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The proteins in free cytoplasmic poly(A)+-ribonucleoprotein complexes from maize
Plant Science Letters, 29 (1983) 61--66
61
Elsevier Scientific Publishers Ireland Ltd.
T H E P R O T E I N S IN F R E E C Y T O P L A S M I C POLY(A)+-RIBONUCLEOPROTEIN COMPLEXES
FROM
MAIZE
J.L. NICHOLS and L. WELDER Department of Biochemistry and Microbiology, University of Victoria, Victoria, B.C. V8W 2Y2 (Canada)
(Received May 20th, 1082) (Accepted August 26th, 1982)
SUMMARY
Free cytoplasmic poly(A)+-ribonucleoprotein (RNP) was isolated from postpolysomal preparations of maize embryos by chromatography on oligo(dT)-cellulose. The protein constituents of the RNP were examined by oneand two-dimensional electrophoresis in polyacrylamide gels. Several proteins (Mr = 68 900, 66 500 and 59 400) exhibited apparent charge heterogeneity. Two proteins appeared to be associated with the poly(A) tract.
K e y words: Messenger ribonucleoprotein -- Polyadenylated m R N A
INTRODUCTION In eucaryotes, m R N A released from polysomes, as well as non-ribosome b o u n d mRNA, is found complexed with proteins in ribonucleoprotein (RNP) particles. There is considerable variation reported in the number and size of the protein constituents of poly(A)+-mRNP, which may reflect the conditions e m p l o y e d in the isolation of the particles and the quantity of material subjected to analysis [ 1 - 3 ] . In the case of animal cells there is general agreement that as many as four of these proteins are specifically associated with the poly(A)-tract [4--9]. The association of proteins with m R N A could affect the transport of the polymer from the nucleus [ 6 ] , the stability of the p o l y m e r in the cytoplasm by protection from nuclease and/or the capacity of the R N A Abbreviations: PMSF, phenylmethanesulfonyl fluoride; RNP, ribonucleoprotein; tc, translationalcontrol. 0304-4211/83/0000--0000/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
62 to function in translation, mRNA-associated proteins, perhaps in association with translational control (tc) RNA [10], may modulate the activities of certain messages in the cytoplasm by affecting their entry into polysomes. In support of this idea, recent studies have shown that the coding capacities of polysomal and free cytoplasmic mRNAs are different [ 11]. Free cytoplasmic mRNA has been shown to be incorporated into polysomes [ 1 1 - 1 3 ] , a process which may involve the loss of proteins from the RNA [12], or an exchange of proteins in the cytoplasm. In this report the protein constituents of maize free cytoplasmic poly(A) ÷RNP are described and evidence is provided for the association of two proteins with the poly(A)-tract. MA T ER I ALS AND METHODS
Embryos were excised from seedlings of Zea mays that had been grown in sterile tap water for 72--96 h at room temperature [14]. Postpolysomal solutions were prepared as described previously [15], except that the extraction buffer contained 0.5 mM phenylmethanesulfonyl fluoride (PMSF, Sigma Chemical Co.} and heparin {100 ug/ml; Sigma Chemical Co.} [9]. After adjusting the solution to 0.5 M KCI, a pellet of postpolysomal particles was obtained by centrifugation at 250 000 × g for 5 h. The pellets were resuspended in buffer (10 mM Tris--HC1, pH 7.6, 1 mM EDTA, 0.5 M NaC1, 100 ~g/ml heparin and 0.5 mM PMSF) and absorbed to oligo(dT)-cellulose (Type T3; Collaborative Research, Inc.) at 4°C as described previously [9]. Poly(A)÷-RNP was eluted from the column with 50% formamide in buffer (10 mM Tris--HCl, pH 7.6, 1 mM EDTA) at room temperature, mRNP was recovered from the eluted fractions by precipitation with two volumes 95% ethanol a t - 2 0 ° C , The isolated poly(A)÷-RNP had A2~0/A2s0 ratios of between 1.1 and 1.3. Phenol extracted RNA obtained from the free cytoplasmic RNP particles exhibited a heterogeneous sedimentation profile in sucrose gradients and was shown to contain a modified nucleotide characteristic of maize poly(A) ÷RNA [16]. The protein constituents of free cytoplasmic RNP were analyzed by electrophoresis on sodium dodecyl sulphate (NaDodSO4)/10% polyacrylamide gels using the procedure of Laemmli [ 17]. Molecular weight markers (BioRad Laboratories) employed were phosphorylase B (92 500), bovine serum albumin (68 000), ovalbumin (45 000), carbonic anhydrase (31 000), soybean trypsin inhibitor (21 500) and lysozyme (14 400). Each sample contained between 0.3 and 1.0 A~60 units RNP. Densitometric scans of Coomassie blue-stained gels (Hoeffer Scientific Co. electrophoresis manual) were performed on a Gilford 2520 gel scanner at a wavelength of 570 nm. Two-dimensional separation of proteins employing isoelectric focusing and NaDodSO4-polyacrylamide gel electrophoresis was performed using the Anderson and Anderson [18] adaptation of the O'Farrell procedure [19].
