Fava F , Raynaud-Messina B , Leung-Tack J , Mazzolini L , Li M , Guillemot JC , Cachot D , Tollon Y , Ferrara P , Wright M .

	Figure 1. Comparison of the amino acid sequence of human 76p (h76p) with its Drosophila (d75p) and Medicago (m85p) orthologues. The alignments have been performed using both amino acid similarities (L/I/V/W/M, K/R/H, A/T/S/P/G, D/E/Q/N, and F/Y) and the conservation of the expected structural motifs ( Callebaut et al. 1997). Similarities are shown by shaded areas, while bold letters indicate identical amino acids. Consensus amino acids between the three orthologue proteins are underlined. The deduced nucleotide sequences of h76p, d75p, and m85p have been deposited in the GenBank/EMBL/DDBJ databases under accession numbers AJ249677, AJ249678, and AJ249679, respectively.
	Figure 2. Characterization of 76p in the γ-tubulin complexes and in cell extracts. (A) Antibodies against h76p labeled the 76-kD polypeptide band. Pig brain γ-tubulin complexes submitted to SDS-PAGE were either stained with Coomassie blue (15 μg of γ-tubulin) or immunoblotted (50 ng of γ-tubulin) with antibodies R72, R801, R190, and R29/30. All antibodies labeled the polypeptide at ≈76 kD. The specificity of the labeling was controlled by preincubation with the immunizing peptide (+P). (B) Presence of 76p and γ-tubulin in the same protein complexes. Pig brain γ-tubulin complexes (250 ng of γ-tubulin) were immunoprecipitated with immune or preimmune h76p antibodies (R801). The immunoprecipitates (protein A–Sepharose beads, lanes b) and the corresponding supernatants (lanes s, one fifth of the initial volume) were analyzed by SDS-PAGE and immunoblotted with γ-tubulin antibodies (C3). A fraction of γ-tubulin specifically coimmunoprecipitated with 76p. (C and D) Detection and determination of the amount of 76p and γ-tubulin in PtK2 cell extracts (C) and Xenopus oocyte extracts (D). Total extracts (50 μg of proteins) were submitted to SDS-PAGE and immunoblotted with antibodies against 76p (R190 and R801) or against γ-tubulin (C3). The specificity was controlled in the presence of the immunizing peptide (+P) and the amounts of 76p (R190) and γ-tubulin (C3) were assessed by comparison with a range of purified recombinant h76p and Xenopus γ-tubulin. Both extracts contained equivalent amounts of 76p and γ-tubulin.
	Figure 3. Biochemical properties of 76p. (A) 76p cosediment with γ-tubulin in Xenopus γ-TuRCs. An oocyte extract (1 mg of protein) was submitted to a sedimentation in a 5-ml linear 5–40% sucrose gradient (160,000 g for 4 h and 15 min at 0°C). The fractions (0.5 ml) were analyzed by SDS-PAGE and immunoblotting with γ-tubulin (C3) and 76p (R801) antibodies. Both proteins cosediment with an apparent mass corresponding to the γ-TuRCs (the arrow corresponds to the sedimentation of thyroglobulin at 669 kD or 19 S). (B) Presence of 76p in Xenopus γ-TuRCs. The presence of 76p in γ-TuRCs was assessed by the immunoprecipitation of γ-tubulin with x76p. Fractions 4–7 of the sucrose gradient (A) corresponding to γ-TuRCs were immunoprecipitated by immune or preimmune 76p antibodies (R190). The protein A–Sepharose beads (b) and a fraction of the supernatants were analyzed by SDS-PAGE and immunoblotted with γ-tubulin antibodies (C3). Protein A–Sepharose beads carrying preimmune antibodies failed to immunoprecipitate γ-tubulin, while a small fraction of γ-tubulin was recovered linked to the beads carrying the immune antibodies. (C) Determination of the amount of 76p in purified mammalian γ-tubulin complexes. Pig brain γ-tubulin complexes, submitted to SDS-PAGE, were immunoblotted with h76p antibodies (R801), and the amount of 76p was determined by comparison with a range of recombinant h76p. (D) Immunodepletion of a small fraction of γ-tubulin complexes by h76p antibodies. High molecular masses purified pig brain γ-tubulin complexes (M), obtained by centrifugation through a 40% sucrose cushion (≈500 ng of γ-tubulin) and a Xenopus oocyte extract (X, ≈2 mg of protein), were submitted to three successive cycles of immunodepletion with h76p antibodies (beads with R801 and R190 for purified mammalian γ-tubulin complexes and beads with R190 for Xenopus extract). Then the successive protein A–Sepharose beads (lanes b1–b3) and supernatant fractions (lanes s1–s3, 1/10 and 1/25 of the initial volume for mammalian γ-tubulin complexes and Xenopus extracts, respectively) were analyzed by SDS-PAGE and immunoblotting with γ-tubulin antibodies (C3).
