Showing papers on "Nuclear DNA published in 1995"
TL;DR: Major unresolved questions concerning the role of mtDNA changes in ageing are addressed and future studies promise to clarify the possible causal relationship between mitochondrial dysfunction, reactive oxygen species production, mtDNA modifications, and ageing.
348 citations
TL;DR: Three lines of investigation demonstrate that the DHBV CCC DNA is stably associated with proteins in the nuclei of infected liver cells and provide compelling evidence that the viral nucleoprotein complex is indeed a minichromosome composed of classical nucleosomes but in arrays that are atypical for chromatin.
Abstract: Replication of hepadnaviruses requires a persistent population of covalently closed circular (CCC) DNA molecules in the nucleus of the infected cell. It is widely accepted that the vital role of this molecule is to be the sole DNA template for the synthesis by RNA polymerase II of all viral transcripts throughout the infection process. Since the transcriptional activity of eukaryotic nuclear DNA is considered to be determined in part by its specific organization as chromatin, the nucleoprotein disposition of the hepadnavirus CCC DNA was investigated. These studies were undertaken on the duck hepatitis B virus (DHBV) CCC DNA present in the liver cell nuclei of DHBV-infected ducks. The organization and protein associations of the DHBV CCC DNA in situ were inferred from sedimentation, micrococcal nuclease digestion, and DNA superhelicity analyses. These three lines of investigation demonstrate that the DHBV CCC DNA is stably associated with proteins in the nuclei of infected liver cells. Moreover, they provide compelling evidence that the viral nucleoprotein complex is indeed a minichromosome composed of classical nucleosomes but in arrays that are atypical for chromatin. When the DHBV chromatin is digested with micrococcal nuclease, a ladder of viral DNA fragments that exhibits a 150-bp repeat is produced. This profile for the viral chromatin is obtained from the same nuclei in which the duck chromatin shows the standard 200-bp ladder. The superhelicity of the DHBV CCC DNA ranges from 0 to 20 negative supertwists per molecule, with all possible 21 topoisomers present in each DNA preparation. The 21 topoisomers of DHBV CCC DNA are inferred to derive from an identically diverse array of viral minichromosomes. In the DHBV minichromosomes composed of 20 nucleosomes, 96.7% of the viral DNA is calculated to be compacted into these chromatin subunits spaced on average by 5 bp of linker DNA; other minichromosomes contain fewer nucleosomes and proportionately more linker DNA. Two major subpopulations of DHBV minichromosomes are detected with comparable prevalence. The two groups correspond to minichromosomes which contain essentially a full or half complement of nucleosomes. The functional significance of this minichromosome diversity is unknown but is suggestive of transcriptional regulation of the viral DNA template.
304 citations
TL;DR: It is hypothesized that the DNA strand breaks produced in the normal transition from a somatic cell histone complex to a protamine complex are not ligated properly, resulting in residualDNA strand breaks and altered chromatin structure, which could lead to the accessibility of the endogenous DNA strands.
Abstract: Sperm from four mammalian species were analyzed by the sperm chromatin structure assay (SCSA) and the terminal deoxynucleotidyl transferase assay (TdTA) using flow cytometry. The SCSA quantitates the susceptibility of sperm nuclear DNA to in situ acid denaturation, while the TdTA quantitates the presence of endogenous DNA strand breaks in sperm nuclear chromatin. Correlations were seen between the percentage of sperm cells showing susceptibility to in situ acid denaturation and the percentage of cells showing the presence of DNA strand breaks for humans (r = 0.56, P = 0.004), rams (r = 0.84, P < 0.001), bulls (r = 0.78, P < 0.001), and stallions (r = 0.65, P < 0.001). No significant differences were seen when using fresh or frozen samples for either assay. These results suggest that sperm cells that are more susceptible to in situ DNA denaturation may have a greater number of accessible endogenous DNA strand breaks. We hypothesize that the DNA strand breaks produced in the normal transition from a somatic cell histone complex to a protamine complex are not ligated properly, resulting in residual DNA strand breaks and altered chromatin structure. Alternatively, altered chromatin structure could lead to the accessibility of the endogenous DNA strand breaks.
