Showing papers on "Nuclear DNA published in 1980"
TL;DR: It is shown that after depleting interphase nuclei of histones and other nuclear proteins by treatment with nonionic detergent and high salt, intact loops of DNA can be visualized as a halo surrounding a nuclear skeleton or matrix, suggesting that the replicating DNA loops are motile with respect to their nuclear matrix anchorage sites.
605 citations
TL;DR: It is proposed that, upon transformation, the host cell ligates incorporated DNA into a large concatameric structure which may at times be as large as 2000 kilobases and from blotting data alone the authors cannot determine whether this structure is chromosomal or extrachromosomal in location.
415 citations
TL;DR: There is no firm evidence for any higher degrees of order that might bring specific functioning genes into close proximity to the nucleus, but in man and other animals the most obvious consequence is satellite association.
Abstract: The factors responsible for producing some degree of order to the arrangement of chromatin in the nucleus are reviewed. They are the following: 1. Chromosomes are attached to the nuclear membrane, nucleolus and intranuclear matrix. As a result they have a relatively fixed position in the nucleus. 2. In some species somatic pairing results in alignment of homologs. This is rare in mammals. 3. The association of ribosomal DNA and 5S DNA with the nucleolous results in the close approximation of the chromosomes carrying these DNA sequences. In man and other animals the most obvious consequence is satellite association. 4. Heterochromatin is condensed onto the inner nuclear membrane and periphery of the nucleolous while genetically active chromatin occupies the more central portion of the nucleus. The results is a peripheral location of late replicating DNA and a central location of early relicating DNA. 5. The DNA replication points tend to be associated with the nuclear matrix. Autoradiography of briefly labelled cells shows a high frequency of grains associated with nuclear matrix material. 6. Heterochromatin association results in chromocenters and ectopic pairing. 7. In addition to all these is the Rabl orientation or alignment of centromeres with centromeres and telomeres with telomeres. This polarization of the chromosomes results from the traction on the centromeres by the spindle fibers. There is no firm evidence for any higher degrees of order that might bring specific functioning genes into close proximity.
272 citations
TL;DR: Examination of subcellular DNA fractions from the crown gall teratoma tumor line BT37 concludes that T-DNA is located not in chloroplasts or mitochondria but rather in the nuclear fraction of this tumor line.
Abstract: The crown gall teratoma tumor line BT37, incited by Agrobacterium tumefaciens strain T37, has been found to contain part of the tumor-inducing plasmid, pTi T37, of the inciting strain. This foreign DNA segment, called T-DNA, is maintained at several copies per diploid tumor cell. We have examined subcellular DNA fractions from this tumor line in an effort to determine whether T-DNA is in chloroplasts, mitochondria, or nuclei. Tumor cell chloroplast DNA exhibited EcoRI and Bst I endonuclease cleavage patterns identical to those of normal tobacco chloroplast DNA. Tumor cell mitochondrial DNA exhibited a complex Bst I cleavage pattern that did not differ detectably from that of normal tobacco mitochondrial DNA. Southern blots of tumor chloroplast and mitochondrial cleavage products did not hybridize with labeled pTi T37 DNA, whereas blots of tumor cell nuclear DNA cleavage products hybridized strongly. We conclude that T-DNA is located not in chloroplasts or mitochondria but rather in the nuclear fraction of this tumor line.
248 citations
TL;DR: Evidence is presented that this reflects the lipophilic character of the derivative and the very high ratio of lipid to DNA in mitochondria, which suggests that mitochondrial DNA may be an important cellular target of chemical carcinogens.
Abstract: When mammalian cell cultures are exposed for 2 hours to (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, a mutagenic and carcinogenic derivative of benzo[a]pyrene, the extent of covalent modificationof mitochondrial DNA is 40 to 90 times greater than that of nuclear DNA. Evidence is presented that this reflects the lipophilic character of the derivative and the very high ratio of lipid to DNA in mitochondria. These results suggest that mitochondrial DNA may be an important cellular target of chemical carcinogens.
229 citations
TL;DR: Cloned DNA complementary to the messenger RNAs for four immunologically distinct VSGs are cloned and hybridised these complementary DNAs with restriction digests of T. brucei nuclear DNA and infer that activation of a VSG gene involves the production of an expression-linked copy of that gene.
