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Address correspondence to Mark Winey, MCD Biology/UCB 347, University of Colorado, Boulder, CO 80309-0347. Tel.: (303) 492-3409. Fax: (303) 492-7744. E-mail: mark.winey{at}colorado.edu
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Abstract |
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Key Words: budding yeast; spindle pole body; MPS1; SPC42; protein kinase
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Introduction |
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The SPB duplication pathway has been described through EM analysis of wild-type cells and mutant cells that fail at different stages of SPB duplication (Byers and Goetsch, 1975; Adams and Kilmartin, 1999). Duplication of the SPB occurs in G1 of the cell cycle, beginning with the accumulation of SPB components (the satellite) onto the cytoplasmic face of the half-bridge, a modification of the nuclear envelope distal to the SPB (Byers and Goetsch, 1975). The amorphous satellite appears to develop into a larger ordered structure called the duplication plaque (Adams and Kilmartin, 1999; O'Toole et al., 1999). Immuno-EM analysis of the satellite and duplication plaque show both structures are composed of the core SPB components, Spc29p, Spc94p/Nud1p, Spc42p, and Cnm67p (Adams and Kilmartin, 1999). Assembly of the new SPB is completed when the duplication plaque is inserted into the nuclear envelope and associates with additional SPB components that will make up the inner (nuclear) plaque layers (Adams and Kilmartin, 1999).
The terminal phenotype of various SPB duplication mutants has suggested when the gene products might be required in the process (Byers and Goetsch, 1975; Rose and Fink, 1987; Winey et al., 1991; Schutz et al., 1997; Schutz and Winey, 1998). For example, yeast containing a mutant SPC42 gene fail in SPB duplication after satellite formation (Donaldson and Kilmartin, 1996). The SPC42 gene product forms the electron-dense (two-dimensional crystalline) central layer of the SPB, and Spc42p is found in the duplication intermediates, the satellite, and duplication plaque (Donaldson and Kilmartin, 1996; Bullitt et al., 1997; Adams and Kilmartin, 1999; O'Toole et al., 1999). Unlike mutant alleles of SPC42, different mutant alleles of MPS1, which encodes a dual specificity protein kinase required for SPB duplication, fail at two distinct points in SPB duplication (Winey et al., 1991; Lauze et al., 1995; Schutz and Winey, 1998). This suggests that Mps1p is required for multiple events in SPB duplication.
Mps1p is unusual in that it has a role in the spindle checkpoint and in SPB duplication (Hardwick and Murray, 1995; Weiss and Winey, 1996). Kinetochores that are not attached to microtubules activate the spindle checkpoint (Wang and Burke, 1995; Pangilinan and Spencer, 1996). Failed SPB duplication also triggers this checkpoint, possibly because a monopolar spindle does not nucleate a sufficient number of microtubules to capture all of the kinetochores and cannot produce tension via bipolar spindle attachment (Winey and O'Toole, 2001). The conditional MPS1 mutants isolated thus far are defective in both pathways; therefore, under restrictive conditions MPS1 mutant cells proceed through mitosis with a monopolar spindle, aberrantly segregate their DNA, and rapidly lose viability (Winey et al., 1991; Schutz and Winey, 1998). Although the role of Mps1p in this checkpoint is not yet clearly defined, it requires kinase activity and is probably accomplished through phosphorylation of another checkpoint component, Mad1p (Hardwick et al., 1996).
Previously, we used a mps1-1 strain in genetic screens to identify interactions that would enhance our understanding of the role of Mps1p in SPB duplication and the spindle checkpoint (Schutz et al., 1997; Jones et al., 1999). We identified genes involved in the spindle checkpoint, spindle function, and those involved in stabilizing the Mps1p kinase (Hofmann et al., 1998; Jones et al., 1999). However, we did not identify SPB components. Here, we characterize a novel conditional allele, mps1-8, that is specifically defective in SPB duplication and use this allele in a dosage suppressor screen. We identified SPC42, a gene that encodes an integral SPB component, as a dosage suppressor of the mps1-8 conditional growth defect. We use genetic and biochemical techniques to investigate the interaction between Mps1p and Spc42p, taking advantage of an Spc42p in vivo assembly assay (Donaldson and Kilmartin, 1996) to show that Mps1p is required for Spc42p assembly.