63
Samples were denatured with NaDodSO4 and slab gels (second dimension) were 10--15% gradients of acrylamide. Poly(A)-protein complexes were isolated from RNP preparations by a procedure that involved nuclease digestion and readsorption to columns of oligo(dT)-ceUulose. RNP particles in 1 ml 0.1 M Tris--HC1 (pH 7.4), 1 mM EDTA, 0.4 M NaC1 and 0.1 mM PMSF were digested with ribonuclease A (0.2 ~g/ml; Worthington Biochemical Corp.). The solutions were incubated at 10°C for 1 h and then readsorbed to, and eluted from oligo(dT)-cellulose columns as described [9]. R E S U L T S A N D DISCUSSION
The polyacrylamide gel electrophoretic separation of free cytoplasmic poly(A)+-RNP proteins is shown in Fig. la. For comparative purposes, the proteins present in the preparation but n o t retained on the affinity column, are shown in Fig. lb. During the preparation of poly(A)÷-RNP, care was taken to avoid proteolysis and nuclease action by the inclusion of PMSF and heparin in the buffers. In addition, the possible formation of artifactual
a
b
Fig. 1. NaDodSO4-polyacrylamide gel electrophoresis of proteins in poly(A)+-RNP. The numbers indicate the molecular weights (× 10 -3) determined by co-electrophoresis with marker proteins. (a) poly(A)÷-RNP eluted from oligo(dT)-cellulose columns, (b) postpolysornal proteins that were not retained on oligo(dT)-cellulose.
64
RNP complexes [20] was reduced by employing high salt (0.5 M NaCl) concentrations in the buffers. Poly(A)÷-RNP proteins that were present in all preparations had molecular weights of 73 400 (73.4P), 68 900 (68.9P), 66 500 (66.5P), 59 400 (59.4P), 47 000 (47P) and 35 100 daltons (35.1P). Although the relative quantities of the proteins varied somewhat between the preparations, the most prevalent were 68.9P, 66.5P, and 35.1P; the former two proteins were not well resolved because of their close molecular weights. Components 68.9P and 66.5P accounted for as much as 30% of the total protein, assuming comparable staining of all proteins with Coomassie blue. Major proteins (70 000, 36 000, 21 000 Mr) have been noted in RNP preparations from radish seeds [21] ; the 70 000-dalton component may be analogous to the 68.9P/ 66.5P maize proteins. Two~limensional electrophoresis was employed to analyze the protein constituents of poly(A)÷-RNP preparations more fully. A representative separation of RNP proteins is shown in Fig. 2. In most of the two-dimensional separations, at least 30 different proteins could be seen. Most of
Fig. 2. Two-dimensional electrophoretic separation of poly(A)*-RNP protein. The acid end is to the left and NaDodSO 4 electrophoresis proceeded downward. The sample origin was at the upper right hand corner. The numbers indicate molecular weights
(x 10-3).
