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Regulation of beta-1,3-glucanase synthesis in Penicillium italicum.

The filamentous fungus Penicillium italicum produced a certain level of beta-1,3-glucanase during active growth in a glucose-supplemented medium; however, at a low glucose concentration (2 to 10 mM), derepression took place and the specific activity of the enzyme increased significantly. Derepressed cells (incubated in a glucose-limited medium) accumulated a capacity for the synthesis of beta-1,3-glucanase, which led to a subsequent increase in the specific activity even when the cells were transferred to a medium with an excess of glucose (180 mM). Two protein synthesis inhibitors, cycloheximide and trichodermin, immediately stopped the increase in specific activity when added to derepressed cells. On the other hand, 8-hydroxyquinoline, an RNA a synthesis inhibitor, acted differently, since it permitted the specific activity to increase for some time after being added to depressed cells. Moreover, the concentration of glucose did not affect the 8-hydroxyquinoline-insensitive synthesis of beta-1,3-glucanase. It is concluded that the glucose repression effect on beta-1,3-glucanase production must be exerted at a pretranslational level that could be either mRNA synthesis or some stage of the process involved in its maturation or stabilization.

Cycloheximide↗

Germ tube induction in Candida albicans.

A reproducible and simple system for the production of germ tubes from yeast cells of Candida albicans using glucose and glutamine as substrates has been described. During germ tube formation there was a doubling of the dry weight but the number of cells remained constant. Although the DNA content did not change for the first 4 h of germ tube formation, the RNA content more than doubled. The DNA and RNA content of C. albicans blastospores are 4.5 x 10(-15) g per cell and 48 x 10(-15) g per cell respectively. Nystatin, phenethyl alcohol, 2,4-dinitrophenol, azaserine, salicylhydroxamic acid, and 5-fluorocytosine were all effective inhibitors of germ tube formation. Cysteine, potassium cyanide, and polyoxin D did not prevent germination. The incorporation of both uracil and leucine occurred rapidly during germ tube formation. The inhibitors of RNA synthesis, actinomycin D. cordycepin, and daunomycin prevented germination and inhibited uracil incorporation. The translational inhibitors, trichodermin, aurin tricarboxylic acid, puromycin, and cyloheximide were effective in inhibiting both germ tube formation and leucine incorporation.

Candida albicans↗

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↗

Characterization and synthesis regulation of Penicillium italicum 1,6-beta-glucanase.

The filamentous fungus Penicillium italicum when grown in a synthetic medium, produced and secreted 1,6-beta-glucanase into the culture medium. This enzyme has been partially purified by gel filtration. After this step the active fractions were free of 1,3-beta-glucanase, alpha-amylase and beta-glucosidase activities. Only four proteins, one associated with the enzyme, were found by polyacrylamide gel electrophoresis under non denaturing conditions. The enzyme behaves as an acidic protein (pI 4.65) with an optimum pH of 5 and an endohydrolytic mode of action. The activity was also found associated with the mycelium. Its synthesis was repressed by glucose or growth-promoting sugars. Derepression in low glucose containing medium required protein synthesis. 8-Hydroxyquinoline, an RNA synthesis inhibitor, added during the derepression period did permit some increase in the specific activity but prevented it when added at the beginning of that period.

Enzyme Repression↗

Nucleo-cytoplasmic interactions in the petite negative yeast Schizosaccharomyces pombe. Inhibition of nuclear and mitochondrial DNA syntheses in the absence of cytoplasmic protein synthesis.

In the petite positive yeast, Saccharomyces cerevisiae, cycloheximide selectively inhibits protein synthesis on cytoplasmic ribosomes, and, as a consequence, nuclear DNA synthesis. Mitochondrial DNA, however, is synthesized for 4-6 h after cessation of protein synthesis. In this paper we show that in contrast to Saccharomyces cerevisiae, synthesis of mitochondrial and nuclear DNA is tightly coordinated in the petite negative yeast Schizosaccharomyces pombe, since inhibition of cytoplasmic protein synthesis leads immediately to cessation of both nuclear and mitochondrial DNA synthesis.

Ascomycota↗

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.

Allophanate Hydrolase↗

Inhibitors of protein synthesis inhibit both La Crosse virus S-mRNA and S genome syntheses in vivo.

The effect of drugs such as puromycin and cycloheximide, which inhibit protein synthesis, on the accumulation of La Crosse virus S genome RNAs in vivo has been examined. We have found that if these drugs are added to the cultures before infection, minuscule amounts of S-mRNA can be detected late in infection. Genome replication, on the other hand, cannot be detected at any time. When these drugs are added later in infection when RNA synthesis is well established, S-mRNA accumulation decreases in a dose-dependent manner proportional to the effect of these drugs on protein synthesis. This decrease cannot be accounted for by increased turnover of the mRNA in the presence of the drug. S genome replication, curiously, was found to be hypersensitive to the effects of these drugs. Our results confirm those of Abraham and Pattnaik (1983) that ongoing protein synthesis is required for the accumulation of complete bunyavirus S-mRNA.

Anisomycin↗

Mitotic recombination within the centromere of a yeast chromosome.

Centromeres are the structural elements of eukaryotic chromosomes that hold sister chromatids together and to which spindle tubules connect during cell division. Centromeres have been shown to suppress meiotic recombination in some systems. In this study yeast strains genetically marked within and flanking a centromere, were used to demonstrate that gene conversion (nonreciprocal recombination) tracts in mitosis can enter into and extend through the centromere.

Alleles↗

Molecular events associated with induction of arginase in Saccharomyces cerevisiae.

Arginase, the enzyme responsible for arginine degradation in Saccharomyces cerevisiae, is an inducible protein whose inhibition of ornithine carbamoyl-transferase has been studied extensively. Mutant strains defective in the normal regulation of arginase production have also been isolated. However, in spite of these studies, the macromolecular biosynthetic events involved in production of arginase remain obscure. We have, therefore, studied the requirements of arginase induction. We observed that: (i) 4 min elapsed between the addition of inducer (homoarginine) and the appearance of arginase activity at 30 degrees C; (ii) induction required ribonucleic acid synthesis and a functional rna1 gene product; and (iii) production of arginase-specific synthetic capacity occurred in the absence of protein synthesis but could be expressed only when protein synthesis was not inhibited. Termination of induction by inducer removal, addition of the ribonucleic acid synthesis inhibitor lomofungin, or resuspension of a culture of organisms containing temperature-sensitive rna1 gene products in a medium at 35 degrees C resulted in loss of ability for continued arginase synthesis with half-lives of 5.5, 3.8, and 4.5 min, respectively. These and other recently published data suggest that a variety of inducible or repressible proteins responding rapidly to the environment may be derived from labile synthetic capacities, whereas constitutively produced proteins needed continuously throughout the cell cycle may be derived from synthetic capacities that are significantly more stable.

Arginase↗