J. PROTOZOOL. 23(3), 458-464 (1976).

Isolation and Identification of Specific Cortical Proteins in Tetrahymmu Pyrif0rni.s strain GL*t PIERRE VAUDAUXi Dkpartement de Biologie animale, Univcrsitt' de Genkre, Genkre, Switzerland, and De$artment of Zoology, University of Zowa, Iowa City, Iowa 52242

SYNOPSIS. Pellicles of the ciliate Tetrahymena pyriformis strain GL (phenoset A) were isolated by a new procedure. Oral apparatuses were also purified by a modification of a previous method. Both preparations were characterized by electron microscopy. Proteins of the isolates were separated by analytical SDS polyacrylamide gel electrophoresis. The isolated pellicles, which included oral apparatuses, contained only 6 major proteins (gel bands), designated A through F. Bands A, B, and C, were found in the pellicle fraction, but not in the oral apparatu fraction. Therefore, these proteins are believed to be present in the somatic cortex of Tetrahymena. Bands D and E were greatly enriched in the oral apparatus fraction; these proteins are therefore believed to be present primarily in the oral apparatus. Band F, identified as tubulin, was present in both preparations. Molecular weight determinations and some selective solubilization experiments are also presented. Index Key Words: Tetrahymena pyriformis GL; ciliate cortex; oral apparatus ; cell fractionation ; electron microscopy; polyacrylamide gel electrophoresis ;protein analysis.

HE mechanisms of cellular development are still poorly understood at the molecular level. The ciliate cortex has been considered by several investigators (8, 27, 30) as a useful system for studying these mechanisms. Thus far, progress in this area has been limited by the scarcity of accurate molecular data related to the diverse constituents of the ciliate cortex. Several investigators have tried to purify individual cortical structures, such as ciliary basal bodies and associated fibers. Their isolation procedures have not allowed unequivocal conclusions concerning nucleic acids (2, 13, 25) or proteins (23) present in the cortex, due to difficulties in eliminating impurities. Procedures for isolating whole pellicles (4, 7, 12, 14, 17, 29) or oral apparatuses (20, 31), devoid of cilia, have been more successful. Nonetheless, few data about protein composition of these structures are presently available (9, 20), except for tubulin (20). In the present study, a new, convenient, and reproducible method of the isolation of Tetrahymena pellicles is reported. This method yields pellicles without membranes; the integrity of these pellicles is maintained by the epiplasmic layer located just beneath the pellicle membranes in intact cells. Studies using this method have led to the unambiguous identification of several major cortical protein components of Tetrahymena pyriformis, strain GL (phenoset A).

T

MATERIAL AND METHODS Ciliate Strain Tetrahymena pyrijormis, strain GL (phenoset A ) , was a gift from the Laboratoire de Zoologie 2, Centre d'Orsay, UniversitC de Paris. Mass cultures for the preparation of isolated pellicles or oral apparatuses were grown in 6-liter flasks containing 1 liter of growth medium consisting of 1% (w/v) Difco proteose

This investigation was supported in part by Research Grants 3.601.71, from the National Swiss Foundation for Scientific Research (to Prof. GCrard de Haller) and GB 41389 from the U.S. National Science Foundation (to Prof. Norman E. Williams). Part of the work was included in a doctoral dissertation submitted to the University of Geneva. ?During his stay in the U.S.,the author was supported by Fonds Marc Birkigt (Geneva), Geigy Jubilaumstiftung (Basel), and the National Swiss Foundation for Scientific Research. Present address: Division des Maladies Infectieuses, D4partement de MCdecine, 1211 Gentve 4, Switzerland.

