464 The Journal of Cell Biology | Volume 156, Number 3, 2002 cause 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-TR–based MPS1 mutagenized li- brary. 56% (17,920) of the TRP transformants replica plated to 5-fluoro- orotic acid were viable. These mps1 alleles were tested for conditional growth at 36 C, benomyl (10 g/ml; DuPont) sensitivity, and cyclohexim- ide (5 g/ml; Sigma-Aldrich) sensitivity. We isolated mps1-8 as a condi- tional mutation (benomylR and cycloheximideR). We sequenced the mu- tagenized region of mps1-8 using primers MPS1AC2, MPS1X, MPS1Y, and MPS1Z. Sequencing was done by the MCD Biology departmental se- quencing facility (ABI automated sequencer). The mps1-8 allele was integrated at either the URA3 or the LEU2 locus. pRS306–mps1-8 was linearized with NcoI to direct integration at the URA3 gene, and pRS305–mps1-8 was linearized with HpaI to direct inte- gration 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- URA-based yeast genomic li- brary (Connelly and Hieter, 1996) and screened 40,000 URA transfor- mants 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 differ- ent clones each containing MPS1 were isolated multiple times. ORFs con- tained 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. We thank John Kilmartin for SPC42 strains and antibody, Sue Biggins for the myc epitope-tagged MPS1 strain and for her support, the Pat O’Farrell lab for anti-GFP antibody, Danni Vinh and Trisha Davis for Spc42p, the Don Cleveland laboratory for affinity purified anti-myc antibody, and Li- Cor Inc. for reagents and technical assistance. We are grateful to Corine Lau, Susan McBratney, Michele Jones, and Sue Jaspersen for critical read- ing of this article, Tom Giddings for advice on EM, and all Winey lab members for helpful discussions. A.R. Castillo was supported by an Indi- vidual National Research Service Award (National Institutes of Health grant GM19566 to A.R. Castillo). This work was also supported by the Na- tional Institute of Health (GM51312 to M. Winey). Submitted: 7 November 2001 Revised: 19 December 2001 Accepted: 19 December 2001 References Adams, I.R., and J.V. Kilmartin. 1999. Localization of core spindle pole body (SPB) components during SPB duplication in Saccharomyces cerevisiae. J. Cell Biol. 145:809–823. Adams, I.R., and J.V. Kilmartin. 2000. Spindle pole body duplication: a model for centrosome duplication? Trends Cell Biol. 10:329–335. Anderson, C.W., P.R. Baum, and R.F. Gesteland. 1973. Processing of adenovi- rus-2 induced proteins. J. Virol. 12:241–252. Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smoth, and K. Struhl. 1997. Current protocols in molecular biology. John Wiley & Sons, Inc., New York. 50338 pp. Baum, P., C. Furlong, and B. Byers. 1986. Yeast gene required for spindle pole body duplication: homology of its product with Ca2 -binding proteins. Proc. Natl. Acad. Sci. USA. 83:5512–5516. Biggins, S., F.F. Severin, N. Bhalla, I. Sassoon, A.A. Hyman, and A.W. Murray. 1999. The conserved protein kinase Ipl1 regulates microtubule binding to kinetochores in budding yeast. Genes Dev. 13:532–544. Bullitt, E., M. Rout, J. Kilmartin, and C. Akey. 1997. The yeast spindle pole body is assembled around a central crystal of Spc42p. Cell. 89:1077–1086. Byers, B. 1981. Multiple roles of the spindle pole bodies in the life cycle of Saccha- romyces cerevisiae. Molecular Genetics of Yeast. 16:119–133. Byers, B., and L. Goetsch. 1974. Duplication of spindle plaques and integration of the yeast cell cycle. Cold Spring Harb. Symp. Quant. Biol. 38:123–131. Byers, B., and L. Goetsch. 1975. Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae. J. Bacterial. 124:511– 523. Chial, H.J., M.P. Rout, T.H. Giddings, and M. Winey. 1998. Saccharomyces cere- visiae Ndc1p is a shared component of nuclear pore complexes and spindle pole bodies. J. Cell Biol. 143:1789–1800. Connelly, C., and P. Hieter. 1996. Budding yeast SKP1 encodes an evolutionarily conserved kinetochore protein required for cell cycle progression. Cell. 86: 275–285. Donaldson, A., and J. Kilmartin. 