Arg243, invariably critical for the transcriptional activation of yeast Gcn4p

作者：Cho GC; Lee JY; Kim J

来源：Journal of Microbiology, 1999, 37(3): 154-158.

• mutagenesis
• two-step polymerase chain reaction
• Arg243
• Gcn4p

The arginine residue at position 243 (Arg 243) of the yeast transcription factor, Gcn4p, is invariably conserved among bZIP transcription factors. Using site directed oligonucleotide saturation mutagenesis involving two-step polymerase chain reaction (PCR) amplification, random mutations were successfully introduced at the codon of 243 in the basic domain of Gcn4p. This mutant library was transformed into Gcn4p defective yeast strain and selected for the transcriptionally active colonies. All colonies which were transcriptionally active had arginines in the codon 243. In this study, the strand preference by Tag polymerase during mutagenesis was also tested. Oligonucleotides were specially designed to test whether or not the polymerase was preferred using the strand as a template. A population of randomly mutated products were cloned into an appropriate vector and characterized by DNA sequencing analysis. Saturation mutagenesis which was performed efficiently by this method revealed a strong bias in terms of strand preference of Tag polymerase by an approximate ratio of 3 to 1 in this study.

• 出版日期1999-9