245

Biochimica et Biophysica Acta, 474 (1977) 245--253 © Elsevier/North-Holland Biomedical Press

BBA 98799

TOYOCAMYCIN INHIBITION OF RIBOSOMAL RIBONUCLEIC ACID PROCESSING IN AN OSMOTIC-SENSITIVE ADENOSINE-UTILIZING SACCHAROMYCES CEREVISIAE MUTANT

P E N C H O V. V E N K O V , L U B O M I R A

I. S T A T E V A and A S E N A. H A D J I O L O V

Department of Molecular Genetics,Instituteof Biochemistry, Bulgarian Academy of Sciences, 1113 Sofia (Bulgaria) (Received June 8th, 1976)

Summary An adenosine-utilizing mutant of Sacchar~myces cerevisiae (SY15ado) is isolated after remutagenesis of an osmotic-sensitive strain, auxotrophic for adenine, with ethyl methanesulfonate. It is shown that the SY15ado mutant can be used to achieve experimental conditions under which cell growth and RNA synthesis are directly dependent on exogenous adenosine. After starvation for adenosine, toyocamycin is incorporated into pre-rRNA * chains of SY15ado cells replacing adenosine residues. The extent of this replacement depends on the concentration of added toyocamycin. Lower doses slow down processing of pre-rRNA into mature rRNA with an accumulation of 27 S and 20 S prerRNA. At higher concentrations toyocamycin blocks the last steps of pre-rRNA processing i.e. the conversions 27 S pre-rRNA -~ 25 S rRNA and 20 S pre-rRNA -* 18 S rRNA. It appears that the main site of toyocamycin action is at the last steps of ribosome formation, while transcription and the early stages of pre-RNA processing are less affected.

Introduction Toyocamycin is a structural analogue of adenosine, which may be converted in vivo to the respective nucleotides and incorporated into RNA chains [1,2]. Analysis of the effects of toyocamycin incorporation into pre-rRNA chains may be helpful in elucidating the mechanisms of ribosome biogenesis. It was reported that in L cells toyocamycin incorporation inhibits the processing of 45 S primary pre-rRNA, while transcription remains apparently unaltered [3,4]. However, this effect was much less pronounced in chick embryo cells A b b r e v i a t i o n s : p r e - r R N A , p r e c u r s o r s to r i b o s o m a l r i b o n u c l e i c acids.

246 even at markedly higher doses of toyocamycin [5,6]. Recent experiments with Novikoff hepatoma cells supplied evidence that t o y o c a m y c i n inhibits the last stages of pre-rRNA processing, while transcription and initial processing are much less altered [7]. Evaluation of the above experiments with animal cells is made difficult by possible variations in cell permeability and the metabolism of t o y o c a m y c i n among cell types. Further, difficulties in carrying out chase experiments with animal cells limit the attempts to specify unequivocally the site of action of t o y o c a m y c i n on pre-rRNA processing. Previous studies from this laboratory have shown that fragile Sacchromyces cerevisiae mutants [8] are sensitive to a variety of inhibitors of RNA synthesis [9,10] and may be used as a model for studying the mechanisms of their action. In this work, adenosine-utilizing strains of the osmotic-sensitive mutant SY15, auxotrophic for adenine, were obtained. It was shown that, after starvation for adenosine, t o y o c a m y c i n is incorporated into pre-rRNA molecules and inhibits the last steps of pre-rRNA processing. This effect of t o y o c a m y c i n is concentration dependent. Lower doses slow down processing to mature r R N A species. However, at higher concentrations t o y o c a m y c i n blocks the conversions 27 S pre-rRNA -* 25 S rRNA and 20 S pre-RNA -* 18 S rRNA. Materials and Methods

