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PubMed1975

The fate of a bacterial plasmid in mammalian cells.

When hamster cells are infected with the bacterial plasmid colicinogenic factor E1 (ColE1), as much as 5-8% of the input plasmid radioactivity is found in the recipient cell, mainly in the nuclear fraction. Density shift experiments with bromodeoxyuridine labeled ColE1 DNA indicate that part of the input DNA may be replicated in the nucleus. ColE1 specific RNA but no colicin E1, can be detected during the first two generations after the uptake of ColE1 DNA. However, extrachromosomal ColE1 DNA is unstable in the mammalian cells and is degraded to acid soluble fragments after a few generations.

Animals↗
PubMed1976

Mitochondrial DNA in yeast recombination and subsequent modification following mating between a Grande and a suppressive Petite.

The fate of mitochondrial DNA, following mating between a grande and suppressive petite of Saccharomyces cerevisiae, has been followed for up to 60 generations. The buoyant density of the mitochondrial DNA was seen to change in a manner explicable by a combination of recombination and subsequent modification phenomena whilst the suppressivity of the petite zygotic clones always remained high. These findings are consistent with current models of mitochondrial DNA metabolism in which petite strains have been observed to undergo deletion and reamplification of certain parts of their genomes.

Centrifugation, Density Gradient↗
PubMed1976

Effects of different alleles of the E. coli K12 pol A gene on the replication of non-transferring plasmids.

The effects of eight different polA alleles on the replication of six different non-transferring enterobacterial plasmids have been tested. Using phage P1CM transduction, different allelic polA mutations were introduced into E. coli K12 strains carrying one of several antibiotic resistance plasmids. Plasmid stability in the transductants was examined by testing clones for drug resistance after growth under various conditions. From the results, the R factors may be divided into three different classes. One plasmid is only affected by PolA conditions which inhibit host cell growth, 3 plasmids (from the same compatibility group) are unstable under conditions in which the cells are severely deficient in DNA polymerase I and two other plasmids (compatible with each other and with the other 4) are immediately lost from such transductants and are unstable in a number of others. Furthermore, the plasmids which are most dependent on DNA polymerase I have been shown to replicate in the presence of chloramphenicol and therefore typigy a class of plasmids which includes bacteriocinogenic factors such as ColE1 and CloDF13, resistance determinant RSF1030 and the E. coli 15 minicircular plasmid.

Alleles↗
PubMed1976

Characterisation of plasmids coding for the restriction endonuclease EcoRI.

The properties of two plasmids coding for the CcoRI restriction and modification enzymes are described. Both plasmids are non auto-transferring (NTP) but can be mobilised by transfer factors. Strains carrying NTP13 produce colicin E1 and the EcoRI enzymes. This plasmid has a molecular weight of 6 X 10(6) daltons and is present as approximately 12 copies per chromosome. The second plasmid, NTP14, was detected after mobilisation of the EcoRI plasmid with the R factor RI-19. NTP14 codes for ampicillin resistance, synthesis of the EcoRI enzymes and colicin E1. The molecular weight of NTP14 is 10.7 X 10(6) daltons and there are about 14 copies per chromosome. DNA-DNA reassociation experiments were performed to determine the interrelationships of NTP13, NTP14, ColE1 and the R factor R1-19. NTP13 and NTP14 continue to replicate when cellular protein synthesis is inhibited by the addition of chloramphenicol.

Ampicillin↗
PubMed1975

The organization of genes in yeast mitochondrial DNA II. The physical map of EcoRI and HindII + III fragments.

1. We have isolated large fragments of the mtDNA of the yeast Saccharomyces carlsbergensis and digested these with restriction endonucleases. The digestion products were separated by electrophoresis in agarose gels. 2. Endonucleases EcoRI, HindII + III, HpaI, HindIII and HapII yield 9, 11, 6, 0 and greater than 80 fragments, respectively. 3. By analysis of partial digestion products and by redigesting the fragments obtained with one endonuclease with a second, we have established the order of all EcoRI and HindII + III fragments. The map is circular and its contour length is 22.1 +/- 0.35 mum, in good agreement with earlier estimates of the size of yeast mtDNA, using electron microscopy and renaturation kinetics. 4. A comparison of the fragmentation pattern of mtDNAs from S. carlsbergensis and various strains of Saccharomyces cerevisiae with endonuclease HindII + III suggests that the overall gene order is similar.

DNA Restriction Enzymes↗
PubMed1975

Transmission and recombination of extranuclear genes during sexual crosses in Aspergillus nidulans.

Three extranuclear mitochondrial mutations in Aspergillus nidulans, (oliA1), (camA1) and (cs67), were used as markers in sexual crosses to provide information on the frequencies of transmission and recombination of the mitochondrial genome. Any individual perithecium contained ascospores of only one extranuclear genotype. Using mono-, bi- and trifactorial crosses it was found that all three markers could be recovered from the progeny, although the transmission frequencies were different for each marker. This bias was present irrespective of the nuclear background or the presence of selective agents in the medium on which the cross was established. These findings enable a series of "transmission strength" to be established, as shown below:-- (camA1) greater than (cs67, camA1) greater than (+) = (cs67) greater than (oliA1, cs67) greater than (oliA1) greater than (oliA1, camA1). However, the numbers of recombinants isolated were so variable as to make this form of analysis unsuitable for mapping the mitochondrial genome.