*

peptone, 0.5% (w/v) Difco yeast extract, and 0.5% (w/v) NaCl. Fifty ml of a late log-phase culture was inoculated into this medium. The cultures, agitated continually by a magnetic stirrer, were allowed to grow 24 hr at room temperature until they reached a density of 2-3 X 106 cells/ml. The cells were collected by centrifugation at 800 g for 15 min and washed in a 0.25 M sucrose solution. Preparation of Isolated Pellicles

All procedures were performed at 2 C, using ice-cold solutions. The lysis conditions reproduced those already described for the isolation of oral apparatuses (31). Washed cells in 10 ml of a 0.25 M sucrose solution were placed into a 100-ml beaker and agitated continually by a magnetic stirrer until the end of lysis. Fifty ml of a SEMT solution ( 1 M sucrose, 1 mM EDTA, 0.1% 2-mercaptoethanol, 10 I ~ MTris, final pH 9.3) were added to the suspension of cells and followed by 5 ml of a 10% Triton X-100 'solution, one minute later. Isolated pellicles appeared at this stage and lysis of the cells was complete. Ten-ml aliquots of the lysate were then layered over 5-ml aliquots of a solution called SMP ( 1 M sucrose, 0.1% mercaptoethanol in 10 mM sodium phosphate buffer, pH 6.8) in 15-ml centrifuge tubes. The tubes were then centrifuged in a swinging bucket rotor in a HS-25 MSE Centrifuge at 2,800 g for 10 min. Four fractions were then collected with a hand pipette: ( a ) the SEMT layers, ( b ) the interfaces between SEMT and SMP, ( c ) the SMP layers, and ( d ) the pellets. The pellets were resuspended in 2-ml aliquots of a dilute SMP solution (0.5 M sucrose, 0.1% mercaptoethanol in 10 mM sodium phosphate buffer, pH 6.8). Fractions ( c ) and ( d ) were pooled and layered over two 5-ml aliquots of SMP in two 15-ml tubes, which were centrifuged at 2,800 g for 10 min. The 4 fractions described above were again collected separately. The pellets and the SMP layers contained the most purified pellicles. Preparation of Isolated Oral Apparatuses

Conditions for lysis by Triton X-100 in SEMT solution were identical to those described above for the preparation of isolated pellicles. Two procedures, both carried out entirely at 2C, were used to isolate oral apparatuses. The first method, used initially, followed rather closely that of Wolfe (31). After the lysis of the cells, the oral apparatuses were purified by 3 differential centrifugations at 12,000 g for respectively 30, 15

458

CORTICAL PROTEINSIN Tetrahymena and 15 min. The final fraction obtained by that procedure was rich in oral apparatuses, but still contained an appreciable amount of small contaminating particles. The oral apparatuses were purified further by 2 additional steps: each 12,000 g pellet was resuspended in 5 ml douhle-distilled water instead of SEMT, then layered over 5 ml of a 1 M sucrose solution in a 15-ml tube. Centrifugation was performed at 2,800 g for 10 min. Four fractions were then collected with a hand pipette: ( a ) the water layer, ( b ) the interface between water and 1 M sucrose, ( c ) the 1 M sucrose layer, and ( d ) the pellet. The pellet was resuspended in 2 ml of water. Fractions (c) and , ( d ) were pooled and layered over 5 ml of a 1 M sucrose layer, and centrifuged at 2,800 g for 10 min. The 4 fractions were again collected separately. The pellet and the 1 M sucrose layer contained the most purified oral apparatuses. The 2nd method for the isolation of oral apparatuses was used in the selective solubilization experiments. After lysis, the isolated pellicles were homogenized in a Logeman, leaving free oral apparatuses as the only particulate material in the preparation (20). The homogenate was then centrifuged at 2,300 g. The pellets were resuspended in 10 ml of water and the oral apparatuses were spun down again at 2,300 g for 15 min. The final pellets contained oral apparatuses which were as pure as those obtained in the first method described above. The 2nd procedure, however, had the advantage of being much simpler. Cilia were isolated hy the procedure of Gibbons (10). Separation