	Figure 5. Centrosomal localization of GFP-h76p in mammalian cells. Cells were observed 65 h after transfection with a plasmid expressing the fusion protein GFP-h76p. (A) Presence of GFP-h76p in COS-transfected cells. In control (C) cell extracts (20 μg), no polypeptide bands were detected both by anti–GFP (GFP) and anti–76p antibodies (R801). In transfected cells (T) both antibodies labeled the 103-kD fusion protein. The average expression of GFP-h76p in transfected cell extract (T cells) was determined with R801 antibodies by comparison with a range of recombinant h76p. (B–D) Centrosomal localization of GFP-h76p in COS-transfected cells. In mitotic cells, the GFP-h76p (B and C, upper row) localized at the spindle poles as shown by a coimmunofluorescent staining with α-tubulin antibodies (B, lower row), and γ-tubulin antibodies (C, lower row). In interphase cells, the GFP-h76p (D, upper row) colocalized with γ-tubulin (D, lower row) although the fluorescence raised by GFP-h76p appeared broader than the γ-tubulin staining. The transformed cells exhibited numerous multipolar spindles and showed frequently two diplosomes localized in close proximity in interphase, but controls performed with the vector expressing GFP alone suggested that the increase in centrosome number was a consequence of the transfection treatment rather than because of h76p overexpression. (E–F) Appearance of apoptotic figures in transfected HeLa cells. The transfected cells expressing GFP-h76p (E) exhibited a nuclear fragmentation shown by DAPI staining (F).
	Figure 6. The 76p complexes are necessary for aster formation by permeabilized Xenopus spermatozoa. (A) Presence of 76p in sperm basal bodies after incubation in an oocyte extract. (1) Localization of 76p, labeled by antibodies R801 (black spot shown by the arrow), at the extremity of the sperm nuclei stained with DAPI (white). (2) Colocalization of 76p and (3) γ-tubulin at the extremity of the sperm basal body (arrows) (R801 and R75 antibodies, respectively). (B) Kinetics of aster nucleation by the sperm basal bodies. Depletion of 76p (left) and γ-tubulin (right) were conducted in the same experiment. (diamond) Control, spermatozoa incubated in an untreated Xenopus oocyte extract; (open circle) spermatozoa incubated in extract depleted with h76p (R801) or γ-tubulin (R74) antibodies; (open triangle) spermatozoa incubated in extract depleted with h76p or γ-tubulin antibodies in the presence of the corresponding immunizing peptide (500 μg/ml); (closed circle) spermatozoa incubated in extract depleted with h76p and γ-tubulin preimmune antibodies; (inverted triangle) spermatozoa incubated with antibodies unrelated to Xenopus centrosomal proteins (R82 directed against the carboxy-terminal region of Arabidopsis γ-tubulin); (square) spermatozoa incubated in h76p- or γ-tubulin–depleted extract further complemented with purified pig brain γ-tubulin complexes (corresponding to 2 ng of γ-tubulin and 0.4 ng of 76p), and (closed triangle) spermatozoa incubated in a 76p- or γ-tubulin–depleted extract complemented with an extract containing recombinant h76p (1.5 μg of recombinant h76p). No effects were observed when an extract devoid of recombinant protein was used (not shown). (C) Recruitment kinetics of 76p and γ-tubulin to the sperm basal bodies. Spermatozoa (Sp, 40 μg) were incubated in the oocyte extract in the presence of 5 μM nocodazole. Samples (Sp + E) taken at various times were centrifuged at 4°C for 5 min. 76p and γ-tubulin, recruited to spermatozoa, were revealed after SDS-PAGE by immunoblotting (R801 and R74 antibodies, respectively) and compared with untreated spermatozoa (Sp). (D) Specificity of the recruitment of the 76p and γ-tubulin to the sperm basal bodies. γ-tubulin and 76p were detected by immunoblotting with R74 