Herskowitz Lab Protocol
Based on techniques from Roy Parker
Ref : Muhlrad, D., et al. (1992) A rapid method for localized mutagenesis of yeast genes. Yeast 8:79-82

Procedure for generating ts alleles of NUT2

Make a haploid strain with a chromosomal deletion for NUT2 rescued by a pCEN.ARS.URA3.NUT2 plasmid. A diploid heterozygous for a nut2®hisG.URA3.hisG deletion of NUT2 was generated by transformation. This allele was converted to an unmarked allele by streaking on 5FOA. Then the strain was transformed with the plasmid pCEN.ARS.URA3.p

Useful Tip. The URA3 plasmid covering the deletion should have different 5' or 3' region from the gene itself. Otherwise, when the gapped vector is transformed in later it can repair itself and recircularize by recombining with the wildtype locus on the URA3 plasmid. Altering the flanking region greatly reduces this problem. Further if your gene (mine was not) is now regulated by galactose, one can simply screen on dextrose; 5-FOA provides a useful secondary screen.

• Construct another CEN.ARS plasmid containing the NUT2 locus carrying a marker other than URA3 eg. TRP1. DO NOT use the Sikorski CEN.ARS plasmids as these do not reliable maintain a copy number of 1-2 (Joachim Li, personal communication). Instead I used YCplac22, a TRP1.CEN.ARS plasmid from the Gietz series of yeast vectors.

  It is preferrable, if possible, to clone in an insert with the NUT2 flanking sequences, but lacking the NUT2 ORF. We had such a fragment available where the NUT2 ORF is replaced by a unique restriction site. Such a fragment is useful for generating knock out constructs. Now, the unique restriction site again becomes useful. Without this fragment it is possible to proceed provided there are unique restriction sites flanking the ORF, but leaving sufficient homology for recombination. However, if the starting plasmid contains the wildtype ORF, a higher background of wildtype alleles may be obtained.

• Digest the above plasmid with a enzyme(s) that removes the ORF but leaves the sequences flanking the ORF on both sides intact. Gel purify the linear fragment.

• PCR the NUT2 ORF and flanking region so that there is >500bp homology on both sides between the gapped plasmid and the PCR product. I used PCR conditions based on Leung DW, Chen E, Goeddel DV (1989). A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1(1):11-15.

• Transform into yeast cut, gel purified vector alone and the vector with PCR product (I do not clean up the PCR reaction except to remove mineral oil on parafilm) and select on SD-TRP for the vector (use galactose if your GAL construct is regulated). I do not continue selecting for URA3 so that the URA3 based plasmid can be lost. It is essential to titrate the amount of vector used. A 2-10 fold stimulation should be observed on the addition of the PCR product (see sample data below).

• If one is working with an essential gene, one can determine from the vector alone colonies if the vector is recirclurizing without obtaining the gene in question by testing on 5FOA. All these tranformants should be 5FOA sensitive. If the other plasmid in the cell has homology to the gapped vector (see Tip above), the gapped plasmid can frequently repair off of it.

• Plasmid Shuffle. I patched transformants on SD-trp (use galactose if your GAL construct is regulated), then replicaed to two 5FOA plates - one incubated at 25 degrees, the other incubated at 37 degrees. [Note : if your gene is maintained by a GAL construct that is regulated by galactose, one can replica to YEP-Dextrose instead of 5-FOA., Then double check by streaking on 5-FOA].

• Candidates for ts genes were streaked for single colonies on 5-FOA at 25 degrees. Single colonies were then streaked on YEPD plates and incubated at 25 and 37 degrees to verify that the strains were really temperature sensistive.

• Plasmids were rescued from yeast by standard techniques and then subcloned into pRS306, an integrating vector. It is essential to show that when integrated the gene is temperature sensitive and not due to a different mutation in another gene. Also when integrated in single copy, the allele may not provide sufficient function compared to on a plasmid (since plasmid copy numbers can vary) One can attempt to loop in and out the ts allele, although failure of this method does not mean the allele is bad. If it does fail, one can integrate into the URA3 locus in a diploid heterozygous for the deletion, sporulate and deletion spores that are URA3+ should be alive at 25, but dead at 37.

• One can then check how rapidly the cells die or arrest by moving single cells onto a YEPD slab and leaving the slab at 37 overnight, then inspecting the slab microscopically the next morning (compared to a slab at 25 degrees).