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2
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17344388163
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Plasmid pYEX 4T-1 (Clontech, Palo Alto, CA) was modified by the addition of a 140-nucleotide recombination domain, 3′ of its Eco Rl site, linearized within the recombination domain by restriction digestion, and cotransformed with a genomic set of reamplified ORFs that had the same ends as the linearized plasmid [J. R. Hudson Jr. et al., Genome Res. 7, 1169 (1997) ] into strain EJ 758 [MATa his3-Δ200, leu2-3,112, ura3-52, pep4::URA3], a derivative of JHRY-20-2Ca (5). Transformants obtained on synthetic minimal (SD) - Ura drop-out plates [F. Sherman, Methods Enzymol. 194, 3 (1991)] (>100 in all cases, and more than five times the cut vector in 97% of the cases) were eluted in batch and saved In 96-well microtiter plates. The library contains 6080 ORF-containing strains and 64 strains with vector only.
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Crude extract from 430 g of strain JHRY-20-2Ca (5) was chromatographed successively on Blue Sepharose, heparin agarose, hydroxyapatite, and Orange A Sepharose to purify cyclic phosphodiesterase activity 4000-fold in relation to crude extract.
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17
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0344901354
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note
-
We thank M. Dumont, M. Gorovsky, and L Tabak for comments and B. Pinsky for assistance. Supported by the University of Rochester Medical School and the Merck Genome Research Institute (grant 196 to E.M.P. and E.J.G.), NIH (grant GM52347 to E.M.P.), American Cancer Society (grant RPG-95-049-05-MBC to E.J.G.), the National Center for Research Resources (grant P41 RR11B23 to S.F.), and the Merck Genome Research Institute (to S.F.). S.F. is an investigator of the Howard Hughes Medical Institute.
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