Terrance G. Cooper

Nonsense suppression in Schizosaccharomyces pombe: The S. pombe Sup3-e tRNA Ser UGA gene is active in S. cerevisiae

SummaryThe gene encoding the efficient UGA suppressor sup3-e of Schizosaccharomyces pombe was isolated by in vivo transformation of Saccharomyces cerevisiae UGA mutants with S. pombe sup3-e DNA. DNA from a clone bank of EcoRI fragments from a S. pombe sup3-e strain in the hybrid yeast vector YRp17 was used to transform the S. cerevisiae multiple auxotroph his4-260 leu2-2 trp1-1 to prototrophy. Transformants were isolated at a low frequency; they lost the ability to grow in minimal medium after passaging in non-selective media. This suggested the presence of the suppressor gene on the non-integrative plasmid. Plasmid DNA, isolated from the transformed S. cerevisiae cells and subsequently amplified in E. coli, transformed S. cerevisiae his4-260 leu2-2 trp1-1 to prototrophy. In this way a 2.4 kb S. pombe DNA fragment carrying the sup3-e gene was isolated. Sequence analysis revealed the presence of two tRNA coding regions separated by a spacer of only seven nucleotides. The sup3-e tRNASerUGAtRNA gene is followed by a sequence coding for the initiator tRNAMet. The transformation results demonstrate that the cloned S. pombe UGA suppressor is active in S. cerevisiae UGA mutant strains.

Isolation of the CAR1 gene from Saccharomyces cerevisiae and analysis of its expression.

We isolated the CAR1 gene from Saccharomyces cerevisiae on a recombinant plasmid and localized it to a 1.58-kilobase DNA fragment. The cloned gene was used as a probe to analyze polyadenylated RNA derived from wild-type and mutant cells grown in the presence and absence of an inducer. Wild-type cells grown without the inducer contained very little polyadenylated RNA capable of hybridizing to the isolated CAR1 gene. A 1.25-kilobase CAR1-specific RNA species was markedly increased, however, in wild-type cells grown in the presence of inducer and in constitutive, regulatory mutants grown without it. No CAR1-specific RNA was observed when one class of constitutive mutant was grown in medium containing a good nitrogen source, such as asparagine. Two other mutants previously shown to be resistant to nitrogen repression contained large quantities of CAR1 RNA regardless of the nitrogen source in the medium. These data point to a qualitative correlation between the steady-state levels of CAR1-specific, polyadenylated RNA and the degree of arginase induction and repression observed in the wild type and in strains believed to carry regulatory mutations. Therefore, they remain consistent with our earlier suggestion that arginase production is probably controlled at the level of gene expression.

Possible failure of NADP-glutamate dehydrogenase to participate directly in nitrogen repression of the allantoin degradative enzymes in Saccharomyces cerevisiae

Abstract The extent of repression exerted by various nitrogen-containing compounds was measured in wild type and NADP-GDH defective strains of Saccharomyces . We found that in strains carrying the gdhA 6− mutation, repression was relieved only for compounds whose metabolism involved the intermediate generation of ammonia. This raises the possibility that relief of repression observed in these strains is the result of their failure to effectively metabolize ammonia rather than an inability of the NADP-GDH protein to function as a regulator itself.

Effects of inducer addition and removal upon the level of allophanate hydrolase in Saccharomyces cerevisiae.

Summary A number of investigators have shown that 8 to 10 minutes are required before an increase in enzyme activity is observed following addition of various inducers to cultures of Saccharomyces cerevisiae. They have also observed messenger RNA and synthetic capacity half lives of approximately 20 minutes. Our data on the other hand indicate that only 3 minutes elapse between addition of inducer and appearance of active allophanate hydrolase. In addition the capacity of an induced culture to continue producing allophanate hydrolase decays with a half life of 3 minutes following removal of the inducer. These observations raise the possibilities that different species of mRNA possess quite different rates of metabolism or that regulation of the allantoin degradative enzymes may occur at levels other than transcription.