270 citations
TL;DR: The results indicate that the BAC system can be used to clone and manipulate large pieces of plant DNA efficiently and increased with decreasing size of the DNA inserts.
Abstract: A bacterial artificial chromosome (BAC) library consisting of 11,000 clones with an average DNA insert size of 125 kb was constructed from rice nuclear DNA. The BAC clones were stable in E. coli after 100 generations of serial growth. Transformation of the BAC clones by electroporation into E. coli was highly efficient and increased with decreasing size of the DNA inserts. The library was evaluated for the presence of organellar, repeated, and telomeric sequences. A very low percentage (< 0.3%) of the library consisted of chloroplast and mitochondrial clones. Eighteen BACs were identified that hybridized with an Arabidopsis telomere repeat. Sixteen BACs hybridized with the AA genome-specific repetitive sequence pOs48. Twelve clones were isolated that hybridized with three DNA markers linked to the Xa-21 disease resistance locus. The results indicate that the BAC system can be used to clone and manipulate large pieces of plant DNA efficiently.
204 citations
TL;DR: The present results indicate that routine and reliable assessment of the involvement of oxidative DNA damage in the development of various diseases, including cancer, is feasible using a variety of tissue sources.
160 citations
TL;DR: It is estimated that 3–7% of the plant nuclear genomic sequence files contain organellar-derived DNA, suggesting that the random integration of organellar DNA has the potential to influence gene expression patterns.
Abstract: A surprisingly large number of plant nuclear DNA sequences inferred to be remnants of chloroplast and mitochondrial DNA migration events were detected through computer-assisted database searches. Nineteen independent organellar DNA insertions, with a median size of 117 by (range of 38 to >785 bp), occur in the proximity of 15 nuclear genes. One fragment appears to have been passed through a RNA intermediate, based on the presence of an edited version of the mitochondrial gene in the nucleus. Tandemly arranged fragments from disparate regions of organellar genomes and from different organellar genomes indicate that the fragments joined together from an intracellular pool of RNA and/or DNA before they integrated into the nuclear genome. Comparisons of integrated sequences to genes lacking the insertions, as well as the occurrence of coligated fragments, support a model of random integration by end joining. All transferred sequences were found in noncoding regions, but the positioning of organellar-derived DNA in introns, as well as regions 5′ and 3′ to nuclear genes, suggests that the random integration of organellar DNA has the potential to influence gene expression patterns. A semiquantitative estimate was performed on the amount of organellar DNA being transferred and assimilated into the nucleus. Based on this database survey, we estimate that 3–7% of the plant nuclear genomic sequence files contain organellar-derived DNA. The timing and the magnitude of genetic flux to the nuclear genome suggest that random integration is a substantial and ongoing process for creating sequence variation.
102 citations
TL;DR: Present knowledge about chromosomes and nuclear DNA of Crustacea is reviewed and the relationship between taxonomy and cytogenetics appears very complex in some taxa, such as Artemia (Branchiopoda).
Abstract: Summary Present knowledge about chromosomes and nuclear DNA of Crustacea is reviewed. Haploid chromosome numbers range from 3 (Acanthocyclops) to 188 (Astacus). Chromosomes are generally small to medium in size (1–5 μm) and punctiform or rod-shape. Nuclear DNA amounts show a large range, lying between 0.37 (Daphnia) and 22.6 pg (Decapoda) per haploid genome, 5.5 × 108 nt to 1.8 × 1010 nt. Highly repetitive sequences may represent as much as 30% of the genome and show a high degree of conservation in Brachyura, while some intermediate repetitive sequences are under-represented. The relationship between taxonomy and cytogenetics appears very complex in some taxa, such as Artemia (Branchiopoda). This genus includes bisexual sibling species with female heterogamety and parthenogenetic populations with different levels of ploidy, up to triploid and pentaploid. Daphnia presents diploid and polyploid strains, and parthenogenetic reproduction. Chromatin diminution occurs in Copepoda; in this group male and female...
