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Messenger RNA formation: Resistance to inhibition by 3′-deoxycytidine
129
BIOCI-IIMICA ET BIOPHYSICA ACTA
BBA 97301
MESSENGER RNA FORMATION: RESISTANCE TO I N H I B I T I O N BY 3'-DEOXYCYTIDINE HERBERT
T. A B E L S O N AND S H E L D O N P E N M A N
Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts A venue, Cambridge, Mass. o2z39 (U.S.A.) ( R e c e i v e d F e b r u a r y 25th, i972 )
SUMMARY
Cordycepin (3'-deoxyadenosine) has been previously shown to inhibit nucleolar RNA synthesis and mRNA production. 3'-Deoxycytidine shares with cordycepin the ability to inhibit nucleolar RNA synthesis, but it does not affect the appearance of mRNA. Neither compound affects heterogeneous nuclear RNA (HnRNA). The results are interpreted to mean that cordycepin inhibits proper poly(A) metabolism.
INTRODUCTION
The majority of mRNA molecules in mammalian cells have a homopolymer of polyadenylic acid Fpoly(A)] included in their structure, probably at the 3' hydroxyl end 1-5. It has also been shown that the heterogeneous nuclear RNA (HnRNA) contains poly(A) sequences and may be the precursor to cytoplasmic mRNA 1,~,e'7'9. Cordycepin (3'-deoxyadenosine) has been shown to inhibit the appearance of mammalian cell mRNA into the cytoplasm while having little or no effect on the formation of HnRNA 8. This behavior is apparently paradoxical since present evidence indicates that HnRNA may be the precursor to cytoplasmic mRNA. The paradox is resolved if cordycepin acts to prevent the propei processing of HnRNA into mRNA, and evidence has been adduced which suggests that cordycepin may inhibit the post-transcriptional addition of poly(A) to HnRNA 9. 3'-Deoxycytidine is a pyrimidine analogue of cordycepin. In this ieport, it will be shown that 3'-deoxycytidine is metabolically active and shares with cordycepin the ability to inhibit the nucleolar polymerase. It does not, however, interfere significantly with mRNA formation. The results support the hypothesis that cordycepin inhibits proper poly(A) metabolism.
MATERIALS AND METHODS
Cell culture
HeLa cells were grown in suspension culture in Eagle's medium 1° at a density of 4" lO5 cells/ml and concentrated to 2 . IOe cells/ml during labeling. A b b r e v i a t i o n : H n R N A , h e t e r o g e n e o u s n u c l e a r IZNA.
Biochim. Biophys. Acta, 277 (1972) 129-133
13o
H . T . ABELSON, S. PENMAN
Cell labeling and/ractionalion After being concentrated, the cells were labeled with F3H~uridine (spec. act. 20 Ci/mmole, Schwartz Bioresearch, Inc.) for IO min in the case of nRNA and for 9° rain when labeling for cytoplasmic mRNA. In the latter instance, rRNA and mitochondrial RNA synthesis were first suppressed with 0.04/,g/ml of actinomycin D 11 and I #g/ml ethidium bromide 12, respectively, added 25 rain prior to labeling. Cytoplasmic extracts were prepared after harvesting, washing and resuspending cells in I ml of Io mM NaCI-I. 5 mM MgC12-IO mM Tris (pH 7.4) buffer by breaking the cells in a Dounce homogenizer. Nuclei were removed by centrifugation at 800 ×g for 2 min. The cytoplasm was then extracted by the hot phenol-sodium dodecyl sulfate method 13. Nucleoli were separated by the method of Penman 14.
Inhibitors 3'-Deoxycytidine was a generous gift from Dr James H. Hunter, The Upjohn Company, Kalamazoo, Mich. Actinomycin D was a gift from William McCormick, Merck, Sharp and Dohme, Rahway, N. J. Cordycepin was purchased from Sigma Chemical Co., St. Louis, Mo. Ethidium bromide was purchased from Calbiochem, Los Angeles, Calif.
