Synchronizing temperatures and rRNA metabolism in Tetrahymena pyriformis

Comp, Biochem. Phy,siol.. Vol. 64B. pp. 167 to 173

0305-0491/79/1001-0167502.00/0

© Pergamon Press Lid 1979. Printed in Great Britain

SYNCHRONIZING TEMPERATURES A N D rRNA METABOLISM IN T E T R A H Y M E N A P Y R I F O R M I S WALTER KAFFENBERGER,BETTINAHEMMERICHand WERNER A. ECKERT Department of Physiology, Institute of Zoology, University of Heidelberg, Im Neuenheimer Feld 230, D-6900 Heidelberg, Federal Republic of Germany

(Received 20 February 1979) Abstract--1. In Tetrahymena pyriformis pre-rRNA is synthesized and efficientlyprocessed and translocated into the cytoplasm at both a supraoptimal (34°C) and a suboptimal (8°C) synchronizing temperature. 2. At the heat shock temperature (34°C) no substantial differences in the kinetics of the main intracellular events of rRNA-metabolism compared to the optimal growth temperature (28°C}were found. 3. The high temperature, however, induces a strong retardation of uptake of Jail]adenosine into the cells and a reduction of the cellular ATP pool size. 4. At the cold shock temperature (8°C) the rates of transcription and nucleocytoplasmic transport of rRNA as well as of nucleotide pool equilibration are reduced to a similar extent (25-30°4 of the optimal rates at 28°C). 5. The results are discussed and compared with the effects of sub- and supraoptimal temperatures on rRNA synthesis and processing in mammalian cells. INTRODUCTION

In the present paper we present results on the synthesis, processing, nucleocytoplasmic translocation and stability of rRNAs in Tetrahymena pyriformis at the optimal growth temperature (28°C) as well as at the supraoptimal (34°C) and at a low (8°C) synchronizing temperature (cf. Zeuthen, 1964). These results are compared with the effects of sub- and supraoptimal temperatures of rRNA synthesis and processing in mammalian cells.

Cell division can be synchronized in Tetrahymena cultures by repetitive heat (Scherbaum & Zeuthen, 1954; Zeuthen, 1971) or cold shifts (PadiUa & Cameron, 1964; Zeuthen, 1964). The precise mechanism by which these procedures induce synchronization is not yet clear (cf. Zeuthen, 1971). Some investigators have supposed the basic molecular effect to be an interference with RNA metabolism (e.g. Moner & Berger, 1966; Moiler, 1967; Byfield & Scherbaum, 1967a; MATERIAL AND METHODS Christensson, 1970: Lane & Padilla, 1970; Yuyama, 1975) leading to the inhibition of specific proteins ("division proteins", cf. Rasmussen & Zeuthen, 1962, Culture conditions Watanabe & Ikeda, 1965; Watanabe, 1971). There- • Axeniccultures of Tetrahymena pyriformis, (amicronucfore, various authors have studied mainly the alter- leate: formerly strain GL, optimal temperature of growth: ations of unstable, messenger-like RNA fractions in- 28-29°C) were grown in 2Yo(w/v) proteose peptone (Difcol duced by these conditions (e.g. Christensson, 1970; • supplemented with 0.4% (w/v) liver extract {Wilson LaborByfield & Lee, 1970a,b; Yuyama, 1975). Little is atory, Chicago, IL) or in a medium according to Neff et al. (1964:0.1 mM ferric citrate, 2 mM KH2PO4, 0.05 mM known about the effects of temperature shifts on the CaCI2, I mM MgSO4, 1.5% glucose, 0.75% yeast extract metabolism of stable RNA, especially rRNA, and the (Difco), 0.75% proteose peptone). For nucleotide pool sparse data that exists is, at least in part, contradic- determination, cells were grown in chemically defined tory or unlikely. For example, some authors have medium according to Holz et al. 0962) with the following reported that synthesis and processing of rRNA is modifications: Na2-guanosine-Y {2') phosphoric acid x markedly reduced at the supraoptimal synchronizing 2 H20 and uracil were replaced by guanine and uridine. temperature in Tetrahymena pyriformis (34°C: respectively, and adenosine and cytosine {each Yuyama & Zimmerman, 1972; Hermolin & Zimmer- 5mg/100mi) were added. In all experiments logarithmiman, 1976), whereas others conclude from indirect cally growing cells (population density: 50-200,000 results that these processes should not be influenced cells/ml) were used. at all at this temperature (de Barros et al., 1973). On Labellino and fractionatinff of the cells the other hand, a total cessation of nucleocytoplasmic Usually 50-100 ml cultures were labelled with [5'-aH]ur transport of stable RNA was reported to occur when idine or [2'-3H]adenosine {specific radioactivity: 20--30Ci optimally growing cells (28°C) were cooled below mmok The Radiochemical Centre, Amersham, U.K.) b) 10°C (Wunderlich et al., 1974), although the cells direct addition of these compounds to the culture flasks. easily adapt to these low temperatures (Frankel, The specific concentrations are indicated in the legends 1967). In the majority of these investigations either to the figures. In most of the "pulse-chase" experiments. the methods used for separation and characterization cells from 400-600 ml cultures were collected by centrifugaof the different rRNA fractions were insufficient or tion (lO00ff, 3min) and incubated with [3H]uridine (20--25#Ci/mf) in a small volume (20-30 ml} for 8-10 rain. the effects of possible changes of nucleoside uptake then washed in and transferred to fresh culture medium on RNA labelling kinetics during the temperature with unlabelled uridine {"chase"), essentially as described shifts were not considered. previously {Eckert et al., 1975). In some cases, cells were 167

