ISOLATION OF A CHITIN SYNTHASE GENE (CHS1) FROM CANDIDA-ALBICANS BY EXPRESSION IN SACCHAROMYCES-CEREVISIAE
ISOLATION OF A CHITIN SYNTHASE GENE (CHS1) FROM CANDIDA-ALBICANS BY EXPRESSION IN SACCHAROMYCES-CEREVISIAE
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DOI：
10.1111/j.1365-2958.1990.tb00587.x
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发表时间：
1990-02-01
影响因子：
3.6
通讯作者：
ROBBINS, PW
中科院分区：
文献类型：
--
作者：
AUYOUNG, J;ROBBINS, PW
研究了白色念珠菌酵母菌和菌丝体中几丁质合成酶的活性。pH-活性曲线表明，酵母菌和菌丝含有蛋白酶依赖的活性，具有最佳pH 6.8。此外，有一种活性在体外不被蛋白水解激活，并且在pH 8.0时显示出峰值。这表明C.白色念珠菌从C.通过在酿酒酵母chs 1突变体中异源表达来克隆白色念珠菌（CHS 1）。证明克隆的几丁质合成酶是一个C.能够在体外进行几丁质生物合成的白色念珠菌膜结合酶原基于几个标准。(i)CHS 1基因与S.（ii）该酶催化[14 C]-GlcNAc从底物UDP[U-14 C]-GlcNAc掺入到碱不溶性几丁质中;（iii）Southern分析显示C. albicans CHS 1探针仅与C.而与S. albicans DNA无明显关系。（iv）克隆酶的pH曲线显示最适pH为6.8。这与在酵母和菌丝形式的C.白色念珠菌因此，CHS 1仅编码C.白色念珠菌在C.白色念珠菌的最适pH值为8.0。DNA序列分析表明，该基因的开放阅读框为2328个核苷酸，预测其为776个氨基酸的多肽。氨基酸序列比对结果表明，C.白念珠菌（canCHS 1）与S.酿酒酵母（sacCHS 1）。
Chitin synthase activity was studied in yeast and hyphal forms of Candida albicans. pH-activity profiles showed that yeast and hyphae contain a protease-dependent activity that has an optimum at pH 6.8. In addition, there is an activity that is not activated by proteolysis in vitro and which shows a peak at pH 8.0. This suggests there are two distinct chitin synthases in C. albicans. A gene for chitin synthase from C. albicans (CHS1) was cloned by heterologous expression in a Saccharomyces cerevisiae chs1 mutant. Proof that the cloned chitin synthase is a C. albicans membrane-bound zymogen capable of chitin biosynthesis in vitro was based on several criteria. (i) the CHS1 gene complemented the S. cerevisiae chs1 mutation and encoded enzymatic activity which was stimulated by partial proteolysis; (ii) the enzyme catalyses incorporation of [14C]-GlcNAc from the substrate, UDP[U-14C]-GlcNAc, into alkali-insoluble chitin; (iii) Southern analysis showed hybridization of a C. albicans CHS1 probe only with C. albicans DNA and not with S. cerevisiae DNA; (iv) pH profiles of the cloned enzyme showed an optimum at pH 6.8. This overlaps with the pH-activity profiles for chitin synthase measured in yeast and hyphal forms of C. albicans. Thus, CHS1 encodes only part of the chitin synthase activity in C. albicans. A gene for a second chitin synthase in C. albicans with a pH optimum at 8.0 is proposed. DNA sequencing revealed an open reading frame of 2328 nucleotides which predicts a polypeptide of Mr 88281 with 776 amino acids. The alignment of derived amino acid sequences revealed that the CHS1 gene from C. albicans (canCHS1) is homologous (37% amino acid identity) to the CHS1 gene from S. cerevisiae (sacCHS1).