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PubMed1991

Nuclear-organelle interaction in Solanum: interspecific cybridizations and their correlation with a plastome dendrogram.

Alloplasmic compatibility, namely the functional interaction between the nuclear genome of a given species with plastomes and chondriomes of alien species, is of considerable relevance in plant biology. The genus Solanum encompasses a wide spectrum of species and is therefore suitable for a study of this compatibility. We thus chose the nuclear genome of Solanum tuberosum (potato) and organelles (chloroplast and mitochondria) from 14 other Solanum species to initiate an investigation of intrageneric nucleus/organelle interactions. An assessment of the diversity of the chloroplast DNAs from these 15 species resulted in the construction of a plastome dendrogram (phylogenetic tree). In parallel we extended a previous study and performed ten additional fusion combinations by the "donor-recipient protoplast fusion" procedure, using potato protoplasts as recipients and protoplasts from any of ten other Solanum species as donors. We found that two fusion combinations did not yield cybrids and that the chloroplasts of S. polyadenium and the mitochondria (or mitochondrial components) from S. tarijense could not be transferred to cybrids bearing potato nuclei. In general, there is a correlation, albeit not perfect, between the cybridization data and the plastome dendrogram. These results furnish valuable information toward future transfer of plasmone-encoded breeding traits from wild Solanum species into potato. This information should also be useful for the planning of asymmetric protoplast fusion between potato and wild accessions for the improvement of pathogen and stress resistance of potato cultivars.

Blotting, Southern↗
PubMed1981

Molecular structure of maternal RNA.

The presence of a stable maternal mRNA population in mature oocytes of many species is well established. In this paper we show that the mature egg contains, in addition to these mature mRNAs, a structurally more complex population of RNA transcripts. This latter class of RNA consists of polyadenylated transcripts of repetitive and nonrepetitive DNA elements covalently linked into long interspersed molecules. As much as seventy percent of the polyadenylated egg RNA of Xenopus laevis and Strongylocentrotus purpuratus is represented in this interspersed population. Most of the nonrepetitive DNA sequences represented in the mature mRNA population are also present in the interspersed RNA. These transcripts have an organization similar to that of somatic cell nuclear RNA. Data are presented that suggests some of these interspersed maternal transcripts are unprocessed precursor-like molecules. Some possible functions of this novel class of RNA during early development are discussed.

Animals↗
PubMed1979

Characterization of extrachromosomal DNA in the flesh fly Sarcophaga bullata.

The polytene pupal foot pad cells of the flesh fly Sarcophaga bullata contain numerous extrachromosomal DNA containing granules. We have determined both the origin and the nature of the DNA sequences present in these granules. Studies done with quinacrine staining of seven day old pupal foot-pad polytene nuclei showed that the granules fluoresced very brightly while the chromosomal bands to which the granules were attached did not. The only other highly fluorescent regions of the polytene karyotype were the centromeric heterochromatin of chromosomes C and E and several bands associated with the nucleolus of Chromsome A. When polytene nuclei were hybridized in situ with cRNA made from highly repetitive DNA, many of the granules positively labeled. Most of the label on these slides was concentrated on the centromeric heterochromatin of chromosomes C and E. Quinacrine staining of the foot-pad cells at very early stages of pupal development showed that when granules were present, they were always closely associated with the same two centromeric regions, those of chromosomes C and E. Since the highly repetitive DNA located in these centromeric regions is underreplicated, we conclude that the granules result from an extrusion process which takes place early during the polytenization of these cells. The chromosomal integrity of the centromeric heterochromatin of chromosomes C and E is apparently disrupted and repetitive sequences are dissociated from the chromosomes as DNA granules which then secondarily become associated with chromosomal bands throughout the nucleus.

Animals↗
PubMed1994

Presence of double-stranded RNA and virus-like particles in Phaffia rhodozyma.