65
the bands observed in one-dimensional gels are resolved into two or more proteins. However, in the ease of protein bands 68.9P, 66.5P and 59.4P there would appear to be a single protein constituent even though each is resolved into three or four spots having somewhat different charge properties. The charge heterogeneity of proteins 68.9P, 66.5P and 59.4P, has been observed in seven different preparations. The charge differences could be a consequence of deamidation, carbohydrate heterogeneity, acetylation or phosphorylation [19,22]. The more likely of these possibilities is phosphorylation, since protein kinase has been found to be associated with mRNP particles [23,24] and mRNP proteins have been shown to be phosphorylated [23--26]. If the charge differences are not artifactual and reflect the in vivo situation with regard to these proteins, then it is conceivable that they may affect the RNA binding capabilities of the protein and consequently play a role in translational regulation. In support of the association/dissociation capabilities of RNP proteins with RNA, there is now some evidence that mRNA-associated proteins exchange in the cytoplasm with a pool of free proteins [27]. To isolate poly(A)-binding proteins, advantage was taken of the fact that poly(A)-tracts in a medium of high ionic strength are resistant to digestion with ribonuclease A. Poly(A)+-RNP was suspended in a buffer containing 0.5 M sodium chloride and digested with ribonuclease A. The released poly(A)-protein complex was recovered by affinity chromatography on oligo(dT)-cellulose columns and analyzed by NaDodSO4-polyacrylamide gel electrophoresis. Figure 3 shows the densitometric scans of stained O
I Fig. 3. Densitometric scans of NaDodSO4-polyacrylamide gel separations of poly(A)associated proteins. (a), intact poly(A~-RNP, (b), ribonucle~e A digest of poly(A)+-RNP.
66 p r o t e i n s in gels o f s t a r t i n g m a t e r i a l (poly(A)+-RNP) a n d n u c l e a s e - t r e a t e d m a t e r i a l ( p o l y ( A ) - a s s o c i a t e d p r o t e i n s ) . I t is a p p a r e n t t h a t t h e r e is a p r e f e r e n tial r e t e n t i o n o f t w o p r o t e i n s in a s s o c i a t i o n w i t h t h e p o l y ( A ) - t r a c t . T h e s e are 6 8 . 9 P a n d / o r 6 6 . 5 P a n d 59.4P. I n o t h e r s y s t e m s , t h e r e a p p e a r s t o b e o n e m a j o r p r o t e i n (78 0 0 0 Mr) t h a t b i n d s p o l y ( A ) a l t h o u g h lesser a m o u n t s of other proteins have been detected [4-9]. The poly(A)-associated proteins c o u l d p l a y an i m p o r t a n t r e g u l a t o r y role b y a f f e c t i n g t h e a s s o c i a t i o n o f m R N A w i t h t h e r i b o s o m e a n d b y increasing t h e stability o f m R N A t o n u c l e a s e digestion. ACKNOWLEDGEMENTS
