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R Sager

Publications and source records attributed to R Sager.

At least 145 records · Page 8Linked to original sources
PubMed1979

Cytoplasmic genetics of mammalian cells: conditional sensitivity to mitochondrial inhibitors and isolation of new mutant phenotypes.

We report here that glucose, as a carbon source, and pyruvate are required for the phenotypic expression of cytoplasmically transmitted chloramphenicol-resistance (CAP-R) mutations, recovery of CAP-R mutants, and continuous growth in the presence of oligomycin or antimycin. We assume that glucose supplies additional energy when mitochondrial respiration is diminished and that pyruvate provides intermediates when the Krebs cycle is inhibited. Thus, the requirement for pyruvate is fully satisfied by an exogenous source of purines, and partially by alpha-ketoglutarate or a pyrimidine source. Based upon these findings, we have obtained two types of mutations affecting mitochondrial function--oligomycin resistance and pyruvate-independent expression of chloramphenicol resistance. Both are cytoplasmically transmitted and provide new markers for a genetic analysis of mitochondrial biogenesis.

Animals↗
PubMed1979

Methylation of chloroplast DNAs in the life cycle of Chlamydomonas.

Methylation patterns of Chlamydomonas chloroplast DNAs (chlDNAs) were examined in the vegetative, gametic, and zygotic stages of the life cycle. Restriction endo-nuclease fragment patterns produced by EcoRI, BamHI, Hpa II, and Msp I were compared; the last two cleave DNA at the sequence C-C-G-G, but Hpa II is blocked by prior methylation of the internal cytidine whereas Msp I is not. chlDNAs from vegetative cells of both mating types showed no evidence of methylation at C-C-G-G. Gametic mt+ chlDNA was heavily methylated at C-C-G-G, whereas the homologous chlDNA from mt- gametes showed very slight methylation at C-C-G-G. Methylation of additional sites in chlDNA from mt+ gametes but not from mt- gametes was shown by blockage of some EcoRI and BamHI sites that were cleaved in the chlDNA from vegetative cells. chlDNA from 6-hr zygotes was much more methylated than gametic mt+ DNA, as shown by its almost total resistance to cleavage by all four restriction enzymes. These findings support and extend previous evidence that chlDNA of mt+ cells is methylated during gametogenesis and that further methylation occurs after gametic fusion in the young zygotes.

Journal Article↗
PubMed1979

Role of methylation in the modification and restriction of chloroplast DNA in Chlamydomonas.

The different metabolic paths followed by homologous chloroplast DNAs of maternal and paternal origins in zygotes of Chlamydomonas were examined by prelabeling parental cells, before mating them, with [3H]adenine, [3H]thymidine, and [3H]deoxycytidine. Within 6 hr after mating, maternal chloroplast DNA was extensively methylated to 5-methylcytosine and its bouyant density decreased. Paternal chloroplast DNA was largely degraded. Some radioactivity from deoxycytidine of maternal origin reappeared in thymine, and residual paternal DNA contained radioactivity in a base tentatively identified as uracil. These results confirm and extend our previous findings and support our hypothesis that modification (methylation) and restriction enzymes determine maternal inheritance of chloroplast DNA and that the two parental DNAs have different metabolic fates within the zygote.

Adenine↗
PubMed1978

Genetic analysis of tumorigenesis: I. Expression of tumor-forming ability in hamster hybrid cell lines.

Two new fibroblasts cell lines from a male Chinese hamster embryo are described: one tumorigenic in nude mice (CHEF 16-2) and the other not (CHEF 18-1). Both lines have a stable diploid mode and mean of 22 with 10 pairs of homologous autosomes. The cell lines differ unambiguously in cloning ability in methylcellulose, colony morphology, and tumorigenicity; the expression of these traits was examined in a set of 18-1 X 16-2 hybrid clones. The results show initial suppression of tumorigenicity and anchorage independence, followed by growth in the nude mouse of subsets of cells with chromosomes reduced to the diploid range. Further studies are in progress to establish whether ability to grow in methylcellulose and in the nude mouse segregate coordinately at the cellular level, and whether this segregation has an identifiable chromosomal basis.

Cell Line↗
PubMed1978

Polyethylene-glycol-mediated cybrid formation: high-efficiency techniques and cybrid formation without enucleation.