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Results |
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mps1-8 mutants fail in SPB duplication
We have established previously that the essential MPS1 cellular function is in SPB duplication (Weiss and Winey, 1996). To test if the mps1-8 conditional growth defect might reflect this role, we monitored SPB duplication in mps1-8 cells using immunofluorescence microscopy and EM. Asynchronously growing mps1-8 cells were arrested in G1 using the mating pheromone -factor, released to the permissive (25°C) or restrictive (36°C) temperature for 3h, and processed for indirect immunofluorescence. The majority of mps1-8 cells complete SPB (shown in green) duplication at 25°C as expected, but they fail to duplicate their SPB at 36°C (92%, n = 42; Fig. 2, A and B). Instead, these cells show a typical S. cerevisiae mitotic arrest state with large buds in which the single unduplicated SPB is associated with cellular DNA (blue) and a focus of microtubules (red) (Fig. 2 B). By immunofluorescence, the SPB duplication defect in the mps1-8 strain is identical to the SPB defect observed in previously characterized conditional MPS1 mutants (Schutz and Winey, 1998).
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mps1-8 mutants are competent to activate the spindle checkpoint
As mentioned, the previously characterized conditional MPS1 mutants are defective in both SPB duplication and the spindle checkpoint (Winey et al., 1991; Weiss and Winey, 1996; Schutz and Winey, 1998). We show that the mps1-8 mutant fails in SPB duplication at the restrictive temperature. To determine if mps1-8 cells also fail in the spindle checkpoint, we compared the mps1-8 strain with mps1-1, a strain known to be defective in activating the spindle checkpoint, and mps2-1, a strain able to activate the spindle checkpoint with a monopolar spindle (Hardwick et al., 1996; Weiss and Winey, 1996). Asynchronously growing cultures of mps1-8, mps1-1, and mps2-1 cells were synchronized in G1 with -factor, released at 25 or 36°C, and samples for flow cytometry and budding indices were taken after 2 and 3 h. At the restrictive temperature, mps1-8 cells exhibit a mitotic arrest similar to that observed for mps2-1 cells; the majority of the cells have a large budded cell morphology, a G2 DNA content (Fig. 3, A and B), and accumulate hyperphosphorylated forms of the spindle checkpoint protein Mad1p, a molecular marker for spindle checkpoint activation (unpublished data; Hardwick et al., 1996). In contrast, the mps1-1 strain fails to arrest in mitosis when grown at the restrictive temperature and instead accumulates cells that appear aploid or aneuploid by flow cytometry (Fig. 3 C). We conclude that mps1-8 is a novel mutant allele leading to defects in SPB duplication but maintaining a functional spindle checkpoint.
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Mps1p localizes to the SPB and kinetochores
The genetic interaction of MPS1 with a bona fide integral SPB component suggested that Mps1p might localize to SPBs, which we initially tested using immunofluorescence microscopy on whole cells. Asynchronously growing cells containing myc epitope-tagged MPS1 at the MPS1 locus (Mps1p-myc) and SPC42 tagged with green fluorescent protein (GFP) (Spc42-GFP) were analyzed by immunofluorescense (see Materials and methods). We observe a strong signal of Mps1p-myc (red) that partially overlaps Spc42p-GFP (green), primarily in unbudded cells, and diffuse nuclear staining (Fig. 6 A). Similar localization was observed with a ProA-tagged Mps1 protein (Schutz et al., 1997; Steiner, 1998). This Mps1p signal is suggestive of SPB and/or kinetochore localization, since yeast kinetochores are adjacent to the SPB during G1 of the cell cycle (Wigge et al., 1998; Wigge and Kilmartin, 2001).
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Finally, we performed immuno-EM on asynchronously growing strains containing Mps1p-myc. Colloidal gold signal overlapping the SPB in the plane of the nuclear envelope suggests that Mps1p localizes to the Spc42p central plaque region (5 examples in 24 cells examined) (Fig. 6 C). Mps1p signal is also detected at the end of microtubules by immuno-EM as is seen for other kinetochore proteins (Wigge et al., 1998; Wigge and Kilmartin, 2001) (18 examples in 24 cells examined) (Fig. 6 D). This dual localization is consistent with a role for Mps1p in SPB duplication and the spindle checkpoint.
