Showing papers on "Nuclear DNA published in 1985"

Nuclear dna contents, shoot phenology and species co-existence in a limestone grassland community

Abstract: Summary Within a species-rich limestone grassland community in Northern England a correlation has been established between nuclear DNA content and the timing and rate of leaf extension in the spring. The data support the hypothesis that natural selection has operated upon nuclear DNA content through the differential sensitivity of cell division and cell expansion to low temperature. It is suggested that measurements of nuclear DNA content may provide an index of temporal niche differentiation in plant communities.

Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanogaster.

Abstract: In the nurse cells of Drosophila, nuclear DNA is replicated many times without nuclear division. Nurse cells differ from salivary gland cells, another type of endoreplicated Drosophila cell, in that banded polytene chromosomes are not seen in large nurse cells. Cytophotometry of Feulgen stained nurse cell nuclei that have also been labeled with 3H-thymidine shows that the DNA contents between S-phases are not doublings of the diploid value. In situ hybridization of cloned probes for 28S+18S ribosomal RNA, 5S RNA, and histone genes, and for satellite, copia, and telomere sequences shows that satellite and histone sequences replicate only partially during nurse cell growth, while 5S sequences fully replicate. However, during the last nurse cell endoreplication cycle, all sequences including the previously under-replicated satellite sequences replicate fully. In situ hybridization experiments also demonstrate that the loci for the multiple copies of histone and 5S RNA genes are clustered into a small number of sites. In contrast, 28S+18S rRNA genes are dispersed. We discuss the implications of the observed distribution of sequences within nurse cell nuclei for interphase nuclear organization. — In the ovarian follicle cells, which undergo only two or three endoreplication cycles, satellite, histone and ribosomal DNA sequences are also found by in situ hybridization to be underrepresented; satellite sequences may not replicate beyond their level in 2C cells. Hence the pathways of endoreplication in three cell types, salivary gland, nurse, and follicle cells, share basic features of DNA replication, and differ primarily in the extent of association of the duplicated chromatids.

Supercoiled loops and the organization of replication and transcription in eukaryotes

Abstract: The nuclear DNA of eukaryotes is organized into a series of loops each topologically anchored by elements of the nuclear matrix. Evidence is reviewed which indicates that the anchorage points of the loops are formed by transcriptionally active genes and that individual loops function as replicons. The data suggests a specific model for coupling of DNA replication and transcription in eukaryotes.

Senecio L. (Asteraceae) in Australia: Nuclear DNA amounts

Abstract: Nuclear DNA amounts of 28 species of Senecio native to Australia and of four exotic species are reported. DNA amounts per 4C nucleus range from 4.22 pg to 42.90 pg, a 10-fold difference. Some of the variation is due to polyploidy as chromosome numbers range from n = 5 (by dysploid reduction) to n = 50, but DNA amounts per genome also vary from 0.84pg to 4.69 pg, a 6-fold difference. DNA amounts correlate with cell size and, in some instances, the size of structures. There is also a correlation between DNA amount and generation length which corresponds with previous observations. The possible direction of change in DNA amount and the influence of natural selection on this process are discussed.

Modification of nuclear matrix proteins by ADP-ribosylation. Association of nuclear ADP-ribosyltransferase with the nuclear matrix.

Abstract: Nuclear matrices were isolated by treatment of isolated HeLa cell nuclei with high DNase I, pancreatic RNase and salt concentrations. ADP-ribosylated nuclear matrix proteins were identified by electrophoresis, blotting and autoradiography. In one experimental approach nuclear matrix proteins were labeled by exposure of permeabilized cells to the labeled precursor [32P]NAD. Alternatively, the cellular proteins were prelabeled with [35S]methionine and the ADP-ribosylated nuclear matrix proteins separated by aminophenyl boronate column chromatography. By both methods bands of modified proteins, though with differing intensities, were detected at 41, 43, 46, 51, 60, 64, 69, 73, 116, 140, 220 and 300 kDa. Approximately 2% of the total nuclear ADP-ribosyltransferase activity, but only 0.07% of the nuclear DNA, was tightly associated with the isolated nuclear matrix. The matrix-associated enzyme catalyzes the incorporation of [32P]ADP-ribose into acid-insoluble products of molecular mass 116 kDa and above, in a 3-aminobenzamide-inhibited, time-dependent reaction. The possible function of ADP-ribosylation of nuclear matrix proteins and of the attachment of ADP-ribosyltransferase to the nuclear matrix in the regulation of matrix-associated biochemical processes is discussed.

