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C S McLaughlin

Publications and source records attributed to C S McLaughlin.

At least 37 records · Page 2Linked to original sources
PubMed1984

The effects of paromomycin on the fidelity of translation in a yeast cell-free system.

The effects of the aminoglycoside antibiotic paromomycin on the fidelity of translation of the synthetic template poly(U), and two natural mRNAs (rabbit globin mRNA and Brome Mosaic virus RNA), were examined in an mRNA-dependent cell-free system from the yeast Saccharomyces cerevisiae. At antibiotic concentrations that did not inhibit translation (100 microM) optimal mistranslation of all three templates was observed, with the effects declining at higher antibiotic concentrations. Synthesis of the opal termination read-through protein of rabbit beta-globin mRNA was induced by paromomycin, but only in lysates prepared from a [psi+] strain of yeast. The antibiotic did not induce detectable levels of either ochre or amber read-through, but did induce general misreading of Brome Mosaic virus RNA to the same degree in both [psi+] and [psi-] lysates. This misreading was enhanced by addition of the polyamine spermidine.

Animals↗
PubMed1984

The effect of non-permissive temperature on met-tRNA synthetase in Saccharomyces cerevisiae temperature-sensitive mutant ts 7-45.

The effects of incubation of yeast spheroplasts at elevated temperature (40 degrees C) on a number of activities involved in protein biosynthesis have been examined in preparations obtained from wild-type cells (wt A364A ) and a temperature-sensitive mutant (ts 7-45) derived from it. With wild-type cells, preincubation of spheroplasts at the elevated temperature had little or no effect on the following: the ribosomal subunit-polysome pattern; the translation of exogenous natural mRNA in postpolysomal extracts devoid of endogenous mRNA; the translation of poly(U) in postpolysomal extracts; the incorporation of methionine into 40 S preinitiation and 80 S initiation complexes; the synthesis of Met-tRNA in postribosomal (cytosol) extracts; and the formation of eIF-2 X GTP X Met-tRNAf ternary complex in the cytosol. With temperature-sensitive spheroplasts that had not been preincubated at the elevated temperature, the concentration of free, native 40 S subunits appeared to be lower and that of 60 S subunits higher than in wild-type cells; translation of exogenous natural mRNA in postpolysomal extracts was somewhat lower than in wild-type preparations, but all of the other reactions and components measured were comparable to those in wild-type preparations. Preincubation of temperature-sensitive spheroplasts at 40 degrees C resulted in: a further decrease in the level of 40 S subunits; disaggregation of polysomes; loss of ability to translate natural mRNA but not poly(U); decreased ability to form 40 S preinitiation intermediates; and production of an activity, found in the cytosol, that inhibited Met-tRNA synthetase reversibly. The inhibitor had the characteristics of a protein and did not appear to be a proteinase, nuclease, or nucleotidase.

Amino Acyl-tRNA Synthetases↗
PubMed1983

In vitro nonsense suppression in [psi+] and [psi-] cell-free lysates of Saccharomyces cerevisiae.

An homologous in vitro assay for yeast nonsense suppressors was used to examine the effect of the cytoplasmically inherited genetic determinant [psi] on the efficiency of in vitro nonsense suppression. The efficiency of all three types of yeast tRNA-mediated nonsense suppressor (ochre, amber, and UGA) is much greater in cell-free lysates prepared from a sup+ [psi+] strain than in lysates prepared from an isogeneic sup+ [psi-] strain. Lysates prepared from a [psi-] strain, into which the [psi+] determinant was reintroduced by kar1-mediated cytoduction, support efficient suppression. Evidence is also presented that [psi-] lysates contain an inhibitor of in vitro nonsense suppression.

Cell-Free System↗
PubMed1982

Analysis of temperature-sensitive mutant ts 187 of Saccharomyces cerevisiae altered in a component required for the initiation of protein synthesis.

Postpolysomal extracts have been prepared from wild type haploid Saccharomyces cerevisiae cells (wt A364A) and from a temperature-sensitive mutant strain (ts 187, gene prt 1). The extracts, prepared via spheroplasts and depleted of endogenous mRNA with nuclease, translate exogenous natural mRNA and polyuridylic acid. The activity of wt A364A with respect to translation of yeast mRNA, poly(U)-dependent synthesis of polyphenylalanine which measures elongation components, reactions involved in the initiation of protein synthesis, and termination and release of polypeptides, is not significantly affected when spheroplasts are incubated at 39 degrees C for relatively short periods of time, prior to the preparation of the cell-free system. With extracts obtained from ts 187 cells, preincubation of spheroplasts at 39 degrees C prior to the preparations of the cell-free system markedly decreases the ability to translate natural mRNA but not poly(U). Compared to extracts from unheated spheroplasts, the following activities in ts 187 extracts from spheroplasts preincubated at 39 degrees C are not significantly affected: activation of methionine and methionylation of tRNAMet; formation of (eukaryotic initiation factor 2.Met-tRNAf.GTP] ternary complex; binding of mRNA to 40S preinitiation intermediate containing Met-tRNAf, and joining of 60S subunits to form the 80S initiation complex; elongation factor 1- and elongation factor 2-dependent elongations reactions; and termination and release of completed polypeptide chains. However, the interaction between the [eukaryotic initiation factor 2.Met-tRNAf.GTP] ternary complex and 40 S subunits, to form the 40 S preinitiation complex, is drastically inhibited by treatment of the spheroplasts at 39 degrees C.

