Molecular Cloning of Mutant cDNA of PU.1 Gene

PU.1 유전자(cDNA)의 인위적 변이체 클로닝

  • 류종석 (성균관대학교 생명자원과학대학 유전공학과) ;
  • 유시현 (성균관대학교 생명자원과학대학 유전공학과 성균관대학교 생명자원과학대학 유전공학과 성균관대학교 생명자원과학대학 유전공학과 성균관대학교 생명자원과학대학 유전공학과 성균관대학교 생명자원과학대학 유전공학과)
  • Published : 1995.11.01

Abstract

PU.1, a tissue-specific transcription activator, binds to a purine-rich sequence(5'-GAGGAA-3') called PU box. The PU.1 cDNA consists of an open reading frame of 816 nucleotides coding for 272 amino acids. The amino terminal end is highly acidic, while the carboxyl terminal end is highly basic. Transcriptional activation domain is located at the amino terminal end, while DNA binding domain is located at the carboxyl terminal end. Activation of PU.1 transcription factor is supposed to be accomplished by the phosphorylation of serine residue(s). There exist 22 serines in the PU.1. Five(the 41, 45, 132$.$133, and 148th) of the serines(plausible phosphorylation site by casein kinase II), are the primary targets of interest in elucidating the molecular mechanism(s) of the action of the PU.1 gene. In this study, PU.1 cDNA coding for the five serine residues(41th AGC, 45th AGC, 132$.$133th AGC$.$TCA, and 148th TCT), was mutated to alanine codon(41th GCC, 45th GCC, 132$.$133th GCC$.$GCA, and 1481h GCT), respectively, by Splicing-Overlapping-Extension(SOE) using Polymerase Chain Reaction(PCR). And each mutated cDNA fragments was ligated into pBluescript KS+ digested with HindIII and Xba I, to generate mutant clones named pKKS41A, pRKS45A, pMKS132$.$133A, and pMKS148A. The clones will be informative to study the "Structure and Function" of the immu-nologically important gene, PU.1.

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