DNA sequence components of the Pinusstrobus nuclear genome

Abstract: The nuclear DNA of Pinusstrobus L. was characterized by whole-genome hydroxyapatite reassociation kinetics. The genome, which is very large, is not well described by partition into three, four, or ...

A method for producing intact plants containing foreign dna

Abstract: An in vivo method for transforming and regenerating intact plants. According to the method, a plant (P) is infected with an infectious microbial agency comprised of: (1) virulence functions, (2) oncogenic factors capable of inducing a shoot-bearing shooty tumour on plant (P), and (3) a carrier vector containing engineered heterologous transfer DNA capable of being integrated into the nuclear DNA of plant (P) cells. Infected plant (P) is maintained until a shoot-bearing shooty tumour develops at or near the infection site. Those shoots, or progeny thereof, that contain transformed cells having heterologous transfer DNA integrated into their genomes are then selected and utilized to produce whole plants that contain cells having heterologous transfer DNA integrated into their genomes.

Control of cell division by aphidicolin without adverse effects upon resting cells.

Abstract: Aphidicolin, a tetracyclic diterpenoid obtained from the culture filtrates of Cephalosporium aphidicola and other fungi, inhibits the growth of eukaryotic cells and of certain animal viruses (SV40, Herpes and Vaccinia viruses) by selectively inhibiting the cellular replicative DNA polymerase alpha or the viral-induced DNA polymerases. The arrest of cellular or viral growth is thus due to inhibition of cellular or viral replicative DNA synthesis without interference with mitochondrial DNA synthesis, RNA, protein and nucleic acid precursors synthesis or other major metabolic pathways. The inhibition of all sensitive eukaryotic DNA polymerases by aphidicolin is competitive with respect to dCTP. Aphidicolin has thus proved extremely useful in elucidating the functional role of DNA polymerase alpha in nuclear DNA replication, of DNA polymerase gamma in mitochondrial DNA synthesis and both DNA polymerases beta and alpha in DNA repair synthesis. An important laboratory application of aphidicolin is the synchronization of the cell cycle of eukaryotic cells both in culture and in vivo. The properties of aphidicolin have recently aroused considerable interest for its possible exploitation in al practice. The mechanism of action of this drug suggests in fact that it may be useful for controlling excessive cell proliferation in patients with cancer, psoriasis or other dermatitis with little or no adverse effect upon non-multiplying cells. Interestingly, when administered to mice, the highest levels of aphidicolin are found in those tissues most actively proliferating with little or no aphidicolin present in neurons or myocardial cells.

Molecular structures of mitochondrial-DNA-like sequences in human nuclear DNA.

Abstract: Two lambda phage clones carrying mitochondrial-DNA-like (mtDNA-like) sequences isolated from a human gene library were named Lm E-1 and Lm C-2, and their DNA structures were characterized. Lm E-1 contains about 0.4 kb DNA homologous to the 5' portion of the mitochondrial 16S ribosomal RNA (rRNA) gene and Lm C-2, a 1.6 kb DNA homologous to the 3' portion of the 12S rRNA gene and to almost all of the 16S rRNA gene. Comparisons of their nucleotide sequences with those of the corresponding regions of the human mtDNA revealed no detectable DNA rearrangement and their homologies to the human mtDNA are 84% and 80%, respectively. There are neither terminal repeats in the nuclear mtDNA-like sequences nor duplications of the nuclear DNAs flanking the mtDNA-like sequences. Evolutionary relationship between these two human nuclear mtDNA-like sequences and the human and bovine mtDNAs is discussed.

Kinetics of DNA replication in C3H 10T1/2 cells synchronized by aphidicolin.

