Eur. J. Biochem. Y8, 267-273 (1979)

RNA Synthesis in Starved Deciliated Tetrahymena pyriformis Lise MARCAUD and Dona1 HAYES Laboratoire de Chimie Cellulaire, Institut de Biologie Physico-Chimique, Paris (Received January 22, 1979)

Tetrahymena pyriformis which has been starved for 20 h by incubation in buffer, and then deciliated, can regenerate its cilia in about 90 min while still in suspension in non-nutrient medium. The process of reciliation is accompanied by protein synthesis which begins a few minutes after deciliation and by synthesis of ribosomal and messenger RNAs during a period extending from about 1 h to about 3 h after deciliation. Although net synthesis of RNA remains at a very low level until 1 h after deciliation, a qualitative change in the translatable poly(A)-containing messenger RNA content of deciliated cells, and in particular, formation of P-tubulin mRNA can be detected almost immediately after deciliation. Tetrahymena pyriformis is easily deciliated as described by Rosenbaum and Carlson [l] and deciliated cells, which remain viable, can regenerate their cilia and recover motility in 60-90 min. This process requires protein synthesis since it is inhibited by cycloheximide [l 1. In experiments with non-growing cells subjected to 6-h starvation in amino-acid-free medium before deciliation, Nelsen [ 2 ] observed little or no induced synthesis of tubulin during ciliary regeneration and found that an intracellular pool of tubulin was the source of this protein in reformed cilia. Guttman and Gorovsky [ 3 ] using cells deciliated after starvation for 20 h obtained somewhat different results. These authors found that cells deciliated even after prolonged starvation can regenerate cilia as rapidly as cells deciliated during exponential growth and that regeneration of cilia is inhibited completely by cycloheximide though only partially by actinomycin D. By allowing regeneration of cilia to take place in the presence of radioactive amino acids, Guttman and Gorovsky observed de novo synthesis of tubulin in deciliated cells, and incorporation of the product into new cilia; they estimated that about 30 % of the tubulin in reformed cilia was newly synthesized protein [3]. The discrepancy between the results of these two studies may be due to the different durations of starvation used. In the present study we have investigated RNA metabolism in T. pyriformis deciliated after incubation Abbreviation. Pipes, 1,4-piperazinediethanesulfonicacid. Enzymas. DNase 1 (EC 3.1.4.5); micrococcal nuclease

3.1.4.7).

(EC

for 20 h in non-nutrient medium and shown that synthesis of tubulin mRNA is induced by deciliation. MATERIALS AND METHODS Chemicals

Sucrose (Analar grade) was obtained from Hopkin and Williams (England): heparin, cycloheximide and spermidine from Sigma (U.S.A.): Nonidet P40 from Shell (France) ; formamide and Pipes (1,4-piperazinediethanesulfonic acid) from Merck (F.R.G.), poly(uridylic acid) from Miles (U.S.A.) :diethylpyrocarbonate from Fluka (Switzerland), CNBr-activated Sepharose 4B from Pharmacia (Sweden) ; RNase-free DNase and micrococcal nuclease from Worthington (U.S.A.). All other chemicals were reagent-grade products supplied by Prolabo (France) or Merck (F.R.G.). Phenol was redistilled in vacuo in a nitrogen atmosphere before use. [5-3H]Uridine (spec. act. 20Ci/mmol) was supplied by the Commissariat a I’Energie Atomique (France) and ~ - [ ~ ~ S ] m e t h i o n(spec. i n e act. 800 Ci/mmol) by the Radiochemical Centre (England). Buj’fers

Buffer 1 contained 0.1 M Tris-HC1, 0.1 M NaCl, 1 mM EDTA, pH 9.0, 250 pg heparin/ml. Buffer 2 contained 0.01 M Tris-HC1, 0.1 M NaCI, 0.01 M EDTA, pH 7.5, 0.2 sodium dodecyl sulfate. Buffer 3 contained 0.01 M Pipes-HC1, 0.01 M NaC1, 5 mM

