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J R Warner

Publications and source records attributed to J R Warner.

At least 91 records · Page 5Linked to original sources
PubMed1982

Mutation-induced instability of antibiotic-resistant mammalian ribosomes.

Emetine is an antibiotic which inhibits the activity of the 40-S subunits of eukaryotic ribosomes. A number of emetine-resistant mutants of Chinese hamster cells have been isolated. We have studied the ribosomes isolated from strains carrying either of two alleles of an emetine resistance locus, one of which leads to an alteration in the electrophoretic mobility of ribosomal protein S14. Both mutations lead to a loss of specific proteins, including S14, from 40-S subunits isolated in 0.5 M KCl, an effect which is enhanced at elevated temperature. 40-S subunits from wild-type cells retain their proteins even at substantially higher concentrations of KCl. Interestingly, the two mutant alleles lead to the loss of slightly different sets of proteins from the 40-S subunits. These results suggest that protein S14 plays an important role in the structure of the ribosome and raise the possibility that resistance to emetine is accomplished through an alteration in a complex emetine-binding site rather than in a single emetine-binding protein.

Animals↗
PubMed1981

The genes for fifteen ribosomal proteins of Saccharomyces cerevisiae.

We have isolated recombinant lambda phage carrying the genes for 14 of the ribosomal proteins of the yeast Saccharomyces cerevisiae. Analysis of these and of the plasmid carrying the gene tcm1, which codes for the ribosomal protein responsible for resistance to trichodermin, demonstrates that in general the genes for ribosomal proteins are unlinked. One exceptional recombinant carries the genes for two ribosomal proteins within a 2-kilobase region. DNA fragments bearing individual ribosomal protein genes were used to probe restriction digests of the yeast genome to determine whether any of the genes were duplicated. Only 3 of 12 of the genes are present unequivocally as a single copy. Similar fragments were used to probe blots of mRNA separated on denaturing agarose gels to determine the size of the mRNA for each protein. In each case, the mRNA is near the minimum size necessary to code for its protein. In certain temperature-sensitive mutants which fail to synthesize functional mRNA for ribosomal protein, Rosbash et al. (Rosbash, M., Harris, P. K. W., Woolford, J., and Teem, J. L. (1981) Cell, 24, 679-686) have demonstrated the accumulation of a larger RNA molecule, homologous to a ribosomal protein gene, that appears to be a transcript which retains an intervening sequence. We find that for 8 of the 11 ribosomal protein genes examined, a larger molecule accumulates in such a mutant strain, suggesting that in general transcripts of ribosomal protein genes may have introns.

Bacteriophage lambda↗
PubMed1981

Cloning of yeast gene for trichodermin resistance and ribosomal protein L3.

Yeast cells sensitive to the eukaryotic protein synthesis inhibitor trichodermin have been transformed with autonomously replicating recombinant plasmids carrying DNA fragments of the genome of a trichodermin-resistant yeast strain. After selection for trichodermin-resistant cells, several transformants yielded a plasmid containing a 13.5-kilobase (kb) DNA fragment that encodes the trichodermin resistance gene, tcm1, and the gene for ribosomal protein L3, the largest of the yeast ribosomal proteins. Cells carrying this plasmid are resistant to trichodermin and to the related drug verrucarin A as well as to the unrelated drug anisomycin. This pattern of resistance is similar to that exhibited by strains carrying a chromosomal copy of tcm1. Moreover, polyribosomes prepared from transformed cells are resistant to trichodermin when tested in an in vitro protein synthesis assay. Subcloning of the 13.5-kb DNA fragment revealed that the gene for tcm1 and the gene for protein L3 are contained within a 3.2-kb segment. These results suggest that the gene for trichodermin resistance in yeast specifies ribosomal protein L3.

Anisomycin↗
PubMed1981

Coordinate control of syntheses of ribosomal ribonucleic acid and ribosomal proteins during nutritional shift-up in Saccharomyces cerevisiae.

