Journal of Molecular Evolution

J Mol Evol (1985) 22:220-229

9 Springer-Verlag 1985

Tubulin Evolution: Ciliate-Specific Epitopes Are Conserved in the Ciliary Tubulin of Metazoa Andr6 Adoutte, ~ Maurice Claisse, ~ Roger Maunoury, 2 and Janine Beisson ~ Centre de G6n6tiqueMol6culaire, C.N.R.S., 91190 Gif-sur-Yvette,France 2Serviced'Anatomie Pathologique,Centre HospitalierSaint Anne, 1, Rue Cabanis, 75014 Paris, France

Summary.

In spite o f their overall evolutionary conservation, the tubulins of ciliates display electrophoretic and structural particularities. We show here that antibodies raised against P a r a m e c i u m and T e t r a h y m e n a ciliary tubulins fail to recognize the cytoplasmic tubulins o f all the metazoans tested. Immunoblotting o f peptide maps of ciliate tubulins reveals that these antibodies react with one or very few ciliate-specific epitopes, in contrast to polyclonal antibodies against vertebrate tubulins, which are equivalent to autoantibodies and recognize several epitopes in both ciliate and vertebrate tubulins. Furthermore, we show that the anti-ciliate antibodies recognize ciliary and flagellar tubulins of metazoans ranging from sea urchin to mammals (with the exception o f humans). The results support the conclusion that although duplication and specialization of tubulin genes in metazoans may have led to distinct types o f tubulins, the axonemal one has remained highly conserved.

Key words:

Microtubules -- Tubulin antibodies -- Immunoblotting -- Immunocytology -- Autoantibodies -- Gene duplication

Introduction Tubulins have long been considered to be among the most conserved proteins and a range of struc-

Offprint requests to: A. Adoutte, Laboratoirede BiologicCellulaire IV, B~tirnent444, Universit6Paris-Sud, 91405 Orsay Cedex, France

tural, biochemical, and sequencing data support this notion (Cleveland et al. 1980; Alexandraki and Ruderman 1983; reviewed in Roberts and Hyams 1979). Recent evidence obtained at the protein and the D N A level has disclosed, however, some heterogeneity o f a- as well as o f B-tubulins within a species and a number of differences among the tubulins of distantly related species (Little et al. 1982a; Cleveland 1983; Cowan and Dudley 1983; Raft 1984). Within a given species this heterogeneity either originates posttranslationally or, in some biological systems, stems from the expression o f distinct genes (Little et al. 1982a; Cleveland 1983; Cowan and Dudley 1983; Raft 1984). Since microtubules occur in a variety of subcellular locations (cytoplasm, mitotic spindle, ciliary and flagellar axonemes, etc.), there has been much recent interest in establishing whether tubulin diversity is correlated with specific intracellular locations and functions. O f particular interest in the context of the present paper are the findings o f Little et al. (1981, 1982b, 1983), who compared the peptide maps o f cytoplasmic and flagellar tubulins of a number o f protistan and metazoan species, and demonstrated in Metazoa more differences between the two types oftubulins within a species than between flagellar tubulins of different organisms. Their data fit the idea that in Protozoa, a single type of tubulin is used for both axonemal and cytoplasmic microtubules, whereas in Metazoa, duplication and evolution oftubulin genes may have led to the development o f distinct axonemal and cytoplasmic tubulins, with greater constraints on axonemal tubulins. The results reported here, based on an immunological approach, provide further evidence for a higher degree o f conservation o f axonemal tubulin.

221 ulins (Igs) or afffinity-purified anti-tubulin Igs (Karsenti et al. 1978; Hill et al. 198 I) were used, with identical results. The antiturkey brain tubulin antiserum, provided by A. Lemoine and M. Wright (Toulouse, France), was obtained after injection oftubulin purified by SDS acrylamide gel electrophoresis. The serum is highly specific for tubulin. However, in immunoblotting experiments, whatever the sample tested, this serum yields a diffuse, nonspecific reaction at ca. 68 kilodaltons. The intensity of the band is independent of the protein concentration loaded in the well and it therefore appears to correspond to a previously described artifact (Tasheva and Dessev 1983). The two anti-ciliate tubulin antisera were used at dilutions of 1/200 or 1/500 both in blotting and in immunocytochemical experiments. The antiturkey tubulin antiserum was used at a dilution of 1/100 and the anti-pig brain tubulin antiserum at 1/20, and the corresponding purified antibodies were used at 0.08 mg/ml.

