Interaction of actinomycin D with yeast ribosomal RNA

Waltschewa, Liliana W.

doi:10.1016/0014-5793(80)80787-3

Cited by 4 publications

(2 citation statements)

References 11 publications

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“…In vitro RNA pol and primase assays were performed on the DNP/RNP samples after removal of SDS by conventional procedures [Waltschewa, 1980;Heberlein et al, 19851.…”

Section: Isolation Of Subcomplex Dnp/rnpsmentioning

confidence: 99%

Nucleoproteins derived from subnuclear RNA polymerase complexes of metastatic large‐cell lymphoma cells possess transcription activities and regulatory properties in vitro

Rosenberg-Nicolson

Nicolson

1992

J of Cellular Biochemistry

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Intact nuclei derived from poorly or highly liver-metastatic murine large-cell lymphoma cell line RAW117 were digested to discrete subchromatin deoxyribonucleoprotein/ribonucleoprotein (DNP/RNP) complexes with Msp-I. The DNP/RNP complexes were composed of DNP/RNPs which were derived from the DNP/RNP complexes by incubation in the presence or absence of DNase-I and subsequent isolation by two-dimensional [isoelectric focusing/sodium dodecylsulfate (SDS)] polyacrylamide gel electrophoresis (PAGE), electroelution from the gel, and removal of SDS. Approximately 450 DNP/RNPs in the two-dimensional gels corresponding to discrete spots or in some cases streaks were analyzed for the presence of v-abl, p53, c-neu, c-H-ras, beta-casein, 18s rDNA, and mu-chain immunoglobulin genes using a hybridization technique. Ten DNP/RNP complexes contained tightly associated p53 DNA, whereas six contained c- or v-abl, four contained mu-chain gene, two contained c-H-ras, one contained dot-blot beta-casein, two contained 18s rDNA, and c-neu was found in one of the DNP/RNPs. The DNP/RNPs were also analyzed for in vitro RNA polymerase and primase activities. To assess the potential transcription abilities of the isolated DNP/RNPs, individual DNP/RNPs or DNP/RNP mixtures (reconstituted after SDS-PAGE separation) were examined for RNA polymerase initiation and synthesis. When RNA products were formed, these were purified by extracellulose chromatography and used as back-hybridization probes for the genes of interest. The RNA products were also analyzed by RNA gel electrophoresis. RNA formation was inhibitable by actinomycin D, and the RNAs formed ranged in size from approximately 80 kbp to approximately 1 kbp. By mixing various DNP/RNP complexes together, different patterns of RNA synthesis were found. For example, one DNP/RNP of M(r) approximately 140,000, isoelectric point (pl) approximately 5.8 synthesized a high molecular weight RNA in vitro that hybridized with beta-casein cDNA, but beta-casein is not expressed in RAW117 cells, suggesting that the silencing of the beta-casein gene was negated by isolation of the DNP/RNP. Mixing this DNP/RNP with two other specific DNP/RNPs again inhibited the synthesis of beta-casein RNA, suggesting that interactions between DNP/RNP complexes can result in differential RNA expression or regulation of RNA polymerases in vitro.

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“…In vitro RNA pol and primase assays were performed on the DNP/RNP samples after removal of SDS by conventional procedures [Waltschewa, 1980;Heberlein et al, 19851.…”

Section: Isolation Of Subcomplex Dnp/rnpsmentioning

confidence: 99%

Nucleoproteins derived from subnuclear RNA polymerase complexes of metastatic large‐cell lymphoma cells possess transcription activities and regulatory properties in vitro

Rosenberg-Nicolson

Nicolson

1992

J of Cellular Biochemistry

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“…While glucan supports and maintains the rigidity of the cell wall, the mannoprotein determines its permeability (Zlotnik et ul.. 1984). We have isolated and studied cell wall mutants characterized by cell lysis in hypotonic solutions and increased permeability to substances for which yeasts are known to be impermeable (Venkov et at.. 1974;Waltschewa et at., 1989). The genetic analysis of VY 1 160 fragile mutant identified two mutations, srhf and t s f , responsible for the mutant phenotype (Kozhina et at., 1979).…”

Section: Introductionmentioning

confidence: 99%

Glucan structure in a fragile mutant of Saccharomyces cerevisiae

Blagoeva

Stoev

Venkov

1991

Yeast

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The phenotype of VY1160 fragile Saccharomyces cerevisiae mutant is characterized by cell lysis upon transfer to hypotonic solutions and increased permeability of cells growing in osmotically stabilized media. Two mutations, srb1 and ts1, have been identified in VY1160 cells and previous studies have shown that the increased permeability is due to the ts1 mutation which causes a shortening of mannan side-chains. Here we report that the srb1 mutation, which is the genetic determinant of cell lysis, is responsible for quantitative and structural changes of glucans. Experiments with isogenic single mutation strains, genetic studies coupled with quantitative measurements of glucan content per cell, and methylation analysis of glucans provide evidence that srb1 mutation leads to i) formation of mechanically unstable cell wall network made of insoluble glucan fibrils which are shorter and contain beta(1-6) inter-residue linkages and ii) insufficient filling of the space between the fibrils due to a shortage of the alkali-soluble glucan. Although growing exponentially in osmotically stabilized media, the srb1 cells cannot resist an osmotic shock and, hence, burst immediately.

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