Protocols for shuttle mutagenesis/epitope-tagging of a yeast gene with mTn-4xHA/

Screening for in-frame lacZ fusions in yeast

1. Transformant colonies are patched to SC-ura ( SC-ura -leu if you already have pB227/GAL-cre in there).
2. Cells are replica plated to an SC-ura (-leu) plate and a SC-ura plate on which a sterile disc of Whatman 1A filter paper has been placed, and grown overnight at 30^oC. Other media or growth conditions can be substituted as desired. For ade2 strains, any test media should contain 80 mg/l of adenine, as the red pigment can obscure the X-gal result.
3. Filters are lifted from the plates and placed in the lid of a 9-cm glass petri dish. This lid is then placed inside a closed 15-cm glass petri dish containing chloroform, for 10 to 30 minutes. The minimum exposure time necessary for a particular yeast strain can be determined empirically.
4. Filters are placed colony-side up onto X-Gal plates (120 ug/ml 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside, 0.1 M NaPO4 [pH 7] and 1 mM MgSO4 in 1.6% agar) and incubated at 30^oC for up to 2 days.
5. Transformants carrying productive lacZ fusions are recovered from the regrown SC-ura (-leu) plate . It is advisable to subsequently maintain selection for URA3 wherever possible, as some mutations are deleterious even in the heterozygous state.
6. PCR primers designed using the mTn-3xHA/lacZ sequence can be used to determine position of the transposon. The IR elements and palindromic lox regions should be avoided.

Using the excision feature to HAT-epitope tag a protein

A leu2 ura3 GAL yeast strain is required. When transposon insertion has created an in-frame fusion to lacZ in the gene of interest, the transposon can be excized by Cre-mediated recombination to leave a 262-bp insertion (sequence given below) containing four copies of the HA epitope. With the 5 base pair duplication caused by transposon insertion, this gives an in-frame 89 amino acid insertion. The popout event is mediated by cre recombinase and requires induction of the GAL1-10 promoter on galactose. Our strains grow poorly on galactose but give 80 to 100% popouts.

The HA tag can be detected by mouse monoclonal antibodies 12CA5 (Boehringer) or MMS101R (BAbCo, Richmond, California). These antibody recognise cross-reacting yeast proteins of about 55kD or110kD, respectively, and can give a spotty background on immunofluorescence. Despite this drawback, the 3xHA tag has been used extensively and successfully in yeast. A rabbit polyclonal antisera is also available (101c500; BabCo) but this was less reactive in the one instance we tried. Protocols for yeast immunofluorescence can be found here, or in Methods in Enzymology 194 (1991).

1. Transform strain with plasmid pB227/GAL-cre, selecting on SC-leu.
2. Inoculate transformants into 2mls SC-ura-leu with 2% raffinose as carbon source, and grow to saturation.
3. Dilute 1/100 into SC-leu with 2% galactose as carbon source. As a control also dilute 1/100 into SC-leu with 2% glucose as carbon source. Grow for 2 days (some strains induce without growing).
4. If grown, dilute 1/100. Otherwise, proceed with undiluted culture.
   • Spot a 10ul drop onto an FOA plate and streak it for single colonies (non-quantitative approach!).
   • Alternatively, plate dilutions onto SC media and replica to SC-ura to identify Ura- colonies. The induced cultures should give 100-fold more Ura- cells than the control.
5. PCR primers designed using the sequence given below can be used to determine position of the tag. The TR elements and palindromic loxR region should be avoided.

N.B. When tagging essential genes, the original strain transformed should obviously be diploid. You can dissect the HAT-tagged version to see if the tagged gene is functional. To be rigourous, only believe a tag is lethal if it is complemented by the wild-type gene, and if several popout events give the same phenotype.