Fig. IA, B. Electrophoretic properties of various tubulins. Polyacrylamide gels (6% or 7.5 to 15%) were run with either Sigma (A) or British Drug House (13) SDS at all steps and stained With Coomassie stain. In this and the following figures the positions of molecular weight standards are given in kilodaltons • 10-3 on the sides of the gels. The samples correspond to purified cilia from Paramecium tetraurelia, purified cytoplasmic tubulin from Physarum polycephalum (the band at 43 kilodaltons is conlaminating actin), and purified tubulins from chicken, mouse, and pig brain. The Paramecium tubulins remain closely spaced even under optimal conditions

Materials and Methods "~crylamide Gels, Peptide Maps, and Immunoblotting. One-dimensional sodium dodecyl sulfate (SDS) polyacrylamide gels were used according to Laemmli (1970). SDS from Bio Rad (Richmond, CA), British Drug House (Poole, England), or Sigma (St. LOuis, MO) was used (see Results). Peptide maps of a- and O-tubulins excised from one-dimensional SDS acrylamide gels Were prepared using Staphylococcus aureus V8 protease from Miles (Naperville, IL) according to Cleveland et al. (1977) with rainor modifications (Claisse et al. 1980). The gels were stained with either Coomassie Brillant Blue R250 or silver nitrate acCOrding to Merril et al. (1981) or Oakley et al. (1980), using the ~odifications of Eschenbruch and Biirk (1982). Immunoblotting Was carried out as previously described (Cohen et al. 1982).

Antibodies. All the antibodies were raised in rabbits except the one against pig brain tubulin, which was raised in a sheep. The preparation and specificity of the antibodies raised against Paramecium ciliary tubulin have been previously described (Cohen et al. 1982). The anti-Tetrahymena tubulin antibodies were Provided by I. Barahona and C. Rodrigues-Pousada (Oeiras, Portugal) and their preparation and specificity have been reported (Barahona and Rodrigues-Pousada 1983). The anti-pig brain tubulin antibodies were provided by A.-M. Hill and D. Pantaloni (Gif-sur-Yvette, France). Either partially purified immunoglob-

Tubulin Extracts. Purified mouse, chicken, and pig brain tubulins (provided by G. Filiatreau, Saclay, and D. Pantaloni, Gifsur-Yvette, France) were prepared according to Shelanski et al. (1973). Physarum tubulins (provided by M. Wright) were prepared according to Roobol et al. (1980). Paramecium cilia (> 50% tubulin) were obtained by the MnCI2 deciliation method of Fukushi and Hiwatashi (1970). Purified cortex from Paramecium was prepared using several modifications of the method described by Tiggemann and Plattner (1981) and involving, in particular, an equilibrium sedimentation step in sucrose gradients (S. Ng and A. Adoutte, manuscript in preparation). Purified flagella from Euglena (provided by M.-H. Br6, Gif-sur-Yvette, France) were prepared by a method derived from that of Thompson et al. (1974). Ciliated cortices from quail oviduct (provided by N. Bordes and D. Sandoz, Ivry-sur-Seine, France) were prepared as described in Sandoz et al. (1982). Sea urchin spermatozoa flagella (provided by M.-P. Cosson, Villefranche-sur-Mer, France) were prepared according to Gibbons and Fronk (1972). Extracts of whole sea urchin eggs and crude preparations of sea urchin egg mitotic apparatuses (provided by M.-N. Raymond, Orsay, France) were obtained as described by Foucault et al. (1984). Immunocytological Procedures. Paramecium cells were processed as described by Cohen et al. (1982). Cultures of mammalian cells were processed as described by Hill et al. (1981 ) and Osborn and Weber (1982). Histological sections were obtained as follows: The tissues were fixed in a cold (4"C) mixture of 95% ethanol and glacial acetic acid (99: I, v/v) for 1 day and embedded in paraffin. Five-micrometer serial sections were cut, deparaffinized, and processed according to the avidin-biotin peroxidase complex (ABC) method (I-Isu et al. 1981). Kits for the ABC method were obtained from Vector Laboratories (Burlingame, CA).

