Biomedical subjects

K Struhl

Publications and source records attributed to K Struhl.

149 records · Page 9Linked to original sources

PubMed1977

Production of a functional eukaryotic enzyme in Escherichia coli: cloning and expression of the yeast structural gene for imidazole-glycerolphosphate dehydratase (his3).

A cloned segment of yeast DNA containing the structural gene for imidazoleglycerolphosphate dehydratase (D-erythro-imidazoleglycerolphosphate hydro-lase, EC 4.2.1.19) is transcribed and translated in Escherichia coli with sufficient fidelity to produce functional enzyme. This segment of yeast DNA was isolated as a viable molecular hybrid of bacteriophage lambda (lambdagt-Sc2601) which complements a nonrevertible hisB auxotroph of E. coli lacking dehydratase activity. The equivalent segments of DNA cloned from two independent his3 mutants of yeast lacking IGP dehydratase activity do not complement the hisB auxotroph. The two nonfunctional his3 alleles cloned in bacteriophage lambda can be recombined in E. coli to generate a hybrid phage which complements the hisB auxotroph. The dehydratase activity produced in E. coli by the cloned segment of yeast DNA strongly resembles the activity found in yeast.

DNA, Recombinant↗

PubMed1976

Functional genetic expression of eukaryotic DNA in Escherichia coli.

We have isolated a segment of DNA from the eukaryote Saccharomyces cerevisiae (baker's yeast) as a viable molecular hybrid of bacteriophage lambda DNA which, when integrated into the chromosome of an E. coli histidine auxotroph, allows this bacterium to grow in the absence of histidine. The nonrevertable, histidine auxotroph lacks the enzymatic activity of imidazole glycerol phosphate (IGP) dehydratase (EC 4.2.1.19). From genetic experiments, we conclude that expression of the segment of yeast DNA results in the production of a diffusible substance and that transcription necessary for the complementation is most likely initiated from the segment of eukaryotic DNA.

Acetoxyacetylaminofluorene↗

PubMed1976

Ammonia-sensitive mutant of Klebsiella aerogenes.

We have isolated a temperature-sensitive mutant of Klebsiella aerogenes unable to grow aerobically at 42 C in standard glucose minimal medium containing 0.03 M ammonium sulfate as a source of nitrogen. This strain, MK810, will grow at this temperature in significantly lower concentrations of ammonia (1 mM) or when ammonia is replaced by a growth rate-limiting source of nitrogen such as histidine or glutamate. A detailed physiological characterization and preliminary biochemical tests support the contention that the mutant has an altered alpha-ketoglutarate dehydrogenase that at the restrictive condition fails to manufacture sufficient succinyl-coenzyme A. We explain the ammonia sensitivity by the dual role of alpha-ketoglutarate as substrate for the formation of succinyl-coenzyme A and glutamate. A defect in the enzyme necessary for the production of succinyl-coenzyme A makes ammonia an overly effective competitor for alpha-ketoglutarate.

Aerobiosis↗

PubMedDate not supplied

The new yeast genetics.

Gene cloning and yeast DNA transformation techniques have greatly enhanced the power of classical yeast genetics. It is now possible to isolate any classically defined gene, to alter the yeast genome at will by replacing normal chromosomal sequences with mutated derivatives produced in vitro, and to create DNA molecules that behave as autonomous replicons or minichromosomes. These unique features of the new yeast genetics have been used to study many problems in eukaryotic molecular biology.

Chromosomes↗

PubMedDate not supplied

Negative control at a distance mediates catabolite repression in yeast.

In prokaryotic organisms, the control of gene expression is mediated by regulatory proteins that activate or repress transcription. However, the molecular mechanisms of positive and negative control are different. In terms of negative control, repressor proteins bind to sites located within the promoter region and as a consequence sterically interfere with functional binding by RNA polymerase. Here, I examine the properties of a regulatory sequence that specifies catabolite (glucose) repression in the yeast Saccharomyces cerevisiae. Specifically, a DNA segment containing this regulatory site was fused upstream of the intact his3 promoter region and structural gene at several locations. Normally, his3 expression in these derivatives occurs at a basal level which can be induced by conditions of amino-acid starvation. However, in glucose medium, the catabolite regulatory sequence overrides the normal his3 promoter elements and reduces transcription both in normal and starvation conditions. The implication from these results is that in contrast to catabolite repression in Escherichia coli, which is mediated by catabolite-activating protein (CAP), catabolite repression in yeast occurs by a negative control mechanism involving a putative repressor protein. The observation that this regulatory site exerts its repressing effects even when located upstream of an intact promoter region suggests that repression in yeast is not mediated by steric interference between regulatory proteins and the transcriptional apparatus.

DNA, Recombinant↗