268

MgC12, 1 mM CaC12, 1 mM dithiothreitol, pH 6.5, 100 pg spermidine/ml, 250 pg heparin/ml. Cells The amicronucleate strain GL of T. pyrijbrmis was used in all experiments. Culture Conditions Cells were grown at 28 "C in PPY medium [4] in gently shaken cultures (generation time 3.5 h). Starvation Conditions Cells were grown to a density of 4 x 1OS/ml, harvested by low-speed centrifugation at room temperature, washed with sterile 0.01 M Tris-HC1 pH 7.3, resuspended, in the same buffer at a concentration of 2 x 1OS/ml and incubated at 28 'C for 20 h with gentle shaking. Deciliation and Reciliation Starved cells concentrated to 2 x 106/ml by centrifugation were deciliated at 2 ' C as described by Rosenbaum and Carlson [l]. 2.5 ml of the concentrate was mixed with 5 ml of 0.05 M sodium acetate, 0.01 M EDTA, pH 6.0, at 2'C and 30 s later 2.5 ml of cold distilled water was added followed after a further 60 s by 0.25 ml of cold 0.2 M CaC12. The suspension was mixed after each addition and 3.5 min after the initial dilution of the cell concentrate it was passed 2-4 times through an 18-gauge needle and immediately added to 4 vol. 0.01 M potassium phosphate buffer, pH 7.0 at 25 'C. The suspension of deciliated cells thus obtained was incubated at 25 ' C with gentle shaking and regeneration of cilia was monitored by following recovery of motility by light microscope observation. In all experiments with deciliated cells, zero time is the time of dilution of cell suspensions with 4 vol. phosphate buffer. Labelling of' Cells RNA synthesized in reciliating cells was labelled by addition of [5-3H]uridine (5 pCi/ml) to suspensions of deciliated cells prepared as just described. Extraction of RNA All operations were carried out at 2 "C; glassware and solutions were sterilised before use. After collection by centrifugation, normal cells were washed and suspended in buffer 1. Starved or starved and deciliated cells, which are very fragile, were suspended in buffer 1 without washing in order

RNA Synthesis in Deciliated T. p~'r(fbrmis

to avoid premature lysis. Cells were then lysed by addition of sodium dodecyl sulfate to a final concentration of 0.5 'i: and 1 vol. water-saturated phenol containing 0.1 'i: 8-hydroxyquinoline was immediately added. Two phenol extractions were then performed followed by one extraction with 2 vol. chloroform/ isoamyl alcohol (24/1, v/v) and nucleic acids were precipitated ( - 20 'C) from the final aqueous phase by addition of NaCl to a final concentration of 0.2 M and 2 vol. cold ethanol. The precipitate was dissolved in 0.01 M Tris-HC1, 0.005 M MgClz pH 7.5 at a concentration of 1 - 5 mg/ml and treated with RNase-free DNase (60 pg/ml) for 10 min at 0 'C to remove DNA, after this the solution was deproteinized by a single treatment with phenol as before and RNA was recovered and washed by two successive precipitations (- 20°C) by 2 vol. ethanol in the presence of 0.2 M NaCI. The final product was stored under ethanol at - 20°C.

Separation of Poly ( A )-Con raining and Poly ( A )-Free Frucrion.~ of Total RNA Poly(A)-free and poly(A)-containing fractions were separated from total RNA by affinity chromatography on poly(U)-Sepharose [5] in buffer 2 in which poly(A)free RNA is not retained on the column. Poly(A)containing RNA bound to poly(U)-Sepharose was released by elution with 707; formamide in 0.01 M Tris-HC1 pH 7.5. Both RNA fractions were concentrated from column eluates by ethanol precipitation and stored under ethanol at - 20'C. Preparation oj Polysomrs All operations were carried out at 2 'C, glassware and solutions sterilised before used and stock 50 yi sucrose solutions treated with diethylpyrocarbonate before sterilisation. Protein synthesis was stopped in suspensions of growing cells (1OS/ml)or of deciliated cells by addition of cycloheximide (100 pg/ml) followed immediately by 0.25 vol. crushed frozen suspension medium ; cells were harvested by low-speed centrifugation, washed in buffer 3 [6], resuspended in the same buffer and lysed by addition of Nonidet P40 to a final concentration of 0.2%. When lysis was complete (2 min), diethylpyrocarbonate (20 pl/ml) was added to the lysate with shaking, the mixture was centrifuged at 10000 x g for 10 min and the upper two-thirds of the supernatant was removed and analysed by centrifugation for 5 h at 24000 rev./min, 4 ' C on 25-ml 1050 sucrose gradients prepared in buffer 3. After centrifugation gradients were collected and their ultraviolet absorption profiles were recorded automatically.

