Showing papers on "Nuclear DNA published in 1996"

Origin and functional consequences of the complex I defect in Parkinson's disease

Abstract: The mitochondrial electron transport enzyme NADH:ubiquinone oxidoreductase (complex I), which is encoded by both mitochondrial DNA and nuclear DNA, is defective in multiple tissues in persons with Parkinson's disease (PD). The origin of this lesion and its role in the neurodegeneration of PD are unknown. To address these questions, we created an in vitro system in which the potential contributions of environmental toxins, complex I nuclear DNA mutations, and mitochondrial DNA mutations could be systematically analyzed. A clonal line of human neuroblastoma cells containing no mitochondrial DNA was repopulated with mitochondria derived from the platelets of PD or control subjects. After 5 to 6 weeks in culture, these cytoplasmic hybrid (cybrid) cell lines were assayed for electron transport chain activities, production of reactive oxygen species, and sensitivity to induction of apoptotic cell death by 1-methyl-4-phenyl pyridinium (MPP+). In PD cybrids we found a stable 20% decrement in complex I activity, increased oxygen radical production, and increased susceptibility to 1-methyl-4-phenyl pyridinium-induced programmed cell death. The complex I defect in PD appears to be genetic, arising from mitochondrial DNA, and may play an important role in the neurodegeneration of PD by fostering reactive oxygen species production and conferring increased neuronal susceptibility to mitochondrial toxins.

Intraspecific nuclear DNA variation in Drosophila.

Abstract: We have summarized and analyzed all available nuclear DNA sequence polymorphism studies for three species of Drosophila, D. melanogaster (24 loci), D. simulans (12 loci), and D. pseudoobscura (5 loci). Our major findings are: (1) The average nucleotide heterozygosity ranges from about 0.4% to 2% depending upon species and function of the region, i.e., coding or noncoding. (2) Compared to D. simulans and D. pseudoobscura (which are about equally variable), D. melanogaster displays a low degree of DNA polymorphism. (3) Noncoding introns and 3' and 5' flanking DNA shows less polymorphism than silent sites within coding DNA. (4) X-linked genes are less variable than autosomal genes. (5) Transition (Ts) and transversion (Tv) polymorphisms are about equally frequent in non-coding DNA and at fourfold degenerate sites in coding DNA while Ts polymorphisms outnumber Tv polymorphisms by about 2:1 in total coding DNA. The increased Ts polymorphism in coding regions is likely due to the structure of the genetic code: silent changes are more often Ts's than are replacement substitutions. (6) The proportion of replacement polymorphisms is significantly higher in D. melanogaster than in D. simulans. (7) The level of variation in coding DNA and the adjacent noncoding DNA is significantly correlated indicating regional effects, most notably recombination. (8) Surprisingly, the level of polymorphism at silent coding sites in D. melanogaster is positively correlated with degree of codon usage bias. (9) Three proposed tests of the neutral theory of DNA polymorphisms have been performed on the data: Tajima's test, the HKA test, and the McDonald-Kreitman test. About half of the loci fail to conform to the expectations of neutral theory by one of the tests. We conclude that many variables are affecting levels of DNA polymorphism in Drosophila, from properties of nucleotides to population history and, perhaps, mating structure. No simple, all encompassing explanation satisfactorily accounts for the data.

Cleavage of Nuclear DNA into Oligonucleosomal Fragments during Cell Death Induced by Fungal Infection or by Abiotic Treatments.

Abstract: It is often claimed that programmed cell death (pcd) exists in plants and that a form of pcd known as the hypersensitive response is triggered as a defense mechanism by microbial pathogens. However, in contrast to animals, no feature in plants universally identifies or defines pcd. We have looked for a hallmark of pcd in animal cells, namely, DNA cleavage, in plant cells killed by infection with incompatible fungi or by abiotic means. We found that cell death triggered in intact leaves of two resistant cowpea cultivars by the cowpea rust fungus is accompanied by the cleavage of nuclear DNA into oligonucleosomal fragments (DNA laddering). Terminal deoxynucleotidyl transferase-mediated dUTP nick end in situ labeling of leaf sections showed that fungus-induced DNA cleavage occurred only in haustorium-containing cells and was detectable early in the degeneration process. Such cytologically detectable DNA cleavage was also observed in vascular tissue of infected and uninfected plants, but no DNA laddering was detected in the latter. DNA laddering was triggered by [greater than or equal to]100 mM KCN, regardless of cowpea cultivar, but not by physical cell disruption or by concentrations of H2O2, NaN3, CuSO4, or ZnCl2 that killed cowpea cells at a rate similar to that of ladder-inducing KCN concentrations. These and other results suggest that the hypersensitive response to microbial pathogens may involve a pcd with some of the characteristics of animal apoptosis and that DNA cleavage is a potential indicator of pcd in plants.