Polyethylene glycol (PEG) can be used to promote the fusion of enucleated cytoplasts from chloramphenicol (CAP)-resistant mouse cells with intact cells, resulting in the formation of viable cybrids. The techniques are simple and highly efficient, yielding up to one viable cybrid per 20 intact cells fused. It also seems that PEG can be used to induce cybrid formation without the necessity of prior enucleation of the CAP-resistant cells.

Animals↗
PubMed1978

Tumorigenicity and its suppression in cybrids of mouse and Chinese hamster cell lines.

The effect of cytoplasm upon the expression of tumorigenicity was examined with a pair of mouse and a pair of Chinese hamster cell lines, in intraspecies cybrids formed by reciprocal fusions between either tumorigenic or nontumorigenic cells and cytoplasms derived from them. With the mouse cells, 3T3 and the simian virus 40-transformed line SVT2, the cybrid clones were tumorigenic when SVT2 cells were fused with 3T3 cytoplasts, but not in the reciprocal fusion. With the hamster cells, CHEF/18 and the spontaneous transformant CHEF/16, however, tumorigenicity was partially suppressed in cybrid clones formed by fusion of tumorigenic CHEF/16 cells with CHEF/18 cytoplasts; cybrids were nontumorigenic in the reciprocal fusion. Thus, cybrid analysis has shown that tumorigenicity is not cytoplasmically transmitted in these two cell pairs, but suppression of tumor-forming ability may be cytoplasmically transmitted in the hamster cybrids.

Animals↗
PubMed1977

A site-specific single-strand endonuclease from the eukaryote Chlamydomonas.

We have found a unique deoxyribonuclease in extracts of the eukaryotic green alga Chlamydomonas. When incubated with viral DNA from adenovirus-2, this enzyme produces discrete fragments that form bands upon electrophoresis in an agarose gel. Site specificity of the enzymatic cleavage examined by identifying the 5'-terminal nucleotides in cleaved adenovirus-2 DNA and by studies with synthetic polynucleotides of defined sequence, indicates that the initial endonucleolytic cleavage occurs at a site containing a deoxythymidine residue. Electron microscopy of cleaved adenovirus-2 DNA revealed single-strand segments within duplex DNA. We propose that the enzyme acts by making initial site-specific single-strand incisions, followed by subsequent excision on the same strand, producing a gapped duplex molecule; and that double-strand scissions result from limited occurrence of overlapping single-strand gaps on complementary strands.

Base Sequence↗
PubMed1975

Identification of a chloroplast ribosomal protein altered by a chloroplast mutation in Chlamydomonas.

Direct evidence is presented that a chloroplast gene mutation in Chlamydomonas reinhardi alters one of the chloroplast ribosomal proteins. Proteins of 30 S subunits of chloroplast ribosomes from mutant strains, carrying maternally inherited antibiotic resistances, were compared with those from the wild type strain by CM-cellulose column chromatography and gel electrophoresis. When 30 S ribosomal proteins from a [3H]arginine-labeled streptomycin-resistant strain and a [14C]arginine-labeled wild type strain, or vice versa, were cochromatographed on a CM-cellulose column, one peak (Peak 17) was absent from the mutant profile. Instead, a pronounced peak was observed to elute at a slightly lower ionic strength than Peak 17 in the region of Peak 16. The molecular weights in both Peak 16 and Peak 17 regions determined by discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis were indistinguishable, approximately 18,000. Thus, a chloroplast gene mutation to streptomycin resistance has altered the chromatographic behavior of a chloroplast ribosomal protein of the 30 S subunit. We interpret the additional protein in the mutant eluting at Peak 16 as most likely the mutationally altered form of the Peak 17 protein.

Cell Fractionation↗
PubMed1974

Mutations that alter the transmission of chloroplast genes in Chlamydomonas.

Two mutations are described that alter the pattern of inheritance of chloroplast genes in Chlamydomonas. The mutant gene mat-1 linked to the mating type allele mt(-) greatly increases the frequency of exceptional zygotes, i.e., zygotes that transmit chloroplast genes from the mt(-) (male) parent. In some crosses, 80-90% of the zygotes are biparental, transmitting chloroplast genes from both parents. The mat-2 mutation, linked to mt(+), acts to decrease the frequency of exceptional zygotes below the spontaneous level. The mutant effects are discussed in terms of a DNA modification-restriction system, postulated to regulate the transmission of chloroplast DNA in zygotes.

