PCR Based Cloning and Sequence Analysis of the Pichia pastoris Cystathionine beta-Synthase Gene.
The cystathionine beta-Synthase (CBS) gene of Pichia pastoris has been cloned by homology to the CBS gene of Saccharomyces cerevisiae. First, based on the homology alignment of CBS genes from different sources, a pair of degenerate PCR primers were designed to amplify a conservative fragment of Pichia pastoris CBS gene. After the sequence was revealed, 5' RACE and 3' RACE were performed separately. The whole sequence of Pichia pasotris CBS gene was assembled. This gene encodes a polypeptide of 501 residues. Comparison of deduced amino acid sequence of this new gene with that of S. cerevisiae CBS showed 54% identity. Disruption of CBS gene resulted in a Cys(-) auxotrophy in Pichia pastoris. Thesequence was submitted to GenBank/EMBL/DDBJ under Accession No.AF367364.
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Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China. zhongyi@sunm.shcnc.ac.cn
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Yu
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Chen
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Yuan
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