99 citations
TL;DR: A purification scheme for mitochondrial DNA was designed which maximized the yield of all forms of the DNA while minimizing damage to the DNA during its isolation, and at doses of H2O2 which were just lethal, neither increased DNA damage nor inactivation of cytochrome c oxidase was observed.
96 citations
TL;DR: The nuclear DNA content was analyzed in Vitis species, hybrid cultivars, and genera of the Vitaceae using flow cytometry to understand the complexity of the Vitis genome and to establish a relationship between the genetic and physical map for map-based cloning.
Abstract: The nuclear DNA content was analyzed in Vitis species, hybrid cultivars, and genera of the Vitaceae using flow cytometry. Significant variation was found among Vitis species, hybrids, and other genera of the Vitaceae (Ampelopsis and Parthenocissus). DNA content was estimated to range from 0.98 to 1.05 pg/2C within V. labrusca (ns) and 0.86 to 1.00 pg/2C within V. vinifera (ns). Genotypes from Vitis and Parthenocissus were similar in nuclear DNA content (approximately 1.00 pg/2C) whereas they differed significantly from Ampelopsis (1.39 pg/2C). No correlation between DNA content and the center of origin of genotypes of the Vitaceae was noted. Based on the present study, the Vitis genome size is 475 Mbp, 96% of which is non-coding. Knowledge of DNA content is useful in order to understand the complexity of the Vitis genome and to establish a relationship between the genetic and physical map for map-based cloning.
90 citations
89 citations
01 Jan 1995
TL;DR: In this paper, a C-terminal truncated form of p53 (p53 delta 30), which is constitutively active for DNA binding and similar to an alternately spliced form found in vivo, showed a concentration-dependent inhibition of DNA replication in both the soluble SV40 system and eukaryotic nuclei.
Abstract: p53 is a transcriptional activator and repressor, but recent evidence suggests that some of its many biological functions may not be dependent on transcription. To determine whether p53 exerts a direct influence on nuclear DNA replication, purified human p53 was added to a transcription‐free DNA replication extract from Xenopus eggs. Full‐length human p53 that inhibits SV40 DNA replication in vitro had no effect on nuclear DNA synthesis in the Xenopus system. In contrast, a C‐terminal truncated form of p53 (p53 delta 30), which is constitutively active for DNA binding and similar to an alternately spliced form found in vivo, showed a concentration‐dependent inhibition of DNA replication in both the soluble SV40 system and eukaryotic nuclei. This inhibition occurred primarily at initiation of DNA synthesis. Oxidation of p53 delta 30, which eliminates DNA binding activity, also abrogated the protein's ability to inhibit nuclear DNA synthesis. The p53 binding DNA consensus sequence enhanced rather than competed away inhibitory activity of p53 delta 30. Therefore, p53 that is constitutively active for DNA binding can inhibit nuclear DNA replication in the absence of transcription. This inhibition may require binding of p53 to DNA, in addition to interactions between p53 and proteins of the replication complex.
TL;DR: Results indicate that the COX defect in the authors' patient is due to a mutation of a nuclear gene, and the use of cybrids obtained from 'customized', patient-derived rho degree cells can have wide applications in the identification of respiratory chain defects originated by nuclear DNA-encoded mutations, and in the study ofnuclear DNA-mitochondrial DNA interactions.
