Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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The Utility of TAR Vectors Used for Selective Gene Isolation by TAR Cloning.

TAR Cloning에 의한 선별적 유전자 분리에 사용되는 TAR Vectors의 유용성에 관한 연구

  • 박정은 (동아대학교 자연과학대학 생물학과) ;
  • 이윤주 (동아대학교 자연과학대학 생물학과) ;
  • 정윤희 (동아대학교 자연과학대학 생물학과) ;
  • 김재우 (동아대학교병원 임상병리실) ;
  • 김승일 (기초과학지원연구소 프로테옴 분석팀) ;
  • 김수현 (기초과학지원연구소 프로테옴 분석팀) ;
  • 박인호 (동아대학교 자연과학대학 생물학과) ;
  • 선우양일 (동아대학교 자연과학대학 생물학과) ;
  • 임선희 (동아대학교 자연과학대학 생물학과)
  • Published : 2003.12.01
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Abstract

The 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 minimum size of specific hooks required for a single-copy gene isolation and compared the utility of different TAR vectors, radial and unique vectors, by cloning the same single-copy gene. The efficiency of TAR cloning of the hHPRT gene was same using hooks varying from 750 to 63 bp. The number of transformants decreased approximately 20-fold when the TAR vector contained two unique hooks versus using a radial vector, but the percentage of positive recombinants increased over 2-fold when a unique TAR vector was used. Therefore, we suggest that the two-unique TAR vector is suitable for general TAR cloning given its high selectivity, and the radial TAR vector is more suitable when genomic DNA is in limited quantity, for example, DNA isolated from pathological specimens. Moreover, we confirm the minimal length of a unique sequence in a TAR vector is approximately 60 bp for a single-copy gene isolation.

TAR(Transformation-Associated Recombination) cloning 법은 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 하며, spheroplast 형질전환 과정에서 게놈 DNA와 5'-와 3'-표적배열을 지닌 TAR vector 사이에서 일어나는 상동성 재조합과정에 의해 이루어진다. 본 연구에서는 single-copy 유전자의 클론닝에 필요한 specific hook의 최소 크기를 조사하였고, 서로 다른 TAR vector(radial과 unique vector)의 유용성을 조사하기 위해 동일한 single-copy 유전자의 클론닝을 통해 비교하였다. 그 결과, hHPRT 유전자에 대한 TAR cloning의 빈도는 hook의 길이가 750 bp∼63 bp의 범위에서 동일하게 나타났다. radial hook을 사용한 경우보다 unique hook을 사용하였을 경우 형질전환체의 수는 약 20배정도의 감소를 보였으나 목적으로 하는 재조합체의 분리 빈도는 두 배 이상 증가하였다. 그러므로 본 연구에서 two-unique TAR vector는 선별력이 높으므로 일반적 TAR cloning에 사용할 수 있으며, radial TAR vector의 경우는 병리학적 표본과 같이 제한된 게놈 DNA를 사용하는 경우에 더 적합하다고 볼 수 있다. 또한, single-copy 유전자의 분리에 필요로 하는 specific hoot의 최소 길이는 약 60 bp로도 가능하다는 것을 확인하였다.

