Construction of a pilot human YAC library in a recombination-defective yeast strain.
Using high-molecular-weight DNA fragments from a human lymphoblastoid cell line, a pilot collection of 2500 YACs was constructed in YKK115, a recombination-deficient strain of Saccharomyces cerevisiae carrying mutations in both the rad51 and rad52 genes. Analysis of 520 clones from the current library by pulsed-field gel electrophoresis revealed more than 97% single YACs with an insert size averaging 340 kb. Fluorescent in situ hybridization (FISH) performed with 37 clones on metaphase chromosomes suggested a high proportion mapping at centromeric (7) or telomeric (4) locations. The results are consistent with the stabilization of YACs in strains disarmed in recombination functions [Kohno, K., Oshiro, T., Kishine, H., Wada, M., Takeda, H., Ihara, N., Imamoto, F., Kano, Y. and Schlessinger, D. (1997) Human YACs unstable in a rad52 single mutant strain become stable in rad51rad52 double mutant. Gene, 000, 000-000 (GENE 10429)], and further suggest that the YACs may include regions that have been difficult to clone in other strains.
Palmieri
G
G
International Institute of Genetics and Biophysics, Naples, Italy.
Miano
M G
MG
Casamassimi
A
A
Lania
A
A
Kohno
K
K
Schlessinger
D
D
D'Urso
M
M
Featherstone
T
T
D002460:Cell Line; D002874:Chromosome Mapping; D018244:Chromosomes, Artificial, Yeast; D004268:DNA-Binding Proteins / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D015723:Gene Library; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D009154:Mutation; D051135:Rad51 Recombinase; D051721:Rad52 DNA Repair and Recombination Protein; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins; C495151:RAD51 protein, S cerevisiae; C495153:RAD51 protein, human; D051135:Rad51 Recombinase