Abstract: Pathogenic African trypanosomes evade the immune system of their mammalian hosts by the sequential expression of alternative cell-surface glycoproteins (reviewed in refs 1,2). Variant surface glycoproteins (VSGs) purified from cloned variants of Trypanosoma brucei have similar molecular weights (about 60,000), but differ in amino acid composition, N-terminal amino acid sequence and C-terminal structure. We have cloned DNA complementary to the messenger RNA's for four immunologically distinct VSGs and hybridised these complementary DNAs (cDNAs) with restriction digests of T. brucei nuclear DNA, fractionated by gel electrophoresis and transferred to nitrocellulose strips. Each cDNA recognises a unique set of fragments and this basic set is present unaltered in the nuclear DNAs from the four variants. In addition, each probe recognises an extra fragment only in nuclear DNA isolated from cells expressing the VSG corresponding to the cDNA probe. We infer that activation of a VSG gene involves the production of an expression-linked copy of that gene.
195 citations
TL;DR: With six polycyclic aromatic compounds, all of which require metabolic activation and bind to DNA to a much smaller extent than direct-acting alkylating agents, the binding to mitochondrial relative to DNA is dramatically increased by a factor of nearly 50 to over 500.
Abstract: Since the pioneering work of the Millers it has become clear that most chemical carcinogens require metabolism to reactive electrophiles and then exhibit their carcinogenic potential by reacting chemically with, and modifying, cellular macromolecules. At first modification of proteins was considered most likely to be of importance in carcinogenesis. Later, Brookes and Lawley demonstrated that the extent of binding of several polycyclic hydrocarbons to DNA, but not to RNA or protein isolated from the skin of mice treated topically with these compounds, correlated with their known carcinogenic potency to this tissue. Mammalian cells, particularly mouse embryo cells, treated with chemical carcinogens have often been used, and DNA has been involved almost exclusively from whole cells. However, mitochondria possess unique DNA which accounts for 0.1-1% of the total DNA present in mammalian cells, and three studies have shown that carcinogenic alkylating agents modify the michondrial DNA by a factor about five times greater than the nuclear DNA from the same cells. We demonstrate here that with six polycyclic aromatic compounds, all of which require metabolic activation and bind to DNA to a much smaller extent than direct than direct-acting alkylating agents, the binding to mitochondrial relative to DNA is dramatically increased by a factor of nearly 50 to over 500.
192 citations
TL;DR: Following digestion with the restriction endonuclease, EcoRI, cages and associated DNA were filtered free of unattached DNA and a model, based on the premise that replication takes place at the nuclear cage, is presented.
Abstract: Structures resembling nuclei are released when HeLa cells are lysed in a detergent and 2 M salt. These nucleoids, which lack any organized membrane structure, contain all the nuclear DNA packaged within a cage of RNA and protein. Their DNA is supercoiled so that the linear DNA must remain unbroken and looped during lysis. Following digestion with the restriction endonuclease, EcoRI, cages and associated DNA were filtered free of unattached DNA. Pulse-labelled (i.e. newly synthesized) DNA remains preferentially associated with the cages. This association has been confirmed by autoradiography. When nucleoids are prepared for electron microscopy by the Kleinschmidt procedure the DNA spills out to form a skirt around the flattened cage. Labelling, which is restricted to the region of the cage after short pulses, extends out into the skirt as the labelling time increases. A model, based on the premise that replication takes place at the nuclear cage, is presented in the Appendix. The results of the biochemical experiments and electron microscopy both indicate that the average size of the unit of replication is approximately 2 micrometer. This is about one-quarter the size of the average structural unit - the loop. Therefore sequences in the loop must become attached to the nuclear cage prior to the initiation of DNA synthesis.
182 citations
TL;DR: The present work presents a meta-analysis of yeast chromosomol genes that highlights the importance of knowing the carrier and removal status of canine coronavirus, as a source of infection for other animals.