Abstract: Defects of the respiratory chain carrying out oxidative phosphorylation (OXPHOS) are the biochemical hallmark of human mitochondrial disorders. Faulty OXPHOS can be due to mutations in either nuclear or mitochondrial genes, that are involved in the synthesis of individual respiratory subunits or in their post-translational control. The most common mitochondrial disorder of infancy and childhood is Leigh's syndrome, a severe encephalopathy, often associated with a defect of cytochrome c oxidase (COX). In order to demonstrate which genome is primarily involved in COX-deficient (COX(-))-Leigh's syndrome, we generated two lines of transmitochondrial cybrids. The first was obtained by fusing nuclear DNA-less cytoplasts derived from normal fibroblasts, with mitochondrial DNA-less (rho degree) transformant fibroblasts derived from a patient with COX(-))-Leigh's syndrome. The second cybrid line was obtained by fusing rho degree cells derived from 143B.TK- human osteosarcoma cells, with cytoplasts derived from the same patient. The first cybrid line showed a specific and severe COX(-) phenotype, while in the second all the respiratory chain complexes, including COX, were normal. These results indicate that the COX defect in our patient is due to a mutation of a nuclear gene. The use of cybrids obtained from 'customized', patient-derived rho degree cells can have wide applications in the identification of respiratory chain defects originated by nuclear DNA-encoded mutations, and in the study of nuclear DNA-mitochondrial DNA interactions.
TL;DR: To cover a wide range of possible demographic situations in the human lineage since the Miocene, a model is introduced that allows temporal changes in population structure and size and the coalescence process of neutral genes is formulated and used to make quantitative inferences on the origin and history of humans.
Abstract: Recent topics in molecular anthropology are reviewed with special reference to hominoid DNA sequences and population genetics theory. To cover a wide range of possible demographic situations in the human lineage since the Miocene, a model is introduced that allows temporal changes in population structure and size. The coalescence process of neutral genes is formulated and used to make quantitative inferences on the origin and history of humans. Nuclear DNA sequence data support the theory that humans and chimpanzees diverged from each other 4.6 million years (mya) and the gorilla lineage branched off as early as 7.0-7.4 mya. The same data estimate the effective size of the Pliocene hominoid population as i05, a figure similar to that obtained independently from alleles that have persisted in the human population for more than 5 my. Hypotheses about the origin of Homo sapiens, genetic differentiation among human populations, and changes in population size are quantified. None of the hypotheses seems compatible with the observed DNA variation. The effective population size decreased to 104 in the Pleistocene, suggesting an important role of extinction/restoration in H. sapiens populations. Natural selection against protein variation might be relaxed in the Pleistocene. The 'Abbreviation and symbols: kb (kilo base pairs), bp (base pairs), yr (years), my (a) (million years (ago)), COII (cytochrome oxidase subunit II), rDNA (ribosomal DNA), mtDNA (mitochondrial DNA), MHC (major histocompatibility complex)
TL;DR: Analysis of all sequences by the neighbor-joining (NJ) method indicated that mitochondrial DNA sequences and their nuclear counterparts can be divided into two distinct clusters and these results constitute a warning for when working with ancient DNA or performing phylogenetic analysis using mitochondrial DNA as a target sequence.
Abstract: Monkey mummy bones and teeth originating from the North Saqqara Baboon Galleries (Egypt), soft tissue from a mummified baboon in a museum collection, and nineteenth/twentieth-century skin fragments from mangabeys were used for DNA extraction and PCR amplification of part of the mitochondrial 12S rRNA gene. Sequences aligning with the 12S rRNA gene were recovered but were only distantly related to contemporary monkey mitochondrial 12S rRNA sequences. However, many of these sequences were identical or closely related to human nuclear DNA sequences resembling mitochondrial 12S rRNA (isolated from a cell line depleted in mitochondria) and therefore have to be considered contamination. Subsequently in a separate study we were able to recover genuine mitochondrial 12S rRNA sequences from many extant species of nonhuman Old World primates and sequences closely resembling the human nuclear integrations. Analysis of all sequences by the neighbor-joining (NJ) method indicated that mitochondrial DNA sequences and their nuclear counterparts can be divided into two distinct clusters. One cluster contained all temporary cytoplasmic mitochondrial DNA sequences and approximately half of the monkey nuclear mitochondriallike sequences. A second cluster contained most human nuclear sequences and the other half of monkey nuclear sequences with a separate branch leading to human and gorilla mitochondrial and nuclear sequences. Sequences recovered from ancient materials were equally divided between the two clusters. These results constitute a warning for when working with ancient DNA or performing phylogenetic analysis using mitochondrial DNA as a target sequence: Nuclear counterparts of mitochondrial genes may lead to faulty interpretation of results.
