Methods for use with the mTn-3xHA/lacZ-mutagenized library

GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGgcggccattgaaggtagaag

The accession for mTn-3xHA/lacZ is U54828.

Transferring the disruption allele to other strains:

When pRSQ2-LEU2 integrates into mTn-3xHA it creates an 11.8 kb insertion. This element is not cleaved by the following enzymes: AscI, AvrII, BspEI, MscI, NotI, PmeI, PmlI, SacII, SnaBI, SpeI. These enzymes can therefore be used to recover a large plasmid containing sequences both 5' and 3' to the transposon insertion. We have successfully 'moved' disruptions by this strategy.

Using the HAT epitope tagging feature of mTn-3xHA/lacZ

When transposon insertion has created an in-frame fusion to lacZ 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 or 110kD, 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 pB227/GAL-cre, selecting on SC-leu.
2. To derepress the GAL promoter, inoculate transformants into 2 mls 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 (control: SC-leu with 2% glucose as carbon source). Grow for 2 days (some strains induce without growing).
4. 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.
5. 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.

Sequence of HAT tag (3xHA):

TR in upper case. loxR in bold.

GGGGTCTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGgc ggccattgaa ggtagaagag aaaatttgta cttccaaaga aagaaggccg ctatcgcttc ggataactcc tgctatacga agttatgggc ggccgtttac ccatacgatg ttcctgacta tgcgggctat ccctatgacg tcccggacta tgcaggatcc tatccatatg acgttccaga ttacgctccg gccgcCCTTA ACGTGAGTTT TCGTTCCACT GAGCGTCAGA CCCC

GenBank accession

• mTn-3xHA/lacZ is U54828
• pRSQ2-LEU2 is U64693

Antibiotics used: