Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Isolation of Human and Mouse Orthologue HPRT Genes by Transformation-Associated Recombination (TAR) cloning

TAR cloning 법에 의한 인간 및 마우스의 상동성 HPRT 유전자의 분리

  • 도은주 (동아대학교 자연과학대학 생물학과) ;
  • 김재우 (동아대학교병원 임상병리실) ;
  • 정정남 (동아대학교 자연과학대학 생물학과) ;
  • 박인호 (동아대학교 자연과학대학 생물학과) ;
  • 임선희 (동아대학교 자연과학대학 생물학과)
  • Published : 2006.10.01
Download PDF
⟨ Previous Next ⟩

Abstract

The transformation-associated recombination (TAR) cloning technique allows selective isolation of chromosome regions or genes from complex genome. The procedure requires knowledge of relatively small genomic sequences that reside adjacent to the chromosome region of interest. This method involves homologous recombination during spheroplast transformation between genomic DNA and a TAR vector that has 5' and 3' gene targeting sequences (hooks). To examine whether TAR cloning can be applied to the isolation of gene homologues, we chose the HPRT genes from human and mouse genome. As results, the yield of positive clones for HPRT gene from human and mouse genome when using a TAR vector containing mHPRT hook or hHPRT hook was almost same level. Analysis of the gap regions in mHPRT revealed that they contain abnormalities that could result in instability of the sequences. In conclusion, we were able to use the TAR cloning technology to isolate gene homologue (orthologue) from nonidentical genome. Moreover, the use of the TAR cloning system may accelerate work on closing the remaining gaps in mammalian genome to achieve the goal of annotation of all mammalian genes.

TAR (Transformation-Associated Recombination) cloning법은 복잡한 고등생물의 게놈으로부터 유전자나 특정 염색체 부위를 선별적 분리를 가능하게 한다. 이 방법은 목적으로 하는 염색체 부위의 주변에 존재하는 비교적 짧은 게놈 염기서열에 대한 정보를 필요로 한다. 이 기술은 출아효모의 spheroplasts 형질전환 동안 목적 유전자를 포함한 게놈 DNA와 그 유전자의 5' 또는 3' 말단 서열 (hook)을 포함하고 있는 TAR vector 사이에 일어나는 상동성 재조합에 의해 이루어진다. 본 연구에서는 TAR cloning 법을 상동성 유전자의 분리에 사용할 수 있는가를 조사하기 위해, 연간과 마우스 게놈의 HPRT 유전자를 선택하였다. 그 결과, 인간과 마우스의 게놈으로부터의 HPRT 유전자의 분리 빈도는 TAR vector로서 hHPRT hook 혹은 mHPRT hook을 사용한 경우에 거의 동일하게 나타났다. 또한 mHPRT 유전자의 gap 부분의 염기서열을 결정하여, 이 부분에 염기서열의 불안정의 요인이 되는 비정상적 특성을 발견하였다. 결론적으로 TAR cloning법을 이용하여 다른 이종 간의 게놈으로부터 상동성 유전자 즉 orthologue의 분리가 가능하였다. 더욱이 TAR cloning 시스템을 이용하여 고등동물 게놈 상에 남아있는 gap 부분을 메움으로서 고등동물의 모든 유전자들의 확인이 가속화될 수 있을 것으로 사료된다.

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. 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
  3. 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
  4. 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
  5. 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
  6. 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
  7. 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
  8. 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 transformationassociated recombination cloning with a small 3' end target sequence. Proc. Natl. Acad. Sci. USA 95, 4469-4474
  9. 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
  10. 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
  11. 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
  12. 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 transformation-associated recombination cloning. Proc. Natl. Acad. Sci. USA 93, 13925-13930
  13. 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
  14. 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
  15. 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
  16. Leem, S.-H., N. Kouprina, J. Grimwood, J.-H. Kim, M. Mullokandov, Y.-H. Yoon, J.-Y. Chae, J. Morgan, S. Lucas, P. Richardson, C. Detter, T. Glavina, E. Rubin, J. C. Barrett and V. Larionov. 2004. Closing the Gaps on Human Chromosome 19 Revealed Genes with a High Density of Repetitive Tandemly Arrayed Elements. Genome Research, 14, 239-246 https://doi.org/10.1101/gr.1929904
  17. 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
  18. 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
  19. 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
  20. 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
  21. 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
  22. Sherman, F., G. R. Fink and J. B. Hicks. 1986. Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y