Development of a Genome-Wide Random Mutagenesis System Using Proofreading-Deficient DNA Polymerase δ in the Methylotrophic Yeast Hansenula polymorpha

자연과학 > 생물
( Oh Cheol Kim ) , ( Sang Yoon Kim ) , ( Dong Hyeon Hwang ) , ( Doo Byoung Oh ) , ( Hyun Ah Kang ) , ( Oh Suk Kwon )
한국미생물생명공학회(구 한국산업미생물학회)
Journal of Microbiology and Biotechnology 2013년, 제23권 제3호, 304~312페이지(총9페이지)
05212222.pdf [무료 PDF 뷰어 다운로드]
  • ※ 본 자료는 참고용 논문으로 수정 및 텍스트 복사가 되지 않습니다.
  • 구매가격
    135원 (구매자료 3% 적립)
    이메일 발송  스크랩 하기
    자료 다운로드  네이버 로그인
    The thermotolerant methylotrophic yeast Hansenula polymorpha is attracting interest as a potential strain for the production of recombinant proteins and biofuels. However, only limited numbers of genome engineering tools are currently available for H. polymorpha. In the present study, we identified the HpPOL3 gene encoding the catalytic subunit of DNA polymerase δ of H. polymorpha and mutated the sequence encoding conserved amino acid residues that are important for its proofreading 3`→5` exonuclease activity. The resulting HpPOL3* gene encoding the error-prone proofreading-deficient DNA polymerase δ was cloned under a methanol oxidase promoter to construct the mutator plasmid pHIF8, which also contains additional elements for site-specific chromosomal integration, selection, and excision. In a H. polymorpha mutator strain chromosomally integrated with pHIF8, a URA3- mutant resistant to 5-fluoroorotic acid was generated at a 50-fold higher frequency than in the wild-type strain, due to the dominant negative expression of HpPOL3*. Moreover, after obtaining the desired mutant, the mutator allele was readily removed from the chromosome by homologous recombination to avoid the uncontrolled accumulation of additional mutations. Our mutator system, which depends on the accumulation of random mutations that are incorporated during DNA replication, will be useful to generate strains with mutant phenotypes, especially those related to unknown or multiple genes on the chromosome.
    오늘 본 자료
    • 오늘 본 자료가 없습니다.
    본 학술논문은 한국학술정보㈜ 각 학회간에 저작권 계약이 체결된 것으로 HAPPY학술이 제공하고 있습니다.
    본 저작물을 불법적으로 이용시는 법적인 제재가 가해질 수 있습니다.
    사업자등록번호 220-87-87785 대표.신현웅 주소.서울시 서초구 방배로10길 18, 402호 대표전화.070-8809-9397
    개인정보책임자.박정아 통신판매업신고번호 제2017-서울서초-1765호 이메일
    copyright (c) 2009 happynlife. steel All reserved.