Abstract: PROSPECTIVES AND SUMMARY 845 GENETIC ANALYSIS IN yEAST 846 Tetrad Analysis 846 New Advances in Genetic Analysis 850 Construction of recombinant DNA molecules 850 Identification of cloned genes 850 Yeast transformation 854 ORGANIZATION AND REPLICATION OF THE YEAST CHROMOSOMES 856 Chromosome Structure 858 Chromosome Replication 860 Structure of replicating nuclear DNA molecules 860 Control of DNA synthesis 862 Yeast DNA polymerases .. ... ... ........ ... ..... 864 ANALYSIS OF SPECIFIC CLASSES OF CHROMOSOMAL GENES 864 Structural Analysis of Single-Copy Genes 865 Repeating Chromosomal Genes 866 Yeast ribosomal DNA genes 866 Structure of yeast tRNA genes 867 Other repeating yeast chromosomol genes 868 STRUCTURE AND REPLICATION OF THE 2p.m PLASMIDS 869 CONCLUSIONS AND FUTURE PROSPECTS 871
142 citations
TL;DR: A procedure is presented for the rapid isolation of mitochondrial DNA (mtDNA) in high yield from Saccharomyces cerevisiae by a single cycle of bisbenzimide-CsCl buoyant density centrifugation.
128 citations
TL;DR: The isolation by molecular cloning of a ‘border fragment’ T- DNA and flanking plant DNA from the crown gall teratoma BT37 is reported and it is shown that T-DNA is covalently joined to a repeated DNA element of the tobacco nuclear genome.
Abstract: Agrobacterium tumefaciens strains containing tumour-inducing (Ti) plasmids1–3 incite cancerous growths called crown galls when inoculated into wounded dicotyledonous plants. Tumour tissue can be cultured axenically in vitro, and exhibits a transformed phenotype in the absence of the inciting bacterium. Transformed cells grow autonomously, are auxin and cytokinin autotrophic in vitro4 and synthesize opines5–8, novel amino acid derivatives dictated by Ti plasmid genetic information9–11. A small segment of the Ti plasmid, termed T-DNA is maintained in axenic tumour cells12–19. Mitochondrial and chloroplast DNA from a crown gall teratoma are free from T-DNA, whereas nuclear DNA contains T-DNA in amounts similar to that in total tumour cell DNA20,21. T-DNA appears to be attached to what is presumably plant DNA in the crown gall tumour cell: Southern blot analysis22 of tumour DNA digested with restriction endonucleases reveals T-DNA fragments that are not fully homologous to Ti plasmid DNA14,17,21. We report here the isolation by molecular cloning of a ‘border fragment’ T-DNA and flanking plant DNA from the crown gall teratoma BT37 and show that T-DNA is covalently joined to a repeated DNA element of the tobacco nuclear genome.
TL;DR: In this paper, a quantitative application of the Southern blotting technique was used to find that residual DNA from SV40 infected 3T3 cells were enriched in SV40 sequences, indicating that they reside near matrix-DNA attachment points.
Abstract: Recent studies indicate that eukaryotic DNA is organized into supercoiled loop domains. These loops appear to be anchored at their bases to an insoluble nuclear skeleton or matrix. Most of the DNA in the loops can be released from the matrix by nuclease digestion; the residual DNA remaining with the nuclear matrix represents sequences at the base of the loops, and possibly other sequences which are intimately associated with the nuclear matrix for other reasons. Using a quantitative application of the Southern blotting technique, we have found this residual DNA from SV40 infected 3T3 cells to be enriched in SV40 sequences, indicating that they reside near matrix-DNA attachment points. An enrichment of 3-7 fold relative to total cellular DNA, was found in each of three different lines of SV40 infected 3T3 cells. Control experiments with globin genes showed no such enrichment in this residual matrix DNA. This sequence specificity suggests that the spatial organization of DNA sequences within loops may be related to the functionality of these sequences within the cell.
TL;DR: The specificity of inhibition and structure of the drug suggest that aphidicolin must recognize a site common only to the replicative DNA polymerases of eucaryotes and different from the binding site for deoxyribonucleic triphosphates and DNA, which should be similar in reparative and procaryote-type DNA polymerase.