T h e a u t h o r s are grateful t o B. Walker w h o p a r t i c i p a t e d in s o m e o f these e x p e r i m e n t s a n d Dr. T. P e a r s o n f o r p e r f o r m i n g t h e t w o - d i m e n s i o n a l electrop h o r e t i c runs. T h i s w o r k was s u p p o r t e d b y a g r a n t f r o m t h e N a t u r a l Sciences a n d E n g i n e e r i n g R e s e a r c h C o u n c i l o f Canada. REFERENCES 1 2 3 4 5
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
A. Preobrazhensky and A. Spirin, Prog. Nucleic Acid. Res. Mol. Biol., 21 (1978) 1. J.R. Greenberg, J. Cell Biol.,65 (1975) 269. D. Rittschof and J.A. Traugh, Eur. J. Biochem., 115 (1981) 45. G. Blobel, Proc. Natl. Acad. Sci. U.S.A., 70 (1973) 924. A. Barrieux, H.A. Ingraham, D.N. David and M.G. Rosenfeld, Biochemistry, 14 (1975) 1815. H. Schwartz and J.E. Darnell, J. Mol. Biol., 104 (1976) 833. V.M. Kish and T. Pederson, J. Biol. Chem., 251 (1976) 5888. W.R. Jeffery, J. Biol. Chem., 252 (1977) 3525. S.K. Jain and S. Sarkar, Biochemistry, 18 (1979) 747. D.S. Kennedy, E. Siegel and S.M. Heywood, FEBS Lett., 90 (1978) 209. G.T-Y. Lee and D.L. Engelhardt, J. Mol. Biol., 129 (1979) 221. D.S. Adams, D. Noonan and W.R. Jeffery, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 83. T. Geoghegan, S. Cereghini and G. Brawerman, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 5587. J.L. Nichols and L. Welder, Biochim. Biophys. Acta, 608 (1980) 1. D.P.S. Verma and G.A. Maclaehlan, Plant Physiol., 58 (1976) 405. J.L. Nichols, Biochim. Biophys. Acta, 652 (1981) 99. U.K. Laemmli, Nature, 227 (1970) 680. N.G. Anderson and N.L. Anderson, Anal. Biochem., 85 (1978) 331. P.H. O'Farrell, J. Biol. Chem., 256 (1975) 4007. D. Baltimore and A.S. Huang, J. Mol. Biol., 47 (1970) 263. A. Fetter, M. Delseny and Y. Guitton, Plant Sci. Lett., 14 (1979) 31. L. Anderson and N,G. Anderson, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 5421. C. Quirin-Stricker and M. Schmitt, Biochim. Biophys. Acta, 477 (1981) 414. J. Bag and B.H. Sells, J. Biol. Chem., 254 (1979) 3137. C. Morel, E.S. Dander, M. Nerzberg, J. Dubochet and K. Scherrer, Eur. J. Biochem., 36 (1973) 455. S. Auerbach and T. Pederson, Biochem. Biophys. Res. Commun., 63 (1975) 149. J. Greenberg, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 2923.
61
Elsevier Scientific Publishers Ireland Ltd.
T H E P R O T E I N S IN F R E E C Y T O P L A S M I C POLY(A)+-RIBONUCLEOPROTEIN COMPLEXES
FROM
MAIZE
J.L. NICHOLS and L. WELDER Department of Biochemistry and Microbiology, University of Victoria, Victoria, B.C. V8W 2Y2 (Canada)
(Received May 20th, 1082) (Accepted August 26th, 1982)
SUMMARY
Free cytoplasmic poly(A)+-ribonucleoprotein (RNP) was isolated from postpolysomal preparations of maize embryos by chromatography on oligo(dT)-cellulose. The protein constituents of the RNP were examined by oneand two-dimensional electrophoresis in polyacrylamide gels. Several proteins (Mr = 68 900, 66 500 and 59 400) exhibited apparent charge heterogeneity. Two proteins appeared to be associated with the poly(A) tract.
