Isolation of cDNA clones coding for peroxisomal proteins of Candida tropicalis: identification and sequence of a clone for catalase.
A cDNA library, complementary to mRNAs of alkane-grown Candida tropicalis, was screened by differential DNA dot-blot hybridization with [32P]cDNA reverse-transcribed from mRNA of alkane-grown cells or from cells in which peroxisome formation was repressed by growth on glucose. 9% of the library encodes alkane-induced sequences. The cell-free translation products of eight hybrid-selected mRNAs were characterized by SDS-polyacrylamide gel electrophoresis and fluorography: most of them are probably peroxisomal proteins. Among these, a catalase clone was identified by immunoprecipitation of the translation product with anti-catalase. The clone was sequenced: the inferred amino acid sequence is homologous to the carboxytermini of mammalian and Saccharomyces cerevisiae catalases. C. tropicalis catalase mRNA is 1.7-1.8 kb long by Northern analysis, of which 1.5-1.6 kb is required to code for the 57 kDa polypeptide. Catalase mRNA (assayed by dot-blot hybridization) is strikingly induced in C. tropicalis by growth on alkanes, suggesting that peroxisome induction is transcriptionally regulated. This sublibrary of alkane-induced, mostly peroxisomal clones, together with a recently developed cell-free peroxisome protein import assay, will permit investigation of the targeting of proteins to peroxisomes.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002175:Candida / Q000201:enzymology / Q000235:genetics*; D002374:Catalase / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D005796:Genes; D005800:Genes, Fungal; D008830:Microbodies / Q000201:enzymology*; D009693:Nucleic Acid Hybridization; D014176:Protein Biosynthesis
D004247:DNA; D002374:Catalase