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FUNCTIONAL COMPLEMENTATION OF AN ARGININE AUXOTROPHIC YEAST MUTANT BY AN ARGININOSUCCINATE SYNTHETASE FROM PORPHYRA YEZOENSIS (RHODOPHYTA) 1
Author(s) -
Endo Hirotoshi,
Ootsuka Shuuji,
Fukuda Satoru,
Kitade Yukihiro,
Saga Naotsune
Publication year - 2006
Publication title -
journal of phycology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.85
H-Index - 127
eISSN - 1529-8817
pISSN - 0022-3646
DOI - 10.1111/j.1529-8817.2006.00264.x
Subject(s) - biology , complementation , auxotrophy , mutant , gene , shuttle vector , saccharomyces cerevisiae , yeast , genetics , protein fragment complementation assay , microbiology and biotechnology , vector (molecular biology) , recombinant dna
The objective of this study was to develop a tool for studying Porphyra yezoensis Ueda gene function using the yeast transformation system. The gene encoding argininosuccinate synthetase (EC 6345) of P. yezoensis ( PyARG1 ) was obtained by PCR using an expressed sequence tag clone as a template, and subcloned into the yeast expression vector pYES2. The gene was expressed when the vector harboring PyARG1 was introduced into an ARG1 ‐deficient strain of Saccharomyces cerevisiae , which resulted in successful complementation of the mutant phenotype. The transformed cells could survive on a selective medium lacking arginine, and transcripts of PyARG1 were detected by reverse transcription‐PCR. A quantitative comparison showed that the rescued mutant cells grew in the selective liquid medium with a minor reduction in growth rate relative to wild‐type cells. This is the first report in which the function of a P. yezoensis gene has been directly demonstrated. This technique will provide new opportunities for further investigations into the functions of various genes in P. yezoensis and other macroalgal species.