CHARACTERIZATION OF AN INDUCIBLE EXPRESSION SYSTEM IN ASPERGILLUS-NIDULANS USING ALCA AND TUBULIN-CODING GENES
CHARACTERIZATION OF AN INDUCIBLE EXPRESSION SYSTEM IN ASPERGILLUS-NIDULANS USING ALCA AND TUBULIN-CODING GENES
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DOI：
10.1016/0378-1119(89)90097-8
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发表时间：
1989-06-30
期刊：
影响因子：
3.5
通讯作者：
MORRIS, NR
中科院分区：
文献类型：
--
作者：
WARING, RB;MAY, GS;MORRIS, NR
我们已经构建了一种质粒，在这种质粒中，编码酒精脱氢酶I的alcA基因的诱导启动子可以控制基因的表达。此外，还构建了携带pyr4的简化穿梭载体，以补充pyrG89突变。这些是基于pUC19和保留。alpha。肽表达。度量。将-微管蛋白基因tubC和benA置于alcA调控下，研究其表达。表达水平可以通过表型分析，因为β的合成增加。-微管蛋白抑制营养生长。无性孢子形成对抗微管药物苯甲酰的敏感性提供了一种检测嵌合基因非常低水平表达的手段。葡萄糖几乎完全抑制了嵌合基因。诱导迅速，并在一小时内达到最大。当携带7个alcA::tubC基因融合的菌株在诱导条件下生长时，总硫酸盐标记蛋白的6.5%由tubC产物组成。环戊酮是固体培养基上嵌合基因最有效的诱导剂，但也部分抑制了嵌合基因的生长。嵌合alcA::tubC和alcA::benA基因的表达水平非常相似，尽管tubC使用了许多罕见的密码子。
Plasmids have been constructed in which expression of gene can be placed under the control of the inducible promoter of the alcA gne encoding alcohol dehydrogenase I in Aspergillus nidulans. Simplified shuttle vectors carrying pyr4 which complements pyrG89 mutations have also been constructed. These are based on pUC19 and retain .alpha.-peptide expression. The .beta.-tubulin genes, tubC and benA, have been placed under the control of alcA and their expression studied. Levels of expression can be assayed phenotypically because increased synthesis of .beta.-tubulin inhibits vegetative growth. Sensitivity of asexual spore formation to the anti-microtubule drug benomyl provides a means of detecting very low levels of expression of the chimeric genes. Glucose almost completely represses the chimeric genes. Induction is rapid and is maximal within an hour. When a strain carrying seven copies of an alcA::tubC gene fusion was grown under inducing conditions, 6.5% of total sulfate labelled protein consisted of tubC product. Cyclopentanone was the most potent inducer of the chimeric genes on solid media but it also partially inhibited growth. Chimeric alcA::tubC and alcA::benA genes were expressed to very similar levels despite the fact that tubC utilizes many rare codons.