Showing papers on "Nuclear DNA published in 1981"
TL;DR: The results show that the distribution of UV light-induced cyclobutane dimers within a defined sequence is similar whether the DNA is exposed to UV light as part of the chromosome of intact cells or as naked DNA, however, the cellular environment shields the nuclear DNA, resulting in about 50% decrease in apparent dose.
Abstract: The distribution of UV light-induced damage to the highly reiterated alpha sequence of human DNA was investigated. The results show that the distribution of UV light-induced cyclobutane dimers within a defined sequence is similar whether the DNA is exposed to UV light as part of the chromosome of intact cells or as naked DNA. However, the cellular environment shields the nuclear DNA, resulting in about 50% decrease in apparent dose. A new type of UV photodamage was detected. Treatment of UV light-irradiated DNA with hot alkali results in strand breaks at positions of cytidine located 3' to pyrimidine nucleosides. The chemical nature and biological significance of the pyrimidine nucleoside-cytidine lesion is discussed.
231 citations
TL;DR: These findings suggest that the DNA replicational sites are continuously bound to the nuclear matrix, and are remarkably sensitive to release from the matrix structure by exogenous nucleases including pancreatic DNase I and micrococcal nuclease.
214 citations
TL;DR: These experiments suggest that the RAD1, RAD2, RAD3, RAD4 (and probably RAD14) genes are all required for the incision of UV-irradiated DNA during pyrimidine dimer excision in vivo.
Abstract: A group of genetically related ultraviolet (UV)-sensitive mutants of Saccharomyces cerevisiae has been examined in terms of their survival after exposure to UV radiation, their ability to carry out excision repair of pyrimidine dimers as measured by the loss of sites (pyrimidine dimers) sensitive to a dimer-specific enzyme probe, and in terms of their ability to effect incision of their deoxyribonucleic acid (DNA) during post-UV incubation in vivo (as measured by the detection of single-strand breaks in nuclear DNA). In addition to a haploid RAD+ strain (S288C), 11 different mutants representing six RAD loci (RAD1, RAD2, RAD3, RAD4, RAD14, and RAD18) were examined. Quantitative analysis of excision repair capacity, as determined by the loss of sites in DNA sensitive to an enzyme preparation from M. luteus which is specific for pyrimidine dimers, revealed a profound defect in this parameter in all but three of the strains examined. The rad14-1 mutant showed reduced but significant residual capacity to remove enzyme-sensitive sites as did the rad2-4 mutant. The latter was the only one of three different rad2 alleles examined which was leaky in this respect. The UV-sensitive strain carrying the mutant allele rad18-1 exhibited normal loss of enzyme-sensitive sites consistent with its assignment to the RAD6 rather than the RAD3 epistatic group. All strains having mutant alleles of the RAD1, RAD2, RAD3, RAD4, and RAD14 loci showed no detectable incubation-dependent strand breaks in nuclear DNA after exposure to UV radiation. These experiments suggest that the RAD1, RAD2, RAD3, RAD4 (and probably RAD14) genes are all required for the incision of UV-irradiated DNA during pyrimidine dimer excision in vivo.
155 citations
TL;DR: Kinetic analysis of the poly(A)+-RNA population indicates it is composed of at least three classes of RNA's of different abundancy levels: two sequences which occur about 20 times per cell, and ca.
91 citations
Journal Article•
TL;DR: The absence of nucleosome-like structures from purified nuclei of the primitive dinoflagellate Prorocentrum micans was demonstrated, and the evolutionary position of the din oflageLLate protists with respect to the procaryotes and eucaryotes is discussed.
90 citations
TL;DR: G+C-rich satellite DNA, representing about 19% of total nuclear DNA, was isolated from various tissues of the monocotyledonous plant, Scilla siberica, by using Ag(+)-Cs(2)SO(4) gradient techniques and suggests that this constitutively methylated sequence is evolutionarily stable.