Results

Ciliate Tubulins Display Distinctive Electrophoretic and Structural Properties Figure 1 compares the electrophoretic migrations in o n e - d i m e n s i o n a l S D S g e l s o f t u b u l i n s f r o m Para-

mecium, Physarum, pig, mouse, and chicken. In all cases, the extent of separation of the two tubulin bands depends on the brand of SDS, i.e., on the percentage of contaminating hexa- and tetradecyl sulfate, as demonstrated by Best et al. (1981). Par-

222

Fig. 2. Silver*stained peptide map of vertebrate and protistan tubulins. Bands of a- and ~-tubulin were excised from gels similar to those shown in Fig. 1 and digested for 30 min with 0.2/~g of Staphylococcus aureus V8 protease per slot. All conditions were exactly as described in Claisse et al. (1980). The gel was stained according to Merril et al. (1981). The bands of vertebrate tubulins are labeled conventionally (a = slower migrating,/~ = faster migrating). The bands from Paramecium and Physarum tubulins are labeled according to peptide pattern by reference to those of vertebrates. The arrow points to the 33-kilodalton band typical of protistan c~-tubulin. Note that the Paramecium ~-tubulin is probably contaminated with some a-tubulin. "Vs" corresponds to a slot loaded with the protease only

Fig. 3A-C. Immunological reactivity oftubulins from Paramecium, Physarum, and vertebrates toward anti-Paramecium and antiturkey tubulin antibodies. A Coomassie-Blue-stained gel (7.5 to 15% acrylamide) containing purified cilia from Paramecium, purified cortex from Paramecium, and purified tubulins from Physarum, pig, mouse, and chicken. A gel identical to the one displayed in A (except that the two adjacent slots labeled "Physarum" contained the Physarum tubulin sample) was blotted, yielding two identical nitrocellulose filters. One of the filters (B) was first incubated with anti-turkey tubulin antibody (1/100 dilution); the other (C), with anti-Paramecium tubulin antibodies (1/200 dilution). Both filters were then incubated with ca. 0.4 ~Ci [~25I]protein A and autoradiographed for 3 days at -80*(2. The diffuse band located at ca. 68 kilodaltons in B corresponds to a parasitic reaction (see Materials and Methods)

amecium and Physarum tubulins are better separated in British Drug House SDS than in the Sigma brand, whereas the reverse is true for vertebrate tubulins. Of the two Paramecium tubulin bands, the upper one comigrates with vertebrate/~-tubulin, and the lower one migrates ahead of it. Peptide maps of the

two tubulin bands (Fig. 2) show that, as in the case of Physarum (Clayton et al. 1980), Paramecium /~-tubulin corresponds to the slower-migrating band. This reversed order of migration with respect to vertebrate tubulins accounts for the reversed positions o f tubulin dots in t w o - d i m e n s i o n a l gels (Adoutte et al. 1980; Cohen et al. 1982). Figure 2

223

l~|g. 4A-D. Reactivity of peptides from a- and /~-tubulins of Paramecium and pig toward four types of anti-tubulin antibodies. l~eptide maps obtained from digestion of Paramecium and pig a- and/~-tubulin bands with increasing amounts of V8 protease were blotted and the filters incubated with anti-Paramecium (A) or anti-Tetrahymena (13) tubulin antibodies or with anti-turkey (C) or auti-pig (D) tubulin antibodies, respectively. Numbers at tops of columns indicate amount (in micrograms) of V8 protease used per slot. Reactivity was detected either with [~25I]protein A (A, C) or peroxidase-coupled second antibody (/3, D). The short arrows point to the position of undigested tubulins. The long arrow points to the single peptides of a- and/~-tubulin of Paramecium, respectively, that react with anti-Paramecium and anfi-Tetrahymena tubulin antibodies when very mild proteolytic digestion is carried out. Note the similarity of the patterns obtained with the anti-Paramecium and anti-Tetrahymena antibodies