We investigated the regulation of ribosome synthesis in Saccharomyces cerevisiae growing at different rates and in response to a growth stimulus. The ribosome content and the rates of synthesis of ribosomal ribonucleic acid and of ribosomal proteins were compared in cultures growing in minimal medium with either glucose or ethanol as a carbon source. The results demonstrated that ribosome content is proportional to growth rate. Moreover, these steady-state concentrations are regulated at the level of synthesis of ribosomal precursor ribonucleic acid and of ribosomal proteins. When cultures growing on ethanol were enriched with glucose, the rate of ribosomal ribonucleic acid synthesis, measured by pulsing cells with [methyl-3H]methionine, increased by 40% within 5 min, doubled within 15 min, and reached a steady state characteristic of the new growth medium by 30 min. Labeling with [3H]leucine reveal a coordinate increase in the rate of synthesis of 30 or more ribosomal proteins as compared with that of total cellular proteins. Their synthesis was stimulated approximately 2.5-fold within 15 min and nearly 4-fold within 60 min. The data suggest that S. cerevisiae responds to a growth stimulus by preferential stimulation of the synthesis of ribosomal ribonucleic acid and ribosomal proteins.

Cell Division↗
PubMed1981

Hierarchy of elements regulating synthesis of ribosomal proteins in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells respond to a heat shock by temporarily slowing the synthesis of ribosomal proteins (C. Gorenstein and J. R. Warner, Proc. Natl. Acad. Sci. U.S.A. 73:1574-1551, 1976). When cultures growing oxidatively on ethanol as the sole carbon source were shifted from 23 to 36 degrees C, the synthesis of ribosomal proteins was coordinately inhibited twice as rapidly and 45% more severely than in comparable cultures growing fermentatively on glucose. Within 15 min, the relative rates of synthesis of at least 30 ribosomal proteins declined to less than one-sixth their initial values, whereas the overall rate of protein synthesis increased at least threefold. We suggest that this is due primarily to controls at the level of synthesis of messenger ribonucleic acid for ribosomal proteins but may also involve changes in messenger ribonucleic acid stability. In contrast, a nutritional shift-up causes a stimulation of the synthesis of ribosomal proteins. Experiments designed to determine the hierarchy of stimuli affecting the synthesis of these proteins demonstrated that temperature shock was dominant to glucose stimulation. When a culture growing on ethanol was shifted from 23 to 36 degrees C and glucose was added shortly afterward, the decline in ribosomal protein synthesis continued unabated. However, in wild-type cells ribosomal protein synthesis began to recover within 15 min. In mutants temperature sensitive for ribosome synthesis, e.g., rna2, there was no recovery in the synthesis of most ribosomal proteins, suggesting that the product of rna2 is essential for the production of these proteins under all vegetative conditions.

Ethanol↗
PubMed1979

Hybrid mammalian cells assemble hybrid ribosomes.

Hybrid cell lines formed by fusion of mouse 3T3 cells and Chinese hamster ovary (CHO) cells resistant to emetine, which have an altered 40S ribosomal protein, are generally sensitive to emetine. From most hybrid lines it was possible to select sublines resistant to emetine. The ribosomal components of three lines were studied: A34, an emetine-sensitive hybrid; A34/R3, an emetine-resistant derivative of A34; and A72, an emetine-sensitive hybrid that did not give rise to emetine-resistant sublines. Genetic and biochemical evidence suggests that in A34 both the mouse emetine sensitivity gene and the hamster emetine resistance gene are active, whereas in A34/R3 only the hamster emetine resistance gene is active and in A72 only the mouse emetine sensitivity gene is active. The ribosomes of all three sublines contained both mouse and hamster RNA, predominantly mouse. However, the 60S subunits had roughly equal amounts of the three mouse and hamster proteins that could be distinguished by two-dimensional electrophoresis, suggesting the association of mouse RNA with hamster ribosomal proteins. The emetine-resistant and emetine-sensitive 40S subunits could be separated by sedimentation in 0.5 M KCl. Resistant subunits contained predominantly mouse RNA, presumably associated with the hamster protein conferring emetine resistance. We conclude that hybrid cells can form hybrid ribosomes and that the amounts of ribosomal RNA and ribosomal protein of each species are not closely coupled.