TL;DR: Levels of mtDNA in skeletal muscle from some patients with other forms of muscle disease were also found to be low, suggesting that mtDNA depletion, possibly related to depletion of mitochondria, may be a relatively non-specific response of muscle to various pathological processes.
Abstract: Recent studies have identified a group of patients with cytochrome oxidase (COX) deficiency presenting in infancy associated with a deficiency of mtDNA in muscle or other affected tissue (Moraes et al 1991). We used a novel approach to compare the level of mitochondrial (mtDNA) compared to nuclear DNA in skeletal muscle from a group of patients and controls, based on dot blots that were hybridized with a mtDNA probe labelled with35S[dCTP] and a reference nuclear DNA probe labelled with [32P]dCTP.
TL;DR: Both methods demonstrated a preferential binding of cisplatin to mtDNA versus gDNA, and DELFIA analysis of cisPlatin-DNA adducts in gDNA and mtDNA showed a six-fold higher incorporation of drug into mtDNA as compared to gDNA.
Patent•
28 Feb 1995
TL;DR: In this paper, a plant's nuclear genome is transformed with a first foreign DNA sequence, which comprises a fertility-restorer DNA which encodes a first RNA, a protein or polypeptide that can inactivate a second RNA, protein, or polyprotein in cells of the plant.
Abstract: A plant, the nuclear genome of which is transformed with a first foreign DNA sequence. The first foreign DNA sequence comprises a fertility-restorer DNA which encodes a first RNA, a protein or polypeptide that can inactivate a second RNA, protein or polypeptide in cells of the plant. The second RNA, protein or polypeptide is encoded by a sterility DNA in a second foreign DNA sequence in the plant's nuclear genome. Expression of the sterility DNA, in the absence of expression of the fertility-restorer DNA, would disturb significantly the metabolism, functioning and/or development of cells of the plant's flowers, particularly, the plant's reproductive organs, or cells of the plant's seeds or embryos in which the sterility DNA is selectively expressed, thereby rendering the plant male- or female-sterile. The first and second foreign DNA sequences also can encode suitable markers.
TL;DR: Results indicate that the egg and sperm nuclei are at the 1C DNA level (basic haploid amount) at the time of karyogamy, which is fundamental to a better understanding of fertilization and zygote maturation in flowering plants.
Abstract: Quantities of DNA were estimated in the nuclei of mechanically isolated egg and zygote protoplasts in two cultivars of barley using 4[prime],6-diamidino-2-phenylindole staining and microfluorometry. Unlike many previous studies on DNA amounts within the sex cells of flowering plants, we obtained consistent and unambiguous results indicating that the egg and sperm nuclei are at the 1C DNA level (basic haploid amount) at the time of karyogamy. Karyogamy was initiated within 60 min postpollination, and the male chromatin became completely integrated into the egg nucleus within 6 to 7 hr postpollination (hpp). Zygotic nuclear DNA levels began to increase at ~9 to 12 hpp in cultivar Alexis and at 12 to 15 hpp in cultivar Igri. The 4C DNA complement was reached in most zygotes by 22 to 26 hpp in cultivar Alexis and by 23 to 29 hpp in cultivar Igri. These data are fundamental to a better understanding of fertilization and zygote maturation in flowering plants. They are also relevant to studies in which the timing of zygotic DNA replication is of interest, such as ongoing investigations on genetic transformations in barley using the microinjection technique.