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WALTER KAFFENBERGER,BE'rTINA HEMMERICHand WERNERA. ECKERT

labelled under optimal growth conditions directly in the culture flasks. Cells were usually fractionated into pure macronuclei and microsomes as described in detail previously (Eckert & Franke, 1975). For the in vitro experiments, macronuclei were prepared by a modification of the method of Gorovsky (1970) as described elsewhere (Kaffenberger & Eckert, 1979). Determination of [3H]uridine incorporation into total cellular R N A and of [3H]UTP into isolated macronuclei [3H]uridine incorporation into acid-precipitable material of Tetrahymena cells was determined as described (Eckert et al., 1975). For determination of [3H]UTP incorporation into isolated macronuclei an in vitro system modified from Byfield et al. (1970) was used. The final concentrations of the constituents were 5 mM MgCI2, 60raM each KC1 and NaCI, 1 mM dithiothreitol, 0.5 mM ATP, 0.05 mM each GTP and CTP, 0.025 mM UTP, 0.5 mM phosphoenolpyruvate, 10pg/ml pyruvate kinase and 30mM Tris-HCl, pH8.0. To this solution was added [3H]UTP (uridine-[5'-6'-3H]triphosphate, ammonium salt; specific radioactivity: 41 Ci/mmol; The Radiochemical Centre, Amersham, U.K.) to give a final radioactivity of 2-3/~Ci/ml as well as ct-amanitin in some of the assays to a concentration .of 5 pg/ml. The volume per assay was 0.8 ml. The reaction was started by addition of 0.2 ml of a suspension of isolated macronuclei (5 x 106-10 ~, pretreated or not with 5/~g/ml ~-amanitin for 5 min, in 40?/0 (w/v) sucrose with 50 mM Tris-HCl, pH 8.0). The reaction was stopped after various times by a 5-fold excess of an ice-cold 10To solution of trichloroacetic acid with 20 mM sodium pyrophosphate and 300,ug/ml unlabelled UTP. The precipitate was placed on glassfibre filters (Whatman GF/C), washed several times with ice-cold 5~o trichloroacetic acid, and then with a large volume of 30~ ethanol. Subsequently, the filter discs were dried, put into counting vials, and incubated with 0.5 ml NCS (Nuclear Chicago Solubilizer, Amersham-Searle) for 2 hr at 50°C. Toluenebased scintillation fluid (8 ml) was added, and radioactivity was determined in a Packard Tri-Carb liquid scintillation spectrometer, DNA contents of the nuclei were determined according to Burton (1956). Determination of the size and .specific radioactivity of the A T P pool After specific labelling times with [3H]adenosine, aliquots (usually 5(~75 ml) were removed from the cultures, rapidly cooled, and centrifuged (1000 0 for 3 min) at 2°C to a loose'pellet, The cells were immediately resuspended in 