Showing papers on "Nuclear DNA published in 1982"

In vitro transformation of plant protoplasts with Ti-plasmid DNA

Abstract: Genetic engineering requires a procedure for introducing DNA into host cells, followed by integration into the host genome and gene expression. Although several procedures for DNA-medi-ated gene transfer in mammalian cells, yeast and bacteria have been reported, no such methods are yet available for plant cells. The major obstacle to DNA uptake in plant cells is the cell wall, but this can be circumvented by using plant protoplasts, cells freed of their cell walls by enzymatic digestion. However, none of the reports on the uptake of DNA into plant protoplasts1,2 has produced conclusive evidence for the integration of DNA into the host genome, that is, that stable transformation occurs. The tumour-inducing bacterium Agrobacterium tumefaciens, which causes crown gall disease, is a natural system for the introduction of foreign DNA into plants. This bacterium introduces part of its tumour-inducing (Ti) plasmid, called T-DNA, into plant cells, where it becomes integrated into the nuclear DNA of the host3,4 and is transcribed into mRNA5,6. T-DNA encodes tumour-specific enzymes responsible for the formation of amino acid derivatives such as octopine or nopaline7, which the bacterium can use as a sole source of carbon and nitrogen. The transformed cells have also acquired the ability to grow in the absence of phytohormones (autotrophy). An in vitro system for infection of Nicotiana tabacum protoplasts by A. tumefaciens has already been reported8. Transformants are selected by their ability to divide and grow in tissue culture without the addition of plant phytohormones to the synthetic culture medium. Here, we report a reproducible method for the stable transformation of tobacco protoplasts with Ti-plasmid DNA, using a similar selection procedure.

Preferential attack of mitochondrial DNA by aflatoxin B1 during hepatocarcinogenesis

Abstract: Administration of the hepatic carcinogen aflatoxin B1 to experimental animals results in covalent binding to liver mitochondrial DNA at concentrations three to four times higher than nuclear DNA. The concentration of carcinogen adducts in mitochondrial DNA remains unchanged even after 24 hours, possible because of lack of excision repair. Similarly, mitochondrial transcription and translation remain inhibited up to 24 hours suggesting long-term effects of aflatoxin B1 on the mitochondrial genetic system.

Dna isolated from DNA/anti-DNA antibody immune complexes in systemic lupus erythematosus is rich in guanine-cytosine content.

Abstract: DNA fragments were isolated from DNA/anti-DNA antibody immune complexes of systemic lupus erythematosus. Hybridization experiments indicated that DNA isolated from immune complexes originates from human nuclear DNA. Two classes of DNA were identified by polyacrylamide gel electrophoresis. The m.w. of small fragments was 25,000 with 30 to 40 base pairs, and that of large fragments was 100,000 with 150 base pairs. The average guanine-cytosine (G-C) content in the small fragments was 55% of total bases, and that in the large fragments was 45%. Compared with the average 38% G-C content of total human DNA, the antigen DNA is rich in G-C content. Such unusually high G-C content can alter DNA structure, which may serve as the target for antibody production.

Discontinuous dna variation in the evolution of plant species: the genus lathyrus.

Abstract: The genus Lathyrus is closely related to Vicia and belongs to the family Leguminosae. Most species in this genus are diploids with 2n = 14. The divergence and evolution within this genus is accompanied by a three-fold increase in chromosome size which is directly correlated with a four-fold increase in their nuclear DNA amounts. The fraction of DNA responsible for the variation in total amounts of DNA in this genus consists of repetitive and non-repetitive DNA in a consistent ratio of 4. 1 to 1.0. Quantitative comparisons of the total DNA and of DNA constituents show discontinuity in the pattern of variation among the species of this genus. The constraints upon the composition of DNA among species and the discontinuity in the pattern of variation would suggest genome evolution by a succession of spasmodic changes rather than by a continuous progression. This investigation was carried out with two major objectives in mind, 1) to determine the nature of DNA variation accompanying the evolution of this genus and 2) to compare among species the relative changes in nuclear components which are closely correlated with DNA changes. Multivariate analysis was used to classify the data. MATERIALS AND METHODS

Interaction of benzo(a)pyrene and its dihydrodiol-epoxide derivative with nuclear and mitochondrial DNA in C3H10T 1/2 cell cultures.