Mps1p and Spc42p physically interact
The genetic interactions between MPS1 and SPC42 and their colocalization at the SPB prompted us to investigate their physical interaction in the cell. Extracts were prepared from cells containing (a) Mps1p-myc, (b) Ndc1p-myc, as a control for nonspecific interaction with the myc epitope, and (c) no tag. These proteins were immunoprecipitated with anti-myc antibody conjugated to agarose beads and resolved using SDS-PAGE. The presence of Mps1p-myc and Ndc1p-myc was detected using an anti-myc antibody (Fig. 7 B, lanes 5 and 6). Polyclonal anti-Spc42p antibody was used in a duplicate Western analysis and detected a band migrating at the expected molecular weight (4651 kD) for Spc42p in the Mps1p immunoprecipitate but not the Ndc1p immunoprecipitate (Fig. 7 A, lanes 1 and 2). Indeed, this band migrates at approximately the same position as Spc42p-myc, isolated from a strain overexpressing Spc42p-myc (Fig. 7 A, lane 4). Thus, Mps1p and Spc42p both localize at the SPB and physically interact with each other.
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Misassembly of Spc42p in MPS1 mutant cells
Overexpression of Spc42p-myc from an inducible promoter results in the lateral expansion of the central layer of the SPB so that organized layers of Spc42p extend from the SPB in all directions and appear to rest on the nuclear envelope (Donaldson and Kilmartin, 1996; Bullitt et al., 1997; O'Toole et al., 1999). This "super plaque" structure appears by EM to be organized into a two-dimensional crystalline lattice identical to what is observed for Spc42p in the central plaque of a normal SPB (Bullitt et al., 1997). Spc42p isolated from both of these structures is phosphorylated; however, it is not yet clear what role phosphorylation of Spc42p might play in their assembly (Donaldson and Kilmartin, 1996). Here, we analyze formation of the "super plaque" upon SPC42 overexpression in MPS1 mutants. The assembly assay was performed in MPS1 and mps1-1 strains at permissive (25°C) and restrictive temperatures (30 and 34°C) for mps1-1. These strains also contained a myc epitope-tagged version of SPC42 under the control of a galactose-inducible promoter (GAL-SPC42-myc).
Proper formation of the "super plaque" was first analyzed using immunofluorescence. The MPS1 GAL-SPC42-myc and mps1-1 GAL-SPC42-myc strains were grown in noninducing media (no galactose), arrested in G1 using -factor, and then released into galactose-containing media at both 25 and 30°C for 3 h to induce expression of GAL-SPC42-myc. At the permissive temperature, the mps1-1 (Fig. 9 A) and MPS1 (unpublished data) strains form Spc42p-mycdependent dome-like structures (super plaque in green) that are associated with DNA (blue) and microtubule (red) signal. The "super plaque" formed in the wild-type strain at 30°C is identical to what we observe for this strain at 25°C (Fig. 9 C). By contrast, in the mps1-1 strain at 30°C we observe a decrease in Spc42p-myc signal at the SPB, and it has a much less organized appearance (Fig. 9 E). To characterize these structures at higher resolution, we analyzed them using EM (see Materials and methods).
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Discussion |
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mps1-8 is a unique allele
Mutations in the noncatalytic region of MPS1 generated a conditional mutant, mps1-8, that is specifically defective in SPB duplication. We targeted the noncatalytic region of MPS1 for mutagenesis, believing that this region might confer spatial or temporal regulation of Mps1p for each of its roles. In fact, our screen also identified a MPS1 allele that appears to be specifically defective in the spindle checkpoint (to be described elsewhere). In this paper, we show that the molecular defect associated with mps1-8 is likely distinct from our previously characterized conditional MPS1 mutants. Mutations are in the noncatalytic region of mps1-8 and do not affect kinase activity of the mutant protein. By contrast, other conditional MPS1 mutants previously characterized in our lab have mutations in the catalytic region of the gene that affect kinase activity measured in vitro (Schutz and Winey, 1998).
Consistent with the notion that the mps1-8 defect is distinct from other conditional MPS1 mutants, mps1-8 shows unique genetic interactions. We show that mps1-8 but not the other conditional MPS1 mutants is suppressed by an increased dosage of the Mps1p in vitro substrate, Spc42p. By contrast, several of the MPS1 kinase mutants but not mps1-8 are suppressed by an increased dosage of the molecular chaperone CDC37 (Schutz et al., 1997). Although it is likely that CDC37 suppresses through stabilization of the jeopardized Mps1p kinase, it is less clear how SPC42 suppression occurs. We propose that suppression by SPC42 may be through stabilizing an interaction between Spc42p and Mps1-8p. Alternatively, extra Spc42p may help localize Mps1-8p to the SPB or another site of action required for SPB duplication. Future localization studies and coimmunoprecipitation experiments using Mps1-8p should address these questions.
