Cell, Vol. 65, 625-636,

May 31, 1991, Copyright

0 1991 by Cell Press

y=Tubulin Is a Highly Conserved of the Centrosome Tim Stearns, Louise Evans, and Marc Kirschner Department of Biochemistry and Biophysics University of California Medical School San Francisco, California 94143

Summary We have cloned and characterized y-tubulin genes from both X. laevis and S. pombe, and partial genes from maize, diatom, and a budding yeast. The proteins encoded by these genes are very similar to each other and to the original Aspergiilus protein, indicating that y-tubuilnsarean ubiquitous and highly conserved subfamily of the tubulln family. A null m’utatlon of the S. pombe gene is lethal. y-tubulin is a minor protein, present at less than 1% the level of a- and j34ubuiln, and is limited to the centrosome. In particular, y-tubuiin is associated with the pericentrlolar material, the microtubule-nucleating material of the centrosome. y-Tubulin remains associated with the centrosome when microtubules are depolymerized, suggesting that it Is an integral component that might play a role in microtubule organization. introduction The microtubule cytoskeleton of most animal cells is organized by the centrosome. The centrosome has the properties of both nucleating microtubule growth and anchoring the resulting microtubule array. Nucleation occurs with a specific polarity: the slow-growing, or minus, end of the microtubule is attached to the centrosome, and the rapidly growing, or plus, end is distal to the centrosome (Heidemann and McIntosh, 1980). Nucleation also constrains the lattice structure of microtubules to the 19protofilament structure most often seen in vivo (Evans et al., 1985). Concentration of the microtubule-nucleating capacity of the cell in the centrdsome results in a geometrically stable, radial array of microtubules with a specific polarity in interphase, and contributes to the formation of a bipolar spindle in mitosis. Maintaining the position and orientation of themicrotubule array isclearly important forthefunction of the microtubule cytoskeleton, as many of the events in which microtubules are involved are polar in nature, such as the segregation of chromosomes during mitosis and the transport of vesicles and organelles in the axons of neuronal cells. The presence of localized microtubulenucleating material is common to all eukaryotic cells, although the form that it takes is not. In fungi, for example, the microtubule-nucleating material is localized to the spindle pole body, an organelle embedded in the nuclear envelope that is functionally homologous to the centrosome, yet is morphologically quite different. The role of the centrosome in organizing the mitotic and interphase microtubule cytoskeleton has been appreciated for over a century (reviewed in Wilson, 1928), yet

Component

we know little of how it functions. Morphologically, the centrosome consists of a pair of centrioles, themselves composed of microtubules arranged in nine triplets, surrounded by electron-dense, osmiophilic pericentriolar material. It is this ill-defined pericentriolar material that is the site of microtubule nucleation (Gould and Borisy, 1977). Centrioles, though widely distributed in animals, are not essential for maintaining localized microtubule-nucleating material; most plant cells and certain animal cells lack centrioles and yet all have microtubule-organizing centers that share antigenic determinants (Calarco-Gillam et al., 1983; Clayton et al., 1985). The spindle pole body of fungi is a laminar structure embedded in the nuclear envelope that also lacks any structures resembling centrioles (Peterson and Ris, 1978) but does have material that crudely resembles the pericentriolar material on the surfaces from which microtubule growth is nucleated. Because of the divergent morphology, it is still unclear whether microtubule-nucleating material is a conserved component of all eukaryotic cells. If it were, we could combine the strengths of genetics in yeast and cell biology in animal cells to understand how the microtubule cytoskeleton is organized by the centrosome or its equivalent. Very little is known about the composition of centrosomes and spindle pole bodies, though a number of associated antigens have been identified (reviewed in Vandre and Borisy, 1989; Rout and Kilmartin, 1990). Functionally, centrosomes and spindle pole bodies have been purified and shown to nucleate microtubules in vitro (Bornens et al., 1987; Hyams and Borisy, 1978; Mitchison and Kirschner, 1984), but these experiments have revealed little about the mechanisms of nucleation and replication. A genetic approach to microtubule function in AspergilIus nidulans has recently identified a protein that may play a key role in the microtubule-organizing center. Weil et al. (1986) identified the gene mipA as a suppressor of a conditional-lethal p-tubulin mutation. Strikingly, the sequence of the protein encoded by this suppressor was found to be related to both a- and 8-tubulin, leading Oakley and Oakley (1989) to name this protein y-tubulin. The a- and f3-tubulin proteins are approximately 350/o-40% identical to each other (see Little and Seehaus, 1988 for compilation) and form a heterodimer that is the main constituent of microtubules. The A. nidulans y-tubulin is approximately 35% identical to both a- and f3-tubulin, and thus is clearly a distinct member of the tubulin family and not an isotype of either a- or 8-tubulin. More recently, Oakley et al. have shown that y-tubulin is an essential protein in A. nidulans and that it appears to be localized to the spindle pole body of that fungus-not to microtubule structures in general (Oakley et al., 1990). The discovery of y-tubulin raises several important questions. First, isy-tubulin, like a- and 8-tubulin, present in all cells that have microtubule cytoskeletons, or is it a specialized molecule, found only in organisms with spindle pole bodies? Second, if it is universal, is it always associated with the microtubule-organizing center, or might it be a

Cdl 826

mipA

MPREIITIQAGQCGNNVGSQFWQQLCLEHGISQDGNLEEFATEGGD~~FYQSDDTRY

tug1 Xgam

-G-----L-------QI--------------GP--T--S-----”---------------

60

Figure 1. Comparison Schizosaccharomyces, bulin Sequences

mipA tug1 xgam

IPRAILLDLEPRVLNGIQSGPYKNIYNPENFFIGQQGIGAGNNWGAGYAAGEWQEEVFD

120

mipA

MIDREADGSDSLEGFMFLHSIAGGTGSGLGSFLLERMNDRPKKL1QTYSVFPDTQAA.D

179

The mipA, fug7+, and Xgam sequences were manually aligned. Dashes indicate amino acid identity, and dots indicate gaps. The numbers refer to the Aspergillus sequence.

tug1

---------------SL-------------------L---Y---I--------NS-S”S-

-------L-L-----QI-FE--K---A-----PE-I”-------T---------A--E”-

xgam

I--------------VLC-------------y-----L---y----”-------NQDEMS”

mipA

VVVNPYNSLLAMRRLTQNADsvvJLDNAALSRIVADRLHVQEPSFQQTNRLVSTVMSAST

tug1

---Q-------LK---L-------------AH-A-------T-N-T-“-Q-Q----------

xgam

---Q------TLK-------C----T--T--N--AT----I-N---S-I-Q----I-----

mipA

TTLRYPGYMHNDLVGIIASLIPTPRSHFLLTSYTPFTGDNIDQAKT~TTVLD~RRLL

tug1

---------N----S-------S--C-----------NQQ~E--AI-------------

xgam

---------N---I-L---------L--M-G---L-T-QS,.--------------

mipA

QPKNRMVSINPSKSS..CYISILNIIQGERDPTDVHKSLLRIRERLASFIPWGPASIQV

239

299

357

tug1

L---Q---V----K-..-F----D--------A-------------y-------------

Xgam

----V---TGRDRQTNH---A--------V---Q-----Q-----K--N-----------

mipA

417

xgam

ALTKKSPYIQNTHRVSGLMLANHTSVATLFKRIVQQM)RLLEQYKKEAPFQDGL --S------KTN-------------I-S----TLD-------------------I-E-D--SR----LPSA-------M----NISS--E-TCR---K----E-----FR--DI-K-NF

