Selective inhibition of ribosomal RNA synthesis in mammalian cells

BIOCHIMICA ET BIOPHYSICA ACTA

33

BBA 95873

SELECTIVE INHIBITION OF RIBOSOMAL RNA SYNTHESIS IN MAMMALIAN CELLS

A R M A N D T A V I T I A N ' , S T A N L E Y C. U R E T S K Y AND G E O R G E ACS

Institute for Muscle Disease, New York, N. Y. Ioo21 (U.S.A.) (Received December ISth, I967)

SUMMARY

I. Toyocamycin, in low concentrations, completely inhibits 28-S and I8-S RNA synthesis; it permits, however, the synthesis of a normally methylated 45-S RNA that accumulates in the cells' nucleoli. This RNA contains toyocamycin. The cells resume rRNA synthesis after the removal of the antibiotic from the medium; the 45-S RNA synthesized in the presence of toyocamycin is not converted under these conditions into 28-S and I8-S RNA, whereas the normally pulse-labeled 45-S RNA is converted to rRNA regardless of the presence of toyocamycin. We postulate that the altered structure of the 45-S RNA does not permit this conversion. Synthesis of tRNA is not inhibited by toyocamycin, as shown by the ratio of methylation to nucleoside incorporation and the ratio of pseudo-uridine to uridine in the 4-S RNA that was synthesized in the presence of toyocamycin. 2. The concentration of toyocamycin that impairs RNA synthesis is cytotoxic, but permits normal DNA and protein synthesis for 6 h.

INTRODUCTION

The data of several laboratories l-s, based on kinetic evidence, strongly suggest that an RNA having a sedimentation coefficient of 45 S is the precursor of rRNA. However, the mechanism responsible for the conversion of this heavy RNA into rRNA has not yet been elucidated. Toyocamycin, 4-amino-5-c yano-7-fl-D-ribofuranosyl-p yrr olo [2,3-dj=pyrimi dine (Fig. I), when used in low concentration, selectively inhibits rRNA synthesis. Moreover, in the presence of this antibiotic, the cells for 6 h accumulate in their nucleoli an RNA sedimenting at 45 S. Upon incubating the cells with tritiated toyocamycin, we showed that toyocamycin is incorporated into this heavy RNA. Furthermore, Abbreviations: rRNA, ribosomal RNA; t R N A , transfer RNA. * Career scientist of the French National I n s t i t u t e of Health a n d Medical Research, supported b y a fellowship from the I n t e r n a t i o n a l Union Against Cancer (Eleanor Roosevelt Cancer Foundation).

Biochim. Biophys. Acta, 157 (1968) 33-42

34

a. TAVITIAN, S. C. URETSKY, C-. ACS

on removing the antibiotic from the culture, we found that its inhibitory effect on RNA synthesis is reversible, but that the 45-S RNA accumulating in the presence NH2

OH

OH

Fig. i. S t r u c t u r e of toyocamycin.

of toyocamycin is not converted to rRNA. We therefore suggest that structural changes owing to the incorporation of toyocamycin into 45-S RNA do not permit the conversion of RNA containing toyocamycin into 28-S and I8-S RNA. Although toyocamycin completely abolishes rRNA synthesis, it has no effect on tRNA synthesis. This selective inhibitory effect of toyocamycin is quite specific. Incubation of cells with 8-azaguanine, 5-fluorouracil, puromycin aminonucleoside, formycin, and actinomycin D reveals a more severe inhibition of rRNA than of tRNA synthesis; however, despite the use of a wide range of concentrations with each of the above-mentioned antimetabolites, the specific effect of toyocamycin, i.e. the accumulation of 45-S RNA, could not be reproduced. Tubercidin (7-deazaadenosine), like toyocamycin, causes an accumulation of 45-S RNA, completely inhibits rRNA synthesis, and permits tRNA synthesis, but its effect on RNA synthesis is not reversible. Moreover, tubercidin also inhibits synthesis of DNA and protein at the same rate that it inhibits synthesis of RNA, whereas in the presence of toyocamycin, synthesis of DNA and protein is much less inhibited than is synthesis of RNA.