Methods of Genome Analysis in Plants

Abstract: Part 1: genome analysis - a prologue, P.P. Jauhar chromosome pairing as a tool in genome analysis - merits and limitations, P.P. Jauhar and L.R. Joppa chromosome banding and genome analysis in diploid and cultivated polyploid wheats, B. Friebe and B.S. Gill numerical meiotic models for the inference of genomic affinity in polyploids, C.F. Crane limitations and pitfalls in the use of quantitative polyploid meiotic models for genome analysis, J. Sybenga molecular-meiotic analysis of cotton, D.M. Stelly et al. Part 2 Characteristics of nuclear DNA and genome analysis: analysis of plant DNA content by feulgen microspectrophotometry and flow cytometry, H.J. Price and J.S. Johnston genome analysis of polyploid species employing variation in repeated nucleotide sequences, J. Dvorak and J. Dubcovsky development and application of simple sequence repeat (SSR) loci for plant genome analysis, S.M. Brown et al. Part 3 "In situ" hybridization and genomic painting: genomic southern and "in situ" hybridization for plant genome analysis, J.S. Heslop-Harrison and T. Schwarzacher multicolour fluorescence "in situ" hybridization - a new tool for genome analysis, Y. Mukai. Part 4 Isozyme and molecular markers in genome analysis: genome analysis in the triticeae using isozymes, G. Hart genome analysis in polyploids using molecular markers, J.A.G. da Silva and M.E. Sorrells barley genome mapping and its applications, P.M. Hayes et al RAPD analysis of plant genomes, G. Penner. Part 5 Cytoplasmic genome analysis - phylogenetic inferences: plasmon analysis as the counterpart of genome analysis, K. Tsunewaki chloroplast DNA analysis of the monogenomic triticeae - phylogenetic implications and genome-specific markers, R.J. Mason-Gamer and E.A. Kellogg chromosome microdissection and microcloning - application to genome analysis, F.-T. Kao numerical taxonomy and genome analysis, B.R. Baum and P.K. Gupta. Part 6 Genomic constitution of an important crop plant as revealed by these different techniques: genomic constitution of bread wheat - current status, Y. Yen et al.

[43]Cytochemistry and immunocytochemistry of mitochondria in tissue sections

Abstract: Publisher Summary This chapter presents the cytochemical and immunohistochemical methods that, in the experience, appear to be the most reliable for the correct identification of mitochondria on frozen tissue sections, to illustrate their potential using specific examples, and to provide an updated version of the methods. Although the described protocols refer to muscle mitochondria, the methods described can be applied to any cell type. The mitochondria are the primary adenosine triphosphate (ATP)-generating organelles in all mammalian cells and they contain their own DNA (mtDNA), which is maternally inherited. The human mitochondrial genome contains genes encoding for thirteen subunits of different respiratory complexes. These include seven subunits of complex I (NADH dehydrogenase–ubiquinone oxidoreductase), one subunit of complex III (ubiquinone–cytochrome-c oxidoreductase), three subunits of complex IV (cytochrome-c oxidase (COX)), and two subunits of complex V (ATP synthase). Although mitochondria have their transcriptional and translational machinery, most of the proteins located within mitochondria are encoded by the nuclear DNA (nDNA). These nuclear gene products are synthesized on cytoplasmic ribosomes and are subsequently imported into the mitochondria.

Non-linear accumulation of 8-hydroxy-2'-deoxyguanosine, a marker of oxidized DNA damage, during aging.