Kinetics↗
PubMed1982

Synthesis of specific identified, phosphorylated, heat shock, and heat stroke proteins through the cell cycle of Saccharomyces cerevisiae.

The methods of centrifugal elutriation, two-dimensional gel electrophoresis, and dual isotopic labeling were applied to the study and identification of a number of purified yeast proteins. The location of polypeptide spots corresponding to specific proteins was determined on two-dimensional gels. A dual-label method was used to determine the rates of synthesis through the cell cycle of the identified proteins as well as to confirm the results of previous studies from our laboratory on unidentified proteins. The identified proteins, and the more generally defined phosphorylated, heat shock, and heat stroke proteins were found to follow the general pattern of exponential increase in rate of synthesis through the cell cycle. In addition, colorimetric enzyme activity assays were used to examine the catabolic enzyme alpha-glucosidase (EC 3.2.1.20). Both the activity and synthesis of alpha-glucosidase were found to be nonperiodic with respect to the cell cycle. These data contrast with earlier reports of periodicity, which employed induction and selection synchrony to study enzyme expression through the yeast cell cycle.

Cell Cycle↗
PubMed1982

Endogenous read-through of a UGA termination codon in a Saccharomyces cerevisiae cell-free system: evidence for involvement of both a mitochondrial and a nuclear tRNA.

Globin mRNA, translated in a Saccharomyces cerevisiae cell-free protein synthesizing system prepared from a [psi+ rho+] strain, primarily directed the synthesis of alpha- and beta-globin. A third globin mRNA-specific polypeptide was also synthesized, representing approximately 10% of the total translation products. This polypeptide (beta') was synthesized by translational read-through of the beta- globin mRNA UGA terminator and was mediated primarily by an endogenous tRNA coded for by the mitochondria. This mitochondrial tRNA, when charged, could be preferentially bound, in high salt, to benzoylated DEAE-cellulose, a characteristic of a tRNATrp. The synthesis of beta- mediated by this mitochondrial tRNATrp was significantly reduced when the translation system was prepared from an isogenic [psi-] strain. Evidence for a nuclear-coded tRNA, also able to suppress the beta-globin mRNA UGA terminator in [psi+] but not [psi-] lysates, was also obtained. The presence of these endogenous UGA suppressor activities in the yeast cell-free system should allow successful in vitro translation of mitochondrial mRNAs.

Cell Nucleus↗
PubMed1981

An homologous in vitro assay for yeast nonsense suppressors.

A cell-free translation system, from the yeast Saccharomyces cerevisiae, has been used to develop an in vitro assay for yeast UGA, ochre and amber suppressors. Amber suppression was assayed by read-through of the brome mosaic virus coat protein cistron UAG terminator. UGA suppression was assayed by read-through of the rabbit beta-globin UGA terminator and ochre suppression by read-through of the rabbit alpha-globin mRNA UAA terminator. Ochre suppression was increased 3-fold when the globin mRNA was heat denatured prior to translation; this was due to an increase in the synthesis of alpha-globin relative to beta-globin. Amber suppression was more efficient in vitro (46%) than ochre suppression (14%). UGA suppression was also highly efficient in vitro, reaching almost 100% using a purified UGA suppressor tRNA from Schizosaccharomyces pombe. Unfractionated yeast tRNA, from a sup+ strain, contained a tRNA species able to suppress UGA termination codons in vitro, but no tRNA species able to suppress either UAA or UAG was found. This homologous in vitro assay for yeast nonsense suppressors will allow, for the first time, an approach to the biochemical analysis of yeast mutants that modify the efficiency of nonsense suppression in vivo.

Cell-Free System↗
PubMed1981

Cell-cycle regulation of yeast histone mRNA.

The levels of H2A and H2B mRNAs as a function of cell-cycle stage were determined by hybridization methods. The analysis was extended to H3 and H4 mRNAs by in vitro translation. Cells were partitioned into cell-cycle stages either by centrifugal elutriation or by G1 synchronization with the yeast mating pheromone, alpha factor. The data lead to the following conclusions. First, histone mRNA can be detected in significant quantities only in S-phase cells. Second, the point of maximal accumulation of histone mRNA is not coincident with the point of maximal DNA synthesis; rather, histone mRNA begins accumulating very early in S, reaching a maximum when less than one half of the DNA has replicated. From this point in the cell cycle the histone mRNA levels decrease, reaching basal levels at the end of S. Third, in spite of the fact that the rate of histone mRNA accumulation is not coincident with the rate of DNA synthesis, the two processes are coupled; inhibition of DNA synthesis results in an extremely rapid disappearance of histone mRNA that is much shorter than the normal histone mRNA half-life. Fourth, there is no visible accumulation of mRNA precursors at any cell-cycle stage. We can conclude that, in yeast, histone mRNA levels are tightly and coordinately regulated throughout cell division and that this regulation most likely occurs at both transcriptional and posttranscriptional levels. We also show that the two genetically unlinked H2B genes present in yeast are both expressed at comparable levels and are regulated. The regulation is probably sequence-specific, since genes in close proximity to the histones are not subject to cell-cycle control.

