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TAR cloning: insights into gene function, long-range haplotypes and genome structure and evolution

Nature Reviews Genetics volume 7, pages 805–812 (2006)Cite this article

Abstract

The structural and functional analysis of mammalian genomes would benefit from the ability to isolate from multiple DNA samples any targeted chromosomal segment that is the size of an average human gene. A cloning technique that is based on transformation-associated recombination (TAR) in the yeast Saccharomyces cerevisiae satisfies this need. It is a unique tool to selectively recover chromosome segments that are up to 250 kb in length from complex genomes. In addition, TAR cloning can be used to characterize gene function and genome variation, including polymorphic structural rearrangements, mutations and the evolution of gene families, and for long-range haplotyping.

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Figure 1: Isolation of genes and large chromosomal fragments by TAR cloning.
Figure 2: Organization of the SPANX genes in primates.
Figure 3: Mutational analysis of the SPANX gene cluster in different individuals.
Figure 4: Amplification of synthetic alphoid DNA repeats for HAC construction by TAR cloning.

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References

  1. Larionov, V., Kouprina, N., Solomon, G., Barrett, J. C. & Resnick, M. A. Direct isolation of human BRCA2 gene by transformation-associated recombination in yeast. Proc. Natl Acad. Sci. USA 94, 7384–7387 (1997).

    Article  CAS  Google Scholar 

  2. Kouprina, N. et al. Functional copies of a human gene can be directly isolated by TAR cloning with a small 3′ end target sequence. Proc. Natl Acad. Sci. USA 95, 4469–4474 (1998).

    Article  CAS  Google Scholar 

  3. Ketner, G., Spencer, F., Tugendreich, S., Connelly, C. & Hieter, P. Efficient manipulation of the human adenovirus genome as an infectious yeast artificial chromosome clone. Proc. Natl Acad. Sci. USA 91, 6186–6190 (1994).

    Article  CAS  Google Scholar 

  4. Larionov, V. et al. Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination. Proc. Natl Acad. Sci. USA 93, 491–496 (1996).

    Article  CAS  Google Scholar 

  5. Bhargava, J. et al. Direct cloning of genomic DNA by recombinogenic targeting method using a yeast-bacterial shuttle vector, pClasper. Genomics 62, 285–288 (1999).

    Article  CAS  Google Scholar 

  6. Burke, D. T., Carle, G. F. & Olson, M. V. Cloning of large segments of DNA into yeast by means of artificial chromosome vectors. Science 236, 806–812 (1987).

    Article  CAS  Google Scholar 

  7. Noskov, V. et al. Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning. Nucleic Acids Res. 29, e62 (2001).

    Article  Google Scholar 

  8. Leem, S. H. et al. Optimum conditions for selective isolation of genes from complex genomes by transformation-associated recombination cloning. Nucleic Acids Res. 31, e29 (2003).

    Article  Google Scholar 

  9. Leem, S. H. et al. Closing the gaps on human chromosome 19 revealed genes with a high density of repetitive tandemly arrayed elements. Genome Res. 14, 239–246 (2004).

    Article  CAS  Google Scholar 

  10. Stinchomb, D. T., Thomas, M., Kelly, I., Selker, E. & Davis, R. W. Eukaryotic DNA segments capable of autonomous replication in yeast. Proc. Natl Acad. Sci. USA 77, 4559–4563 (1980).

    Article  Google Scholar 

  11. Noskov, V. et al. A novel genetic system allowing to directly select gene-positive clones during the gene capture by transformation-associated recombination in yeast. Nucleic Acids Res. 30, e8 (2002).

    Article  Google Scholar 

  12. Raymond, C. K. et al. Genetic variation at the O-antigen biosynthetic locus in Pseudomonas aeruginosa. J. Bacteriol. 184, 3614–3622 (2002).

