Restriction digests of the clone give the following sizes (kb): SacI--6.2; EcoRI--6.2; BamHI--6.2; PvuII--5.8, 0.4. SUP11 in the vector suppresses the red colony phenotype of ade2-101 ochre allele host strains, grown on limiting adenine media. In the absence of plasmid selection, plasmids are lost at a frequency of 1 to 3%, giving rise to red/white sectoring. One strategy for mutation scanning is to clone the normal allele of a gene into a SUP11 vector and transform into an ade2-101 host with a null mutation in the same gene, thus selecting for plasmid maintenance and producing white colonies. Vectors pUN20 (ATCC 77263), pUN25 (ATCC 77264), pUN60 (ATCC 77266) and pUN65 (ATCC 77267) contain SUP11. A second, potentially mutated copy of the gene can then be cloned into a second centromeric shuttle vector (pUN30, ATCC 77265; pUN70, ATCC 77268; pUN90, ATCC 77269; pUN100, ATCC 77270) for transformation into the SUP11 plasmid containing host. A normal gene in plasmid #2 can relieve the selective pressure on the SUP11 plasmid, restoring the red/white sectoring; a null allele in plasmid #2 will result in SUP11 plasmid maintenance and white colonies. YC-type shuttle vector useful for the sectoring-shuffle mutagenesis assay. Contains promoters for in vitro RNA synthesis and permits visual detection of recombinants by lacZalpha complementation. |