Yeast strains, media and mutagenesis Saccharomyces cerevisiae SY15 (an osmotic-sensitive mutant obtained from A364 strain) is remutagenized with ethyl methanesulfonate according to the procedure described earlier [8]. All buffers and solutions are supplemented with 10% sorbitol to prevent lysis of the fragile SY15 cells. The mutagenized cells are plated on complete synthetic medium [11] supplemented with 10% sorbitol but containing 20 pg/ml adenosine instead of adenine. After incubation at 30°C for 5 days, a b o u t 10% of the cells are growing on this medium. A fast-growing clone is isolated and designated further SY15ado. The SY15ado cells are cultivated in liquid complete synthetic medium, supplemented with 10% sorbitol and 20 pg/ml adenosine, until reaching the midlog phase of growth (about 5 × 106 cells/ml). In experiments for studying the effect of toyocamycin, the cultures are briefly chilled to 4°C, the cells collected on Millipore filters, washed extensively with cold 10% sorbitol and resuspended in prewarmed to 30°C complete synthetic medium plus 10% sorbitol, without adenosine. After 3 h starvation for adenosine, t o y o c a m y c i n (or adenosine) at different concentrations are added and 4 min later the cells are labelled with [ 3H] uracil or [ 3H] adenosine as specified in the text. Isolation o f ribosomal RNA and analysis of RNA At the end of the labelling period the cultures are poured over an equal volume of ice-crushed 10% sorbitol and the cells collected and washed with cold 10% sorbitol on Millipore filters. The cells are lysed directly in 10 mM NaC1/ 10 mM EDTA (pH 7.0) at 4°C and RNA extracted with an equal volume of water-saturated phenol (pH 6.0) for 15 min at 50°C. RNA is further deproteinized with a mixture of phenol/chloroform (1 : 1) and precipitated with

247 ethanol containing 1% potassium acetate, as described [9]. Using this procedure mainly rRNA and tRNA are isolated from the fragile yeast mutants. Poly(A)-containing mRNA could not be detected in these RNA preparations by using the oligo(dT)-cellulose technique [12]. For measuring the specific activity, rRNA is further separated from tRNA by precipitation in 1.5 M NaC1 for 12 h at --10°C and the trichloroacetic acid-insoluble radioactivity in rRNA determined. RNA samples are analysed by electrophoresis in agar gels [13]. The absorbance of the dried agar plates is recorded at 260 nm with a recording spectrodensitometer. The dried agar plates are cut in 1-mm slices, RNA hydrolysed with 5% NH4OH for 18 h at 37°C and the radioactivity counted with toluenePPO-dimethyl-POPOP phosphor containing 30% Triton X-100 in a Packard TriCarb 3320 spectrometer. Ethyl methanesulfonate is obtained from Eastman Organic Chemicals, New York. Sorbitol, agar and Yeast Nitrogen Base without amino acids are from Difco Laboratories, Michigan, U.S.A. Adenine and adenosine are from Pabst Laboratories, Wisc., U.S.A. [5-3H]Uracil (spec. act. 28 Ci/mmol) is from the Radiochemical Centre, Amersham, U.K. [ 2-3H] Adenosine (spec. act. 8 Ci/mmol) is from New England Nuclear, Mass., U.S.A. All other reagents are analytical grade. Toyocamycin was a gift from Dr H.B. Wood, National Cancer Institute, Bethesda, Md., USA. Results Isolation o f adenosine-utilizing strains of Saccharomyces cerevisiae SY15 The osmotic-sensitive S. cerevisiae mutant SY15 is auxotrophic for adenine (adl, ad2), uracil (url) and several aminoacids [8]. In this mutant, adenine cannot be replaced by adenosine, a finding in line with observations for other S. cerevisiae mutants auxotrophic for adenine [14]. Therefore, we remutagenized SY15 cells and selected the clones capable of fast growth on complete synthetic medium containing 10% sorbitol and 20 pg/ml of adenosine instead of adenine. One of these clones, designated SY15ado, was partly characterized and used in subsequent experiments. The SY15ado cells, like the parental strain, are osmotic sensitive and sorbitol dependent. They grow exponentially in media supplemented with 10% sorbitol. Resuspension in buffers lacking sorbitol causes immediate lysis and release of 50--70% of the cellular content [8]. The SY15ado mutant requires exogenous adenine or adenosine for growth (Table I). The growth of the parental SY15 strain can be supported by adenine only, while the SY15ado strain grows on media containing either adenine or adenosine. The growth rate of the SY15ado mutant increases with increasing concentration of adenosine and at 20 pg/ml of adenosine it grows exponentially with a generation time of 3.5 h. To specify the conditions of macromolecular synthesis in the absence of adenosine, we studied the growth of SY15ado cells after removal of adenosine from the medium (Fig. 1). As can be seen, transfer of SY15ado cells to a medium lacking adenosine stops completely their growth within 2 h. Obviously, this effect reflects the depletion of the intracellular pool of adenosine and its nucleotide derivatives, since addition of adenosine results in a rapid resumption of

