Thermosensitive mutants generation and suppressor screen for MAK11

GIM

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Reference

Muhlrad D, Hunter R, Parker R. "A rapid method for localized mutagenesis of yeast genes" Yeast 1992 Feb;8(2):79-82.
... and precious advice from Olivier Gadal, Micheline Fromont-Racine, Alice Lebreton, Gwenael Breard, Alain Jacquier and Laurence Decourty.

Introduction

This document describes in detail the diverse steps used in obtaining thermosensitive ts mutants of the essential MAK11 gene as well as the use of the Mak11-2 mutant to search for suppressors.

Background information

An essential gene is placed on a plasmid that contains the URA3 marker. A second plasmid containing a mutagenized version of the essential gene is introduced in the same yeast cell. On a medium containing 5-fluoro-orotic acid (5FOA) the URA3 plasmid is lost and only the mutagenic plasmid remains to support growth. These strains are then tested at two temperatures (25C and 35C for example) and the clones that do grow at the permissive temperature but do not grow at the nonpermissive temperature are recovered (Figure 1).
Schematics drawing
Figure 1: Ts generation by plasmid shuffling (replacement) using a mutagenized version of the gene - in this case MAK11.

Cloning MAK11 in pFL38CII

This step is required to generate the matrix for the mutagenic PCR and the plasmid that will serve to complement the deletion of the endogenous MAK11 gene. The oligonucleotides used in a genomic (FY1679 strain) PCR (pfu Turbo polymerase) were:
Note: the intergenic regions around MAK11 are unusually short.
The PCR product was phenol-chloroform extracted and digested with AscI and XbaI. After purification on low melting agarose, digested pFL38CII and the digested PCR product were ligated using Quick Ligase (NEB) and the ligation product transformed in DH5a calcium competent E. coli cells.
Seven (7) clones were obtained and tested by EcoRV digestion - all positive. Two of the plasmids obtained were sequenced using primers GB035 and AJ48 and the C2 miniprep was used subsequently.

Obtaining the LMA260 strain

This strain contains the plasmid pFL38CII-MAK11 which complements the deletion of the genomic MAK11 in a haploid MatA context. It was obtained by transformation of the diploid Y24870, heterozygous for MAK11 deletion (Euroscarf strain) followed by sporulation and tetrad dissection. The efficiency of sporulation for this strain was increased by a short, 10 hours, growth on rich fresh GNA medium plate. About 3 days of growth on a SPO -URA plate were needed for efficient sporulation.
Replica plates of the tetrads on YPD+G418 - to verify the marker of genomic MAK11 deletion, -URA - to verify the presence of the plasmid and 5FOA - to verify the requirement of the plasmid for the spore viability allowed the selection of the LMA260 strain. Note: In two out of three complete tetrads the plasmid was lost in the wild-type spores.

Mutagenic PCR methods

Background information

The use of the PCR reaction to induce random mutations in genes of interest is based on the ability of the Taq polymerase to incorporate a different deoxynucleotide in place of another during the elongation step. To "improve" the levels of mutagenesis by the PCR reaction manganese (Mn2+) ions are included in the reaction mix and one of the deoxynucleotides, usually dA, is included at much lower concentration than the others. The concentration of the deoxynucleotides is increased.

Mutagenic PCR - two versions, three reactions

Three PCR products were obtained under one non-mutagenic condition and two highly mutagenic PCR conditions - one involving the use of manganese and the other one involving the use of DMSO.
  1. Non-mutagenic PCR:
  2. Manganese mutagenesis PCR
  3. DMSO mutagenesis PCR
Each PCR reaction was performed in 6 tubes on the Eppendorf Mastercycler Gradient PCR machine using a gradient program around 56C:
1. 3' at 94C;
2. 30" 94C;
3. 30" 56C 10C;
4. 2'30" 72C;
5. GO TO 2 REPEAT 30 TIMES;
6. 5' 72C;
7. HOLD 4C;
The positions used in the gradient were 5 to 9 (between 51.5 and 62C)
With non-mutagenic conditions the highest yield of PCR product was obtained at all the annealing temperatures. About half of the amount was obtained with the manganese conditions. DMSO had an important effect on the yield of the PCR product depending on the temperature, the highest amount was obtained at 59.5C with lower amounts at lower temperatures and no PCR product at 62C. An estimate of the amount is that we had 600, 400 and 60 ng PCR product obtained under the three tested conditions.
The PCR products were pooled (only the 56.8 and 59.5 fraction for the DMSO conditions) precipitated with PEG/Mg2+ and resuspended in 30 ml TE for the non-mutagenic PCR, 20 ml TE for the manganese condition and 5ml TE for the DMSO conditions. The estimated concentration of these purified PCR products was 20 ng ml.