Abstract: G+C-rich satellite DNA, representing about 19% of total nuclear DNA, was isolated from various tissues of the monocotyledonous plant, Scilla siberica, by using Ag+-Cs2SO4 gradient techniques. This satellite DNA had an unusually high melting point and a high methylcytosine (m5C) content (≈25% of total bases; m5C/cytosine ratio ≈1.5) and was localized, by in situ hybridization, in the heterochromatin regions of the chromosomes. Digestion with restriction endonuclease Hae III yielded a series of fragments ranging from 35 to several hundred nucleotide pairs. The major fragments, I-IV (35, 50, 59, and 69, nucleotide pairs, respectively), were isolated, and their nucleotide sequences were determined. The dominant fragment I was a highly symmetrical molecule, with a basically palindromic arrangement. This sequence represented the basic unit of Scilla satellite DNA and was tandemly repeated many times, with some base substitutions and multiple successive insertions of the tetranucleotide G-T-C-C. The dinucleotide CpG was the commonest nearest-neighbor sequence. Thin layer chromatography, DNA sequence analysis, and gas chromatography combined with mass spectrometry showed the high m5C content (m5C/Cyt = 2.2 and 2.8, respectively, for fragments II and III). Identical cleavage fragments were found in satellite DNAs from two other species of this genus (S. amoena and S. ingridae), which suggests that this constitutively methylated sequence is evolutionarily stable. The sequence arrangement of this plant satellite DNA is compared with those reported for several animal satellite DNAs.
86 citations
TL;DR: Electron microscopy, restriction enzyme analysis and Southern hybridization experiments allowed us to establish that SEN-DNAs obtained from independent senescent cultures, both from the same strain and from different strains, can differ in the size of their monomer unit.
Abstract: The non-nuclear DNA of a number of independent senescent cultures of Podospora anserina was extracted and studied. In all cases, a specific repetitive DNA (SEN-DNA) arranged in multimeric sets of circular molecules, was identified. Depending on the senescent culture, the SEN-DNA was found either in a band of about same density as the mitochondrial DNA from young mycelia (1.694 g/cm3) or in a band of higher density (1.699 g/cm3). Electron microscopy, restriction enzyme analysis and Southern hybridization experiments allowed us to establish that: (1) SEN-DNAs obtained from independent senescent cultures, both from the same strain and from different strains, can differ in the size of their monomer unit (from 2.5 to 6.3 kb). (2) All SEN-DNAs hybridize with mitochondrial DNA of a young culture and not with nuclear DNA. (3) These SEN-DNAs belong to two classes which hybridize with two non-overlapping regions of the mitochondrial chromosome.
80 citations
TL;DR: Proteins resistant to sarcosyl-Cs2SO4 treatment are associated exclusively with skeleton-attached DNA both in interphase nuclei and metaphase chromosomes and a group of tightly bound low molecular weight polypeptides seem to be randomly distributed throughout the whole DNA loop.
79 citations
TL;DR: In this paper, annealing studies were performed on DNA fragments associated with rat and mouse liver interphase nuclear matrix and the metaphase scaffold of Chinese hamster DON cells.
Abstract: Annealing studies were performed on DNA fragments associated with rat and mouse liver interphase nuclear matrix and the metaphase scaffold of Chinese hamster DON cells. Matrix and scaffold bound DNA fragments, reassociated with an excess of total genomic DNA, displayed kinetics virtually identical with total nuclear DNA probes. Moreover, both the extent and kinetics of these hybridizations were independent of the matrix DNA fragment size (less than 350--5000 base pairs) and the method of nuclease digestion used in their preparation (DNase I, micrococcal nuclease or endogenous digestion). The repetitive DNA component of the matrix DNA was examined by reacting discrete sizes of matrix DNA fragments (less than 350--5000 base pairs) from mouse liver with a library of cloned repetitive sequence DNA fragments which included mouse major satellite sequences. Our results demonstrate that short DNA fragments anchored to the nuclear matrix contain these cloned sequences is similar proportion of total nuclear DNA and, when viewed in light of the annealing results, indicate that matrix DNA is not enriched in either repetitive or unique sequences. Furthermore, the matrix DNA fragments appear to contain the entire sequence complexity of the genome. Finally, we hybridized both matrix and total nuclear DNA fragments with cDNA to total nuclear polyadenylated RNA. The kinetics and extent of hybridization indicate that most, if not all, of the actively transcribed DNA sequences are present in similar concentrations. We conclude that in the overall organization of eukaryotic DNA within the nucleus, the repeating domains or loops which have been demonstrated by a number of investigators are not anchored at specific attachment sequences in interphase cells or during mitosis. These findings are discussed with regard to current concepts of eukaryotic DNA loop organization.