RESULTS
The structures of cordycepin and 3'-deoxycytidine resemble adenosine and cytidine except for the altered 3' position in the ribose moiety where a hydrogen is substituted for the hydroxyl group. One of the major targets of cordycepin is rRNA synthesis. The nucleolar polymerase is inhibited by the compound in a characteristic manner 15. After administration of the drug, completed 45-S molecules are no longer formed, but rather sholtened pieces of RNA ale found in the nucleolus. This result has been interpreted to imply that after cordycepin is phosphorylated within the cell, it actually enters the growing ribosomal precursor polynucleotide. Having once been inserted by the polymerase, the subsequent 5'-3' phosphodiester bond cannot be formed because of the blocked 3' position in the cordycepin molecule. Kinetic studies of nucleolar function suggest further that these prematurely terminated molecules are released from the template and break down. 3'-Deoxycytidine has a similar mode of action. The results in Fig. I show pulse-labeled nucleolar RNA from normal and drug-treated cells. After a Io-min pulse of incorporation, 45-S RNA is the principal RNA species observed in normal nucleoli. In contrast, a spectrum of smaller molecules is observed in the nucleoli from drug-treated cells. It was previously shown that the average size of the molecules labeled in the presence of cordycepin was dependent on the concentration of the drug 15. The distribution of shortened RNA molecules shown in Fig. I resembles those obtained with cordycepin at the same concentration. Thus 3'-deoxycytidine has an effect similar to that of cordycepin on nucleolar RNA formation, and furthermore has approximately the same level of activity as the adenosine analogue. The results further suggest that 3'-deoxycytidine, like cordycepin, enters cells and is phosphorylated by the appropriate enzymes. A remarkable property displayed by coldycepin is its selective effect on some Biochim. Biophys. Acta, 277 (1972) 129-133
RESISTANCE TO 3'-DEOXYCYTIDINE IN
mRNA
FORMATION
131
[
15~-
I~
T
Nucteoli
I! \.
i
I0 20 FrGcfion number
50
Fig. i. Effect of 3'-deoxycytidine on nucleolar RI~A synthesis. One culture received 3'-deoxycytidine (25 #g/ml) for io min. 13oth cultures were t h e n pulse-labeled for io rain with [SH]uridine (io #Ci/ml), Nucleoli were prepared as in Materials and Methods. The R N A was analyzed by zonal density centrifugation t h r o u g h a 15-3o % (w/w) sodium dodecyl sulfate-sucrose gradient at 20 ooo r e v . / m i n for 15.5 h using a Spinco SW 27 rotor. 0 - 0 , control; O - O , 3'-deoxy cytidine.
polymerases. In particular, the polymerase which forms HnRNA, presumably polymerase II, is unaffected by the compound 8. Apparently some enzymes are able to detect the aberrant sugar and do not attempt to incorporate the compound in place of A T E Similarly, 3'-deoxycytidine has no affect on the formation of HnRNA. The previously reported effect of cordycepin on the appearance of mRNA on the cytoplasm is shown in Fig. 2As. The bell-shaped distribution ranging from 25-- 5,
Cor~ycep,n
'
B
5-Deexycytidir'e
-~
,~ ,or i
b
g 5; -
28S
I
¢
I0
18S
20
30 Frocllon number
IO
20
50
Fig. 2. Effect of cordycepin a n d 3'-deoxycytidine on the appearance of cytoplasmic mRl~IA. A. Ribosomal a n d mitochondrial R N A s y n t h e s i s were inhibited as in Materials and Methods. One culture t h e n received cordycepin (25/zg/ml) for io rain. B o t h cultures were pulse-labeled for 9o min with [*H]uridine (io/zCi/ml). C y t o p l a s m was isolated a n d extracted with phenol. The R N A was analyzed as in Fig. i, except centrifugation was a t 24 ooo r e v . / m i n for 16 h in a Spinco SW 27 rotor. O - O , control; O - O , cordycepin. ]3. As in A, except t h a t 3'-deoxycytidine (25 ffg/ml) was used r a t h e r t h a n cordycepin. 0 - 0 , control; O - O , 3'-deoxycytidiue.
Biochim. Biophys. Acta, 277 (1972) 129-133
i32
H . T . ABELSON, S. PENMAN
approx. 6 to 30 S with a m a x i m u m at about I8-S has been shown in m a n y previous studies to constitute the m R N A fraction of H e L a cells 16. The appearance of this RNA in the cytoplasm is dramatically suppressed by cordycepin leaving only a flat distribution of heterogeneous RNA whose nature is as yet unknown. In contrast, the same level of 3'-deoxycytidine, which has been shown to drastically effect nucleolar RNA synthesis, has little effect on the appearance of the messenger fraction in H e L a cell cytoplasm. This is shown in Fig. 2B. In addition, as is the case with cordycepin, 4-S RNA is also inhibited, but to a far lesser degree.