248 TABLE I G R O W T H O N A D E N O S I N E O F T H E A D E N O S I N E - U T I L I Z I N G S. C E R E V I S I A E AND THE PARENTAL STRAIN SY15

SY15ado STRAIN

Cells g r o w n o n c o m p l e t e s y n t h e t i c m e d i a c o n t a i n i n g 10% s o r b i t o l are i n o c u l a t e d i n t o the s a m e m e d i a w h e r e a d e n i n e is r e p l a c e d b y a d e n o s i n e at the i n d i c a t e d c o n c e n t r a t i o n s . T h e c u l t u r e s are s h a k e n f o r 4 8 h at 3 0 ° C . Strain

G r o w t h (cells X 1 0 6 p e r m l c u l t u r e ) Controls (20 pg/ml adenine)

SY15ado SY15

Adenosine (#g/ml)

5.2 6.1

0

20

40

160

0.02 0.01

3.5 --

7.4 --

11.6 0.4

growth. This conclusion is further supported by analyses of [3H]adenosine incorporation into RNA of SY15ado cells at 3 and 5 h after removal of adenosine from the medium (Fig. 1, inset). The obtained results demonstrate the immediate resumption of RNA synthesis upon addition of adenosine. In summary, the characterization of the SY15ado mutant reveals that it can be used to achieve experimental conditions under which cell growth and RNA synthesis will be directly dependent on exogenous adenosine.

• 20nm 18

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Time (h) Fig. 1. D e p e n d e n c e o f t h e g r o w t h of S. cerevisiae S Y 1 5 a d o cells o n a d d e d a d e n o s i n e . T h e S Y 1 5 a d o ceils are g r o w i n g e x p o n e n t i a l l y in c o m p l e t e s y n t h e t i c m e d i u m c o n t a i n i n g 10% s o r b i t o l a n d 2 0 # g / m l ' o f a d e n o s i n e . A t p o i n t A, t h e cells are chilled, w a s h e d a n d r e s u s p e n d e d in c o m p l e t e s y n t h e t i c m e d i u m plus 10% s o r b i t o l , p r e w a r m e d t o 3 0 ° C w i t h o u t (o o) o r w i t h ( a A) 2 0 # g / m l o f a d e n o s i n e . A t p o i n t B, t o p a r t o f t h e c u l t u r e d cells, s t a r v e d f o r a d e n o s i n e , a d e n o s i n e is a d d e d t o 2 0 # g / m l final c o n c e n tration (e . . . . . -e). Cell g r o w t h i s m e a s u r e d as t h e a b s o r b a n c e o f t h e c u l t u r e a t 4 2 0 n m ( a n A 4 2 0 n m v a l u e o f 0.1 c o r r e s p o n d s t o 1 X 106 cells p e r m l ) . I n s e t : I n c o r p o r a t i o n o f [ 3 H ] a d e n o s i n e i n t o 5% c o l d t r i c h l o r o a c e t i c acid-insoluble m a t e r i a l o f w h o l e cells a t d i f f e r e n t t i m e i n t e r v a l s a f t e r a d e n o s i n e r e m o v a l f r o m the m e d i u m ( p o i n t A). 5 #Ci o f [ 3 H ] a d e n o s i n e p e r m l are a d d e d t o t h e c u l t u r e a n d t h e r a d i o a c t i v i t y d e t e r m i n e d in 0 . 5 - m l a l i q u o t s a t d i f f e r e n t t i m e i n t e r v a l s (× X ).

249

Synthesis of rRNA in the presence of toyocamycin To investigate the consequences of toyocamycin incorporation into RNA chains on rRNA synthesis and processing, we carried out experiments in which adenosine~lepleted SY15ado cells were incubated in the presence of toyocamycin. In a first series of experiments SY15ado cells, 3 h after removal of adenosine from the medium, were incubated with different concentrations of adenosine or toyocamycin. The cells were labelled for 30 or 120 min with [3H]uracil and the specific radioactivity of rRNA determined (Fig. 2). As expected, addition of adenosine results in the resumption of rRNA synthesis. Incorporation of added [3H]uracil into rRNA follows an exponential kinetics of labelling, which most probably reflects the participation of cold uracil present in the medium and the intracellular pool. These experiments show also that toyocamycin can partly replace adenosine as a precursor utilized in rRNA synthesis. Incorporation of [3H]uracil into rRNA in the presence of toyocamycin is lower than with equal concentrations of adenosine, which suggests that the analogue is less readily involved in phosphorylation and/or polymerization reactions than the natural nucleoside (see also ref. 1). Nevertheless, toyocamycin permits a substantial synthesis of rRNA, which at the highest concentration tested (20 pg/ml) amounts to about 60% of the rRNA made in the presence of adenosine (Fig. 2A). Further, the linear increase of 30 min [3H]uracil incorporation into rRNA with increasing concentrations of toyocamycin suggests that transcription is only slightly, if at all affected by this analogue. On the other hand, with longer labelling times the difference between rRNA synthesis in the presence of toyocamycin or adenosine is markedly more pronounced. These results indicate that in yeast cells, as in other eukaryotes [5,7], the major