78 citations
Journal Article•
TL;DR: In the overall organization of eukaryotic DNA within the nucleus, the repeating domains or loops which have been demonstrated by a number of investigators are not anchored at specific attachment sequences in interphase cells or during mitosis.
Abstract: Annealing studies were performed on DNA fragments associated with rat and mouse liver interphase nuclear matrix and the metaphase scaffold of Chinese hamster DON cells. Matrix and scaffold bound DNA fragments, reassociated with an excess of total genomic DNA, displayed kinetics virtually identical with total nuclear DNA probes. Moreover, both the extent and kinetics of these hybridizations were independent of the matrix DNA fragment size (less than 350--5000 base pairs) and the method of nuclease digestion used in their preparation (DNase I, micrococcal nuclease or endogenous digestion). The repetitive DNA component of the matrix DNA was examined by reacting discrete sizes of matrix DNA fragments (less than 350--5000 base pairs) from mouse liver with a library of cloned repetitive sequence DNA fragments which included mouse major satellite sequences. Our results demonstrate that short DNA fragments anchored to the nuclear matrix contain these cloned sequences is similar proportion of total nuclear DNA and, when viewed in light of the annealing results, indicate that matrix DNA is not enriched in either repetitive or unique sequences. Furthermore, the matrix DNA fragments appear to contain the entire sequence complexity of the genome. Finally, we hybridized both matrix and total nuclear DNA fragments with cDNA to total nuclear polyadenylated RNA. The kinetics and extent of hybridization indicate that most, if not all, of the actively transcribed DNA sequences are present in similar concentrations. We conclude that in the overall organization of eukaryotic DNA within the nucleus, the repeating domains or loops which have been demonstrated by a number of investigators are not anchored at specific attachment sequences in interphase cells or during mitosis. These findings are discussed with regard to current concepts of eukaryotic DNA loop organization.
69 citations
TL;DR: The DNA-rich matrices were virtually identical with previously isolated DNA-depleted matrices in recovery of total nuclear protein and overall polypeptide composition on sodium dodecyl sulfate-acrylamide gels as discussed by the authors.
Abstract: Rat liver nuclear matrices isolated by a method which limits DNA degradation contain a major portion of the total nuclear DNA. A majority of the DNA sediments at greater than or equal 100 S on alkaline sucrose gradients, which represents an estimated single strand size of greater than or equal to 500 kilobases. These DNA-rich matrices were virtually identical with previously isolated DNA-depleted matrices in recovery of total nuclear protein and overall polypeptide composition on sodium dodecyl sulfate-acrylamide gels. Thin-sectioning electron microscopy revealed a structure similar to the DNA-depleted matrices with the addition of a prominent meshwork of DNA fibrils extended throughout the matrix interior. In vivo labeling of regenerating livers showed a continuous association of newly replicated DNA with DNA-rich matrices (greater than or equal to 80% of total labeled DNA) which is independent of the pulse period (1 min to 4 h). Moreover, the matrix-associated DNA is highly enriched in replicating intermediates after a 1-min in vivo pulse including a small amount of the primary Okazaki fragments. The matrix-associated replicating intermediates (4-50 S) are effectively chased into DNA of replicon size and larger (100 S) following a 1-h pulse. DNA-rich nuclear matrices may therefore provide a useful in vitro system for studying DNA replication in correlation with the higher order, intranuclear arrangement of eukaryotic DNA.
Journal Article•
TL;DR: Rat liver nuclear matrices isolated by a method which limits DNA degradation contain a major portion of the total nuclear DNA and may provide a useful in vitro system for studying DNA replication in correlation with the higher order, intranuclear arrangement of eukaryotic DNA.