Abstract: The inhibition in vitro of herpes simplex virus 1 and vaccinia virus DNA polymerases by aphidicolin is primarily noncompetitive with dGTP, dATP, dTTP, DNA, and Mg2+ and competitive with dCTP in analogy with the mode of inhibition of cellular alpha-polymerase. The degree of inhibition of viral or cellular growth in vivo can be quantitatively predicted by the degree of inhibition of the isolated replicative DNA polymerases at the same concentration of aphidicolin in suitable conditions (limiting dCTP concentration). Thus, the only in vivo target for aphidicolin is probably the replicative DNA polymerase, and aphidicolin is a highly specific inhibitor of replicative nuclear DNA synthesis in eucaryotes. This, coupled with the lack of mutagenic effect, represents a valuable property for an anticancer drug. The specificity of inhibition (contrary to the aspecific effect on almost all DNA polymerases by a true competitive inhibitor, such as 1-beta-D-arabinofuranosylcytidine 5'-triphosphate) and the structure of the drug, which does not resemble that of the triphosphates, suggest that aphidicolin must recognize a site common only to the replicative DNA polymerases of eucaryotes and different from the binding site for deoxyribonucleic triphosphates and DNA, which should be similar in reparative and procaryote-type DNA polymerase; the aphidicolin binding site is probably very near to, or even overlaping with, the binding site for dCTP so that the drug mimics a competitive effect with this nucleotide.
TL;DR: A precise technique to measure DNA content in Microseris douglasii is reported, and differences among individual plant specimens in the 2.5%-5% range are detectable with this procedure.
Abstract: A precise technique to measure DNA content in Microseris douglasii is reported. Relative absorbancy (560 nm) of Feulgen-stained spherical nuclei, arrested at the 2C DNA level, was measured microspectrophotometrically. Relative DNA contents were determined by placing epidermis of an inbred line on the same slide with epidermis from the unknown sample prior to hydrolysis and staining. The DNA values were adjusted to the internal standard; direct comparisons were made of values collected from different slides and staining batches. Differences among individual plant specimens in the 2.5%-5% range are detectable with this procedure. Intraspecific variation up to 20% in DNA content was apparent
TL;DR: The ability to map these genes implies that sequences cannot be attached at random to the cage; rather, specific sequences must be attached, so looping the DNA.
Abstract: Nuclear DNA is organised into loops, probably by attachment to a supramolecular structure. We describe a method which enables us to map the position of sequences within a loop relative to the point of attachment. Nuclear DNA is isolated unbroken by lysing HeLa cells in 2M NaCl to release structures which retain many of the morphological features of nuclei. Their DNA is supercoiled and so must remain unbroken and looped during lysis. Nucleoids are digested to various degrees with a restriction endonuclease and the cages - and any associated DNA - sedimented free from unattached DNA. The cage-associated DNA is purified and completely fragmented using the same restriction endonuclease. Equal weights of fragmented DNA are separated by gel electrophoresis, transferred to a filter and the relative amounts of the alpha, beta and gamma globin genes on the filter determined by hybridisation to the appropriate probes. The alpha genes, unlike the beta and gamma genes, resist detachment from the cage and so must lie close to the point of attachment to the cage. Our ability to map these genes implies that sequences cannot be attached at random to the cage; rather, specific sequences must be attached, so looping the DNA.
TL;DR: Using 125I-labeled ribosomal RNA (rRNA) as a probe, it is found that the genes for rRNA are significantly enriched in the residual matrix DNA, indicating that matrix DNA does not represent any specific sequence class.
Journal Article•
TL;DR: No evidence was obtained for repair-induced long-range nucleosomal movement in normal fibro Blasts or constitutive movement in the absence of excision repair in xeroderma pigmentosum fibroblasts, and the initial differences in adduct concentration between linker DNA and core DNA diminished during posttreatment incubation.
Abstract: The excision and persistence of covalent DNA adducts formed by N -acetoxy-2-acetylaminofluorene (AAAF) were studied in human skin fibroblasts. The changes in adduct concentration as a function of posttreatment incubation were measured in purified nucleosomal core DNA and in total nuclear DNA, and from these data the changes in adduct concentration in nucleosomal linker DNA were calculated. Immediately after N -acetoxy-2-acetylaminofluorene treatment which introduced 20 to 38 µmol adducts per mol DNA-P, the adduct concentration was 4 to 5 times higher in linker DNA than in core DNA. Adduct removal was rapid during the first 8 hr of incubation and occurred 3.5 to 4 times more efficiently from linker DNA than from core DNA. After 24 hr incubation, adduct removal continued only at a very low rate, leaving a substantial fraction of adducts unexcised. This fraction of persistent adducts had a value of 0.5 and was independent of the initial adduct concentration in the range of 12 to 115 µmol adducts per mol DNA-P. Approximately 65% of the persisting adducts were located in nucleosomal cores. The initial differences in adduct concentration between linker DNA and core DNA diminished during posttreatment incubation. This was entirely due to preferential early adduct excision from linker DNA. No evidence was obtained for repair-induced long-range nucleosomal movement in normal fibroblasts or constitutive movement in the absence of excision repair in xeroderma pigmentosum fibroblasts. Nucleosomal movement would tend to diminish the concentration differences between linker DNA and core DNA.