mipA tug1 Xgam

DEFDEARAVVMDLVGEYEAAERENYLDPDAGKDEVGV N---SS-D--A--IN----C-Dp---SL --L-NS-EI-QQ-ID--H--T-PD-ISWGTQDK

454

tug1

component of.functionally different microtubules in cells? If it is limited to the centrosome, does it play a role in microtubule nucleation and/or microtubule anchoring? To ask about the universality of y-tubulin, we report on the use of the polymerase chain reaction (PCR) to clone y-tubulin genes from evolutionarily diverse sources. The Schizosaccharomyces pombe and Xenopus laevis y-tubulin genes are analyzed in detail, and are shown to be highly homologous to the original A. nidulans gene. As to its distribution and function, we demonstrate that y-tubulin is a rare protein compared with a- and f&tubulin, making up less than 1% of total tubulin, and that it is localized exclusively to the centrosome throughout the cell cycle. This centrosomal localization is not dependent on the presence of microtubules. By immune-gold electron microscopy, y-tubulin appears to be a component of the pericentriolar material, the microtubule-nucleating material of the centrosome. These findings have implications for understanding both the structure of tubulin as a protein family and the organization of the microtubule cytoskeleton. Results To determine whether y-tubulin is widespread in eukaryotes, we made use of the ability of PCR to amplify specific sequences using degenerate oligonucleotides as primers (Lee et al., 1988; Wilks, 1989). Four PCR primers were made, corresponding to regions of y-tubulin that are conserved in the Aspergillus sequence (Oakley and Oakley, 1989) and a recently isolated Drosophila y-tubulin sequence (kindly provided prior to publication by B. Oakley). Three of these primers were from regions of y-tubulin that are divergent from a- and f3-tubulin, whereas the fourth, corresponding to the sequence GGTGSG, is conserved in a-, 5-, and y-tubulin. The primers were used in PCRs on DNA templates from various sources. PCR products of

of the Aspergillus, and Xenopus Y-Tu-

approximately the expected size were obtained with one or more of the possible primer combinations from Xenopus cDNA and S. pombe genomic DNA. The Xenopus and S. pombe products were sequenced and found to be y-tubulin gene fragments, based on the sequence homology described below, and were used to clone the full-length genes. We have named the Xenopus gene Xgam, and the S. pombe gene tugl’. The tugl’ gene has six putative introns, each having the sequence elements common to other S. pombe introns. Interestingly, the first four introns in the S. pombe rugl+ gene are in identical positions as introns 2-5 in the A. nidulans y-tubulin gene mipA, although there is no sequence homology within the introns. The Xgam gene encodes a protein of 451 amino acid residues, with a predicted molecular weight of 51,175 daltons. The fugI+ gene encodes a protein of 448 amino a6id residues, with a predicted molecular weight of 49,972 daltons. The mipA, tugl’, and Xgam y-tubulin sequences are compared in Figure 1. The Xenopus and Aspergillus y-tubulin proteins are 87% identical, representative of the sequence identity found between a-tubulins or f3-tubulins from vertebrate and fungal species (Little and Seehaus, 1988). The S. pombe and Aspergillus y-tubulins are 77% identical, also representative of the homology between a- or b-tubulins from these species. The homology between the y-tubulins extends throughout the length of the proteins, but is weakest at the C-terminus, and most of the difference in length between the sequences is due to variation in the C-terminal extensions. More recently, we have used the degenerate y-tubulin PCR primers on templates from more diverse species and have recovered fragments of y-tubulin genes from rat cDNA, maize cDNA, and diatom genomic DNA (T. S., M. K., and Z. Cande, unpublished data). In each case, the level of homology among these y-tubulin sequences is similar to that among a- or t3-tubulins. Thus, y-tubulin

;:;bulin

in the Centrosome

genes have been found in vertebrates, invertebrates (B. Oakley, personal communication), plants, and fungi. This wide distribution suggests that it is universal in eukaryotes. Surprisingly, attempts to isolate ay-tubulin gene from Saccharomyces cerevisiae, using this set of primers and others, repeatedly failed. In PCR experiments on a selection of other budding yeasts, a y-tubulin gene fragment was isolated from Yarrowia lipolytica, a budding yeast that is relatively divergent from S. cerevisiae. The predicted amino acid sequence from this fragment was 67% identical to the Aspergillus sequence in a region in which the Xenopus and Aspergillus proteins are 69% identical. Disruption of the S. pombe fugI+ Gene y-Tubulin is an essential protein in Aspergillus (Oakley et al., 1990). To determine whether y-tubulin is similarly required for the viability of S. pombe cells, we disrupted the coding sequence of the tug7’ gene by homologous recombination with the genomic sequence. A DNA fragment containing the wild-type S. pombe ura4+ gene was inserted into an Sphl site in the middle of the tugl+ coding region. This construction was then cut with restriction enzymes to release a fragment that was used to disrupt one of the chromosomal copies of tugl+ in a ura4-DlBlura4-D18 homozygous diploid strain. Four of four transformants checked by DNA blot hybridization had the disruption construction integrated correctly. Two of these transformants were sporulated and a total of 40 tetrads were dissected. Both transformants yielded only two viable spores, and the viable spore colonies were all Ura-, lacking the tugl::ura4+ allele. This demonstrates that tugl+ is an essential gene in S. pombe. The spores bearing the disruption were able to germinate, but unable to divide, similar to the phenotype of A. nidulans y-tubulin disruptions (Oakley et al., 1990). y-Tubulin Is a Minor Protein Relative to a-Tubulin and P-Tubulin Antibodies against the Xenopus and S. pombe y-tubulin proteins were made by first using the cloned y-tubulin coding sequences to construct bacterially produced fusion proteins. Initial fusions were between fragments of the Xgam and tugl’ genes and the glutathione S-transferase (GST) gene. The resulting GST-Xgam fusion protein and two GST-tug7 fusion proteins(consistingof thetwo largest Ug7+ exons) were used to immunize rabbits. Polyclonal antibodies were also made against the peptide IIDREADGSDSLEGF, corresponding to residues 121-135 of Xgam, a sequence that is conserved in y-tubulins. Antibodies against the GST fusion proteins were affinity purified against different fusion proteins consisting of the bacterial TrpE protein fused to the Xenopus and S. pombe y-tubulin proteins. Figure 2 shows the result of probing total protein extracts from unfertilized Xenopus eggs, XTC cells (an X. laevis cell line), and Chinese hamster ovary (CHO) cells with anti-Xgam antibody. A single band at 50-55 kd is seen in the crude extract lanes. The faint background bands are not specific to the primary antibody. A single band is also seen in S. pombe crude extract probed with anti-tug7 antibody(data not shown). The frog and mammalian y-tubulins