MATERIALS AND METHODS

Cells: Mouse fibroblasts (strain L-929) were propagated in suspension culture in minimal Eagle's medium 4 supplemented with IO% fetal bovine serum. The average generation time was 16-18 h. HeLa Ss cells (generation time 20-24 h) were grown similarly in a medium supplemented with 5 % calf serum. Experiments were performed with exponentially growing cells at a concentration of 4-5 × lO5 cells/ml. Ehrlich ascites cells were maintained by intraperitoneal injection into HAICR Swiss white mice; the cells were collected 6 days after transplantation, centrifuged, washed in Earle's saline, suspended, and incubated in vitro under conditions similar to those for cultltre cells. Antibiotics: Tubercidin was a gift of Dr. C. SMITH. Toyocamycin and formycin were generously provided by Dr. H. UMEZAWA. 5-Fluorouracil was purchased from Biochim. Biophys. Acta, 157 (1968) 33-42

INHIBITION OR rRNA SYNTHESIS

35

Calbiochem. Corp., 8-azaguanine from Mann Research Laboratories, and puromycin aminonucleoside from Nutritional Biochemical Corp. Radioisotopes: Uniformly labeled [SH]guanosine, [SH]adenosine, [SH]thymidine, [14Cjguanosine, and [2-14C]uridine were purchased from Nuclear-Chicago Corp.; L [14C]valine and L- [MeJ4C]methionine from New England Nuclear Corp. ;L-[Me-SHs] methionine from Schwarz Bio Research Inc. RNA, DNA and protein: Determinations of total RNA, Db~A, and protein synthesis were performed as described previously 5. Cell/ractionation: Nucleoli were isolated from H e L a SS cells according to the method of PENMAN,SMITH AND HOLTZMAN6. The fibroblast cell walls were more resistant to the hypotonic medium. Three successive homogenizations of the pellet were required to isolate 99 % pttre nuclei. Further ffactionation was followed according to the method of PENMAN. Ribosomes and polyribosomes were prepared in HOAGLAND'S isotonic medium 7 from the postmitochondrial supernatant fraction according to a procedure adapted for cultured cells s. Characterization o[ RNA : Approximately 8 × lO 7 cells (18o-2oo ml) were harvested b y centrifugation and resuspended in a small volume of 0.o2 M sodium acetate, p H 5.2. R N A was extracted from the cells with o.4 % sodium lauryl sulfate (Mann, enzyme grade) and then with an equal volume of 90 % freshly distilled phenol in the same acetate buffer. This procedure was carried out at 4°; the aqueous layer was precipitated with 2 vols. of IOO ~o cold ethanol and kept at --20 ° for 2 h. The precipitate was collected b y centrifngation and resnspended in acetate buffer, then analyzed b y zone sedimentation on a 20-5 % sucrose gradient containing 0.02 M sodium acetate, p H 5.2. Nuclear R N A was extracted according to PENMAN'S technique with phenol and a mixture of chloroform and isoamyl alcohol S. Sucrose gradient centrifugations of RN A were carried out at 23 ooo rev./min for 14 h in the Spinco SW 25.I rotor. Paper chromatography: The t R N A isolated b y sucrose gradient was precipitated with 2 vols. of IOO % ethanol, resuspended in I M HC1, and heated at IOO° for i h. An aliquot of the hydrolysate was spgtted on W h a t m a n No. I paper and chromatograpbed in two dimensions: 7o % isopropanol and 30 0% water in an atmosphere of ammonia and isobuty¢ic acid-ammonia 9.

RESULTS AND DISCUSSION

Cytotoxicity Inhibition of growth b y toyocamycin was measured on L cells grown in monolayers. Fig. 2 shows that toyocamycin at a concentration as low as 0.002 #g/ml inhibits growth. At a concentration of 0.I #g/ml, the cells develop nuclear alteration and cytoplasmic vacuolation; however, when lower concentrations are used, the growth inhibition is less marked, and cells do not develop the same toxic appearance.

E/]ect on synthesis o] total protein, DNA, and RNA Fig. 3A, B, and C shows that within 8 h toyocamycin inhibits the synthesis of all three macromolecules; however, the inhibition of R N A synthesis is strikingly greater and occurs earlier than that of either DNA or protein synthesis.

