DNA.
Tubes are vortexed to mix the contents thoroughly, then incubated at 45oC for 30 minutes.
Cells are pelleted and resuspended in 400 ul of SC-ura. 200 ul is plated onto SC-ura medium. Plates are incubated at 30oC for 3 to 4 days.
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 BDF1, 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.
To determine the site of transposon insertion, genomic DNA imediately adjacent to the transposon sequences must be rescued. We have not yet constructed a rescue vector for mTn-3XHA/GFP. If demand for the library is high we will construct one. Otherwise, we will be happy to provide reagents and information to another laboratory who wishes to construct it. Inverse PCR on genomic DNA could be used to recover the site of insertion. Alternatively, Carl Friddle has developed a 'vectorette PCR' rescue protocol for lacZ-based transposons. I have transcribed Carl's protocol and modified the suggested enzymes and primers, to make it suitable for PCR.html">vectorette PCR of the mTn-3xHA-based transposons.
When transposon insertion has created an in-frame fusion to GFP in the gene of interest, the transposon can be excized 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 in the protein. 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).
- Transform strain with pB227/GAL-cre, selecting on SC-leu.
- To derepress the GAL promoter, inoculate transformants into 2 mls SC-ura-leu with 2% raffinose as carbon source and grow to saturation.
- Dilute 1/100 into SC-leu with 2% galactose as carbon source (control: SC-leu with 2% glucose as carbon source). Grow for 2 days (some strains induce without growing).
- If grown, dilute 1/100. Spot a 10ul drop onto an FOA plate and streak it for singles (non-quantitative approach!). Or plate dilutions onto SC media and replica to identify ura- colonies. The induced cultures should give 100x more Ura- cells than the control.
- 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 popped-out version to see if the tagged gene is functional. Only believe a tag is lethal if it is complemented by the wild-type gene, and if several popout events give the same phenotype.
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