reveals many differences between Paramecium and Vertebrate tubulins, especially for a-tubulin, which displays a slow-migrating intense band of ca. 33 kilodaltons also present in Physarum (Clayton et al. 1980; Fig. 2) and Tetrahymena (Little et al. 1982b) ~'tubulins and absent in vertebrate a-tubulin. This band (or closely spaced set of bands) apparently characterizes the a-tubulins of lower eukaryotes and Plants (Little et al. 1982b; Morejohn and Fosket 1982).

"lnti-Ciliate Tubulin Antibodies Have a Narrow Reaction Spectrum The specificity o f our anti-Paramecium tubulin ar~tiserum has been previously described: In im-

munocytochemical studies it decorates all microtubular structures in Paramecium, and in immunoblotting of one- or two-dimensional SDS gels of different cell fractions, it binds only to tubulins (Cohen et al. 1982). Figure 3C shows that this antiserum fails to react with cytoplasmic tubulins of vertebrates. The anti-Tetrahymena tubulin antiserum gave an identical result. In contrast, polyclonal antibodies against pig brain or turkey brain tubulin yielded positive responses with all the species tested in immunoblotting experiments (Fig. 3B). With these antibodies, the intensity of the reaction appeared generally to be proportional to the amount of tubulin present in the gels. The only exceptions were the weaker reactivity

224

Fig. 5A,B. Immunological reactivity of extensively digested tubulins. Paramecium and pig a- and ~-tubulins were digested with 0.2 /zg protease per slot, blotted as previously, and challenged with anti-pig tubulin antibodies. A very mildly digested (ca. 0.002 #g per slot) pig tubulin sample is included for orientation. A Coomassie-stained gel. B Corresponding filter (peroxidase detection). Note the persistence of two and four to five bands in fully digested a-tubulins of Paramecium and pig, respectively. Longer digestion with the protease (2 h instead of 30 rain) and Coomassie staining (instead of silver staining) account for the differences in pattern of the 0.2-#g pig tubulin slots with respect to those of Fig. 2

o f Physarum tubulins toward b o t h types o f antibodies, and the weaker reactivity o f Paramecium tubulin toward the anti-pig brain tubulin antibodies. The antibodies generally reacted with b o t h tubulin bands, as can be seen better on blots o f either well-separated tubulins (not shown) or peptide m a p s o f a- and ~-tubulins excised from gels in which they were well separated (Fig. 4). However, the anti-tur-

key antibodies reacted exclusively with Physarum a-tubulin (Fig. 3B), and the anti-pig antibodies reacted preferentially with the a-tubulin o f Paramecium (Fig. 5). As detailed elsewhere (Adoutte et al. 1984), both antisera were negative on all m e t a z o a n cytoplasmic tubulins tested, including those o f insects, molluscs, and lower and higher vertebrates.

225

~'18.6A-F. Specificityof the anti-Paramecium tubulin antibodies for ciliary tubulins. A Coomassie-stainedgel (7.5 to 15% acrylarnide) containing the followingsamples: 1, Paramecium cilia; 2, pig brain tubulin; 3, sea urchin spermatozoa flagella;4, Euglena gracilis flagella.B Coomassie-stained gel identical to A after blotting. C Autoradiogram &one of the filters obtained from B tested Withanti-turkey tubulin antibody. D Same as C but tested with anti-Paramecium tubulin antibody. E, F Autoradiograms of filters obtained from another gel that contained the following:5, Paramecium cilia; 6, Euglena flagella; 7, sea urchin spermatozoa flagella; 8, quail oviduct cortex; 9, pig brain tubulin; 10, sea urchin egg soluble tubulin; 11, sea urchin egg mitotic apparatus. Note that all the Sanaplesreact with anti-turkey tubulin antibody (C, E), whereas only ciliary or flagellartubulins react with anti-Paramecium tubulin antibody (D, F)

Anti-Ciliate Tubulin Antibodies Probably Recognize Very Few Epitopes The anti-Paramecium and anti-Tetrahymena tubulin antibodies were tested against blots o f peptide maps of purified Paramecium a- and /3-tubulins. Parallel experiments were conducted with anti-turkey and anti-pig tubulin antibodies against peptide maps o f Paramecium and pig tubulins. Figure 4 shows that for Paramecium tubulins, Only peptides of a relatively large size, obtained under mild digestion conditions (0.002 or 0.02 ~g protease), are recognized by the antibodies. When digestion is carded out with 0.2 t~g protease, yielding laeptides averaging 15 kilodaltons in size (see Figs. 2 and 5), no antigenic determinant is preserved (Fig. 4). U n d e r mild digestion, single peptides o f a- and of B-tubulin of approximately 22 and 21 kilodaltons, respective/y, react with the anti-ciliate antibodies (Fig. 4A,B). These peptides are not recognized by anti-pig or anti-turkey antibodies (results not shown). Under similarly mild digestion, several peptides from pig brain tubulins are recognized (although With different affinities) by anti-pig or anti-turkey tUbulin antibodies (Fig. 4C, D). Under further digestion (Fig. 5) antigenic determinants on several pep-

tides from pig a-tubulin are still recognized by the anti-pig tubulin antibodies, which also react with two peptides from Paramecium a-tubulin. These results demonstrate that the two types of anti-vertebrate antibodies react with several epitopes in both ciliate and vertebrate tubulins, whereas the two anti-ciliate antisera react with fewer epitopes restricted to a single 21- to 22-kilodalton peptide.

Anti.Paramecium Tubulin Antibodies React Exclusively with Axonemal Tubulin o f Metazoa The reactivity o f the anti-ciliate tubulin antibodies on metazoan ciliary or flagellar tubulins was tested. Some o f the results obtained by immunoblotting are illustrated in Fig. 6. Positive reactions were obtained with sea urchin flagella but not with sea urchin eggs; similarly, tubulin from quail oviduct cilia, but not that from chicken brain, was positive. The weak result obtained with Euglena flagellar tubulin probably reflects the wide evolutionary distance separating ciliates from euglenoids (Adoutte et al. 1984). Quail oviduct cilia were also found to react positively in immunocytological tests at the light- and

PARAMECIUM ANTIBODIES

PIG BRAIN ANTIBODIES

Fig. 7A-D. Immunoperoxidasestainingof two serial transverse sections of the rat bronchus after incubationwith antibodiesto tubulins from Paramecium (A, B) and pig brain (C, D). In A and B note the selectivestaining for the periphery of epithelial ciliatedcells (C); in C and D note staining for the nerve fibers (N), musclecells (SM), and cilia (C). L, bronchial lumen

electron-microscope levels. Histological sections from a number of invertebrates and vertebrates (mussel, earthworm, lizard, frog, and mouse) were analyzed by light microscopy after incubation with either anti-Paramecium or anti-pig brain tubulin antibodies and subsequent reaction with peroxidase-labeled second antibody. Whereas the anti-pig brain tubulin antibodies stained all microtubulecontaining cells (particularly neurons), the anti-Paramecium antibodies decorated only ciliary structures. As an example, Fig. 7 shows sections through rat bronchial lumen in which the only structure decorated with anti-Paramecium antibodies is the ciliated epithelium. All ciliary structures except those of human reacted positively in this broad spectrum of species.

Finally, immunocytological tests were conducted on rat, sheep, and human spermatozoa. Rat and sheep reacted positively with both the anti-Paramecium and the anti-pig antibodies, whereas human spermatozoa reacted only with the anti-pig antibodies. Table 1 summarizes the results.

