Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
권호 표지

Effect of Non-homologous Spacing in Target DNA Sequence on the Frequency of Cloning Based Homologous Recombination

Target DNA 염기서열 내에 존재하는 비상동성 간격이 상동성재조합을 이용한 클로닝 빈도에 미치는 영향

  • Kim Jae-Woo (Department of Clinical Pathology, Dong-A University Hospital) ;
  • Do Eun-Ju (Department of Biology, Dong-A University) ;
  • Yoon Se-Lyun (Department of Biology, Dong-A University) ;
  • Jeong Yun-Hee (Department of Biology, Dong-A University) ;
  • Yoon Young-Ho (Department of Biology, Dong-A University) ;
  • Leem Sun-Hee (Department of Biology, Dong-A University) ;
  • Sunwoo Yangil (Department of Biology, Dong-A University) ;
  • Park In-Ho (Department of Biology, Dong-A University)
  • 김재우 (동아대학교병원 임상병리실) ;
  • 도은주 (동아대학교 자연과학대학 생물학과) ;
  • 윤세련 (동아대학교 자연과학대학 생물학과) ;
  • 정윤희 (동아대학교 자연과학대학 생물학과) ;
  • 윤영호 (동아대학교 자연과학대학 생물학과) ;
  • 임선희 (동아대학교 자연과학대학 생물학과) ;
  • 선우양일 (동아대학교 자연과학대학 생물학과) ;
  • 박인호 (동아대학교 자연과학대학 생물학과)
  • Published : 2005.12.01
Download PDF
⟨ Previous Next ⟩

Abstract

Transformation-Associated Recombination (TAR) cloning technique allows selective isolation of chromosomal regions and genes from complex genomes. The procedure requires knowledge of relatively small genomic sequences that reside adjacent to the chromosomal region of interest. This technique involves homologous recombination during yeast spheroplast transformation between genomic DNA and a TAR vector that has 5' and 3' gene targeting sequences. In this study, we examined the effect of non-homologous spacing sequence in target hooks on homologous recombination using a plasmid model system. The efficiency of homologous recombination between the modified his3-TRP1-his3 fragments and HlS3 gene on plasmid were analyzed by the characterization of $Ura^+$ transformants. The numbers of $Ura^+$ transformant showed same level when seven different modified his3-TRP1-his3 fragments were used. But the percentage of positive recombinants. $Trp^+His^-$, dramatically decreased when used the modified his3-TRP1-his3 fragments contained incorrect spacing of nonhomologous region. As a result, we suggest that incorrect spacing inhibits the homologous recombination between target hook and substrate DNA. Therefore, we should consider the correct spacing in target hook when the target hook are used for cloning of orthologue gene.

Transformation-associated recombination (TAR) 클로닝 법은 복잡한 게놈으로부터 염색체 내의 특정부위나 유전자를 선택적으로 분리할 수 있다. 이 방법은 목적 유전자에 근접한 작은 게놈DNA 염기서열 정보를 필요로 한다. 이 기술은 효모의 spheroplast transformation을 시키는 동안 목적으로 하는 유전자의 5' 또는 3' 서열을 포함하고 있는 TAR vector와 게놈DNA사이에서 일어나는 상동성재조합에 의해 이루어진다. 본 연구에서는 plasmid 모델시스템을 이용하여 target hooks 내에 존재하는 비상동성 염기서 열이 상동성재조합에 미치는 영향을 조사하였다. plasmid에 존재하는HIS3유전자와 변형시킨 his3-TRP1-his3 단편 사이의 상동성재조합의 효율은 $Ura^+$ 형질전환체의 형질분석에 의해 이루어졌다. $Ura^+$ 형질전환체의 수는 7종류의 서로 달리 변형된 his3-TRP1-his3 단편들을 사용하였을 매 거의 동일하게 나타났다. 그러나 $Trp^+His^+$ positive recombinants의 빈도는 변형된 his3-TRP1-his3 단편 내에 비상동성 영역에 부정확한 간격을 지닐 때 현저한 감소를 나타내었다. 이러한 결과로서, 부정확한 간격이 target hook과 substrate DNA 사이에 일어나는 상동성재조합을 방해하는 것으로 사료된다. 그러므로 이종간의 상동유전자를 클로닝 할 때에는 target hook내의 비상동성 염기서열이 존재한다면 이것이 정확한 간격을 지니는지 여부를 중요란 요인으로 고려해야 한다.

