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西方许旺酵母NADP依赖型谷氨酸脱氢酶基因的克隆、测序及表达

Cloning, sequencing and expression of the Schwanniomyces occidentalis NADP-dependent glutamate dehydrogenase gene.

作者信息

De Zoysa P A, Connerton I F, Watson D C, Johnston J R

机构信息

Department of Microbiology, University of Reading, England, UK.

出版信息

Curr Genet. 1991 Aug;20(3):219-24. doi: 10.1007/BF00326236.

DOI:10.1007/BF00326236
PMID:1934128
Abstract

The cloned NADP-specific glutamate dehydrogenase (GDH) genes of Aspergillus nidulans (gdhA) and Neurospora crassa (am) have been shown to hybridize under reduced stringency conditions to genomic sequences of the yeast Schwanniomyces occidentalis. Using 5' and 3' gene-specific probes, a unique 5.1 kb BclI restriction fragment that encompasses the entire Schwanniomyces sequence has been identified. A recombinant clone bearing the unique BclI fragment has been isolated from a pool of enriched clones in the yeast/E. coli shuttle vector pWH5 by colony hybridization. The identity of the plasmid clone was confirmed by functional complementation of the Saccharomyces cerevisiae gdh-1 mutation. The nucleotide sequence of the Schw. occidentalis GDH gene, which consists of 1380 nucleotides in a continuous reading frame of 459 amino acids, has been determined. The predicted amino acid sequence shows considerable homology with GDH proteins from other fungi and significant homology with all other available GDH sequences.

摘要

构巢曲霉（gdhA）和粗糙脉孢菌（am）的克隆的NADP特异性谷氨酸脱氢酶（GDH）基因已被证明在降低的严格条件下与西方许旺酵母的基因组序列杂交。使用5'和3'基因特异性探针，已鉴定出一个包含整个许旺酵母序列的独特的5.1 kb BclI限制性片段。通过菌落杂交从酵母/大肠杆菌穿梭载体pWH5中的富集克隆库中分离出携带独特BclI片段的重组克隆。通过酿酒酵母gdh-1突变的功能互补证实了质粒克隆的身份。已确定西方许旺酵母GDH基因的核苷酸序列，其由1380个核苷酸组成，处于459个氨基酸的连续阅读框中。预测的氨基酸序列与来自其他真菌的GDH蛋白具有相当的同源性，并且与所有其他可用的GDH序列具有显著同源性。

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