Protocols for shuttle mutagenesis/epitope-tagging of a yeast gene with mTn-3xHA/

Screening for in-frame GFP fusions in yeast

We have not done assays of GFP activity in yeast.

See Niedenthal et al (1996) for their methods.

Analyzing GFP fusion protein localization in yeast

We tested mTn-3xHA/GFP by mutagenesis of the BDF1 gene, which encodes a chromatin-associated protein. We grew individual bdf1::mTn-3xHA/GFP transformants to a density of 10⁷ cells/ml in SC-ura. The last four hours of growth were at room temperature, to allow formation of the GFP chromophore. Then we examined cells directly using a Leitz microscopy with a system 13 filter (this may not be optimal). In 4 of 38 transformants, we saw green fluorescence of the nucleus. Fixation and spheroplasting of the cells improved the signal-to-noise ratio.

Using the exision 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 GFP in the gene of interest, the transposon can be excized by Cre-mediated recombination to leave a 274 bp insertion (sequence given below) containing the 3xHA tag. With the 5 base pair duplication caused by transposon insertion, this gives an in-frame 93 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 triple 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.
5. 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 100x more Ura- cells than the control.
6. PCR primers designed using the sequence given below can be used to determine position of the tag. The IR 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 rigorous, only believe a tag is lethal if it is complemented by the wild-type gene, and if several popout events give the same phenotype.

Sequence of HAT tag (3xHA):

TR in upper case. loxR in bold.

GGGGTCTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGgc ggccattgaa ggtagaagag aaaatttgta cttccaaaga aagaaggccg ctatcgcttc ggataactcc tgctatacga agttat