PubMed Health⌕ Search

Biomedical subjects

J R Shuster

Publications and source records attributed to J R Shuster.

22 records · Page 2Linked to original sources
PubMed1987

High level expression of proinsulin in the yeast, Saccharomyces cerevisiae.

Human proinsulin (PI) has been expressed to a high level (100 mg/liter) as a human superoxide dismutase-PI fusion protein in the yeast, Saccharomyces cerevisiae. At the junction of the two proteins is a methionine residue, allowing PI to be released from the fusion by reaction with cyanogen bromide. The fusion is expressed using a regulated, hybrid promoter containing the regulatory region of the alcohol dehydrogenase II promoter and the 3' end of a glyceraldehyde-3-phosphate dehydrogenase promoter, allowing the recombinant yeast cells to be stably maintained. Production of the fusion protein is induced by growth in medium lacking a fermentable carbon source. The heterologous fusion protein is probably insoluble within the cell, since electron microscopy reveals the presence of 'inclusion bodies'. In a cell-free extract the fusion protein is also insoluble, but can be solubilized with sodium dodecyl sulfate, and cleaved with cyanogen bromide. The PI that is produced contains incorrect disulfide bonds. After sulfitolysis, the product can be easily purified, renatured, and processed to yield insulin.

Chromosome Mapping↗
PubMed1982

Mating-defective ste mutations are suppressed by cell division cycle start mutations in Saccharomyces cerevisiae.

Temperature-sensitive mutants which arrest in the G1 phase of the cell cycle have been described for the yeast Saccharomyces cerevisiae. One class of these mutants (carrying cdc28, cdc36, cdc37, or cdc39) forms a shmoo morphology at restrictive temperature, characteristic of mating pheromone-arrested wild-type cells. Therefore, one hypothesis to explain the control of cell division by mating factors states that mating pheromones arrest wild-type cells by inactivating one or more of these CDC gene products. A class of mutants (carrying ste4, ste5, ste7, ste11, or ste12) which is insensitive to mating pheromone and sterile has also been described. One possible function of the STE gene products is the inactivation of the CDC gene products in the presence of a mating pheromone. A model incorporating these two hypotheses predicts that such STE gene products will not be required for mating in strains carrying an appropriate cdc lesion. This prediction was tested by assaying the mating abilities of double mutants for all of the pairwise combinations of cdc and ste mutations. Lesions in either cdc36 or cdc39 suppressed the mating defect due to ste4 and ste5. Allele specificity was observed in the suppression of both ste4 and ste5. The results indicate that the CDC36, CDC39, STE4, and STE5 gene products interact functionally or physically or both in the regulation of cell division mediated by the presence or absence of mating pheromones. The cdc36 and cdc39 mutations did not suppress ste7, ste11, or ste12. Lesions in cdc28 or cdc37 did not suppress any of the ste mutations. Other models of CDC and STE gene action which predicted that some of the cdc and ste mutations would be alleles of the same locus were tested. None of the cdc mutations was allelic to the ste mutations and, therefore, these models were eliminated.

Cell Division↗
PubMedDate not supplied

The alcohol dehydrogenase system in the yeast, Kluyveromyces lactis.

We have studied the alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis. Southern hybridization to the Saccharomyces cerevisiae ADH2 gene indicates four probable structural ADH genes in K. lactis. Two of these genes have been isolated from a genomic bank by hybridization to ADH2. The nucleotide sequence of one of these genes shows 80% and 50% sequence identity to the ADH genes of S. cerevisiae and Schizosaccharomyces pombe respectively. One K. lactis ADH gene is preferentially expressed in glucose-grown cells and, in analogy to S. cerevisiae, was named K1ADH1. The other gene, homologous to K1ADH1 in sequence, shows an amino-terminal extension which displays all of the characteristics of a mitochondrial targeting presequence. We named this gene K1ADH3. The two genes have been localized on different chromosomes by Southern hybridization to an orthogonal-field-alternation gel electrophoresis-resolved K. lactis genome. ADH activities resolved by gel electrophoresis revealed several ADH isozymes which are differently expressed in K. lactis cells depending on the carbon source.

Alcohol Dehydrogenase↗