Biomedical subjects
D B Finkelstein
Publications and source records attributed to D B Finkelstein.
Improvements in fungal product synthesis by recombinant DNA technology.
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hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures.
hsp82 is one of the most highly conserved and abundantly synthesized heat shock proteins of eucaryotic cells. The yeast Saccharomyces cerevisiae contains two closely related genes in the HSP82 gene family. HSC82 was expressed constitutively at a very high level and was moderately induced by high temperatures. HSP82 was expressed constitutively at a much lower level and was more strongly induced by heat. Site-directed disruption mutations were produced in both genes. Cells homozygous for both mutations did not grow at any temperature. Cells carrying other combinations of the HSP82 and HSC82 mutations grew well at 25 degrees C, but their ability to grow at higher temperatures varied with gene copy number. Thus, HSP82 and HSC82 constitute an essential gene family in yeast cells. Although the two proteins had different patterns of expression, they appeared to have equivalent functions; growth at higher temperatures required higher concentrations of either protein. Biochemical analysis of hsp82 from vertebrate cells suggests that the protein binds to a variety of other cellular proteins, keeping them inactive until they have reached their proper intracellular location or have received the proper activation signal. We speculate that the reason cells require higher concentrations of hsp82 or hsc82 for growth at higher temperatures is to maintain proper levels of complex formation with these other proteins.
Sharp boundaries demarcate the chromatin structure of a yeast heat-shock gene.
In both induced and basally transcribed states, the chromatin structure of the yeast HSP82 heat-shock locus exhibits a remarkable degree of organization with respect to DNA sequence. The promoter region contains a constitutive DNase I hypersensitive site. The transcription unit is markedly sensitive to DNase I, and exhibits a sharp transition from a phased half- to a whole nucleosomal cleavage periodicity at the 3' end. Distant upstream and downstream regions are also organized into distinct arrays of phased nucleosomes. Each array is demarcated by DNase I hypersensitive sites that display internal protected regions, suggesting the presence of DNA binding proteins. In addition, since these sites are of mononucleosomal DNA length, they may acquire a nucleosomal structure under certain environmental conditions without disrupting flanking nucleosomal phasing frames. Thus, the HSP82 locus is organized into specific, phased, chromatin structures that appear to function in transcriptional initiation, RNA polymerase passage, transcriptional termination, and the establishment of chromatin-domain microenvironments.
Improvement of enzyme production in Aspergillus.
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Metabolism of alpha-factor by a mating type cells of Saccharomyces cerevisiae.
When a mating type cells of Saccharomyces cerevisiae are exposed to the mating pheromone alpha-factor in liquid cultures, there is a time-dependent loss of alpha-factor activity from the culture fluid. This loss of biological activity can be directly correlated with the proteolysis of the pheromone by a mating type cells. The metabolism of alpha-factor by a mating type cells may be measured by using either in vitro 125I-labeled or in vivo 35S-labeled pheromone. Addition of chloroquine to growing cultures of a mating type cells at concentrations which cause no detectable alterations in cell growth produces a potentiation of alpha-factor mediated cell cycle arrest. This potentiation of alpha-factor activity is directly correlated with the inhibition of alpha-factor proteolysis. Thus, while proteolytic digestion of alpha-factor appears to be related to the mechanism whereby a mating type cells "detoxify" alpha-factor and recover from cell cycle arrest, proteolysis of the mating factor is not necessary for alpha-factor mediated cell cycle arrest.
Reaction order of Saccharomyces cerevisiae alpha-factor-mediated cell cycle arrest and mating inhibition.
Alpha-factor-mediated cell cycle arrest and mating inhibition of a mating-type cells of Saccharomyces cerevisiae have been examined in liquid cultures. Cell cycle arrest may be monitored unambiguously by the appearance of morphologically abnormal cells after administration of alpha factor, whereas mating inhibition is determined by comparing the mating efficiency in the absence or presence of added alpha factor. For both cell cycle arrest and mating inhibition, a dose-dependent response may be observed at limiting concentrations of the pheromone. If cell cycle arrest and mating inhibition require a small number of alpha-factor molecules, one might expect that responsive/nonresponsive cells = K(alpha factor)(N) where N is the order of dependence of cell cycle arrest (or mating inhibition) on alpha-factor concentration. The value of N has been determined to be 0.98 +/- 0.18 (standard error of the mean) for cell cycle arrest and 1.08 +/- 0.32 for mating inhibition. These results support the notion that saturation of a single site by alpha factor is sufficient to cause cell cycle arrest or mating inhibition of a mating-type cells.
DNA-binding proteins in yeast. Effect of growth phase and mitochondrial function.
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DNA-binding proteins related to the dosage of specific yeast chromosomes.
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