Abstract: Aphidicolin is an inhibitor of DNA polymerase alpha and blocks nuclear DNA replication without interfering with mitochondrial DNA synthesis. The efficacy of this mycotoxin as a tool in cell synchronization was evaluated in C3H 10T1/2 clone 8 cells. At concentrations of 1-2 micrograms/mL, aphidicolin quickly reduced the [3H]thymidine uptake to less than 5% of control levels in the first 5 min of incubation. This inhibition was easily reversed by washing and refeeding cells with fresh medium. The synchronization protocol consisted of first blocking cells by confluence arrest, replating them at lower density, and then treating the cells with aphidicolin for 24 h. Once the inhibitor was removed, DNA replication started without any delay. The cell population traversed the S phase in about 8 h and synchronously doubled in cell number. Autoradiography studies revealed a labeling index of 89-93% during the S phase. However, it was also observed that 10T1/2 cells were able to enter S phase in the presence of aphidicolin. The extent of the ensuing replication in the nucleus was dependent on the time that cells remained arrested in early S phase. Analyses of the newly replicated DNA in alkaline sucrose gradients revealed a fairly homogeneous distribution of sizes of nascent DNA in synchronized cells pulse-labeled at the beginning of the S phase. Upon chase in nonradioactive medium, the average molecular weight of the nascent DNA increased linearly with time of DNA synthesis for 2 h. The apparent rate of DNA chain growth determined from pulse and chase experiments was 1.2 micron/min. This rate was strongly inhibited (93%) by aphidicolin at a concentration of 2 micrograms/mL.

Distribution of initial and persistent 2-acetylaminofluorene-induced DNA adducts within DNA loops

Abstract: The intranuclear distribution of initial and persistent DNA adducts induced in vivo after four weekly injections of the hepatocarcinogen 2-acetylaminofluorene was examined in rat liver by using a protocol that fractionates chromatin from various regions of each of the multiple nuclear DNA loops. Ten hours after the initial dose, two acetylated [(N-acetyl-N-(deoxyguanosin-8-yl)-2-aminofluorene and 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene] and one deacetylated [N-(deoxyguanosin-8-yl)-2-aminofluorene] adduct were detected by a 32P-labeling assay and were found to have a random genomic distribution, as evident by their relative concentrations in various chromatin fractions. These data suggest that all regions of the DNA loops are equally susceptible to adduct formation. A nonrandom persistence of the deacetylated adduct in the regions where the DNA loops are constrained by the nuclear matrix was evident by 6 days after the last dose and was markedly apparent by 60 days. In contrast, all chromatin fractions had equally inefficient removal of the N2-acetylated adduct by 6 days as well as 60 days but had complete removal of the C8-acetylated adduct. These findings suggest that pronounced regional differences in adduct repair along the DNA loops may play a role in chemically induced hepatocarcinogenesis.

Isolation of a photoreactivation-deficient mutant of Chlamydomonas.

Abstract: A mutant deficient in photoreactivation has been isolated following mutagenesis of Chlamydomonas reinhardi with N-methyl-N'-nitro-N'-nitrosoguanidine. The mutant is deficient in the photorepair of pyrimidine dimers from nuclear DNA but appears to be normal in the rate of photorepair of dimers from chloroplast DNA. Cell-free extracts prepared from the photoreactivation-deficient mutant have about 17% of the DNA photolyase activity of wild-type cells. These results are consistent with the hypothesis that nuclear and chloroplast DNA photolyases are controlled by two separate genes.

Plastid and nuclear DNA synthesis are not coupled in suspension cells ofNicotiana tabacum

Abstract: The relationship between nuclear and plastid DNA synthesis in cultured tobacco cells was measured by following3H-thymidine incorporation into total cellular DNA in the absence or presence of specific inhibitors. Plastid DNA synthesis was determined by hybridization of total radiolabeled cellular DNA to cloned chloroplast DNA.

Chromosomal DNA variation in Cucumis

Abstract: Variation in nuclear DNA amounts found in different species of Cucumis was surveyed The DNA amounts varied from 1373 to 2483 pg in diploids and from 2846 to 3886 pg in tetraploids DNA amount was not correlated with chromosome number and periodicity Tetraploids were found to have double the quantity of nuclear DNA of diploids A positive linear relationship was established between the nuclear DNA amounts and volume of chromosomes The botanical varieties within a particular species do not differ significantly for 2C DNA amounts A comparison of the distribution of DNA amounts among different chromosomes of haploid complement in different species revealed that the quantitative DNA changes associated with speciation affected all chromosomes DNA changes were not however, of the same magnitude in all chromosomes of the complement Speciation in Cucumis thus seems to have occurred through amplification or diminution of DNA proportionate to the size of chromosomes The relationship between the basic numbers, x=7 and x=12, will have to be considered relative to the high DNA amount noticed in some species with x=12

DNA ligase activities during hepatocarcinogenesis induced by N-2-acetylaminofluorene.

