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Example sentences for: pmet

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To construct [ pMET3-CDC15, URA3 ] (pWS100) and [ pMET3-cdc15-2, URA3 ] (pWS109), the corresponding genes (from start codon to 300 bps downstream of the stop codon) were amplified by PCR from genomic DNA of wild type (RJD381) or cdc15-2 (RJD619) cells, and cloned into the SpeI and SacII sites of RDB620 (a derivative of pRS316 containing the MET3 promoter inserted between the HindIII and EcoRI sites).
The same crosses were also used to determine if Tab +was recessive or dominant: for every tab mutant, haploid cdc15Δ tab [pMET3-cdcl5-2, URA3] and diploid cdc15Δ/cdc15Δ tab/+ [pMET3-cdc15-2, URA3 ] cells were pre-grown on YPD (1% yeast extract/2% peptone + 2% glucose) at 25°C for 2-3 days, spotted in 5-fold serial dilutions onto SD+5-FOA plates, and the number of 5-FOA resistant colonies from the diploid was divided by that from the haploid to obtain ratio P. The mutant was defined to be dominant if P= 0.1-1, semi-dominant if P= 0.01-0.
PCR analysis confirmed that all twenty five colonies had lost the [ pMET3-cdc15-2, URA3 ] plasmid, indicating that they were true bypassers of cdc15Δ.
The scheme is outlined in Figure 2. Ninety independent cultures of cdc15Δ [pMET3-cdc15-2, URA3 ] (WY221) were grown at 25°C in synthetic minimal medium + 2% glucose (SD) in the absence of methionine.
The transformants lost their Tab +phenotype, and when crossed to cdc15Δ::TRP1 [pMET3-CDC15, URA3], the diploid yielded no spores that could bypass cdc15Δ (n = 40-50).