Site-directed saturation mutagenesis of yeast Gcn4p at codon 242

Jae Yung Lee, Yu Byung Bae, Jung Ae Kim, Jae Mahn Song, Muhyeon Choe, Ick Young Kim, Joon Kim

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Gcn4p, a transcriptional activator protein of the yeast, Sacchromyces cerevisiae, binds to the specific sequence in the promoters of many amino acid biosynthetic genes for general control. The serine residue (Set 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 oligonucleotides 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 be a simple, fast, and economical method for constructing a library of all 20 amino acids at specific codon.

Original languageEnglish
Pages (from-to)122-125
Number of pages4
JournalJournal of microbiology and biotechnology
Volume9
Issue number1
Publication statusPublished - 1999 Feb

Keywords

  • Gcn4p
  • Mutagenesis
  • Polymerase chain reaction
  • Saccharomyces cerevisiae

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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