Cloning of cDNA coding for peroxisomal acyl-CoA oxidase from the yeast Candida tropicalis pK233.
Cloning of cDNA coding for peroxisomal acyl-CoA oxidase from the yeast Candida tropicalis pK233.
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从热带假丝酵母 pK233 中克隆编码过氧化物酶体酰基辅酶 A 氧化酶的 cDNA。
DOI：
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发表时间：
1985
影响因子：
11.1
通讯作者：
P. Lazarow
中科院分区：
文献类型：
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作者：
R. Rachubinski;Y. Fujiki;P. Lazarow
以正构烷烃为碳源培养热带假丝酵母pK233细胞，诱导过氧化物酶体蛋白的合成和过氧化物酶体的增殖。分离Poly-(A)+ RNA，将双链逆转录物插入pBR322的Pst I位点，并在大肠杆菌中克隆，构建cDNA文库。利用从葡萄糖生长细胞（含少量过氧化物酶体）或烷烃生长细胞的poly(A)+ RNA反转录的[32P]cDNA，通过差异DNA斑点分析选择与烷烃上生长诱导的mrna互补的克隆。在这些克隆中，通过杂交选择翻译和免疫沉淀鉴定了一个含有1.7千碱基插入编码酰基辅酶a氧化酶（过氧化物酶体β -氧化途径的第一酶）的克隆。通过RNA印迹分析，估计酰基辅酶a氧化酶mRNA大约等于2.2千碱基长，其中2.1千碱基需要编码大约等于76 kda的蛋白质。由于mRNA是聚腺苷化的，因此似乎没有额外的非翻译区域。无细胞mRNA翻译和RNA斑点杂交分析表明，葡萄糖培养的热带草含有很少或不含酰基辅酶a氧化酶mRNA，而烷烃培养的细胞含有如此多的这种mRNA，使酰基辅酶a氧化酶成为体外翻译的主要产物之一。
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.