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

• Published in: Proceedings of the National Academy of Sciences - PNAS 1985-06, Vol.82 (12), p.3973-3977
• Main Authors: Rachubinski, Richard A., Fujiki, Yukio, Lazarow, Paul B.
• Format: Article
• Language: English
• Subjects: Acyl-CoA Oxidase; adn; Biological and medical sciences; Biotechnology; candida; Candida - enzymology; Candida - genetics; Candida tropicalis; Cell growth; clone; clones; Cloning, Molecular; code genetique; codigo genetico; Complementary DNA; DNA; DNA - genetics; DNA Restriction Enzymes; enzimas; enzyme; enzymes; Flavoproteins - genetics; Fundamental and applied biological sciences. Psychology; genetic code; Genetic engineering; Genetic technics; Messenger RNA; Methods. Procedures. Technologies; Microbodies - enzymology; Molecular cloning; Molecular Weight; Open reading frames; Oxidases; Oxidoreductases - genetics; Peroxisomes; Plasmids; RNA; RNA, Messenger - genetics; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.82.12.3973

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 β -oxidation pathway) was identified by hybridization-selection translation and immunoprecipitation. By RNA blot analysis, the acyl-CoA oxidase mRNA was estimated to be ≈ 2.2 kilobases long, of which 2.1 kilobases are required to code for the ≈ 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.