Orientation of enzymic domains in tryptophan synthase of Neurospora crassa: an immunoblot analysis of TRP3 mutant products.
Orientation of enzymic domains in tryptophan synthase of Neurospora crassa: an immunoblot analysis of TRP3 mutant products.
复制标题
粗糙脉孢菌色氨酸合酶中酶结构域的方向：TRP3 突变产物的免疫印迹分析。
DOI：
10.1007/bf00328130
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
DeMoss,JA
中科院分区：
文献类型：
--
作者：
Matchett,WH;Lacy,AM;DeMoss,JA
通过电泳印迹分析的方法测试了粗糙脉孢菌52TRP3突变体的提取物中血清学交叉反应物质的存在。 SDS-聚丙烯酰胺凝胶电泳后，切下变性的纯色氨酸合酶的浅染色条带，获得测试抗原。针对该抗原产生的兔抗血清中和并沉淀了天然色氨酸合酶。在52个菌株中，19个菌株在电泳印迹上表现出与野生型相似的条带图案，25个菌株没有给出明显的条带，8个菌株显示出独特的条带图案。这一证据和相关的遗传精细结构图谱数据表明，表现出与野生型相似的条带模式的菌株携带错义突变。没有表现出任何明显条带的菌株可能是由某些类型的错义或无义突变或移码突变或延伸缺失引起的。在电泳印迹上表现出独特条带模式的菌株被解释为携带链终止突变或缺失。遗传精细结构图谱数据将这些菌株的突变损伤按线性顺序排列，对应于它们表现出的交叉反应蛋白片段的表观分子量。从这些关系推断出脉孢子虫中TRP3基因座的转录和翻译方向。脉孢菌TRP3基因的表观组织与酵母属TRP5系统的表观组织一致，并且表明该蛋白质的N末端部分对应于大肠杆菌系统的“A”蛋白质，并且该蛋白质的C末端部分对应于细菌系统的“B”蛋白质。
Extracts of 52TRP3mutants ofNeurospora crassawere tested for the presence of serologically cross-reacting material by the method of electrophoretic blot analysis. The test antigen was obtained by excision of lightly stained bands of denatured pure tryptophan synthase after SDS-polyacrylamide gel electrophoresis. Rabbit antisera raised against this antigen neutralized and precipitated native tryptophan synthase. Of the 52 strains, 19 exhibited banding patterns similar to wild type on electrophoretic blots, 25 strains gave no apparent bands, and 8 strains showed unique banding patterns. This evidence and related genetic fine structure mapping data indicate that strains exhibiting banding patterns similar to wild type carry missense mutations. Strains which did not exhibit any obvious bands may have resulted from certain kinds of missense or nonsense mutations or from frameshift mutations or extended deletions. Strains exhibiting unique banding patterns on electrophoretic blots were interpreted as carrying chain-terminating mutations or deletions. Genetic fine structure mapping data place the mutant lesions of these strains in a linear order corresponding to the apparent molecular weights of the crossreacting protein fragments which they exhibit. The direction of transcription and translation of theTRP3locus inNeurosporawas inferred from these relationships. The apparent organization of theNeurospora TRP3gene is consistent with that ascribed to theSaccharomyces TRP5system and suggests that the N-terminal portion of the protein corresponds to the “A” protein of theEscherichia colisystem and that the C-terminal portion of the protein corresponds to the “B” protein of the bacterial system.