Cloning of cDNA coding for peroxisomal acyl-CoA oxidase from the yeast Candida tropicalis pK233

Abstract

Candida tropicalis pK233 cells were grown with n-alkanes as carbon source to induce the synthesis of peroxisomal proteins and the proliferation of peroxisomes. Poly-(A)+ RNA was isolated and used to construct a cDNA library by insertion of double-stranded reverse transcripts into the Pst I site of pBR322 followed by cloning in Escherichia coli. Clones complementary to mRNAs induced by growth on alkanes were selected by differential DNA dot-blot analysis using [32P]cDNA reverse-transcribed from poly(A)+ RNA of glucose-grown cells (which contain few peroxisomes) or of alkane-grown cells. Among these clones, one containing a 1.7-kilobase insert coding for acyl-CoA oxidase (the first enzyme in the peroxisomal Beta-oxidation pathway) was identified by hybridization-selection translation and immunoprecipitation. By RNA blot analysis, the acyl-CoA oxidase mRNA was estimated to be approximately equal to 2.2 kilobases long, of which 2.1 kilobases are required to code for the approximately equal to 76-kDa protein. Since the mRNA is polyadenylylated, there appears to be little additional nontranslated region. Cell-free mRNA translation and RNA dot-blot hybridization analyses demonstrated that, whereas glucose-grown C. tropicalis contained little or no acyl-CoA oxidase mRNA, alkane-grown cells contained so much of this mRNA as to make acyl-CoA oxidase one of the major in vitro translation products.