Multiple requirements for Mps1p in SPB duplication
This and previous analyses suggest that Mps1p is required for multiple steps in SPB duplication (Winey et al., 1991; Schutz and Winey, 1998). Here, we show that mps1-8 mutant cells fail very early in SPB duplication when grown at their restrictive temperature. By EM analysis, the unduplicated SPB in mps1-8 cells is similar to those observed in cdc31 and kar1 mutants (Baum et al., 1986; Rose et al., 1986). The unduplicated SPB of mps1-8 does not have an extended half-bridge that is characteristic of several other MPS1 mutants (Winey et al., 1991; Schutz and Winey, 1998). This suggests that there are two distinct requirements for Mps1p early in SPB duplication. Alternatively, the functions defined by mps1-8 and the other conditional MPS1 mutants are not mutually exclusive; proper spatial regulation of Mps1p and Mps1p kinase activity may both be required for its early SPB function. Presumably, the MPS1 kinase domain mutants proceed further in SPB duplication because the mutant proteins localize properly or make appropriate physical interactions, though they are unable to function; the extended half-bridge might be a result of failed attempts to initiate SPB duplication.
MPS1 also functions late in SPB duplication. mps1-737 mutants assemble the central and outer layers of the new SPB but like ndc1, mps2, and bbp1 mutants fail to insert this structure into the nuclear envelope (Winey et al., 1991, 1993; Schramm et al., 2000). Whereas these proteins may function to insert the duplication plaque into the nuclear envelope by providing an opening in the nuclear envelope, it is unclear how Mps1p is involved in this process (Chial et al., 1998; Munoz-Centeno et al., 1999; Schramm et al., 2000). One possibility is that Mps1p facilitates insertion of the duplication plaque into the nuclear envelope by controlling assembly of the inner plaque, a structure not formed in the mps1-737 mutant. Two of the six proteins that make up the inner plaque, Spc110p and Spc98p, are substrates for Mps1p in vitro, and their phosphorylation is dependent on Mps1p in vivo (Pereira et al., 1998; Friedman et al., 2001). The mps1-737 mutant phenotype might reflect a disrupted interaction with Spc110p and Spc98p or the inability of Mps1-737p to phosphorylate Spc110p and Spc98p. The multiple MPS1 mutant phenotypes are consistent with Mps1p, playing a regulatory role in SPB duplication.
Mps1p is required for Spc42p assembly
We investigated the role of Mps1p as a regulator of SPB assembly using a Spc42p in vivo assembly assay. Overexpression of Spc42p at very high levels causes the central plaque of the SPB (normal site of Spc42p localization) to extend laterally and form a structure we call the "super plaque." We believe that Mps1p might function in assembly of the central plaque during SPB duplication based on MPS1 mutant phenotypes and because Mps1p physically interacts with and colocalizes with Spc42p in vivo and regulates Spc42p phosphorylation. When Spc42p was overexpressed in a mps1-1 mutant at restrictive temperatures, we observed a defect in assembly of the "super plaque." This structure is not vertically confined to the two-dimensional crystalline lattice typically seen in MPS1 cells overexpressing SPC42 (Donaldson and Kilmartin, 1996; Adams and Kilmartin, 1999). A similar structure is detected when spc42-S:A, a mutant allele in which 34 serines have been mutated to alanines, is overexpressed at similar levels (Adams and Kilmartin, 1999). Since spc42-S:A is functional at endogenous levels, we believe this common overexpression phenotype reveals the importance of phosphorylation for proper plaque assembly that apparently results in different phenotypes when some phosphorylation is lost (the spc42-S:A allele with threonines intact) versus when most phosphorylation is lost (mutations in the kinase) (Adams and Kilmartin, 1999). In fact, Mps1-1p is severely compromised for kinase activity, and the mps1-1 mutant accumulates fewer phosphorylated forms of Spc42p. We suspect that the phenotype we observe results from the inability of Mps1-1p to properly phosphorylate Spc42p. Failure of Mps1-1p to phosphorylate Spc42p during SPB duplication should then prevent assembly of the central plaque of the new SPB; consistent with this prediction, mps1-1 cells do not show accumulation of satellite material at the half-bridge (Winey et al., 1991).