Animals↗
PubMed1979

Insulin-stimulated protein phosphorylation in 3T3-L1 preadipocytes.

A protein of molecular weight 31,000 became labeled with 32P within 5 min after addition of insulin to differentiated 3T3-L1 preadipocytes previously incubated for 55 min with 32Pi. The effect was mimicked by antisera directed against the insulin receptor and was eliminated by anti-insulin antiserum. Incorporation of 32P into this protein was more than 20-fold greater in insulin-treated cells than in cells not exposed to the hormone. At concentrations greater than required with insulin, epidermal growth factor and serum (1-5%) also stimulated phosphorylation whereas 1-isoproterenol, a beta-adrenergic agonist that increases intracellular accumulation of cyclic AMP, was without effect. The 31,000-dalton protein has been tentatively identified as ribosomal protein S6 by two-dimensional polyacrylamide gel electrophoresis. Incorporation of 32P into S6 could be detected within the same time period (5 min) and at the same insulin concentrations (0.1-1.0 nM) as are required to stimulate hexose uptake in both 3T3-L1 cells and mature mammalian adipocytes. The mechanism by which this phosphorylation either mediates or reflects the intracellular actions of insulin remains to be elucidated.

Adipose Tissue↗
PubMed1979

Distribution of newly formed ribosomal proteins in HeLa cell fractions.

The distribution of newly formed ribosomal proteins between cytoplasmic, nucleoplasmic, and nucleolar fractions of HeLa cells was determined. All but a few of the newly formed ribosomal proteins were concentrated 10- to 50-fold in the nucleolus and two- to fivefold in the nucleoplasm. Nevertheless, substantial amounts were found in the cytoplasm. Pretreatment of cells with actinomycin D to deplete the nucleolar pool of ribosomal precursor RNA had no effect on the concentration of newly formed ribosomal proteins in the nucleus, but did lead to an increased amount in the nucleoplasm at the expense of the nucleolus.

Cell Nucleolus↗
PubMed1977

Synthesis and turnover of ribosomal proteins in the absence of 60S subunit assembly in Saccharomyces cerevisiae.

We have measured the synthesis and stability of ribosomal proteins in a temperature sensitive strain of yeast which at the restrictive temperature is specifically blocked in the processing of 27S ribosomal precursor RNA. We find that in the absence of 60S ribosomal subunit assembly, the synthesis of all the ribosomal proteins studied continued. However, the proteins of the 60S subunit fail to accumulate and are rapidly degraded.

Electrophoresis, Polyacrylamide Gel↗
PubMed1977

Noncoordinated transcription in the absence of protein synthesis in yeast.

In the yeast Saccharomyces cerevisiae we have carried out a detailed study of the response of transcription to the inhibition of translation. Measurement of the incorporation of labeled bases and nucleosides into the nucleoside triphosphate pools revealed that amino acid deprivation brings about a 10- to 50-fold inhibition of such labeling. Therefore accurate comparisons of RNA synthesis using such precursors are difficult to obtain. To overcome this problem we have turned to the use of L-[methyl-3H] methionine as a precursor, because the labeling of the S-adenosylmethionine pool is relatively unaffected by the rate of protein synthesis. Using this precursor, we have observed that in the absence of protein synthesis the transcription of ribosomal RNA is reduced by 80%, the transcription of messenger RNA is reduced by about 25%, and the transcription of transfer RNA is reduced by less than 20%. These results are obtained when protein synthesis is inhibited either by deprivation of an amino acid or by the addition of cycloheximide. Ribosomal precursor RNA synthesized in the absence of protein synthesis is fully methylated. We conclude that the transcription of rRNA is the primary target of stringent control. Furthermore the inhibition of protein synthesis, itself, may be the trigger for this response.

Adenine↗