Biochim. Biophys. Acta, 157 (I968) 33-42

36

A. TAVITIAN, S. C. URETSKY, G. AC,~

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Fig. 3. E f f e c t of t o y o c a m y c i n on s y n t h e s i s of A, R N A ; B, D N A ; a n d C, protein. L cells (5 x zoS/ml) were i n c u b a t e d w i t h o.25/zC//~mole of [3H]guanosine, [ 3 H ] t h y m i d i n e , or L-[14C] valine, respectively. in t h e p r e s e n c e or a b s e n c e of z / z g / m l of t o y o c a m y c i n . A l i q u o t s (io ml) were t a k e n a t t h e i n d i c a t e d i n t e r v a l s , a n d 1RNA, DNA, a n d p r o t e i n s were isolated as described in MI~THODS f r o m c o n t r o l cells, O, a n d f r o m t o y o c a m y c i n - t r e a t e d cells, A.

Characterization and localization o/the RNA synthesized in the presence o~ toyocamycin Fig. 4 represents the sucrose gradient centrifugation profile of RNA extracted from the intact cells. The cells in their logarithmic phase were preincubated for 15 ,

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Fig. 4. Sucrose g r a d i e n t a n a l y s i s of R N A . A, cells were e x p o s e d to o. i/~g of t o y o c a m y c i n a n d 0. 5 / , C / m l of [3H]guanosine for 3 h. ]3, t h e y were i n c u b a t e d u n d e r t h e s a m e c o n d i t i o n s e x c e p t for t o y o e a m y c i n . R N A w a s e x t r a c t e d as d e s c r i b e d in METHODS. A b s o r b a n c e , O - O ; r a d i o a c t i v i t y ,

0-0. Biochim. Biophys. Acta, 157 (1968) 33-42

INHIBITION OR

rRNA

37

SYNTHESIS

min with o.I #g/ml of toyocamycin; thereafter, o.5/,g/ml of guanosine and o.5/,C/ml of [3Hlguanosine were added, and the incubation was continued for 3 h. No radioactivity was detected at the regions corresponding to the 28-S and I8-S RNA, whereas two sharp radioactive peaks appeared at the regions of 4-S and 45-S RNA. The radioactivity appearing at these regions is acid-precipitable, but becomes acidsoluble after treatment with 0.5/zg/ml of ribonuclease; it is not affected by 50/~g/ml of deoxyribonuclease. Methylation of RNA was investigated by labeling the cells with methionine tritiated in the methyl group under incubation conditions similar to those described above. Radioactivity was again restricted to the 45-S and 4-S region, and no methyl groups were found in the region corresponding to the 28-S and I8-S RNA (Fig. 5)This experiment emphasizes the similarity between the pulse-labeled 45-S RNA and

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Fig. 5. I n c o r p o r a t i o n of [Me-3H3]methionine into R N A . Sucrose g r a d i e n t of R N A e x t r a c t e d f r o m cells i n c u b a t e d w i t h o . I / , g / m l of t o y o c a m y c i n a n d 8 # C / m l of [ 3 H ] m e t h i o n i n e in a m e t h i o n i n e poor m e d i u m for 3 h. R N A w a s e x t r a c t e d as described in METHODS. A b s o r b a n c e , O - O ; radioactivity, 0-0. Fig. 6. N u c l e o l a r R N A of t o y o c a m y c i n - t r e a t e d cells. Sucrose g r a d i e n t c e n t r i f u g a t i o n of t h e R N A e x t r a c t e d f r o m t h e nucleoli a c c o r d i n g to PENMAN'S procedure. T h e cells were i n c u b a t e d for 3 h in t h e p r e s e n c e of o . i / ~ g / m l of t o y o c a m y c i n . U n l a b e l e d 28-S a n d I8-S 1RNA's were c e n t r i f u g e d together with the nucleolar RNA. Absorbance, O-O; radioactivity, 0-0.

the 45-S RNA that accumulates in the presence of toyocamycin. This similarity is further demonstrated by their identical intracellular localization. Figs. 6 and 7 show that the 45-S RNA synthesized in the presence of toyocamycin is found in the nucleoli; moreover, except for the 4-S region, there are no acid-precipitable counts in the cytoplasmic fraction. Fig. 8 shows that the radioactivity in the nucleoli representing 45-S RNA accumulates for 6 h in the presence of toyocamycin. These results are corroborated by the findings that the specific activity of polyribosomes extracted from toyocamycin- and [aHlguanosine-treated cells is one-tenth that of control cells, whereas monosomes extracted from similarly treated cells show negligible activity compared to those of untreated cells (Table I). However, while the pulse-labeled 45-S RNA can be chased to 28-S and I8-S RNA even in the presence of actinomycin 1°, the 45-S RNA that accumulates in the presence of toyocamycin cannot be chased into the rRN'A even after removing the antibiotic from the medium by washing the cells several times, resuspending them, and further incubating them in a toyocamycin-free medium for 18 h. When the 45-S RNA is labeled with [3HJguanosine, we see some radioactivity Biochim. Biophys. Acta, 157 (1968) 33-42

