Stabilization of yeast artificial chromosome clones in a rad54-3 recombination-deficient host strain.
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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Department of Biochemistry, Faculty of Medicine, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada.
Dobson
M J
MJ
D018244:Chromosomes, Artificial, Yeast; D004265:DNA Helicases; D045643:DNA Repair Enzymes; D004276:DNA, Satellite / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D005838:Genotype; D006801:Humans; D000072235:Mating Factor; D008540:Meiosis; D008938:Mitosis; D016296:Mutagenesis; D010455:Peptides / Q000235:genetics; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic
D004276:DNA, Satellite; D005656:Fungal Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D000072235:Mating Factor; C075468:RAD54 protein, S cerevisiae; D004265:DNA Helicases; D045643:DNA Repair Enzymes