Journal Article•
TL;DR: The successful extraction and amplification of nuclear DNA from the beta-globin region from 5 of 10 bone specimens up to 12,000 years old shows that the systematic study of nuclearDNA polymorphisms of ancient populations is feasible.
Abstract: Analyzing the nuclear DNA from ancient human bones is an essential step to the understanding of genetic diversity in current populations, provided that such systematic studies are experimentally feasible. This article reports the successful extraction and amplification of nuclear DNA from the P-globin region from 5 of 10 bone specimens up to 12,000 years old. These have been typed for P-globin frameworks by sequencing through two variable positions and for a polymorphic (AT){sub x}(T){sub y} microsatellite 500 bp upstream of the P-globin gene. These specimens of human remains are somewhat older than those analyzed in previous nuclear gene sequencing reports and considerably older than those used to study high-copy-number human mtDNA. These results show that the systematic study of nuclear DNA polymorphisms of ancient populations is feasible. 34 refs., 3 figs., 2 tabs.
TL;DR: DNA damage induced by Adriamycin further supported the possible correlation between hydroxyl radical-mediated cleavage of DNA and nuclear translocation of hsp70 protein and results suggest that preexisting hsp 70 protein translocated into the nucleus to either protect chromatin DNA from further damage or to facilitate the repair of DNA damage in some unknown ways.
TL;DR: Comparisons with plants that produce multiflagellated or pollendispersed sperm indicate operation of a nucleotypic effect in archegoniates with biflageLLated sperm, which may be on sperm cell functioning, which in turn influences reproductive success.
Abstract: Nuclear DNA contents of developing sperm were estimated for 17 species of bryophytes by cytophotometry in squash preparations of antheridia after Feulgen staining. Genome sizes are in the lower end of the range for land plants. Two hornwort C-values have the lowest recorded for bryophytes at 0.17 and 0.26 pg DNA per nucleus. In liverworts, C-values range from 0.49 pg in Blasia pusilla to 4.05 pg in Pellia epiphylla, while moss genome sizes are less variable, ranging from 0.38 pg in Takakia ceratophylla to 0.92 pg in Atrichum oerstedianum. DNA content is not correlated with chromosome number in these bryophytes, but sperm cell size and cellular complexity are directly related to C-value. Structural variations in the locomotory apparatus are viewed as evolutionary modifications associated with changes in genomic complexity, with a generalized increase in complexity of the motile assemblage accompanying increases in DNA content. Nuclear DNA values are not as variable in bryophytes as they are in pteridophytes and seed plants. We suggest that in plants producing biflagellated gametes, lower DNA contents afford a selective advantage. Comparisons with plants that produce multiflagellated or pollendispersed sperm indicate operation of a nucleotypic effect in archegoniates with biflagellated sperm. This effect may be on sperm cell functioning, which in turn influences reproductive success.
TL;DR: Data presented suggested that DNA content is a useful descriptor for characterization of plant genetic resources and the occurrence of significant levels of variation within and between Paspalum species indicates that estimates of DNA contents may be useful for characterization and identification of cytotypes.
Abstract: DNA contents in nuclei isolated from 81 accessions ofPaspalum, representing 35 species, was determined by laser flow cytometry. Nuclei were isolated by chopping leaf base tissue in Tris/HCl buffer containing 1 % Triton X-100 and fixed in EtOH. Fixation, or no fixation, of nuclei in 70% or 95% EtOH did not affect estimates of DNA contents in DAPI-stained nuclei. DNA contents, among the species examined, varied approximately four-fold (1.02 – 3.86 pg/2C nucleus). Coefficients of variation ranged from less than 1% to approximately 5% and did not increase following fixation. Variation in DNA contents between plants within accessions was less than 0.1%. Significant differences in DNA contents estimates within and between species were detected. Data presented suggested that DNA content is a useful descriptor for characterization of plant genetic resources. The occurrence of significant levels of variation within and betweenPaspalum species indicates that estimates of DNA contents may be useful for characterization and identification ofPaspalum cytotypes. In addition, the identification and maintenance of variability for DNA contents withinPaspalum spp. might be useful as a means to capture genomic adaptations to ecological variation.