D E

- 31

Figure

2. Crude

Protein

Extracts

Probed

with y-Tubulin

Antibody

Protein samples from Xenopus egg extract (lane A), Xenopus XTC cultured cells (lane B), CHO cells (lane C), and Xenopus tubulin prepared by cycling (lane D) were probed with anti-Xgam antibody. The position of the (x- and p-tubulin bands in the purified tubulin is shown by Coomassie blue staining (lane E). Lanes A-C contained 50 pg of crude protein and lanes D and E contained 1 Kg of purified tubulin. Molecular size markers are in kilodaitons.

differ slightly in mobility. By comparing the signal seen in the crude extracts containing Xenopus y-tubulin with that of known concentrations of TrpE-Xgam fusion protein, it can be estimated that y-tubulin comprises about 0.005% of total protein in XTC cells and about 0.01 O/oin unfertilized frog eggs. The total tubulin concentration in cultured cells and frog eggs is about 2.5% of total protein (Gard and Kirschner, 1967; Hiller and Weber, 1978). As a- and p-tubulin are present in approximately equal amounts, y-tubulin is present at less than 1% the level of either a- or B-tubulin. a- and fl-tubulin can be crudely purified from other soluble proteins by cycles of polymerization and depolymerization driven by heating and cooling. To determine whether r-tubulin copurifies with a- and P-tubulin prepared in this way, soluble protein from frog eggs was subjected to three cycles of polymerization and depolymerization, resulting in a sample in which a- and P-tubulin are the major proteins (Figure 2, lane E). There was no detectable y-tubulin in this purified tubulin (Figure 2, lane D). Interestingly, almost all of the a-, fi-, and y-tubulin, as well as more than half of the total protein, sedimented in the first polymerization. At least half of the a- and P-tubulin could be solubilized from this pellet and polymerized again, whereas all of the y-tubulin remained insoluble. Thus, it is possible to separate a- and P-tubulin from y-tubulin simply by cycling, and this relatively y-tubulin-free tubulin is able to polymerize normally. It is not clear whether the sedimentation of y-tubulin in the first cycle is significant. Though it is possible that v-tubulin forms a cold-stable polymer that remains insoluble, the fact that more than half of all protein sediments in this step suggests that the y-tubulin sedimentation might be nonspecific.

Figure

3. Triple

Labeling

of a-Tub&,

PTubulin,

and DNA in CHO Calls

CHO cells wera fixed and stained with DMla, an anti-a-tubulin antibody (A, D, G. and J). anti-Xgam antibody (B, E, Ii, and K), and DP rPI, a DNA-binding dye (C, F, I, and L). The call in (A-C) is in interphase, the cell in (D-F) is in prophass, the cell in (G-i) is in metaphase, and th, e cell in (J-L) is in late anaphase. Bar, IO pm.

y-Tubulin Is Localized to the Centrosome To determine the location of y-tubulin in cells, we performed immunofluorescence experiments with the antibodies described above. Each of the antibodies made against the Xgam and tug7’ proteins stained a small perinuclear dot in cultured animal cells that could be seen to be the focus of a microtubule aster in double labeling

experiments with anti-tubulin antibody. This staining I pattern is characteristic of the centrosome. All further ex :periments were performed with affinity-purified anti-X :gam antibody. Many cell types were examined by imm fluorescence with these antibodies, including HeLa, 3T3, FtATl, CHO, Pt&, primary bovine endothelial cells, and primary chick embryo fibroblasts. In all cases the anti-

y-Tubulin 829

in the Centrosome

Xgam antibody stained the centrosome exclusively. Figure 3 shows a triple labeling experiment in which CHO cells were stained with anti-a-tubulin antibody, anti-y-tubulin antibody, and DAPI, a fluorescent DNA-binding dye. The figure depicts a cell cycle progression: interphase (Figures 3A-3C), prophase (Figures 3D-3F), metaphase (Figures 3G-31) and anaphase (Figures3J-3L). Within interphase, y-tubulin staining was seen as either a single dot or two associated dots, representing duplication of the centrosome. y-Tubulin staining was strictly limited to the centrosome at all times in the cell cycle; no y-tubulin staining of microtubules was apparent even in metaphase when there was extremely bright microtubule staining of the spindle with the anti-a-tubulin antibody. Although it is not evident in the figure, there was consistently somewhat more y-tubulin staining in metaphase and less in telophase, when compared with interphase. The method of fixation did not affect the localization of y-tubulin; methanol and glutaraldehyde fixed samples had an identical staining pattern. We conclude that y-tubulin is localized to the centrosome and is not present in appreciable quantities in microtubules themselves. Association of yTubulln with Microtubule-Nucleating Material Is Independent of Microtubules Centrosomes are known to retain their distinctive morphology even when the microtubules attached to them are depolymerized (Mitchison and Kirschner, 1984). Structural elements of the centrosome might be expected to remain associated with the centrosome after depolymerization, whereas peripherally associated proteins, or proteins associated with microtubules near the centrosome, might be expected to disperse. To determine the nature of the association of y-tubulin with the centrosome, cells that had been treated with the microtubule-depolymerizing drug nocodazole were stained for both a-tubulin and y-tubulin. Some samples were released from the nocodazole treatment for a short period before fixation, making it possible to see microtubule nucleation from the centrosome more clearly. Xenopus XTC cells were treated with 5 pg/ ml nocodazole for 4 hr, then released from nocodazole for 0,5, and 10 minTThe results are shown in Figure 4. In the untreated cells, a-tubulin staining shows a normal microtubule cytoskeleton and y-tubulin staining is limited to the centrosome. In cells treated without release from the drug, the microtubules were completely depolymerized, with only background staining due to tubulin monomer; they-tubulin staining of the centrosome appeared identical to that of untreated cells. In cells released from nocodazole for either 5 or 10 min, a-tubulin staining showed a bright microtubule aster nucleated from the centrosome, as well as free microtubules in the cytoplasm. v-Tubulin staining was limited to the center of the microtubule asters. A particularly interesting example of this was seen in the cell shown in the 5 min time point; there was a small amount of microtubule nucleation from a point separate from the main point of nucleation. This smaller point of nucleation also stained with the y-tubulin antibody, although faintly compared with the centrosome. We believe that this smaller nucleation center resulted from fragmentation of the cen-

