Temperature-Sensitive Nonsense Mutations in Essential Genes of Escherichia coli

Abstract

Cells containing nonsense mutations in essential genes have been isolated in a strain of Escherichia coli that carried the su4ts gene which specifies a temperature-sensitive tyrosine transfer ribonucleic acid. Such cells are unable to form colonies at temperatures which inactivate this suppressor transfer ribonucleic acid. A screening procedure for the identification of mutants that carry temperature-sensitive nonsense mutations in essential genes is described, and certain properties of two such mutants are reported.

Use of temperature-sensitive mutants to study gene expression during sporulation in Bacillus subtilis

“…references 2 and 18), and, second, that the gene product is not reversibly thermolabile. Among the possible explanations that remain are thermal denaturation or temperature-sensitive synthesis of the gene product (4).…”

“…Most temperature-sensitive mutants are thought to result from missense mutations (11) that cause a single amino acid substitution in a polypeptide (34,35); the resultant misfolding or misaggregation of the polypeptide changes its biological activity (15,27). However, temperature-sensitive nonsense mutants, which have an altered transfer ribonucleic acid (RNA) species, have also been found (4,24,29). Since the products of most sporulation-specific genes await identification, the product of the defective gene in a newly isolated temperature-sensitive sporulation mutant is unlikely to be known.…”

“…Since the products of most sporulation-specific genes await identification, the product of the defective gene in a newly isolated temperature-sensitive sporulation mutant is unlikely to be known. Moreover, before the mechanism of action of the defective gene can be understood, several types of temperature sensitivity should be distinguished (4). The gene product may be (i) reversibly thermolabile, in which case the conformation changes spontaneously between active and inactive forms as the temperature is altered, or (ii) irreversibly thermolabile, in which case thermal denaturation occurs as the temperature is raised.…”

Genetic aspects of bacterial endospore formation.

“…Alternatively, the gene product may be temperature labile at all times and, in this case, the ts period coincides with the period during which the gene product is required. These possibilities complicate interpretation of results (see references 27,155,335). A large number of nonsense-like suppressible spo mutations are known (137), and it is possible that some of these problems could be overcome by the isolation of a temperature-sensitive sup-pressor mutant ofB.…”

“…This would effectively make all nonsense mutations available for temperature shift studies, and the sensitive step would be at the point of synthesis. Such mutants are known in other species (27,98,236,279), and should be obtainable in B. subtilis.…”