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A sample of colonies from the Clarke-Carbon Co1E1-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, Δ(aroP aceE aceF lpd). Two Co1E1-lpd + hybrid plasmids were identified: pGS2 (Co1E1-ace lpd +; 24 kb) and pGS5 (Co1E1-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 futher 13 sites (six enzymes) were defined in a 5·4 kb sub-segment containing the lpd gene. γ 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.
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