Four double-stranded RNA (dsRNA) molecules were isolated from Phaffia rhodozyma UCD 67-385. Their molecular sizes were approximately 4.3, 3.1, 0.9 and 0.75 kilobase pairs (kbp) as determined by agarose-gel electrophoresis and they were designated as L, M, S1 and S2, respectively. By differential centrifugation in sucrose gradients, these dsRNAs copurified with isometric virus-like particles 36 nm in diameter. A cured strain, UV-S2, lacking the S2-dsRNA was obtained from P. rhodozyma UCD 67-385 by ultraviolet (UV) light treatment. UV-S2 strain contains identical virus-like particles to those from the wild-type strain, as determined by electron microscopy, suggesting that the S2-dsRNA was not essential for the expression of mycovirus structural polypeptides. On the other hand, both the UCD 67-385 and UV-S2 strains were able to kill P. rhodozyma UCD 67-383, a strain without dsRNAs. These results suggest that the dsRNA molecules also encode a killer system. Finally, the UV-S2 strain maintains killer ability, which suggests that S2-dsRNA is not involved in the killer phenotype expression.

Extrachromosomal Inheritance↗
PubMed1976

Replication of small plasmids in extracts of Escherichia coli.

Sakakibara and Tomizawa (1974a) have described a soluble in vitro system that can carry the semi-conservative replication of the Co1 E1 plasmid. However, the usefulness of this system is restricted by its rapid inactivation during storage. This paper describes a stable soluble system prepared by freeze-thaw lysis of chloramphenicol-treated E. coli cells which replicates added Co1 E1 and C1o DF13 DNA. It differs from the system employed by Sakakibara and Tomizawa in two important points: (1) Its replicative capacity for Co1 E1 DNA is by an order of magnitude higher and (2) it can be stored in liquid nitrogen for several months without loss of activity Plasmid replication in vitro is dependent of DNA polymerase I and requires de novo RNA synthesis. It is completely inhibited by rifampicin, oxolinic acid, and novobiocin. The DNA synthesized during a 60 min incubation at 30 degrees C consists mostly of monomeric supercoils. If Co1 E1 DNA is used as template, a minor portion of the label is also found in closed dimeric catenanes. Density labelling experiments indicate that plasmid DNA synthesis occurs by a semi-conservative replication process.

Cell-Free System↗
PubMed1976

Expression of the plasmid pKM101--determined DNA repair system in recA- and lex- strains of Escherichia coli.

pKM101, a plasmid R factor of the N compatibility group increases methylmethane sulfonate mutagenesis and diminishes UV-killing in recA+ LEX+ and recA+ lex- strains, , but not in recA-lex+ strains. The induction of a "reclex" dependent colicin is not present in lex- strains carrying the pKM101 factor. These facts indicate that pKM101 acts through an error-prone DNA repair system which is recA+ dependent, but not lex+ dependent.

Conjugation, Genetic↗
PubMed1991

Characterization of a novel open reading frame, urf a, in the mitochondrial genome of fission yeast: correlation of urf a mutations with a mitochondrial mutator phenotype and a possible role of frameshifting in urf a expression.

Between the genes for tRNA(gin) and tRNA(ile) an open reading frame of 227 amino acids has been identified which is unique among known mitochondrial genomes and which has been termed urf a (Lang et al. 1983; Kornrumpf et al. 1984). It uses the "mitochondrial" genetic code, i.e., it contains a TGA codon, whereas all other protein-encoding genes, and all but one intronic open reading frame, use the "standard" genetic code (UGG for tryptophan). A previous paper has demonstrated that "mutator" strains show an increased formation of mitochondrial drug-resistant and respiration-deficient mutants (including deletions). In this paper we show that the mutator activity is correlated with mutations in urf a. A detailed analysis of one urf a mutant is presented (anar-6), where the deletion of an A residue leads to a frameshift mutation and consequently to premature termination of the putative protein. The phenotype of colonies originating from a single mutant clone varies from no growth up to full growth on non-fermentable substrate. This phenomenon of phenotypic segregation can be explained by the ability of the cell to perform translational frameshifting. A detailed analysis of the DNA sequence and the putative urf a protein will be presented and a possible function of the protein will be discussed.