trosome, induced by the long nocodazole treatment. Although rare (found in -1% of cells), fragmented nucleating material was consistently seen in nocodazole-treated cells, with as many as four small fragments in a single cell, each with a microtubule aster and each staining with antiy-tubulin antibody. Fragmented centrosomes were clearly distinct from duplicated centrosomes, which usually stained equally intensely with anti-y-tubulin antibody. Fragmentation of mitotic centrosomes has previously been reported to occur after recovery of CHO cells from colcemid treatment (Keryer et al., 1984). Immuno-Gold Electron Microscopic Localization of y-Tubulin to the Pericentriolar Material We wished to correlate our immunofluorescence results, showing that y-tubulin is localized to the centrosome, with the more detailed structural information available by electron microscopy. CHO cells were fixed such that microtubule structure and y-tubulin antigenicity were preserved, incubated with anti-y-tubulin primary antibody and goldconjugated secondary antibody, and processed for transmission electron microscopy. In the micrographs shown in Figure 5, the centrosome is visible as a dense body adjacent to the nucleus. In favorable sections, the triplet microtubules of the centrioles are clearly visible, as well as the surrounding pericentriolar material. Most sections had a number of microtubules extending from the centrosome region, although this was variable. Virus-like particles were often associated with the centrosome; these have been reported previously in CHO and other cells (Wheatley, 1974, 1982). The virus-like particles were usually embedded in dense material resembling the pericentriolar material. The gold particles revealing the location of the anti-y-tubulin antibody were localized to the pericentriolar region of the centrosome, on the outer surface of the pericentriolar material. The dense material associated with the virus-like particles occasionally had y-tubulin staining as well. The centriole in Figure 58 is replicating; the parental centriole has abundant pericentriolar material and y-tubulin staining, whereas the smaller daughter centriole has neither. In contrast, the centrioles in Figure 5C are of equal size and both have pericentriolar material and y-tubulin staining. Similar asymmetrical distributionof pericentriolar material in replicating centrioles has been reported previously (Rieder and Borisy, 1982; Vorobjev and Chentsov, 1982). In no case was y-tubulin staining clearly seen along the length of a microtubule, even when microtubules seemed to end in the pericentriolar material, whereas a-tubulin antibody stained microtubules in similar preparations (not shown). No y-tubulin staining of the centriole microtubules was observed, although, given the density of the centrosome, it seems likely that antibody access to the inside of the centrosome might be impaired. Indeed, in experiments with anti-a-tubulin antibody, we also did not observe staining of the centriole microtubules (not shown). Discussion We have found that y-tubulin, initially described in the filamentous fungus A. nidulans (Oakley and Oakley, 1989) is

Cell s30

Figure 4. Nocodazole XTC Cells

Arrest

and Release

of

Xenopus XTC cells were fixed and stained with DMla, an anti-a-tub&in antibody(A, C, E, and G), and anti-Xgam antibody (6, D, F, and H). The ceils were fixed before nocodaxole treatment (A and B), after a 4 hr incubation with 5 uglml nocodazole (C and D), and after a 5 (E and F) or 10 min (G and H) release from the nocodaxole incubation. Bar, 10 urn.

also present in animals, plants, and other fungi, and is as highly conserved among these groups as are the a-tub&n and fi-tubulin proteins. It seems likely then that y-tubulin, though present at much lower levels than a- and f%tubulin, is a fundamental component of the microtubule cytoskeleton, playing an important role in microtubule function. The localization of y-tubulin within cells reveals something of the nature of this role; the protein is located in the centrosome, and is not present in other microtubule structures. This location probably reflects a conserved role in

the function of microtubule-organizing centers in general, as y-tubulin in A. nidulans is localized to the spindle pole body (Oakley et al., 1999) a fungal organelle that is functionally homologous to, but morphologically distinct from, the centrosome. We have also found that the localization of y-tubulin to the centrosome does not depend on intact microtubules, suggesting that y-tubulin is bound to the centrosome by interactions with proteins other than the a-tubulin and f3-tubulin subunits of microtubules. At a more detailed level, y-tubulin is associated with the pericentrio-

;-$ubulin

Figure

in the Centrosome

5. ImmuneGold

Electron

Microscopic

Cells were permeabilized, fixed, and incubated 63,000 x in (A); 104,400 x in (C) and (D).

Localization

of y-Tubulin

with anti-Xgam

in CHO Cell Centrosomes

antibody

lar material, the microtubule-nucleating material of the centrosome. We discuss the consequences that these findings have for understanding tubulin and microtubules. The Tubulin Family In this paper we have presented the sequences of y-tubulin genes from S. pombe and X. laevis. Comparison of the predicted y-tubulh protein sequences from these organisms with that of the original y-tubulin from A. nidulans reveals a high level of sequence conservation. The frog and yeast y-tubulins are almost 70% identical. This is the same level of sequence conservation found when comparing a-tubulins or P-tubulins from vertebrates and yeast (Little and Seehaus, 1988) indicating that y-tubulins are a distinct and highly conserved subfamily within the tubulin family. The similar degree of conservation also suggests that the same constraints that have resulted in the evolutionary conservation of a- and 3-tubulin have acted upon y-tubulin. Among the constraints for a- and P-tubulin must be binding of GTP, binding to each other to form a heterodimer, binding to other heterodimers to form a microtubule, and interactions with microtubule-organizing centers, kinetochores, and microtubule-associated proteins. Of these, y-tubulin is only known to interact with microtubule-organizing centers. We suggest, however, that the level of sequence conservation indicates that y-tubulin will

and 5 nm gold-conjugated

goat anti-rabbit

antibody.

Magnifications:

be found to have other interactions as complex as those found with a- and 9-tubulin. The finding that the y-tubulins are a conserved tubulin subfamily allows meaningful comparisons between a-, p-, and y-tubulin in order to identify structural elements that define tubulin as a family. In Figure 8 we have aligned the a-, p-, and y-tubulin sequences from S. pombe (S. pombe has two a-tubulin genes; only the major one is shown) and boxed identical residues and conservative changes often found in related proteins. All of the tubulins are approximately 450 amino acid residues in length (or about 50 kd, molecular mass). The tubulins are about 35% identical to each other, and 60% similar considering conservative changes. In most cases the sequence conservation is in regions where all three tubulins are similar; there are few stretches where two of the tubulins are similar and the other is divergent. There are several long stretches that show a high degree of conservation. One of particular interest, starting at position 143 of a-tubulin, contains the sequence GGTGSG; a glycine-rich region of this length is a common feature of nucleotide-binding proteins, and might be involved in tubulin GTP binding, although this has not yet been demonstrated. The C-termini of the tubulins show the most divergence, both within groups of particular tubulins and when comparing all tubulins. Though the amino acid sequence is not conserved, a common feature

cell 632

beta

288

Figure

6. Comparison

of a-, p-, and y-Tubulin

Sequences

from S. pombe

Sources: a-tubulin (nda2’ (Toda et al., 1994)). p-tubulin (nda3’ [Hiraoka et al., 19&l]), and y-tubulin Identical residues and consemative changes are boxed, and dashes indicate gaps. Conservative D, E; N, Q; H. R. K; I. L, V, M; F, W, Y; C; P.