Aspergillus nidulans↗
PubMed1976

The isolation of IS1 and IS2 DNA.

DNA of the IS-elements IS1 and IS2 was prepared by digestion of appropriate heteroduplex molecules with endonuclease S1, followed by sucrose gradient centrifugation or gel electrophoresis. The material obtained is homogeneous with regard to size. The length of IS1 DNA is 820 +/- 65 nucleotides, the length of IS2 DNA is 1,350 +/- 70 nucleotides. IS1 DNA is not cleaved by the restriction endonucleases Eco R1, Hind II or Hind III. IS2 DNA is cleaved once by each of the two latter enzymes. The buoyand density determined by equilibrium centrifugation of Hg-complexes in Cs2so4 corresponds to a GC content of approximately 50%. Labelling with polynucleotide kinase indicates that both IS DNA's have a guanosyl residue at both of their 5'-termini.

Centrifugation, Density Gradient↗
PubMed1976

A method for the deletion of restriction sites in bacterial plasmid deoxyribonucleic acid.

A general method has been developed for the deletion of restriction endonuclease sites in bacterial plasmid DNA. The procedure involves partial digestion of the covalently closed circular plasmid DNA with an appropriate restriction endonuclease under conditions which allow accumulation of unit-length linear DNA molecules, a controlled digestion of the exposed 5' ends with the lambda 5'-exonuclease, and in vivo recircularization of the resulting linear DNA in a bacterial host cell. The method has been used for the deletion of one of the two EcoRI sites in the plasmid pML2 (colE1-Km). Two of the resulting plasmids, pCR1 and pCR11, have a single EcoRI cleavage site, but retain genetic determinants specifying resistance to colicin E1 and kanamycin, and thus may be useful as vectors for the cloning and amplification of DNA in bacteria.

Coliphages↗
PubMed1976

Pattern of somatic segregation of the cytoplasmic drug-resistance factors in yeast.

The pattern of somatic segregation of the cytoplasmic factors confering resistances to chloramphenicol, erythromycin and oligomyin in S. cerevisiae was studied. The fractions of the zygotes heterozygous for the chloramphenicol-resistance factor and for the erythromycin-resistance factor decreased exponentially with generation number of zygotes. The rate of the segregation was highest for the chloramphenicol-resistance factor and lowest for the oligomycin-resistance factor. The segregation rate as well as the transmission polarity of the chloramphenicol-resistance factor varied with different carbon sources with which the parental haploids were grown prior to mating.

Chloramphenicol↗
PubMed1976

Biogenesis of mitochondria. XLII. Genetic analysis of the control of cellular mitochondrial DNA levels in Saccharomyces cerevisiae.

The proportion of total cell DNA which is mitochondrial DNA was measured in haploid, diploid and tetraploid strains of S. cerevisiae grown under a standard set of conditions. For all strains tested the mitochondrial DNA level was in the range 16%-25% of total cell DNA. Repeated measurements of the cellular level of mitochondrial DNA in two haploid strains showed that these strains have measurably different cellular mitochondrial DNA levels (17% and 24% of total DNA, respectively) under our conditions. These two grande strains were used to investigate the role of the mitochondrial and nuclear genomes in the regulation of the mitochondrial DNA level. We have shown by genetic analysis that the difference between these two strains is determined by at least two nuclear genes. The mitochondrial genome is not involved in the regulation of cellular mitochondrial DNA levels. A number of purified petite clones derived from independent spontaneous petite isolates of the grande strain which contained 24% mitochondrial DNA were also studied. The mitochondrial DNA levels in all but one of these petites fell in the range 20-25% of total cell DNA. From these results we conclude that, in general, the mitochondrial DNA level in petite strains is controlled by the same mechanism as operates in grande strains. We propose a general model for the control of the cellular mitochondrial DNA level, in which the amount of mitochondrial DNA per cell is determined by regulation of the number of mitochondrial DNA molecules per cell. This regulation is mediated through the availability of a set of nuclear coded components, possibly a mitochondrial membrane site, which are required for the replication of mitochondrial DNA.

Aneuploidy↗
PubMed1976

Octopine and nopaline synthesis and breakdown genetically controlled by a plasmid of Agrobacterium tumefaciens.

Several nopaline degrading strains and one octopine degrading strain are shown to loose oncogenicity as well as the ability to utilize these guanidine compounds when they are cured of their TI plasmid. To investigate whether the specific genes involved in the utilization of one or the other compound are located on the plasmid, plasmid-transfer experiments have been performed. The plasmid from a nopaline degrading strain has been transferred to a naturally non oncogenic Agrobacterium namely A. radiobacter. Furthermore, the plasmid from an octopine degrading strain has been transferred to a plasmid-cured strain which originally had the capacity to utilize nopaline. Both kinds of experiments prove that the TI plasmid determines the strain specificity with regard to the utilization of either octopine or nopaline. They also demonstrate that the synthesis of either octopine or nopaline in crown gall cells is also determined by genes located on the TI plasmid harboured by the transforming A. tumefaciens strains.

Extrachromosomal Inheritance↗