TL;DR: The number of nuclear pores appears to be associated with some aspect of nuclear metabolic activity, e.g., transcriptional capacity or release of products to the cytoplasm, and further evidence for such a view comes from studies of chick embryo erythroblasts.
Abstract: To assess the functional significance of nuclear pore complexes, we have investigated whether the number of pores per nucleus is determined by such factors as the nuclear volume, nuclear surface area, DNA content, or aspects of nuclear activity. Comparisons were made between cell types chosen to permit observation of differences in nuclear pore number as a function of differences in the other qualities measured. The number of nuclear pores was determined by freeze-etching and measurements of nuclear surface and nuclear volume by electron and light microscopy. Pairs of cell strains in culture that contained different numbers of chromosome sets were investigated to examine the relation of pore number to total DNA content. Tetraploid cells of the rat kangaroo (Potorous tridactylus) have almost exactly twice the number of pores found in the parental diploid strain. However, the pore number in diploid grassfrog (Rana pipiens) cells was only 65% greater than in the parental haploid cells. In addition, a polyploid series of nucleated RBC had a 62% pore number increase with each successive increase in ploidy. Diploid cell strains from the canyon mouse (Peromyscus crinitus) and from the cactus mouse (P. eremicus) were compared to test whether a difference reflecting the 36% additional DNA in cells of the latter, associated with extra heterochromatin, existed. Although both were found to have the same number of pores and nuclear surface area, the cells differed in nuclear volume. These observations suggest that the number of nuclear pores is independent of the total amount of nuclear DNA, the nuclear surface area (and, thus, presumably the fraction of DNA that is bound to the nuclear membrane), the nuclear volume, and the size of the genome. Rather, the number of nuclear pores appears to be associated with some aspect of nuclear metabolic activity, e.g., transcriptional capacity or release of products to the cytoplasm. Further evidence for such a view comes from studies of chick embryo erythroblasts. In these, nuclear pore number was found to be lower in associated with the decreasing nuclear transcriptional activity and longer generation times that characterize the successive cell divisions leading to the fully differentiated state. The number of pore complexes reconstructed in the last cell cycles declined in a manner consistent with reutilization of previously formed pores in the absence of new pore synthesis. Challenging this interpretation is the increase in pore number at lower metabolic activity when Xenopus laevis cells are grown at different temperatures. The speculation that pore complexes have a longer half-life in cooler grown Xenopus cells could resolve the discrepancy.
TL;DR: The evidence shows that, among related species, the DNA lost or gained during divergence is of a remarkably consistent and restricted composition in respect of the ratio of repetitive to non-repetitive base sequences.
Abstract: Speciation in eukaryotes is often accompanied by massive changes in nuclear DNA amount resulting from the accumulation or deletion of DNA base sequences within chromosomes. Our evidence shows that, among related species, the DNA lost or gained during divergence is of a remarkably consistent and restricted composition in respect of the ratio of repetitive to non-repetitive base sequences. It is argued that the restriction may in part at least be imposed by natural selection, in the sense that the only changes tolerable are confined to DNA fractions of particular composition and organisation.
TL;DR: By using this method, a heavily methylated fragment was identified in nuclear DNA fom Chinese hamster cells, the methylation pattern of calf thymus satellite I DNA was examined, and chloroplast DNAs that were extracted from various stage of the Chlamydomonas life cycle were compared.