of

Proteins

Analytical separation of proteins was performed by gel electrophoresis in a 12-tube system (Buchler equipment). The procedure of Laemmli (15) for sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis was used with the following minor modifications. The gels were most often 7.5% polyacrylamide and 0.2% N, N methylene bisacrylamide (Bis). For some experiments, the gels contained either 10% polyacrylamide and 0.27% Bis, or 12.5% polyacrylamide and 0.33% Bis. A constant current of 24 mA (2 mA/tube) was applied during electrophoresis. After the gels were removed from their tubes, they were stained overnight in a 0.25% (w/v) Coomassie brilliant blue G-250 (Serva) solution in a methanol/water/ acetic acid mixture (5:5: 1). The gels were destained for 24 hr in the methanol/water/acetic acid mixture, then for 48 hr in a 7% (v/v) acetic acid solution. In the selective solubilization experiments, the gels were fixed in the methanol/water/ acetic acid mixture overnight, then stained in 1% (w/v) Fast Green in 7% (v/v) acetic acid for 1 hr. They were then destained in 7% acetic acid. The gels stained with Fast Green were scanned at 600 nm on a Beckman spectrophotometer. The molecular weights of the proteinate-SDS complexes were estimated by comparison of their migrations relative to those of known proteinate-SDS standards (26). The following standards were used for molecular weight ( M W ) calibration: glyceraldehyde phosphodehydrogenase (MW 36,000), ovalbumin (MW 43,000), catalase (MW 60,000), bovine serum albumin ( M w 68,000), and phosphorylase A (MW 94,000). Selective Solubilization Tests The purified pellicles, oral apparatuses, or cilia were spun down and resuspended overnight in 2 ml of a high ionic strength solution ( l o ) , consisting of 0.6 M KCl, 1 mM EDTA and 10 mM Tris-HCI, pH 8.8. The extracted material then was centrifuged at 15,000 g for 15 min. The pellets were resuspended in 2 ml of water. Proteins in the supernatant fluids and in the pellets

459

were precipitated by the addition of 5 volumes of absolute ethanol, then dialyzed against water to remove ethanol and KCl, which precipitated in the presence of ethanol. Ethanol precipitation was used to avoid degradation during prolonged dialysis. The protein precipitates were collected hy a 15-min centrifugation at 15,000 g and resolubilized directly in the sample buffer (15) used for SDS gel electrophoresis. Electron Microscopy Sections.-Isolated pellicles or oral apparatuses were fixed in 2 ml of 1% (v/v) glutaraldehyde and 0.5% (w/v) OsO, in 0.1 M sodium phosphate buffer, p H 7.0 (20) for 20 min. After fixation, the isolated structures were centrifuged at 2,000 g for 5 rnin and resuspended in 5 ml 0.1 M sodium phosphate buffer, p H 7.0 for 6 hr at 2 C. They were then centrifuged at 3,000 g for 10 min to make a compact pellet. This pellet was fragmented into smaller pieces and embedded in agar ( 6 ) . The specimens were dehydrated, embedded in Epon, and sectioned on a manual Porter Blum microtome. The thin sections were doubly-stained with uranyl acetate and lead citrate (22). Whole-mounts.-One drop of the suspension of isolated structures was applied to the surface of a thin Formvar film made on a 400 mesh copper grid. After 5 min, the grid was drained, then stained with 0.5% uranyl acetate, or unneutralized phosphotungstic acid for 30 sec, then drained. All preparations were examined in a Hitachi HS-7S electron microscope. RESULTS Structure of Isolated Cortical Structures The following features were observed in the ultrathin sections of isolated pellicles. Pellicles have no endoplasmic contaminants (Figs. 1, 2). Mucocysts are absent. The cilia are absent, except in the oral area where some membranellar cilia remain attached to their basal bodies. No evidence of membranes is found in the isolated pellicles; the epiplasmic layer is the only continuous sheet between basal bodies (Figs. 1, 2). Some isolated cilia contaminate the preparation (Fig. 2 ) , but none has any membranes. The different microtubular systems (transverse, posterior, and longitudinal) found in intact cells (1, 19) are well preserved in isolated pellicles, except for the basal microtubules ( 1), which are consistently absent. Kinetodesmal fibers ( 1, 19) are also present (Figs. 1, 3 ) . Basal bodies are generally well aligned in kineties (Fig. 3 ) , but some tilting of the bodies may occur (Fig. 1 ) ; the C microtubules (24) and central core (24) are frequently absent. Isolated oral apparatuses also lack membranes. Basal bodies are held together by microfilamentous arrays (16, 31 ) or by microtubular connectives (16, 20, 31). Most of the oral ribs and the deep fibers (16) are missing. Analysis of Cortical Proteins Isolated pellicles or oral apparatuses are quickly solubilized in the SDS sample buffer. The protein patterns of the SDS gels are shown in Fig. 4. The main protein bands have been designated by the letters A to F, starting from the top of the gels. Bands A to C are present only in preparations of isolated pellicles, while bands D to F are present in preparations of the oral apparatus and of the pellicle. All these bands are removed by pretreatment with pronase or trypsin, which suggests their proteinaceous nature. Different tests were performed to verify that the proteins of bands A to F are true cortical proteins rather than some endoplasmic contaminant not revealed by the electron microscope.

