Chicken triosephosphate isomerase complements an Escherichia coli deficiency

Abstract

We present the sequence of full-length chicken triosephosphate isomerase (D-glyceraldehyde 3-phosphate ketol-isomerase, EC 5.3.1.1) mRNA based on the analysis of cDNA and genomic clones. To isolate cDNA clones encoding the enzyme, we screened a muscle cDNA library with radioactively labeled cDNA made from RNA that had been enriched by immunoselection of polysomes. We blocked the signal caused by contaminating species in the probe with cloned DNA corresponding to the contaminants. Screening a chicken genomic library with cDNA coding for triosephosphate isomerase led to the isolation of phage containing the entire gene, which we used to map the transcriptional start. When placed downstream from a hybrid trp-lac promoter, the cDNA encoding the chicken enzyme programs the synthesis of functional protein, as judged by enzymatic criteria and by complementation of an Escherichia coli mutant that is deficient in bacterial triosephosphate isomerase.