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Concerltretion of cldded Qdenosine or toyoc(:lmyEin (#g/ml) Fig. 2, I n c o r p o r a t i o n o f [31-I]uracil i n t o r i b o s o m a l R N A o f S. cerevlmJae S Y 1 5 a d o cells in t h e p r e s e n c e o f a d e n o s i n e (o o) o r t o y o c a m y c i n ( e -'). T h e cells are starved f o r a d e n o s i n e f o r 3 h a n d labelled w i t h 1 0 ~ C i / m l o f [ 3 H ] u r a c i l f o r 3 0 m i n ( A ) o r 1 2 0 m i n (B) in the p r e s e n c e o f d i f f e r e n t c o n c e n t r a t i o n s o f a d e n o s i n e or t o y o c a m y c i n . A t e a c h e x p e r i m e n t a l p o i n t the R N A f r o m a l i q u o t s o f t h e e u l ~ r e s is isol a t e d a n d its r a d i o a c t i v i t y d e t e r m i n e d as d e s c r i b e d u n d e r M e t h o d s .

250 effect of toyocamycin on rRNA synthesis is post-transcriptional. The fact that toyocamycin permits some limited synthesis of rRNA in SY15ado cells starved for adenosine, suggests that, as with animal cells [3,4], the analogue is incorporated into rRNA chains. The limited amount of rRNA synthesized in the presence of toyocamycin did not allow a direct estimate of the percentage of adenosine residues in rRNA replaced by toyocamycin residues. Therefore, we attempted to obtain some information on this point by studying the incorporation of [3H]adenosine into rRNA of SY15ado cells, starved for adenosine, in the presence of increasing concentrations of toyocamycin or adenosine. The results (data not shown) show that toyocamycin competes with [3H]adenosine for incorporation sites into rRNA chains and that the extent of replacement in rRNA of adenosine residues by toyocamycin residues depends on the concentration of toyocamycin in the medium. Summarizing, the results obtained show that toyocamycin is incorporated into rRNA chains of the SY15ado mutant, most probably replacing adenosine residues. The extent of this replacement depends on the concentration of added toyocamycin.

Inhibition of pre-rRNA maturation by toyocamycin The results described above suggest that in yeasts, like in other eukaryotes [4,5,7] the major site of toyocamycin action is posttranscriptional. To specify the site of toyocamycin action we analyzed the gel electrophoretic pattern of pre-rRNA and rRNA species synthesized by adenosine-starved SY15ado cells in the presence of increasing concentrations of toyocamycin. In all experiments, the cells were labelled for 30 min with 5 pCi/ml of [3H]adenosine. Control experiments showed that under these conditions the label is located exclusively in the mature 25 S and 18 S rRNA peaks. The rRNA labelled in the presence of 1 pg/ml of toyocamycin is also represented by the two mature rRNA species (Fig. 3A). However, at toyocamycin concentrations of 3 pg/ml and higher, the rRNA labelled for 30 min is represented exclusively by pre-rRNA species (Fig. 3B,D,G). The 3H-labelled peaks of 37 S, 27 S and 20 S pre-rRNA characteristic for yeast cells [15,16] are neatly delineated. It is noteworthy that the labelling of 27 S and 20 S pre-rRNA is markedly higher than that of 37 S pre-rRNA. Therefore, these results show that toyocamycin inhibits the last steps of prerRNA maturation, i.e. the conversions 27 S pre-rRNA -~ 25 S rRNA and 20 S pre-RNA -~ 18 S rRNA, while transcription and processing of 37 S pre-rRNA is less affected by this analogue. It was considered important to clarify if maturation of the pre-rRNA species synthesized in the presence of toyocamycin is slowed down or is completely blocked. Therefore, we carried out experiments in which the SY15ado cells, labelled with [3H]adenosine for 30 min in the presence of different concentrations of toyocamycin, were collected on Millipore filters, washed with 10% sorbitol and resuspended in prewarmed complete synthetic medium containing 100 pg/ml of cold adenosine. Control experiments have shown that under these conditions, the cells continue to grow without any lag period, while further incorporation of [ 3H]adenosine into RNA is abolished. At different times after the chase, rRNA was isolated and analyzed by agar gel electrophoresis. The results obtained (see Fig. 3) show that the processing of pre-labelled pre-

