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

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Abstract
C. tropicalis pK233 cells were grown with n-alkanes as C source to induce the synthesis of peroxisomal proteins and the proliferation of peroxisomes. Poly(A)+ RNA was isolated and used to construct a c[complementary]DNA 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 1st 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 as .apprxeq. 2.2 kilobases long, of which 2.1 kilobases are required to code for the .apprxeq. 76-kDa [kilodalton] protein. Since the mRNA is polyadenylylated, there apparently is 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.