Basic amino acid inhibition of growth inSaccharomyces cerevisiae

Abstract Growth ofSaccharomyces cerevisiaeon poor nitrogen sources such as allantoin or proline is totally inhibited by addition of lysine to the medium. The same result is observed with ornithine if its degradation to glutamic semialdehyde is prevented. Since inhibition occurs even when arginine is used as nitrogen source it is not likely that cessation of growth is due to arginine limitation as previously suggested. Preliminary observations suggest that addition of basic amino acids to slowly growing cultures ofSaccharomycesinhibits some process associated with the cell cycle.

Lomofungin inhibition of allophanate hydrolase synthesis in Saccharomyces cerevisiae

SummaryThe RNA polymerase inhibitor, lomofungin has been used to determine the half life of specific synthetic capacities (invertase and α-glucosidase) as well as that for gross protein synthesis. In both cases the studies conclude that cognate messenger RNAs decay with a half life of approximately 20 minutes. This antibiotic has been used to determine the half life of allophanate hydrolase specific synthetic capacity. We find that it decays with a half life of about three minutes; a value that agrees with the decay rates of allophanate hydrolase synthetic capacity following removal of inducer. These observations argue that mRNA may be metabolized by two separate routes in Saccharomyces.

Selective gene expression and intracellular compartmentation: two means of regulating nitrogen metabolism in yeast

Abstract The system that degrades allantoin has been used to investigate the control of nitrogen metabolism in Saccharomyces cerevisiae . Studies of this system are beginning to provide an appreciation of the complementary roles played by gene expression and intracellular compartmentation in the acquisition and utilization of nitrogenous metabolites.

Mutants of Saccharomyces cerevisiae possessing fully induced levels of urea amido-lyase in the absence of added inducer.

Abstract We have isolated strains of S. cerevisiae that are resistant to the growth inhibitory effects of methylamine and found them to be insensitive to nitrogen repression. However, our mutants could additionally be divided into three groups with respect to inducibility of urea amido-lyase. The first class of strains resembled the wild-type and had very little enzyme activity in the absence of inducer. The second class had significantly increased basal levels of activity while the third class possessed fully induced levels of urea amido-lyase in the absence of added inducer. The procedures we used may be applicable to the generation of similar mutants in other inducible-repressible transport and degradative enzyme systems as well.

Post-translational processing of urea amidolyase in Saccharomyces cerevisiae.

Urea amidolyase catalyzes the two reactions (urea carboxylase and a allophanate hydrolase) associated with urea degradation in Saccharomyces cerevisiae. Past work has shown that both reactions are catalyzed by a 204-kilodalton, multifunctional protein. In view of these observations, it was surprising to find that on induction at 22 degrees C, approximately 2 to 6 min elapsed between the appearance of allophanate hydrolase and urea carboxylase activities. In search of an explanation for this apparent paradox, we determined whether or not a detectable period of time elapsed between the appearance of allophanate hydrolase activity and activation of the urea carboxylase domain by the addition of biotin. We found that a significant portion of the protein produced immediately after the onset of induction lacked the prosthetic group. A steady-state level of biotin-free enzyme was reached 16 min after induction and persisted indefinitely thereafter. These data are consistent with the suggestion that sequential induction of allophanate hydrolase and urea carboxylase activities results from the time required to covalently bind biotin to the latter domain of the protein.

Factors influencing the observed half-lives of specific synthetic capacities in Saccharomyces cerevisiae.

We have identified a variety of factors affecting the stability of allophanate hydrolase-specific and gross cellular protein synthetic capacities. These synthetic capacities have been extrapolated by many laboratories to represent functional messenger RNAs. Synthetic capacity turnover rates that we measured were greater in diploid organisms than in haploid strains and were proportional to the temperature of the culture medium. The stability of allophanate hydrolase-specific synthetic capacity was not influenced by alterations in the nitrogen source provided in the culture medium, but was increased up to 15-fold by the total inhibition of protein synthesis. Cultures in which protein synthesis was inhibited as little as 20% exhibited hydrolase-specific synthetic capacities more than 2-fold greater than those observed in the absence of inhibition.