50 ml of cold inorganic medium (Hamburger & Zeuthen, 1957) and an aliquot was removed for cell counting in a Fuchs-Rosenthal counting chamber (Hecht, Sondheim, GFR). The cells were then collected quantitatively by high speed centrifugation (3000 g, 5 min) and treated twice each with pre-cooled ( - 2 f f C ) acetone and ether and dried overnight at -80°C in a dessicator essentially as described by Plesner (1964). The dried cell powder was extracted three times with 0.5 N HCIO,~ in the cold (4°C) with the aid of a small glass homogenizer. The acid extract was neutralized by successive addition of IN, 3N and 6N KOH in the presence of 0.05 M Tris-HCl, pH 7,2 and the precipitated potassium perchlorate (KCIO,0 was removed by centrifugation. For separation of ATP, 100-200#1 of the neutralized extracts were chromatographed in two dimensions on poly(ethylenimine)-cellulose sheets (20 × 20cm, PEI, CEL 300; Macherey-Nagel, Diiren, GFR) with a procedure modified from Randerath & Randerath (1967). The plates were developed in the first dimension with 0.2 M LiCI for 2 min, 1 M LiCI for 6rain and 1.6M LiC1 up to the top. After washing with methanol, the plates were developed in the second dimension with 0.5 M (NH4)2SO4 in the cold (4"C). The ATP spots were localized under u.v. light, cut

out and eluted with 1 ml of 0.7 M MgCI2-2.0 M Tris- HCI, pH 7.4 (100:1 v/w) for 0.5 hr at room temperature (cf. Randerath & Randerath, 1967). After removal of the contaminants by centrifugation at 2000 g for 5 min, the absorbance of the eluate at 260 nm was measured and radioactivity was determined in a Packard Tricarb scintillation spectrometer after adding 10 ml of lnsta-gel (Packard Instruments) to the samples in counting vials. Preparation and fraetionation of R N A RNA was extracted by a modification of Kirby's cold phenol-cresol procedure {Kirby, 1968, cf. Eckert et al.. 1975). Macronuclei were first digested with protemase K. and DNA was removed by desoxyribonuclease 1 treatment before the "phenol-cresol'-extraction procedure (cf. Eckert et al., 1978). RNA was separated either on 2.2-2.4°<~ as well as 7.5% cylindrical polyacrylamide gels according to Loening (1969; cf. Eckert et al.. 1978) or on slabs of 0.5Q~, agarose-2.25% acrylamide composite gels according to Ringborg et al. (1970; cf. Eckert et al., 1975). Unlabelled E. coli rRNA was added to some of the RNA samples as internal tool. wt standard (23S: 1.05 × 1 0 6 and 16S: 0.525 × 106 daltons; cf. Brosius et al., 1978). Gels were run and assayed for absorbance at 260 nm and radioactivity was determined as described previously (Eckert et al., 1975, 1978).