Cell Nucleus↗
PubMed1974

Localization of five antibiotic resistances at the subunit level in chloroplast ribosomes of Chlamydomonas.

The chloroplast ribosomes from five antibiotic resistant strains of Chlamydomonas, each carrying one mutant gene mapping in chloroplast DNA, have been shown to be resistant to the corresponding antibiotic in a poly(U)-directed amino-acid incorporating assay system. The alteration conferring resistance was localized to the 30S subunit in ribosomes from streptomycin, neamine, and spectinomycin resistant strains, and to the 50S subunit in ribosomes from cleocin and carbomycin resistant strains. Spectinomycin resistant ribosomes showed no cross-resistance to any other drugs, but limited cross-resistance was noted with the other mutant ribosomes. The similarity between these findings and results reported by others with bacterial ribosomes supports our hypothesis that at least some chloroplast ribosomal proteins are coded by genes in chloroplast DNA.

Chlamydomonas↗
PubMed1974

Regulation of nuclear DNA replication by thechloroplast in Chlamydomonas.

The experiments described in this paper implicate chloroplast protein synthesis in the regulation of nuclear DNA replication. The inhibition of nuclear DNA replication in the lower eukaryote, Chlamydomonas reinhardi strain 21gr, was examined after growth of cells with a series of antibiotics (streptomycin, neamine, spectinomycin, cleocin, chloramphenicol, and rifampicin) each of which has a known effect upon chloroplast RNA or protein synthesis in this organism. Each antibiotic inhibited nuclear DNA replication at drug concentrations at which there was little or no inhibition of adenine incorporation into chloroplast DNA. That chloroplast DNA was replicating under these conditions rather than merely being repaired, was shown first by the high incorporation rates and second by a (14)N-(15)N density transfer experiment in which chloroplast DNA doubled in the presence of streptomycin, while no incorporation into nuclear DNA was detected. A small DNA peak, Component III, located between nuclear and chloroplast DNA's in CsCl gradients, possibly mitochondrial, was more pronounced in DNA from antibiotic-inhibited cultures than from controls.

Adenine↗
PubMed1972

Mutation of a cytoplasmic gene in Chlamydomonas alters chlorplast ribosome function.

A mutation, car, determining resistance to several macrolide antibiotics, including carbomycin, has been identified in the alga Chlamydomonas as cytoplasmic, and mapped in the known cytoplasmic linkage group close to genes determining resistance to other antibiotics, including streptomycin, erythromycin, and spectinomycin. The effect of the car mutation on chloroplast ribosome function was demonstrated with an in vitro system incorporating amino acids especially developed to assess activity of 70S chloroplast ribosomes. In an S-30 extract containing both 70S chloroplast and 80S cytoplasmic ribosomes, low concentrations of Mg(++) and spermidine favored 80S ribosome activity, and high concentrations activated 70S ribosomes and reversibly inactivated the 80S component. Under conditions favoring chloroplast ribosome activity, carbomycin inhibited incorporation by an S-30 extract, and by purified 70S ribosomes from wild-type but not from car cells. These results show that cytoplasmic genes are directly involved in chloroplast ribosome function and they suggest that the car gene product is a ribosomal protein; the results further strengthen the evidence that the cytoplasmic linkage group is located in chloroplast DNA.

Amino Acids↗
PubMed1972

Molecular basis of maternal inheritance.

The mechanism of preferential transmission (i.e., maternal inheritance) of cytoplasmic genes was investigated with chloroplast DNA of Chlamydomonas as a model system. The behavior of nuclear and chloroplast DNAs were compared in the sexual cycle; DNAs from male and female parents were distinguished by labeling with (14)N- or (15)NH(4)Cl and then by making the crosses: (14)N (female) x (15)N (male) and the reciprocal. Chloroplast DNAs from the two parents followed different paths in the zygote, but nuclear DNAs showed no differences. Chloroplast DNA from the female parent persists in the zygote, but undergoes a density shift of 0.003-0.005 g/cm(3) to a lighter buoyant density, whereas that from the male disappears soon after zygote formation. The possibility is discussed that a modification-restriction system may be involved.

Alleles↗