460

CORTICAL PROTEINS IN Tetrahymena

Figs. 1-3. [Electron micrographs of isolated pellicles.] 1. Thin section. Note the basal bodies (bb), kinetodesmal fibers (kd), ppsterior microtubules (mp), and transverse microtubules (mt). The continuom layer of epiplasm is indicated by arrows. Basal bodies

CORTICAL PROTEINS IN Tetrahymena

461

Fig. 5. Bands obtained by SDS gel electrophoresis of purified pellicles in 10% polyacrylamide ( a ) . The supernatant fluid remaining after harvesting the pellicles was subjected to ultracentrifugation ( 100,000 g for 1 hr) . The proteins of the high speed pellet are shown in b, and those of the supernatant fluid are shown in c. Fig. 4. Bands obtained by SDS polyacrylamide gel electrophoresis of preparations of purified pellicles and oral apparatuses. Only 6 major protein bands are evident in the preparation of the pellicle (a). Three of these proteins (D, E and F) are found in the oral apparatus. The proteins found in the supernatant fluid from the last step in purification of the oral apparatus are seen in c. The gels were 7.5% polyacrylamide. The tests involved comparisons of the proteins in the purified structures with the contaminants removed during purification. A comparison of purified oral proteins with those of the water layer of the first low speed centrifugation is shown in Fig. 4. This layer contains the contaminants which remain at the last step of Wolfe’s (31) procedure, but are effectively removed (Fig. 4 ) by the low speed centrifugations in 30% sucrose. Bands D and E are absent from the water layer, which is poor in oral apparatuses, but are conspicuous in the purified oral apparatus fractions. Thus, these bands can be considered as true oral proteins. Band F is present in both the water layer and the purified oral apparatus fractions. By coelectrophoresis with isolated cilia and extraction in a high ionic strength solution (10, 20), band F can be identified as the tubulin component of the oral apparatuses and of the contaminants (containing

many isolated cilia). The doublet nature of the band, not apparent in Fig. 4, can be seen in Figs. 5 and 9. Unlike band F, D and E cannot be extracted by the high ionic strength solution (Fig. 9 ) . Purified pellicular proteins and those of contaminants removed during the first low speed centrifugation are compared in Fig. 5. Protein bands corresponding to the positions of A, B, and C are absent from the SEMT layer (subfractionated into sedimentable and soluble proteins). The gel concentrations used for the experiments shown in Figs. 4 and 5 did not resolve proteins with MW lower than 20,000. Gels of higher polyacrylamide content (Fig. 6) revealed that no major cortical protein of MW under 20,000 was present in either the oral or pellicular purified fractions. MW determinations are shown in Figs. 7 and 8. A fairly linear plot was obtained with the 5 protein standards. MW values for D and E were found to be 87,500 and 83,500, respectively; for the 2 sub-bands of F, 51,000 and 49,000. MWs of the proteins in bands A to C exceeded those of the available standards and could not be determined. A less rigorous MW determination of these proteins was obtained in SDS gels after their solubilization in the absence of 2-mercaptoethanol. Under