Keywords

References

  1. Annab, L.A., N. Kouprina, G. Solomon, P.L. Cable, D.E. Hill, J.C. Barrett, V. Laronov, and C. A. Afshari. 2000. Isolation of a functional copy of the human BRCA1 gene by transformation-associated recombination in yeast. Gene 250, 201-208 https://doi.org/10.1016/S0378-1119(00)00180-3
  2. Berben, G., J. Dumont, V. Gilliquet, P.-A. Bolle and F. Hilger. 1991. The YDp plasmids: a uniform set of vectors bearing versatile gene disruption cassettes for Saccharomyces cerevisiae. Yeast 7, 475-477 https://doi.org/10.1002/yea.320070506
  3. Burke, D.T., G.F. Carle, and M.V. Olson. 1987. Cloning of Large Segments of Exogenous DNA into Yeast by means of Artificial Chromosome Vectors. Science 236, 806-812 https://doi.org/10.1126/science.3033825
  4. Cancilla, M.R., K.M. Tainton, A.E. Barry, V. Larionov, N. Kouprina, M.A. Resnick, D. Dustart, and K.H.A. Choo. 1997. Direct Cloning of Human 10q25 Neocentromere DNA Using Transformation- Associated Recombination(TAR) in Yeast. Genomics 47, 399-404 https://doi.org/10.1006/geno.1997.5129
  5. Hua, S.-B., M. Qui, E. Chan, L. Zhu, and Y. Luo. 1997. Minimum Length of Sequence Homology Required for in Vivo Cloning by Homologous Recombination in Yeast. Plasmid 38, 91-96 https://doi.org/10.1006/plas.1997.1305
  6. Jinks-Robertson, S., M. Michelitch, and S. Ramchran. 1993. Substrate length requirements for efficient mitotic recombination in Saccharomyces cerevisiae. Mol. Cell. Biol. 13, 3937-3950 https://doi.org/10.1128/MCB.13.7.3937
  7. Kim, J.-H., S.-H. Leem, Y. Sunwoo, and N. Kouprina. 2003. Separation of long-range human TERT gene haplotypes by transformation- associated recombination cloning in yeast. Oncogene, 22, 2452-2456 https://doi.org/10.1038/sj.onc.1206316
  8. Kim, J.-H., Y.-S. Shin, Y.-H. Yoon, H.-J. Jang, E.-A. Kim, K.-S. Kim, C.-N. Chung, I.-H. Park, S.-H. Leem, and Y. Sunwoo. 2003. Effect of GC content on target hook required for gene isolation by transformation -associated recombination cloning. Kor. J. Microbiol. 39, 128-134
  9. Kouprina, N., L. Annab, J. Graves, C. Afshari, J.C. Barrett, M.A. Resnick, and V. Larionov. 1998. Functional copies of a human gene can be directly isolated by transformation-associated recombination cloning with a small 3' end target sequence. Proc. Natl. Acad. Sci. USA 95, 4469-4474
  10. Kouprina, N., M.R. Cancilla, J. Graves, M.A. Resnick, and V. Larionov. 1997. Specific isolation of human rDNA genes by TAR cloning. Gene 197, 269-276 https://doi.org/10.1016/S0378-1119(97)00271-0
  11. Kouprina, N. and V. Larionov. 1999. Selective Isolation of mammalian Genes by TAR cloning, In Current Protocols in Human Genetics, UNIT 5.17, John Wiley & Sons, Inc
  12. Larionov, V., N. Kouprina, J. Graves, X.-N. Chen, J. Korenberg, and M.A. Resnick. 1996a. Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination. Proc. Natl. Acad. Sci. USA 93, 491-496
