Science_He_2001 - REPORTS NHEJ, using screens for genes...

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NHEJ, using screens for genes up-regulated in haploids versus diploids ( 22, 23 ) or using two hybrid-screens with Lif1p ( 24 ). hap3 mutants also showed a high average (mean 6 SD) percentile (99.3 6 0.4% ) in diploid microarray analyses (Fig. 1). Prelim- inary results suggest that diploid hap3 mu- tants have modest linear-plasmid transforma- tion defects. However, when diploid hap3 mutants were transformed with Eco RI–di- gested pSO98 (see the legend to Fig. 2A) and plated onto SC-Leu to select for precisely rejoined transformants, diploid hap3 mutant transformation efficiency was not distin- guishable from that of wild-type cells, where- as yku80 mutants had a fivefold-reduced transformation efficiency. We have shown here that pools of thou- sands of mutants generated by DNA trans- formation can be analyzed in parallel. This approach is likely to have many important applications in any genetic screen requiring a plasmid. Furthermore, this approach makes feasible genetic screens for quanti- tative phenotypes such as NHEJ frequency, mutation rate, and gross chromosomal re- arrangement rate, which are physically too labor-intensive to be carried out by conven- tional screening methods. Finally, databas- es containing quantitative phenotypic infor- mation of this type will provide an impor- tant resource for mapping genetic-interac- tion networks. References and Notes 1. E. A. Winzeler et al ., Science 285 , 901 (1999). 2. D. D. Shoemaker, D. A. Lashkari, D. Morris, M. Mitt- man, R. W. Davis, Nature Genet. 14 , 450 (1996). 3. The UPTAG and DOWNTAG features allow large num- bers of deletion strains to be pooled and analyzed in parallel. The pool of mutants was transformed with either circular or Eco RI–linearized pRS416 and plated onto SC-Ura plates. Genomic DNA was isolated from pooled Ura 1 transformants and used as a PCR template to amplify the DOWNTAGs or UPTAGs of the strains present. Genomic DNA from the circular pRS416-trans- formed cells was amplified with Cy5 (red)-labeled uni- versal primer, whereas that of the Eco RI–linearized pRS416-transformed cells was amplified with Cy3 (green)-labeled universal primers. These fluorescently labeled probes were then cohybridized to a DNA mi- croarray bearing each tag in triplicate. An NHEJ-defec- tive deletion strain would be underrepresented in the Eco RI–linearized plasmid-transformed pool and would have a reduced signal in the Cy3 channel ( Web fig. 1B). 4. G. Giaever et al ., Nature Genet. 21 , 278 (1999). 5. D. C. van Gent, J. H. Hoeijmakers, R. Kanaar, Nature Rev. Genet. 2 , 196 (2001). 6. D. B. Roth, M. Gellert, Nature 404 , 823 (2000). 7. L. K. Lewis, M. A. Resnick, Mutat. Res. 451 , 71 (2000). 8. S. U. Åstrom, S. M. Okamura, J. Rine, Nature 397 , 310 (1999). 9. S. E. Lee, F. Paques, J. Sylvan, J. E. Haber, Curr. Biol. 9 , 767 (1999). 10. S. J. Boulton, S. P. Jackson, Nucleic Acids Res. 24 , 4639 (1996). 11. Construction of the deletion strain pools: To con- struct the deletion pools, collections of 4647 MAT a haploid mutants (Research Genetics) or 3546 ho- mozygous diploid mutants (gift of M. Johnston) were pinned as individual 5-mm patches onto YPD plates containing G418 (200 m g/ml) and incubated at 30°C for 3 days. The mutants were scraped into ; 300 ml of 15% glycerol. The optical densities at 600 nm
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Science_He_2001 - REPORTS NHEJ, using screens for genes...

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