미생물학회지 (Korean Journal of Microbiology)

  • 제39권3호
  • /
  • Pages.128-134
  • /
  • 2003
  • /
  • 0440-2413(pISSN)
  • /
  • 2383-9902(eISSN)
한국미생물학회 (The Microbiological Society of Korea)
권호 표지

Transformation-associated recombination cloning에 의한 유전자 분리에 사용되는 target hook에 대한 GC content의 영향

Effect of GC Content on Target Hook Required for Gene Isolation by Transformation-Associated Recombination Cloning

  • 김중현 (동아대학교 자연과학대학 생물학과) ;
  • 신영선 (동아대학교 자연과학대학 생물학과) ;
  • 윤영호 (동아대학교 자연과학대학 생물학과) ;
  • 장형진 (동아대학교 자연과학대학 생물학과) ;
  • 김은아 (동아대학교 자연과학대학 생물학과) ;
  • 김광섭 (동아대학교 자연과학대학 생물학과) ;
  • 정정남 (동아대학교 자연과학대학 생물학과) ;
  • 박인호 (동아대학교 자연과학대학 생물학과) ;
  • 임선희 (동아대학교 자연과학대학 생물학과)
  • 발행 : 2003.09.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

Transformation-associated recombination (TAR) 클로닝법은 목적 유전자를 포함한 게놈 DNA와 그 유전자의 5' 또는 3' 말단 서열을 포함하고 있는 선형의 TAR vector를 동시에 출아효모의 spheroplast내로 co-penetration 시켜 상동부위에서 일어나는 재조합에 의해 환형의 Yeast Artification Chromosome(YAC)으로 분리되는 방법이다. 일반적으로 TAR 클로닝법에 의한 목적의 single-copy 유전자 분리 빈도는 전체 형질전환체의 0.01~1% 정도이다. 이러한 TAR 클로닝법을 개선하기 위하여 Tg.AC transgenic mouse를 모델계로 사용하여 유전자 분리에 대한 target hook 내의 GC content 가 미치는 영향을 조사하였다. 이러한 목적으로 한쪽에는 다양한 GC content(18~45%)를 지닌 transgene 특이적 hook을 포함하고 다른 한쪽은 B1 반복서열을 가지는 radial TAR vector를 사용하여 transgene 분리 빈도를 측정하였다. 그 결과 target hook의 GC content는 23% 이하의 경우, ~40%인 경우에 비해 두 배 정도 클로닝 빈도가 감소하였다. 따라서 TAR vector를 제작할 때, 유전자 분리에 이용되는 target hook의 GC content는 약 40% 일때 가장 적정한 것으로 나타났다. 또한 높은 target hook 내의 GC content(65%)위치분포에 의한 차이는 클로닝 빈도에 큰 영향을 미치지 않는 것으로 나타났다.

Transformation-associated recombination (TAR) cloning is based on co-penetration into yeast spheroplasts of genomic DNA along with TAR vector DNA that contains 5'- and 3'-sequences (hooks) specific for a gene of interest, followed by recombination between the vector and the human genomic DNA to establish a circular YAC. Typically, the frequency of recombinant insert capture is 0.01-1% for single-copy genes by TAR cloning. To further refine the TAR cloning technology, we determined the effect of GC content on target hooks required for gene isolation utilizing the $Tg\cdot\AC$ mouse transgene as the targeted region. For this purpose, a set of vectors containing a B1 repeated hook and Tg AC-specific hooks of variable GC content (from 18 to 45%) was constructed and checked for efficiency of transgene isolation by radial TAR cloning. Efficiency of cloning decreased approximately 2-fold when the TAR vector contained a hook with a GC content ～${\leq}23$% versus ～40%. Thus, the optimal GC content of hook sequences required for gene isolation by TAR is approximately 40%. We also analyzed how the distribution of high GC content (65%) within the hook affects gene capture, but no dramatic differences for gene capturing were observed.