Abstract: A method to identify 5-methylcytosine (m5Cyt) in DNA immobilized on nitrocellulose paper by using antibody against m5Cyt raised in rabbits is described. Immobilized restriction fragments of DNA are incubated first with purified antibody against m5Cyt and then with goat anti-rabbit IgG labeled with 125I. Restriction fragments containing m5Cyt are visualized by autoradiography. By using this method, a heavily methylated fragment of about 1700 base pairs was identified in nuclear DNA fom Chinese hamster cells, the methylation pattern of calf thymus satellite I DNA was examined, and chloroplast DNAs that were extracted from various stage of the Chlamydomonas life cycle were compared. Little if any methylation was detected in chloroplast DNA from vegetative cells or from male gametes, whereas homologous DNAs from female gametes and from zygotes were heavily methylated. The sensitivity of the method was examined with calf thymus satellite I DNA (which contains approximately 40 m5Cyt residues per repeat unit of 1400 base pairs) and with phi X174 virion DNA (which contains a single m5Cyt per molecule). The presence of m5Cyt was detected with as little as 40 ng of phi X174 DNA containing 0.02 pmol of m5Cyt and with 100 ng of satellite DNA containing about 0.5 pmol of m5Cyt. Thus, the method makes possible the identification of individual methylated sites in purified DNAs in the size range of single genes.
TL;DR: Results indicate that receptor-EGF interaction at the cell surface leads to the intracellular generation of protein that are capable of stimulating quiescent nuclei into activity.
Abstract: This paper explores the pathway from nuclear quiescence to mitogenesis. It describes an in vitro assay for an activator of DNA replication induced by epidermal growth factor (EGF) in responsive cells. Cytoplasmic extracts from EGF-treated 3T3 cells were found to contain substances that can stimulate DNA synthesis in isolated nuclei from spleen cells of adult frogs. Extracts from untreated resting 3T3 cells lack this activity, and EGF itself is incapable of stimulating DNA synthesis in these cell-free systems. The extract-induced stimulation of incorporation of [3H]dTTP into nuclear DNA is ATP dependent and requires the presence of the four deoxyribonucleoside triphosphates, suggesting the occurrence of replication rather than repair synthesis. This cell-free assay has been used to obtain some initial insights into the mechanism of induction and biochemical characterization of the intermediate in EGF action. Half-maximal induction of the active intracellular substance is achieved at about 0.08 nM EGF, a concentration that correlates well with the concentration required for half-maximal mitogenesis. Studies on the biochemical characteristics of this active substance strongly suggest that the activity is associated with a protein. The activity is nondialyzable and sensitive to trypsin and heat. Sucrose gradient centrifugation of the extract revealed three peaks of activity with molecular weights of 46,000, 110,000, and 270,000 (sedimentation coefficients: 3.7 S, 6.6 S, and 12 S, respectively). These results indicate that receptor-EGF interaction at the cell surface leads to the intracellular generation of protein that are capable of stimulating quiescent nuclei into activity.
TL;DR: It is concluded that mild micrococcal-nuclease treatment of nuclei causes their separation into transcribed and non-transcribed DNA as determined by a number of very different criteria.
Abstract: Extremely mild treatment with micrococcal nuclease of isolated nuclei yields subnuclear fractions in which the majority of RNA polymerase II transcriptional complexes formed in vivo are segregated [Tata & Baker (1978) J. Mol. Biol. 118, 249-272]. We now describe different approaches followed to established whether or not the nuclei are thus resolved into transcribed and non-transcribed DNA. First, we have compared the sensitivity to deoxyribonuclease I, which is known to digest preferably expressed genes as present in nuclei or chromatin, of three micrococcal-nuclease-derived fractions from nuclei of different transcriptional activities. In transcriptionally active nuclei (rat liver, hen liver and oviduct, and Xenopus liver), the DNA in a polynucleosomal fraction comprising 6-15% of DNA and the majority of template-engaged RNA polymerase II (fraction P2) was 10-50 times as sensitive to deoxyribonuclease I as the DNA in the other two fractions (fractions P1 and S, comprising 78-88% of total nuclear DNA as large polynucleosomal aggregates and 2-6% of DNA mostly as mononucleosomes, respectively). In transcriptionally inactive nuclei obtained from hen erythrocytes, micrococcal nuclease did not separate DNA into fractions exhibiting such differential sensitivities. Second, we have monitored the partition of an expressed gene. Hybridization of complementary DNA to Xenopus albumin mRNA revealed a 5-10-fold enrichment of the albumin (but not the globin) gene in the P2 fraction of nuclei from Xenopus liver in which this gene is fully expressed. Third, a large part of the nascent rapidly labelled RNA synthesized in vivo in rat liver nuclei was recovered in the micrococcal-nuclease-derived fraction that is more susceptible to digestion with deoxyribonuclease I. It is concluded that mild micrococcal-nuclease treatment of nuclei causes their separation into transcribed and non-transcribed DNA as determined by a number of very different criteria.
