|TR||Tn3 terminal inverted repeats|
|Xa||Factor Xa cleavage recognition site|
|loxR||lox site, target for Cre recombinase|
|GFP||gene encoding Green Fluorescent Protein mutant p11|
|URA3||URA3 gene from S. cerevisiae|
|tet||Tetracycline resistance gene|
|res||Tn3 site for resolution of transposition intermediate|
|loxP||lox site, target for Cre recombinase|
|3xHA||Hemagglutinin (HA) triple epitope tag|
Uses: Gene disruption, analysis of gene expression, creating fusion to GFP, HAT epitope-tagging protein at range of sites, creating conditional alleles.
In more detail: mTn-3xHA/GFP can be used easily to create a library of insertions, each at a different site in a given gene. The mutagenized DNA is then transformed into yeast, where it replaces the chromosomal locus by homologous recombination. The transposon insertions create a pool of insertion/disruption alleles. Insertions that generate in-frame fusion of the coding region to GFP can be used to monitor and quantify gene expression, via assays for fluorescence activity. Localization of the GFP fusion protein can be examined by fluorescence microscopy. The transposon can also be excized by Cre-mediated recombination to leave a 5 base-pair duplication caused by transposon insertion plus a 274-bp insertion containing sequences encoding the 3xHA tag and the factor Xa protease cleavage recognition site. When GFP is fused in-frame to the gene of interest, the excision event results in an in-frame insertion of 93 amino acids, called a HAT tag, into the encoded protein. The HAT tag allows immunodetection of the protein. Insertion of the HAT tag also has the potential to create conditionally-defective forms of the protein.
The accession number for mTn-3xHA/GFP is U54830.
A kit for mutagenesis of a yeast gene with mTn-3xHA/GFP is available.
Please read this whole document before you start!
We have not done assays of GFP activity in yeast.
See Niedenthal et al (1996) for their methods.
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.
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).
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.
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
|R1123||Strain XL1-blue carrying vector pHSS6.|
|R1236/B211||Strain RDP146 (F- recA' dlac-pro) rpsE; spectinomycin resistant) with plasmid pLB101 (pACYC184 with tnpA; active transposase, chloramphenicol resistant)(F. Heffron)|
|#111/B428||Strain RDP146 with pOX38 F factor derivative carrying mTn3 derivative mTn-3xHA/GFP (GFP, URA3, tet; tetracycline resistant)|
|#70/B425||Strain NG135 (K12 recA56 gal-delS165 strA; streptomycin resistant) with plasmid pNG54 (pACYC184 with mTn3 res and tnpR seqs; active resolvase, chloramphenicol resistant)(N. Grindley)|
|B227||Strain DH5-alpha carrying pB227/GAL-cre (amp, ori, CEN, LEU2) (B. Sauer)|
The accession for pHSS6 is M84115
|Tetracycline HCl, Tet (Sigma T3383)||12 mg/ ml in 50% ethanol. Use at 3 ug/ml (Tet3)|
|Kanamycin, Kan (Sigma K800)||10 mg/ ml in water. Use at 40 ug/ml (Kan40)|
|Chloramphenicol, Cm (Sigma C0378, I think)||34 mg/ml in ethanol. Use at 34 ug/ml (Cm34)|
|Streptomycin, Sm (Sigma S6501)||10 mg/ml in water. Use at 50 ug/ml (Sm50)|
|Ampicillin, Amp (Sigma A9518)||50 mg/ml in water. Use at 50 ug/ml (Amp50)|
NB. When only a few plates of each type are used, it's convenient to chop an LB plate up with a sterile toothpick, put the bits in a sterile flask, and melt the agar by microwave. Add appropriate amounts of antibiotic and repour plates.