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Incorporation of copy-number control elements into yeast artificial chromosomes by targeted homologous recombination

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Abstract

We have developed a pair of vectors for exchanging yeast artificial chromosome (YAC) arms by targeted homologous recombination. These conversion vectors allow the introduction of copy-number control elements into YACs constructed with pYAC4 or related vectors. YACs modified in this way provide an enriched source of DNA for genetic or biochemical studies. A LYS2 gene on the conversion vector provides a genetic selection for the modified YACs after transformation with appropriately prepared vector. A background of Lys+ clones that do not contain modified YACs is also present. However, clones with converted YACs can be distinguished from this background by counter-screening for loss of the original p YAC4 TRP1 arm (Trp- phenotype). The elimination of yeast replication origins (ARS elements) from the conversion vectors increased the frequency of Lys+ Trp- clones, but resulted in weaker amplification. Several YACs have been converted with these vectors, and the fate of the transformed DNA and of the resident YAC DNA has been systematically investigated.

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Authors and Affiliations

  1. Collaborative Research, Inc., 1365 Main Street, 02154, Waltham, Massachusetts, USA

    Douglas R. Smith, Adrienne P. Smyth & Donald T. Moir

  2. Whitehead Institute for Biomedical Research, 9 Cambridge Center, 02134, Cambridge, Massachusetts, USA

    William M. Strauss

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  1. Douglas R. Smith
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  2. Adrienne P. Smyth
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  3. William M. Strauss
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  4. Donald T. Moir
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Smith, D.R., Smyth, A.P., Strauss, W.M. et al. Incorporation of copy-number control elements into yeast artificial chromosomes by targeted homologous recombination. Mammalian Genome 4, 141–147 (1993). https://doi.org/10.1007/BF00352229

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  • DOI: https://doi.org/10.1007/BF00352229

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