TL;DR: Comparison with the rates of accumulation of cytochrome c, b, and aa3 indicates that during the first 24 hours of cardiac enlargement the inner mitochondrial components accumulate faster then mtDNA, but during the six subsequent days the rate of mtDNA increment far outstrips that of the cytochromes.
Abstract: Mitochondrial proliferation was studied in mature female rats following aortic constriction. Mitochondrial DNA (mtDNA) was assayed by a fluorometric method. The conditions for removal of nuclear DNA were developed and verified by assessment of molecular conformation of DNA. The mtDNA concentration in mitochondria increased 2,4, and 7 days post-operatively by 11, 72 and 117% respectively. Comparison with the rates of accumulation of cytochrome c, b, and aa3 indicates that during the first 24 hours of cardiac enlargement the inner mitochondrial components accumulate faster then mtDNA, but during the six subsequent days the rate of mtDNA increment far outstrips that of the cytochromes. These data indicate that the amount of available mtDNA template is not the only factor regulating the transcriptional and translational processes in the enlarging myocardium.
Journal Article•
TL;DR: The relationship between DNA distribution pattern and estrogen receptor (ER) content was investigated in primary mammary carcinomas from 56 female patients and results strongly indicate a correlation between nuclear DNA distribution patterns and ER levels in primary breast carcinomas.
Abstract: An investigation was conducted to determine if a relationship exists between the estrogen receptor (ER) content and the type of nuclear DNA distribution patterns in human mammary carcinomas. Primary breast carcinomas from 56 female patients (52 postmenopausal and 4 premenopausal women) were examined. All cases were treated by mastectomy or local excision and none of the patients had received any treatment for their disease before cell sampling for DNA and ER analysis was performed. The DNA histograms of these tumors could be grouped into 4 distinctly different classes or types: type 1 is characterized by a single modal DNA value in the normal diploid or near-diploid region; histograms of type 2 have a distinct single modal value in the normal tetraploid or near-tetraploid region or pronounced peaks in the 2c region as well as in the 4c region; type 3 populations differ from type 2 populations by showing a sizable number of cells in the normal DNA synthesis region; and the type 4 histograms are characterized by pronounced irregular aneuploidy with individual DNA values ranging from levels near 2c up to values beyond 6c or even 8c. The data showed that mammary carcinomas in which the DNA amounts of the tumor cells do not deviate conspicuously from diploid or tetraploid DNA values of normal mammary epithelium were characterized by high levels of ER. Mammary carcinomas which contained a sizable number of cells with DNA amounts in the normal DNA synthesis region or with high degrees of aneuploidy were distinguished by low or even unmeasurable ER levels. The findings indicate a correlation between nuclear DNA distribution patterns and ER levels in primary breast carcinoma.
TL;DR: Using this procedure, the mtDNA sequences of CAP’ interspecific cybrids, or reconstituted cells, obtained by fusion of rat CAPr cytoplasts to hypoxanthine-aminopterin-thymidine-resistant (HAT?
TL;DR: The location of proteins on the mitochondrial DNA of Drosophila melanogaster was investigated by trimethylpsoralen photoreaction of embryos disrupted by gentle homogenization and protection of this part of the mtDNA from crosslinking was interpreted to be the result of association with proteins in the mitochondrion.
Abstract: The location of proteins on the mitochondrial DNA (mtDNA) of Drosophila melanogaster was investigated by trimethylpsoralen photoreaction of embryos disrupted by gentle homogenization. After photoreaction, the mtDNA was isolated and the pattern of DNA crosslinking was determined by electron microscopy of the DNA under totally denaturing conditions. In contrast to nuclear DNA, which showed periodic crosslinks indicative of a nucleosome structure, most of each mtDNA molecule exhibited uniformly heavy crosslinking. A 10% region of the mtDNA was, however, protected from psoralen crosslinking in a distinctive manner: five uncrosslinked segments were closely clustered in the mtDNA. Four were 394 +/- 13 (SD) base pairs in size, while the fifth measured about 200 base pairs. These protected segments mapped within the A+T-rich region of the mtDNA, extending from the end of the A+T-rich region near the Bg1 II cleavage site to the center of the A+T-rich region. Protection of this part of the mtDNA from crosslinking was interpreted to be the result of association with proteins in the mitochondrion because mtDNA that was deproteinized before the photoreaction was uniformly crosslinked over its entire length. The origin of replication of the mtDNA is also located at the center of the A+T-rich region, which suggests that the protection from the psoralen photoreaction may be due to proteins involved in membrane attachment or replication.
