Molecular cloning and characterization of the STA2 glucoamylase gene of Saccharomyces diastaticus.
Molecular cloning and characterization of the STA2 glucoamylase gene of Saccharomyces diastaticus.
复制标题
糖化酵母 STA2 葡糖淀粉酶基因的分子克隆和表征。
DOI：
10.1007/bf00330380
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Marmur,J
中科院分区：
文献类型：
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作者：
Pretorius,IS;Chow,T;Modena,D;Marmur,J
通过对一株酵母菌的互补克隆，获得了编码胞外糖淀粉酶(1，4-α-d-葡聚糖水解酶，EC 3.2.1.3)的糖化结构基因STA2。以酵母表达载体pYCL中的一株STA2酵母为模板，初步构建了一个基因组文库。进一步将StA+互补功能限定为8.3kbBglII片段，其限制性内切酶图谱与STA1和STA3的相关基因组区域相似。从8.3 kbBglII片段衍生的几个DNA片段与TruncatedE.Coliβ-半乳糖苷酶基因产生两个重叠的片段，可指导大融合蛋白的生产。Coli.这些融合蛋白可被抗糖淀粉酶II抗体免疫沉淀，证实StA+互补融合蛋白是由于编码酵母糖化酶的基因表达所致。以克隆的STA2基因的一个内部片段为探针，通过RNA-DNA杂交检测STA1、STA2和STA3RNA转录本，结果表明每个STA1、STA2和STA3RNA转录本都有一个2.5kb的共同转录本。利用体外构建的供体基因片段将URA3基因插入到克隆的糖淀粉酶基因的编码区，通过同源重组将STA2酵母菌株转化到StA−中，从而实现了STA2基因的整合破坏。对携带被破坏的STA2和功能STA2的菌株之间的杂交的四分体分析证实了这一点。用BamHI消化的15个四分体的基因组DNA进行Southern杂交分析，表明StA−表型与STA2：：URA3一致的共分离和孟德尔遗传。这些数据进一步证实，克隆的具有StA+互补活性的DNA携带有功能的STA2基因，编码酵母胞外糖淀粉酶II。
TheSaccharomyces diastaticusstructural geneSTA2, encoding an extracellular glucoamylase (1,4-α-d-glucan glycohydrolase, EC 3.2.1.3.), has been cloned by complementation of astaostrain. A genomic library was initially constructed from aSTA2yeast strain in the yeastEscherichia colishuttle cosmid vector pYCl. The Sta+complementing function was further delimited to an 8.3 kbBglII fragment whose restriction map was found to be similar to related genomic regions ofSTA1andSTA3. Fusions of several DNA fragments derived from the 8.3 kbBglII fragment with a truncatedE. coliβ-galactosidase gene resulted in two overlapping fragments that could direct the production of large fusion proteins inE. coli. These fusion proteins were immunoprecipitable by anti-glucoamylase II antibodies, confirming that the Sta+complementing fusion was due to the expression of a gene that coded for a yeast glucoamylase. Measurements of theSTA1, STA2andSTA3RNA transcripts by RNA-DNA hybridization using an internal fragment of the clonedSTA2gene as the probe indicated that a common transcript of 2.5 kb is produced by each of theSTAgenes. Integrative disruption of theSTA2gene through homologous recombination was achieved by transforming aSTA2yeast strain to Sta−using an in vitro constructed donor DNA fragment that has theURA3gene inserted within the coding region of the cloned glucoamylase gene. This was confirmed by tetrad analysis of crosses between strains carrying a disruptedSTA2and a functionalSTA2. Southern blot analysis usingBamHI digested genomic DNA from 15 tetrads demonstrated consistent co-segregation and Mendelian inheritance of the Sta−phenotype withSTA2::URA3. These data further confirm that the cloned DNA that showed Sta+complementing activity carries a functionalSTA2gene that encodes the yeast extracellular glucoamylase II.