Is Mps1p function required for assembly of other satellite components? We suspect that Mps1p is required indirectly for assembly of Nud1p, Cnm67p, and Spc29p; Mps1p is required for assembly of Spc42p that may serve as a scaffold upon which the other satellite components assemble. This is supported by the unique suppression of mps1-8 by increased dosage of SPC42. NUD1, CNM67, and SPC29 were not identified in our screen as dosage suppressors of mps1-8 or earlier as dosage suppressors of the mps1-1 (Schutz et al., 1997). We tested SPC29 directly and verified that increased dosage of SPC29 does not suppress mps1-8 (unpublished data). This does not preclude that the function of Mps1p is required for assembly of the other satellite components; the development of assembly assays for Nud1p, Cnm67p, and Spc29p will allow for investigation of the requirement for Mps1p in assembling these components. Regardless of other requirements for Mps1p in SPB duplication, the specific interaction established between Mps1p and Spc42p in this analysis indicates that their association is essential for assembly of the central plaque, an event that may be critical for launching SPB duplication.
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Materials and methods |
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We determined relative specific activity for each fusion protein by using the Storm860 phosphorImager and ImageQuaNT analysis package to measure 32P incorporation and by using the Odyssey Infrared Imager and analysis software to measure protein amounts. The number representing 32P incorporation was divided by the number representing protein amount to yield relative specific activity.
In the coimmunoprecipitate experiment, the immunoprecipitated material was resolved on an 8.5% laemmli SDS-PAGE gel (Ausubel et al., 1997). Gels were subjected to electrophoretic transfer onto a polyvinylidene difluoride membrane (Millipore). Polyvinylidene difluoride membranes were blocked as in Chial et al. (1998). Mps1p-13Xmyc and Ndc1p-3Xmyc were detected using an anti-myc primary antibody (1:1,000; Santa Cruz Biotechnology, Inc.) and a sheep antimouse antibody conjugated to HRP (1:10,000; Sigma-Aldrich). Spc42p was detected using the anti-Spc42p polyclonal primary antibody (a gift from John Kilmartin, Medical Research Council Laboratory of Molecular Biology, London, UK) and a donkey antirabbit antibody conjugated to HRP (1:10,000; Sigma-Aldrich).
Alkaline phosphatase treatment of samples and two-dimensional gel electrophoresis were performed by Kendrick Labs, Inc. as described by Donaldson and Kilmartin (1996) and O'Farrell (1975), respectively. Isoelectric focusing was performed in glass tubes using 2% pH 48 ampholines (BDH ampholines Gallard Schlesinger) for 9,600 V/h. The tube gel was sealed to the top of a stacking gel on top of a 10% acrylamide slab gel. Samples were treated with 10 U of calf intestinal alkaline phosphatase (New England Biolabs, Inc.) at 30°C for 30 min.
Cytological techniques
Flow cytometric analysis of cells was performed as described using the DNA stain propidium iodide (Sigma-Aldrich) (Hutter and Eipel, 1979). Samples were analyzed on a Becton Dickinson FACScan® flow cytometer using CELL QUEST software (Becton Dickinson).
Indirect immunofluorescence was performed as described in Chial et al. (1998), and chromosome spreads were performed as in Biggins et al. (1999). Tubulin was visualized using rat anti-tubulin primary antibody, YOL1/34 (1:150; Accurate Chemical), and antirat antibody conjugated to Texas red (1:400; Scientific). An affinity purified primary polyclonal anti-myc antibody (1:1,000; a gift from the Don W. Cleveland lab, University of California, San Diego, La Jolla, CA) and a donkey antirabbit Cy3-conjugated antibody (1:1,000; Scientific) were used to detect Mps1p-myc. In some experiments, a mouse anti-GFP primary antibody (1:40; a gift from the Pat O'Farrell lab, University of California, San Francisco, CA) and an antimouse FITC-conjugated antibody (1:800; Scientific) were used to detect Spc42-GFP. A monoclonal mouse anti-myc primary antibody (1:450) (9E10; Santa Cruz Biotechnology, Inc.) and antimouse FITC-conjugated antibody (1:800) (Jackson ImmunoResearch Laboratories) were used to detect Spc42p-myc. In chromosome spreads, a mouse anti-HA primary antibody (1:500) was used with an antimouse FITC-conjugated antibody (1:800). Fluorescent microscopy was performed using a Leica DMRXA/RF4/V automated microscope with a Cooke SensiCam digital camera and Slidebook software (Intelligent Imaging Innovations).