  13. Larionov, V., N. Kouprina, J. Graves, and M.A. Resnick. 1996b. Highly selective isolation of human DNAs from rodent-human hybrid cells as circular yeast artificial chromosomes by transfor-mation-associated recombination cloning. Proc. Natl. Acad. Sci. USA 93, 13925-13930
  14. Larionov, V., N. Kouprina, G. Solomon, J.C. Barrett, and M.A. Resnick. 1997. Direct isolation of human BRCA2 gene by transformation- associated recombination in yeast Proc. Natl. Acad. Sci. USA 94, 7384-7387
  15. Leem, S.-H., J.A. Londono-Vallejo, J.-H. Kim, H. Bui, E. Tubacher, G. Solomon, J.-E. Park, I. Horikawa, N. Kouprina, J.C. Barrett, and V. Larionov. 2002. The human telomerase gene: complete genomic sequence and analysis of tandem repeat polymorphisms in intronic regions. Oncogene, 21, 769-777 https://doi.org/10.1038/sj.onc.1205122
  16. Leem, S.-H., V.N. Noskov, J.-E. Park, S. I. Kim, V. Larionov, and N. Kouprina. 2003. Optimum conditions for selective isolation of genes from complex genomes by transformation-associated recombination cloning. Nucleic Acid Research, 31, e29 https://doi.org/10.1093/nar/gng029
  17. Ma, H., S. Kunes, P.J. Schatz, and D. Botstein. 1987. Plasmid construction by homologous recombination in yeast. Gene 58, 201-216 https://doi.org/10.1016/0378-1119(87)90376-3
  18. Neil, D.L., A. Villasante, R.B. Fisher, D. Vetrie, B. Cox, and C. Tyler-Smith. 1990. Structural instability of human tandemly repeated DNA sequences cloned in yeast artificial chromosome vectors. Nucleic Acids Res. 18, 1421-1428 https://doi.org/10.1093/nar/18.6.1421
  19. Noskov, V.N., M. Koriabine, G. Solomone, M. Randolph, J. C. Barrett, S.-H. Leem, L. Stubbs, N. Kouprina, and V. Larionov. 2002. Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning. Nucleic Acids Res., 29, e32
  20. Noskov, V.N., S.-H. Leem, G. Solomone, M. Mullokandov, J.-Y. Chae, Y.-H. Yoon, Y.-S. Shin, N. Kouprina, and V. Larionov. 2003. A novel strategy for analysis of gene homologues and segmental genome duplications. J. Mol. Evol. 56, 702-710 https://doi.org/10.1007/s00239-002-2442-x
  21. Park, J.-E., Y.-J. Lee, Y.-H. Jeong, J.-W. SI. Kim, S. Kim, I.-H. Park, Y. Sunwoo, and S.-H. Leem. 2003. The utility of tar vectors used for selective gene isolation by TAR cloning. Kor. J. Microbiol. Biotechnol. 31, 322-328
  22. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y
  23. Sherman, F., G.R. Fink, and J.B. Hicks. 1986. Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y
  24. Sikorski, R., and P. Philip. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19-27
  25. Stinchomb, D.T., M. Thomas, I. Kelly, E. Selker, and R.W. Davis. 1980. Eukaryotic DNA Segments Capable of Autonomous Replication in Yeast. Proc. Natl. Acad. Sci. USA 77, 4559-4563
  26. Theis, J.F., and C.S. Newlon. 1997. The ARS309 chromosomal replicator of Saccharomyces cerevisiae depends on an exceptional ARS consensus sequence. Proc. Natl. Acad. Sci. USA 94, 10786- 10791