Mps1p regulates assembly of Spc42p | Castillo et al. 463 Table I. Yeast strain list Strain SBY650 LPY5 HC12-2b WX257-14c ACY17-16a WX257-8b ACY47-2a DB8WX257-5c ACY71-14b ACY66-4 WX241-3b WX241-17a SMY7-1b WX241-20c ACY54-9b WX241-10c ACY139 ACY140 ACY141 ACY142 JM7 ACY123-10a ACY122-1c JM16 JM43 Genotype MATa,MPS1-13Xmyc::KanMX,ade2-1,bar1-1,can1-100 MATa,ade2-1,can1-100,his3-11,leu2-3,112,trp1-1,ura3-1 MATa,NDC1-3Xmyc,ura3-52,trp1 1,his3 200, his4-539,ade2 MATa,ura3-52,his3 200,leu2-3,112,trp1 1 MATa,mps1 ::KanMX,cin8 ::HIS3,trp1 1 (pURA-MPS1, pLEU-CIN8-cyhs) MAT ,ura3-52,his3 200,leu2-3,112,trp1 1 MATa,mps1 kanMX,ura3-52,his3 200,leu2-3,112,trp1 1 pRS314-MPS1 MATa,mps1 ::HIS3,ura3-52,trp1 1 pRS314-mps1-8 MATa,mps1 ::KanMX,URA3::mps1-8,spc42 ::LEU2,(TRP::SPC42-GFP)3X, leu2-3,112 his3 200,ade2- 101,can1-100/GALpsi ,ssd1-d2 MATa/MAT ,mps1 ::KanMX/mps1 ::KanMX,URA3::mps1-8/URA3::mps1-8,his3 200/his3 200,leu2- 3,112/leu2-3,112,trp1 1/trp1 1 MATa,mps1-1,ura3-52,his3 200,leu2-d MAT ,mps1-1,ura3-52,his3 200,trp1 1 MATa,mps2-1,his3 200,trp1 1,ura3-52,leu2-3,112 MATa,mps1-1,ura3-52,his3 200 MATa,mps1 ::KanMX,URA3::mps1-8,his3 200,leu2-3,112,trp1 1 MATa,mps1-1,ura3-52,his3 200 MAT ,spc42 ::LEU2,ura3-52,trp1 1,his3 200,leu2-3,112 pRS202-SPC42 and pRS313-spc42-10 MAT ,ura3-52,trp1 1,his3 200,leu2-3,112 pRS202-SPC42 and pRS313-spc42-10 MATa,mps1 ::KanMX::mps1-8-TRP1,ura3-52,trp1 1,his3 200,leu2-3,112 pRS202-SPC42 and pRS313- spc42-10 MATa,mps1 ::KanMX::mps1-8-TRP1,spc42 ::LEU2,ura3-52,trp1 1,his3 200,leu2-3,112 pRS202- SPC42 and pRS313-spc42-10 MATa,MPS1-13Xmyc-KanMX, spc42 ::LEU2,TRP::SPC42-GFP(3X) MATa ,mps1-1,TRP::GAL-SPC42-3Xmyc,ura3-1,his3-11,leu2-3 MATa,TRP::GAL-SPC42-3Xmyc,ura3-1,his3-11,leu2-3 MATa/MAT , MPS1-13Xmyc:KAN/MPS1,NDC10/NDC10:HA3:URA3:,leu- trp- his- MAT ,NDC10:HA3:URA3,spc42 :LEU2,TRP1:SPC42-GFP(3X),his- Source S. Bigginsa L. Pillusb H.J. Chialc Jones et al., 1999 This study This study This study Lauze et al. 1995 This study This study This study Schutz et al., 1997 S. McBratneyd Jones et al., 1999 This study Winey et al., 1991 This study This study This study This study This study This study This study This study This study aFred Hutchinson Cancer Research Center, Seattle, WA. bUniversity of California at San Diego, La Jolla, CA. cUniversity of Colorado. dUniversity of Colorado. branes 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 anti–mouse 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 anti–rabbit 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. Iso- electric focusing was performed in glass tubes using 2% pH 4–8 ampho- lines (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 anti–rat 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 anti–rabbit Cy3-con- jugated 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 anti–mouse FITC-conjugated antibody (1:800; Scientific) were used to de- tect Spc42-GFP. A monoclonal mouse anti-myc primary antibody (1:450) (9E10; Santa Cruz Biotechnology, Inc.) and anti–mouse 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 anti–mouse FITC-conjugated antibody (1:800). Fluorescent microscopy was performed using a Leica DMRXA/RF4/V auto- mated 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 Digi- tal 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- l 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 liga- tion 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 be- cause MPS1 is essential, and pLEU-CIN8-cyhs-CEN was maintained be-