c bbl and bb2 appear tilted in relation to the remaining ones. X 30,000. 2. Thin section at a higher magnification. In addition to the structures shown also in Fig. 1, longitudinal microtubules (ml) are seen located between epiplasm (arrows) and the pellicular membrane. Note the absence of a membrane distal to these microtubules. No membrane is evident around the cilium that contaminated this preparation. x 42,000. 3. Whole-mount preparation of isolated pellicle stained in 1% phosphotungstic acid. Heavily stained basal bodies (bb) and microtubule ribbons (arrows) are seen in this type of preparation. X 12,000.

CORTICAL PROTEINS IN Tetrahymena

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rf Fig. 6. Bands obtained by SDS gel electrophoresis of purified pellicles (a) and oral apparatuses (b) in 12.5% polyacrylamide. F indicates tubulin and V represents a previously identified contaminating protein of relatively low MW (see Fig. 4 ) . these conditions, the MW of the proteins in bands A to C could be compared to those of ovalbumin polymers (Fig. 8) which give a fairly linear plot. Under these conditions, M W values were found to be 230,000,165,000,and 145,000 for the proteins of bands A, B, and C, respectively. The relative migration of bands A to C in SDS gels was found to be identical after solubilization of the cortical proteins in the presence or absence

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Fig. 7. Determinations of the MWs of cortical proteins D, E, and tubulin (F1 and F2) in 7.5% polyacrylamide SDS gels. Standards used were glyceraldehyde phosphodehydrogenase (Gl), ovalbumin (Ov), catalase (Ca), bovine serum albumin (BSA), and and phosphorylase A (Ph) .

Fig. 8. Determination of the MW of cortical proteins A, B, and C. Polymers of ovalbumin ranging from the trimer (111) to the heptamer (VII)were used to formulate the calibration curve. of 2-mercaptoethanol. This result indicates the absence of disulfide bridges linking different or similar chains of the proteins present in bands A to C. The absence of interchain bridges can also be demonstrated for the proteins of bands D, E, and F.

Localization of Cortical Proteins Band F, present in isolated pellicles and oral apparatuses, has been identified as the tubulin component of these structures, on the basis of coelectrophoresis with isolated ciliary axonemes and solubility in a high ionic strength solution (10, 20) (Fig. 9 ) . Bands D and E are obviously parts of some oral components which remain unextracted in a high ionic strength solution. Electron microscopic examination of whole mounts confirms previous observations (31) that whereas microtubules are extracted by a high ionic strength solution, microfilaments remain present. In fact, the so-called microfilamentous fraction (31)is far from homogeneous and contains also the terminal plates of the basal bodies. The exact relationships between each protein of bands D and E and each component of the microfilamentous-terminal plates complex remains unknown. Are D and E also present in non oral areas of the cortex? If so, they probably contribute very little to the somatic structures of the cortex, because their intensity decreases significantly in the stained SDS gels when the preparation of cortical structures contains a high proportion of intact pellicles and a low proportion of isolated oral apparatuses. Bands A to C contain proteins located exclusively in the somatic region of the pellicles, because these proteins are absent from isolated oral apparatuses. The main structures present in the somatic pellicular region and absent from the oral region are the epiplasm and the kinetodesmal fibers. Proteins of bands A to C might then be located in these structures. Selective solubilization of the isolated pellicles by a high ionic strength solution (10) is shown in Fig. 9. Bands A and C appear to be more soluble in the high ionic strength solution than band B. Whole mounts were prepared from the insoluble residue remaining after the selective solubilization treatment and examined with the electron microscope. Micotubules and kineto-