Abstract: Damage to DNA seems to be involved in aging and the etiology of age-associated degenerative diseases. The purpose of this study is to examine changes in DNA damage during aging. An oxidized nucleoside, 8-hydroxy-2'-deoxyguanosine (8-OHdG), is a proposed biomarker for DNA damaged by oxidative stress. The content of 8-OHdG in nuclear DNA isolated from brain, heart, liver, and kidneys of male Fischer 344 rats of different ages was measured, 8-OHdG can be detected selectively and sensitively at the fmol level by high performance liquid chromatography-electrochemical detection at an applied potential of +350 mV. The amount of 8-OHdG, expressed as the ratio to deoxyguanosine in nuclear DNA, in heart, liver, and kidney remained steady from 2 to 24 months and then increased progressively. The content of 8-OHdG in the DNA in brain showed no changes from 2 to 27 months, but was significantly higher in 30 month-old rats. There was a significant 2-fold increase in the amount of 8-OHdG in the nuclear DNA of all organs tested in 30 month-old rats as compared to 2-24 month-old rats. These results indicate that the accumulation of 8-OHdG in the DNA of rat organs begins at ages above 24 months.

DNA adducts and chronic degenerative diseases. Pathogenetic relevance and implications in preventive medicine

Abstract: Chronic degenerative diseases are the leading causes of death in developed countries. Their control is exceedingly difficult due to their multiplicity and diversity, the interconnection with a network of multiple risk factors and protective factors, the long latency and multistep pathogenesis, and the multifocal localization. Adducts to nuclear DNA are biomarkers evaluating the biologically effective dose, reflecting an enhanced risk of developing a mutation-related disease more realistically than the external exposure dose. The localization and accumulation of these promutagenic lesions in different organs are the composite result of several factors, including (a) toxicokinetics (first-pass effect); (b) local and distant metabolism; (c) efficiency and fidelity of DNA repair; and (d) cell proliferation rate. The last factor will affect not only the dilution of DNA adducts but also the possible evolution towards either destructive processes, such as emphysema or cardiomyopathies, or proliferative processes, such as benign or malignant tumors at various sites. They also include heart tumors affecting fetal myocytes after transplacental exposure to DNA-binding agents, blood vessel tumors, and atherosclerotic plaques. In this article, particular emphasis is given to molecular alterations in the heart, which is the preferential target for the formation of DNA adducts in smokers, and in human aorta, where an extensive molecular epidemiology project is documenting the systematic presence of adducts to the nuclear DNA of smooth muscle cells from atherosclerotic lesions, and their significant correlation with known atherogenic risk factors. Exocyclic DNA adducts resulting from lipid peroxidation, and age-related indigenous adducts (I-compounds) may also originate from endogenous sources, chronic infections and infestations, and inflammatory processes. Type II I-compounds are bulky DNA lesions resulting from oxidative stress, whereas type II-compounds are presumably normal DNA modifications, which display positive correlations with median life span and are decreased in cancer and other pathological conditions. Profiles of type II-compounds strongly depend on diet and are related to the antidegenerative effects of caloric/ dietary restriction. Even broader is the possible meaning of adducts to mitochondrial DNA, which have been detected in rodents exposed to genotoxic agents and complex mixtures, as well as in untreated rodents, in larger amounts when compared to the nuclear DNA of the same cells. Mutations in mitochondrial DNA increase the number of oxidative phosphorylation-defective cells, especially in energy-requiring postmitotic tissues such as brain, heart and skeletal muscle, thereby playing an important role in aging and a variety of chronic degenerative diseases. A decreased formation of DNA adducts is an indicator of reduced risk of developing the associated disease. Therefore, these molecular dosimeters can be used as biomarkers in the prevention of chronic degenerative diseases, pursued either by avoiding exposure to adduct-forming agents or by using chemopreventive agents. Interventions addressed to the human organism by means of dietary measures or pharmacological agents have encountered a broad consensus in the area of cardiovascular diseases, and are deserving a growing interest also in cancer prevention. The efficacy of chemopreventive agents can be assessed by evaluating inhibition of nuclear DNA or mitochondrial DNA adduct formation in vitro, in animal models, and in phase II clinical trials in high-risk individuals.

Mitochondrial DNA migration events in yeast and humans: integration by a common end-joining mechanism and alternative perspectives on nucleotide substitution patterns.