RESUt,TS When Tetrahymena pyriformis cells growing at the optimal temperature (28°C) are cooled below 14-16°C or warmed up to 33-34°C cell division is strongly delayed ("excess delay"; cf, Thomar, 1959; FrankeL 1967; Zeuthen, 1971) or inhibited. This effect is reversible upon returning the culture to the optimal temperature of growth and is the basis for the different temperature shock synchronization methods developed for Tetrahymena cells (for refs see Introduction). It has already been shown that the rate of [3HJuridine incorporation into total cellular R N A is significantly reduced at both the supraoptimal (34°C) and suboptimal (8°C) synchronizing temperatures (e.g. Byfield & Lee, 1970a, b; de Barros et al,, 1973; Wunderlich et al., 1974}. In our experiments we found a reduction of this rate to less than 505~ and 10~°~, at 34 and 8°C, respectively, compared to the optimal temperature of growth (Fig. 1). The lower rate of labelling of cellular RNA at 34~C can be roughly explained by the similar large reduction of the initial equilibration rate of exogenous nucleosides with the cellular nucleotide pools (specifically of [3H]adenosine with the ATP-pool: Fig. 2). Interestingly, the pool size of A T P is reduced by about 50 °/ after 30 min incubation of the cells at 34°C (Table 1), which indicates that utilization of nucleotides initially is greater than uptake and phosphorylation of nucleosides at this temperature. At 8"C the lower rate of uptake of exogeneous nucleosides into the intracellular nucleotide pools (about 25°,~;of that at the optimal temperature!), however, can account only in part for the strongly reduced RNA labelling kinetics. To study directly the effects of these synchronizing temperatures on RNA synthesis, the relative rates of RNA transcription were determined in the absence of cytoplasmic nucleoticle pools by [ 3 H ] U T P incorporation into isolated macronuclei. Figure 3 shows that under our in vitro conditions macronuclei isolated from logarithmically growing Tetrahymena cells efficiently incorporate [ 3 H ] U T P into polynucleotide

169

Synchronizing temperatures and rRNA metabolism in Tetrahymena Table 1. Size of the ATP pool in Tetrahymena cells at optimal growth conditions (28°C) and after a 30 rain shift to a supraoptimal temperature (34°C) Growth temperature

Pool size (pmols/106 cells)

28°C 34°C

15700 + 3850(4) 8530 + 1420 (4)

0

E E x

Logarithmically growing Tetrahymena cultures (200 ml) in chemically defined medium were divided. One half was left at the optimal growth temperature (28°C), the other was heated to 34°C. After 30 min the cells from both portions were harvested and counted. Nucleotides were extracted and separated by two-dimensional chromatography on PEl-cellulose sheets. The ATP spots were cut out, eluted and determined quantitatively as described in Methods. The results are mean values (+ SE) from four determinations.

chains for at least 60 min at 28°C. Of the total RNApolymerase activity measured in these macronuclei 60-70% is resistant to 5 #g/ml ~-amanitin (Fig. 3), which approximately corresponds to the proportion of silver grains associated with the numerous extrachromosomal nucleoli in electron microscope autoradiographs of likewise incubated isolated macronuclei (Kaffenberger & Eckert, 1979). That the polynucleotide chains synthesized (dongated!) in vitro in the presence of ~t-amanitin are actually pre-rRNAs and thus due to the activity of RNA-polymerase A is also evident from their electrophoretic pattern, their high specific methylation and their exclusive hybridization to the r D N A region in Cs2SO,,-Hg 2+ gradients of macronuclear D N A (Kaffenberger & Eckert, 1979). In Table 2 the transcriptional activities of isolated macronuclei incubated for 20 min (a period in which

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Fig. 2. Labelling kinetics of cellular ATP pool at different growth temperatures. A logarithmically growing Tetrahymena culture in chemically defined medium at the optimal growth temperature (28°C) was divided into 3 portions. One portion was warmed up to 34°C (O O), another rapidly cooled to 8°C (A A) and the last one left at 28°C (O O), and all were incubated after 30 min with [~H]adenosine (1 pCi/ml; zero time). At the times indicated samples were removed, nucleotides extracted and separated by two-dimensional chromatography on PEIcellulose sheets as described in the Experimental section. The ATP spots were localized, cut out and eluted, and absorbance at 260 nm as well as radioactivity of the eluate was determined. the [ 3 H ] U T P incorporation rate is very high and degradation can be almost excluded or neglected, cf. Fig. 3) at the different temperatures are given. At the supraoptimal temperature (34°C) the total amount of