463

CORTICAL PROTEINS IN Tetrahymena

desmal fibers were found to be generally absent from the preparation. In addition, the original pattern of the isolated pellicles had been destroyed. It was not possible to determine if epiplasm had been partially solubilized by the high ionic strength solution. Thus far, any precise correlation could not be established between each protein of bands A to C and each component of the somatic zone in isolated pellicles. DISCUSSION

Fig. 9. [Selective solubilization of oral, pellicular, and ciliary proteins by a high ionic strength solution (0.6 M KCI, 1 mM EDTA, 10 mM Tris-HC1, pH 8.8 at 2 C) . In each trace, the proteins are resolved on 7.5 % polyacrylamide SDS gels of the indicated fractions. All gels were stained with Fast Green and read at 600 nm.] a. Soluble extract of oral apparatuses. b. Insoluble residue of oral apparatuses. e. Soluble extract of pellicles. d. Insoluble residue of pellicles. f . Soluble extract of cilia. [The position of dynein (Dy)is indicated.]

The results obtained by the new procedure for pellicle isolation, and the improved method for isolation of the oral apparatus, provide for the first time general information about the main proteins located in the cortex of Tetrahymena. This description of cortical proteins does not include those of the mucocysts and cortical membranes, which are absent from the isolated pellicles. Nonetheless, the procedures presented here may be useful in a search for the membrane proteins; a comparison of pellicles with membranes ( 1 7 ) and those without could allow the recognition, by subtraction, of the main membrane protein components. The presence of a small number of main protein classes, as visualized by SDS gel electrophoresis, may provide some insight into the organization and morphogenesis of cortical structures in Tetrahymena. This finding leads to the hypothesis, for example, that some of these cortical structures might be composed of homogeneous protein subunits arranged in quasi-crystalline networks. Microfilaments and kinetodesmal fibers are good candidates for such a pattern of organization. Microtubules are already known to have this organization ( 5 ) , despite the fact that their building blocks are somewhat heterogeneous at the molecular level ( 18). Microfilaments and kinetodesmal fibers might consist also of similar, but not identical proteinaceous building blocks. Gel bands A through E identified in the present study may contain more than one protein, having identical MWs but with different charges. More sophisticated technics of protein separation will be required to elucidate further the definite number of cortical proteins and their composition. The presence of a large proportion of high MW singlechained proteins in isolated pellicles, although at first surprising, is not without precedent; a very high MW single-chained protein has been found recently in the cortical zone of Paramecium (11, 21). The possibility of anomalous behavior of proteins from bands A through E in SDS gel electrophoresis, giving erroneously high MWs ( 3 ) , has been checked by a study of their relative mobilities in different concentrations of polyacrylamide (Vaudaux, unpublished results). These proteins have high friction coefficients in SDS gels and there is no evidence of their anomalous relative mobility (3). When the isolated pellicles were first solubilized in 5 M guanidine-HC1 and 0.1 M mercaptoethanol, then dialyzed against adequate sample buffer for SDS gel electrophoresis, the usual pattern of bands A through E was found in the gels. Proteins of bands A through E also were eluted in the void volume of a Sephadex G-200 column in the presence of 5 M guanidine-HC1 (Vaudaux, unpublished experiments). These results indicate that the MWs remain at least above 70,000 under strong denaturing conditions. One of the most striking features of the isolated pellicles is their stability in the absence of membranes; this stability must be due to the strength of the epiplasmic layer. Some further stabilization might occur at the junction points between epiplasm and the ends of kinetodesmal fibers, transverse microtubules, or posterior microtubules. These connections, first described from thin sections of whole cells ( 1 ) , have been confirmed with iso-