K e y words: Messenger ribonucleoprotein -- Polyadenylated m R N A
INTRODUCTION In eucaryotes, m R N A released from polysomes, as well as non-ribosome b o u n d mRNA, is found complexed with proteins in ribonucleoprotein (RNP) particles. There is considerable variation reported in the number and size of the protein constituents of poly(A)+-mRNP, which may reflect the conditions e m p l o y e d in the isolation of the particles and the quantity of material subjected to analysis [ 1 - 3 ] . In the case of animal cells there is general agreement that as many as four of these proteins are specifically associated with the poly(A)-tract [4--9]. The association of proteins with m R N A could affect the transport of the polymer from the nucleus [ 6 ] , the stability of the p o l y m e r in the cytoplasm by protection from nuclease and/or the capacity of the R N A Abbreviations: PMSF, phenylmethanesulfonyl fluoride; RNP, ribonucleoprotein; tc, translationalcontrol. 0304-4211/83/0000--0000/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
62 to function in translation, mRNA-associated proteins, perhaps in association with translational control (tc) RNA [10], may modulate the activities of certain messages in the cytoplasm by affecting their entry into polysomes. In support of this idea, recent studies have shown that the coding capacities of polysomal and free cytoplasmic mRNAs are different [ 11]. Free cytoplasmic mRNA has been shown to be incorporated into polysomes [ 1 1 - 1 3 ] , a process which may involve the loss of proteins from the RNA [12], or an exchange of proteins in the cytoplasm. In this report the protein constituents of maize free cytoplasmic poly(A) ÷RNP are described and evidence is provided for the association of two proteins with the poly(A)-tract. MA T ER I ALS AND METHODS
Embryos were excised from seedlings of Zea mays that had been grown in sterile tap water for 72--96 h at room temperature [14]. Postpolysomal solutions were prepared as described previously [15], except that the extraction buffer contained 0.5 mM phenylmethanesulfonyl fluoride (PMSF, Sigma Chemical Co.} and heparin {100 ug/ml; Sigma Chemical Co.} [9]. After adjusting the solution to 0.5 M KCI, a pellet of postpolysomal particles was obtained by centrifugation at 250 000 × g for 5 h. The pellets were resuspended in buffer (10 mM Tris--HC1, pH 7.6, 1 mM EDTA, 0.5 M NaC1, 100 ~g/ml heparin and 0.5 mM PMSF) and absorbed to oligo(dT)-cellulose (Type T3; Collaborative Research, Inc.) at 4°C as described previously [9]. Poly(A)÷-RNP was eluted from the column with 50% formamide in buffer (10 mM Tris--HCl, pH 7.6, 1 mM EDTA) at room temperature, mRNP was recovered from the eluted fractions by precipitation with two volumes 95% ethanol a t - 2 0 ° C , The isolated poly(A)÷-RNP had A2~0/A2s0 ratios of between 1.1 and 1.3. Phenol extracted RNA obtained from the free cytoplasmic RNP particles exhibited a heterogeneous sedimentation profile in sucrose gradients and was shown to contain a modified nucleotide characteristic of maize poly(A) ÷RNA [16]. The protein constituents of free cytoplasmic RNP were analyzed by electrophoresis on sodium dodecyl sulphate (NaDodSO4)/10% polyacrylamide gels using the procedure of Laemmli [ 17]. Molecular weight markers (BioRad Laboratories) employed were phosphorylase B (92 500), bovine serum albumin (68 000), ovalbumin (45 000), carbonic anhydrase (31 000), soybean trypsin inhibitor (21 500) and lysozyme (14 400). Each sample contained between 0.3 and 1.0 A~60 units RNP. Densitometric scans of Coomassie blue-stained gels (Hoeffer Scientific Co. electrophoresis manual) were performed on a Gilford 2520 gel scanner at a wavelength of 570 nm. Two-dimensional separation of proteins employing isoelectric focusing and NaDodSO4-polyacrylamide gel electrophoresis was performed using the Anderson and Anderson [18] adaptation of the O'Farrell procedure [19].
63
Samples were denatured with NaDodSO4 and slab gels (second dimension) were 10--15% gradients of acrylamide. Poly(A)-protein complexes were isolated from RNP preparations by a procedure that involved nuclease digestion and readsorption to columns of oligo(dT)-ceUulose. RNP particles in 1 ml 0.1 M Tris--HC1 (pH 7.4), 1 mM EDTA, 0.4 M NaC1 and 0.1 mM PMSF were digested with ribonuclease A (0.2 ~g/ml; Worthington Biochemical Corp.). The solutions were incubated at 10°C for 1 h and then readsorbed to, and eluted from oligo(dT)-cellulose columns as described [9]. R E S U L T S A N D DISCUSSION
The polyacrylamide gel electrophoretic separation of free cytoplasmic poly(A)+-RNP proteins is shown in Fig. la. For comparative purposes, the proteins present in the preparation but n o t retained on the affinity column, are shown in Fig. lb. During the preparation of poly(A)÷-RNP, care was taken to avoid proteolysis and nuclease action by the inclusion of PMSF and heparin in the buffers. In addition, the possible formation of artifactual
a
b
Fig. 1. NaDodSO4-polyacrylamide gel electrophoresis of proteins in poly(A)+-RNP. The numbers indicate the molecular weights (× 10 -3) determined by co-electrophoresis with marker proteins. (a) poly(A)÷-RNP eluted from oligo(dT)-cellulose columns, (b) postpolysornal proteins that were not retained on oligo(dT)-cellulose.