    Article  CAS  Google Scholar 

  13. Noskov, V. N. et al. A general transformation-associated recombination cloning system to selectively isolate any eukaryotic or prokaryotic genomic region. BMC Genomics, 4, 16–23 (2003).

    Article  Google Scholar 

  14. Kouprina, N. & Larionov, V. in Current Protocols in Human Genetics (ed. Boyle, A. L.) (John Wiley & Sons, New York, 2000).

    Google Scholar 

  15. Kouprina, N., Noskov, V. N., Koriabine, M., Leem, S. H. & Larionov, V. Exploring transformation-associated recombination cloning for selective isolation of genomic regions. Methods Mol. Biol. 255, 69–90 (2004).

    CAS  PubMed  Google Scholar 

  16. Shizuya, H. et al. Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl Acad. Sci. USA 89, 8794–8797 (1992).

    Article  CAS  Google Scholar 

  17. Ioannou, P. A. et al. A new bacteriophage P1-derived vector for the propagation of large human DNA fragments. Nature Genet. 6, 84–89 (1994).

    Article  CAS  Google Scholar 

  18. Raymond, C. K. et al. Targeted, haplotype-resolved resequencing of long segments of the human genome. Genomics 86, 759–766 (2005).

    Article  CAS  Google Scholar 

  19. Annab, L. et al. Isolation of a functional copy of the human BRCA1 gene by TAR cloning in yeast. Gene 250, 201–208 (2000).

    Article  CAS  Google Scholar 

  20. Kouprina, N. et al. Segments missing from the draft human genome sequence can be isolated by TAR cloning in yeast. EMBO Rep. 4, 257–262 (2003).

    Article  CAS  Google Scholar 

  21. Nihei, N. et al. Functional evidence for a metastasis suppressor gene for rat prostate cancer within a 60 kb region on human chromosome 8p21–p12. Cancer Res. 62, 367–370 (2002).

    CAS  PubMed  Google Scholar 

  22. Leem, S. H. et al. Cloning of the human telomerase gene: complete genomic sequence and analysis of tandem repeats polymorphisms in intronic regions. Oncogene 21, 769–777 (2002).

    Article  CAS  Google Scholar 

  23. Kouprina, N. et al. Dynamic structure of the SPANX gene cluster mapped to the prostate cancer susceptibility locus HPCX at Xq27. Genome Res. 15, 1477–1486 (2005).

    Article  CAS  Google Scholar 

  24. Horikawa, I. et al. Differential cis-regulation of human versus mouse TERT gene expression in vivo: identification of a human-specific repression element. Proc. Natl Acad, Sci. USA 102, 18437–18442 (2005).

    Article  CAS  Google Scholar 

  25. Ayabe, F. et al. A novel expression system for genomic DNA loci using a human artificial chromosome vector with transformation-associated recombination cloning. J. Hum. Genet. 50, 592–599 (2005).

    Article  CAS  Google Scholar 

  26. Noskov, V. N. et al. A novel strategy for analysis of gene homologs and segmental genome duplications. J. Mol. Evol. 56, 702–710 (2003).

    Article  CAS  Google Scholar 

  27. Pavlicek, A. et al. Evolution of the tumor suppressor BRCA1 locus in primates: implications for cancer predisposition. Hum. Mol. Genet. 13, 2737–2751 (2004).

    Article  CAS  Google Scholar 

  28. Kouprina, N. et al. Accelerated evolution of the ASPM gene controlling brain size begins prior to human brain expansion. PLoS Biol. 2, 653–663 (2004).

    Article  CAS  Google Scholar 

  29. Kouprina, N. et al. The SPANX gene family of cancer-testis specific antigens: rapid evolution, an unusual case of positive selection and amplification in African great apes and hominids. Proc. Natl Acad. Sci. USA 101, 3077–3082 (2004).

    Article  CAS  Google Scholar 

  30. Kouprina, N. et al. The microcephaly ASPM gene is expressed in proliferating tissues and encodes for a mitotic spindle protein. Hum. Mol. Genet. 14, 2155–2165 (2005).

