Biomedical subjects
Publications and source records attributed to B Magasanik.
The correct transcription of the hut (histidine utilization) operon DNA of Salmonella typhimurium requires activation either by 3':5'-cyclic adenosine monophosphate and catabolite-activating protein or by nonadenylylated glutamine synthetase (EC 6.3.1.2.). Thus, glutamine synthetase plays a regulatory role distinct from its enzymatic function.
A mutant of Klebsiella aerogenes lacking glutamate synthase activity (asm-200) is blocked in only one pathway of glutamate synthesis and can still use glutamate dehydrogenase to produce glutamate when ammonia in sufficient concentration, i.e., higher than 1 mM, is provided in the medium. However, a mutant that has neither glutamate synthase nor glutamate dehydrogenase activities (asm-200, gdhD1) requires glutamate. Transductants obtained by phage grown on wild-type cells of this double mutant, selected on medium containing less than 1 mM ammonia, regain glutamate synthase but not glutamate dehydrogenase. Surprisingly, these gdhD1 transductants grow as well in a variety of media as does a strain with glutamate dehydrogenase activity. Furthermore, transductions with these and other mutants indicate that the genes encoding glutamate synthase, glutamate dehydrogenase, glutamine synthetase, and citrate synthase are not closely linked.
Different levels of beta-galactosidase are found in various trp-lac fusion strains. These levels of beta-galactosidase fall within a 60-fold range. The amount of thiogalactoside transacetylase activity detected in these same strains only varies 10-fold and is found in amounts greater than those predicted from the beta-galactosidase levels. The observation that the beta-galactosidase and thiogalactoside transacetylase levels are not directly proportional, that the lacZ messenger ribonucleic acid (mRNA) levels are not proportional to the beta-galactosidase activity, that, at least for the one fusion strain tested, the SuA polarity suppressor does not affect the beta-galactosidase level, and that, in all but one strain, the beta-galactosidase activity appears to reside in normal beta-galactosidase molecules suggests that the disproportionately low production of beta-galactosidase is due to a decrease in the frequency of translation initiation of lacZ mRNA in these strains. Several mechanisms are proposed to explain this decrease. Some possible bases for the disproportional production of beta-galactosidase and thiogalactoside transacetylase are also described. The preferred explanation for these disproportional enzyme levels is that only a fraction of the full complement of ribosomes need initiate translation at lacZ for the functional synthesis of lac mRNA to occur and that once the lac ribonucleic acid is made a full complement of ribosomes can bind at internal translation initiation sites at Y and A.
A mutation of Salmonella typhimurium was obtained that results in the failure of cells to synthesize the enzyme l-histidine ammonia-lyase (histidase). The mutation mapped within the hutH gene and in merodiploid strains was dominant over the wild-type allele. Extracts from cells bearing the trans-dominant histidase-negative allele were shown to contain material that reacts immunologically with antiserum against purified wild-type histidase. It is proposed that the trans-dominant allele results in the synthesis of defective histidase subunits that can combine with, and partially inactivate, wild-type histidase subunits. This subunit mixing presumably does occur, as the enzyme synthesized in a hybrid merodiploid strain is abnormally heat sensitive.
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In Salmonella typhimurium the structural genes of the enzymes responsible for histidine utilization (hut) are clustered in two adjacent operons. A single repressor regulates both operons. The repressor itself is a member of one of the hut operons and, thus, regulates its own synthesis. We have assayed the hut repressor by its ability to bind radioactive DNA to nitrocellulose filters. The binding is specific for DNA bearing the hut operons, and the binding is abolished by the inducer, urocanate. As a member of one of the hut operons, the repressor is inducible, subject to catabolite repression, and affected by a promoter mutation.
In addition to carrying determinants conferring resistance to at least two antibiotics, chloramphenicol and streptomycin, a Klebsiella aerogenes strain contains a plasmid responsible for increased beta-galactosidase activity. The plasmid can be transferred to Escherichia coli and Salmonella typhimurium strains. K. aerogenes segregants without the plasmid grow on lactose one-half as fast as the parent strain and contain only one-tenth to one-fifth as much beta-galactosidase.
The catabolite repression caused by glucose and glucose-6-phosphate has been studied for both beta-galactosidase and thiogalactoside transacetylase, the products of the operator proximal and distal cistrons of the lac operon, respectively. We find that both cistrons are affected coordinately by this form of repression. We also find that a single alteration at the lac promoter region is sufficient to abolish sensitivity to repression of both cistrons. From this, we conclude that there is only one target site for catabolite repression in the lac operon.
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