Abstract: Rat liver nuclear matrices isolated by a method which limits DNA degradation contain a major portion of the total nuclear DNA. A majority of the DNA sediments at greater than or equal 100 S on alkaline sucrose gradients, which represents an estimated single strand size of greater than or equal to 500 kilobases. These DNA-rich matrices were virtually identical with previously isolated DNA-depleted matrices in recovery of total nuclear protein and overall polypeptide composition on sodium dodecyl sulfate-acrylamide gels. Thin-sectioning electron microscopy revealed a structure similar to the DNA-depleted matrices with the addition of a prominent meshwork of DNA fibrils extended throughout the matrix interior. In vivo labeling of regenerating livers showed a continuous association of newly replicated DNA with DNA-rich matrices (greater than or equal to 80% of total labeled DNA) which is independent of the pulse period (1 min to 4 h). Moreover, the matrix-associated DNA is highly enriched in replicating intermediates after a 1-min in vivo pulse including a small amount of the primary Okazaki fragments. The matrix-associated replicating intermediates (4-50 S) are effectively chased into DNA of replicon size and larger (100 S) following a 1-h pulse. DNA-rich nuclear matrices may therefore provide a useful in vitro system for studying DNA replication in correlation with the higher order, intranuclear arrangement of eukaryotic DNA.
TL;DR: Measurement of total cell protein and cell volumes during culture indicated that a relationship existed between these parameters and the initiation of mitosis, and the significance of these observations is discussed.
Abstract: A microfluorimetric procedure, employing the fluorescent stain 33258 Hoechst, has been developed for the investigation of the process of DNA synthesis during the initial stages of culture of tobacco (N. tabacum cv. Xanthi) leaf protoplasts.
In this system, the freshly-isolated protoplasts exhibited a unimodal distribution of nuclear DNA content characteristic of the diploid state. The almost immediate onset of DNA synthesis during culture resulted in a doubling of nuclear DNA levels prior to the first mitoses. Although the majority of the protoplasts subsequently entered into synchronous mitosis and cell division, a proportion of the remainder developed into large polyploid cells. Upon further culture, the polyploid cells became subdivided into clusters of small diploid cells. Measurement of total cell protein and cell volumes during culture indicated that a relationship existed between these parameters and the initiation of mitosis. The significance of these observations is discussed.
TL;DR: It is concluded that the undermethylation found in the tumor genomes of this system is not due to inactivation nor a significant deficiency of the activity of this enzyme relative to the demand in tumors for methylation of de novo synthesized DNA.
Abstract: The status of DNA methylation, as measured by the 5-methylcytosine content of nuclear DNA, was examined in normal livers and in chemically induced or spontaneous primary hepatocellular carcinoma (PHC) arising in three strains of mice. The DNA from spontaneous tumors of genetic origin in C3H mice and also from acetylaminofluorene, chlordane, or 3'-methyl-4-dimethylaminoazobenzene-induced tumors in C57Bl and B6C3 mice was undermethylated compared to the levels in background and normal liver samples. The DNA methylase activities from normal liver, background liver, and PHC were assayed in C3H mice to determine whether the observed genomic undermethylation is related to a dysfunction of this enzyme and were compared to the rates of DNA synthesis in these tissues. Since DNA methylase levels from tumor nuclei were elevated compared to background, it is concluded that the undermethylation found in the tumor genomes of this system is not due to inactivation nor a significant deficiency of the activity of this enzyme relative to the demand in tumors for methylation of de novo synthesized DNA.
TL;DR: It is concluded that extensive DNA rearrangement has taken place during amplification in methotrexate-resistant mouse lymphoma EL4 cells and mouse melanoma PG19 cells.
TL;DR: The structure of adenovirus deoxyribonucleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococcal nuclease was investigated and a repeating unit which was distinctly different from the cellular nucleosomal repeat was identified.
Abstract: We investigated the structure of adenovirus deoxyribonucleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococcal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parenteral DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with [3H]thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure we identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. We found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.
TL;DR: The known hepatocarcinogenicity of clofibrate cannot be based upon an initiating, DNA damaging, mode of action but must be due to other, nongenotoxic, mechanisms such as peroxisome proliferation, hepatomegaly, or cytotoxicity due to protein binding.