Abstract: A progressive accumulation of DNA breaks has been reported to occur in nuclear DNA obtained from putative premalignant hepatic lesions induced by carcinogens. To determine if this alteration resulted from a defect in the level of, or functional activity of DNA ligases, we compared these enzymes in normal rat liver, 24-h regenerating liver, and hepatic nodules at intervals after cessation of N-2-acetylaminofluorene (AAF) treatment. Nuclear extracts of hepatocytes were separated into soluble and chromatin fractions, and multiple forms of DNA ligase activity were obtained by AcA34 gel filtration chromatography. In activities of the two largest species, DNA ligase Ia (480 kd) and DNA ligase Ib (240 kd), were present exclusively in soluble, nuclear fractions and were increased 4-fold and 2-fold, respectively, in 24-h regenerating livers. In AAF-induced nodules, these species were increased 3-fold and 1.5-fold, respectively, above those of normal rat liver, somewhat higher than predicted from the rate of cell division. In all of the test tissues, these ligase species demonstrated identical sensitivity to inhibition with 0.1 M NaCl or heating at 50 degrees C. DNA ligase II (80 kd) was found in both soluble nuclear fractions and chromatin at approximately identical levels in all tissues tested. Ligase II from all tissues also demonstrated identical responses to salt and heat. These data support the concept that DNA ligases Ia and Ib are related to DNA replication and suggest that ligase II may be a repair enzyme. The failure to detect significant alterations from expected values in the hepatic nodules and the lack of alteration in sensitivity to salt and heat indicate that the accumulation of DNA damage (presumably breaks) previously observed in carcinogen-induced altered hepatocytes is not due to an alteration in the level or the biochemical properties of DNA ligase.

DNA Content of Diffusely Infiltrative Carcinomas in the Stomach

Abstract: Summary DNA content of diffusely infiltrative carcinomas of 15 Japanese patients, ranging in age from 38 to 65 years, was determined by cytofluorometry, using paraffin sections which were available for histological examination. Sections were stained with 0.0025% propidium iodide. By measuring the fluorescence intensity of at least 50 mitotic cells, ploidy patterns of the cancers were determined, whereas a control of the diploid DNA content was set by measuring the fluorescence intensity of mitotic cells in the non-cancerous gastric mucosa; only metaphase nuclei in the sections were considered to have a whole amount of nuclear DNA in the cytofluorometric measurement. In the present study, it was found that most of the diffusely infiltrative carcinomas consisted of a heteroploid cell line. Their stem DNA contents ranged between 2.8C and 4.8C (2C corresponds to a diploid amount of nuclear DNA). Of 15 cancers, four comprised a mosaic cancer of two different heteroploid cell lines. We encountered only one carcinoma containing a diploid cell line. However, this cancer also contained a heteroploid cell line. Six cancers contained a polyploid cell population, the DNA content of which was double of the stem DNA content. In most cases, the cancer cells distributed in the mucosal layer were also heteroploid, thereby suggesting a heteroploid origin of the diffusely infiltrative carcinomas.

Role of mitochondrial DNA in cell death induced by 125I decay.

Abstract: SummaryThe role of mitochondrial DNA in radiation-induced cell death was determined by selective [125I]iododeoxyuridine (125IUdR) incorporation into exclusively nuclear sites compared to labelling in both nuclear and mitochondrial DNA of Chinese hamster cells. Such selectivity was achieved by using berenil (25 µg/ml for 24 h), a drug which inhibits mitochondrial DNA synthesis without affecting incorporation of 125IUdR into nuclear DNA but does not result in reduced clonogenicity or cell cycle perturbations or alteration in the X-ray response of cells. There was no difference in cell killing between cells with nuclear labelling alone compared with nuclear plus mitochondrial labelling. The absence of decays in mitochondrial DNA does not affect the ability of 125I to induce lethal cell damage. The two treatment groups have superimposable curves with a Do of 96 decays/cell. These findings indicate that mitochondrial DNA is not the most sensitive target for radiation-induced cell death from 125I decay.