of all known a- and 5-tubulins is a very acidic C-terminus (Little and Seehaus, 1988); in S. pombe 11 of the last 25 residues of a-tubulin and 12 of 25 in 5-tubuiin are aspartate or glutamate. The C-terminus of S. pombe y-tubulin is less acidic, with 7 of 25 acidic residues, and the frog r-tubulin has only 3 of 25 acidic residues, suggesting that the C-terminus might be a region where functional differences exist between r-tubulin and a- and f34ubulin. One of the fundamental properties of a- and (Mubulin is that they associate to form a heterodimer that can assemble to form microtubules (Luduefia et al., 1977). There is no evidence that a- or 5-tubulin acts individually in any cell process. The similarity of y-tubulin to a-and 5-tubulin leads to the obvious suggestion that y-tubulin might form a heterodimer with another tubulin. Liile is known about the interaction of a-tubulin with 5-tubulin, however, and it is imposstbfe to predii whether y-tubulin might bind to a- or 5-tubulin based on sequence homologies alone. It has been suggested that the pattern of genetic interactions between mipA alleles and mutant f34ubulin alleles in A. nidulans argues for interaction between y-tubulin and 5-tubulin (Oakley and Oakley, 1989; Weil et al., 1988), but this is a very indirect assay; determination of the subunit composition of y-tubulin must await biochemical analysis. Given that y-tubulin was discovered only recently, despite many years of research on microtubules, it is tempting to speculate that other tubulins exist and that they, like y-tubulin, are minor yet essential components of the microtubule cytosketeton. Such novel tubulins might account for some of the microtubule behaviors that have been observed in vivo but are not mechanistically understood (Schulze and Kirschner, 1988) or might be partners

(tugf’). The sequences were manually aligned. amino acids are grouped as follows: S, T; A, G;

with y-tubulin in a heterodimer. One way of isolating these putative tubulin family members would be to perform PCRs using degenerate oligonucleotides against the regions conserved in a-, p-, and y-tubulin. For example, the sequences GGTGSG and (Y,L)(V,I)DLEP are present in a-, 5-, and y-tubulin, and are of sufficient length to make PCR primers. Preliminary PCR experiments with such primers have yielded only the known a-, 5-, and y-tubulin genes from the S. pombe genome and a- and 54ubulin genes from the S. cerevisiae genome (unpublished data). y-Tubulin and the Centrosome Using antibodies that are specific for y-tubulin, we have shown that y-tubulin is localized to the centrosome of cultured cells through all stages of the mitotic cell cycle. This correlates well with the finding that y-tubulin in Aspergillus is localized to the spindle pole body (Oakley et al., 1999) and strongly suggests that y-tubulin plays a role in one of the functions that these microtubule-organizing centers have in common, such as microtubule nucleation. The y-tubulin staining that we describe is a discrete dot or two dots, depending on the stage of the cell cycle. By electron microscopy, we have shown that this staining corresponds to the pericentriolar material that is tightly apposed to the centrioles and is the site of microtubule nucleation. The question of whether y-tubulin is a component of the centriale microtubules as well has not yet been adequately addressed, as the dense centrosomal material probably limits access by antibodies. We are attempting to probe samples with antibodies after sectioning to circumvent this problem. The centrosome is a complex organelle that undoubt-

y-Tubulin 833

in the Centrosome

edly contains many proteins. A growing number of antigens have been reported as being centrosomally localized, yet these antigens differ greatly in extent of staining beyond the centrioles, in cell cycle-specific changes in staining, and in the properties of their association with the centrosome. In extent, the y-tubulin staining that we have described seems to represent the most limited staining of the centrosome that has been observed, apart from staining of the centrioles themselves. An antibody that has a similar limited staining pattern is the 5051 human autoimmune serum (Calarco-Gillam et al., 1983; S. Doxsey, personal communication). The y-tubulin staining is also relatively constant throughout the cell cycle, remaining limited to the centrosome. In contrast, antibodies against kinesin stain the centrosome in interphase and mitosis, but stain portions of the spindle as well in mitosis (Neighbors et al., 1988); in addition, a number of centrosomal antigens isolated from Drosophila,-embryos vary greatly in distribution and intensity of staining during the cell cycle (Kellogg et al., 1989). y-Tubulin remains associated with the centrosome even when microtubules are completely depolymerized, suggesting that it interacts with other structural elements of the centrosome; similar properties have been described for other centrosomal antigens (Moroi et al., 1983). Some centrosomal antigens disperse after depolymerization (Buendia et al., 1990; Rao et al., 1989), perhaps indicating an interaction with microtubules rather than the centrosome itself. Although the comparison of localization results in different cell types and organisms must be interpreted with caution, we believe that the wide variety of staining patterns and properties indicates the following: First, the centrosome is a highly dynamic organelle, the composition of which changes during the cell cycle. Second, functional assays will be important in distinguishing those antigens that are important for the function of the centrosome from those that are localized to the centrosome for other reasons, such as segregation at division. There are two basic aspects to the function of the centrosome as a microtubule-organizing center: nucleation of microtubule growth and anchoring of the resulting microtubule array. Corollaries of these main functions are the role of the centrosome in determining the number, polarity, and lattice structure of nucleated microtubules, and the strict coupling of centrosome duplication to the cell cycle. How does our knowledge of y-tubulin help to explain these properties? The homology of y-tubulin to a- and P-tubulin makes it likely that y-tubulin interacts in some way with a- and/or P-tubulin. Yet, despite this homology, y-tubulin is bound to the centrosome through interactions that are independent of microtubules, and is not a component of microtubule polymer. These properties suggest that y-tubulin might function as a link between the centrosome and nucleated microtubules. Nucleation of microtubules requires the elimination of the lag phase associated with polymerization of microtubules from free tubulin subunits. y-Tubulin may be thought of as a tethered tubulin, maintained at high local concentration at the centrosome. Assuming that a centrosome has a volume of 1 pm3, and that all y-tubulin is bound to the centrosome, the local

concentration of y-tubulin at the centrosome would be about 5 mglml. This high concentration might drive the formation of y-tubulin-containing oligomers that function as seeds to which a-P-tubulin subunits could add to form microtubules, anchored to the centrosome by their interaction with y-tubulin. In fact, y-tubulin might form very stable oligomers, since a nucleating component need not have the dynamic properties of tubulin in the bulk polymer lattice. By their structure, these y-tubulin oligomers might constrain nucleated microtubules to the 13-protofilament lattice structure. From the concentration of y-tubulin, we estimate that there might be lo4 to 1 05y-tubulin molecules per centrosome, a reasonable number to be involved in the formation of the ml000 microtubules that a single centrosome can nucleate. As we currently have no functional information about y-tubulin, it is certainly possible that it does not play a part in these fundamental centrosome functions; yet we believe that the sequence, evolutionary conservation, and localization of y-tubulin argue strongly for such a direct role as a link between the centrosome and the microtubule. Experimental Growth

Procedures

of Cells

X. laevis XTC cells were grown in 70% L-15 medium with 10% fetal calf serum at 25OC in ambient atmosphere. CHO cells were grown in MEMa with 10% fetal calf serum at 37OC in a 5% COn atmosphere. Cells for immunofluorescence experiments were plated on sterile 12 mm glass coverslips at least 1 day prior to the experiment. Nocodazole was added from a 10 mglml stock solution in dimethyl sulfoxide to a concentration of 5 Kg/ml. Cells were released from nocodazole arrest by transferring coverslips to new medium and washing extensively with medium. S. pombe cells were grown in YE liquid and solid medium (0.5% yeast extract and 3% glucose, + 2% agar) (King, 1974). Strain SP818 h’lh- ade6-210/ade6-216 leul-32//eul-32 ura4-DlBlura4-D18 was used for transformation experiments. SP818 was transformed with DNAfragments by a lithium acetate method as described for S. cerevisiae transformation (Stearns et al., 1990). Ura’ transformants were selected on SD minimal plates (Sherman et al., 1974) with leucine and 0.25% casamino acids added. S. pombe transformants were sporulated by incubation in SPA (King, 1974) for 2 days, and dissected on YE plates. Other yeast species were grown in YEPD (Sherman et al., 1974).