TL;DR: The present results for centromeres provide probably the first example to indicate that variation in the total DNA content of small segments present on each chromosome sometimes varies directly in proportion to large interspecific variation in nuclear DNA C-value.
Abstract: The total volume of centromeres per nucleus varies widely within Triticum aestivum cv. Chinese Spring (7-fold) and between 11 higher plant species (24-fold). Such variation is closely correlated with nuclear DNA content, nuclear volume and, to a lesser extent, the total volume of nucleoli per nucleus. Centromere volume reflects minor intraspecific developmental fluctuations in nuclear size independent of variation in nuclear DNA content, but variation in nuclear DNA plays the major role in determining centromere volume. Thus, in general a given total volume of centromeric material is apparently characteristic of an approximately constant nuclear volume and mass of nuclear DNA, but largely independent of chromosome number. The range of volume of single centromeres in 4 taxa corresponds with the ranges of their single chromosome lengths or chromosome DNA contents. The centromere is, therefore, not a unit structure of constant size and mass but a chromosome segment whose highly variable volume closely reflects the volume and mass of the chromosome to which it belongs. The correlation between centromere size and chromosome size and DNA content is potentially useful for identifying single centromeres in unsquashed interphase and dividing nuclei; thereby facilitating studies of the intranuclear disposition of chromosomes. The present results for centromeres provide probably the first example to indicate that variation in the total DNA content of small segments present on each chromosome sometimes varies directly in proportion to large interspecific variation in nuclear DNA C-value. The close correlation between centromere volume, and nuclear DNA content is probably nucleotypic in origin. The functional significance of the variation in centromere volume is unknown, as is the nature of the mechanism which determines that centromere volume closely reflects nuclear and chromosome size and mass.
Journal Article•
TL;DR: Analysis of nuclear DNA in squamous cell carcinomas showed in most tumours a peak value near 2c (diploid) or 3 c (aneuploid), and a wide distribution of other nuclear DNA values, which may prove useful as adjuncts in the diagnosis and in selection of treatment.
Abstract: Cytophotometric analysis of nuclear DNA in 30 squamous cell carcinomas showed in most tumours a peak value near 2c (diploid) or 3 c (aneuploid), and a wide distribution of other nuclear DNA values. A follow-up study showed that the prognosis was best for patients with diploid tumour cells and worst for patients with aneuploid tumour cells. The DNA histogram patterns give information about the dominating DNA ploidy of the tumour cells; in some tumours information about the tumour proliferation activity is also provided. These data are useful in prognostic evaluation and may prove useful as adjuncts in the diagnosis and in selection of treatment.
TL;DR: It is shown that nuclear matrices from bovine cells are in fact enriched in satellite DNA, and the satellite DNAs of bovian cells meet these criteria.
Abstract: The skeletal framework of the eukaryotic nucleus is a protein matrix1,2 which maintains the physical shape of the nucleus. Attached to the matrix is a fraction of DNA resistant to deoxyribonuclease I (DNase I) digestion. Ribosomal DNA sequences also are enriched on nuclear matrices from rat liver3. The question thus arises whether other repeated DNAs are preferentially attached to the nuclear matrix. This question could be resolved if the repeated DNA was available in sufficient quantity for biochemical studies and differed in its physical or sequence characteristics from the rest of the nuclear DNA. The satellite DNAs of bovine cells meet these criteria. Here we show that nuclear matrices from bovine cells are in fact enriched in satellite DNA.
TL;DR: Experiments with the DNA fluorochrome Hoechst dye 33258 (bisbenzimide) demonstrate that plastids isolated from all phases of cell growth each possess a ring-shaped nucleoid containing detectable DNA.