Inhomogeneity of the nucleus to 125IUdR cytotoxicity.

Abstract: Synchronized suspension cultures of Chinese hamster ovary (CHO) cells were used to determine the lethal effects produced by the decay of 125I incorporated into different subfractions of the nuclear genome. Such a shift in nuclear incorporation pattern was achieved by using the drug aphidicolin, which inhibits 95% of all nuclear DNA synthesis, is nontoxic to cells in a colony-forming assay, and does not modify the radiation response of CHO cells to X irradiation. In addition to shifting incorporation of 1251 to only 5% of the nuclear genome, both nuclease digestions to characterize the molecular location of 125I and electron microscope autoradiography show an inhomogeneous distribution of sites of 125I incorporation in the presence of 5 ,g/ml aphidicolin. These data in combination with survival curves of CHO cells labeled with 125Iiododeoxyuridine (125IUdR) either with or without aphidicolin showed a dramatic change in the survival response (Do: 30 decays/cell and 96 decays/cell, respectively). It is concluded, therefore, that the nucleus is not a homogeneous target for radiation-induced cell death because when subfractions of the nuclear genome are labeled, radically different levels in cell survival

DNA content in Tradescantia

Abstract: There is a wide variation in the nuclear DNA content and chromosome size between the species belonging to the T. crassifolia and T. virginiana alliances (all the species but one are native to Centr...

Nuclear and mitochondrial DNA have sequence homology with a chloroplast gene.

Abstract: A PstI 7.7 kbp fragment from chloroplast (ct) DNA of spinach shows homology to an EcoRI 8.3 kbp fragment of mitochondrial (mt) DNA and in turn, both are homologous to a number of common regions of nuclear (n) DNA. The common area of homology between the chloroplast and mitochondrial fragments is between a KpnI 1.8 segment internal to the PstI sites in the ctDNA and an EcoRI/BamHI 2.9 kbp fragment at one end of the mitochondrial 8.3 kbp fragment. The KpnI 1.8 kbp ctDNA fragment is within a structural gene for the P700 chlorophyll a apoprotein. Further analysis of this KpnI 1.8 kbp fragment confined the homologous region in mtDNA to a ct 0.8 kbp HpaII fragment. These smaller pieces of the organellar genomes share homologies with nuclear DNA as well as displaying unique hybridization sites. The observations reported here demonstrate that there is a common or closely related sequence in all three genetic compartments of the cell.

Properties of adenoviral DNA bound to the nuclear matrix.

Abstract: The association of adenoviral DNA with the high salt (2 M NaCl) resistant nuclear fraction, termed the nuclear matrix, has been investigated in HeLa cells at different times after infection with adenovirus type 5. When nuclear matrices were prepared in the absence of exogenously added nucleases, Ad5 DNA was quantitively associated with the matrix throughout the infection period (0-24 h). Moreover, early in infection (0-10 h) Ad5 DNA was severalfold enriched in DNase I digested nuclear matrices (8-15% of total nuclear DNA) compared to the high salt soluble chromatin fraction (85-90% of total nuclear DNA). At later times after infection, progressively more Ad5 DNA appeared in the chromatin fraction until, at 24 h, the nuclear matrix was strikingly depleted in Ad5 DNA. A large proportion of the Ad5 DNA in nuclear matrices prepared early in infection, e.g., 4 h, was full length in size. At later times (12-24 h) most of the viral DNA was fragmented to a size equivalent to total matrix DNA (100-1000 base pairs). The apparent switch of the matrix-associated viral DNA from a relatively DNase I resistant to sensitive state was initiated approximately at the time when viral DNA replication began (12 h). Since no discrete portion of the Ad5 genome was significantly enriched at the sites of attachment to the nuclear matrix throughout the infection period, the switch in DNase I sensitivity is not mediated by a change in DNA sequence attachment to the matrix.(ABSTRACT TRUNCATED AT 250 WORDS)