Large human YACs constructed in a rad52 strain show a reduced rate of chimerism.
Current YAC libraries are plagued by a high frequency of chimeras--that is, clones containing fragments from multiple genomic regions. Chimeras are thought to arise largely through recombination in the yeast host cell. If so, the use of recombination-deficient yeast strains, such as rad52 mutants, might be expected to alleviate the problem. Here, we report the construction of megabase-sized human YACs in the rad52 strain MHY5201 and the determination of their rate of chimerism by fluorescence in situ hybridization analysis. Examination of 48 YACs showed a rate of chimerism of at most 8%, whereas YACs constructed in the wildtype host AB1380 showed a rate of about 50%. These results show that it is possible to significantly decrease the rate of YAC chimerism through the use of appropriate yeast host strains.
Haldi
M
M
Whitehead Institute for Biomedical Research, Nine Cambridge Center, Massachusetts 02142.
Perrot
V
V
Saumier
M
M
Desai
T
T
Cohen
D
D
Cherif
D
D
Ward
D
D
Lander
E S
ES
D001483:Base Sequence; D002678:Chimera / Q000235:genetics*; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D002877:Chromosomes, Human; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051721:Rad52 DNA Repair and Recombination Protein; D011995:Recombination, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D051721:Rad52 DNA Repair and Recombination Protein