TL;DR: In agreement with the cytophotometric findings, a significant, positive correlation was determined between the DNA contents of populations and the copy numbers of DNA sequences related to FokI repeats, and significant differences between accessions were found in the length of the chromosome complement at metaphase.
Abstract: Findings obtained using different approaches indicated the occurrence of genomic size variations within V. faba. Significant differences in the basic amount of nuclear DNA (up to 34.6 per cent) between 39 local populations collected from the Mediterranean Basin were observed by means of Feulgen/DNA cytophotometry. By contrast, no difference in genome size was found when five commercial varieties were compared. Dot blot hybridization of FokI V. faba repeats to genomic DNAs showed up to fourfold differences in the redundancy of these sequences in the nuclear DNA of different accessions. In agreement with the cytophotometric findings, a significant, positive correlation was determined between the DNA contents of populations and the copy numbers of DNA sequences related to FokI repeats. Significant differences between accessions were found in the length of the chromosome complement at metaphase, and these differences were particularly apparent in certain chromosome pairs. A positive correlation was found between the length of the complement and the genome size of the populations. These results are discussed in relation to other data in the literature on intraspecific nuclear DNA changes.
TL;DR: The results are consistent with the hypothesis that topoisomerase II is essential for the segregation of chromosomes in mammals and other eukaryotes.
TL;DR: To identify and characterize nuclear proteins which interact with the pcbAB-pcbC intergenic promoter region, crude and partially purified nuclear extracts were used in mobility shift and DNA footprinting assays.
Abstract: The biosynthesis of penicillin, a secondary metabolite produced by Penicillium chrysogenum, is subject to sophisticated genetic and metabolic regulation. The structural genes, pcbC and pcbAB, which encode two of the penicillin biosynthetic enzymes are separated by a 1.16-kb intergenic region and transcribed divergently from one another. To identify and characterize nuclear proteins which interact with the pcbAB-pcbC intergenic promoter region, crude and partially purified nuclear extracts were used in mobility shift and DNA footprinting assays. Multiple DNA-binding proteins appear to bind to different regions of this DNA segment. An abundant nuclear protein, nuclear factor A (NF-A), binds at a single site in the intergenic promoter region and recognizes an 8-bp sequence, GCCAAGCC. Penicillin production is sensitive to nitrogen catabolite repression. The global-acting nitrogen regulatory protein NIT2 of Neurospora crassa binds strongly to the intergenic promoter region of the pcbAB and pcbC genes at a single site that contains two closely spaced GATA sequences.
TL;DR: The rDNA analysis indicated evolutionary relatedness of the various taxa within the Narbonensis species complex, and provides new evidence that none of the species within the complex can be considered to be putative allies of broad bean.
Abstract: DNA restriction endonuclease fragment analysis was used to obtain new information on the genomic organization of Vicia ribosomal DNA (rDNA), more particularly among V. faba and its close relatives and the taxa within three (Narbonensis, Villosa, Sativa) species' complexes. Total genomic DNA of 90 accessions representing 49 Vicia species was restricted with 11 enzymes, and the restriction fragments were probed with three ribosomal clones. Twenty-eight repeat unit length classes were identified. The number of length classes (1–2) per accession did not correspond to the number of nucleolar organizing regions (NORs). The number of rRNA genes was independent of the 2C nuclear DNA amount present in the taxon. Each of the 90 accessions had 2 (rarely 1)-4 DraI sites. Those taxa with the same number of DraI sites generally could be distinguished from each other by different configurations. Probing of the DNA samples digested with tetranucleotide recognition restriction endonucleases emphasized differences between divergent spacer regions and enabled relative homologies between the coding regions to be established. Overall, rDNA restriction site variation among the species showed a good correlation with taxonomic classification. The rDNA analysis indicated evolutionary relatedness of the various taxa within the Narbonensis species complex. rDNA diversity within two other species complexes (Villosa, Sativa), on the other hand, was more extensive than expected. With few exceptions, data on the two complexes give evidence of taxon-specific divergences not seen with other approaches. The restriction site variability and repeat length heterogeneity in the rDNA repeat exhibited startling differences between V.faba and its close wild relatives included in the Narbonensis species complex. This analysis provides new evidence that none of the species within the complex can be considered to be putative allies of broad bean.
