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Cloning of a Cdna Encoding Human Centrin, an EF-Hand Protein of Centrosomes and Mitotic Spindle Poles

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Cloning of a Cdna Encoding Human Centrin, an EF-Hand Protein of Centrosomes and Mitotic Spindle Poles Journal of Cell Science 107, 9-16 (1994) 9 Printed in Great Britain © The Company of Biologists Limited 1994 Cloning of a cDNA encoding human centrin, an EF-hand protein of centrosomes and mitotic spindle poles Ramesh Errabolu, Mark A. Sanders and Jeffrey L. Salisbury* Laboratory for Cell Biology, Department of Biochemistry and Molecular Biology, Mayo Clinic Foundation, Rochester, Minnesota 55905, USA *Author for correspondence SUMMARY A human cDNA expression library was screened using human centrin is a protein of ~20,000 Mr as predicted from anti-centrin antibodies to obtain a cDNA clone encoding the cDNA clone. Indirect immunofluorescence analysis of human centrin. The cDNA clone contains an open reading HeLa cells demonstrates that centrin is localized at the cen- frame of 516 base pairs and predicts a product of 172 trosome of interphase cells and that it redistributes to the amino acids with a calculated molecular mass of 19,528 and region of the spindle poles during mitosis. When taken a pI of 4.61. Sequence analysis demonstrates that human together with earlier genetic studies, these results demon- centrin and centrins from higher plants, protozoa, algae, strate that centrin is a ubiquitous component of centro- Xenopus and the yeast CDC31 gene product are closely somes and mitotic spindle poles of diverse organisms and related members of a subfamily of the EF-hand superfam- suggest that centrin plays a role in centrosome separation ily of calcium-binding proteins. The human centrin at the time of mitosis. sequence has four putative calcium-binding domains as defined by the EF-hand consensus. Immunoprecipitation Key words: centrosome, centriole, centrin, EF-hand protein, and western blot studies from HeLa cells confirm that microtubule, mitosis INTRODUCTION eukaryotes have demonstrated that centrin plays important roles in the determination of centrosome position and segre- The centrosome plays a key role in the organization of cyto- gation, and in the process of microtubule severing (McFadden plasmic microtubules, in the determination of cell polarity, and et al., 1987; Salisbury et al., 1988; Sanders and Salisbury, in the establishment of the bipolar mitotic spindle (Kupfer et 1989; Schulze et al., 1987). al., 1982; McIntosh and Koonce, 1989; Mitchison and Kirschner, 1984). During the cell cycle the centrosome dupli- cates, and at prophase the duplicated centrosomes separate into MATERIALS AND METHODS the nascent poles of the forming mitotic spindle (Rieder and Borisy, 1982). Mitotic spindle poles nucleate a larger number Cell culture of microtubules that show relatively rapid turnover when HeLa cells (American Type Culture Collection, Rockville, MD) were compared to the microtubules of interphase centrosomes grown in bicarbonate-buffered modified Eagle’s medium (MEM), pH (Salmon et al., 1988; Saxton et al., 1984; Snyder and McIntosh, 7.3, supplemented with 10% fetal bovine serum at 37˚C and 5% CO2 in air. All culture reagents were purchased from Gibco Laboratories 1975). Centrosome dynamics during the cell cycle are thought (Grand Island, NY). to be influenced by changes in the activity of the principle regulator of cell cycle progression, p34cdc2 kinase (Alfa et al., Isolation of cDNA clones encoding human centrin 1990; Bailly et al., 1989; Riabowol et al., 1989), and through A human testis cDNA expression library in λgt11 (HL 1010b, the action of γ-tubulin, a centrosome-specific tubulin isotype Clontech Laboratories, Palo Alto, CA), was screened with anti-centrin involved in microtubule nucleation (Bass and Joshi, 1992; polyclonal serum 26/14-1 according to the procedures outlined by Joshi et al., 1992; Oakley and Oakley, 1989; Oakley et al., Sambrook and coworkers (1989). λ particles (105 pfu/plate) were r− 1990). Biochemical and genetic studies suggest that the cen- plated with E. coli Y 1090 cells onto 150 mm plates and induced trosome’s protein composition is complex (Bornens et al., with IPTG. The induced plaques were lifted onto nitrocellulose membranes (HATF type HA, Millipore Corp., Bedford, MA) and 1987; Lux and Dutcher, 1991). Here, we identify and sequence screened for reaction against anti-centrin polyclonal serum 26/14-1 a cDNA clone encoding human centrin and demonstrate that using alkaline phospatase-conjugated secondary antibodies and the centrin is a component of centrosomes and mitotic spindle reaction procedures described below for western blots. Screening of poles in cultured human cells. Previous studies in lower fifty filters resulted in the identification of two positive plaques, which 10 R. Errabolu, M. A. Sanders and J. L. Salisbury were plaque-purified through seven additional screenings. These two PWB (three changes, 5 minutes each), and immediately blocked to cDNA clones proved to contain identical inserts and were also eliminate nonspecific