464

CORTICAL PROTEINSIN Tetrahymena

lated pellicles (Figs 1, 2). In the absence of such bonds, the kinetodesmal fibers and microtubular bundles had a tendency to rotate from their original orientations. There are 2 main obstacles associated with selective solubiliiation methods for determining the localizations of the cortical proteins present in bands A through E. First, it is difficult to quantify at the electron microscope level how much of a given structure has been removed by selective solubilization, especially when a continuous layer like epiplasm is only partially removed. Second, it is possible for a structure to disappear at the electron microscope level without being solubilized. I n this situation the structural proteins may be converted into amorphous aggregates by denaturation following the solubilization treatment, and these can remain undetected by electron microscope sampling technics. There has been only one previous report of the selective solubilization of cortical structures in Tetrahymena. Rubin & Cunningham, who used different concentrations of potassium phosphotungstate (PTA), concluded that there is a single “peptide” of molecular weight 21,000 in the kinetodesmal fiber (23). I have been unable to reproduce their result, using their solubilization procedures on preparations of isolated pellicles. Furthermore, the SDS gels of PTA extracts revealed the presence of some artifacts, even after careful dialysis of the extracts against water (23). This fact militates against the use of PTA solubilization for identification purposes. Finally, it must be pointed out that no major protein with a MW of 21,000 was found in the pellicles studied in the present investigation, even though kinetodesmal fibers were present in quantity. I t must be concluded either that the 21,000 h4W protein is not the unique constituent of the kinetodesmal fiber, or that it is a degradation product of a larger protein. More recently, Stephens (28) characterized the protein composition of the ciliary rootlets of molluscan gill epithelium. These structures are similar, though not identical, to the kinetodesmal fibers of Tetrahymena. The rootlets are believed to be composed of 2 high MW proteins, and the MWs of the 2 are similar to that of band A in Tetrahymena pellicles. A comparison of peptide maps and amino acid compositions of the ciliary rootlets of molluscan epithelium and ciliate kinetodesmal fibers would be of great interest from an evolutionary viewpoint. It should be mentioned also that strain GL, used in the present study, is probably not the most favorable material for isolating the cortical proteins of Tetrahymena. Recent isolation attempts have been made using the B strain of syngen 1. These investigations have shown that this strain gives much higher yields of isolated pellicles. The B strain should be preferred for a continued investigation and preparative separation of the main cortical proteins of Tetrahymena. ACKNOWLEDGMENTS

I would like to acknowledge with gratitude the advice and help of Professors GCrard de Haller, Norman E. Williams, and Joseph Frankel. REFERENCES 1. Allen RD. 1967. Fine structure, reconstruction and possible functions of components of the cortex of Tetrahymena pyriformis. J. Protozool. 14, 553-65. 2. Argetsinger J. 1965. The isolation of ciliary basal bodies (kinetosomes) from Tetrahymena pyriformis. J . Cell Biol. 24, 154-7. 3. Banker GA. Cotman C \ 6 1972. Measurement of free electrophoretic mbbility and retardation coefficient of proteinsodium dodecylsulfate complexes by gel electrophoresis. A method to validate molecular weight estimates. J. Biol. Chem. 247, 585661.

4. Child FM, Mazia D. 1956. A method for the isolation of parts of Ciliates. Experienta 12, 161-2. 5. Cohen C, Harrison S, Stephens RE. 1971. X-ray diffraction from microtubules. J. Mol. Biol. 59, 375. 6. de Haller G, Ehret CF, Naef R. 1961. Technique d’inclusion et d‘ultramicrotomie, destinCe A 1’Ctude du dCveloppement des organelles dans une cellule isolke. Experientia 17, 524-6. 7. Flavell RA, Jones IG. 1971. DNA from isolated pellicles of Tetrahymena. J . Cell Sci. 9, 719-26. 8. Frankel J. 1974. Positional information in unicellular organisms. ]. Theor. Biol. 47, 439-81. 9. Gavin RH. 1974. The oral apparatus of Tetrahymena pyriformis, mating type 1, variety 1. I. Solubilization and electrophoretic separation of oral apparatus proteins. Ex#. Cell Res. 85. 212-6. 10. Gibbons IR. 1965. Chemical dissection of cilia. Arch Biol. (LiJge) 76, 317-52. 11. Hansma HG. 1975. The immobilization antinen of Paramecium aurelia is a single polypeptide chain. ]. Pyotozool. 22,

