Incorporation of copy-number control elements into yeast artificial chromosomes by targeted homologous recombination |
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Authors: | Douglas R. Smith Adrienne P. Smyth William M. Strauss Donald T. Moir |
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Affiliation: | (1) Collaborative Research, Inc., 1365 Main Street, 02154 Waltham, Massachusetts, USA;(2) Whitehead Institute for Biomedical Research, 9 Cambridge Center, 02134 Cambridge, Massachusetts, USA |
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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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