269

L. Marcaud and D. Hayes

Protein Synthesis in vitro Micrococcal-nuclease-treated rabbit reticulocyte lysate was prepared as described by Pelham and Jackson [7]. Incubation mixtures for protein synthesis (final volume 12 pl) contained 5 p1 of reticulocyte lysate, 85 mM KCl, 1.2 mM MgC12, 0.5 mM spermidine, 1 mM ATP, 0.2 mM GTP, 0.5 mM CTP, 20 mM creatine phosphate, 0.5 mM dithiothreitol, 0.8 pg of T. pyriformis tRNA, 0.25 pg of T. pyriformis poly(A)-containing RNA, 0.6 nmol of all amino acids except methionine, and 4 pCi of ~-[~'S]methionine. They were incubated for 1 h at 30 "C and their protein content was then recovered by acetone precipitation, denatured, and analysed by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate [8]. After electrophoresis gel slabs were stained with Coomassie blue, dried and autoradiographed. Samples of a and fl tubulins (gift of M. M. Portier), isolated from Tpyriformis cilia as described by Renaud et al. [9], were included in all gel slabs as controls. Incorporation of ~ - [ ~ ~ S ] m e t h i o ninto i n e protein per whole incubation mixture was 2 - 4 x lo4 counts/min without, and 46 x lo5 counts/min with added poly(A)-containing RNA.

RESULTS RhTA Content of Tetrahymena in Difffrent Physiological States Several studies have shown that the cellular contents of total RNA [lo, 111 and ribosomes [12,13] fall considerably during starvation of Tetrahymena. Measurement of the amounts of total and poly(A)-containing RNA in Tetrahymena in the various physiological states examined in the present study (Table 1) confirm this observation and show that it extends to poly(A)-containing RNA. As expected, the cellular RNA contents of total and poly(A)-containing RNA do not change significantly during reciliation of starved deciliated cells (incubation in non-nutrient medium).

Table I . R N A conten/ of Tetrahymena pyriformis C L bi i w k m s pliysiohgiwl states Total RNA was extracted from cell samples (2-11 x 10' cells) harvested in various physiological states and divided into poly(A)containing and poly(A)-free fractions as described in Materials and Methods. The yields of the two fractions were mesaured as per cell sample and are expressed as the weight RNA per lo6 cells Cell sample

RNA content total RNA

poly(A)-containing RNA

pg/1O6 cells (?,, total) ~

Exponential

370

5 3 (1 4)

Starved for 24 h

130 136

1 09 (0 84) 1 3 6 (1 0)

120 100 88 89

1.3 1.5 1.1 1.1

Starved for 24 h, deciliated (time after deciliation) 0 min 20 min 75 min 150 min

(1.1) (1.5) (1.25) (1.2)

Table 2. R N A Sytithesi.r in deciliated T. pyriformis A preparation of T. pyrifovmis starved by incubation for 20 h in 0.01 M Tris-HCI p H 7.3 was divided into four equal aliquots. Cells in three of the aliquots were deciliated (Materials and Methods) and the fourth was adjusted to the volume of the suspensions of the deciliated cells with 0.01 M potassium phosphate bulrer pH 7 (control cells). At zero time of reciliation (see Materials and Methods) and immediately after dilution of control cells. each suspension was divided into four equal samples, actinomycin (20 pg/ml) was added to one set of four samples of deciliated cells and cycloheximide (5 pg/ml) t o a second set, and all samples were incubated at 25°C with gentle shaking. [3H]Uridine (5 pCi,nil) was added to one sample of each set four at zero time of deciliation and to the three remaining samples after I , 2 and 3 h of incubation. In corporation of [3H]uridine was allowed to proceed for 1 h in all cases, cells were then harvested and total RNA was extracted and its specific activity measured as described in Materials and Methods Duration of labelling after deciliation