and R801 antibodies, whereas the polypeptide band at 33 kD, observed after Coomassie blue staining, was used as a standard to check the recovery of sperm heads. (control, lane 1, Sp) Nonincubated spermatozoa (Sp, 40 μg), (lane 2, Sp + E) spermatozoa incubated for 20 min in the oocyte extract (E), (lane 3, Sp + E + IS) spermatozoa incubated in oocyte extract depleted with R801 immune serum (IS), (lane 4, Sp + E + IS + p) spermatozoa incubated in oocyte extract depleted with the R801 immune serum in the presence of the immunizing peptide (p), (lane 5, Sp + E + pIS) spermatozoa incubated in the oocyte extract depleted with R801 preimmune serum (pIS), and (lane 6, E) oocyte extract incubated alone showing that the recruitment is linked to the presence of spermatozoa. (E) Quantification of the amount of 76p in immature and mature sperm basal bodies. Spermatozoa (Sp, 160 μg) and spermatozoa incubated for 20 min in the oocyte extract in the presence of 5 μM nocodazole (Sp + E, 40 μg) were immunoblotted with R801 antibodies and the amount of 76p was assessed by comparison with a range of purified recombinant h76p.
	Figure 7. Comparison of the amino acid sequences of human 76p, 103p (h97p) and 104p (h98p). (A) Regions of homology of the three proteins (shaded area). (B) Sequence comparison in the regions of homology. Identical and homologue amino acids are shown by bold letters and by shaded areas, respectively. Consensus amino acids between h76p, h103p and h104p are underlined. Comparison between Fig. 1 and Fig. 7 shows that the consensus amino acids between h76p, d75p, and m85p and the consensus amino acids between h76p, h103p, and h104p are 18% identical. The alignments have been performed using both the similarities of sequences and the conservation of the expected structural motifs ( Callebaut et al. 1997).

Blow, Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. 1986, Pubmed, Xenbase

Blow, Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. 1986, Pubmed , Xenbase
Bullitt, The yeast spindle pole body is assembled around a central crystal of Spc42p. 1997, Pubmed
Burns, Analysis of the gamma-tubulin sequences: implications for the functional properties of gamma-tubulin. 1995, Pubmed
Byers, Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae. 1975, Pubmed
Callebaut, Deciphering protein sequence information through hydrophobic cluster analysis (HCA): current status and perspectives. 1997, Pubmed
Debec, Polar organization of gamma-tubulin in acentriolar mitotic spindles of Drosophila melanogaster cells. 1995, Pubmed
Detraves, Protein complexes containing gamma-tubulin are present in mammalian brain microtubule protein preparations. 1997, Pubmed , Xenbase
Doxsey, Pericentrin, a highly conserved centrosome protein involved in microtubule organization. 1994, Pubmed , Xenbase
Erickson, Protofilaments and rings, two conformations of the tubulin family conserved from bacterial FtsZ to alpha/beta and gamma tubulin. 1996, Pubmed
Félix, Centrosome assembly in vitro: role of gamma-tubulin recruitment in Xenopus sperm aster formation. 1994, Pubmed , Xenbase
Geissler, The spindle pole body component Spc98p interacts with the gamma-tubulin-like Tub4p of Saccharomyces cerevisiae at the sites of microtubule attachment. 1996, Pubmed
Horio, The fission yeast gamma-tubulin is essential for mitosis and is localized at microtubule organizing centers. 1991, Pubmed
Hyman, Preparation of modified tubulins. 1991, Pubmed
Joshi, Gamma-tubulin is a centrosomal protein required for cell cycle-dependent microtubule nucleation. 1992, Pubmed
Joshi, gamma-Tubulin and microtubule organization in plants. 1996, Pubmed
Julian, gamma-Tubulin participates in the formation of the midbody during cytokinesis in mammalian cells. 1993, Pubmed , Xenbase