    Article  CAS  Google Scholar 

  31. Salem, R. M., Wessel, J. & Schork, N. J. A comprehensive literature review of haplotyping software and methods for use with unrelated individuals. Hum. Genomics 2, 39–66 (2005).

    Article  CAS  Google Scholar 

  32. International HapMap Consortium. A haplotype map of the human genome. Nature 437, 1299–1320 (2005).

  33. Kim, J. H., Leem, S. H., Sunwoo, Y. & Kouprina, N. Separation of long-range human TERT gene haplotypes by transformation-associated recombination cloning in yeast. Oncogene 22, 2443–2456 (2003).

    Article  Google Scholar 

  34. Westbrook, V. A. et al. Genomic organization, incidence, and localization of the SPANX family of cancer-testis antigens in melanoma tumors and cell lines. Clin. Cancer Res. 10, 101–112 (2004).

    Article  CAS  Google Scholar 

  35. Baffoe-Bonnie, A. B. et al. A major locus for hereditary prostate cancer in Finland: localization by linkage disequilibrium of a haplotype in the HPCX region. Hum. Genet. 117, 307–316 (2005).

    Article  CAS  Google Scholar 

  36. Sharp, A. J. et al. Segmental duplications and copy-number variation in the human genome. Am. J. Hum. Genet. 77, 78–88 (2005).

    Article  CAS  Google Scholar 

  37. Shaw, C. J. & Lupski, J. R. Implications of human genome architecture for rearrangement-based disorders: the genomic basis of disease. Hum. Mol. Genet. 13, R57–R64 (2004).

    Article  CAS  Google Scholar 

  38. Lockwood, W. W., Chari, R., Chi, B. & Lam, W. L. Recent advances in array comparative genomic hybridization technologies and their applications in human genetics. Eur. J. Hum. Genet. 1, 139–148 (2005).

    Google Scholar 

  39. Perry, G. H. et al. Hotspots for copy number variation in chimpanzees and humans. Proc. Natl Acad. Sci. USA 103, 8006–8011 (2006).

    Article  CAS  Google Scholar 

  40. Feuk, L., Marshall, C. R., Wintle, R. F. & Scherer, S. W. Structural variants: changing the landscape of chromosomes and design of disease studies. Hum. Mol. Genet. 15, R57–R66 (2006).

    Article  CAS  Google Scholar 

  41. Eichler, E. E., Clark, R. A. & She, X. An assessment of the sequence gaps: unfinished business in a finished human genome. Nature Rev. Genet. 5, 345–354 (2004).

    Article  CAS  Google Scholar 

  42. Grimwood, J. et. al. The DNA sequence and biology of human chromosome 19. Nature, 428, 529–535 (2004).

    Article  CAS  Google Scholar 

  43. Grimes, B. R. & Monaco, Z. L. Artificial and engineered chromosomes: developments and prospects for gene therapy. Chromosoma 114, 230–241 (2005).

    Article  CAS  Google Scholar 

  44. Basu, J. & Willard, H. F. Artificial and engineered chromosomes: non-integrating vectors for gene therapy. Trends Mol. Med. 11, 251–258 (2005).

    Article  CAS  Google Scholar 

  45. Ikeno, M. et al. Generation of human artificial chromosomes expressing naturally controlled guanosine triphosphate cyclohydrolase I gene. Genes Cells 10, 1021–1032 (2002).

    Article  Google Scholar 

  46. Mejia, J. E. et al. Efficiency of de novo centromere formation in human artificial chromosomes. Genomics 79, 297–304 (2002).

    Article  CAS  Google Scholar 

  47. Kotzamanis, G. et al. Construction of human artificial chromosome vectors by recombineering. Gene 351, 29–38 (2005).

    Article  CAS  Google Scholar 

  48. Kakeda, M. et al. Human artificial chromosome (HAC) vector provides long-term therapeutic transgene expression in normal human primary fibroblasts. Gene Ther. 12, 852–856 (2005).