64
RNP complexes [20] was reduced by employing high salt (0.5 M NaCl) concentrations in the buffers. Poly(A)÷-RNP proteins that were present in all preparations had molecular weights of 73 400 (73.4P), 68 900 (68.9P), 66 500 (66.5P), 59 400 (59.4P), 47 000 (47P) and 35 100 daltons (35.1P). Although the relative quantities of the proteins varied somewhat between the preparations, the most prevalent were 68.9P, 66.5P, and 35.1P; the former two proteins were not well resolved because of their close molecular weights. Components 68.9P and 66.5P accounted for as much as 30% of the total protein, assuming comparable staining of all proteins with Coomassie blue. Major proteins (70 000, 36 000, 21 000 Mr) have been noted in RNP preparations from radish seeds [21] ; the 70 000-dalton component may be analogous to the 68.9P/ 66.5P maize proteins. Two~limensional electrophoresis was employed to analyze the protein constituents of poly(A)÷-RNP preparations more fully. A representative separation of RNP proteins is shown in Fig. 2. In most of the two-dimensional separations, at least 30 different proteins could be seen. Most of
Fig. 2. Two-dimensional electrophoretic separation of poly(A)*-RNP protein. The acid end is to the left and NaDodSO 4 electrophoresis proceeded downward. The sample origin was at the upper right hand corner. The numbers indicate molecular weights
(x 10-3).
65
the bands observed in one-dimensional gels are resolved into two or more proteins. However, in the ease of protein bands 68.9P, 66.5P and 59.4P there would appear to be a single protein constituent even though each is resolved into three or four spots having somewhat different charge properties. The charge heterogeneity of proteins 68.9P, 66.5P and 59.4P, has been observed in seven different preparations. The charge differences could be a consequence of deamidation, carbohydrate heterogeneity, acetylation or phosphorylation [19,22]. The more likely of these possibilities is phosphorylation, since protein kinase has been found to be associated with mRNP particles [23,24] and mRNP proteins have been shown to be phosphorylated [23--26]. If the charge differences are not artifactual and reflect the in vivo situation with regard to these proteins, then it is conceivable that they may affect the RNA binding capabilities of the protein and consequently play a role in translational regulation. In support of the association/dissociation capabilities of RNP proteins with RNA, there is now some evidence that mRNA-associated proteins exchange in the cytoplasm with a pool of free proteins [27]. To isolate poly(A)-binding proteins, advantage was taken of the fact that poly(A)-tracts in a medium of high ionic strength are resistant to digestion with ribonuclease A. Poly(A)+-RNP was suspended in a buffer containing 0.5 M sodium chloride and digested with ribonuclease A. The released poly(A)-protein complex was recovered by affinity chromatography on oligo(dT)-cellulose columns and analyzed by NaDodSO4-polyacrylamide gel electrophoresis. Figure 3 shows the densitometric scans of stained O
I Fig. 3. Densitometric scans of NaDodSO4-polyacrylamide gel separations of poly(A)associated proteins. (a), intact poly(A~-RNP, (b), ribonucle~e A digest of poly(A)+-RNP.
