title: Large human YACs constructed in a rad52 strain show a reduced rate of chimerism.
abstract: 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.
authors.affiliation: Whitehead Institute for Biomedical Research, Nine Cambridge Center, Massachusetts 02142.
authors.forename: M
authors.initials: M
authors.lastname: Haldi
authors.forename: V
authors.initials: V
authors.lastname: Perrot
authors.forename: M
authors.initials: M
authors.lastname: Saumier
authors.forename: T
authors.initials: T
authors.lastname: Desai
authors.forename: D
authors.initials: D
authors.lastname: Cohen
authors.forename: D
authors.initials: D
authors.lastname: Cherif
authors.forename: D
authors.initials: D
authors.lastname: Ward
authors.forename: E S
authors.initials: ES
authors.lastname: Lander
mesh_terms: 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
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D051721:Rad52 DNA Repair and Recombination Protein