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Fig. 1. Incorporation of [3H]uridine into total RNA of cells grown at 28°C (optimal temperature), 34°C and 8°C, respect!vely. A logarithmically growing culture in complex medium was divided into 3 portions, which were incubated with [3H]uridine (1 pCi/ml) at 28°C ( x x ), 34°C (O O) and 8°C (O O), respectively. Aliquots were removed at the times indicated, precipitated with ice-cold 10% w/v trichloroacetic acid and radioactivity and protein was determined.

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Fig. 3. Kinetics of [~H]UTP incorporation into isolated macronuclei from Tetrahymena at 28°C and 8°C, respectively. Macronuclei (Mn) isolated from logarithmically growing cells were incubated with i-3HIUTP in an in vitro system with and without ct-amanitin (see Materials and Methods). At the times indicated aliquots were removed from the assays, treated with trichloracetic acid (10% w/v), and the precipitated material was collected on glass-fibre filters. The filters were washed and prepared for radioactivity determination as described in the Methods (S 0, Mn incubated at 28°C without ~-amanitin; O O, 28°C plus ~t-amanitin (5/~g/ml); • • 8°C without ct-amanitin; [] [] 8°C plus a-amanitin).

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WALTER KAFFENBERGER.BETTINA HEMMERICHand WERNER A. ECKER'I

Table 2. Effect of various temperatures on the in vitro RNA-transcription rate in isolated Tetrahymena macronuclei Specific conditions of in ritro incubation

:t-Amanitin resistance (°i~)

[3H]UMP incorporation (cpm/pg DNA)

28 C 28 C + ~-amanitin 34 C 34C + ct-amanitin 8C 8 C + ~-amanitin

2306 1408 2075 1393 719 437

64.4 66.6 61.2

Macronuclei isolated from logarithmically growing cells were incubated in vitro with [3H]UTP for 20 min at 28, 34 and 8°C, respectively. The assays were with or without ~t-amanitin (5 ,ug/ml), essentially as described in the legend to Fig. 3.

radioactivity incorporated into polynucleotide chains is slightly less than at the optimal growth temperature (28°C), whereas the ~t-amanitin-resistant incorporation is nearly identical at both temperatures. At the low temperature (8°C) the ~-amanitin-resistant RNA synthesis is somewhat less than 30~o of the rate at optimal growth temperature which corresponds to an average Ql0 of about 1.7 for the in vitro rRNA transcription process within the range of 8-28°C. In order to study the effects of the low and high synchronizing temperatures on processing as well as nucleocytoplasmic translocation of pre-rRNA, pulsechase experiments were performed. When Tetrahymena cells were pulse-labelled with [3H]uridine for 5-8 rain at 28°C, radioactivity is essentially associated with the nuclear pre-rRNA fractions (cf. Eckert et al., 1978) and almost no radioactivity is found in cytoplasmic mature rRNAs (not shown; cf. Eckert et al., 1975). Figure 4 shows the gelelectrophoretic pattern of nuclear and microsomal RNA of such pulselabelled cells after a chase of 60 min at the optimal growth temperature (28°C) and 8°C, respectively. Only a small amount of radioactivity is still found in the region of nuclear rRNA precursors (fraction A D; cf. Eckert et al., 1978) from the cells "chased" at 28°C (mainly associated with the direct nuclear precursor to the cytoplasmic 26SrRNA; fraction B, Fig. 4A) and the mature cytoplasmic rRNAs (26 and 17S) possess high specific radioactivities (Fig. 4B), indicating that under optimal conditions most of the newly synthesized nuclear pre-rRNA has been efficiently processed and transported to the cytoplasm during this chase. After a chase at the low temperature, however, a substantial retention of labelled prerRNA fractions can be recognized in the macronucleus (Fig. 4A), correlated with a reduced appearance of newly synthesized mature rRNAs in the cytoplasm (about 25~o of the control, Fig. 4B). When similar chase experiments were done at 34 and 28°(3, the gelelectrophoretic radioactivity patterns showed no substantial differences during the whole chase period (not shown), indicating that the velocity and efficiency of processing and nucleocytoplasmic translocation of RNA is very similar at both temperatures (for the influence of the nucleotide pools on the chase kinetics see Eckert et al., 1975). It has been reported that exposure of Tetrahymena cells to supraoptimal temperature leads to a degrada-

