome size variation in the two groups did not overlap. Geographically, these groups with smaller or larger genome size are distributed in the west and in the east of eastern Europe, respectively.

Quartz exposure of the rat lung leads to a linear dose response in inflammation but not in oxidative DNA damage and mutagenicity.

Abstract: Exposure to quartz and high concentrations of other poorly soluble particles can lead to the development of lung tumors in the rat. The mechanisms involved in particle-induced carcinogenesis seem to include inflammation-associated production of reactive oxygen species (ROS) and DNA damage. ROS induce 8-oxoguanine (8-oxoGua) and a panel of other oxidation products in DNA. In proliferating cells such DNA lesions can lead to various types of mutations, which might be critical for cancer-related genes with respect to tumor formation. Quartz is known to mediate the induction of 8-oxoGua in the nuclear DNA of lung cells when applied to the lung of rats. We have investigated the time- and dose-dependent biologic effects of quartz and, as a control, corundum, on cell proliferation and various pulmonary inflammation and toxicity markers in rat bronchoalveolar lavage fluid (BALF); on the induction of 8-oxoGua in the DNA of rat lung cells; and on the cellular levels of p53 wild-type and p53 mutant (mut) protein. Rat...

Nuclear intron sequences for phylogenetics of closely related mammals: an example using the phylogeny of mus

Abstract: DNA sequence data from 2 nuclear introns (mitochondrial malate dehydrogenase intron 7 and acid phosphatase type V intron 2) were collected for 9 taxonomic units of rodents, 8 of which were in the genus Mus. Data were used to infer phylogenetic history of the 9 taxa, which was then compared with a consensus phylogeny taken from previous literature. In general, the nuclear intron data provided strong support (bootstrap proportions > 80) for relationships accepted in the consensus tree. The intron data disagreed with previous mitochondrial data in that 2 M. m. domesticus sequences were paraphyletic with respect to M. m. castaneus and M. m. musculus. This paraphyly, if real, likely reflects the longer lineage-sorting time of the nuclear genome compared with the mitochondrial genome. Introns seem to provide an attractive source of nuclear DNA sequence data for phylogenetic analysis, but mitochondrial data are likely to be superior for very recent divergences.

Measurement of DNA damage associated with apoptosis by laser scanning cytometry.

Abstract: Background Phosphatidylserine (PS) binding by annexin V (AV) is an early membrane marker of apoptosis. Using laser scanning cytometry (LSC) and the comet assay, we showed that the DNA of AV+ cells is so highly fragmented that it cannot be quantified by the comet assay (Bacso et al.: Cancer Res 60:4623–8, 2000). Methods The “halo” assay was used instead of the comet assay to quantify DNA damage associated with apoptosis. The LSC was used to measure both AV fluorescence and DNA damage on the same Jurkat cells following treatment with anti-Fas. The data from both sets of measurements were merged, allowing direct correlation of membrane and nuclear markers of cell death. Results AV+ cells had significant DNA damage determined by the ratio between nuclear DNA and peripheral (migrated) DNA. Cells in the early and late stages of apoptosis could be discriminated on the basis of DNA content. In addition, it was possible to distinguish between apoptotic and necrotic cells in the AV+ propidium iodide-positive population based on DNA content and DNA damage. The addition of specific inhibitors for caspases-8, 9, and 3 blocked both PS externalization and DNA fragmentation, indicating these events are downstream from caspase activation. Conclusions This technique allows accurate distinction between apoptotic and necrotic cells and cytometric grading of apoptosis. Cytometry 45:180–186, 2001. © 2001 Wiley-Liss, Inc.