| Abstract: | A sample of colonies from the Clarke-Carbon ColE1-Escherichia coli DNA plasmid gene bank was screened by conjugation for complementation of the lipoamide dehydrogenase lesion of a deletion strain lacking all components of the pyruvate dehydrogenase complex, delta (aroP aceE aceF lpd). Two ColE1-lpd+ hybrid plasmids were identified: pGS2 (ColE1-ace lpd+; 24 kb) and pGS5 (ColE1-lpd+; 14 kb). Enzymological studies confirmed that pGS2 expressed all the activities of the pyruvate dehydrogenase complex, whereas pGS5 expressed the lipoamide dehydrogenase and acetyltransferase activities (the latter from a ColE1 promoter). These and other plasmids were used to construct a 47-site (15 enzymes) restriction map for a 24.2 kb segment of bacterial DNA in the nadC-lpd region. A further 13 sites (six enzymes) were defined in a 5.4 kb sub-segment containing the lpd gene. lambda phage derivatives containing specific fragments were constructed and used in transduction studies which located the ace and lpd genes in a 7.78 kb sub-segment flanked by AccI and NruI sites. |
