Rachubinski R A, Fujiki Y, Lazarow P B
Biochim Biophys Acta. 1987 Jun 6;909(1):35-43. doi: 10.1016/0167-4781(87)90044-3.
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.
用从烷烃培养细胞的mRNA或从在葡萄糖上生长而使过氧化物酶体形成受到抑制的细胞的mRNA反转录得到的[32P]cDNA，通过差异DNA斑点杂交筛选了与烷烃培养的热带假丝酵母mRNA互补的cDNA文库。该文库的9%编码烷烃诱导序列。通过SDS-聚丙烯酰胺凝胶电泳和荧光自显影对8种杂交选择的mRNA的无细胞翻译产物进行了表征：其中大多数可能是过氧化物酶体蛋白。其中，通过用抗过氧化氢酶对翻译产物进行免疫沉淀鉴定出一个过氧化氢酶克隆。对该克隆进行了测序：推导的氨基酸序列与哺乳动物和酿酒酵母过氧化氢酶的羧基末端同源。通过Northern分析，热带假丝酵母过氧化氢酶mRNA长1.7 - 1.8 kb，其中1.5 - 1.6 kb用于编码57 kDa的多肽。通过斑点杂交测定，过氧化氢酶mRNA在热带假丝酵母中通过在烷烃上生长而显著诱导，表明过氧化物酶体诱导是受转录调控的。这个烷烃诱导的、主要是过氧化物酶体克隆的亚文库，连同最近开发的无细胞过氧化物酶体蛋白导入测定法，将允许研究蛋白质向过氧化物酶体的靶向运输。