Discussion Some immunological methods, such as microcom" plement fixation, can yield quantitative estimates of the degree of sequence divergence between homologous globular proteins (Prager and Wilson 1971; Benjamin et al. 1984). In contrast, immunocyto"

227

Table 1. Summaryof reactionswith antibodiesto Paramecium tubulin

Antigenic reaction Cyto-

S~cies Vertebrates Lizard (Lacerta muralis) Toad (Buffo buffo) Quail (Coturnix coturnix) Mouse (Mus musculus) Rat (Ratus norvegicus) Pig (Sus scrofus) Man (Homo sapiens) Invertebrates Sea Urchin (Trypneustes gratilla) Earthworm (Lombricus terrestris) Fruit Fly (Drosophila melanogaster) Mussel (Mytilus edulis) Ciliates Paramecium tetraurelia Tetrahymena pyriformis Stylonichia mytilus Euplotes eurystomus Blepharisma musculus

Fla-

plas- Cili- gelmic ary lar -

+ + +

nt nt nt

-

+ +

+ +

-

nt

nt

-

-

-

-

+ +

nt nt nt nt

-

-

+

+ + + + +

+ + + + +

+, Positive reaction; -, negativereaction; nt, not tested; blank, organellenot present. Antibodiesto vertebratetubulin yieldpositive results throughout the whole table chemistry and immunoblotting essentially provide qualitative information: They enable the identification o f specific epitopes in a conserved protein among various species or among various subcellular Organelles. We have used the latter approach here in a comparative analysis of ciliary and cytoplasmic tubulins o f Protozoa and Metazoa. The results indicate that the tubulins of P a r a m e c i u m display electrophoretic and structural differences with respect to those of Metazoa and that antibodies raised against P a r a m e c i u m and T e t r a h y m e n a ciliary tubulins fail to react with the cytoplasmic tubulins o f Metazoa, but do react with the ciliary tubulins o f Metazoa ranging up to mammals. We will examine the significance of these immunological data before discussing the implications of Our results for the evolution o f tubulin genes. T h e Specificity o f A n t i - T u b u l i n A n t i b o d i e s

The evolutionary conservation o f tubulins, which has been well demonstrated by protein and D N A sequence data, is also supported by immunological data. Most o f the polyclonal anti-tubulin antibodies described in the literature were raised against vertebrate tubulin (typically brain tubulin), but some Were raised against flagellar tubulin from sea urchin

(Weber et al. 1977) or N a e g l e r i a (Fulton and Lai 1982) or against ciliary tubulin o f T e t r a h y m e n a ( V a n de Water et al. 1982). These antibodies react with the tubulins o f all species (reviewed in Osborn and Weber 1982; Van de Water et al. 1982); the antipig and anti-turkey antibodies used in our experiments belong to this category. Such broad spectrum anti-tubulin antibodies can be considered equivalent to autoantibodies against rabbit tubulin; i.e., they are directed not against species-specific determinants but against some epitope(s) likely to be present in most or all tubulins. As previously demonstrated in a similar case (Osborn and Weber 1982), we have ascertained that the anti-turkey antibodies raised in a rabbit reacted with rabbit brain tubulins. We have also shown that such antibodies react with several distinct epitopes o f tubulins. In contrast, the structural particularities o f ciliate tubulins suggest that they might be recognized as foreign proteins by a rabbit, yielding antibodies o f restricted specificity, as seems also to be the case for plant tubulin (Morejohn et al. 1984). These anticiliate tubulin antibodies, which appear to react with a single peptide of a- and/~-tubulin of P a r a m e c i u m and to discriminate between axonemal and cytoplasmic tubulins of various metazoans, resemble the monoclonal or anti-peptide antibodies that discriminate among microtubule subpopulations within a single cell type (Gallo and Anderton 1983; Gundersen et al. 1984). It is then reasonable to assume that our anti-ciliate antibodies recognize only ciliate-specific epitope(s) and can therefore be used as a qualitative tool to detect the presence of these particular epitopes in other tubulins.