Abstract: To analyze the distribution of radioactive carcinogens and [3H]thymidine between nuclear DNA (nDNA) and mitochondrial DNA (mtDNA), we have developed a simple and rapid method for the separation of nDNA and mtDNA using gel electrophoresis of cell lysates. Using this method, we found that, when C3H10T 1/2 cells are exposed to either 0.5 microM [3H]-(+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene ([3H]BPDE) or 1 microM [3H]benzo(a)pyrene, the mtDNA contains a major fraction of the total adducts formed with cellular DNAs. Deoxynucleoside adducts formed between benzo(a)pyrene and mtDNA in intact C3H10T 1/2 cells or between BPDE and isolated rat liver mtDNA were analyzed by high-performance liquid chromatography, and were found to be much more heterogeneous than those present in nDNA of C3H10T 1/2 cells. The extensive modification of mtDNA in BPDE in C3H10T 1/2 cells is associated with preferential inhibition of the incorporation of [3H]thymidine into mtDNA, when compared to incorporation of [3H]thymidine into nDNA. To analyze the factors responsible for the extensive modification of mtDNA by BPDE, we investigated the role of a lipid phase utilizing liposome:DNA complexes as a model system. We found that the liposomes protect BPDE from spontaneous hydrolysis and enhance the extent of DNA modification at low DNA concentrations. These findings extent previous evidence suggesting that the mitochondria may be important cellular targets in the process of chemical carcinogenesis.

Identification and cloning of a yeast nuclear gene (CBP1) involved in expression of mitochondrial cytochrome b

Abstract: Nuclear pet mutants of Saccharomyces cerevisiae deficient in mitochondrial respiration have been studied genetically and biochemically. Seven noncomplementing mutations leading to a deficiency of mitochondrial cytochrome b have been assigned to a single complementation group (group 60). Examination of mitochondrial RNA by blot hybridization on diazobenzyloxymethyl-paper has revealed that group 60 mutants produce a large number of novel apocytochrome b transcripts not detected in wild-type yeast. The product of the gene affected in the mutants, therefore, appears to be required either for correct transcription or for processing of apocytochrome b premessenger RNA. The gene has been designated CBP1. A representative mutant from complementation group 60 (N5-26) has been transformed to respiratory competency with a recombinant plasmid pool consisting of random fragments of wild-type yeast nuclear DNA inserted into a vector capable of replicating in yeast and Escherichia coli. The complementation of the N5-26 mutation has been shown for a number of independent transformants to be due to the presence of plasmid DNA. The plasmid pG60/T10 was further characterized to have a nuclear DNA insert of 6.7 kilobase pairs. This plasmid complements the mutations of all group 60 mutants, thus confirming that it contains the CBP1 gene.

Crown gall disease and prospects for genetic manipulation of plants.

Abstract: Agrobacterium tumefaciens incites crown gall tumors when bacterial DNA integrates into plant nuclear DNA. Plant cells can express these integrated bacterial genes. Following insertion of desired genes into bacterial DNA using recombinant DNA techniques, this system permits introduction of these new genes into plant DNA. We discuss the potential for genetic manipulation of plants using Agrobacterium tumefaciens and the related organism Agrobacterium rhizogenes.

Genome size and heterochromatin variation in rodents

Abstract: Nuclear DNA amounts are determined in 16 species and the C-banding patterns for 19 species of rodents. A list of rodent DNA amounts is compiled. The fraction of heterochromatin in the genome is determined as the length of C-banded chromosome material relative to the total karyotype length. Among all rodents, heterochromatin amounts tend to be larger in the larger genomes. However, this relationship is not exact and does not hold true for individual genera. In general the notion of a basic rodent genome of defined size to which various amounts of heterochromatin have been added is untenable.

The nuclear cell cycle in Chlamydomonas (Chlorophyceae).

