Incorporation of copy-number control elements into yeast artificial chromosomes by targeted homologous recombination.
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.
Smith
D R
DR
Collaborative Research, Inc., Waltham, Massachusetts 02154.
Smyth
A P
AP
Strauss
W M
WM
Moir
D T
DT
D015825:Chromosomes, Fungal; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics*; D010957:Plasmids; D011995:Recombination, Genetic / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics