Biomedical subjects
Publications and source records attributed to C S McLaughlin.
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Centrifugal elutriation was used to separate cells in different stages of the cell cycle from a culture of Saccharomyces cerevisiae in balanced exponential growth. The rate of DNA and RNA synthesis was determined using a pulse--long-term label technique that is capable of distinguishing between exponential, linear, and periodic variations in the rate of synthesis through the cell cycle. It was found that while the rate of DNA synthesis varies periodically through the cell cycle, the rate of synthesis of mRNA, rRNA, and tRNA increases exponentially through the cell cycle. The implications of these findings for the control of RNA synthesis are discussed.
Centrifugal elutriation was used to separate yeast cells by their cell cycle position. The rate of synthesis of ribosomal proteins showed a constant exponential increase through the cell cycle.
We have used a novel technique to study the synthesis, modification and degradation of proteins during the cell cycle in Saccharomyces cerevisiae. Logarithmically growing cells were pulse-labeled twice, with the pulses separated in time by more than one generation. Subsequently, the cells were fractionated as to their position in the cell cycle by centrifugal elutriation, and for different proteins the ratio of radioactive material from the two pulses was then determined. Periodic degradation, synthesis, or modification would produce periodic variations in the ratio of counts. Two-dimensional gel electrophoresis was used to examine 110 different proteins at different times of the cell cycle. All but two proteins had a constant ratio of counts through the cell cycle. This indicates that the rate of synthesis of individual proteins increases exponentially during the cell cycle and that periodic degradation or modification of proteins is not a general feature of the cell cycle in S. cerevisiae.
Arsenate, a competitive inhibitor with phosphate in phosphorylation reactions, has been used to lower adenine and guanine nucleotide levels in Saccharomyces cerevisiae to study nucleotide effects on protein synthesis. By measuring polysome levels, we have shown that initiation of protein synthesis is much more sensitive than elongation or termination to inhibition when the ATP/ADP, GTP/GDP ratios are low. When the arsenate-phosphate molar ratio was 0.27, protein synthesis was inhibited by about 85% and the kinetics of polysome decay was similar to that observed with the initiation inhibitor, verrucarin-76, or with the protein synthesis initiation mutant, ts187, at the restrictive temperature. With this level of arsenate, the adenylate energy charge dropped from 0.9 to 0.7 and the ATP/ADP and GTP/GDP ratios dropped from 6 to 2. The observed correlations between nucleotide ratio changes and inhibition of protein synthesis suggest that the former may be a control signal for the latter. The significance of these in vivo correlations will have to be tested with an in vitro protein synthesizing system. Higher arsenate levels resulted in even lower ATP/ADP, GTP/GDP ratios and in a slower decay of polysomes, implying that, eventually, elongation (in addition to initiation) was being inhibited.
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Centrifugal elutriation was used to separate cells of Saccharomyces cerevisiae in balanced exponential growth according to position in the cell cycle. Macromolecular synthesis was examined. DNA synthesis was found to be periodic, but RNA and protein synthesis showed an exponential increase in rate. Two-dimensional electrophoresis was used to determine the rate of synthesis of individual proteins, with 111 of the more abundant cellular proteins selected for analysis from among the more than 1000 proteins that migrate in the system. All the examined proteins showed an exponentially increasing rate of synthesis.
A study has been made of the effects of a casamino acids shift-up on a prototrophic strain of yeast growing under conditions of ammonium repression. The shift-up produced an increase in growth rate some 120 min after the addition of amino acids to the medium. This growth rate increase was slightly preceded by an increase in the rate of accumulation of DNA. In contrast, the rate of accumulation of protein increased immediately and that of RNA 15-20 min after the shift. RNA was initially accumulated at a rate greater than that required to sustain the new steady state. This was shown to be due to an increase in the rate of synthesis of the rRNA species derived from the 35S precursor. The rate of synthesis of 5S rRNA and of tRNA increased much later and to a lesser extent than that of the 35S derived species. The implications of these results for general theories of regulation of RNA synthesis are discussed.