38

A. TAVITI&N, S. C. URETSKY, G. ACS

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Fig. 7. C y t o p l a s m i c R N A of t o y o c a m y c i n - t r e a t e d cells. Sucrose g r a d i e n t c e n t r i f u g a t i o n of c y t o p l a s m i c R N A . T h e cells were i n c u b a t e d a n d d i s r u p t e d as described in t h e legend to Fig. 6. Abs o r b a n c e , @-f._); r a d i o a c t i v i t y , 0 - 0 . Fig. 8. A c c u m u l a t i o n of 45-S R N A in t h e nucleoli. I n s e p a r a t e e x p e r i m e n t s , cells (5 × IO5/ml) were i n c u b a t e d w i t h o . I / z g / m l of t o y o c a m y c i n a n d o. 5 / z C / m l of [3H]guanosine. T h e 45-S R N A w a s e x t r a c t e d f r o m t h e isolated nucleoli, t r e a t e d w i t h 25 # g / m l of d e o x y r i b o n u c l e a s e ( W o r t h i n g ton, e l e c t r o p h o r e t i c a l l y pure), p r e c i p i t a t e d in 5 9o trichloroacetic acid, a n d c o u n t e d . E a c h t i m e p o i n t r e p r e s e n t s 5 × lO7 cells. O , ©, /x r e p r e s e n t t h e c o u n t s in s e p a r a t e e x p e r i m e n t s .

TABLE I SPECIFIC RADIOACTIVITY OF POLYRIBOSOMES AND MONORIBOSOMES U n t r e a t e d a n d t r e a t e d cells were labeled w i t h [3H]guanosine (o. 5 / , C / m l ) for 3 h.

Ribosomes

Untreated cells (counts/rain per A260 m#)

Cells treated with (o.z #g/ml) toyocamycin

Poly Mono

65 500 76 800

6 430 590

after 18 h in the 28-S and I8-S RNA. However, this incorporation represents the fact that the 45-S RNA has been broken down to mononucleotides and that the cells, after recovering from the effect of the antibiotic, utilize these breakdown products for the synthesis of rRNA. Three lines of evidence suggest that the 45-S RNA synthesized in the presence of toyocamycin is not converted directly to 28-S and I8-S RNA. I. Upon adding actinomycin to the washed and resuspended cells, no radioactivity can be observed in the peaks corresponding to the 28-S and I8-S RNA, even after prolonged chasing. On the contrary, actinomycin enhances the breakdown of this 45-S RNA to mononucleotides. 2. When the 45-S RNA is labeled with [Me-3Hslmethionine, no radioactivity can be observed in the regions of the 28-S and I8-S RNA after removing the antibiotic and further incubating the cells. 3. When the cells are labeled with ~3Hltoyocamycin, acid-precipitable counts occur only in the 45-S and 4-S region of the sucrose gradient (Fig. 9). However, as in the case in which we labeled the 45-S RNA with ~Me-3Hs]methionine, the radioactivity accumulated in the 45-S RNA cannot be chased into the 28-S and I8-S RNA. Thus, the 45-S RNA accumulated in the presence of toyocamycin resembles the pulse-labeled 45-S RNA with respect to its sedimentation coefficient 1,3, intraBiochim. Biophys. Acta, 157 (1968) 33-42

INHIBITION OR r R N A SYNTHESIS i

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Fig. 9. Incorporation of tritiated toyocamycin into RNA. Sucrose gradient centrifugation of 1RNA extracted from L cells incubated for 3 h with o.i/~g/ml of tritiated toyocamycin; specific activity, 1.2 X lO9 counts/min per #mole. Absorbance, O - O ; radioactivity, O - Q .