Abstract: In contrast to extensive infiltration of plant nuclear genomes by mitochondrial and chloroplast DNA fragments, a computer assessment method could only detect seven mitochondrial DNA integration events in Saccharomyces cerevisiae chromosomes and five examples of DNA migration into mammalian nuclear genes. No evidence could be detected for mitochondrial DNA insertion into chromosome III of Caenorhabditis elegans or in nuclear DNA sequences of Drosophila sp. or Plasmodium falciparum. Thus, the quantity of organellar DNA in the nucleus appears to vary amongst organisms and is lower in Saccharomyces cerevisiae than suggested by experimental plasmid systems. As in plants, migratory mitochondrial DNA fragments in yeast and mammals are found in intergenic regions and introns. Although many of these insertions are located near retroelements, mitochondrial DNA incorporation appears to be independent of retroelement insertion. Comparison of the mitochondrial DNA fragments with mitochondrial transcription maps suggest that two fragments may have transposed through DNA-based and one through RNA-based mechanisms. Analyses of the integration sites indicate that organellar DNA sequences are incorporated by an end-joining mechanism common to yeast, mammals, and plants. The transferred sequences also provide a novel perspective on rates and patterns of nucleotide substitution. Analysis of the D-loop region including a nuclear copy of mitochondrial DNA supports a progressive reduction in D-loop length within both monkey and great apes mitochondrial lineages. Relative distance tests polarized with nuclear copies of the mitochondrial 12S/16S rRNA region suggest that a constant number of transversions has accumulated within the great ape clade, but the number of transitions in orangutan is elevated with respect to members of the human/chimp/gorilla clade. In addition to DNA migration events, 29 nuclear/mitochondrial genes were identified in GenBank that appear to result from inadvertent ligation of nuclear and mitochondrial mRNA transcripts during the cloning process.

Distinguishing fall armyworm (lepidoptera : noctuidae) strains using a diagnostic mitochondrial dna marker

Abstract: The fall armyworm, Spodoptera frugiperda (J. E. Smith), includes morphologically indistinguishable corn and rice strains. The two strains were surveyed for diagnostic restriction patterns in mitochondrial DNA (mtDNA) using 25 restriction endonucleases. Polymorphic mtDNA restriction patterns were identified for BstNI, HinfI and MspI. The MspI pattern was the most distinctive since the molecular size of each DNA fragment differed between the two strains. Analyses of laboratory and field-collected insects showed the MspI mtDNA pattern to be a diagnostic marker for corn and rice strain insects. Strain identification by the MspI mtDNA profile correlated exactly with nuclear DNA markers. Since no HaeIII sites are present in fall armyworm mtDNA, a double-digest of total fall armyworm DNA using HaeIII and MspI allowed the direct detection of mtDNA restriction fragments from total DNA on a stained agarose gel. In contrast to conventional techniques utilizing mtDNA markers, this rapid and simple procedure does not require the isolation of mtDNA, or avoids the use of DNA blots and labeled mtDNA.

Up-regulation of Bcl-2 is associated with neuronal DNA damage in Alzheimer's disease.

Abstract: Cell death and neurofibrillary tangle formation are prominent features of Alzheimer's disease (AD) It has been suggested that DNA damage may reflect neuronal vulnerability In this context, the Ced homologue Bcl-2 is able to repress a number of cell death programs Recently we found both numerous nuclei exhibiting DNA damage within neurons in the AD brain and increases in Bcl-2 immunoreactivity In this study, we examined the relationship between Bcl-2 expression and nuclear DNA damage or tangle formation Nuclei exhibiting DNA damage were associated with an up-regulation of Bcl-2 expression, whereas tangle-bearing neurons were associated with a down-regulation of Bcl-2 expression

Nuclear accumulation of the E2F heterodimer regulated by subunit composition and alternative splicing of a nuclear localization signal

Abstract: The cellular transcription factor E2F plays a critical role in integrating cell cycle progression with the transcription apparatus by virtue of a physical interaction and control by key regulators of the cell cycle, such as pRb, cyclins and cyclin-dependent kinases. Generic E2F DNA binding activity arises when a member of two families of proteins, E2F and DP, form heterodimeric complexes, an interaction which results in co-operative transcriptional and DNA binding activity. Here, we characterise a new and hitherto unexpected mechanism of control influencing the activity of E2F which is mediated at the level of intracellular location through a dependence on heterodimer formation for nuclear translocation. Nuclear accumulation is dramatically influenced by two distinct processes: alternative splicing of a nuclear localization signal and subunit composition of the E2F heterodimer. These data define a new level of control in the E2F transcription factor whereby interplay between subunits dictates the levels of nuclear DNA binding activity.