251

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Fig. 3. Agar gel e l e c t r o p h o r e s i s p a t t e r n o f r i b o s o m a l R N A f r o m S. cerevisiae S Y 1 5 a d o cells s y n t h e s i z e d in the p r e s e n c e o f t o y o c a m y e i n . T h e S Y 1 5 a d o cells are starved o f a d e n o s i n e for 3 h and labelled w i t h 5 # C i / m l [ ~ I ] a d e n o s i n e for 3 0 rain in the p r e s e n c e o f 1 ( A ) , 3 (B), 5 ( D ) and 1 0 ( G ) # g per m l o f t o y o c a m y c i n . A t the end o f the [ ~ - I ] a d e n o s i n e labelling p e r i o d parts o f the c u l t u r e s are c h a s e d b y w a s h i n g the cells on Millipore m e m b r a n e filters and r e s u s p e n s i o n in fresh c o m p l e t e s y n t h e t i c m e d i u m c o n t a i n i n g 1 0 % s o r b i t o l and 1 0 0 # g / m l o f c o l d a d e n o s i n e . The cells are i n c u b a t e d f o r 3 0 rain (C, E and H) or 6 0 rain ( F and I) after the chase. T h e r R N A is e x t r a c t e d f r o m the separate c u l t u r e s and a n a l y s e d b y agar gel e l e c t r o p h o r e s i s as d e s c r i b e d u n d e r M e t h o d s . - . . . . . , a b s o r b a n c e at 2 6 0 n m ; , r a d i o a c t i v i t y . T h e a r r o w s i n d i c a t e the s values assigned to the separate y e a s t p r e - r R N A or r R N A s p e c i e s . For details see the t e x t .

rRNA depends on the concentration of added toyocamycin before the chase. Maturation of pre-rRNA pulse labelled for 3 min with Jail]adenosine (without toyocamycin) is completed within 10 min under our experimental conditions (data not shown). In contrast, processing of [3H]adenosine labelled pre-rRNA, accumulated for 30 min in the presence of toyocamycin (Fig. 3B, D and G) is

252 markedly slowed down. Pre-rRNA accumulated in the presence of 3 pg/ml of t o y o c a m y c i n is fully processed within 30 min (Fig. 3C), while at 5 pg/ml toyocamycin, even with a 60-min chase, the processing of pre-rRNA is not comppleted (Fig. 3F). Processing of pre-rRNA labelled in the presence of 10 pg/ml of t o y o c a m y c i n appears to be blocked completely since even with a 60-min chase there is no shift of labelled RNA to mature rRNA species (Fig. 3I). At all concentrations of t o y o c a m y c i n tested the small amount of accumulated labelled 37 S pre-rRNA is fully processed during the chase. This finding supports the conclusion that the main site of t o y o c a m y c i n action is at the last stages of prer R N A processing. It is also of interest that despite the block in pre-rRNA processing observed at higher t o y o c a m y c i n concentrations, the accumulated 27 S and 20 S pre-rRNA species are not degraded during the extended chase periods tested and appear to be stabilized by t o y o c a m y c i n incorporation. Summarizing, it may be stated that t o y o c a m y c i n incorporation into RNA chains inhibits the last steps of pre-rRNA processing. This inhibition is dependent on the concentration of t o y o c a m y c i n in the medium. At higher concentrations of toyocamycin, processing of pre-rRNA is blocked, while at lower concentrations of the analogue it is only delayed. Discussion