Kilmartin, Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. 1982, Pubmed
Knop, The spindle pole body component Spc97p interacts with the gamma-tubulin of Saccharomyces cerevisiae and functions in microtubule organization and spindle pole body duplication. 1997, Pubmed
Knop, Receptors determine the cellular localization of a gamma-tubulin complex and thereby the site of microtubule formation. 1998, Pubmed
Knop, Spc98p and Spc97p of the yeast gamma-tubulin complex mediate binding to the spindle pole body via their interaction with Spc110p. 1997, Pubmed
Lajoie-Mazenc, Recruitment of antigenic gamma-tubulin during mitosis in animal cells: presence of gamma-tubulin in the mitotic spindle. 1994, Pubmed , Xenbase
Li, gamma-tubulin is a minus end-specific microtubule binding protein. 1995, Pubmed
Liu, gamma-Tubulin in Arabidopsis: gene sequence, immunoblot, and immunofluorescence studies. 1994, Pubmed
Marschall, Analysis of Tub4p, a yeast gamma-tubulin-like protein: implications for microtubule-organizing center function. 1996, Pubmed
Martin, Xgrip109: a gamma tubulin-associated protein with an essential role in gamma tubulin ring complex (gammaTuRC) assembly and centrosome function. 1998, Pubmed , Xenbase
Melki, Chaperonin-mediated folding of vertebrate actin-related protein and gamma-tubulin. 1993, Pubmed
Morin, The proteolysis-dependent metaphase to anaphase transition: calcium/calmodulin-dependent protein kinase II mediates onset of anaphase in extracts prepared from unfertilized Xenopus eggs. 1994, Pubmed , Xenbase
Moritz, Microtubule nucleation by gamma-tubulin-containing rings in the centrosome. 1995, Pubmed
Moudjou, gamma-Tubulin in mammalian cells: the centrosomal and the cytosolic forms. 1996, Pubmed
Murphy, The mammalian gamma-tubulin complex contains homologues of the yeast spindle pole body components spc97p and spc98p. 1998, Pubmed , Xenbase
Murray, Cyclin synthesis drives the early embryonic cell cycle. 1989, Pubmed , Xenbase
Nguyen, A genetic analysis of interactions with Spc110p reveals distinct functions of Spc97p and Spc98p, components of the yeast gamma-tubulin complex. 1998, Pubmed
Oakley, Identification of gamma-tubulin, a new member of the tubulin superfamily encoded by mipA gene of Aspergillus nidulans. 1989, Pubmed
Oakley, Gamma-tubulin is a component of the spindle pole body that is essential for microtubule function in Aspergillus nidulans. 1990, Pubmed
Oegema, Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules. 1999, Pubmed , Xenbase
Pereira, Spc98p directs the yeast gamma-tubulin complex into the nucleus and is subject to cell cycle-dependent phosphorylation on the nuclear side of the spindle pole body. 1998, Pubmed
RODRIGUEZ, Preparation of a semipermanent mounting medium for fluorescent antibody studies. 1960, Pubmed
Sagliocco, Identification of proteins of the yeast protein map using genetically manipulated strains and peptide-mass fingerprinting. 1996, Pubmed
Shu, Gamma-tubulin can both nucleate microtubule assembly and self-assemble into novel tubular structures in mammalian cells. 1995, Pubmed
Spang, gamma-Tubulin-like Tub4p of Saccharomyces cerevisiae is associated with the spindle pole body substructures that organize microtubules and is required for mitotic spindle formation. 1996, Pubmed , Xenbase
Stearns, Gamma-tubulin is a highly conserved component of the centrosome. 1991, Pubmed , Xenbase
Stearns, In vitro reconstitution of centrosome assembly and function: the central role of gamma-tubulin. 1994, Pubmed , Xenbase
Tassin, Characterization of the human homologue of the yeast spc98p and its association with gamma-tubulin. 1998, Pubmed , Xenbase
Tassin, Identification of an Spc110p-related protein in vertebrates. 1997, Pubmed , Xenbase
Zheng, Nucleation of microtubule assembly by a gamma-tubulin-containing ring complex. 1995, Pubmed , Xenbase