Defective Repair of Oxidative Damage in the Mitochondrial DNA of a Xeroderma Pigmentosum Group A Cell Line

Abstract: Recent evidence has linked mitochondrial DNA (mtDNA) damage to several disease processes, including cancer and aging An important source of such damage is reactive oxygen species These molecules can be generated endogenously via the electron transport system or may arise from a host of exogenous sources It has been reported that extracts from cells of individuals with xeroderma pigmentosum group A (XP-A) do not repair some types of oxidative DNA damage The current experiments were designed to determine whether there is a correlation between the inadequate repair of oxidatively damaged nuclear DNA in XP-A cells and the capacity of such cells to repair similar damage to their mtDNA The ability of karyotypically normal human fibroblasts (WI-38) and XP-A fibroblasts to repair alloxan-generated oxidative damage to nuclear and mtDNA was assessed using a quantitative Southern blot method in conjunction with the repair enzymes endonuclease III and formamidopyrimidine DNA glycosylase The data indicate that both nuclear and mtDNA repair of each damage type investigated is more efficient in the WI-38 cells These findings suggest a similarity between the process(es) used to repair oxidative damage to nuclear and mtDNA in that both are inhibited by the defect in XP-A

UV-B induces DNA damage and DNA synthesis delay in the marine diatom Cyclotella sp

Abstract: The effect of UV-B on the occurrence of DNA damage and consequences for the cell cycle were studied in the marine diatom Cyclotella sp. DNA damage was quantified by immunofluorescent detection of thymine dimers in nuclear DNA of single cells using flow cytometry. A total UV-B dose (biologically effective dose BE(DNA300nm)) of 1.05 kJ m(-2) caused detectable damage in about 20% of the exposed population. In the period after the UV-B treatment, when the culture was exposed to PAR (450 mu mol m(-2)s(-1)) only, thymine dimers were removed; after 8 hours none of these photoproducts remained. Cellular DNA content measurements and quantification of the fraction of recently divided cells revealed that the DNA synthesis rate as well as the rate of cell division were reduced during and shortly after UV-B exposure. Apparently, UV-B irradiation extends the cell cycle of Cyclotella sp. in the S (DNA synthesis) phase until the dimers are removed. [KEYWORDS: cell cycle; DNA damage; flow-cytometry; marine diatom; thymine dimers; UV-B Ultraviolet-radiation; antarctica; dosimeter; dimers; ocean;cells]

Variation in chromosomal DNA associated with the evolution of Arachis species.

Abstract: The 2C nuclear DNA amounts were determined for 99 accessions, representing 23 Arachis species from 8 of 9 taxonomic sections, and two synthetic amphidiploids. Mean 2C DNA amounts varied by 15.20%, ranging from 10.26 to 11.82 pg, between accessions of Arachis hypogaea (2n = 4x = 40). Nuclear DNA content variation (5.33–5.91 pg) was also detected among Arachis duranensis (2n = 2x = 20) accessions. The intraspecific variation in the two species may have resulted from indirect selection for favourable genome sizes in particular environmental conditions. The accessions belonging to A. hypogaea ssp. hypogaea (mean value 11.27 pg) with longer life cycle had significantly larger mean DNA content than the accessions of A. hypogaea ssp. fastigiata (mean value 10.97 pg). For 20 diploid (2n = 2x = 20) species of the genus, 2C nuclear DNA amounts ranged from approximately 3 to 7 pg. The diploid perennial species of section Arachis have about 12% more DNA than the annual species. Comparisons of DNA amounts show that ev...

Phylogenetic analysis of restriction-site variation in wild and cultivated Amaranthus species (Amaranthaceae).

