Yeast strains and plasmid constructs.
The yeast strains, plasmids, and oligonucleotide primers used in this work are listed in Tables and . The sequences of the oligonucleotides used in this study are available upon request.
Yeast strains used in this study
Plasmid constructs used in this study
Most of the strains were derived from the W303 background (JHY200) (1
). Initially, phenotypic characterization, including cellular responses to various stresses (e.g., see Fig. ) and suppression by 2μm SGO1
, was conducted in parallel with both JHY200 and its S288C mutant counterpart JHY205 (1
). Both strains exhibited literally identical phenotypes. Subsequent studies were thus focused on the W303 background strains.
FIG. 1. The G44S mutation confers pleiotropic phenotypes. (A) Yeast cells bearing the G44S allele as the sole copy of H3 were tested on YPD medium under the indicated conditions. Left panel, sixfold serially diluted log-phase cells were spotted for growth tests. (more ...)
To delete SGO1
, primers O361 and O362 were used to PCR amplify the Kluyveromyces lactis TRP1
selective marker from plasmid pBS1479 (47
), and the PCR product was transformed into yeast cells for tryptophan prototroph selection.
To tag endogenous Pds1p with 13×Myc, O323 and O324 were used to PCR amplify pFA6a-13Myc-TRP1 for yeast integrative transformation. Integration was verified with PDS1
-specific primers O325 and O326 and TRP1
-specific primers O375 and O376. Mcd1p-13Myc was created in a scheme identical to that used for Pds1p-13Myc, except for the use of MCD1
-specific primers OJL21, OJL22 (for integration), and OJL23 (for verification). To introduce a carboxyl-terminal six-hemagglutinin (6×HA) tag into Sgo1p, yeast strain A10652 (22
) was used for a genomic PCR (primers O363 and O364) that amplified the 6×HA-TRP1 fragment flanked by SGO1
sequences. The resultant PCR product was transformed into yMK1243 and yJL145 to knock in the HA tag and the TRP1
marker, creating yJL343 and yJL344, respectively. Contrary to Kitajima et al. (25
), who analyzed Myc-tagged SGO1
, we did not observe discernible phenotypes associated with carboxyl-terminal HA tagging.
To place the MCD1
gene under the control of the GAL1
promoter (i.e., yJL171 and yJL172), the His3MX6-PGAL1 sequence (42
) was amplified with primers O388 and O389. The PCR fragment was agarose gel purified and transformed into yMK1329 for histidine prototroph selection. Yeast genomic PCR was conducted using primers O390 and O391 (derived from the MCD1
locus) against O392 or O393 (derived from the His3MX6 sequence) for verification. The correct integration of the GAL
promoter into the MCD1
5′ untranslated region was further verified by the inability of cells to grow in the presence of glucose.
To introduce the G44S mutation into a green fluorescent protein (GFP)-marked strain, SBY214, the (hht1-hhf1)Δ::KanMX allele from JHY200 was PCR amplified using primers O396 and O397 and transformed into SBY214 to knock out HHT1-HHF1 (creating yJL118). Correct integration was verified by genomic PCR using primers O398, O399 (from the HHT1/HHF1 locus), and mk133 (from KanMX). To replace the remaining copy of H3 and H4, i.e., HHF2-HHT2, with either wild-type (WT) H3 or the G44S mutant with the URA3 selective marker, plasmid pMK622 (WT or G44S mutant) was cleaved with SnaBI and EcoRI for integrative transformation. Ura+ colonies were isolated for genomic PCR to verify the integration. Genomic PCR using primers O404 and O405 was done to amplify an HHT2 fragment for ScaI digestion (indicative of the G44S mutation) and sequencing to rule out the existence of any unwanted mutations. To create congenic strains that differ only at the HHT2 locus, both the WT and G44S mutant versions of pMK622 were digested for yJL118 transformation, resulting in yJL292 (HHT2-HHT2::URA3-KTR5) and yJL293 (hht2 G44S-HHF2::bpURA3-KTR5).
The yeast chromosomal copy of HHT2
was mutated by integrating pMK622 bearing the desired mutation. To create pMK622, pMK621 was first generated by PCR to amplify from yeast genomic DNA part of HHF2
with primers O400 and O401. The PCR fragment was digested with EcoRI and SphI and cloned into the same sites of pJJ244 (18
). pMK622 was made by inserting the SpeI (blunted) and AatII fragment from pQQ18 bearing the G44S mutation into the NarI (blunted) and AatII sites of pMK621, resulting in pMK622 with two 140-bp direct repeats spanning the URA3
Histone mutations were generated by two-step PCR site-directed mutagenesis. Briefly, the desired mutations were incorporated into two complementary oligonucleotides, and each was used for PCR against O17 (downstream of the HHT2 open reading frame [ORF]) or O19 (downstream of the HHF2 ORF) that hybridized outside the HHT2/HHF2 genes in pQQ18. The two PCR fragments (amplified using pQQ18 as the template by Pfu Turbo DNA polymerase [Stratagene]) were agarose gel purified and subjected to a second round of PCR. The complementary sequence at the 3′ ends of these two PCR fragments allowed annealing and extension. Primers O17 and O19 were included in the reaction mixture to exponentially amplify the entire HHT2/HHF2 gene with the mutation. The final PCR fragments were then digested with SalI and SpeI and used to replace the WT SalI/SpeI sequence in pQQ18. The entire H3 and H4 genes were sequenced for verification. This second-step PCR usually did not work well with Pfu Turbo polymerase. Taq polymerase was used to circumvent this problem. However, Taq polymerase frequently introduced unwanted mutations that had to be revealed by sequencing and, if detected, set aside. The original G44S mutation was obtain fortuitously in this manner.