Abstract: The timing of replication and division of the Chlamydomonas Ehrenberg nucleus in the vegetative cell cycle and at gametogenesis was examined, using fluorescence microspectrophotometry with two fluorochromes, mithramycin and 4′,6-diamidino-2-phenylindole (DAPI). Under appropriate conditions, these bind specifically to DNA, and the fluorescence of the DNA fluorochrome complex is a quantitative measure of the DNA content. The alga is a haplont, which produces 2n daughter cells at the time of vegetative reproduction; cytokinesis and daughter cell release lag behind karyokinesis. No nucleus was found to contain more than the 2c quantity of DNA. Hence daughter cell production proceeds by doubling of the nuclear DNA followed by karyokinesis, in a repetitive sequence. As reported previously for C. reinhardtii Dangeard, the gametes of C. moewusii Gerloff contain the 1c amount of nuclear DNA. Several conflicting interpretations of the cell cycle sequence proposed in the literature were resolved.

Nonrandom distribution of repeated DNA sequences with respect to supercoiled loops and the nuclear matrix.

Abstract: The DNA in a eukaryotic nucleus is arranged into a series of supercoiled loops that are anchored at their bases to the nuclear matrix. We have analyzed the DNA sequences that are closest to the matrix attachment points for their relative content of specific repeated sequences. Sequences were enriched (mouse satellite, human Alu family) or depleted (mouse EcoRI repeat, monkey alpha component), depending on the specific sequence and species examined. These results can be understood in terms of a nonrandom arrangement of DNA sequences with respect to nuclear DNA loops.

Clustering of spore-specific genes in Aspergillus nidulans.

Abstract: We have investigated the chromosomal organization of genes that are expressed specifically in the asexual spores (conidia) of the Ascomycete fungus Aspergillus nidulans, using two experimental approaches. In the first, 30 different recombinant clones, containing long nuclear DNA inserts and at least one spore-specific gene, were selected randomly. The total number of spore-specific genes present in each clone was then determined by RNA blot analysis. In the second approach, several chromosomal recombinant DNA libraries, having average insert lengths ranging from 1 to 15 kilobases, were constructed. The fraction of clones in each library having one or more spore-specific poly(A)+RNA-coding regions was then determined by colony or plaque filter hybridization with radiolabeled, spore-specific, complementary DNA. The results from these experiments were compared to statistical predictions based on the assumption that the spore-specific genes are randomly distributed in the Aspergillus genome. In both cases, the experimental values deviated significantly from the predicted values, demonstrating that the spore-specific genes are nonrandomly arranged in the genome. Rather, they appear frequently to occur in tightly linked clusters.

Multiple transcripts of T-DNA detected in nopaline crown gall tumors.

Abstract: Crown gall plant tumors contain neoplastic cells transformed by incorporation of a foreign DNA element, T-DNA, derived from a large tumor-inducing plasmid in the inciting Agrobacterium strain. T-DNA is covalently joined to the nuclear DNA of the tumor cell, and RNA transcripts from T-DNA are present in polyadenylated form on polysomes. This paper presents a detailed analysis of those parts of T-DNA transcribed in a nopaline-type tobacco teratoma, BT37, whose T-DNA has been mapped and cloned. Northern blots of polyA+ RNA were probed with 21 different nick-translated T-DNA fragments, and at least 13 well-defined transcripts were visualized.

Clonogenicity of multiple populations of human glioma cells in vitro sorted by DNA content.

Abstract: Malignant human gliomas have a highly variable distribution of cell nuclei, consisting of diploid and/or other populations in terms of nuclear DNA content. In order to study in vitro clonogenicity of each population, dissociated or cultured human glioma cells were stained with 20 microM/ml of Hoechst 33342 dye (which stains viable DNA with minimal cell kill), and were sorted sterile into separate populations, based on specific nuclear DNA content, for clonogenicity assay. The colony-forming efficiency (CFE) of tumor cells plated immediately after disaggregation of the biopsy specimens ranged from 0.0044 to 0.149%, and the CFEs increased dramatically with successive passages (to 5 to 40%). The CFEs of the individual populations sorted according to DNA content were similar within individual tumors. These results suggest not only that malignant gliomas are composed of multiple populations in terms of DNA content, but also that each of these populations contain clonogenic cells. The morphologic structure of cells within and among colonies did not appear to relate to DNA content.