Abstract: The kinetic complexity of chloroplast DNA isolated from the chromophytic alga Olisthodiscus luteus has been determined. Using optical reassociation techniques, it was shown that the plastid DNA of this alga reacted as a single component with a second order rate constant of 4.1 molar−1 and second−1 (Cot½ 0.24 molar second) under conditions equivalent to 180 millimolar Na+ and 60°C. Given the 92 × 105 dalton complexity calculated for this chloroplast genome, an Olisthodiscus cell contains 650 plastome copies. Although this complement remains constant throughout the growth cycle of the organism, the ploidy level of an individual chloroplast shows significant plasticity and is dependent upon the number of chloroplasts present per cell. Experiments with the DNA fluorochrome Hoechst dye 33258 (bisbenzimide) demonstrate that plastids isolated from all phases of cell growth each possess a ring-shaped nucleoid containing detectable DNA. Olisthodiscus chloroplast DNA showed no sequence mismatch when thermal denaturation profiles of reassociated chloroplast DNA were examined, thus all plastome copies are essentially identical. Finally, reassociation studies demonstrated that no foldback (short inverted repeat) sequences were present in the plastid genome although significant hairpin loop structures were observed in control nuclear DNA samples.
TL;DR: Comparisons of the 'fingerprint' patterns of total Physarum DNA and isolated M+ DNA after digestion using different restriction endonucleases and probing for the presence of specific repetitive sequences in Southern blots of M+ and M- DNA indicate that many repetitive sequences are shared by both compartments, though some repetitive sequences appear to be considerably enriched, or are present exclusively, either in M+.
Abstract: Nuclear DNA from the slime mould Physarum polycephalum is digested by the restriction endonuclease HpaII to generate a high molecular weight and a low molecular weight component. These are referred to as the M+ and the M- compartment, respectively. Sequences that are present in the M+ compartment are cleaved by MspI, the restriction enzyme isoschizomer of HpaII, thus showing that the recognition sequences for these enzymes in M+ DNA contain methylated CpG doublets. The distribution of repetitive sequences in the M+ and M- DNA compartments was investigated by comparison of the 'fingerprint' patterns of total Physarum DNA and isolated M+ DNA after digestion using different restriction endonucleases, and by probing for the presence of specific repetitive sequences in Southern blots of M+ and M- DNA by the use of cloned DNA segments. Both types of experiment indicate that many repetitive sequences are shared by both compartments, though some repetitive sequences appear to be considerably enriched, or are present exclusively, either in M+ DNA or in M- DNA.
TL;DR: The decline in mitochondrial DNA content occurred earlier and proceeded faster than it did for the longer-lived Oregon R strain, and the buoyant density of nuclear DNA was 1.693 and the mitochondrial 1.680, with no age-related changes.
Abstract: Mitochondrial and nuclear DNA were quantitated in cesium chloride equilibrium density gradients for the Swedish C strain of Drosophila melanogaster Total nuclear DNA per fly remained unchanged between 7 and 43 days of age, whereas the mitochondrial DNA content decreased dramatically reaching a value of zero at 43 days of age The decline in mitochondrial DNA content occurred earlier and proceeded faster than it did for the longer-lived Oregon R strain The buoyant density of nuclear DNA was 1693 and the mitochondrial 1680, with no age-related changes
TL;DR: A comparison of the total nuclear DNA reassociation kinetics of the two species indicates a reduction of lowly repetitive sequences in M. m.
Abstract: The extent of nuclear single-copy DNA divergence between Muntiacus reevesi and Muntiacus muntjak vaginalis (Cervidae), a species pair showing extreme karyotype differences but striking morphological similarity, is 2%, as judged from the thermal stability of interspecific DNA-DNA hybrids. A comparison of the total nuclear DNA reassociation kinetics of the two species indicates a reduction of lowly repetitive sequences in M. m. vaginalis.
TL;DR: Comparison of the amounts of known infective units by animal assay indicated that CJ specific DNAs should be detectable in these fractions with the methods used, and results appear to rule out usual forms of double-stranded or partially double- Stranded DNAs as components of transmissible encephalopathy agents.
TL;DR: Results establish that in u.v.-irradiated HeLa cells most of the DNA repair synthesis is not due to DNA polymerase alpha, but they do not preclude a significant role for this enzyme in DNA repair processes.