Amino Acid Sequence↗
PubMed1978

Free genes for rRNAs in the macronuclear genome of the ciliate Stylonychia mytilus.

When separated on an agarose gel, macronuclear DNA of the hypotrichous ciliate Stylonychia mytilus gives rise to many well-defined bands ranging in molecular weight from 0.3 x 10(6) to 14 x 10(6) dalton. Hybridization of 25 S rRNA, 17S rRNA or 5 S RNA to such a gel revealed sharp hybridization bands. This suggests that this banding pattern is not an artefact due to nonspecific degradation of macronuclear DNA but that the DNA in the macronucleus of Stylonychia occurs in discrete fragments, each coding for at least one gene. The size of the DNA fragment coding for rRNA was found to be 4.5 x 10(6) dalton, the fragment coding for 5 S RNA has a molecular weight of 150,000-250,000 dalton.

Animals↗
PubMed1985

Effect of concentration on the subsequent fate of plasmid DNA in human fibroblasts.

The physical fate of plasmid DNA after entry into human fibroblasts was studied using Southern hybridisation and electron microscopy. Exposure of the cells (5 X 10(5) per well) to pC194 DNA-CaPi, containing 50 micrograms plasmid DNA, resulted in the occasional formation of interlocked molecules. Exposure to a co-precipitate containing 100 micrograms pC194 plasmid DNA per well resulted in an increase of interlocked molecules by a factor of 10-20 relative to the number of monomers. In addition, new classes of molecules were observed. After prolonged incubation of the cells exposed to the higher DNA concentration, the plasmid DNA was partly contained in structures with a very low electrophoretic mobility. Upon restriction endonuclease digestion of the re-extracted DNA, a pattern of bands was observed, suggesting the involvement of illegitimate recombination between non-random plasmid DNA sequences in the formation of the new classes of molecules.

Cells, Cultured↗
PubMed1989

Extrachromosomal circular DNAs in Drosophila melanogaster: comparison between embryos and Kc0% cells.

We established the size distribution of extrachromosomal covalently closed circular DNA molecules from embryos of various Drosophila melanogaster strains and from Kc0% tissue culture cells. In embryos, more than 80% of the circular DNA molecules are smaller than 2.5 kb and all the distributions show a peak of molecules of between 200 and 400 bp. The Kc0% cell distribution differs mainly from that of embryos in that 48% of the molecules have a size between 4 and 8 kb. Correlating with this, circular molecules homologous to copia, 412 and 297 were detected only in Kc0% cells. The three tandemly repeated families containing the 5S genes, the histone genes and the 240 bp repeat of the ribosomal DNA intergenic spacer, which had previously been identified in circular DNAs from embryos, were also found in cultured cells. A fourth tandemly repeated family corresponding to the 1.688 g/cm3 satellite DNA was detected, both in embryos and Kc0% cells. It consists of circular multimeric molecules containing multiple copies of the 359 bp repeated unit. No circular DNA molecules homologous to the actin genes, the type I ribosomal DNA insertion, or the F and I transposable elements were found in embryos or Kc0% cells. Thus it appears that the extrachromosomal circular DNA molecules from embryos and from tissue culture cells differ mainly in the presence of circular copies of the copia-like transposable elements.

Animals↗
PubMed1975

Studies on a temperature sensitive nuclear petite mutant of Saccharomyces cerevisiae: phenotypic reversibility of the mitochondrial functions.

1. We have studied the pleiotropic effect of a single-gene mutation of the pts mutant strain 1511 grown at 23 degrees C and 36 degrees C. 2. Growth of the mutant at the non-permissive temperature results in a decrease of respiration rate to about 50% after one generation and to less than 5% after five generations. The cytochrome spectra analysis revealed that only cytochrome c was present after growth at 36 degrees C. 3. Mitochondrial protein synthesis experiments in vivo demonstrated that the protein synthesizing system was not as rapidly inactivated by high temperature as the respiratory system. 4. The recovery of the respiratory capacity of the cells at 23 degrees C is complete but dependent on the de novo synthesis of a temperature sensitive protein.

Cell Nucleus↗