EM
Cells for EM were prepared for thin sectioning by high pressure freezing and freeze substitution (Winey et al., 1995) or by chemical fixation (Byers and Goetsch, 1975). Serial thin sections were viewed on a Philips CM10 electron microscope (Philips Electronic Instruments), and images were captured on film or with a Gatan digital camera and viewed with the Digital Micrograph Software package (Gatan Inc.). Immuno-EM was performed using high pressure frozen and freeze-substituted cells as described by Giddings et al. (2001). Myc-tagged Mps1p was detected with polyclonal anti-myc antibody described earlier and 10 nM colloidal gold-conjugated secondary antibodies.
Isolation of new MPS1 allele
Primers MPS1AC3 and MPS1AC4 were used to amplify the NH2-terminus of MPS1 (2.2kb) under mutagenic PCR conditions: dATP = 0.1 mM, MnCl2 = 0.5 mM, and MgCl2 = 1.5 mM. 25, 20-µl PCR reactions were pooled, cut with EcoRI and BamHI, and ligated to EcoRI- and BamHI-digested pRS314-MPS1 to replace the wild-type NH2 terminus. This ligation was used to transform E. coli, and transformants (805) were collected, grown for 2 h in Luria broth at 37°C, and prepared for DNA to make the MPS1 mutagenized library.
We screened the MPS1 mutagenized library in a strain (ACY17-16A) (Table I), mps1::KanMX cin8
::HIS3 pRS316-pac8-1, pLEU2-CIN8-cyhs-CEN. pRS316-pac8-1 (pac8-1 is an allele of MPS1) was maintained because MPS1 is essential, and pLEU-CIN8-cyhs-CEN was maintained because of the lethal interaction between pac8-1 and cin8
::HIS3 (Geiser et al., 1997). mps1
::KanMX cin8
::HIS3 pRS316-pac8-1 plus pLEU-CIN8-cyhs-CEN was transformed with the CEN-TRbased MPS1 mutagenized library. 56% (17,920) of the TRP+ transformants replica plated to 5-fluoroorotic acid were viable. These mps1 alleles were tested for conditional growth at 36°C, benomyl (10 µg/ml; DuPont) sensitivity, and cycloheximide (5 µg/ml; Sigma-Aldrich) sensitivity. We isolated mps1-8 as a conditional mutation (benomylR and cycloheximideR). We sequenced the mutagenized region of mps1-8 using primers MPS1AC2, MPS1X, MPS1Y, and MPS1Z. Sequencing was done by the MCD Biology departmental sequencing facility (ABI automated sequencer).
The mps1-8 allele was integrated at either the URA3 or the LEU2 locus. pRS306mps1-8 was linearized with NcoI to direct integration at the URA3 gene, and pRS305mps1-8 was linearized with HpaI to direct integration at the LEU2 gene. Proper integration at the URA3 and LEU2 loci was verified by PCR, using a primer internal to MPS1 (MPS1AC7), and one within the URA3 gene (ACURA3) or the LEU2 (ACLEU2B).
Dosage suppressor screen
We transformed a mps1-8 strain with a 2-µ URA-based yeast genomic library (Connelly and Hieter, 1996) and screened 40,000 URA+ transformants for growth at 36°C. 86 transformants grew at 36°C. We determined that 60 transformants exhibited plasmid-dependent growth at 36°C and isolated clones as done in Schutz et al. (1997). 22 clones conferred growth to mps1-8 at 36°C upon retransformation. We used restriction digests to determine the number of unique, nonMPS1, clones. Seven clones (S14, S36, S45, S47, S79, S81, S89) with unique genomic inserts and two different clones each containing MPS1 were isolated multiple times. ORFs contained in these clones were identified as described in Jones et al. (1999). S81 contained three ORFs from chromosome XI, YKL044W, PHD1, and SPC42. SPC42 was subcloned into pRS202, and we transformed the mps1-8 strain with this clone and found that it conferred growth to mps1-8 at 36°C. The other clones identified in this screen will be reported elsewhere.
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Footnotes |
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* Abbreviations used in this paper: GFP, green fluorescent protein; GST, glutathione S-transferase; SPB, spindle pole body.
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Acknowledgments |
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Submitted: 7 November 2001
Revised: 19 December 2001
Accepted: 19 December 2001
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References |
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