The results obtained in this work demonstrate that in the SY15ado mutant, starved for adenosine, t o y o c a m y c i n can substitute for adenosine and permit limited pre-rRNA synthesis. However, incorporation of t o y o c a m y c i n residues in the pre-rRNA chains inhibits their processing. Our results reveal that in S. cerevisiae the last steps in pre-rRNA processing, i.e. the conversions 27 S prer R N A -~ 25 S r R N A and 20 S pre-rRNA -* 18 S rRNA, may be specified as the main sites of t o y o c a m y c i n action. These results are in agreement with experiments on the effect of t o y o c a m y c i n on pre-rRNA processing in Novikoff hepatoma cells [7]. Thus, the earlier observations with L cells leading to the conclusion that t o y o c a m y c i n inhibits the first stages of 45 S pre-rRNA maturation [3,4] should be reconsidered. Differences among cell types in specifying variations in the site of t o y o c a m y c i n action remain a possibility [5,6]. However, we consider the earlier conclusion that t o y o c a m y c i n blocks 45 S pre-rRNA maturation as reflecting specific experimental conditions and technical imperfections in pre-rRNA fractionation, rather than revealing the major site of action of the drug. It appears more likely that the action of t o y o c a m y c i n is identical for all eukaryotic cells, the most pronounced effect being the blocking of the last stages in the formation of mature ribosomes. In this respect the effect of t o y o c a m y c i n is similar to the action of a broad variety of inhibitors of ribosome biogenesis [17]. Thus, our present results supply further evidence that the last stages in the formation of mature ribosomes pose markedly more stringent requirements to the structure of pre-rRNA than the preceding ones. Apparently, pre-rRNA chains, containing t o y o c a m y c i n residues, may be synthesized and processed until the stage of ribonucleoprotein particles containing the immediate precursors to the mature 25 S and 18 S rRNAs. The last stage of ribosome formation is also possible, although at a reduced rate. Our observation that this effect depends on the concentration of t o y o c a m y c i n suggests that above a

253 critical substitution of adenosine residues by toyocamycin, the formation of mature ribosomes is totally blocked. A quantitative analysis of the extent and character of toyocamycin substitution in pre-rRNA chains may be helpful in elucidating the control mechanisms in ribosome biogenesis. Acknowledgement The authors are greatly indebted to Dr. H.B. Wood, Drug Development Branch, Division of Cancer Treatment, National Cancer Institute, USA, for the supply of toyocamycin. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Suhadolnik, R.J. (1970) Nucleoside Antibiotics, Wiley-Interscience, New York Langen, P. (1975) Antimetabolites of Nucleic Acid Metabolism, Gordon and Breach, New York Tavitian, A., Uretsky, S.C. and Acs, G. (1968) Biochim. Biophys. Acta 157, 33--42 Tavitian, A., Uretsky, S.C. and Acs, G. (1969) Biochim. Biophys. Acta 179, 50--57 Sverak, L., Bonar, R.A., Langlois, A.J. and Beard, J.W. (1970) Biochim. Biophys. Acta 224,441---450 Riman, J., Sverak, L., Langlois, R., Bonar, J. and Beard, J. (1969) Cancer Res. 29, 1707--1716 Weiss, J.W. and Pitot, H.C. (1974) Cancer Res. 34, 581--593 Venkov, P.V., Hadjiolov, A.A., Battaner, E. and Schlessinger, D. (1974) Biochem. Biophys. Res. Commun. 56, 599--604 Venkov, P.V., Milchev, G.I. and Hadjiolov, A.A. (1975) Antimicr. Agents Cbemother. 8, 6 2 7 - 6 3 2 Waltschewa, L., Stoyanova, B.B. and Venkov, P.V. (1975) C.R. Acad. Sci. Bulgaria 28, 1 2 5 7 - - 1 2 6 0 Sherman, F., Fink, G.R. and Lukins, H.B. (1970) Methods in Yeast Genetics, a l a bora t ory manual, p. 54, Cold Spring Harbor Laboratory, New York Edmonds, M. and Caramela, M.G. (1969) J. Biol. Chem. 244, 1314--1324 Tsanev, R. (1965) Biochim. Biophys. Acta 103, 374--378 Anderson, J.M. and Roth, R.M. (1974) Biochim. Biophys. Acta 335, 285--289 Udem, S.A. and Warner, J.R. (1972) J. Mol. Biol. 65, 227---242 Brand, R.C. and Planta, R.J. (1975) Mol. Biol. Rep. 2, 3 21--325 Hadjiolov, A.A. and Nikolaev, N. (1976) Prog. Biophys. Mol. Biol. 31, 1--50