|Genome Mapping Section
DOE Human Genome Program Contractor-Grantee Workshop
59. New Host Strains for Stabilization and Modification of YAC Clones
Natalay Kouprina, Maxim Koriabine, and
The recent development of a new approach (TAR cloning) for the selective isolation of specific regions and genes from complex genomes as large linear or circular YACs greatly advanced YAC cloning technology1,2. While TAR cloning provides many opportunities for studying mammalian genomes, some YAC isolates containing multiple repeats may be mitotically unstable. Recently we systematically studied the contribution of several RAD genes to the stability of human YAC clones in yeast. Using a variety of linear and circular internally marked YACs, we demonstrated that rad52 substantially stabilizes human DNA inserts, decreasing YAC instability 25- to 400-fold compared to a recombination-proficient host strain. In contrast, other rad mutant strains analyzed (rad1, rad50, rad51, rad54 and rad55) had a minor affect (2- to 5-fold reduction) on YAC instability. Thus, if YAC stabilization is desired, propagation in a rad52-deficient strain is strongly advisable. However, there is no opportunity to manipulate YACs by recombination in rad52 strains. Moreover, rad52-deficient strains cannot be used for specific gene isolation by TAR cloning. Therefore, we chose to develop a rad52-based system that could be used for stable maintenance of any YAC, while providing the opportunity for recombinational manipulation. We constructed a set of kar1 strains that have a conditional RAD52 gene under the control of the galactose-inducible GAL1/GAL10 promoter. These strains are rad52-deficient on glucose-containing medium and recombination-proficient on medium containing galactose. A YAC from any genetic background can be efficiently and accurately transferred into new hosts during mating with karyogamy-deficient kar1 strains3. To expand more the utility of a new YAC transfer system, the RNA telomerase gene TLC1 in RAD52-conditional strains was modified to produce (TTAGGG)n repeats specific to human telomere sequences4. The kar1-induced transfer of a YAC into the strains with the modified TLC1 gene resulted to replacement of yeast-specific telomeric repeats by human-specific repeats in the YAC.
1Larionov et al. (1997)
Proc. Natl. Acad. Sci. USA 94: 7384-7387.
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