Loss of nuclear DNA in leaves of rye.

Abstract: The nuclear DNA content of rye leaf cells was cytophotometrically determined. At the commencement of differentiation nuclei in rye leaves remain standing at G1 phase. With further differentiation a remarkable diminution of nuclear DNA content occurs in diploid cells. The largest number of cells showing a loss of nuclear DNA content were found in the top of the leaf. The age of the leaf and the extent of diminution in nuclear DNA content are correlated.

Cell Growth and Nuclear DNA Increase by Endoreduplication and Differential DNA Replication

Abstract: Somatic variations in the amount of nuclear DNA due to somatic polyploidization and differential DNA replication are events, which occur much more frequently than thought by many biologists and biochemists. This might be, in part, because there is particular interest in the genetic information of the DNA rather than on its “nucleotypical” (1) or “nucleoskeletal” effects (2). Actually, it is widely believed that the genome is an extremely stable constant, and that the genetic information stored in the nucleus is identical in all cells of an individual (dogma of DNA constancy). Classical cytologists, however, discovered already in 1939 the process of endomitosis (3), and somewhat later that of DNA endoreduplication (4). Recently, it was shown in a review that some kind of somatic polyploidy can be found in nearly every taxon investigated (5), and that often up to 70% of the cells of an organism may be polyploid (6). Hence, we should no longer ignore this fact, but search for the biological significance of somatic DNA increase.

Replicator Regions of the Yeast Mitochondrial DNA Active In Vivo and in Yeast Transformants

Abstract: The replication of the mtDNA in Saccharomyces cerevisiae is independent from that of the nuclear DNA. It is continuous throughout the cell cycle (Williamson and Moustacchi 1971; Sena et al. 1975), it is active in the cdc mutants deficient for the initiation of nuclear DNA synthesis (Cottrell et al. 1973; Wintersberger et al. 1974; Newlon and Fangman 1975), and it is catalyzed by a specific mtDNA polymerase (Wintersberger and Blutsch 1976). Furthermore, in most density-labeling experiments, gradual shifts in mtDNA densities are observed (Sena et al. 1975Sena et al. 1976; Williamson and Fennell 1974). This apparent dispersive mode of replication of the mtDNA probably results from the high rate of genetic recombination between the different nonsynchronous mtDNA molecules of each yeast cell, each molecule undergoing a semiconservative replication. But the exact mechanisms of the initiation and control of yeast mtDNA replication remain poorly understood. This situation is at contrast with the precise descriptions of the process of mtDNA replication now available for some other organisms, such as the vertebrates (in this case, a distinctive D-loop mechanism with specific origins for the replication of the H and L strands has been described [Arnberg et al. 1971; Berk and Clayton 1974, 1976; Kasamatsu and Vinograd 1974; Crews et al. 1979; Martens and Clayton 1979; Nass 1980; Sekiya et al. 1980]) and the insects (in this case, the replication is initiated within the A + T-rich segment [Goddard and Wolstenholme 1980]). In the case of yeast, the difficulty in isolating intact mtDNA molecules from the...

Unusual variations in nuclear DNA contents in the marine copepod Pseudocalanus.

Abstract: Variations in DNA/nucleus within adult females are similar, when normalized for mean differences, in the copepods Pseudocalanus sp. and Eurytemora herdmani. However, Pseudocalanus shows great variation among individuals, with no evidence of significant groupings (species?), except perhaps for a few individuals with abnormally small DNA values. Body lengths of adult female Pseudocalanus are approximately proportional to the cube root of DNA/nucleus. It is inferred that nuclear size, not nuclear number, determines body size, and this is supported by generally similar nuclear counts in newly hatched nauplii and among first copepodid stages. Although body size and development rate of Pseudocalanus had earlier been shown to be strongly heritable, an offspring-parent regression reflected no heritability of DNA/nucleus (and some abnormally small offspring values) under the probably suboptimal conditions of rearing. Some variability in DNA content of adult somatic nuclei may be related to chromatin diminution dur...