CONCLUSIONS At present, experimental evidence indicates that H n R N A serves as the precursor to cytoplasmic m R N A 1,~,8,:,9. The strongest current support for this model comes flom experiments which show that poly(A) stretches, diagnostic for cytoplasmic mRNA, are also found in high molecular weight nRNA. Poly(A) synthesis appears to occur as a separate event from transcription 9. In the case of adenovirus, m R N A is formed with a poly(A) homopolymer even though adenovirus DNA itself has no significant amount of poly(T) stretch. In this latter case, at least, it seems that the poly(A) is added at a post-transcriptional stage of m R N A processing 17. Coldycepin does not inhibit the formation of the H n R N A which is the presumptive precursor of messenger RNA. its inhibition of the appearance of cytoplasmic m R N A is presumably due to interference with the processing and export of m R N A in the nucleus. A possible target for the action of cordycepin would be the proper formation of the poly(A) sequence in H n R N A (or pre-mRNA), indeed, it has been shown that cordycepin greatly suppresses the formation of poly(A) in mammalian cells 9. Mendecki et al. is have reported that the poly(A) which is formed in the presence of the drug appears truncated but apparently does not terminate in a cordyeepin moiety. There is no evidence at present as to the exact mechanism of action of cordycepin and the suppression of poly(A) formation m a y not be a primary effect of the compound. The experiments reported here show that the 3'-deoxy analogue of cytidine is active in cells and exhibits much the same effect as cordycepin on nucleolar synthesis. However, it has no effect on the formation of m R N A which suggests that only analogues of purines and possibly only of adenosine are active against this process. The findings are consistent with, but do not prove, the hypothesis that formation of poly(A) is the target responsible for m R N A inhibition by cordycepin.
ACKNOWLEDGMENT This work was supported by Awards CA-o8416-o6 from the National Institutes of Health and GB-27684 of the National Science Foundation. S. P. is a Career Development Awardee of the United States Public Health Service, GM-I6127-o5. H.T.A. was a Fellow of the Jane Coffin Childs Memorial Fund for Medical Research. Carol Hahnfeld provided excellent technical assistance. Biochim. Biophys. Acta, 277 (1972) 129-133
RESISTANCE TO 3'-DEOXYCYTIDINE IN
m R N A FORMATION
133
REFERENCES J. E. Darnell, R. Wall and R. J. Tushinski, Proc. Natl. Acad. Sci. U.S., 68 (1971) I32I. M. E d m o n d s , M. H. V a u g h a n , J r and H. N a K a z a t o , Proc. Natl. A cad. Sci. U.S., 68 (1971 ) 1336. S. Y. Lee, J. Mendecki a n d G. Brawerman, Proc. Natl. Acad. Sci. U.S., 68 (I97 I) I33 I. J. Kates, Cold Spring Harbor Syrup. Quant. Biol., 35 (I97 °) 743L. L i m a n d E. S. Canellakis, Nature, 227 (I97 o} 71o. M. E d m o n d s a n d M. G. Caramelo, J. Biol. Chem., 244 (I969) 1314. U. Lindberg and J. E. Darnell, Jr, Proc. Natl. Acad. Sci. U.S., 65 (I97 o) IO89. S. P e n m a n , M. R o s b a s h and M. P e n m a n , Proc. Natl. Acad. Sci. U.S., 67 (197 o) 1878. J. E. Darnell, L. Philipson, R. Wall and M. Adesnik, Science, 174 (1971) 507. I-[. Eagle, Science, 13o (1959) 432. R. P. Perry, Exp. Cell Res., 29 (1963) 400. E. Zylber, C. Vesco and S. P e n m a n , J. Mol. Biol., 44 (1969) 195. E. K. Wagner, L. K a t z a n d S. P e n m a n , Biochem. Biophys. Res. Commun., 28 (1967) 152. S. P e n m a n , Fundamental Techniques in Virology, Academic Press, New York, 1969, p. 35M. Siev, R. Weinberg and S. P e n m a n , J. Cell Biol., 41 (1969) 520. S. P e n m a n , C. Vesco a n d iVL P e n m a n , J. Mol. Biol., 34 (1968) 49L. Philipson, R. Wall, R. Glickman and J. E. Darnell, Proc. Natl. 3cad. Sci. U.S., 68 (1971) 2806. 18 J. Y[endecki, S. Y. Lee and G. Brawerman, Biochemistry, i i (1972) 792.
