No abstract

SUMMARY Mutants of Escherichia coli K12 with deletions in the nadC-Zpd region of the chromosome were obtained for use in studies on the expression of the ace (pyruvate dehydrogenase complex, specific components) and Zpd (lipoamide dehydrogedase) genes. These were isolated by selecting spontaneous aroP mutants (lacking the general aromatic amino-acid permease and thus resistant to inhibitory aromatic amino-acid analogues) and screening for auxotrophy due to deletions extending into neighbouring genes. From 2892 isolates tested, the AroP-phenotypes of 2322 were confirmed and, of these, 28 stable and independentlyderived auxotrophs were designated as deletion mutants.Six nutritionally-distinct categories were recognized : Nad-(8 strains) ; Nad-Ace- (7); Nad-'Ace-' (3); Ace-(8); 'Ace-' (I); Lpd-(I). The Ace-phenotypes of four isolates designated 'Ace-' were leaky and emymological studies confirmed that they had less than 7 % of parental pyruvate dehydrogenase complex activity.Enzymological studies showed that the 15 Ace-or Nad-Ace-strains all lacked the pyruvate dehydrogenase complex and pyruvate dehydrogenase (EIP) activities and only three retained detectabledihydrolipoamide acetyltransferase (Ezp). The one Lpd-strain lacked pyruvate dehydrogenase, dihydrolipoamide acetyltransferase and lipoamide dehydrogenase (E3) activities as well as the activities of the pyruvate and a-ketoglutarate dehydrogenase complexes.The results confirmed the gene order nadC-aroP-aceSaceF-Zpd and indicated that no other essential functionsaredetermined by genes within the nadC-Zpd region. Resistance to lactate during growth ofpps mutants on acetate was directly related to the specific activity of the pyruvate dehydrogenase complex. None of the deletions promoted the high degree of resistance characteristically associated with constitutive expression of the dehydrogenase complex. Six pps mutants having Ace+ or 'Ace-' phenotypes were more sensitive than the parental strains and expression of their ace operons appeared to be affected; most sensitive were the Ace-strains which lacked pyruvate dehydrogenasecomplexand phosphoenolpyruvate synthetase activities.The lipoamide dehydrogenase activities of the deletion strains (Lpd+) ranged between 30 % and 100 % of parental levels indicating that expression of the Zpd gene may be affected by the ace operon but can be independent.

A new one-step procedure for the isolation of bacterial RNA, involving lysis by proteinase K in the presence of sodium dodecyl sulfate, is described.Pulse-labeled RNA isolated by this procedure from Bacillus brevis, Bacillus subtiZis, and Escherichia coZi B has been found to contain a substantial fraction (15-40%) of polyadenylated RNA as determined by adsorption to oligo (dT)-cellulose. This contrasts with RNA isolated by procedures involving phenol extraction, a process which appears to lead to the selective loss of polyadenylated RNA. The presence of polyadenylated RNA in E. coZi was confirmed by an independent method which involved hybridization with [3H]polyuridylic acid. Using the proteinase K method for RNA isolation, it was possible to demonstrate the in vitro synthesis of polyadenylated RNA by toluene-

A substantial fraction (30--40%) of pulse-labeled RNA from exponentially growing cells of Bacillus brevis contains polyadenylate sequences, as measured by adsorption to oligo(dT)-cellulose. The weight-average length of poly(A) tracts obtained after digestion with pancreatic and T1 ribonucleases is 60 nucleotide residues. Susceptibility to degradation by snake venom phosphodiesterase after ribonuclease degradation indicates that the poly(A) sequences are located near the 3' ends of the RNA chains, but that in 40% of the material at least one internal pyrimidine nucleotide residue intervenes between the poly(A) tract and the 3'-hydroxyl terminus. These pyrimidine nucleotides consist of 65% cytidylate and 35% uridylate residues. In the remaining RNA chains, the poly(A) sequence is directly at the 3'-terminus, but the possibility cannot be excluded that a small fraction of this material may contain a 3'-hydroxyl terminal guanylate residue. The weight-average sedimentation coefficient of poly(A)-containing RNA is 12.5 S, corresponding to a polynucleotide chain length of 800--900 residues. This is in a size range expected for messenger RNA, a possibility which is also supported by the observation that pulse-labeled RNA has a considerably higher poly(A) content than long-term labeled RNA.