66 p r o t e i n s in gels o f s t a r t i n g m a t e r i a l (poly(A)+-RNP) a n d n u c l e a s e - t r e a t e d m a t e r i a l ( p o l y ( A ) - a s s o c i a t e d p r o t e i n s ) . I t is a p p a r e n t t h a t t h e r e is a p r e f e r e n tial r e t e n t i o n o f t w o p r o t e i n s in a s s o c i a t i o n w i t h t h e p o l y ( A ) - t r a c t . T h e s e are 6 8 . 9 P a n d / o r 6 6 . 5 P a n d 59.4P. I n o t h e r s y s t e m s , t h e r e a p p e a r s t o b e o n e m a j o r p r o t e i n (78 0 0 0 Mr) t h a t b i n d s p o l y ( A ) a l t h o u g h lesser a m o u n t s of other proteins have been detected [4-9]. The poly(A)-associated proteins c o u l d p l a y an i m p o r t a n t r e g u l a t o r y role b y a f f e c t i n g t h e a s s o c i a t i o n o f m R N A w i t h t h e r i b o s o m e a n d b y increasing t h e stability o f m R N A t o n u c l e a s e digestion. ACKNOWLEDGEMENTS
T h e a u t h o r s are grateful t o B. Walker w h o p a r t i c i p a t e d in s o m e o f these e x p e r i m e n t s a n d Dr. T. P e a r s o n f o r p e r f o r m i n g t h e t w o - d i m e n s i o n a l electrop h o r e t i c runs. T h i s w o r k was s u p p o r t e d b y a g r a n t f r o m t h e N a t u r a l Sciences a n d E n g i n e e r i n g R e s e a r c h C o u n c i l o f Canada. REFERENCES 1 2 3 4 5
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
A. Preobrazhensky and A. Spirin, Prog. Nucleic Acid. Res. Mol. Biol., 21 (1978) 1. J.R. Greenberg, J. Cell Biol.,65 (1975) 269. D. Rittschof and J.A. Traugh, Eur. J. Biochem., 115 (1981) 45. G. Blobel, Proc. Natl. Acad. Sci. U.S.A., 70 (1973) 924. A. Barrieux, H.A. Ingraham, D.N. David and M.G. Rosenfeld, Biochemistry, 14 (1975) 1815. H. Schwartz and J.E. Darnell, J. Mol. Biol., 104 (1976) 833. V.M. Kish and T. Pederson, J. Biol. Chem., 251 (1976) 5888. W.R. Jeffery, J. Biol. Chem., 252 (1977) 3525. S.K. Jain and S. Sarkar, Biochemistry, 18 (1979) 747. D.S. Kennedy, E. Siegel and S.M. Heywood, FEBS Lett., 90 (1978) 209. G.T-Y. Lee and D.L. Engelhardt, J. Mol. Biol., 129 (1979) 221. D.S. Adams, D. Noonan and W.R. Jeffery, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 83. T. Geoghegan, S. Cereghini and G. Brawerman, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 5587. J.L. Nichols and L. Welder, Biochim. Biophys. Acta, 608 (1980) 1. D.P.S. Verma and G.A. Maclaehlan, Plant Physiol., 58 (1976) 405. J.L. Nichols, Biochim. Biophys. Acta, 652 (1981) 99. U.K. Laemmli, Nature, 227 (1970) 680. N.G. Anderson and N.L. Anderson, Anal. Biochem., 85 (1978) 331. P.H. O'Farrell, J. Biol. Chem., 256 (1975) 4007. D. Baltimore and A.S. Huang, J. Mol. Biol., 47 (1970) 263. A. Fetter, M. Delseny and Y. Guitton, Plant Sci. Lett., 14 (1979) 31. L. Anderson and N,G. Anderson, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 5421. C. Quirin-Stricker and M. Schmitt, Biochim. Biophys. Acta, 477 (1981) 414. J. Bag and B.H. Sells, J. Biol. Chem., 254 (1979) 3137. C. Morel, E.S. Dander, M. Nerzberg, J. Dubochet and K. Scherrer, Eur. J. Biochem., 36 (1973) 455. S. Auerbach and T. Pederson, Biochem. Biophys. Res. Commun., 63 (1975) 149. J. Greenberg, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 2923.