Cell Cycle↗
PubMed1981

Replication of double-stranded RNA of the virus-like particles in Saccharomyces cerevisiae.

The mode of replication of the L double-stranded RNA (dsRNA) present in virus-like particles in Saccharomyces cerevisiae was examined by density transfer experiments. After transfer to light medium, significant amounts of fully heavy dsRNA persisted over a number of cell doublings. In addition, very little material of hybrid density was ever formed, and the accumulation of fully light material began as early as 0.5 doubling after transfer to light medium. Our results are compatible with a conservative mode of replication or with a semiconservative mode of replication carried out by a small portion of the total dsRNA population. In additional experiments the synthesis of dsRNA relative to the cell cycle was studied. This was done by determining the ratio of short-term to long-term radioactive label in size-separated cell fractions of a prelabeled exponential culture. The ratio of short-term to long-term label remained constant for all fractions, implying that dsRNA is synthesized throughout the cell cycle, increasing through the cell cycle at an exponential rate.

Cell Cycle↗
PubMed1980

Faithful and efficient translation of homologous and heterologous mRNAs in an mRNA-dependent cell-free system from Saccharomyces cerevisiae.

A cell-free protein synthesizing system from the yeast Saccharomyces cerevisiae has been optimized for the translation of both homologous yeast mRNA and for a number of heterologous eukaryotic mRNAs. A significant increase in protein synthesis was observed when K(OAc) rather than KCl was used as the source of K+ in the in vitro translation system. This was due primarily to an inhibitory effect oif Cl-. The polyamine putrescine hydrochloride stimulated protein synthesis only at low Mg2+ concentrations. Protein synthesis directed by both yeast mRNA and several eukaryotic mRNAs examined in the system was sensitive to the mRNA 5'-cap analogue, 7-methylguanosine 5'-monophosphate. One-dimensional and two-dimensional polyacrylamide gel analysis of polypeptides synthesized in response to yeast polysomal RNA demonstrated faithful translation in vitro. Translational control and post-translational modifications appear to operate normally in vitro. RNA from several eukaryotic viruses (brome mosaic virus, turnip yellow mosaic virus, and tobacco mosaic virus) were found to be faithfully translated in vitro yielding discrete polypeptides. Reticulocyte polysomal RNA directed the synthesis of a single protein that co-migrated with rabbit globin. The prokaryotic RNAs of Q beta and MS2 were translated with a very low efficiency. The yeast cell-free system programmed with yeast polysomal RNA provides an excellent model for the study of translational control in a eukaryote.

Animals↗
PubMed1980

The metabolic properties of acid soluble polyphosphates in Saccharomyces cerevisiae.

Tripolyphosphate was found to be the predominant species of soluble polyphosphate in yeast. Evidence is presented which shows that under normal growth conditions tripolyphosphate had little or no turnover. The amounts of the various polyphosphates decreased as the chain length increased. Tetrapolyphosphate was shown to be synthesized more rapidly than tripolyphosphate. These observations suggest that short chain polyphosphates arise by degradation of longer chain length polyphosphates with tripolyphosphate the ultimate degradation product. During nitrogen starvation, the normal accumulation of tripolyphosphate rapidly ceased even though the cells continued normal growth for at least two hours. After the addition of L-amino acids or (NH4)2SO4 to nitrogen starved cells, there was a dramatic increase in the accumulation of tripolyphosphate and tetrapolyphosphate which occurred at the same time as the increase in growth rate. Implications of this result are discussed in terms of possible functions of polyphosphate.

Chromatography↗
PubMed1979

The preparation and characterization of a cell-free system from Saccharomyces cerevisiae that translates natural messenger ribonucleic acid.

A cell-free protein-synthesizing system has been prepared from Saccharomyces cerevisiae by differential centrifugation of lysed spheroplasts. The preparation, a modified 100,000 x g supernatant fraction, contains ribosomes and monosomes, ribosomal subunits, translation factors, and aminoacyl-tRNA synthetases, but no polysomes. After removal of small amounts of remaining mRNA with micrococcal nuclease, protein synthesis is stringently dependent on the addition of mRNA, as well as amino acids and an energy-generating system. The 5'-cap analogue, 7-methylguanosine 5'-phosphate, inhibits translation of several natural mRNAs, but has no effect on chain elongation. Incubation of the polysome-free extract with natural mRNA leads to the formation of protein-synthesizing polysomes and eventually, to the release of protein; the molecular weight of the protein synthesized in the presence of BMV (brome mosaic virus) RNA is consistent with that of BMV coat protein.

Cations, Monovalent↗