Cloning

the yTubulin

Genes

Three PCR primers were derived from regions of y-tubulin that are conserved between the Aspergillus (Oakley and Oakley, 1989) and Drosophila@. Oakley, personal communication) y-tubulin sequences, but are distinct from those regions conserved in all tubulins. A fourth was derived from the sequence GGTGSG that is highly conserved in a- and P-tubulin as well as y-tubulin. The primers were completely degenerate for the amino acid sequences against which they were directed. TGA.l:

GCCGAGCTC~AY~TN~~;AY~R

TGA.2: GCCTCTAGA;CC;SW;CC;GT;CC;CC TGA.3: GCCGAGCTCp$TNtTNFNrY$AY TGA.4: GCCTCTAGA~C;CC~G~T~T[lT

(coding strand) (noncoding

strand).

(coding strand). (noncoding

strand).

N = A, C, G, T; R = A, G; Y = C, T; S = G, C; W = A, T. Templates used for PC!+ were S. pombe genomic DNA prepared by glass bead disruption (Hoffman and Winston, 1987), and Xenopus first strand cDNA, prepared from Xenopus oocyte mRNA as described

Cell 034

(Sambrook et al., 1989). The primers were used in the combinations TGA.l + TGA.2, TGA.1 + TGA.4. TGA.3 + TGA.4. Fragments derived from the PCRs were cloned into ml 3mpl8 and m13mp19 vectors and sequenced by the diixy chain termination method using Sequenase DNA polymerase (United States Biochemical Corp., Cleveland, OH). These fragments were radioactively labeled by a random hexamer priming procedure (Feinberg and Vogelstein, 1963) and used to clone the entire genes by hybridization to DNA libraries (Sambrook et al., 1989). The full-length Xenopus cDNA was cloned from a Xenopus oocyte cDNA library in the vector SgtlO (Rebagliati et al., 1985). The entire S. pombe gene, except for the 3’ 200 bp, was cloned from an S. pombe genomic DNA library in the plasmid pWH5 (Wright et al., 1986). The 3’end of the S. pombe T-tubulin gene was cloned by RACE PCR (Frohman et al., 1988). S. pombe mRNA was purified from total RNA, prepared by glass bead disruption (Hoffman and Winston, 1987) by poly(dT) chromatography (Sambrooket al., 1969). RACE PCR was performed on this mRNA as described (Frohman et at., 1988), except that two rounds of PCR were performed, using nested primers. The sequence derived from the RACE PCR fragment was then used to isolate the corresponding fragment from genomic DNA by PCR with specific primers. Both strands of the Xenopus and S. pombe T-tubulin genes were sequenced. Genomic DNA from budding yeast species (supplied by H. Roiha) was prepared by glass bead disruption (Hoffman and Winston, 1987). The Mgl+ gene disruption construction was made by inserting the 1.8 kb Hind81 fragment containing the S. pombe ura4+ gene, modified so that it is bounded by Sphl sites, into the Sphl site at position 883, in exon 4 of fug7’. Production of T-Tubulln Antlbodleo Protein fusions between GST and fragments of the Xenopus and S. pombe y-tubulin genes were created by inserting PCR fragments into the vector pGEX2 (Amrad Corp., Australia). The Xgam fusion protein, GST-Xgam, included amino acids 49-337. Two tugI+ fusion proteins were constructed, GST-tugla and GST-tug1 b, which included amino acids 73-231 and 232-386, respectively. The resulting fusion proteins were insoluble and were purified on this basis from Escherichia coli as described (Kleid et al., 1961). Protein fusions to the bacterial TrpE proteinwerecreated by inserting PCRfragmentsin thevectorpATHI1, a derivative of the original PATH vectors (Dieckmann and Tzagoloff, 1985). The Xgam fusion protein, TrpE-Xgam, included the entire codingsequence,andthefugl+fusionprotein,TrpE-tugl, includedamino acids l-437. The tugl+ fragment was isolated by PCR using an S. pombe cDNA library as the template (Fikes et al., 1999). Polyacrylamide gel slices containing the GST fusion proteins were used to produce antibodies in rabbits (Babco, Inc., Emeryville, CA). Anti-y-tubulin antibodies were purified from the rabbit serum by affinity purification against the TrpE protein fusions. Large-scale affmity purification was by cotumn chromatography; small-scale affinity purification was by binding to nitrocellulose strips bearing the fusion protein (Harlow and Lane, 1988). TrpE fusion protein to be used in column chromatography was purified from polyacrylamide gel slices by electroelution. The eluted protein was coupled to Affigel 10 matrix in 0.1 M HEPES (pH 6.8). Both etectroelutttn and coupling buffers contained 0.05% SDS. Antibodies were bound to, and eluted from, the column as da scribed (Harfow and Lane, 1986). Anti-peptide antibodies were made by coupling a 16 residue peptide of sequence IIDREADGSDSLEGFC (synthesized by the UCSF Biomolecular Resource Center), which includes a cysteine residue for coupling, to keyhole limpet hemocyanin as described (Green et al., 1982). and immunizing rabbits with the resulting conjugate. Protein Blotting Protein samples were made from CHO and XTC cells by adding SDS sample buffer to a confluent monolayer of cells, scraping cells from the plate, boiling, and then sonicating briefly to fragment chromosomal DNA. Crude Xenopus egg extract was made as described (Murray and Kirschner, 1989) diluted 1:9 in SDS sample buffer, and boiled. Tubulin was prepared from Xenopus eggs by three cycles of warm polymerization and cold depolymerixation. A high speed supernatant was prepared from eggs as described by Gard and Kirschner (1987). Dimethyl sulfoxide was added to lo%, GTP to 1 mM, and the sample was warmed to 35°C and incubated for 1 hr. Microtubules were sedimented by spinning at 200,000 x g for 1 hr at 35OC. The resulting pellet was