R N A Synthesis in Deciliated Cells

h

Table 2 shows the results of measurement of the incorporation of tritiated uridine into RNA in starved deciliated and in control starved non-deciliated cells during four successive 1-h periods after deciliation. It can be seen that incorporation of uridine into the RNA of control cells remains at a very low level throughout the 4-h incubation. In contrast, incorporation in deciliated cells, which is comparable to that in control cells during the first hour after deciliation, then increases substantially reaching a maximum between 2 and 3 h after deciliation. Table 2 also shows

0- 1 1-2 2-3 3-4

Specific activity of total RNA --

control cells

~~

deciliated cells

counts 3H min-' pg-' ___~ 530 390 170 6400 175 9300 130 41 00 ~~

deciliated cells + actinomycin

deciliated cells + cycloheximide

~~

-

10 ~

32 10 5 ~

that uridine incorporation in deciliated cells is completely inhibited by both cycloheximide and actinomycin. Although these results can be interpreted as evidence that deciliation of T. pyriformis induces RNA synthesis after a lag of 1 h they could also be

210

RNA Synthesis in Deciliated T. pyriformis

the table show that the relative amounts of radioactivity incorporated into the poly(A)-containing and poly(A)-free RNA fractions change detectably during the first hour after deciliation. The ratio of incorporation into these two RNA fractions continues to change as the reciliation process proceeds, eventually (2 - 3 h) reaching a value comparable to that observed in normally growing cells.

explained as the result of refeeding of the starved cells brought about by lysis of a fraction of the preparation during the deeiliation process and consequent release of cell constituents into the reciliation medium. In order to test this possibility, a suspension of deciliated cells prepared in the standard manner was incubated for 10 min at 25 "C (Materials and Methods) and the suspension medium was then recovered by filtration through a nitrocellulose membrane under sterile conditions. This medium was then used to repeat the control experiment using starved non-deciliated cells the results of which are given in Table 2. The results obtained were identical to those observed when the starved non-deciliated cells were suspended in untreated regeneration medium. We therefore conclude that refeeding of deciliated cells by material released from any cells which are lysed during the deciliation process does not contribute significantly to the RNA synthesis observed in Tpyriformis during the later stages of ciliary regeneration.

Polysome Metabolism in Starved Deciliated Cells Comparison of the polysome contents of exponentially growing, starved, and starved deciliated cells harvested at various times during reciliation gave the results shown in Fig. 1. The polysome content of starved cells is very small (compare data in Fig. 1 A and B) but increases rapidly after deciliation (compare sedimentation profiles in Fig. 1 B). Polysomes of all size classes are formed progressively during the reciliation process, and after 45 min of incubation more than half the total ribosome content of the cells is present in polysomes whose sedimentation profile does not differ qualitatively from that observed in exponentially growing cells (data in Fig. 1A and D). Moreover the discontinuous profiles in Fig. l C and D show that reformation of polysomes in deciliated cells is only partially inhibited by the presence of actinomycin. Since it has already been shown that actinomycin completely inhibits RNA synthesis in deciliated cells (Table 2), it follows that polysome formation in actinomycin-treated deciliated cells must take place by mobilisation of pre-existing messenger RNAs. The results obtained in these experiments show that protein synthesis (polysome formation) is induced by deciliation of starved cells and is directed, at least in part, by mRNAs synthesized before deciliation. They agree with those of studies of the effects of cycloheximide and actinomycin on regeneration of cilia which showed

Nature o j R N A Synthesized in Deciliated Cells Table 3 contains the results of analyses of RNA synthesized in deciliated cells during regeneration of cilia. Both poly(A)-containing and poly(A)-free RNA are formed throughout the reciliation process. Polyacrylamide gel analysis of these RNA fractions showed that, as expected, the poly(A)-free material contained essentially ribosomal and transfer RNAs together with the 39-S rRNA precursor previously described [14] and the poly(A)-containing material contained RNA species migrating in a broad peak centred between 16-S and 25-S rRNAs (results not shown). The results in Table 3 show that, as already discussed, RNA synthesis in deciliated cells does not significantly exceed that observed in non-deciliated control cells during the first hour after deciliation. However, later data in