257-9. 12. Hartman H, Puma JP, Gurney T. 1974. Evidence for the association of RNA with the ciliary basal bodies of Tetrahymena. J. Cell Sci. 16, 241-59. 13. Hoffman EJ. 1965. The nucleic acids of basal bodies isolated from Tetrahymena pyriformis. J. Cell Biol. 25, 217-28. 14. Hufnagel LA. 1969. Cortical ultrastructure of Paramecium aurelia. Studies on isolated pellicles. J. Cell Biol. 40, 779-801. 15. Laemmli UK. 1971. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-5. 16. Nilsson JR, Williams NE. 1966. An electron microscope study of the oral apparatus of Tetrahymena Qyriformis. C.R. Trav. Lab. Carlsberg 35, 119-41. 17. Nozawa Y, Thompson GA. 1971. Studies on membrane formation in Tetrahymena pyriformis. 11. Isolation and lipid analysis of cell fractions. J. Cell Biol. 49, 712-21. 18. Olmsted JB, Borisy GG. 1973. Microtubules. Annu. Rev. Biochem. 42, 507-40. 19. Pitelka DR. 1961. Fine structure of the silverline and fibrillar system of three tetrahymenid ciliates. J . Protozool. 8, 75-89. 20. Rannestad J, Williams NE. 1971. The synthesis of microtubule and other proteins of the oral apparatus in Tetrahymena pyriformis. J. Cell B i d . 50, 709-20. 21. Reisner AH, Rowe J, Macindoe HM. 1969. The largest known monomeric globular proteins. Biochim. Biophys. Acta 188, 196-206. 22. Reynolds ES. 1963. The use of lead citrate a t high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208-12. . 23. Rubin RW, Cunningham WP. 1973. Partial purification of basal bodies and kinetodesmal fibers from Tetrahymena pyriformis. I . Cell Biol. 57, 601-12. 24. Satir B, Rosenbaum JL. 1965. The isolation and identification of kinetosome-rich fractions from Tetrahymena pyriformis. 1. Protozool. 12. 397-405. * 25. Seaman-GR. 1660. Large-scale isolation of kinetosomes from the ciliated protozoan Tetrahymena pyriformis. Exp. Cell Res. 21. 292-302. 26. Shapiro AL, Vinuela E, Maize1 JV. 1967. Molecular weight estimation of polypeptide chains by electrophoresis in SDSpolyacrylamide gels. Biochem. Biophys. Res. Commun. 28, 815-20. 27. Sonneborn TM. 1973. Ciliate morphogenesis and its bearing on general cellular morphogenesis, in de Puytorac P de, Grain J, eds., Actualitks Protozoologiques, 4th Int. Congr. Protozool., Umv. de Clermont, Clermont-Ferrand, France, 1, 327-55. 28. Stephens RE. 1975. The basal apparatus. Mass isolation

from the molluscan ciliated gill epithelium and a preliminary characterization of striated rootlets. J. Cell Biol. 64,408-20. 29. Vaudaux P. 1972. Purification of cortical structures in Tetrahymena pyriformis. Protistologica 8, 509-17. 30. Williams NE, Frankel J. 1973. Cortical development in Tetrahymena, in Elliott AM, ed., Biology of Tetrahymena, Dowden, Hutchinson 8 Ross, Inc., pp. 375-409. 31. Wolfe J. 1970. Structural analysis of basal bodies of the isolated oral apparatus of Tetrahymena pyriformis. J . Cell Sci. 6 fi79.7nn -I