Words similar to uau
Example sentences for: uau
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We selected 25 Arg +transformants and analyzed them by Southern blotting for integration of the UAU1 cassette into one of the two CaCET1 chromosomal loci to yield the heterozygote CaCET1/cacet1::UAU1 configuration depicted in Figure 1. Briefly, genomic DNA was isolated from the 25 Arg +strains, then digested with Sca I (which cuts neither CaCET1 nor UAU1 ). The digests were resolved by agarose gel-electrophoresis and transferred to membranes, which were probed with a radiolabeled DNA corresponding to the 5' segment of CaCET1 (probe A in Figure 1).
Correct insertion via homologous recombination into one copy of the CaCET1 gene, resulting in cacet1::UAU1 (Figure 1), was confirmed by Southern blotting of genomic DNA digested with diagnostic restriction endonucleases.
The blots were probed with a 5' specific CaCET1 fragment (probe A in Figure 1), which detects both the wild-type CaCET1 allele and the cacet1::UAU1 allele, and with a 3'-specific CaCET1 fragment (probe B in Figure 1) derived from the segment deleted during construction of the cacet1::UAU1 disruption cassette.
5-kbp fragment corresponding to cacet1::UAU1 (data not shown).
Third, we grew 54 independent liquid cultures of the heterozygotes in nonselective medium and then selected for cells that were Arg +and Ura +. Uracil prototrophy requires restitution of the integrity of the disrupted ura3 gene of the UAU1 cassette by recombination between the overlapping regions of the ura3Δ3' and ura3Δ5' fragments with excision of the intervening ARG4 gene [ 16 ] . If CaCET1 were nonessential, then recombination of UAU1 into the second copy of CaCET1 (to generate cacet1::UAU1/cacet1::UAU1 ) followed by excisional recombination of ARG4 in one allele to restore URA3 (generating cacet1::UAU1/cacet1::URA3 ) would result in the selected Arg +Ura +phenotype with complete loss of the wild-type CaCET1 locus.
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