Table 3. Synthesis oJpol,v(A)-contuininR undpoly(A)-free R N A in control and deciliated T. pyriformis Samples of deciliated, and starved non-deciliated T. pyrifirmis were prepared and labelled with [3H]uridine during successive 1-h periods of reciliation (see legend to Table 2 for full details). Non-deciliated control cells were labelled for 1 h immediately after dilution into reciliation medium. Total RNA extracted from each sample of labelled cells was separated into poly(A)-containing and poly(A)-free fractions by chromatography on poly(U)-Sepharose (Materials and Methods) and the specific activities of the two R N A fractions and the percentage of the total radioactivity present in each were determined RNA source

Specific activity of ~~~

Radioactivity in -

~

PIY(A)free R N A

POIY(A)containing R N A

counts min-' pg-' -

Non-deciliated starved cells Deciliated cells labelled from 0 to 1h Deciliated cells labelled from 1 to 2 h Deciliated cells labelled from 2 to 3 h

4400 6 200 33 500 70 000

-

~

~

POlY(A)free RNA %total

-

~~~

70000 66 000 250000 330000

55 65 71 80

~

~~

POlY(A)containing RNA

-~ ~~~

45 35 29 20

L. Marcaud and D. Hayes

271

0

1.c

Q5

C +GI

1.c

+B

a5 0

-l 0

8 u

2 1.0 8 1? D a

0.5

I

II:

75

45

20

0

0

Time after deciliation (rnin) 0 1D

A

a5

0

Fig. 1. Sedimentation profiles ofpolysomes extractedfrom T. pyriformis in variousph~siological.~tates. Cycloheximide(lO0 pg/ml) followed immediately by 0.25 vol. of crushed frozen suspension medium was added to aliquots of exponentially growing cultures or of suspensions of starved, or starved deciliated T. pyrijormis containing about l o 7 cells; the cells were then harvested, washed, and lysed, and polysomes were prepared as described in Materials and Methods. The polysome suspensions were adjusted to a concentration of 15 AZ6" units/ml and 1-ml aliquots were analysed by centrifugation on 10-50x linear sucrose gradients as described in Materials and Methods. (A) Polysomes from exponentially growing cells (---); (B) polysomes from 20-h starved cells (----) and from starved deciliated cells harvested after 10 min of reciliation (--); (C) polysomes from starved deciliated cells harvested after 20 rnin of reciliation in the absence (-) and in the presence of actinomycin (20 pg/ml) in the reciliating medium (----); (D) polysomes from starved deciliated cells harvested after 45 rnin of reciliation in the absence (-----) and in the presence of actinomycin (20 pg/ml) in the reciliating medium (----). Direction of sedimentation is from left to right

that the former completely inhibits reciliation [l - 31 whereas the latter inhibits it only partially [3].

Messenger R N A Species Synthesized during Reciliation As just shown, the amount of polysomal mRNA in starved cells begins to increase very soon after deciliation, some of this increase being caused by mobilisation of pre-existing messenger RNAs and some by entry of newly synthesized mRNA into poly-

Fig. 2. Pol~~acrylarnide/dode~ylsu~ate gel electrophoresis patterns qf the products of translution in vitro of poly(A)-containing R N A extractedfrorn exponentially growing and 20-h starved T. pyriformis and from starved deciliated cells harvested at various times during reciliation. Total RNA extracted from 7:pyriformis (5 x 10' cells) harvested during exponential growth (I) after starvation of cells for 20 h (11) and at increasing times (0, 20, 45, 75 min) during reciliation of starved deciliated cells, was separated into poly(A)containing and poly(A)-free fractions by chromatography on poly(U)Sepharose (Materials andMethods). Samples of the poly(A)containing RNA preparations (0.25 pg) were used to direct protein synthesis in a reticulocyte lysate system in the presence of L-[~'SS]methionine (4 pCi per incubation mixture, final volume 12 PI). Synthesized proteins were analysed by electrophoresis in 12 "; polyacrylamide/dodecylsulfate gel slabs (4 x 10' counts/min in each sample) and after electrophoresis gel slabs were dried stained and autoradiographed. Control samples of non-radioactive tubulins isolated from Tpyrijormis cilia were included in gel slabs. The radioactive bands designated x and p (arrows) in the autoradiograph of the dried gel slab (a photograph of which is shown in the figure) were identified by reference to the positions of the stained bands of GI and J/' tubulin. Direction of electrophoresis if from top t o bottom

