Yeast kar1 mutants provide an effective method for YAC transfer to new hosts.
Yeast artificial chromosome (YAC) clones propagate large segments of exogenous DNA in a host organism with well-developed classical and molecular genetics. Most extant YAC clones are from libraries created in a single yeast host (AB1380). The application of techniques allowing the manipulation and/or restructuring of these cloned DNA segments often requires a change in the yeast genetic background to introduce desirable genetic markers. Transfer methods in current use require extremely high yeast transformation efficiencies or require access to equipment for yeast tetrad analysis. We have developed an alternative method for moving YAC clones from one yeast strain to another, taking advantage of the properties of kar1 mutants altered in a gene required for normal karyogamy (nuclear fusion) during mating. Transfer by this method requires generally accessible methods, including yeast cell culture, replica plating, and pulsed-field gel electrophoresis. We present data demonstrating efficient transfer of nine different YACs from their original host (AB1380) to a kar1 recipient strain (YPH925) with genetic markers that facilitate the use of existing homologous recombination-based modification methods. The enhanced ability to transfer clones to this new host will accelerate the pace of refinement and fine-structure mapping of the YAC contigs currently under construction and facilitate gene manipulation on YACs for subsequent functional analysis.
Center for Medical Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-4922.
F
F
Spencer
Y
Y
Hugerat
G
G
Simchen
O
O
Hurko
C
C
Connelly
P
P
Hieter
D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D003001:Cloning, Molecular; D004171:Diploidy; D018014:Gene Transfer Techniques; D005800:Genes, Fungal; D005819:Genetic Markers; D005822:Genetic Vectors; D006238:Haploidy; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D005819:Genetic Markers