tion of newly synthesized nuclear as well as cytoplasmic RNAs (e.g. Byfield & Scherbaum, 1966, 1967b; Byfield & Lee, 1970a). In the pattern of nuclear and cytoplasmic rRNA fractions, however, we found no substantial changes even during long-time exposures (3 hr) of the cells at 34°C (Fig. 5). In particular, the relative steady-state proportions (!) of the nuclear preRNA fractions (A-D) are very similar at both temperatures, except for a somewhat higher background of heterogenous label at 34°C (Fig. 5A, B), and the specific radioactivities of the cytoplasmic rRNAs (17S and 26S) are nearly identical after continuous labelling with [3H]uridine for 3 hr (Fig. 5C, D). To further substantiate this point, we specifically analysed the stability of cytoplasmic rRNA at both 8

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Fig. 4. Gelelectrophoretic analysis of nuclear and microsomal RNA from pulse-labelled cells after a chase at 8 and 28°C, respectively. Logarithmically growing cells in complex organic medium were collected by centrifugation and incubated in 20ml of culture medium containing [3H]uridine (25 ~uCi/ml) at 28°C for 7 min. The cells were then washed, divided into two portions and transferred to fresh culture medium containing unlabelled uridine ("chase"). One portion was held at 28°C, the other at 8~C. After 60 min of chase macronuclei and microsomes were prepared and RNA was isolated. Total nuclear RNA (A) and 20ug of microsomal RNA (B) of both portions were run in parallel. The arrows along the nuclear RNA profile indicate the position of parallel run Tetrahymena mature rRNAs ( 0 - - 0 , 8"C: @ - - @, 28'C).

171

Synchronizing temperatures and rRNA metabolism in Tetrahymena

The subsequent decrease of radioactivity in two portions of these cells held for extended times at 28 and 34°C, respectively, is presented in Fig. 6 and reveals that there is no significant difference of RNA stability during the first 4-6 hr, the half-life of decrease of label being about 30hr at both temperatures. The obviously beginning deviation after 6 hr is most probably due to the fact that at the high temperature the cells begin to die after this period.

