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yeast strains frequently used for Y2H assays

MaV103 and MaV203

S. cerevisiae strains MaV103 (MATa) andMaV203 (MATα) contain deletions of the GAL4 and GAL80 genes encoding Gal4p and its repressor Gal80p, respectively. In the absence of Gal80p, galactose is not required for activation of Gal4p-inducible promoters. The strains contain three Gal4p inducible reporter genes, providing four phenotypes to identify true interactors. The reporter genes are HIS3, providing for growth on plates lacking histidine, LacZ, for colorimetric detection of Gal4p activity, and URA3, providing growth on media lacking uracil, as well as sensitivity to the drug 5-FOA. In addition, the strains are resistant to canavanine and cycloheximide, aiding in plasmid shuffling.

MaV103 and MaV203 were derived by Marc Vidal from a cross between two non-isogenic strains, PCY2 and MaV99, and are described in more detail in Chevray and Nathans 1992, Vidal et. al. 1996, and in "The Reverse Two-Hybrid system, M. Vidal" in "The yeast two-hybrid system" ed. P. L. Bartel and S. Fields.

Y8800 and Y8930

S. cerevisiae strains Y8800 (MATa) and Y8930 (MATα) contain deletions of the GAL4 and GAL80 genes encoding Gal4p and its repressor Gal80p, respectively. In the absence of Gal80p, galactose is not required for activation of Gal4p-inducible promoters. The strains contain three Gal4p inducible reporter genes, providing four phenotypes to identify true interactors. The reporter genes are HIS3, providing for growth on plates lacking histidine, LacZ, for colorimetric detection of Gal4p activity, and ADE2, providing growth on media lacking adenine, as well as a range of color from red to white depending on the strength of Gal4p expression. In addition, the strains are resistant to cycloheximide, aiding in plasmid shuffling.

Y8800 and Y8930 were generated by X. Xin in C. Boone's laboratory by adding cycloheximide resistance to the PJ69-4 Y2H strains. PJ69-4 is described in P. James et. al. 1996.

The differences

Both strains are very similar. Both are intended for Gal4 based two-hybrid assays, and feature three reporter genes. Each uses leu2 and trp1 as auxotrophic markers to select for cells containing bait and prey plasmids. As a consequence, the same plasmids and libraries can be used in either strain. Both use HIS3 and LacZ reporter genes , but differ in the third reporter. MaV102 and MaV203 use URA3, which has the added benefit of conferring sensitivity to 5-FOA. This is essential for the reverse two-hybrid system, in which mutations are selected that prevent an interaction. Instead of URA3, Y8800 and Y8930 use ADE2. Both URA3 and ADE2 yield less positives than the HIS3 marker. This does not necessarily equate to being more stringent or identifying fewer false positives, as certain interactions can show stronger induction of URA3 or ADE2 than HIS3. It is always advisable to test all 3 reporter genes.

Although the strains share the HIS3 and LacZ reporters, the phenotypes resulting from them differ. For the HIS3 reporter, Y8800 and Y8930 need less 3-AT in the -His plates than MaV103 and MaV203 (0-5 mM compared to 20-100 mM). The reason for this is not known, since the HIS3 reporter locus is the same. The LacZ reporter is much more useable in MaV103/MaV203. In Y8800/Y8930, even negative controls turn blue, making it difficult to identify true positives. This is especially the case for filter based X-gal assays, which are not suitable for Y8800/Y8930. An agar overlay X-gal assay works better.

Genotypes

MaV103/203

Auxotrophic markers

Description

leu2-3,112 Need for Leu in medium
trp1-901 Need for Trp in medium
his3Δ200 Need for His in medium
ura3-52 Need for Ura in medium
ade2-101 Reverted back to wild-type through an unknown mechanism
Other Description
gal Deletion of endogenous Gal4p transcription factor
gal80Δ Deletion of Gal4p repressor.
cyh2R Resistance to Cycloheximide
can1R Resistance to Canavinine
Reporters Description
GAL1::HIS3@LYS2 125 bp GAL1UAS in the HIS3 promoter driving HIS3, integrated at the LYS2 locus (Durfee et. al. 1993)
GAL1::lacZ The GAL1 promoter driving LacZ, integrated at an unknown locus.
SPAL10::URA3@ura3 SPO13 promoter with 10 Gal4p binding sites driving URA3, integrated at ura3-52 locus (Vidal et. al. 1996)
Y8800 / Y8930
Auxotrophic markers Description
leu2-3,112 Need for Leu in medium
trp1-901 Need for Trp in medium
his3Δ200 Need for His in medium
ade2-101 Need for Ade in medium
ura3-52 Need for Ura in medium
Other Description
gal Deletion of endogenous Gal4p transcription factor
gal80Δ Deletion of Gal4p repressor.
cyh2R Resistance to Cycloheximide
Reporters Description
GAL2::ADE2 ade2-101 locus replaced by GAL2 promoter driven ADE2
GAL1::HIS3@LYS2 GAL1 promoter driven HIS3 gene downstream of LYS2 gene
GAL7::LacZ@met2 GAL7 promoter driven LacZ gene inserted in the MET2 locus