cellular localization TM, and methyl acceptor activity TM, but it cannot be converted to rRNA. This failure in conversion is due probably to the fact that this RNA at least partly contains toyocamycin instead of adenosine. Data from E. REICH AND G. Acs (unpublished) show that in a cell-free system with Escherichia coli RNA polymerase, toyocamycin 5'-triphosphate can replace ATP; furthermore, it has been shown that RNA pyrophosphorylase can incorporate toyocamycin 5'-triphosphate in lieu of ATP into the terminal end of the tRNA 1~. The possibility that conversion of 45-S RNA into 28-S and I8-S RNA is inhibited b y toyocamycin or its derivatives was investigated in the following way: L cells were pulse-labeled for 20 min in the presence of [Me-SH3]methionine in a methionine-poor medium. Further incubation was performed with toyocamycin in the presence or absence of actinomycin D. As Fig. IO shows, the conversion of the pulselabeled 45-S RNA into 28-S and I8-S RNA was not inhibited by toyocamycin. In the absence of actinomycin, we observed an additional radioactive peak at the 45-S region. This radioactivity represents the 45-S RNA newly synthesized in the presence of toyocamycin that cannot be converted into rRNA. As seen in Figs. 4, 5, and 9, toyocamycin at a concentration of o.I #g/ml completely abolishes 28-S and I8-S RNA synthesis, whereas the incorporation of [Me3Ha]methionine or L3H]guanosine into the 4-S RNA region is unaltered. Moreover, using E3H]-labeled toyocamycin, we observed that in addition to the 45-S RNA, the 4-S RNA also became labeled. Two lines of evidence suggest that the radioactivity observed in the 4-S region represents incorporation into tRNA and that it is not due to breakdown products, which are indistinguishable from the tRNA, in sucrose gradients. i. After incubating cells with [2-14C]uridine in the presence of toyocamycin, we fractionated the RNA on sucrose gradient. Two-dimensional chromatography of the acid hydrolysate of the RNA sedimenting at 4 S reveals that the ratio of uridylic acid to pseudouridylic acid is the same in the tRNA whether derived from untreated or toyocamycin-treated cells (Table II). Since this ratio is characteristic of tRNA, we feel that the radioactivity appearing in the 4-S region in the presence of toyocamycin represents tRNA synthesis. Biochim. Biophys. Acla, 157 (1968) 33-42

40

A. TAVITIAN, S. C. URETSKY, G. A('.~ 'A

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Fig. Io. Pulse-chase e x p e r i m e n t s w i t h [Me-3Ha]methionine. A, sucrose gradient centrifugation of R N A f r o m cells exposed to io ~uC/ml of [Me-'~H:~]methionine for 2o min in a methionine-free m e d i u m . B, sucrose gradient centrifugation o f R N A f r o m cells labeled as in (A) and f u r t h e r i n c u b a t e d w i t h o . i / ~ g / m l of t o y o c a m y c i n and 3/~g/ml of unlabeled methionine for 3 h. C, s a m e e x p e r i m e n t as in (B) except t h a t cells were f u r t h e r incubated with o . i / , g / m l of t o y o c a m y c i n , I / , g / m l of a c t i n o m y c i n D, and 3 / , g / m l of unlabeled methionine for 3 h. Absorbance, O - Q ; radioactivity, 0 - 0 .

TABLE

II

DETERMINATION OF PSEUDOURIDINE AND URIDINE IN THE "4-8'" R N A U n t r e a t e d and t r e a t e d cells were labeled with o. 5/~C/ml of [2-14C]uridine for 4 h. The 4-S R N A w a s isolated, hydrolyzed, and c h r o m a t o g r a p h e d as described in METHODS

Sources

Uridylic acid (counts~rain)

Pseudouridylic acid

Ratio o/uridylic to pseudouridylic acid

U n t r e a t e d cells Cells t r e a t e d w i t h toyocamycin

8 55 °

i 635

5.2

7 039

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4-9

TABLE

III

COMPARISON OF INC ORPORATI ON OF [ M e - a H 3 ] M E T H I O N I N E AND ? 4 C ] G U A N O S I N E INTO

4-S R N A

T r e a t e d and u n t r e a t e d cells were i n c u b a t e d for 3 h w i t h 4/~C/ml of [Me-aH3]methionine and 0.05/2C/ml of [14C]guanosine. The 4-S regions of the sucrose gradients were pooled and incubated at p H 9 for 15 m i n at 37 °. Samples were t h e n precipitated in 5 % trichloroacetic acid, collected on Millipore H A filter, a n d counted.