somes. In addition, although a large proportion of the tubulins incorporated into newly formed cilia in reciliating cells is derived from a pre-existing intracellular pool of these proteins [2,3], de novo synthesis of tubulin takes place during reciliation [3]. It can therefore be deduced that reciliating cells contain functional mRNAs for tubulins. In order to determine whether these mRNAs are conserved in starved cells or synthesized after deciliation the products of translation in vitro of poly(A)-containing RNAs, extracted from starved and from starved deciliated cells, were analysed by polyacrylamide/dodecyl sulfate gel electrophoresis. Fig. 2 compares the products of translation of poly(A)-containing RNA of exponentially growing cells, of cells subjected to 20 h of starvation, and of starved deciliated cells harvested after various

212

periods of incubation in reciliation medium. It can be seen that prolonged starvation of cells reduces their content of mRNAs corresponding to almost all the major protein bands observed in the products of translation of poly(A)-containing RNA of exponentially growing cells, including z and p tubulins. A notable exception is the mRNA or mRNAs whose translation products form the very intense band in the lower half of the electrophoresis pattern of the products of translation of poly(A)-containing RNA of starved cells. The products of translation of poly(A)-containing RNA extracted from starved deciliated cells 1.25 h after deciliation contain increased amounts of several of the bands which are prominent in the electrophoretic pattern of proteins produced by translation of poly(A)-containing RNA of exponentially growing cells, the increase being particularly noticeable in the case of the p-tubulin band. Thus deciliation of starved T. pyrifwmis seems to lead to selective formation of a group of mRNAs including p-tubulin mRNA. Fig. 2 also shows that enhanced synthesis of p tubulin relative to the amount formed by translation of poly(A)containing RNA of starved cells is in fact detectable in the products of translation of mRNA extracted from cells at the experimental zero time of reciliation, i.e. about 4- 5 min after the beginning of the deciliation process (see Materials and Methods). Furthermore 20 min after deciliation the P-tubulin mRNA content of deciliated cells has reached the level found in cells 1.25 h after deciliation. Thus deciliation of T. pyrijormis causes a very rapid change in the pattern of translatable mRNAs in starved cells and, in particular, induces synthesis of new (or activation of preexisting inactive) p-tubulin mRNA.

DISCUSSION The experiments described here establish the following characteristics of RNA metabolism in starved, and in starved deciliated cells. a) RNA synthesis in starved Tpyrijormis is reduced to a very low level and cells contain only small residual amounts of polysomes. The ratio of the incorporation of radioactive precursors into poly(A)free RNA (rRNA, tRNA) and poly(A)-containing RNA (mRNA) in these cells is about 1. b) During the first hour after deciliation, regeneration of cilia and recovery of motility are largely completed and extensive reformation of polysomes occurs, although RNA synthesis remains at a very low level. Polysome reformation is due partly to mobilisation of conserved mRNA since it is only partially inhibited by actinomycin. Although RNA synthesis remains unchanged during the first hour after deciliation the ratio of incorporation of radioactive precursor into poly(A)-free and poly(A)-containing RNAs rises to

R N A Synthesis in Deciliated T. pyriformis