resuspended in cold BRB80 (80 mM potassium PIPES [pH 6.8],1 mM MgCI,, 1 mM EGTA) containing 1 mM DTT, incubated on ice for 40 min. and spun at 200,000 x g for 30 min at 4OC. This procedure was repeated twice more, yielding a preparation in which (I- and 8-tubulin were the major proteins. Protein samples (50 ug of total protein for the crude extracts, and 1 ug of purified tubulin) were electrcphoresed through 5%-15% gradient SDS-polyactytamide gels and electrophoretically transferred to nitrocetlutose membranes. The membranes were stained with Ponceau S (Sigma), and blocked with 5% dried milk in tris-buffered saline (TBS) with 0.1% NP-40. Affinity-purified primary antibodies were incubated with the Mot for 1 hr. The primary antibody was detected with horseradish peroxid ase-conjugated donkey antiECL reagents. We were able to detect processing with An&ham 100 pg of T-tubulin fusion protein per lane under these wnditions. Exposure to Kodak XAR film was for approximately 1 min. Immunotluoreecence Animal cells were fixed with methanol by immersing wverslips bearing cells in -2O“C methanol for 5 min. Coverslips were then rehydrated with PBS, and washed three times with Ab buffer (PBS, 3% BSA, 0.1% Triton X-100,0.02% azide). Primary antibodies (either affinity-purified anti+ubulin or DMla, a mouse monoctonaf anti-tubulin [Blase et al., 19641), diluted in Ab buffer, were added to wverslips and incubated in a humid chamber for 30 min. Secondary antibodies (affinity-purified rhodamineconjugated goat anti-rabbit or fluorescein-conjugated rat anti-mouse (Cappel, Inc.]) were diluted 150 in Ab buffer and incubated on wverslips for 30 min. Triple labeling of y-tubulin, a-tubulin, and DNA was accomplished by incubating sequentially with anti-y-tubulin, rhodamine goat anti-rabbit, DMla, fluorescein rat anti-mouse, and DAPI. The DAPI incubation was at a concentration of 1 ug/ml for approximately 30 s. Coverslips were washed three times with Ab buffer between incubations and mounted in 90% glycerol, PBS @H 9.0) with 1 mg/ml p-phenylenediamine. Glutaraldehyde fixation was as described previously (Schulze and Kirschner, 1986). Micrographs were taken with a Zeiss Axiophot microscope and Kodak TMY film. Immune-Gold Electron Mkroecopy CHO cells grown on 12 mm coverstips were rinsed with PBS at 37OC, permeabllized for 15 s in permeabilization buffer (80 mM PIPES [pH 6.81, 5 mM EGTA, 1 mM MgCl,, 0.5% Trlton X-100). and fixed in the same buffer with 2% formaldehyde and 0.4% glutaraldehyde for 10 min. All subsequent steps were performed at rwm temperature. The fixed cells were treated with 1 mglml sodium borohydride in PBS for 7 min, then washed three times over 20 min with Ab buffer. Cells were incubated with affinity-purified anti-Xgam antibody diluted in Ab buffer for 1 hr, then washed three times with TBS @H 7.4). 0.1% Triton X-l 00, and three times with TBS (pH 8.2). 1% BSA. Cells were then incubated with 5 nm gold-conjugated goat anti-rabbit antibody (Janssen) diluted 1:3 in TBS (pH 8.2), 1% BSA for 4 hr, washed three times with TBS (pH 8.2), and incubated with cacodylate buffer (0.1 M sodium cacodytate [pH 7.21) for 10 min. The cells were poetfixed in 2% glutaratdehyde incacodylatebufferfor 15min,andwashedthreetimeswtthcacodytate buffer. The coverstips were incubated in 2% OsC, in cacodytate buffer forl5min, thendehydratedinaeerlesofEtOH(7O%,95%,andlOO%), propylene oxide, 113 propylene oxide, and 2/3 Araldite (British Grade) over 20 min, and embedded in Aratdlte. The cetts for Figure 58 were treated as above except that the blacking solutiin and the gotd antibody solution contained 0.1% fish gelatin (Janssen). The cells for Figure 5C were treated as above except that the sodium borohydride treatment was eliminated and replaced with 0.1 M L-lysine in TBS for 60 min, and cells were blocked with 3% normal goat serum, 3% SSA, 1% fish gelatin in PBS. The gold antibody in this experiment was diluted 1 :l. In all experiments gold sectiins were cut with a diamond knife and stained with 2% uranyl acetate in 50% MeOH for 30 min and 0.4% lead citrate in 0.1 N NaOH for 2 min. Grids were examined in a Philips 400 electron microscope. Acknowledgments We are very grateful to Berl Oakley for making the Drosophila T-tubulin sequence available prior to publication, and to Mitsuhiro Yanagida for sharing preliminary information on the S. pombe y-tubulin gene. We

;-&ubulin

in the Centrosome

thank Bob Booher and Tom Musci for providing cDNA, libraries, strains, and helpful advice, Josh Miller for assistance with RACE PCR, and Linda Hicke for advice on purification of Xenopus tubulin. We also thank Paul ‘Goldie” Goldsmith for helpful suggestions on immuno-gold electron microscopy, and Ray Deshaies and Michael Glotzer for comments on the manuscript. This work was supported by a grant to M. K. from the National Institute of General Medical Sciences. T. S. was supported by a postdoctoral fellowship from the Helen Hay Whitney Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

February

28, 1991; revised

March

References Blase, S. H., Meltzer, D. I., and Feramisco, J. R. (1984). IO-nm filaments are induced to collapse in living cells microinjected with monoclonal and polyclonal antibodies against tubulin. J. Cell Biol. 98,847858. Bornens, M., Paintrand, M., Berges, J.. Marty, M., and Karsenti, E. (1987). Structural and chemical characterization of isolated centrosomes. Cell Motil. Cytoskel. 8, 238-249. Buendia, B., Antony, C., Verde, F., Bornens, M., and Karsenti, E. (1990). A centrosomal antigen localized on intermediate filaments and mitotic spindle poles. J. Cell Sci. 97, 259-271. Calarco-Gillam. P. D., Siebert, M. C., Hubble, R., Mitchison. T., and Kirschner, M. (1983). Centrosome development in early mouse embryos as defined by autoantibody against pericentriolar material. Cell 35, 621-829. nucleatpericen-

Dieckmann, C. L., and Tzagoloff, A. (1985). Assembly of the mitochondrial membrane system. J. Biol. Chem. 260, 1513-1520. Evans, L., Mitchison, T., and Kirschner, M. (1985). Influence of the centrosoms on the structure of nucleated microtubules. J. Cell Biol. 700,1186-1191. Feinberg, A., and Vogelstein, DNA restriction endonuclease Biochem. 132.8-13.

B. (1983). fragments

A technique for radiolabeling to high specific activity. Anal.

Fikes, J. D., Becker, D. M., Winston, F., and Guarente, L. (1990). Striking conservation of TFIID in Schizosaccharomyces pornbe and Saccharomycws cerevisiae. Nature 346, 291-294. Frohman, M. A., Dush, M. K., and Martin, G. R. (1968). Rapid production of full-length cDNAs from rare transcripts: amplification using a singlegene-specificdigonucleotideprimer. Proc. NatLAcad. Sci. USA 85,8998-9002. Gard, D. L., and Kirschner, M. W. (1987). Microtubule assembly in cytoplasmic extracts of Xeflopus oocytes and eggs. J. Cell Biol. 705, 2191-2201. Gould, R. R., and Borisy, G. G. (1977). The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation. J. Cell Biol. 73, 801-615. Green, N., Alexander, H., Olson, A., Alexander, S., Shinnick, Sutcliffe, J. G., and Lerner, R. A. (1982). lmmunogenicstructureof influenza virus hemagglutinin. Cell 28, 477-487. Harlow, E., and Lane, D. (1988). Antibodies: A Laboratory (Cofd Spring Harbor, New York Cold Spring Harbor Laboratory Heidemann, S. R., and McIntosh, structural polarity of microtubules.

