PMC:4502370 / 21835-23718 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/4502370","sourcedb":"PMC","sourceid":"4502370","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4502370","text":"RPO26 and RPO31 are dosage suppressors of tgs1Δ cs growth\nThe dosage suppressor screen entailed transformation of S. cerevisiae tgs1∆ cells with a 2-μ URA3 plasmid-based wild-type genomic DNA library and selection for Ura+ colonies that grew at 18°. Plasmid DNA was recovered from individual yeast colonies grown at 18° and then transformed into E. coli. Candidate suppressors were retransformed into the original tgs1∆ strain and tested for growth at 18°. Sequencing the insert junctions of the plasmids that retested faithfully revealed that the rescuing clones contained either TGS1 (as was to be expected) or one of two distinct extragenic suppressor loci, which we provisionally named DTS1 and DTS2, respectively (DTS = deletion of TGS1 suppressor). Note that whereas the 2-µ DTS1 or 2-µ DTS2 plasmids restored growth at 18°, compared to tgs1∆ cells carrying the empty 2-µ vector, neither 2-µ DTS1 nor 2-µ DTS2 was as effective as a TGS1 plasmid, as gauged by colony size (Figure 3).\nFigure 3 RPO26 and RPO31 are dosage suppressors of tgs1Δ cold sensitivity. DTS1 (8.6 kb) and DTS2 (12.4 kb) are the two genomic inserts in the 2-μ URA3 plasmids that were isolated in the dosage suppressor screen for reversal of tgs1Δ cs growth. Individual genes with complete ORFs within the DTS1 and DTS2 inserts including PZF1, RPO26, MLC2, SKI3, AZF1, RPO31, TRS33, and YOR114w were cloned into a 2-μ URA3 vector. These plasmids, an empty CEN URA3 vector (negative control), a CEN URA3 plasmid containing wild-type TGS1 (positive control), and the plasmids containing DTS1 or DTS2 were transformed into tgs1Δ cells. Ura+ transformants were selected and grown at 30° in liquid medium lacking uracil. The cultures were adjusted to an A600 of 0.1 and aliquots of serial 10-fold dilutions were spotted on agar medium lacking uracil. The plates were photographed after incubation for 7 d at 18°.","divisions":[{"label":"title","span":{"begin":0,"end":57}},{"label":"p","span":{"begin":58,"end":988}},{"label":"label","span":{"begin":989,"end":997}}],"tracks":[]}