Restriction digests of the clone give the following sizes (kb): BamHI/EcoRI--2.7, 3.2; BglI--2.5, 1.3, 1.1, 0.9; HindIII--3.1, 1.7, 0.8, 0.2. The GenBank accession number for the adh insert is M15394. Expression appears to be regulated by the lac promoter, and growth is increased under anaerobic conditions. Constructed by inserting a 1.4 kb EcoRI/SalI fragment from pLOI284 containing the promoterless adh coding sequence into the BamHI site of pLOI276. The pdc reading frame begins 163 bp downstream of the lac promoter and ends with two stop codons 85 bp upstream of the adhB reading frame. The adhB gene has a single stop codon followed by a palindromic sequence which serves as a terminator. pLOI284 consists of two DraI fragments of 0.12 and 1.23 kb cloned into the SmaI site of pUC18. The insert starts at the DraI site 402 nt downstream of the 5' DraI site of the reported sequence, with the coding region from nt 30 to 1181 of the insert. The (5')EcoRI/BamHI (3') fragment contains both genes. |