I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17
Biochim. Biophys. Acta, 277 (1972) 129-133
BIOCI-IIMICA ET BIOPHYSICA ACTA
BBA 97301
MESSENGER RNA FORMATION: RESISTANCE TO I N H I B I T I O N BY 3'-DEOXYCYTIDINE HERBERT
T. A B E L S O N AND S H E L D O N P E N M A N
Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts A venue, Cambridge, Mass. o2z39 (U.S.A.) ( R e c e i v e d F e b r u a r y 25th, i972 )
SUMMARY
Cordycepin (3'-deoxyadenosine) has been previously shown to inhibit nucleolar RNA synthesis and mRNA production. 3'-Deoxycytidine shares with cordycepin the ability to inhibit nucleolar RNA synthesis, but it does not affect the appearance of mRNA. Neither compound affects heterogeneous nuclear RNA (HnRNA). The results are interpreted to mean that cordycepin inhibits proper poly(A) metabolism.
INTRODUCTION
The majority of mRNA molecules in mammalian cells have a homopolymer of polyadenylic acid Fpoly(A)] included in their structure, probably at the 3' hydroxyl end 1-5. It has also been shown that the heterogeneous nuclear RNA (HnRNA) contains poly(A) sequences and may be the precursor to cytoplasmic mRNA 1,~,e'7'9. Cordycepin (3'-deoxyadenosine) has been shown to inhibit the appearance of mammalian cell mRNA into the cytoplasm while having little or no effect on the formation of HnRNA 8. This behavior is apparently paradoxical since present evidence indicates that HnRNA may be the precursor to cytoplasmic mRNA. The paradox is resolved if cordycepin acts to prevent the propei processing of HnRNA into mRNA, and evidence has been adduced which suggests that cordycepin may inhibit the post-transcriptional addition of poly(A) to HnRNA 9. 3'-Deoxycytidine is a pyrimidine analogue of cordycepin. In this ieport, it will be shown that 3'-deoxycytidine is metabolically active and shares with cordycepin the ability to inhibit the nucleolar polymerase. It does not, however, interfere significantly with mRNA formation. The results support the hypothesis that cordycepin inhibits proper poly(A) metabolism.
MATERIALS AND METHODS
Cell culture
HeLa cells were grown in suspension culture in Eagle's medium 1° at a density of 4" lO5 cells/ml and concentrated to 2 . IOe cells/ml during labeling. A b b r e v i a t i o n : H n R N A , h e t e r o g e n e o u s n u c l e a r IZNA.
Biochim. Biophys. Acta, 277 (1972) 129-133
13o
H . T . ABELSON, S. PENMAN
Cell labeling and/ractionalion After being concentrated, the cells were labeled with F3H~uridine (spec. act. 20 Ci/mmole, Schwartz Bioresearch, Inc.) for IO min in the case of nRNA and for 9° rain when labeling for cytoplasmic mRNA. In the latter instance, rRNA and mitochondrial RNA synthesis were first suppressed with 0.04/,g/ml of actinomycin D 11 and I #g/ml ethidium bromide 12, respectively, added 25 rain prior to labeling. Cytoplasmic extracts were prepared after harvesting, washing and resuspending cells in I ml of Io mM NaCI-I. 5 mM MgC12-IO mM Tris (pH 7.4) buffer by breaking the cells in a Dounce homogenizer. Nuclei were removed by centrifugation at 800 ×g for 2 min. The cytoplasm was then extracted by the hot phenol-sodium dodecyl sulfate method 13. Nucleoli were separated by the method of Penman 14.
Inhibitors 3'-Deoxycytidine was a generous gift from Dr James H. Hunter, The Upjohn Company, Kalamazoo, Mich. Actinomycin D was a gift from William McCormick, Merck, Sharp and Dohme, Rahway, N. J. Cordycepin was purchased from Sigma Chemical Co., St. Louis, Mo. Ethidium bromide was purchased from Calbiochem, Los Angeles, Calif.