pMK573, a 2μm URA3 SGO1
plasmid, was created by PCR amplifying the SGO1
ORF from yeast genomic DNA with primers O329 and O330 that included 42 bp of homology to the vector pMK572 (see below) at the 5′ ends. The PCR fragment was cotransformed with HindIII- and EcoRI-digested pMK572 into yeast strain yMK839 (32
colonies were subjected to DNA isolation and bacterial transformation. Miniprep DNA was analyzed by restriction digestion and sequencing across the insertion junctions for confirmation of a correct insert.
To create pMK572 (a multicloning sequence flanked by the ADH1
promoter and terminator), ADH-Ras-ΔBamHI (4
) was digested with SmaI and self-ligated to remove the Ras sequence. The resultant plasmid, pMK322, then was used as the template for a PCR using primers O327 and O328 to amplify the ADH1
sequence and the multicloning sites. The PCR fragment was cotransformed with HindIII- and EcoRI-digested YEplac195 (a 2μm URA3
]), resulting in pMK572. The multicloning sequence contains unique HindIII, SmaI, SalI, BssHII, MluI, SacI, NotI, EagI, SfiI, BalI, and EcoRI restriction sites.
To create a construct for glutathione S-transferase (GST)-HA-Sgo1p in Escherichia coli, the SGO1 ORF was PCR amplified with primers OJL25 and OJL26. The PCR fragment obtained was digested with NotI and ligated to NotI-linearized pMK595, resulting in in-frame fusion of 3×HA and SGO1 (pJL51). To further generate a GST fusion of HA-SGO1 for bacterial production, 3×HA-SGO1 was isolated from pJL51 and inserted into pSP72 (Promega) at the HindIII and XhoI sites. The BamHI-XhoI fragment was then isolated and ligated to the BamHI and XhoI sites of pGEX4T-1, generating pJL55.
Yeast growth media, conditions, and transformation were based on standard procedures (49
). When appropriate, a 5% concentration of Casamino Acids (CAA) was used to substitute for synthetic amino acid mixtures as a selective medium for a uracil, tryptophan, or adenine prototroph. Yeast transformation was done by the lithium acetate method (9
Chromosome stability of the WT and G44S mutant strains was examined according to Spencer et al. (51
), using plasmid pYCF1/CEN3.L cut with BglII and transformed into yMK1243 and yJL145. Ura+
transformants were grown in CAA-Ura medium overnight and then plated directly onto yeast extract-peptone-dextrose (YPD) plates to allow colony formation and scoring. A tension-sensing test using the pGAL
mutant strains was done according precisely to reference 15
, using strains yJL171 and yJL172.
For Western analyses of yeast proteins, yeast extracts were prepared by directly boiling cell pellets in appropriate volumes of 2× sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) loading dye for 5 min, followed by vortexing with 1 lysate volume of glass beads (0.45 μm in diameter; Sigma) at room temperature for 5 min. The mixtures were boiled again for 5 min and then centrifuged at 14,000 × g at room temperature for 1 min. The supernatant was transferred to another tube for SDS-PAGE.
Recombinant Sgo1p preparation.
To express and purify GST-HA-Sgo1p from E. coli, 125 ml of BL21 codon+ cells (Stratagene) were subjected to induction (optical density at 600 nm of 0.5 to 0.6 in LB-ampicillin medium) with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 37°C for 4 h. Cells were pelleted (5,000 × g for 5 min) at 4°C and sonicated in 5 ml of HEMGT buffer (25 mM HEPES, pH 7.9, 12.5 mM MgCl2, 150 mM KCl, 0.1 mM EDTA, 0.1% Tween 20, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) six times for 20 s each time with 1-min chilling intervals. The soluble fraction was obtained by centrifugation at 10,000 × g for 15 min at 4°C. Sgo1p was purified by incubating the cytosol with 200 μl of reduced glutathione Sepharose 4B beads (Amersham) at 4°C for 1 h. Bound Sgo1p was washed twice with 5 ml of binding buffer for 5 min each time, followed by another wash with 1.5 ml of binding buffer, and transferred to a microcentrifuge tube. The elution was done by gently rocking beads in 200 μl of 50 mM glutathione in the binding buffer for 30 min at 4°C. Eluate was collected, and the elution was repeated once under the same conditions. Two batches of eluate were separately dispensed and stored at −70°C. The yield and purity of GST-HA-Sgo1p were estimated by SDS-PAGE.