Methods for use with the mTn-lacZ/LEU2-mutagenized library

Screening for gene expression using lacZ fusions

OVERVIEW: Transformant strains carrying in-frame fusions between yeast genes and lacZ are identified by a color assay for beta-galactosidase activity.

• To maximize detection of lacZ fusions expressed at a low level, transformant colonies are patched to YPAD plates at a density of 100 per plate.
• To identify vegetatively expressed genes, cells are replica plated to an SC-leu 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.
• 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.
• Filters are placed colony-side up onto X-Gal plates (120 ug/ml 5-bromo-4-chloro-3-indolyl-beta-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. These plates can be very thin; their use increases the signal over that obtained by simply soaking the filters in a buffered X-gal solution.
• Transformants carrying productive lacZ fusions are recovered from the regrown YPAD plates. It is advisable to subsequently maintain selection for LEU2 wherever possible, as some mutations are deleterious even in the heterozygous state.

Identification of the genomic site of transposon insertion

OVERVIEW: To determine the site of transposon insertion, genomic DNA imediately adjacent to the lacZ sequences is rescued in Escherischia coli. To introduce an origin of replication (ori), a plasmid marked with URA3(pRSQ2-URA3, U64694) replaces part of the transposon by recombination between plasmid- and transposon-borne copies of lacZ sequences. This procedure is oulined in this figure. Yeast DNA is recovered from these transformants and cut with a 'recovery' enzyme (EcoRI, HindIII EcoRV, PstI, ClaI, SalI, XhoI, KpnI). This releases as a linear segment the bacterial origin of replication, the beta-lactamase gene and a portion of the lacZ gene with adjacent yeast DNA; this fragment is then circularized and recovered in bacteria. pRSQ2 is high copy number in E. coli. Plasmids are sequenced using a primer complementary to the 5' end of the transposon. This process requires the three protocols given below.

Alternative method: C. Friddle (http://genome-www.stanford.edu/group/botlab) has developed a vectorette rescue protocol for lacZ-based transposons.PCR

CAUTION! Two or more insertion events may have occurred in up to 10% of the population. These can be identified by examination of segregation of the transposon-borne LEU2 marker upon tetrad dissection. You should be sure that the strain has only one transposon before proceeding to recovering a plasmid containing genomic DNA. If you have used the library for mutagenesis, you are strongly advised to make sure that your phenotype is linked to the transposon insertion, since spontaneous mutations can arise at other sites. You can waste time recovering junk if you don't check.

Transformation of yeast strains

We suggest transforming the yeast with 1-5ug of BamHI-digested pRSQ2-URA3 plasmid DNA, selecting the transformants on SC-leu-ura. This is a targetted replacement, so efficiency will depend on your strain. The method of Chen et al. (1992) given above can be used. If an amp^R plasmid is present in the yeast strain to be transformed, a different marker could be cloned into the pRSQ2 polylinker to enable its recovery.

Recovery of genomic DNA from yeast strains

For a detailed discussion of genomic DNA preparation from yeast, seePhilippsen et al. (1991). Here is the method that we use.

1. Yeast strains are grown to saturation at 30^oC in 2 ml of YPAD. We suggest using a couple of transformants for each strain.
2. Cells are recovered by centrifugation at 13,000 r.p.m. for 1 minute. The supernatant is removed by aspiration and cells are resupended in 250 ul of 0.1 M EDTA (pH 7.5), 14 mM beta-mercaptoethanol containing 150 ug/ml zymolyase. Cells are incubated at 37^oC until spheroplasted. Overspheroplasting does not affect recovery.
3. 50 ul of miniprep mix (0.25 M EDTA (pH 8.5), 0.5 M Tris base, 2.5% SDS) is added to each tube. Samples are mixed by inversion, then incubated in a water bath at 65^oC for 30 minutes.
4. 63 ul of 5 M KAc is added to each sample. Samples are mixed by inversion and incubated on ice for 30 minutes.
5. Samples are spun at 13000 r.p.m. in a microfuge for 10 minutes. Supernatants are transferred by pouring each sample into a new tube containing 720 ul of 100% ethanol. A DNA precipitate should be visible. Samples are mixed by inversion and spun for 5 minutes as above.
6. Tubes are drained thoroughly, and 130 ul TE containing 1 mg/ml RNAase A is added to the undried pellets. Resupension of DNA is gradual and occurs during subsequent incubation at 37^oC for 35 minutes with occasional vortexing, DNA is reprecipitated by addition of 130 ul of isopropanol. Samples are mixed by inversion and spun for 5 minutes as above.
7. Tubes are drained. A 70% ethanol wash may be performed to remove salt. Finally, pellets are air dried and resupended in 40 ul of TE with incubation at 37^oC. About 10ug of genomic DNA is obtained.

Plasmid rescue

1. 5 ug of yeast genomic DNA is digested overnight at 37^oC with 5 units of 'recovery' enzyme (EcoRI, HindIII, SalI, EcoRV, PstI, ClaI, XhoI, or KpnI) in a total volume of 40 ul.
2. 20 ul of the sample is run on a gel to check digestion. This gel may also be used for Southern analysis (see below). The remainder is heated to 65^oC for 25 minutes to inactivate the restriction enzyme, and 215 ul of H2O, 25 ul of 10X ligase buffer and 1 ul of ligase (400 units) are added. To favour intramolecular reactions, the DNA concentration in the ligation should not be over 10 ug/ml, and can be as low as 2 ug/ml.
3. After ligation at 16^oC for 4 to 16 hours, DNA is precipitated by addition of 125 ul of 7.5 M NH4Ac and 375 ul of isopropanol and recovered by centrifugation at 2190g (or more) for 20 minutes.
4. The DNA pellet is washed once with 70% ethanol, then resuspended in 6-20 ul of TE. 3 ul of this is transformed into E. coli, (we use electroporation) selecting for ampicillin resistance. Do minipreps of several colonies for each strain.
5. Rescued plasmids can be analyzed by double-digestion with BamHI and the 'recovery' enzyme. Desired plasmids display a 2.85-kb band containing vector sequences (see Figure 1b) plus additional band(s) from genomic DNA. If you get 'mystery' plasmids, try a different transformant/ recovery enzyme.
6. DNA preparations may be sequenced using a primer from lacZ sequences inside the terminal repeat, eg the -40 primer (#1212, New England Biolabs). Only trust your sequence up to the first site for the recovery enzyme, as other fragments can get cloned in during circularization.

Sequence of lacZ end of mTn3-lacZ/LEU2:

Bases 1-38 are the terminal repeat, also present on the vector.

GGGGTCTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGgg ggatcCCGTC GTTTTACAAC GTCGTGACTG GGAAAACCCT GCGTTACCCA ACTTAATCG

The accession number for mTn-lacZ/LEU2 is U35112.

The accession number for pRSQ2-URA3 is U64694.

Alternative rescue strategies

pRSQ

• Diagram outlining use of pRSQ
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