RESULTS
The structures of cordycepin and 3'-deoxycytidine resemble adenosine and cytidine except for the altered 3' position in the ribose moiety where a hydrogen is substituted for the hydroxyl group. One of the major targets of cordycepin is rRNA synthesis. The nucleolar polymerase is inhibited by the compound in a characteristic manner 15. After administration of the drug, completed 45-S molecules are no longer formed, but rather sholtened pieces of RNA ale found in the nucleolus. This result has been interpreted to imply that after cordycepin is phosphorylated within the cell, it actually enters the growing ribosomal precursor polynucleotide. Having once been inserted by the polymerase, the subsequent 5'-3' phosphodiester bond cannot be formed because of the blocked 3' position in the cordycepin molecule. Kinetic studies of nucleolar function suggest further that these prematurely terminated molecules are released from the template and break down. 3'-Deoxycytidine has a similar mode of action. The results in Fig. I show pulse-labeled nucleolar RNA from normal and drug-treated cells. After a Io-min pulse of incorporation, 45-S RNA is the principal RNA species observed in normal nucleoli. In contrast, a spectrum of smaller molecules is observed in the nucleoli from drug-treated cells. It was previously shown that the average size of the molecules labeled in the presence of cordycepin was dependent on the concentration of the drug 15. The distribution of shortened RNA molecules shown in Fig. I resembles those obtained with cordycepin at the same concentration. Thus 3'-deoxycytidine has an effect similar to that of cordycepin on nucleolar RNA formation, and furthermore has approximately the same level of activity as the adenosine analogue. The results further suggest that 3'-deoxycytidine, like cordycepin, enters cells and is phosphorylated by the appropriate enzymes. A remarkable property displayed by coldycepin is its selective effect on some Biochim. Biophys. Acta, 277 (1972) 129-133
RESISTANCE TO 3'-DEOXYCYTIDINE IN
mRNA
FORMATION
131
[
15~-
I~
T
Nucteoli
I! \.
i
I0 20 FrGcfion number
50
Fig. i. Effect of 3'-deoxycytidine on nucleolar RI~A synthesis. One culture received 3'-deoxycytidine (25 #g/ml) for io min. 13oth cultures were t h e n pulse-labeled for io rain with [SH]uridine (io #Ci/ml), Nucleoli were prepared as in Materials and Methods. The R N A was analyzed by zonal density centrifugation t h r o u g h a 15-3o % (w/w) sodium dodecyl sulfate-sucrose gradient at 20 ooo r e v . / m i n for 15.5 h using a Spinco SW 27 rotor. 0 - 0 , control; O - O , 3'-deoxy cytidine.
polymerases. In particular, the polymerase which forms HnRNA, presumably polymerase II, is unaffected by the compound 8. Apparently some enzymes are able to detect the aberrant sugar and do not attempt to incorporate the compound in place of A T E Similarly, 3'-deoxycytidine has no affect on the formation of HnRNA. The previously reported effect of cordycepin on the appearance of mRNA on the cytoplasm is shown in Fig. 2As. The bell-shaped distribution ranging from 25-- 5,
Cor~ycep,n
'
B
5-Deexycytidir'e
-~
,~ ,or i
b
g 5; -
28S
I
¢
I0
18S
20
30 Frocllon number
IO
20
50
Fig. 2. Effect of cordycepin a n d 3'-deoxycytidine on the appearance of cytoplasmic mRl~IA. A. Ribosomal a n d mitochondrial R N A s y n t h e s i s were inhibited as in Materials and Methods. One culture t h e n received cordycepin (25/zg/ml) for io rain. B o t h cultures were pulse-labeled for 9o min with [*H]uridine (io/zCi/ml). C y t o p l a s m was isolated a n d extracted with phenol. The R N A was analyzed as in Fig. i, except centrifugation was a t 24 ooo r e v . / m i n for 16 h in a Spinco SW 27 rotor. O - O , control; O - O , cordycepin. ]3. As in A, except t h a t 3'-deoxycytidine (25 ffg/ml) was used r a t h e r t h a n cordycepin. 0 - 0 , control; O - O , 3'-deoxycytidiue.
Biochim. Biophys. Acta, 277 (1972) 129-133
i32
H . T . ABELSON, S. PENMAN
approx. 6 to 30 S with a m a x i m u m at about I8-S has been shown in m a n y previous studies to constitute the m R N A fraction of H e L a cells 16. The appearance of this RNA in the cytoplasm is dramatically suppressed by cordycepin leaving only a flat distribution of heterogeneous RNA whose nature is as yet unknown. In contrast, the same level of 3'-deoxycytidine, which has been shown to drastically effect nucleolar RNA synthesis, has little effect on the appearance of the messenger fraction in H e L a cell cytoplasm. This is shown in Fig. 2B. In addition, as is the case with cordycepin, 4-S RNA is also inhibited, but to a far lesser degree.