1996. Spc42p: a phosphorylated component of the Saccharomyces cerevisiae spindle pole body (SPB) with an essential func- tion during SPB duplication. J. Cell Biol. 132:887–901. Flory, M.R., M.J. Moser, R.J. Monnat, Jr., and T.N. Davis. 2000. Identification of a human centrosomal calmodulin-binding protein that shares homology with pericentrin. Proc. Natl. Acad. Sci. USA. 97:5919–5923. Friedman, D.B., J.W. Kern, B.J. Huneycutt, D.B.N. Vinh, D.K. Crawford, E. Steiner, D. Scheilts, J. Yates, III, K.A. Resing, N.G. Ahn, et al. 2001. Mps1p phosphorylates the spindle pole component Spc110p in the N-terminal do- main. J. Biol. Chem. 276:17958–17967. Geiser, J., E. Schott, T. Kingsbury, N. Cole, L. Totis, G. Bhattacharyya, L. He, and A. Hoyt. 1997. Saccharomyces cerevisiae genes required in the absence of the CIN8-encoded spindle motor act in functionally diverse mitotic path- ways. Mol. Biol. Cell. 8:1035–1050. Geissler, S., G. Pereira, A. Spang, M. Knop, S. Soues, J. Kilmartin, and E. Schiebel. 1996. The spindle pole body component Spc98p interacts with the gamma- tubulin-like Tub4p of Saccharomyces cerevisiae at the sites of microtubule at- tachment. EMBO J. 15:3899–3911. Giddings, T.H., Jr., E.T. O’Toole, M. Morphew, D.N. Mastronarde, J.R. McIn- tosh, and W. Winey. 2001. Using rapid freeze and freeze-substitution for preparation of yeast cells for electron microscopy and three-dimensional analysis. Methods Cell Biol. 67:27–42. Hardwick, K., and A. Murray. 1995. Mad1p, a phosphoprotein component of the spindle assembly checkpoint in budding yeast. J. Cell Biol. 131:709–720. Hardwick, K., E. Weiss, F.C. Luca, M. Winey, and A. Murray. 1996. Activation of the budding yeast spindle assembly checkpoint without mitotic spindle dis- ruption. Science. 273:953–956. Hofmann, C., I.M. Cheeseman, B.L. Goode, K.L. Mcdonald, G. Barnes, and D.G. Drubin. 1998. Saccharomyces cerevisiae Duo1p and Dam1p, novel proteins involved in mitotic spindle function. J. Cell Biol. 143:1029–1040. Hutter, K.J., and H.E. Eipel. 1979. Microbial determination by flow cytometry. J. Gen. Microbiol. 113:369–375. Jones, M.H., J.B. Bachant, A.R. Castillo, T.H. Giddings, Jr., and M. Winey. 1999. Yeast Dam1p is required to maintain spindle integrity during mitosis and in- teracts with the Mps1p kinase. Mol. Biol. Cell. 10:2377–2391. Kilmartin, J., S. Dyos, D. Kershaw, and J. Finch. 1993. A spacer protein in the Sac- charomyces cerevisiae spindle pole body whose transcript is cell cycle–regu- lated. J. Cell Biol. 123:1175–1184. Knop, M., and E. Schiebel. 1997. Spc98p and Spc97p of the yeast -tubulin com- plex mediate binding to the spindle pole body via their interaction with Spc110p. EMBO J. 16:6985–6995. Knop, M., G. Pereira, S. Geissler, K. Grein, and E. Schiebel. 1997. The spindle pole body component Spc97p interacts with the gamma-tubulin of Saccha- romyces cerevisiae and functions in microtubule organization and spindle pole body duplication. EMBO J. 16:1550–1564. Kochanski, R., and G. Borisy. 1990. Mode of centriole duplication and distribu- tion. J. Cell Biol. 110:1599–1605. Lauze, E., B. Stoelscker, F.C. Luca, E. Weiss, A. Schutz, and M. Winey. 1995. Yeast spindle pole duplication gene MPS1 encodes an essential dual specific- ity protein kinase. EMBO J. 14:1655–1663. Marshall, I., and K. Wilson. 1997. Nuclear envelop assembly after mitosis. Trends Cell Biol. 997:69–74. Middendorp, S., A. Paoletti, E. Schiebel, and M. Bornens. 1997. Identification of a new mammalian centrin gene, more closely related to Saccharomyces cerevi- siae CDC31 gene. Proc. Natl. Acad. Sci USA. 94:9141–9146. Moritz, M., M. Braufeld, J. Sedat, B. Alberts, and D. Agard. 1995. Microtubule nucleation by -tubulin-containing rings in the centrosome. Nature. 378: 638–640. Munoz-Centeno, M.C., S. McBratney, A. Monterrosa, B. Byers, C. Mann, and M. Winey. 1999. Saccharomyces cerevisiae MPS2 encodes a membrane protein localized at the spindle pole body and the nuclear envelope. Mol. Biol. Cell. 10:2393–2406. O’Farrell, P.H. 1975. High resolution two-dimensional electrophoresis of proteins.