Duplication and Specialization of Tubulin Genes

As suggested by Little et al. (1982a,b), one can assume that in Protozoa, a single type of or- and /3-tubulin is used in both axonemal and cytoplasmic microtubules [not taking into account possible posttranslational modifications ofaxonemal tubulin (e.g., see MacKeithan and Rosenbaum 1981; Brunke et al. 1982; L'Hernault and Rosenbaum 1983)]. Although we do not yet know the status of P a r a m e c i u m , several studies have established that the copy number o f a- and/3-tubulin genes is low in unicellular nonparasitic eukaryotes (Cleveland 1983; Raft 1984). In C h l a m y d o m o n a s , which has two genes for a- and two genes for/3-tubulin, it was recently shown by D N A sequencing that the two 13-tubulin genes differ only by silent substitutions; i.e., their predicted amino acid sequences are identical (Youngblom et al. 1984). Most importantly, it has been reported that T e t r a h y m e n a has a single gene for

228 a - t u b u l i n ( C a l l a h a n et al. 1984). I n c o n t r a s t , m e t a zoans display substantial redundancy of tubulin genes. T h e greater c o n s e r v a t i o n o f a x o n e m a l t u b u 1ins, as o p p o s e d to c y t o p l a s m i c ones, t h r o u g h o u t e v o l u t i o n (Little 1982a,b, 1983) suggests t h a t s o m e o f t h e g e n e s b e c a m e s p e c i a l i z e d for c y t o p l a s m i c t u b u l i n s a n d d i v e r g e d w h i l e o t h e r s r e m a i n e d specific for a x o n e m a l t u b u l i n s a n d were m o r e c o n s e r v e d . O u r d a t a p r o v i d e e v i d e n c e i n f a v o r o f this h y p o t h esis b y s h o w i n g t h a t e p i t o p e s p r e s e n t i n b o t h c i l i a r y and cytoplasmic tubulins of "ancient" unicellular e u k a r y o t e s are lost i n the c y t o p l a s m i c t u b u l i n s o f M e t a z o a , b u t are c o n s e r v e d i n c i l i a r y a n d flagellar t u b u l i n s i n taxa r a n g i n g all the w a y u p to m a m m a l s . T h e c o n c o m i t a n t absence of cross-reaction of ciliary a n d flagellar a x o n e m e s o f m a n w i t h the a n t i - P a r a m e c i u m a n t i b o d i e s suggests t h a t b o t h t y p e s o f axo n e m e s are b u i l t f r o m the s a m e t u b u l i n s , a n d t h a t t h e loss o f the r e a c t i v e epitope(s) o c c u r r e d at a late stage o f m a m m a l i a n e v o l u t i o n . T h e greater c o n s e r v a t i o n o f a x o n e m a l t u b u l i n m a y be correlated with the considerable conservatism of the a x o n e m a l f u n c t i o n a n d s t r u c t u r a l design. T h e " 9 + 2 " m o t i v e is d i s p l a y e d i n its e l a b o r a t e f o r m b y v e r y a n c i e n t e u k a r y o t e s a n d h a s u n d e r g o n e few m o d i f i c a t i o n s i n the c o u r s e o f e v o l u t i o n . T h i s i n t u r n m a y b e r e l a t e d to t h e fact t h a t t h e a x o n e m e is an elaborate functional assembly of proteins made u p o f o v e r 150 i n t e r a c t i n g p o l y p e p t i d e s ( P i p e r n o et al. 1977; A d o u t t e et al. 1980). M a n y o f these p r o t e i n s are k n o w n o r s u s p e c t e d to b e e n g a g e d i n specific a s s o c i a t i o n s w i t h a x o n e m a l m i c r o t u b u l e s . A x o n e m a l t u b u l i n m a y t h e r e f o r e h a v e b e e n left w i t h v e r y few " d e g r e e s o f f r e e d o m . " Acknowledgments. This work could not have been carried out without the generous gifts of samples and antibodies provided by the many colleagues listed in the Materials and Methods section, to whom we are most grateful. We thank C6cile Couanon, Jacqueline Brizard, and Annie Charrier for their help in preparing the manuscript. This work was supported by grant no. 82 V 1268 from the D616gation G6n6rale de la Recherche Scientifique et Technique and a grant from the Ligue Nationale Franfaise contre le Cancer.

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Received March 12, 1985/Revised May 24, 1985