Abstract: Amaranthus includes approximately 60 species, of which three are cultivated as a grain source. Many wild Amaranthus species possess agriculturally desirable traits such as drought and salt tolerance, and pathogen resistance. We examined relationships among wild and cultivated Amaranthus species based upon restriction-site variation in two chloroplast DNA regions and in a nuclear DNA region. The chloroplast regions consisted of (1) an intergenic spacer in transfer RNA genes and (2) the ribulose-1,5-bisphosphate carboxylase gene with a flanking open reading frame. The nuclear region was the internal transcribed spacers ITS-1 and ITS-2 flanking the 5.8S gene in the ribosomal DNA. These regions were amplified by the polymerase chain reaction and digested with a total of 38 restriction endonucleases. We detected 11 potentially informative restriction-site mutations and seven length-polymorphisms among the 28 Amaranthus species. Parsimony analysis was used to find the shortest tree for each separate data set (chloroplast, nuclear, and length) and for two combined matrices (chloroplast/nuclear and all data sets). Overall, there was a low level of variation which generated poorly resolved trees among the 28 species. Congruence analyses revealed that the chloroplast and nuclear data sets were congruent with each other but not to the length data set. The congruence of the chloroplast and nuclear data sets suggested that cytoplasmic gene flow may not be a confounding factor in our analyses. The phylogeny also suggested that drought tolerance evolved independently several times. The molecular phylogeny provides a basis for selection of species pairs for crop development.

In Differentiating Mouse Myoblasts DNA Methyltransferase Is Posttranscriptionally and Posttranslationally Regulated

Abstract: Upon the onset of mouse myoblast differentiation there is a rapid drop in DNA methyltransferase activity followed by a genome wide demethylation [Jost and Jost (1994) J. Biol. Chem. 269, 10040-10043]. Here we show by using specific antibodies directed against DNA methyltransferase that upon differentiation there was a rapid drop in nuclear DNA methyltransferase whilst the internal control histone H1 remained constant. The loss of nuclear methyltransferase was not due to a translocation of the enzyme from the nucleus to the cytoplasm where there was an increase in creatine phosphokinase protein. In vitro run on experiments carried out with growing and differentiating myoblast nuclei showed no difference in the rate of DNA methyltransferase mRNA synthesis. As measured by Northern blot hybridization the relative half life of DNA methyltransferase mRNA in growing and differentiating cells in the presence of Actinomycin D was 5 h and 1 h 30 min respectively, whereas in the same cells the half life of histone H4 mRNA was in both cases 80 min. As measured by a combination of pulse chase experiments with labeled leucine and immunoprecipitation, the relative half-life of DNA methyltransferase in growing and differentiating cells was approximately 18 h and 4 h 30 min respectively.

Mutations which block the binding of calmodulin to Spc110p cause multiple mitotic defects

Abstract: We have generated three temperature-sensitive alleles of SPC110, which encodes the 110 kDa component of the yeast spindle pole body (SPB). Each of these alleles carries point mutations within the calmodulin (CaM) binding site of Spc110p which affect CaM binding in vitro; two of the mutant proteins fail to bind CaM detectably (spc110-111, spc110-118) while binding to the third (spc110-124) is temperature-sensitive. All three alleles are suppressed to a greater or lesser extent by elevated dosage of the CaM gene (CMD1), suggesting that disruption of CaM binding is the primary defect in each instance. To determine the consequences on Spc110p function of loss of effective CaM binding, we have therefore examined in detail the progression of synchronous cultures through the cell division cycle at the restrictive temperature. In each case, cells replicate their DNA but then lose viability. In spc110-124, most cells duplicate and partially separate the SPBs but fail to generate a functional mitotic spindle, a phenotype which we term ‘abnormal metaphase’. Conversely, spc110-111 cells initially produce nuclear microtubules which appear well-organised but on entry into mitosis accumulate cells with ‘broken spindles’, where one SPB has become completely detached from the nuclear DNA. In both cases, the bulk of the cells suffer a lethal failure to segregate the DNA.

The nuclear targeting and nuclear retention properties of a human DNA repair protein O6-methylguanine-DNA methyltransferase are both required for its nuclear localization: the possible implications.