binding of antibodies in blocking buffer, which positive for reaction with monoclonal anti-centrin antibody 20H5. consisted of 5% FBS, 5% glycerin, 0.04% sodium azide in PBS (10 Sequence analysis demonstrated an open reading frame encoding the mM KH2PO4 and K2HPO4, 150 mM NaCl), pH 7.2, for 1 hour at complete human centrin sequence. The approximately 1100 bp insert 37˚C. After blocking all specimens were triple-labeled with mono- was transferred from λgt11 into pBluescript SK+ (Stratagene Cloning clonal anti-α-tubulin (Sigma Chem. Co.) antibody, polyclonal anti- Sys., La Jolla, CA) and the recombinant plasmid psk+:333 was used centrin (26/14-1) antiserum, and the DNA-binding fluorochrome 4,6- for sequencing of both strands of the cDNA according to Sanger’s diamidino-2-phenylindole (DAPI; Sigma Chem. Co.). Coverslips dideoxy chain termination method (Sanger et al., 1977) using a were: (1) incubated in rabbit anti-centrin immune serum 26/14-1 Sequenase sequencing kit (United States Biochem. Corp., (1:6000 dilution) overnight at 37˚C, washed with PBS; (2) incubated Cleveland, OH). in mouse monoclonal anti-tubulin ascites fluid (1:500 dilution; Sigma Chem. Co.) for 4 hours at 37˚C, washed with PBS; (3) incubated in Sequence analysis secondary antibodies (goat anti-mouse fluorescein-conjugated IgG DNA and protein sequence analysis was performed using the and goat anti-rabbit rhodamine-conjugated IgG; 1:1000 dilution of sequence analysis software package GCG (University of Wisconsin). each; Caltag Laboratories, San Francisco, CA) for 4 hours at 37˚C, The Motif routine was used to search for consensus patterns defined washed with PBS, rinsed with deionized water to remove salts, and in the Prosite Dictionary of Protein Sites and Patterns. The multiple finally, mounted with Fluoroseal mountant (Microfluor Ltd, Stony sequence alignment routine, Pileup, was used to determine cluster Brook, NY) containing 0.1 µg/ml DAPI to label DNA. relationships among the sequences of interest and to construct den- Micrographs were taken on a Nikon Microphot FXA microscope dograms representing cluster relationships. Sequences for comparison (Nikon Inc., Instrument Div., Garden City, NY) equipped with epi- were obtained from the GenBank sequence data bank (cited in refer- fluorescence illumination using a ×60, 1.4 NA, oil immersion ences) and from Dr T. Stearns (personal communication) for Xenopus objective. Images were recorded on HyperTech film (Microfluor Ltd, centrin and Dr C. Fulton and Y. Levy (personal communication) for Stony Brook, NY) and developed with D-19 for 6 minutes at 20˚C. Naegleria centrin. Immunoprecipitation and western blots A HeLa culture grown in a 100 mm Petri dish was harvested by RESULTS scraping and washing cells in PBS. After centrifugation in a microfuge tube, the cell pellet was lysed in 0.1 ml lysis buffer con- Isolation and sequence of a cDNA clone encoding taining 2.5% SDS, 0.5% deoxycholate, and 0.5% NP40 with brief son- human centrin ication. The cell lysate was diluted with 9 volumes of immunopre- A cDNA clone was obtained by screening 1×106 recombinants cipitation buffer containing 190 mM NaCl, 50 mM Tris HCL (pH from a human testis cDNA λgt11 expression library with anti- 7.4), 6 mM EDTA, and 2.5% NP40 and then clarified by spinning in centrin antibodies raised against bacterially expressed Chlamy- a microfuge for 3 minutes. Anti-centrin serum 26/14-1 (6 µl) was added to the supernatant and allowed to incubate at 4˚C overnight with domonas centrin. Two positive cDNA clones were identified gentle rocking. The preparation was then treated with 30 µl of Protein by reaction with a polyclonal anti-centrin serum (26/14-1). A/G-Sepharose (Pharmacia Biotech Inc, Piscataway, NJ) for one hour These clones were also shown to react with monoclonal anti- at 4˚C, and the immune complexes collected by centrifugation. After centrin antibody (20H5). Sequencing of the cDNA clones SDS-PAGE according to the standard methods described by Laemmli proved them to be identical. An approximately 1170 base pair (1970), the gels were analyzed by western blotting according to Hulen insert, designated Hcen-1, was restriction mapped and and coworkers (1991). The gels were soaked for 15 minutes in 25 mM sequenced, and found to contain an open reading frame of 516 KH2HPO4 buffer, pH 7.0 (KP buffer), followed by transfer to bases beginning with an initiation codon (ATG) starting at bp Immobilon P membranes (Millipore Corp.) in KP buffer at 20 V ′ overnight at 4˚C using a Hoeffer TE Transphor unit (Hoeffer Sci. 49 from the 5 end of the cDNA insert. The complete Instr., San Francisco, CA). After transfer, the membranes were nucleotide sequence of the cDNA insert and the deduced amino incubated for 45 minutes at room temperature in 0.2% (v/v) glu- acid sequence of the encoded protein are shown in Fig. 1. The taraldehyde freshly prepared in KP buffer and then blocked in 5% putative methionine initiation codon is preceded at position, nonfat dry milk (Carnation Co., Los Angeles, CA) in TBS for 1 hour −3 (3 nucleotides upstream of the ATG) by an A residue.
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