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 RNase H deficiency in an Escherichia coli rnhA mutant by using a genomic DNA library from the trypanosomatid Crithidia fasciculata. The encoded 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 the 155-residue E. coli RNase HI (41% identity) and the 166-residue Saccharomyces cerevisiae RNase HI (33% identity). The recombinant protein has been purified as a six-histidine-tagged fusion protein by metal chelate chromatography and was shown to have RNase H activity. Antibodies against the recombinant protein recognize proteins of approximately 65 kDa and 56 kDa on Western blots of C. fasciculata extracts. These results demonstrate the feasibility of cloning trypanosome genes by complementation of appropriate E. coli mutants with genomic DNA libraries.
Campbell
A G
AG
Molecular Biology Institute, University of California, Los Angeles 90024-1570.
Ray
D S
DS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016831:Crithidia fasciculata / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology*; D005800:Genes, Fungal; D017238:Genes, Helminth; D005814:Genes, Viral; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016914:Ribonuclease H / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015678:Viral Structural Proteins / Q000235:genetics
D009838:Oligodeoxyribonucleotides; D015678:Viral Structural Proteins; D016914:Ribonuclease H