J. R. (1980). Visualization Nature 286, 517619.

Hyams, J. S., and Borisy, G. G. (1978). Nucleation of microtubules in vitro by isolated spindle pole bodies of the yeast Saccharomyces cerevisiae. J. Cell Biol. 78, 401-414. Kellogg, D. R., Field, C. M., and Alberts, B. M. (1989). Identification of microtubule-associated proteins in the centrosome, spindle, and kinetochore of the early Drosophile embryo. J. Cell Biol. 109, 29772991. Keryer, G., Ris, H., and Borisy, during tripolar mitosis in Chinese 2222-2229. King, R. C. (1974).

29, 1991

Clayton, L., Black, C. M.. and Lloyd, C. W. (1985). Microtubule ing sites in higher plants identified by an auto-antibody against triolar material. J. Cell Biol. 101, 319-324.

Hoffman, C. S., and Winston, F. (1987). A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia co/i. Gene 57, 267-272.

T. M., the Manual Press). of the

Hiller, G., and Weber, K. (1978). Radioimmunoassay for tubulin: a quantitative comparison of the tubulin content of different established tissue culture cells and tissues. Cell 14, 796-804. Hiraoka, Y., Toda, T., and Yanagida, M. (1984). The MIA3 gene of fission yeast encodes 8tubulin: a cold-sensitive ndad mutation reversibly blocks spindle formation and chromosome movement in mitosis. Cell 39. 349-366.

Handbook

G. G. (1984). Centriole distribution hamster ovary cells. J. Cell Biol. 98,

of Genetics

(New York:

Plenum

Press).

Kleid, D. G., Yansura, D., Small, B., Dowbenko, D., Moore, D. M., Grubman, M. J., McKercher, P. D., Morgan, D. O., Robertson, 8. H., and Bachrach, H. L. (1981). Cloned viral protein vaccine for foot-andmouth disease: responses in cattle and swine. Science 214, 1125 1129. Lee, C. C., Wu, X., Gibbs, R. A., Cook, R. G., Muzny, D. M., and Caskey, C. T. (1988). Generation of cDNA probes directed by amino acid sequence: cloning of urate oxidase. Science 239, 1288-1291. Little, M., and Seehaus, T. (1968). Comparative analysis sequences. Comp. B&hem. Physiol. 90, 855-870. Ludueria, R. F., Shooter, E. M., and Wilson, L. (1977). tubulin dimer. J. Biol. Chem. 252, 7006-7014.

of tubulin

Structure

of the

Mitchison, T., and Kirschner, M. W. (1984). Microtubule assembly cleated by isolated centrosomes. Nature 312, 232-237.

nu-

Moroi, Y., Murata, I., Takeuchi, A., Kamatani, N., Tanimoto, K., and Yokohari, R. (1983). Human anticentriole autoantibody in patients with scleroderma and Raynaud’s phenomenon. Clin. Immunol. Immunopathol. 29, 381-390. Murray, A. W., and Kirschner, the early embryonic cell cycle.

M. W. (1989). Cyclin synthesis Nature 339, 275-280.

drives

Neighbors, B. W., Williams, R. C., and McIntosh, J. R. (1988). Localization of kinesin in cultured cells. J. Cell Biol. 108, 1193-1204. Oakley, B. R., Oakley, C. E., Yoon, Y., and Jung, M. K. (1990). T-Tubulin is a component of the spindle pole body that is essential for microtubule function in Aspergillus nidulans. Cell 67, 1289-1301. Oakley, C. E., and Oakley, B. R. (1989). Identification of y-tubulin, a new member of the tubulin superfamily encoded by the mipA gene of Aspergillus nidulans. Nature 338, 662-864. Peterson, J. B., and Ris. H. (1976). spindle and chromosome movement visiae. J. Cell Sci. 22, 219-242.

Electron-microscopic study in the yeast Saccharomyces

of the cere

Rao. P. N., Zhao, J.-Y., Ganju, R. K., and Ashorn, C. L. (1989). Monoclonal antibody against the centrosome. J. Cell Sci. 93, 83-89. Rebagliati, M. R., Weeks, D. L., Harvey, R. P., and Melton, D.A. (1985). Identification and cloning of localized maternal RNAs from Xenopus eggs. Cell 42, 789-777. Rieder, C. L., and Borisy, G. G. (1982). The centrosome cycle in PtKz cells: asymmetric distribution and structural changes in the pericentriolar material. Biol. Cell 44, 117-132. Rout, M. P., and Kilmartin, J. V. (1990). Components of the yeast spindle and spindle pole body. J. Cell Biol. 771, 1913-1927. Sambrwk, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press). Schulze, E., and Kirschner, M. W. (1986). Microtubule interphase cells. J. Cell Biol. 102, 1020-1031. Schulze, behavior

E., and Kirschner, observed in vivo.

M. (1988). New features Nature 334, 356-359.

dynamics

in

of microtubule

Sherman, F., Fink, G., and Lawrence, C. (1974). Methods in Yeast Genetics (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press). Stearns, genome Toda,

T., Ma, H., and Botstein, D. (1990). Manipulating the yeast using plasmid vectors. Meth. Enzymol. 785, 280-296. T., Adachi,

Y., Hiraoka,

Y., and Yanagida,

M. (1984).

Identifica-

Cdl 636

tion of the pleiotropic cell division cycle gene NDAP as one of the two different a-tubulin genes in Schizosaccharomyces pombe. Cell 37, 233-242. Vandre, D. D., and Borisy, G. G. (1969). The centrosome cycle in animal cells. In Mitosis: Molecules and Mechanisms, J. S. Hyams and B. R. Brinkley, eds. (San Diego: Academic Press), pp. 39-l 16. Vorobjev, I. A., and Chentsov, Y. S. (1962). Centrioles I. Epithelial cells. J. Cell Biol. 98, 936949.

in the cell cycle.

Weil, C. F., Oakley, C. E., and Oakley, B. R. (1966). Isolation of mip (microtubule-interacting protein) mutations of Asper@us nidulans. Mol. Cell. Biol. 8, 2963-2966. Wheatley, D. N. (1974). Pericentriolar virus-like hamster ovary cells. J. Gen. Virol. 24, 396-399.

particles

in Chinese

Wheatley, D. N. (1962). The Centriole: ACentral (Amsterdam: Elsevier Biomedical Press).

Enigmaof

Cell Biology

Wilks, A. F. (1969). Two putative protein-tyrosine application of the polymerase chain reaction. USA 88, 1603-1607.

kinases identified by Proc. Natl. Acad. Sci.

Wilson, E. 8. (1926). The Cell in Development edition (New York: Macmillan, lnc).

and Heredity,

Third

Wright, A., Maundrell, K., Heyer, W. D., Beach, D., and Nurse, P. (1966). Vectors for the construction of gene banks and the integration of cloned genes in SoMzosaccMromycespornbe and Saccheromyces cefevisiae. Plasmid 75, 156-158. GenBank

Accesslon

Numbers

The accession numbers for the nucleotide sequences rug7’ genes are M63446 and M63447, respectively.

of the Xgam and