TL;DR: This work has shown that numerous reactive metabolites of benzo[a] pyrene generated by microsomes are capable of binding to exogenous DNA in vitro, but in isolated organs or in cells and intact tissues most of these reactive forms are detoxified or otherwise “trapped” before they reach nuclear DNA.
Abstract: Although the necessity for metabolic activation of chemical carcinogens is well established, the critical target(s) for their action is not known. Many recent investigations have been based on the assumption that carcinogens act through somatic mutation and have focused on carcinogen‐nucleic acid interactions. Methods have been developed for detecting biological consequences of DNA damage caused by carcinogenic metabolites (for example, mutagenesis in prokaryotes or eukaryotes) or quantitating specific metabolite‐DNA adducts. Such methods have made it possible to elucidate probable ultimate carcinogenic forms and study cellular factors affecting carcinogen‐DNA interactions. It has been shown that numerous reactive metabolites of, for example, benzo[a] pyrene generated by microsomes are capable of binding to exogenous DNA in vitro, but in isolated organs or in cells and intact tissues most of these reactive forms are detoxified or otherwise “trapped” before they reach nuclear DNA. After binding, DNA repair...
01 Jan 1980
TL;DR: Since immunological methods are perhaps the most powerful tools available for probing the heterogeneity, diversity, and specificity of macromolecular components, it can be anticipated that their application to the research on chromosomal proteins will contribute substantially to the authors' knowledge of chromatin biology and biochemistry.
Abstract: Assuming that the genetic information of the eukaryotic cell is contained within the nuclear DNA, differentiation can be viewed as transcriptional restriction of DNA, specific for each differentiated cell type. Consequently, detailed knowledge of biological and biochemical properties of nuclear proteins and their functional associations with DNA is essential for our understanding of mechanisms governing cellular proliferation and differentiation. Since immunological methods are perhaps the most powerful tools available for probing the heterogeneity, diversity, and specificity of macromolecular components, it can be anticipated that their application to the research on chromosomal proteins will contribute substantially to our knowledge of chromatin biology and biochemistry.
TL;DR: Protein synthesis was necessary within the first one and a half hours after irradiation for the survival of wild-type cells to be unaffected and the results provide additional evidence for an inducible repair process in U. maydis.
Abstract: The size of nuclear DNA from wild-type Ustilago maydis was determined to be approximately 6·09 ± 0·3 × 108 daltons from neutral sucrose gradient sedimentation analysis. Following exposure to ionizing radiation the nuclear DNA size was reduced due to the production of double-strand breaks in the DNA. These breaks were repaired when the irradiated cells were incubated in medium for at least one hour after irradiation. The repair was seen as a shift in the DNA profile from a low molecular weight region where the control DNA sedimented. Inhibition of protein synthesis by cycloheximide prevented this type of repair. Blocking protein synthesis also decreased the survival of irradiated wild-type cells but not radiation-sensitive mutants. Protein synthesis was necessary within the first one and a half hours after irradiation for the survival of wild-type cells to be unaffected. The results provide additional evidence for an inducible repair process in U. maydis.
TL;DR: Studies of nuclear and chloroplastic-DNA repair after ultraviolet irradiation of Euglena gracilis show that photoreactivation is very efficient at both the nuclear andchloroplastic level, and it is indicated that an important degradation follows UV irradiation during liquid holding in the dark.
Abstract: Studies of nuclear and chloroplastic-DNA repair after ultraviolet irradiation of Euglena gracilis show that photoreactivation is very efficient at both the nuclear and chloroplastic level. Liquid-holding or split-dose experiments and treatment with caffeine reveal, furthermore, that dark-repair is very efficient in nuclear DNA but not in chloroplastic DNA (ctDNA). The possibility of a chloroplastic dark-repair of restricted efficiency is discussed.