키워드

참고문헌

  1. Mol. Cell. Biol. v.4 Most highly repeated dispersed DNA families in the mouse genome Bennett,K.L.;R.E.Hill;D.F.Pieras;M. Wood worth-Guta;C.Kane-Haas;J.M.Houston;J.K.Heath;N.D.Hastie https://doi.org/10.1128/MCB.4.8.1561
  2. Science v.236 Cloning of large segments of 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. Nature Genet. v.1 Construction of a mouse yeast artificial chromosome library in a recombination-deficient strain of yeast Chartier,F.L.;J.T.Keer;M.J.Sutcliffe;D.A.Henriques;P.Mileham;S.D.Brown https://doi.org/10.1038/ng0592-132
  4. Nature v.287 Isolation of a yeast centromere and construction of a functional small circular chromosome Clarke,L.;J.Carbon https://doi.org/10.1038/287504a0
  5. Nucleic Acids Res. v.29 Effect of chromosomal locus, GC content and length of homology on PCR-mediated targeted gene replacement in Saccharomyces Gray,M.;S.M.Honigberg https://doi.org/10.1093/nar/29.24.5156
  6. Genomics v.11 Detection and characterization of chimeric yeast artificial-chromosome clones Green,E.D.;H.C.Riethman;J.E.Dutchik;M.V.Olsen https://doi.org/10.1016/0888-7543(91)90073-N
  7. 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
  8. Genomics v.21 A model system to assess the integrity of mammalian YACs during transformation and propagation in yeast Kouprina,N.;M.Eldarov;R.Moyzis;M.Resuick;V.Larionov https://doi.org/10.1006/geno.1994.1218
  9. Proc. Natl. Acad. Sci. USA v.88 Yeast artificial chromosome libraries containing large inserts from mouse and human DNA Larin,Z.;A.P.Monaco;H.Lehrach https://doi.org/10.1073/pnas.88.10.4123
  10. Nucleic Acids Res. v.22 Recombination during transformation as a source of chimeric mammalian artificial chromosomes in yeast(YACs) Larionov,V.;N.Kouprina;N.Nikolaishvili;M.A.Resnick https://doi.org/10.1093/nar/22.20.4154
  11. Proc. Natl. Acad. Sci. USA v.93 Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination Larionov,V.N.;N.Kouprira;J.Graves;X.N.Chen;J.R.Korenberg;M.A,Resnick https://doi.org/10.1073/pnas.93.1.491
  12. Nucleic Acids Res. v.29 no.6 Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning Larionov,V.N.;M.Koriabine;G.Solomon;M,Randolph;J.C.Barrett;S.H.Leem;L.Stubbs;N.Kouprina;V.Larionov https://doi.org/10.1093/nar/29.6.e32
  13. Genet. Eng. Princ. Methods v.21 Direct isolation of specific chromosomal regions and entire genes by TAR cloning Larionov,V.
  14. 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.Solomom;J.E.Park;I.Horikawa;N.Kouprina;J.C.Barrett;V.Larionov https://doi.org/10.1038/sj.onc.1205122
  15. Nucleic Acid Res. v.31 Optimum conditions for selecive 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
  16. 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
  17. Molecular cloning a laboratory manual Maniatis,T.;E.F.Fritsch;J.Sanbrook
  18. Gene v.155 Construction of a human DNA library in a circular centromere-based yeast plasmid McGonigal,T.;P.Bodell;C.Schopp;A.V.Sarthy https://doi.org/10.1016/0378-1119(94)00887-X
  19. Nature v.305 Construction of artificial chromosomes n yeast Murray,A.W.;J.W.Szostak https://doi.org/10.1038/305189a0
  20. Nucleic Acids Res. v.18 Structrual 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
  21. Genetics v.162 Context dependence of meiotic recombination hotspots in yeast: The relationship between recombination activity of a reporter construct and base composition Petes,T.D.;J.D.Merker
  22. Laboratory course of manual for methods in yeast genetics Sherman,F.;G.R.Fink;J.B.Hicks
  23. Proc. Natl. Acad. Sci. USA v.86 Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector Shizuya,H.;B.Birren;U.J.Kim;V.Mancine;T.Slepak;Y.Tachiiri;M.Simon
  24. Proc. Natl. Acad. Sci. USA v.77 Eukaryotic DNA segments capable of autonomous replication in yeast Stinchomb,D.T.;M.Thomas;Kelly,I.;E.Selker;R.W.Davis https://doi.org/10.1073/pnas.77.8.4559
  25. Proc. Natl. Acad. Sci. USA v.94 The ARS 309 chromosomal replicator of Sacchromyces cerevisiae depends on an exceptional ARS consensus sequence Theis,J.F.;C.S.Newlon https://doi.org/10.1073/pnas.94.20.10786