Abstract: Human O6-methylguanine-DNA methyltransferase (MGMT) protects human cells from the mutagenic effects of alkylating agents by repairing the O6-alkylguanine residues formed by these agents in the nuclear DNA. We report here a study showing a possible two-step model for the nuclear localization of the 21 kDa human protein. The first step is the translocation of the protein from the cytosol to the nucleus. This appears to require the nuclear targeting property associated with the holoprotein in combination with a cellular factor(s) to effect the nuclear translocation of MGMT. The second step involves the nuclear retention of MGMT (to prevent its export from the nucleus). This requires a basic region (PKAAR, codons 124-128) that can bind to the non-diffusible DNA elements in the nucleus. Supporting data for such mechanisms are: (i) the holoprotein can target the cytosolic 110 kDa beta-galactosidase into the nucleus; (ii) purified recombinant MGMT requires a cellular factor for transport across the nuclear membrane; (iii) nuclear MGMT can be removed selectively by DNase I; (iv) the repair-positive K125L mutant, which alters the PKAAR motif, remains in the cytosol and fails to bind DNA in vitro; and (v) polypeptide containing the PKAAR motif has no nuclear targeting property. Interestingly, mutants in another basic region, KLLKVVK (codons 101-107) are DNA binding and repair deficient but entirely nuclear. As these substitutions affect the functional properties of human MGMT, they are potential targets for genetic screening of individuals for risk assessment to alkylating agents.

Presence of a DNA-4236 bp deletion and 8-hydroxy-deoxyguanosine in mouse cardiac mitochondrial DNA during aging.

Abstract: A particular mitochondrial DNA (mtDNA) deletion, the so-called “common deletion”, accumulates progressively with age in human post-mitotic cells. We investigated the presence of age-related mtDNA deletions in mouse heart and, according to the free-radical theory of aging, the potential involvement of reactive oxygen species (ROS). Hearts from young, adult and old male Balb/c mice were homogenized and centrifuged in order to discard nuclear DNA. The supernatant was then utilized to prepare mtDNA by SDS-proteinase K digestion. The presence of a mtDNA4236 deletion was estimated by PCR analysis, by separating the amplificated segment on agarose gel. The incidence of the mtDNA4236 deletion was 16%, 28% and 78% in young, adult and old mice, respectively. 8-hydroxy-2′-deoxyguanosine (8-OH-dG), a marker of DNA oxidation, was also determined by HPLC-electrochemical analysis. 8-OH-dG was not detectable in young mice, while its concentrations (moles 8-OH-dG / 106 moles dG; mean±SD) were 59.0±1.41 and 31.0±4.24 (p<0.02) in adult and old mice, respectively. These data indicate that a mtD-NA4236 deletion is progressively associated with aging in mouse hearts, and that oxidative damage to mtDNA is greater in middle-aged than senescent animals.

An improved procedure for the isolation of nuclear DNA from leaves of wild grapevine dried with silica gel

Abstract: An efficient and convenient method is presented for the isolation of nuclear DNA from leaves of wildVitis species that have been dried with silica gel. The nuclear DNA obtained with this method is suitable for both PCR amplification and digestion with restriction endonucleases.

Nucleotype and phenotype in Vicia faba

Abstract: Following the results of a previous investigation which showed significant differences (up to 34.6 per cent) in the basic amount of nuclear DNA between local populations of Vicia faba, the development of plants obtained from seeds collected from different populations was studied. Both cell proliferation in the meristems and cell enlargement in differentiated tissues are affected by the genome size. DNA content and the mitotic index in the root meristem are negatively correlated. The higher proportion of cells entering mitosis in the meristems of plants with a lower amount of DNA is not the result of alterations of the duration of the mitotic cycle, which was found to be quite comparable in two populations with largely differing genome sizes. Cell growth was studied in the epicotyl cortex and the leaf epidermis. In both differentiated tissues, cells were longer or had larger surface areas in populations with higher amounts of DNA than in populations with lower amounts of DNA. By studying plant development, positive correlations were found between the genome size and both the germination power of the seeds and the growth rate of the epicotyl. In contrast, negative correlations were found between the basic amount of nuclear DNA and both the height of the main stem and the fresh weight of plants at anthesis. The possible role that intraspecific alterations of the nucleotype may play in environmental adaptation and species evolution is briefly discussed.

Reproductive development and nuclear DNA content in angiosperms

Abstract: Correlates of nuclear DNA content in angiosperms have been noted previously for a range of features, cellular to geographic. A new hypothesis, the correlation between nuclear DNA content and reproductive developmental features (after Cavalier-Smith, Journal of Cell Science 34, 247-268, 1978) is posed and tested here. Of three features tested (megasporogenesis, microsporogenesis, and endosperm development), megasporogenesis alone was shown to be correlated with nuclear DNA amount. The hypothesis was examined in 107 families of angiosperms using nonparametric statistics, and in 53 families of monocotyledons and outgroups using a phylogenetic test of association. A correlation was found between large genomes and successive microsporogenesis for all angiosperms, but not for monocots and dicots analyzed separately, thus underlining the importance of taking into account phylogenetic relationships in such studies. A correlation between cellular endosperm and large genomes in dicotyledons needs to be confirmed in a phylogenetic context. A tendency for deviations from monosporic megasporogenesis to occur in taxa that have a nuclear DNA content of over 9.0 pg/C was demonstrated using both phylogenetic and nonphylogenetic tests. It is hypothesized that cytoskeleton dynamics are affected in reproductive cells, enabling decoupling between nuclear and cytoplasmic cell cycles and leading to variation in reproductive development.

