Isolation of yeast artificial chromosomes free of endogenous yeast chromosomes: construction of alternate hosts with defined karyotypic alterations.
An intrinsic feature of yeast artificial chromosomes (YACs) is that the cloned DNA is generally in the same size range (i.e., approximately 200-2000 kb) as the endogenous yeast chromosomes. As a result, the isolation of YAC DNA, which typically involves separation by pulsed-field gel electrophoresis, is frequently confounded by the presence of a comigrating or closely migrating endogenous yeast chromosome(s). We have developed a strategy that reliably allows the isolation of any YAC free of endogenous yeast chromosomes. Using recombination-mediated chromosome fragmentation, a set of Saccharomyces cerevisiae host strains was systematically constructed. Each strain contains defined alterations in its electrophoretic karyotype, which provide a large-size interval devoid of endogenous chromosomes (i.e., a karyotypic "window"). All of the constructed strains contain the kar1-delta 15 mutation, thereby allowing the efficient transfer of a YAC from its original host into an appropriately selected window strain using the kar1-transfer procedure. This approach provides a robust and efficient means to obtain relatively pure YAC DNA regardless of YAC size.
Diagnostic Development Branch, National Center for Human Genome Research, National Institutes of Health, Bethesda, MD 20892, USA.
L
L
Hamer
M
M
Johnston
E D
ED
Green
D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular / Q000379:methods*; D016521:Electrophoresis, Gel, Pulsed-Field; D005822:Genetic Vectors; D007621:Karyotyping; D016296:Mutagenesis; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*