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. 1995 Dec 11;23(23):4850–4856. doi: 10.1093/nar/23.23.4850

A new vector for recombination-based cloning of large DNA fragments from yeast artificial chromosomes.

M S Bradshaw 1, J A Bollekens 1, F H Ruddle 1
PMCID: PMC307474  PMID: 8532528

Abstract

The functional analysis of genes frequently requires manipulation of large genomic regions embedded in yeast artificial chromosomes (YACs). We have designed a yeast-bacteria shuttle vector, pClasper, that can be used to clone specific regions of interest from YACs by homologous recombination. The important feature of pClasper is the presence of the mini-F factor replicon. This leads to a significant increase in the size of the plasmid inserts that can be maintained in bacteria after cloning by homologous recombination in yeast. The utility of this vector lies in its ability to maintain large fragments in bacteria and yeast, allowing for mutagenesis in yeast and simplified preparation of plasmid DNA in bacteria. Using PCR-generated recombinogenic fragments in pClasper we cloned a 27 kb region from a YAC containing the Hoxc cluster and a 130 kb region containing the entire Hoxb cluster. No rearrangements were seen when the recombinants in the shuttle vector were transferred to bacteria. We outline the potential uses of pClasper for functional studies of large genomic regions by transgenic and other analyses.

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Selected References

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  1. Bentley K. L., Bradshaw M. S., Ruddle F. H. Physical linkage of the murine Hox-b cluster and nerve growth factor receptor on yeast artificial chromosomes. Genomics. 1993 Oct;18(1):43–53. doi: 10.1006/geno.1993.1425. [DOI] [PubMed] [Google Scholar]
  2. Bradshaw M. S., Ruddle F. H. Identification of the murine Hox-c12 and Hox-c13 homeoboxes on yeast artificial chromosomes. Genomics. 1994 Jul 1;22(1):234–236. doi: 10.1006/geno.1994.1371. [DOI] [PubMed] [Google Scholar]
  3. Burke D. T., Olson M. V. Preparation of clone libraries in yeast artificial-chromosome vectors. Methods Enzymol. 1991;194:251–270. doi: 10.1016/0076-6879(91)94020-d. [DOI] [PubMed] [Google Scholar]
  4. Campbell C., Gulati R., Nandi A. K., Floy K., Hieter P., Kucherlapati R. S. Generation of a nested series of interstitial deletions in yeast artificial chromosomes carrying human DNA. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5744–5748. doi: 10.1073/pnas.88.13.5744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen D. C., Yang B. C., Kuo T. T. One-step transformation of yeast in stationary phase. Curr Genet. 1992 Jan;21(1):83–84. doi: 10.1007/BF00318659. [DOI] [PubMed] [Google Scholar]
  6. Coletta P. L., Shimeld S. M., Chaudhuri C., Müller U., Clarke J. P., Sharpe P. T. Characterisation of the murine Hox-3.3 gene and its promoter. Mech Dev. 1991 Sep;35(2):129–142. doi: 10.1016/0925-4773(91)90063-c. [DOI] [PubMed] [Google Scholar]
  7. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Erickson J. R., Johnston M. Direct cloning of yeast genes from an ordered set of lambda clones in Saccharomyces cerevisiae by recombination in vivo. Genetics. 1993 May;134(1):151–157. doi: 10.1093/genetics/134.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Garza D., Ajioka J. W., Burke D. T., Hartl D. L. Mapping the Drosophila genome with yeast artificial chromosomes. Science. 1989 Nov 3;246(4930):641–646. doi: 10.1126/science.2510296. [DOI] [PubMed] [Google Scholar]
  10. Hieter P., Mann C., Snyder M., Davis R. W. Mitotic stability of yeast chromosomes: a colony color assay that measures nondisjunction and chromosome loss. Cell. 1985 Feb;40(2):381–392. doi: 10.1016/0092-8674(85)90152-7. [DOI] [PubMed] [Google Scholar]
  11. Hosoda F., Nishimura S., Uchida H., Ohki M. An F factor based cloning system for large DNA fragments. Nucleic Acids Res. 1990 Jul 11;18(13):3863–3869. doi: 10.1093/nar/18.13.3863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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 U S A. 1994 Jun 21;91(13):6186–6190. doi: 10.1073/pnas.91.13.6186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kline B. C. A review of mini-F plasmid maintenance. Plasmid. 1985 Jul;14(1):1–16. doi: 10.1016/0147-619x(85)90027-7. [DOI] [PubMed] [Google Scholar]
  14. McGonigal T., Bodelle P., Schopp C., Sarthy A. V. Construction of a human DNA library in a circular centromere-based yeast plasmid. Gene. 1995 Apr 3;155(2):267–271. doi: 10.1016/0378-1119(94)00887-x. [DOI] [PubMed] [Google Scholar]
  15. Miller L. M., Gallegos M. E., Morisseau B. A., Kim S. K. lin-31, a Caenorhabditis elegans HNF-3/fork head transcription factor homolog, specifies three alternative cell fates in vulval development. Genes Dev. 1993 Jun;7(6):933–947. doi: 10.1101/gad.7.6.933. [DOI] [PubMed] [Google Scholar]
  16. Murray A. W., Schultes N. P., Szostak J. W. Chromosome length controls mitotic chromosome segregation in yeast. Cell. 1986 May 23;45(4):529–536. doi: 10.1016/0092-8674(86)90284-9. [DOI] [PubMed] [Google Scholar]
  17. Orr-Weaver T. L., Szostak J. W. Yeast recombination: the association between double-strand gap repair and crossing-over. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4417–4421. doi: 10.1073/pnas.80.14.4417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pavan W. J., Hieter P., Reeves R. H. Generation of deletion derivatives by targeted transformation of human-derived yeast artificial chromosomes. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1300–1304. doi: 10.1073/pnas.87.4.1300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Peterson K. R., Li Q. L., Clegg C. H., Furukawa T., Navas P. A., Norton E. J., Kimbrough T. G., Stamatoyannopoulos G. Use of yeast artificial chromosomes (YACs) in studies of mammalian development: production of beta-globin locus YAC mice carrying human globin developmental mutants. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5655–5659. doi: 10.1073/pnas.92.12.5655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rockmill B., Lambie E. J., Roeder G. S. Spore enrichment. Methods Enzymol. 1991;194:146–149. doi: 10.1016/0076-6879(91)94012-2. [DOI] [PubMed] [Google Scholar]
  21. Rossant J., Nagy A. Genome engineering: the new mouse genetics. Nat Med. 1995 Jun;1(6):592–594. doi: 10.1038/nm0695-592. [DOI] [PubMed] [Google Scholar]
  22. Rossi J. M., Burke D. T., Leung J. C., Koos D. S., Chen H., Tilghman S. M. Genomic analysis using a yeast artificial chromosome library with mouse DNA inserts. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2456–2460. doi: 10.1073/pnas.89.6.2456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
  24. Shizuya H., Birren B., Kim U. J., Mancino V., Slepak T., Tachiiri Y., Simon M. 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 U S A. 1992 Sep 15;89(18):8794–8797. doi: 10.1073/pnas.89.18.8794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Spencer F., Hugerat Y., Simchen G., Hurko O., Connelly C., Hieter P. Yeast kar1 mutants provide an effective method for YAC transfer to new hosts. Genomics. 1994 Jul 1;22(1):118–126. doi: 10.1006/geno.1994.1352. [DOI] [PubMed] [Google Scholar]
  27. Tolun A., Helinski D. R. Separation of the minimal replication region of the F plasmid into a replication origin segment and a trans-acting segment. Mol Gen Genet. 1982;186(3):372–377. doi: 10.1007/BF00729456. [DOI] [PubMed] [Google Scholar]

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