FUNCTIONAL COMPLEMENTATION OF AN ESCHERICHIA-COLI RIBONUCLEASE-H MUTATION BY A CLONED GENOMIC FRAGMENT FROM THE TRYPANOSOMATID CRITHIDIA-FASCICULATA

A gene designated Cfa RNH1 has been cloned by complementation of an RN ase H deficiency in an Escherichia coli rnhA mutant by using a genomic DNA library from the trypanosomatid Crithidia fasciculata. The encode d RNase H is predicted to have 494 amino acid residues and a molecular mass of 53.7 kDa. The carboxyl half of the protein is homologous to t he 155 -residue E. coli RNase HI (41% identity) and the 166-residue Sa ccharomyces cerevisiae RNase HI (33% identity). The recombinant protei n has been purified as a six-histidine-tagged fusion protein by metal chelate chromatography and was shown to have RNase H activity. Antibod ies against the recombinant protein recognize proteins of approximatel y 65 kDa and 56 kDa on Western blots of C. fasciculata extracts. These results demonstrate the feasibility of cloning trypanosome genes by c omplementation of appropriate E. coli mutants with genomic DNA librari es.