Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
권호 표지

Stability of Human Centromeric Alphoid DNA Repeat during Propagation in Recombination-Deficient Yeast Strains

효모의 재조합 변이주를 이용한 인간 Centromeric Alphoid DNA Repeat의 안정성에 관한 연구

  • Kim, Kwang-Sup (Department of Biological Science, Dong-A University) ;
  • Shin, Young-Sun (Department of Biological Science, Dong-A University) ;
  • Lee, Sang-Yeop (Department of Biological Science, Dong-A University) ;
  • Ahn, Eun-Kyung (Department of Biological Science, Dong-A University) ;
  • Do, Eun-Ju (Department of Biological Science, Dong-A University) ;
  • Park, In-Ho (Department of Biological Science, Dong-A University) ;
  • Leem, Sun-Hee (Department of Biological Science, Dong-A University) ;
  • SunWoo, Yang-Il (Department of Biological Science, Dong-A University)
  • 김광섭 (동아대학교 자연과학대학 생명과학과) ;
  • 신영선 (동아대학교 자연과학대학 생명과학과) ;
  • 이상엽 (동아대학교 자연과학대학 생명과학과) ;
  • 안은경 (동아대학교 자연과학대학 생명과학과) ;
  • 도은주 (동아대학교 자연과학대학 생명과학과) ;
  • 박인호 (동아대학교 자연과학대학 생명과학과) ;
  • 임선희 (동아대학교 자연과학대학 생명과학과) ;
  • 선우양일 (동아대학교 자연과학대학 생명과학과)
  • Published : 2007.12.30
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Abstract

The centromere is a highly differentiated structure of the chromosome that fulfills a multitude of essential mitotic and meiotic functions. Alphoid DNA (${\alpha}$-satellite) is the most abundant family of repeated DNA found at the centromere of all human chromosomes, and chromosomes of primates in general. The most important parts in the development of Human Artificial Chromosomes (HACs), are the isolation and maintenance of stability of centromeric region. For isolation of this region, we could use the targeting hook with alphoid DNA repeat and cloned by Transformation-Associated Recombination (TAR) cloning technique in yeast Saccharomyces cerevisiae. The method includes rolling-circle amplification (RCA) of repeats in vitro to 5 kb-length and elongation of the RCA products by homologous recombination in yeast. Four types of $35\;kb{\sim}50\;kb$ of centromeric DNA repeat arrays (2, 4, 5, 6 mer) are used to examine the stability of repeats in homologous recombination mutant strains (rad51, rad52, and rad54). Following the transformation into wild type, rad51 and rad54 mutant strains, there were frequent changes in inserted size. A rad52 mutant strain showed extremely low transformation frequency, but increased stability of centromeric DNA repeat arrays at least 3 times higher than other strains. Based on these results, the incidence of large mutations could be reduced using a rad52 mutant strain in maintenance of centromeric DNA repeat arrays. This genetic method may use more general application in the maintenance of tandem repeats in construction of HAC.

Centromere는 채세포분열과 생식세포분열 등 맡은 주요 기능을 담당하는 고도로 분화된 구조이다. Alphoid DNA (${\alpha}$-satellite)는 인간뿐 아니라 모든 영장류의 염색체 내 centromere에서 발견되는 반복서열의 대부분을 차지한다. 인간 인공염색체(Human Artificial Chromosome, HAC)의 개발에서 가장 핵심적인 부분은 centromere의 분리 및 안정적인 유지에 있다. 이 영역은 출아효모에서 alphoid DNA 반복서열을 hook으로 이용하여 Transformation-associated recombination (TAR) cloning법을 사용하여 선택적으로 분리할 수 있다. 이러한 실험방법으로 먼저 repeat array를 rolling-circle amplication (RCA)를 통하여 약 5 kb까지 길이를 연장시킨 후, 효모내에서 상동성재 조합을 이용한 TAR cloning법을 사용하여 분리할 수 있다. 이렇게 분리된 35 kb-50 kb 길이의 4종류의 centromeric DNA repeat arrays (2,4,5,6 mer)를 사용하여, 반복서열의 안정성 유지를 조사하기 위해 상동성재조 합 변이주인 rad51, rad52, rad54를 사용하여 비교 분석하였다. 야생주, rad51과 rad54 변이주를 이용하여 형질전환을 수행한 결과, 반복서열의 크기에 있어서 많은 변화를 나타내었다. 반면, rad52 변이주는 야생주와 다르게 형질전환빈도가 매우 낮은 비율로 나타났으나, centromeric DNA repeat array의 안정성은 3배 이상으로 높게 나타냈다. 이러한 결과들을 미루어, rad52 변이주를 사용하여 centromeric DNA repeat arrays의 형질전환실험에서 발생하는 맡은 변이를 줄일 수 있을 것으로 보인다. 이러한 유전적 방법은 HAC 제작에서 반복서열의 유지에 훨씬 효율적으로 사용할 수 있을 것으로 사료된다.

Keywords

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