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Nucleic Acids Research, Vol 25, Issue 6 1248-1253, Copyright © 1997 by Oxford University Press

ARTICLES

Stabilization of yeast artificial chromosome clones in a rad54-3 recombination-deficient host strain

Y Le and MJ Dobson
Department of Biochemistry, Faculty of Medicine, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada.

The cloning and propagation of large fragments of DNA on yeast artificial chromosomes (YACs) has become a routine and valuable technique in genome analysis. Unfortunately, many YAC clones have been found to undergo rearrangements or deletions during the cloning process. The frequency of transformation-associated alterations and mitotic instability can be reduced in a homologous recombination- deficient yeast host strain such as a rad52 mutant. RAD52 is one member of an epistatic group of genes required for the recombinational repair of double-strand breaks in DNA. rad52 mutants grow more slowly and transform less efficiently than RAD + strains and are therefore not ideal hosts for YAC library construction. We have investigated the ability of both null and temperature-sensitive alleles of RAD54 , another member of the RAD52 epistasis group, to prevent rearrangements of human YAC clones containing tandemly repeated DNA sequences. Our results show that the temperature-sensitive rad54-3 allele blocks mitotic recombination between tandemly repeated DYZ3 satellite sequences and significantly stabilizes a human DYZ5 satellite- containing YAC clone. Yeast carrying the rad54-3 mutation can undergo meiosis, have growth and transformation rates comparable with RAD + strains, and therefore represent improved YAC cloning hosts.
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