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The data presented shows that in Tetrahymena pyriformis pre-rRNA is synthesized and efficiently processed and translocated into the cytoplasm at both g -0S the supra- and suboptimal synchronizing temperatures. Moreover, we found no substantial differences > in the kinetics of the main intracellular events of ;.7. g rRNA-metabolism at the high temperature (34°C) a 0 O when compared to the optimal growth temperature 0 C "O (28°C). However, a strong inhibitory effect of the high O temperature on the incorporation of exogenous nucill: lC leosides into the cellular nucleotide pools was observed (cf. also de Barros et al., 1973; Byfield & Lee, 1970b), a process which is normally rate-limiting for RNA synthesis (Plageman & Roth, 1969; Wolfe, 1974). The finding that synthesis and processing of rRNA are not primarily impaired at 34°C is a verification of the prediction made by de Barros et al. (1973) from their results of the kinetics of exhaustion of preformed (at 28°C) nucleotide pools at the supraoptimal temperature (34°C). This fact of an t _ 2 0 obviously unimpaired rRNA metabolism together l o 2 o 3 o ~ o s o o 10 2o~o~0s~ with a strongly reduced uptake of nucleosides, could Slice n u m b e r at least in part, account for the significant reduction Fig. 5. Separation of nuclear (A, B) and microsomal (C, of cellular ATP level during a 30 min heat period. D) RNA from cells labelled with [3H]uridine at 28°C (A, In this context the finding of a normal respiration C) and 34°C (B, D) on 2.2~o and 2.4~o polacrylamide gels, rate and oxydative phosphorylation during 30 min respectively. A logarithmically growing culture in complex heat shocks in Tetrahymena should be mentioned medium was divided. One portion was incubated at 34°C, (Rooney & Eiler, 1969). the other left at 28°C, and both were labelled with [aH]uridine (2,5 #Ci/ml) for 3 hr (beginning at time zero). Macronuclei and microsomes were prepared and RNA was iso? lated. Total nuclear RNA and 15 #g of microsomal RNA were run on cylindrical polyacrylamide gels at 5 mA/tube 30 for 3 hr. Nuclear RNA was coelectrophoresed with unlabelled E. coil rRNA (23S and 16S; 1.05 x 106 and 20 0.525 x 106 dalton mol. wt, respectively). The positions of these molecular weights standard RNAs in the gels as >" 10 > revealed by the absorbance peaks at 260 nm are indicated by arrows. 5 x

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the supraoptimal and the optimal growth temperature during longer time periods. To this end, cells were pulse-labelled with [3H]uridine (10min) and then "chased" in the presence of unlabelled uridine for 60 min at the optimal growth temperature. After this procedure the labelled nucleotide pools in the cells should be completely exhausted (of. Jacobson & Prescott, 1964; Eckert, 1972) and almost all of the labelled nuclear RNA should have been processed and transported into the cytoplasm (el. Fig. 4, Prescott et al., 1971; Eckert et al., 1975). Moreover, after this procedure almost 90% of total cellular label can be assumed to be associated with cytoplasmic rRNA and most of the rest with tRNA (cf. Leick, 1973; Eckert et al., 1975).

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Fig. 6. Degradation kinetics of stable RNA at optimal growth conditions (complex organic culture medium and optimal growth temperature, 28°C, O O, and at the supraoptimal temperature (34°C, x x x ). Logarithmically growing cells were pulse-labelled with [3H]uridine for 10min, sedimented by centrifugation, washed and incubated in fresh organic culture medium and "chased" for 60 min. The culture was then divided and held at 28 and 34°C, respectively. At the times indicated, 5 ml aliquots were removed from the two portions, precipitated and washed with 5% cold trichloroacetic acid, and radioactivity was determined.