about 2 during this period and a considerable change in the composition of the translatable mRNA pool of the cells including, in particular, an increase in the amount of J-tubulin mRNA is observed. c) At about an hour after deciliation RNA synthesis in reciliating cells increases rapidly reaching a peak between 1 and 2 h later, after which it decreases. During the period of increased RNA synthesis between 1 and 3 h after deciliation the ratio of incorporation of radioactivity into poly(A)-free and poly(A)containing RNA rises to 4, a value which is similar to that found in exponentially growing cells. Although the RNA synthesis observed in deciliated cells is not required fur the regeneration of cilia with which it is contemporary, several results show that it is related to this process: (a) it is not induced by those stages of the deciliation process which do not cause loss of cell mobility, but is induced by those that do (results not shown); (b) it is not due to artefactual refeeding of deciliated cells caused by lysis of a fraction of the population during the deciliation process; (c) starved deciliated cells which have reformed cilia can do so again after a second deciliation while still in non-nutrient medium, but a second reciliation is impossible if the first takes place in the presence of actinomycin (unpublished results). The latter result suggests that the mRNA synthesis observed after deciliation is necessary to replenish pools of mRNA species which are depleted during reciliation. However extensive synthesis of poly(A)free RNA (rRNA, tRNA) in deciliated, cells cannot, at present, be accounted for. Induction of synthesis or activation of latent B-tubulin mRNA by deciliation of starved T. pyriformis recalls the similar situation which exists in gametes of Chlamydomonas reinhardi during flagellar regeneration [15] and in sea urchin embryos [16]. Throughout this discussion reference is made only to j-tubulin mRNA for the following reasons. Of the two bands designated CI and p tubulin in the products of translation in vitro of poly(A)containing RNA of T.pyriformis, the p band has been identified with certainty but some doubt still remains concerning the protein or proteins present in the CI band. In addition during translation of 7:pyriformis mRNA in vitro, the band is consistently more highly labelled than the CI band. These observations, which are not central to the argument of the present study, are discussed in detail elsewhere in this journal [17]. Financial support for the work described here was provided by grants from the Centre Nurionul de la Recherche ScientifYque (Equipe de Recherche 101, ATP contract 419905; R.C.P. 386). the Delegation a la Recherche ScientqYque et Technique (ACC 7570199), the Fonds de la Recherche MPdicale Franfaise and the Commissuriut d I’Energie Aromique. The authors thank Drs M . M . Portier and C. E. Sripati for advice and discussion, Mrs M. Milet for expert technical assistance and Mrs Tichonicky for help with the preparation of anemic rabbits.

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REFERENCES 1. Rosenbaum, J . L. & Carlson, K. (1969) J . Cell B i d . 40, 415425. 2. Nelsen, E. M. (1975) Exp. Cell Res. 94, 152- 158. 3. Guttmann, S. D. & Gorovsky, M. A. (1975) J . Cell Bid. 67, 147a. 4. Hjelm, K . K. (1970) €xp. Cell Res. 60, 191-198. 5. Gray, R. E. & Cashmore, A. R. (1976) J . Mol. B i d . 108, 595- 608. 6. Sripati, C. & Warner, J. R. (1978) Methods Cell B i d . 20, 61 -81. 7. Pelham, H. R. B. &Jackson, R. J. (1976) Eur. J . Biochem. 67, 247-256. 8. Studier, F. W. (1973) J . Mol. B i d . 79, 237-248. 9. Renaud, F. L., Rowe. A. J. & Gibbons. J . R. (1968) J . Cell Biol. 36. 79-90

10. Koroly, M. J. & Conner, R. L. (1974) J . Prorozool. 21, 169177. 11. Conner, R. L. & Koroly, M. J. (1974) J . Protosool. 21, 177182. 12. Hallberg, R. L. & Sutton, C. A . (1977) J . Cell B i d . 75, 268276. 13. Kristiansen, K. & Kruger, A. (1979) € . ~ p . Cell Res. 118. 159- 169. 14. Pousada, C. R., Marcaud, L., Portier, M. M. & Hayes, D. H. (1975) Eur. J . Biochem. 56, 177-122. 15. Weeks, D. P. & Collis, P. S. (1976) Cell, 9, 15-27. 16. Merlino, G . T., Chamberlain, J. P. & Kleinsmith, L. J. (1978) J . B i d . Chew. 253, 7078-7085. 17. Portier, M. M., Milet, M. & Hayes, D. H. (1979) Eur. J . Biochem. 97, 161 - 168.

L. Marcaud, Laboratoire de Chimie de la Differenciation, Institute de Recherche en Biologie Moleculaire, Universite de Paris VIII, Tour 43, 2 Place Jussieu, F-75231 Paris-Cedex-05, France D. Hayeb*, Laboratoire de Chimie Cellulaire, lnstitut de Biologie Physico-Chimique, 13 Rue Pierre-et-Marie-Curie, F-75005 Paris, France

* To whom correspondence should be addressed