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Tester for Chromosome III

Before using any of the strains for mapping an unknown gene, check the strain with a known marker located on the specific chromosome for which the strain is being used. There have been reports of two incorrectly marked strains obtained from YGSC and from the Sherman-Wakem lab.

Mapping strains: chromosomal assignment with 2-micron tester strains (AB2)

gal-resistant

Each of the 2-micron strains contains derivatives of the integrated plasmid YEp24 and is URA3+. It has become apparent that several of the 2-micron strains tend to lose the plasmid spontaneously at a rather high rate. To prolong stability of the integrated plasmid, revive the strains from paper replicas on Ura- medium rather than on rich YEPD medium. A study by Y. Kaneko of the Institute for Fermentation in Osaka, Japan, has established approximate percentages of loss of the Ura+ phenotype in the 2-micron strains when replica-plated from YEPD medium to Ura-. The results indicate that strains B-7588, B-7589, B-7175, B-7595, B-7596, B-7599, B-7602, B-7604, B-7608, B-7610, B-7612, and B-7614 become auxotrophic for uracil at a rate between 10 and 39%; the percentage loss of the Ura+ phenotype for all other strains is between 0.22 and 0.49%. For more precise numbers, please contact YGSC.

Temperature: 25°C

Atmosphere: Typical aerobic

Protocol: Protocol for the use of 2-micron tester strains:The method relies on a set of 16 [cir0] tester strains, each containing plasmid DNA integrated at or near the centromere of a different chromosome. The plasmid DNA derived from YEp24 contains the URA3+ gene, the 2-micron inverted repeat sequence, pBR322 sequences, and different yeast segments corresponding to centromeric regions of each of the 16 chromosomes. The 2-micron plasmid DNA remains stably integrated in [cir0] strains since the plasmid DNA lacks the 2-micron FLP gene required for site-specific recombination and the [cir0] cells contain no resident 2-micron plasmid to provide FLP function. Specific loss of integrated 2-micron plasmid DNA and the chromosome into which integration occurred is induced in a [cir0]/[cir+] diploid since FLP function provided by the 2-micron circles of the [cir+] parent catalyzes a site-specific recombination event. This results in the loss of the integrant plus the entire chromosome containing the integrant if the integration occurred at the centromere. The recessive mutation, in a [cir+] strain, can be assigned to its chromosome by crossing to each of the [cir0] tester strains and isolating the [cir0]/[cir+] diploids on selective medium. A subclone of each diploid is resuspended and plated on nonselective medium. Several hundred colonies of each diploid are replica-plated onto a medium that selects for the expression of the unmapped mutation. Each of the [cir0]/[cir+] diploids will lose the 2-micron plasmid DNA as well as the chromosome which contains the integration at a high frequency. The recessive mutation will be manifested only in the diploid containing the [cir0] tester strain in which the integrant is on the same chromosome as the mutation.

Protocol: Protocol for the use of 2-micron tester strains:The method relies on a set of 16 [cir0] tester strains, each containing plasmid DNA integrated at or near the centromere of a different chromosome. The plasmid DNA derived from YEp24 contains the URA3+ gene, the 2-micron inverted repeat sequence, pBR322 sequences, and different yeast segments corresponding to centromeric regions of each of the 16 chromosomes. The 2-micron plasmid DNA remains stably integrated in [cir0] strains since the plasmid DNA lacks the 2-micron FLP gene required for site-specific recombination and the [cir0] cells contain no resident 2-micron plasmid to provide FLP function. Specific loss of integrated 2-micron plasmid DNA and the chromosome into which integration occurred is induced in a [cir0]/[cir+] diploid since FLP function provided by the 2-micron circles of the [cir+] parent catalyzes a site-specific recombination event. This results in the loss of the integrant plus the entire chromosome containing the integrant if the integration occurred at the centromere. The recessive mutation, in a [cir+] strain, can be assigned to its chromosome by crossing to each of the [cir0] tester strains and isolating the [cir0]/[cir+] diploids on selective medium. A subclone of each diploid is resuspended and plated on nonselective medium. Several hundred colonies of each diploid are replica-plated onto a medium that selects for the expression of the unmapped mutation. Each of the [cir0]/[cir+] diploids will lose the 2-micron plasmid DNA as well as the chromosome which contains the integration at a high frequency. The recessive mutation will be manifested only in the diploid containing the [cir0] tester strain in which the integrant is on the same chromosome as the mutation.

ATCC <<--YGSC<<--L.P. Wakem and F. Sherman

Before using any of the strains for mapping an unknown gene, check the strain with a known marker located on the specific chromosome for which the strain is being used. There have been reports of two incorrectly marked strains obtained from YGSC and from the Sherman-Wakem lab.

Each of the 2-micron strains contains derivatives of the integrated plasmid YEp24 and is URA3+. It has become apparent that several of the 2-micron strains tend to lose the plasmid spontaneously at a rather high rate. To prolong stability of the integrated plasmid, revive the strains from paper replicas on Ura- medium rather than on rich YEPD medium. A study by Y. Kaneko of the Institute for Fermentation in Osaka, Japan, has established approximate percentages of loss of the Ura+ phenotype in the 2-micron strains when replica-plated from YEPD medium to Ura-. The results indicate that strains B-7588, B-7589, B-7175, B-7595, B-7596, B-7599, B-7602, B-7604, B-7608, B-7610, B-7612, and B-7614 become auxotrophic for uracil at a rate between 10 and 39%; the percentage loss of the Ura+ phenotype for all other strains is between 0.22 and 0.49%. For more precise numbers, please contact YGSC.