TL;DR: The nuclear DNA content of isolated eggs and zygotes of maize was estimated using DAPI staining and microspectrofluorometry to indicate that egg nuclei contain the 1C level of DNA at the time of karyogamy, and that, by inference, the sperm nuclei are also at 1C.
Abstract: The nuclear DNA content of isolated eggs and zygotes of maize was estimated using 4′,6-diamidino-2-phenylindole (DAPI) staining and microspectrofluorometry. The data indicate that egg nuclei contain the 1C level of DNA (basic haploid amount) at the time of karyogamy, and that, by inference, the sperm nuclei are also at 1C. Fertilization occurred in most ovules by 24–28 h post-pollination (hpp), and DNA synthesis was well underway by 27–31 hpp. By 30–34 hpp, 80% of the zygotes were at the 3C DNA level or above, and many were undergoing mitosis. This study provides information that is pertinent to experiments on the microinjection of exogenous DNA into isolated zygotes of maize, and it will serve as a comparative base for future determinations of the DNA content of zygotes produced and cultured in vitro.
TL;DR: A PCR‐based method that utilizes restriction site variation of element copies within a genome is developed, which allows transposable elements, or even other multiple‐copy nuclear DNA sequences, to be generally utilized as molecular markers in discriminating between other closely related species and subspecies.
Abstract: Transposable elements are mobile sequences found in nuclear genomes and can potentially serve as molecular markers in various phylogenetic and population genetic investigations. A PCR-based method that utilizes restriction site variation of element copies within a genome is developed. These patterns of site variation, referred to as transposon signatures, are useful in differentiating between closely related groups. Signature data using the magellan retrotransposon, for example, is useful in examining relationships within the genus Zea and Tripsacum. This method allows transposable elements, or even other multiple-copy nuclear DNA sequences, to be generally utilized as molecular markers in discriminating between other closely related species and subspecies.
TL;DR: It is suggested that nuclear DNA fragmentation precedes chromatin condensation and nuclear fragmentation in X‐ray‐induced apoptosis and agarose gel electrophoresis of extracted DNA from irradiated brains showed a “ladder” pattern which is typical of internucleosomal DNA fragmentation and endonuclease activation.
TL;DR: Interestingly, the DNA electrophoresis revealed the smear pattern of small DNA fragments shortly after SSA exposure, although it did not show the apoptotic ladder pattern, which seems to support the idea that the sub‐G, peak represents necrosis rather than apoptosis.
Abstract: Saikosaponin A (SSA) induced cell death in the human hepatoma cell line (HuH-7) was investigated. Shortly after exposure to SSA, a DNA histogram showed a 'sub-G1 peak', which was recently reported as suggestive of apoptosis by other researchers. However, the electrophoresis of DNA indicated that such was not the case in the present experiment. The decreased intake of rhodamine 123 and the swelling of mitochondria were remarkable shortly after SSA exposure. These data seem to support the idea that the sub-G1 peak represents necrosis rather than apoptosis. Interestingly, the DNA electrophoresis revealed the smear pattern of small DNA fragments shortly after SSA-exposure, although it did not show the apoptotic ladder pattern. This finding and the appearance of the 'sub-G1 peak' is thought to have been a result of the degradation of the nuclear DNA in the early stages of cell death. Because these findings are different not only from apoptosis, but also from typical necrosis, a distinct mechanism of cell death caused by SSA is suggested.