    Article  CAS  Google Scholar 

  49. Grimes, B. R., Rhoades, A. A. & Willard, H. F. α-Satellite DNA and vector composition influence rates of human artificial chromosome formation. Mol. Ther. 5, 798–805 (2002).

    Article  CAS  Google Scholar 

  50. Basu, J., Compitello, G., Stromberg, G., Willard, H. F. & Van Bokkelen, G. Efficient assembly of de novo human artificial chromosomes from large genomic loci. BMC Biotechnol. 5, 21 (2005).

    Article  Google Scholar 

  51. Kouprina, N. et al. Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes. Nucleic Acids Res. 31, 922–934 (2003).

    Article  CAS  Google Scholar 

  52. Ebersole, T. et al. Rapid generation of long synthetic tandem repeats and its application for analysis in human artificial chromosome formation. Nucleic Acids Res. 33, e130 (2005).

    Article  Google Scholar 

  53. Becker, M. et al. Isolation of the repertoire of VSG expression site containing telomeres of Trypanosoma brucei 427 using transformation-associated recombination in yeast. Genome Res. 14, 2319–2329 (2004).

    Article  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. the Laboratory of Biosystems and Cancer, National Cancer Institute, National Institute of Health, Building 37, Room 5032, 9000 Rockville Pike, Bethesda, 20892, Maryland, USA

    Natalay Kouprina & Vladimir Larionov

Authors
  1. Natalay Kouprina
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  2. Vladimir Larionov
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Correspondence to Natalay Kouprina.

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FURTHER INFORMATION

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Glossary

Alphoid DNA

A 170 bp AT-rich repeat that is present in the centromeric regions of all human chromosomes. The repeat forms large tandem arrays with sizes of up to 5 Mb.

Alu interspersed

A ∼300 bp repeat that is specific to primates. There are approximately 1.5 million copies of this repeat in the human genome.

ARS

Autonomously replicating sequence — a small DNA fragment that can support replication in the budding yeast.

Comparative genomic hybridization

A technology through which tumour and reference DNA are differentially labelled to show copy-number changes in tumour genomes.

Contig

From contiguous. A set of clones that represent a continuous region of DNA. A contig map depicts the relative order of a linked library of contigs that represent a complete chromosome segment.

HAC

Human artificial chromosome — a system for cloning large DNA segments in mammalian cells. Similar to natural chromosomes, HACs have a functional centromere and therefore are stably maintained in a single copy during mitotic cell divisions.

Kinetochore

The protein structure in eukaryotes that assembles on the centromere and links the chromosome to microtubule polymers from the mitotic spindle during mitosis.

Minisatellites

A class of repetitive sequences, each of which is 7–100 nucleotides, that span 500–20,000 bp and are mainly located towards chromosome ends.

Segmental duplications

Large genomic segments of recent origin and nearly identical sequence.

Spermatocyte

A male gametocyte that is derived from a spermatogonium. Initially, a spermatogonium divides by mitosis into two 'primary spermatocytes'. A primary spermatocyte is diploid and, by meiosis, gives place to two secondary spermatocytes, which are haploid.

Synteny

Collinearity in the order of genes (or of other DNA sequences) in a chromosomal region of two species.

Yeast spheroplasts

Yeast cells following the removal of the cell wall. They can take up large DNA molecules (up to 1 Mb), and were used to construct the first genomic YAC libraries.

Z-DNA

A form of DNA in which the double helix winds to the left in a zig-zag pattern (instead of to the right, like the more common B-DNA form).

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Kouprina, N., Larionov, V. TAR cloning: insights into gene function, long-range haplotypes and genome structure and evolution. Nat Rev Genet 7, 805–812 (2006). https://doi.org/10.1038/nrg1943

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