Abstract: A radioautographic examination of nuclear DNA synthesis in unirradiated and u.v.-irradiated HeLa cells, in the presence and in the absence of aphidicolin, showed that aphidicolin inhibits nuclear DNA replication and has no detectable effect on DNA repair synthesis. Although the results establish that in u.v.-irradiated HeLa cells most of the DNA repair synthesis is not due to DNA polymerase alpha, they do not preclude a significant role for this enzyme in DNA repair processes.
TL;DR: The results indicate that neither PSTV nor its complementary strand is transcribed from nuclear DNA but do not rule out the possibility of sequence homology between host DNA and a small portion of PSTV or its complement.
Abstract: High molecular weight tomato nuclear DNA was isolated from uninfected and potato spindle tuber viroid (PSTV)-infected tomato leaves. Restriction digests were fractionated on agarose gels, denatured and transferred to diazobenzyloxymethylpaper, and hybridized to 32P-labeled cloned double-stranded PSTV cDNA. No hybridization to DNA from either uninfected or infected tissue could be detected under conditions that permitted detection of cloned double-stranded PSTV cDNA at a concentration equivalent to one-fifth copy of PSTV-related DNA per haploid tomato genome. Hybridization of tomato DNA to 32P-labeled cloned soybean 18S and 28S ribosomal DNA sequences showed that the restricted nuclear DNA was suitable for hybridization to probes containing homologous sequences. Our results indicate that neither PSTV nor its complementary strand is transcribed from nuclear DNA but do not rule out the possibility of sequence homology between host DNA and a small portion of PSTV or its complement.
TL;DR: Reassociation kinetics analysis has shown that 440-nucleotide DNA fragments from the genome of this alga contain 4% foldback, 58% repetitive, and 34% single-copy sequences.
Abstract: Nuclear DNA of the marine chromophytic alga Olisthodiscus luteus was analyzed in this study. Reassociation kinetics analysis has shown that 440-nucleotide DNA fragments from the genome of this alga contain 4% foldback, 58% repetitive, and 34% single-copy sequences. Precise analysis using isolated single-copy DNA revealed that Olisthodiscus has a large haploid DNA content of 1.66 x 10(-12) g/cell. For determination of the organization of single-copy and repetitive sequences within this genome, DNA fragments 3000 nucleotides in length were reassociated to C0t= 100 M . s. At this low C0t value 89% of the DNA bound to hydroxylapatite, suggesting that single-copy and repetitive elements are interspersed. The lengths of the duplexed repetitive DNA on these 3000-nucleotide fragments were measured by electron microscopy after digestion with S1 nuclease which removed the unreassociated single-copy DNA regions. Of these repetitive sequences, 68% were shorter than 1200 nucleotide pairs in length and had a modal length of 350 nucleotide pairs. A minor class of longer (to 4000 nucleotide pairs) repetitive sequences was also observed.
TL;DR: This chapter discusses the replication of the chloroplast DNA in vivo and explains the mechanism by which both DNAs replicate by a semiconservative mechanism.
Abstract: Publisher Summary Chlamydomonas reinhardtii occupies a central position in cytoplasmic inheritance because it is the simplest organism that contains a chloroplast and non-Mendelian inheritance. Being unicellular and heterothallic, Chlamydomonas can be handled in the laboratory with the same ease as bacteria and yeast. The vegetative, haploid life cycle and the diploid meiotic sexual cycle can be physiologically controlled in large synchronous populations to facilitate either cell cycle biochemistry or genetic analysis. This chapter discusses the replication of the chloroplast DNA in vivo. Chloroplast replication usually is initiated 1–3 hours after the onset of the light period and continues for six hours, whereas nuclear DNA synthesis is initiated 2–3 hours into the dark period and continues for six hours. If cell division results in the production of four daughters, then two rounds of replication of chloroplast DNA synthesis precede two rounds of nuclear DNA synthesis. Both DNAs replicate by a semiconservative mechanism. Chlamydomonas does not appear to have dark repair reactions, so the chloroplast chromosome is stable over the entire cell cycle
TL;DR: Preliminary results of electron microscopy and sucrose gradient centrifugation indicate that nuclear shell DNA accounts for 0.8–1% of total nuclear DNA, and is presumably constrained in highly stable supranucleosomes by a specific protein environment.