Plasmid linearization

Twenty-five micrograms of pFL36CII were digested in a 150ml total volume using 10 ml each of XbaI and HindIII, NEB2, BSA for 2 hours at 37C. Under these conditions the PCR product overlaps with the linearized plasmid on about 200 bases upstream the ORF insertion and 180 nucleotides downstream.
The linearized plasmid was separated from the small insert on an agarose gel and purified from the gel using the Quiaquick purification kit. The resulting plasmid had an estimated concentration of 60 ng ml, the total amount being around 3 micrograms.

Yeast transformation - yield and survivors

Five types of transformation were performed using the usual protocol but with DMSO addition and with a 3 hours incubation at the end in rich YPD liquid medium, finally plated on -LEU medium:
  1. Negative control: no PCR product and no plasmid plated on one plate
  2. No PCR but transformation with the linearized plasmid: 2ml @ 120 ng on 4 plates
  3. Non-mutagenic PCR product 5ml @ 100 ng and 120 ng plasmid on 8 plates
  4. Manganese PCR product and plasmid on 8 plates
  5. DMSO PCR product and plasmid on 8 plates
A control missing from these transformations is the non-linearized plasmid itself. All the plates were incubated at 30C for 36 hours (probably too much). After counting the number of transformants was:
The DMSO and Manganese clones were replica plated in duplicate on -LEU+5FOA plates to eliminate the pFL38CII-MAK11 (URA3) plasmid. The first replica was done on the plate that will be incubated at 35C (the nonpermissive temperature) and the second one on the plate that will be incubated at 25C. A first incubation was done for about 10 hours (from morning to evening) at 25C to allow growth of the cells devoid of the wild-type plasmid. In the evening half of the plates were moved to 35C.
A number of individual clones were also manually transfered on -LEU+5FOA plates to count for the survival of the cells containing the mutagenized plasmid at 25C. Only 10% (22/180) of the restreaked clones were viable when the manganese mutagenesis was used but 50% (95/180 and 100/180) of the clones were viable when DMSO mutagenesis was used. This means that only about 800 clones will be tested for manganese mutagenesis and about 4000 for DMSO mutagenesis.

How to compare 25C and 35C plates?

Even if the replica plating is done carefully, sometimes the number of clones is too high to allow efficient visual inspection of the differences. In this case, the plates are scanned and the images artificially coloured and superimposed in Adobe Photoshop.
This way I found 17 ts candidates (DMSO) and 7 ts candidates (manganese) that were isolated on -LEU5+FOA plates and grown for one day at 35C and 25C. Nine best ts candidates were selected from these plates - 6 DMSO and 3 manganese - Figure 2.
Plates image
Figure 2: Test for the ts phenotype at 35C. The numbered clones were kept and further tested.

Testing the ts mutants

It is important to demonstrate that the observed phenotype is strictly dependent on the plasmid containing the mutagenized version of the tested gene (MAK11 in this case). The plasmid from 9 candidates and three other clones coming either from the transformation with the linearized plasmid or from the transformation with the non-mutagenic PCR product were recovered from yeast (Protocol G of our lab) and retransformed in the original LMA260 strain (which contains the wild type copy of MAK11 on a URA3 plasmid) and selected on -LEU medium.
The plasmids rescued from the ts strains were also tested by restriction enzyme digestion. Using EcoRV, normal profiles were obtained for most of the plasmids with the exception of F7 and F8. In F8 the restriction site was affected at a position at +300 after the ATG and in F7 an insert of about 1200 nucleotides was present in addition to the expected fragments.
To test the phenotype of the LMA260 strain transformed with the pFL36CII-MAK11 ts mutants three independent clones were streaked on -LEU +5FOA plates and grown at 25C for 2 days. One colony was then streaked on -LEU +5FOA plates that were placed at 16C, 25C, 30C and 37C. No phenotype was observed at low temperature but Mak11-2 was dead at 37C and is partially affected at 30C.
Six ts strains were conserved in the freezer (LMA263-1 to 6) and the strain transformed with F10 (no PCR product used in the initial transformation) was conserved as a wild type control (LMA264).
The growth of three Mak11 ts strains was tested at 25C and after shift to 37C and the growth curves are presented in Figure .
Growth curve
Figure 3: Test for the growth of ts strains (2, 3, 7) and wild-type (10 - LMA264) at 37C..
The strain LMA263-2 (MAK11 ts 2) was further used for the suppressor screen.



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On 12 Mar 2008, 15:18.