CONCLUSIONS At present, experimental evidence indicates that H n R N A serves as the precursor to cytoplasmic m R N A 1,~,8,:,9. The strongest current support for this model comes flom experiments which show that poly(A) stretches, diagnostic for cytoplasmic mRNA, are also found in high molecular weight nRNA. Poly(A) synthesis appears to occur as a separate event from transcription 9. In the case of adenovirus, m R N A is formed with a poly(A) homopolymer even though adenovirus DNA itself has no significant amount of poly(T) stretch. In this latter case, at least, it seems that the poly(A) is added at a post-transcriptional stage of m R N A processing 17. Coldycepin does not inhibit the formation of the H n R N A which is the presumptive precursor of messenger RNA. its inhibition of the appearance of cytoplasmic m R N A is presumably due to interference with the processing and export of m R N A in the nucleus. A possible target for the action of cordycepin would be the proper formation of the poly(A) sequence in H n R N A (or pre-mRNA), indeed, it has been shown that cordycepin greatly suppresses the formation of poly(A) in mammalian cells 9. Mendecki et al. is have reported that the poly(A) which is formed in the presence of the drug appears truncated but apparently does not terminate in a cordyeepin moiety. There is no evidence at present as to the exact mechanism of action of cordycepin and the suppression of poly(A) formation m a y not be a primary effect of the compound. The experiments reported here show that the 3'-deoxy analogue of cytidine is active in cells and exhibits much the same effect as cordycepin on nucleolar synthesis. However, it has no effect on the formation of m R N A which suggests that only analogues of purines and possibly only of adenosine are active against this process. The findings are consistent with, but do not prove, the hypothesis that formation of poly(A) is the target responsible for m R N A inhibition by cordycepin.
ACKNOWLEDGMENT This work was supported by Awards CA-o8416-o6 from the National Institutes of Health and GB-27684 of the National Science Foundation. S. P. is a Career Development Awardee of the United States Public Health Service, GM-I6127-o5. H.T.A. was a Fellow of the Jane Coffin Childs Memorial Fund for Medical Research. Carol Hahnfeld provided excellent technical assistance. Biochim. Biophys. Acta, 277 (1972) 129-133
RESISTANCE TO 3'-DEOXYCYTIDINE IN
m R N A FORMATION
133
REFERENCES J. E. Darnell, R. Wall and R. J. Tushinski, Proc. Natl. Acad. Sci. U.S., 68 (1971) I32I. M. E d m o n d s , M. H. V a u g h a n , J r and H. N a K a z a t o , Proc. Natl. A cad. Sci. U.S., 68 (1971 ) 1336. S. Y. Lee, J. Mendecki a n d G. Brawerman, Proc. Natl. Acad. Sci. U.S., 68 (I97 I) I33 I. J. Kates, Cold Spring Harbor Syrup. Quant. Biol., 35 (I97 °) 743L. L i m a n d E. S. Canellakis, Nature, 227 (I97 o} 71o. M. E d m o n d s a n d M. G. Caramelo, J. Biol. Chem., 244 (I969) 1314. U. Lindberg and J. E. Darnell, Jr, Proc. Natl. Acad. Sci. U.S., 65 (I97 o) IO89. S. P e n m a n , M. R o s b a s h and M. P e n m a n , Proc. Natl. Acad. Sci. U.S., 67 (197 o) 1878. J. E. Darnell, L. Philipson, R. Wall and M. Adesnik, Science, 174 (1971) 507. I-[. Eagle, Science, 13o (1959) 432. R. P. Perry, Exp. Cell Res., 29 (1963) 400. E. Zylber, C. Vesco and S. P e n m a n , J. Mol. Biol., 44 (1969) 195. E. K. Wagner, L. K a t z a n d S. P e n m a n , Biochem. Biophys. Res. Commun., 28 (1967) 152. S. P e n m a n , Fundamental Techniques in Virology, Academic Press, New York, 1969, p. 35M. Siev, R. Weinberg and S. P e n m a n , J. Cell Biol., 41 (1969) 520. S. P e n m a n , C. Vesco a n d iVL P e n m a n , J. Mol. Biol., 34 (1968) 49L. Philipson, R. Wall, R. Glickman and J. E. Darnell, Proc. Natl. 3cad. Sci. U.S., 68 (1971) 2806. 18 J. Y[endecki, S. Y. Lee and G. Brawerman, Biochemistry, i i (1972) 792.
I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17
Biochim. Biophys. Acta, 277 (1972) 129-133