Nuclear DNA Content and Floral Evolution in Silene latifolia

Abstract: Nuclear DNA content shows extensive intraspecific variation in plants, attributable to changes in levels of repetitive DNA. Since its discovery, repetitive DNA often has been regarded as `junk' or selfish DNA with little or no evolutionary significance at the individual level, except possibly a deleterious role stemming from its over-replication. However, recent works have pointed out that repetitive DNA may have an important impact on phenotypic selection through local or general modifications of gene expression. Phenotypic evolution has typically been studied using quantitative genetic techniques that treat underlying genetic effects as variance components. In previous work, we identified an among population negative correlation between nuclear DNA content variation, attributable to repetitive DNA, and calyx diameter, a phenotypic character with obvious ecological importance, in Silene latifolia. In the present study, we established a negative genetic relation between an AT-biased flow cytometric measure of nuclear DNA content and calyx diameter across lineages that had been subjected to selection for increases or decreases in calyx diameter. This finding suggests that new attention should be directed toward repetitive DNA as a basis for phenotypic variation and evolution.

Adducts to nuclear DNA and mitochondrial DNA as biomarkers in chemoprevention

Abstract: DNA adducts are biomarkers evaluating the biologically effective dose of carcinogens, which reflects, more realistically than the external exposure dose, an enhanced risk of developing cancer. Likewise, inhibition of DNA adduct formation can be assumed as an indicator of decreased risk. Molecular dosimetry techniques can be exploited in anticarcinogenicity studies in animal models as well as in Phase II clinical chemoprevention trials. We have extensively used these end points in animal studies using individual carcinogens and complex mixtures. As assessed by 32P-postlabelling, DNA adducts were formed in the liver of rats fed a diet supplemented with 2-acetylaminofluorene. DNA adducts were detected by synchronous fluorescence spectrophotometry (SFS) in rat liver, lung, heart and testis following intratracheal (l.t) instillations of benzo[a]pyrene. The whole-body exposure of rats to mainstream cigarette smoke resulted in the appearance of DNA adducts in lung, heart, aorta and kidney, whereas adducts were not detected by SFS in liver, brain, oesophagus and testis. Moreover, typical diagonal radioactive zones and multiple DNA adducts were revealed by 32P-postlabelling in the tracheal epithelium, nasal mucosa, aorta and testis of smoke-exposed rats. Formation of adducts to lung DNA, as assessed by both 32P-postlabelling and SFS, also occurred in rats receiving i.t. instillations of air particulate extracts from polluted urban and rural areas. The oral administration of the thiol N-acetylcysteine (NAC) significantly inhibited the formation of DNA adducts in all organs of the rats exposed to the aforementioned carcinogens, which correlated with the parallel inhibition of biochemical, cytogenetic and histopathological alterations as well as with inhibition of preneoplastic and neoplastic lesions in rodents. Our working hypothesis is that DNA adducts in trachea/lung, heart and aorta may be associated with lung cancer, cardiomyopathies and atherosclerosis, respectively. DNA adducts were consistently detectable in the DNA of smooth muscle cells from abdominal aorta specimens taken at surgery from atherosclerotic patients. Even broader are the consequences of mitochondrial (mt) DNA impairment, which has been associated with aging, cancer, and other degenerative diseases. Our data show that adduct levels are consistently higher in mtDNA than in the nuclear DNA in different organs of rats exposed either to benzo[a]pyrene, 2-acetylaminofluorene or cigarette smoke. NAC significantly decreased the formation of adducts to mtDNA when administered with drinking-water. Inhibition of adducts to nuclear DNA is one of the end points evaluated in ongoing Phase II chemoprevention trials in high-risk individuals.