Immunological and genetic properties of Escherichia coli K12 argE mutants

Abstract

CRM+ nonsense mutations, as described for E. coli K12 lacZ, in theory permit determination of the direction of transcription of an isolated gene. In the hope of utilising this approach to confirm that the E gene of the E. coli K12 argECBH cluster is transcribed in the opposite direction from the CBH unit, 30 argE mutations were investigated immunologically and genetically. Only three, E1, E25 and E26 (which map close together towards the left-hand end of the gene), were found to be CRM+. Co-suppression of each argE mutation with known strongly polar lacZ amber, ochre and UGA mutations was looked for to distinguish missfense from nonsense argE's, Arg+ revertants being screened for Lac+ and Mel+ phenotypes. Of 16 mutations not hitherto characterised as nonsense, frameshift or large deletions, only three, E1, E25 and E26, appeared to be missense. Three of the nonsense mutations were also streptomycin suppressible. It appears, therefore, that among argE mutants so far studied the correlation between CRM+ and missense is complete, so that the projected method for determining the direction of transcription of argE could not be applied.

Cloning and restriction analysis of a bacillus subtilis DNA fragment carrying gene, coding for carbamoyl phosphate synthetase enzyme

“…a-32p-dATP or a-35S-dATP were employed for chasing new DNA synthesis. Both normal and buffer-gradient gels were em,ployed to improve the resolution in the autoradiography as et al, 1980;Biggin et al, 1983).…”