PCR Based Cloning and
Sequence Analysis of the Pichia pastoris
Cystathionine β-Synthase Gene
LI Dong-Yang, JI Xin-Song, YU Jian, CHEN Mei-Juan, YUAN
Zhong-Yi*
( Institute of Biochemistry and Cell Biology, Shanghai Institutes for
Biological Sciences,the Chinese Academy of Sciences, Shanghai 200031,
China )
Abstract The
cystathionine β-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.
Key words cystathionine beta-synthase； Pichia pastoris； RACE； cloning； alignment
*Corresponding author： Tel, 86-21-64374430-5289； Fax, 86-21-64338357； e-mail, [email protected]