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KMID : 0545119990090010122
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Journal of Microbiology and Biotechnology
1999 Volume.9 No. 1 p.122 ~ p.125
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Site-Directed Saturation Mutagenesis of Yeast Gcn4p at Codon 242
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Lee, Jae Yung
Bae, Yu Byung/Kim, Jung Ae/Song, Jae Mahn/Choe, Mu Hyeon/Kim, Ick Young/Kim, Joon
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Abstract
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Gcn4p, a transcriptional activator protein of the yeast, Saechromyces cerevisiae, binds to the specific sequence in the promoters of many amino acid biosynthetie genes for general control. The serine residue (Ser 242) of Gcn4p directly contacts the DNA. Here, for inspecting the DNA binding properties and the level of transcriptional activation of Gcn4p. we introduced a polymerase chain reaction (PCR) site-directed saturation mutation library into the Ser 242 site using 2 outside primers and 2 oligonueleotides with its codons fully degenerated. The sequencing analysis of 146 samples revealed the even nucleotide distribution within the experimental error showing 23, 26. 25, and 26% frequency of U, C A, and G bases, respectively. This method turned out to he a simple. fast. and economical method for constructing a library of all 20 amino acids at specific colon.
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