Keywords

References

  1. Gene v.250 Isolation of a functional copy of the human BRCAI gene by transformation-associated recombination in yeast Annab,L.A.;N.Kouprina;G.Solomon;P.L.Cable;D.E.Hill;J.C.Barrett;V.Laronov;C.A.Afshari https://doi.org/10.1016/S0378-1119(00)00180-3
  2. Science v.236 Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors Burke,D.T.;G.F.Carle;M.V.Olson https://doi.org/10.1126/science.3033825
  3. Genomics v.47 Direct cloning of human 10q25 neocentromere DNA using transformation-associated recombination(TAR) in yeast Cancilla,M.R.;K.M.Tainton;A.E.Barry;V.Larionov;N.Kouprina;M.A.Resnick;D.Dustart;K.H.A.Choo https://doi.org/10.1006/geno.1997.5129
  4. Plasmid v.38 Minimum length of sequence homology required for in vivo cloning by homologous recombination in yeast Hua,S.B.;M.Qui;E.Chan;L.Zhu;Y.Luo https://doi.org/10.1006/plas.1997.1305
  5. Mol. Cell. Biol. v.13 Substrate length requirements for efficient mitotic recombination in Sacharomyces cerevisiae Jinks-Robertson,S.;M.Michelitch;S.Ramchran
  6. Oncogene v.22 Separation of long-range human TERT gene haplotypes by transformation-associated recombination cloning in yeast Kim,J.H.;S.H.Leem;Y.Sunwoo;N.Kouprina https://doi.org/10.1038/sj.onc.1206316
  7. Proc. Natl. Acad. Sci. USA. v.95 Functional copies of a human gene can be directly isolated by transformation-associated recombination cloning with a small 3' end target swquence Kouprina,N.;L.Annab;J.Graves;C.Afshari;J.C.Barrett;M.A.Resnick;V.Larionov https://doi.org/10.1073/pnas.95.8.4469
  8. Gene v.197 Specific isolation of human rDNA genes by TAR cloning Kouprina,N.;M.R.Cancilla;J.Graves;M.A.Resnick;V.Larionov https://doi.org/10.1016/S0378-1119(97)00271-0
  9. Current Protocols in Human Genetics, UNIT 5.17 Selective isolation of mammalian genes by TAR colning Kouprina,N.;V.Larionov
  10. Proc. Natl. Acad. Sci. USA. v.93 Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination Larionov,V.;N.Kouprina;J.Graves;X.N.Chen;J.Korenberg;M.A.Resnick https://doi.org/10.1073/pnas.93.1.491
  11. Proc. Natl. Acad. Sci. USA. v.93 Highly selective isolation of human DNAs from rodent-human hybrid cells as circular yeast artificial chromosomes by transformation-associated recombination cloning Larionov,V.;N.Kouprina;J.Graves;M.A.Resnick https://doi.org/10.1073/pnas.93.24.13925
  12. Proc. Natl. Acad. Sci. v.94 Direct isolation of human BRCA2 gene by transformation-associated recombination in yeast Larionov,V.;N.Kouprina;G.Solomon;J.C.Barrett;M.A.Resnick https://doi.org/10.1073/pnas.94.14.7384
  13. Oncogene v.21 The human telomerase gene: complete genomic sequence and analysis of tandem repeat polymorphisms in intronic regions 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;V.Larionov https://doi.org/10.1038/sj.onc.1205122
  14. Nucleic Acid Res v.31 Optimum conditions for selective isolation of genes from complex genomes by transformation-associated recombination cloning Leem,S.H.;V.N.Noskov;J.E.Park;S.I.Kim;V.Larionov;N.Kouprina https://doi.org/10.1093/nar/gng029
  15. Gene v.58 Plasmid construction by homologous recombination in yeast Ma,H.;S.Kunes;P.J.Schatz;D.Botstein https://doi.org/10.1016/0378-1119(87)90376-3
  16. Nucleic Acids Res v.18 Structural instability of human tandemly repeated DNA sequences cloned in yeast artificial chromosome vectors Neil,D.L.;A.Villasante;R.B.Fisher;D.Vetrie;B.Cox;C.Tyler-Smith https://doi.org/10.1093/nar/18.6.1421
  17. Nucleic Acids Res. v.29 Defining the minimal length of sequence homlogy required for selective gene isolation by TAR cloning Noskov,V.N.;M.Koriabine;G.Solomone;M.Randolph;J.C.Barrett;S.H.Leem;L.Stubbs;N.Kouprina;V.Larionov
  18. Molecular cloning:a laboratory manual(2nd ed.) Sambrook,J.;E.F.Fritsch;T.Maniatis
  19. Methods in yeast genetics Sherman,F.;G.R.Fink;.J.B.Hicks
  20. Proc. Natl. Acad. Sci. v.77 Eukaryotic DNA segments capable of autonomous replcation in yeast Stinchomb,D.T.;M.Thomas;I.Kelly;E.Selker;R.W.Davis https://doi.org/10.1073/pnas.77.8.4559
  21. Proc. Natl. Acad. Sci. USA. v.94 The ARS309 chromosomal replicator of Saccharomyces cerevisiae depends on an exceptional ARS consensus Theis,J.F.;C.S.Newlon https://doi.org/10.1073/pnas.94.20.10786