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The psi+ and psi- phenotypes, which affect the efficiency of ochre suppression in yeast, are inherited in a non-Mendelian fashion. There is no apparent difference in length or in length distribution of 2 micronm circular DNA molecules between psi+ and psi- strains. It seems that the psi genetic determinant is probably not connected with the presence or absence of these small circular DNA molecules.
The synthesis of tRNA in yeast is shown to be under separate control to that of rRNA during amino acid and nitrogen starvation. Inhibitors of the elongation and termination steps of protein synthesis were found to stimulate the synthesis of tRNA in starved yeast cells. This effect appeared to be due to the "trickle-charging" of tRNA. Two inhibitors of early steps in the initiation of protein synthesis were found to be unable to stimulate RNA synthesis in starved cells. It is proposed that yeast tRNA synthesis is under autoregulatory control and that the level of tRNA charging and the mRNA-ribosome complex are important components of this control system.
When a [psi-] strain of yeast mutates to [psi+], the efficiency of suppression by certain ochre suppressors is increased. The [psi+] phenotype is inherited extrachromosomally. There is a nuclear gene, PNM, which, when mutant, causes loss of the [psi+] phenotype. PNM- is dominant to PNM+ and a heterozygous diploid gradually loses the ability over successive generations, to produce PNM+ [psi+] spores. This paper describes the kinetics of this elimination and the data obtained are discussed in relation to two models of the molecular nature of the [psi] genetic determinant--one considering the [psi] determinant as an autonomous nucleic acid, the other treating the possibility that the [psi] nucleic acid is that which codes for rRNA in the nuclear genome.
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A study was made of the virus-like particle (VLP) of Saccharomyces cerevisiae S7. This strain contains elevated amounts of P1 double-stranded ribonucleic acid (dsRNA) but no P2 dsRNA. The amount of dsRNA contained in cells grown on a fermentable carbon source (glucose) was compared with that in cells grown on a nonfermentable carbon source (ethanol). It was found that ethanol-grown cells contain higher levels of dsRNA than glucose-grown cells. In the former, the amount of dsRNA increased during the logarithmic phase of growth, whereas in the latter it increased during the transition from the logarithmic to the stationary phase. A method was devised to isolate VLPs from these cells by using CsCl gradients, and the yield was assessed by monitoring the recovery of dsRNA. Three proteins were found to be tightly associated with these particles. They have molecular weights of 75,000, 53,000, and 37,000. Together they account for almost all of the coding capacity of the P1 dsRNA that the VLP contains.
The kinetics of entry of newly synthesized 40 S and 60 S ribosomal subunits into yeast polysomes is described. The entry times for 40 S and 60 S subunits were found to be 3 and 8 min, respectively. The kinetics of entry of 40 S subunits into large polysomes is found to be different from the kinetics of entry of 60 S subunits into large polysomes.
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A 5.8 S ribosomal RNA (rRNA) is found noncovalently bound to the 25 S RNA in the 60 S subunit of yeast ribosomes. Both molecules appear to be derived from the 27 S RNA, a ribosomal precursor species (Uden, S. A., and Warner, J. R. (1972) J. Mol. biol. 65, 227-242). We have detected two other RNA species by pulse labeling which may also be produced during 27 S RNA processing. The first is approximately 5.9 S and appears by kinetics of synthesis to be a direct precursor of 5.8 S rRNA. The second species is about 7 S, is absent from uniformly labeled RNA preparations, and is produced coincidentally with the 5.8 S rRNA. Both the 5.9 S RNA and the 7 S RNA are not produced in mutants defective in ribosome biosynthesis (Hartwell, L. H., McLaughlin, C. S., and Warner, J. R. (1970) Mol. Gen. Genet. 109, 42-56) which are unable to make the 25 S-5.8 S rRNA complex from 27 S RNA. Thus, the maturation of 27 S RNA appears to involve the production of a 7 S RNA, which could be either a precursor or a nonconserved RNA region, and a 5.9 S RNA, most of which is trimmed to give the mature 5.8 S rRNA in the 25 S-5.8 S rRNA complex of 60 S subunits.