172

WALTER KAFFENBERGER, BETTINA HEMMERICH and WERNER A. ECKERT

At the low temperature (8~'C), transcription, proing temperature shifts on the synthesis and translation cessing and nucleocytoplasmic translocation of rRNA of replication-supporting messengers in Tetrahymena pyriformis. Expl. Cell Res. 61, 42-50. are reduced to a similar extent (25-30% of the optimal BYFIELDJ. E. & LEE Y. C. (1970b) Do synchronizing temrates at 28°C). This indicates that the temperature perature shifts inhibit RNA synthesis in Tetrahymena coefficients for the individual processes involved in pyriJbrmis? J. Protozool. 17, 445-453• the formation of cytoplasmic rRNA are very similar BYHELD J. E. & SCHERBAUMO. H. (1966} Temperature or identical, corresponding to average Q~o-values dependent RNA decay in Tetrahymena. J. Cell Physiol. from 1.7 to about 2.0 in the considered temperature 68, 203-206. range (28-8°C). In general, a tight coupling and correBVFIELD J. E. & SCHERRAUMO. H. 11967a) Temperature lation of transcriptional and post-transcriptional effect on protein synthesis in a heat-synchronized protoevents of r R N A metabolism have been observed in zoan treated with actinomycin D. Science, N.E 156, 1504- 1505. Tetrahymena under various conditions IEckert et al., BWIELD J. E. & SCHERBAUMO. M. (1967b) Temperature1975; Eckert, 1977). A differential cessation of nucleodependent decay of RNA and of protein synthesis in cytoplasmic transport of rRNA below 10°C by a heat-synchronized protozoan. Proc. nam. Acad. Sci. switching the nuclear pores from an "open" to a U.S.A. 57, 602-606. "closed" state as reported by Wunderlich et al. (1974: BYFIELDJ. E., LEE Y. C. & BENNETTL. R. (1970) Similarity cf. also N~igel & Wunderlich, 1977), is thus not comof Tetrahymena and mammalian RNA polymerases patible with our data. This assumption is also in conbased on rifampicin resistance. Biochim. hiophys. Acta flict with earlier findings that Tetrahymena cells ac204, 610-613. cumulate substantial amounts of RNA during 2-9 hr CHRIS'lENS.SONE. G. (1970) Studies of messenger RNA durcold shifts (cf. Zeuthen~ 1964; Padilla et al., 1966). ing the cell cycle in synchronized mass cultures of Tetrahymena pyriformis GL by hybridization. J. Protozool. 17, Altogether our results indicate that the poikilother496-501. mic organism, Tetrahymena is able to maintain a ECKERTW. A. (1972) Uber den Einflu8 transkriptions- und completely functioning rRNA metabolism over a wide translationshemmender Antibiotika auf den Kern-Cytotemperature range and thus a deficiency in supply plasma-Ubertritt yon RNA bei Tetrahymena pyriformis of new rRNA cannot be involved in the inhibition GL. Thesis, Univ,ersity of Freihurg, p. l-158. of cell division during heat and cold shocks in this ECKERT W. A. (1977) Effect of puromycin on synthesis. organism. This is in clear contrast to the situation processing and nucleocytoplasmic translocation of in cells from homeothermic organisms, e.g. human rRNA in Tetrahymena pyriformis. Comp. Biochem. Physiol. 57B, 275-280. cells in culture (HeLa), which grow and divide only ECKERT W. A. & FRANKEW. W. (19751 Changes in fine in a very narrow temperature range (33-40 C; see structure and composition of macronuclei of TetrahyRao & Engelberg, 1966). At supraoptimal temperamena pyriformis induced by drugs interfering with RNA tures (e.g. 42~C) where HeLa cells are viable for at synthesis and processing. Cytobiology !1, 392-418. least 24 hr, pre-rRNA is still synthesized, but its norECKERTW. A., F'RANKEW. W. & SCHEERU. (1975) Nucleomal metabolism to functional rRNA is blocked and cytoplasmic translocation of RNA in Tetrahymena pyrino new r R N A appears in cytoplasmic ribosomes formis and its inhibition by actinomycin D and cyclohex(Waroquier & Scherrer, 1969). A similar impairment imide. Expl. Cell Res. 94, 31-46. of r R N A metabolism in these cells seems to occur ECKERT W. A., KAFEENBERGERW., KROHNEG. & FRANKE W. W. (1978) Introduction of hidden breaks during at suboptimal temperatures: Stevens & Amos (1971) rRNA maturation and ageing in Tetrahymena pyriformis. have observed a successive and differential cessation Eur. J. Biochem. 87, 607-616. of specific processing steps, although 45S pre-rRNA FRANKEL J. (1967) Critical phases of oral primordium deis synthesized down to about 2 0 C . In cultured cells velopment in Tetrahymena pyriformis GL-C: An analysis from homeothermic organisms, therefore, cold and employing low temperature treatment. J. Protozool. 14, heat sensitivity of specific rRNA processing enzymes 639 649. correlated with the inhibition of ribosome formation GROVSKV M. A. (1970) Studies on nuclear structure and could be a major reason for the inhibition of growth function in Tetrahymena pyriformis. II. 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