Transcription and translation in E. coli of hybrid plasmids containing the catabolic dehydroquinase gene from Neurospora crassa

Abstract

Two hybrid plasmids which carry the gene for Neurospora crassa catabolic dehydroquinase (C-DHQase) and complement an aroD6 (dehydroquinase-deficient) auxotroph of Escherichia coli have been analyzed. One of these contains a 2.9 kilobase (kb) fragment cloned in the HindIII site of plasmid pBR322 (pVK57) and the other contains a 6.8 kb fragment cloned in the PstI site (pVK88). Restriction enzyme mapping of these plasmids has demonstrated that the 2.9 kb fragment is totally contained within the 6.8 kb fragment. When the polarity of either the HindIII fragment or PstI fragment was reversed with respect to pBR322 no effect was observed on either the ability of the hybrid to complement an aroD- auxotroph or on the level of C-DHQase activity. In vivo transcription of plasmid pVK88 in both orientations was analyzed by RNA-DNA hybridization and by the techniques developed by Southern (1975). Approx. 40% of the plasmid-directed transcription occurred from the cloned PstI fragment and 60--70% of these N. crassa transcripts were encoded by the 2.9 kb HindIII fragment. The Southern technique allowed a further localization of the region of most extensive transcription to a 1.8 kb HindIII-EcoRI fragment. Biochemical analysis revealed that the C-DHQase protein produced by strains harboring pVK57 and pVK88 in either orientation was identical to the N. crassa enzyme. Furthermore, when these plasmids were segregated into minicells and labeled with 14C amino acids, the C-DHQase protein was synthesized at a level comparable to other plasmid-encoded proteins. Taken together, these experiments demonstrate that transcription is efficiently initiated in E. coli from a site on the cloned N. crassa DNA and that the resulting C-DHQase mRNA is efficiently and accurately translated.