4-S R N A source

[3H]

[1~C]

Ratio o~ ? H ] to [14C]

Cells t r e a t e d w i t h t o y o c a m y c i n U n t r e a t e d cells

i 14 853 I I 3 113

91 o 17 93 475

i. 26 1.21

Biochim. Biophys. Acta, I57 (I968) 33-42

INHIBITION OR r R N A SYNTHESIS

41

2. Since the t R N A contains an unusually high amount of methylated bases, we surmised that the ratio of incorporated nucleosides to incorporated EMe-3H31methi onine in a biosynthetic process will be characteristic of t R N A synthesis. Table I I I shows that the ratio of incorporated ~Me-3H3]methionine to incorporated F14C~guanosine in the 4-S RNA isolated b y sucrose gradient is quite similar regardless of whether the cells have or have not been treated with toyocamycin.

The e//ect o/ other antibiotics As reported previously 14, upon preincubation of cells for 3 h with 8-azaguanine, the synthesis of cytoplasmic RNA is inhibited, whereas nucleolar RNA synthesis is uninhibited. However, using concentrations of 8-azaguanine ranging from o.1-3o #g/ml and varying the time of preincubation, we could not observe the same sucrose gradient profiles of RNA with azaguanine t h a t we did after a short treatment with a low concentration of toyocamycin; puromycin aminonucleoside inhibits r R N A synthesis, but, as with azaguanine, we could not reproduce the effect of toyocamycin. The same is true for 5-fluorouracil, formycin, and actinomycin D. It should be pointed out t h a t if we use the ratio of incorpolated nucleoside to incorporated EMe-ZH3~methionine as an index for t R N A synthesis, all these antibiotics affect r R N A synthesis more markedly than they do t R N A synthesis. Tubercidin, on the other hand, in concentrations of 1-2 #g/ml has an effect similar to that of toyocamycin on RNA synthesis. However, its effect on cellular RNA synthesis is not reversible, i.e. after removal of the antibiotic the cells do not resume normal RNA synthesis.

E//ect o/toyocamycin in other mammalian cells The effect of toyocamycin was assayed in two other mammalian cell systems, H e L a S 3 cells grown in suspension culture and Ehrlich ascites cells incubated in suspension medium in vitro. The conditions of preincubation with toyocamycin and incubation with E3H~guanosine were identical to those used with L-cells. In both cases, we observed similar results, i.e. inhibition of 28-S and I8-S rI~NA with accumulation of 45-S RNA, whereas there was no inhibition of t R N A synthesis.

ACKNOWLEDGMENTS

The skillful technical assistance of Mrs. HANNA KLETT is gratefully acknowledged. This study was aided b y grant CA-o875I from the National Institutes of Health, b y grant P-299 from the American Cancer Society, and b y Muscular Dystrophy Associations of America.

REFERENCES I 2 3 4

K. SCHERRER AND J. E. DARNELL, Biochem. Biophys. Res. Commun., 7 (1962) 486. R. P. PERRY, Proc. Natl. Acad. Sci. U. S., 48 (1962) 2179. S. I~NMAN, J. Mol. Biol., 17 (1966) 117. H. EAGLE;,Science, 13 ° (1959) 432.

Biochim. Biophys. Acta, 157 (1968) 33-42

A. TAVITIAN, S. C. URETSKY, (;. A(7>

42 5 6 7 8 9 io II 12 13 14

G. Acs, E. REICH AND IV[. MORI, Proc. Natl. Acad. Sci. U.S., 52 (1964) 493. S. PENMAN, I. SMITH AND E. I~IOLTZMAN, Science, 154 (1966) 786. M. B. HOAGLAND, Cold Spring Harbor Syrup. Quant. Biol., 26 (1961) 153. A. TAVITIAN AND M. BOIRON, Compt. Rend., 260 (1965) 5967. 1R. LIPSHITZ AND E. CHARGAFF, Biochim. Biophys. Acta, 42 (196o) 544. M. GIRARD, S. PENMAN AND J. E. DARNELL, Proc. Natl. Acad. Sci. U.S., 51 (I964) 205. R. P. PERRY Natl. Cancer Inst. Monograph, 18 (1966) 325 . E. F. ZIMMERMAN AND B. W. HOLLER, .[. Mol. Biol., 23 (1967) 149. S. C. URETSKY, G. AcS, E. REICH, M. MORI AND L. ALTWERGER, J. Biol. Chem., 243 (1968) 3o6. R. P. PERRY', Natl. Cancer Inst. Monograph, 14 (1964) 73.

Biochim. Biophys. Acta, 157 (1968) 33-42