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An electron microscope study of deoxyribonucleoprotamines
296
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96462
AN ELECTRON MICROSCOPE STUDY OF DEOXYRIBONUCLEOPROTAMINES S A D A K O I N O U E * AND M O T O H I R O F U K E ' " , * * "
*Department o[ Biophysics and Biochemistry and ""Department of Physics, Faculty of Science, The University o[ Tokyo, Bunkyo-ku, Tokyo II3 (Japan) (Received October 27th , 1969)
SUMMARY
The nucleoprotamine extracted from the trout testis and the DNA-clupeine complexes reconstituted in solutions have been examined by electron microscopy. Samples were spread on an air-water interphase and transferred onto carbon-coated grids. They were visualized by staining with a uranyl acetate solution or by shadowcasting in vacuum. The electron micrographs showed that both the native nucleoprotamine and the DNA-clupeine complexes were dispersed in solution in the form of extended networks. DNA fibrils appeared to be linked side by side through protamine bridges. No isolated complex, consisting of one DNA double helix with protamine molecules wrapping around it, was observed in these systems. The shapes of DNA-oligo-L-lysine and DNA-oligo-L-arginine complexes were also observed by use of the electron microscope. It was found that the DNA-(Arg)20 complexes form aggregates similar to those formed by the DNA clupeine complexes. However, the former aggregates were smaller than the latter. The DNA-(Arg)8 complexes formed no aggregate. The shape of the aggregates formed by the DNA-(Lys)20 complexes was different from the shape shown by the DNA-clupeine complexes.
INTRODUCTION
In the sperm nuclei of fish, DNA is associated with strongly basic proteins called the protamine. ANDO and co-workers 1-3 have recently determined the amino acid sequences of all three components of clupeine (YI, YII and Z), a typical protamine isolated from the testis of Pacific herring. A clupeine molecule, consisting of a total 3o-3I amino acids, has 20-2I arginine residues and no other basic amino acids. Arginine occurs singly or in sequences comprising a maximum 4 consecutive residues. Between arginine residues, 1-3 neutral amino acid residues are present. A great similarity has been found between the sequences of the three components. At present, clupeine is the only nuclear basic protein of which the amino acid sequence is known and, therefore, is one of the best materials for studying the properties and function of DNA-protein complexes in nuclei. INOUE AND ANDO4 6 have already reported some of the physicochemical properties of DNA-clupeine complexes which *"~ P r e s e n t address: D e p a r t m e n t of Biological Chemistry, H a r v a r d Medical School, Boston. Mass.
Biochim. Biophys. Acta, 204 (197 o) 296"--303
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are reconstituted in solutions, and SUZUKI AND ANDO~ showed an inhibitory effect of clupeine on a DNA-dependent RNA-synthesizing system. This paper reports an electron microscope study of the nucleoprotamine complexes. MATSUO et al. s also studied b y electron microscopy the shapes of the complexes formed between poly-Llysine and nucleic acids. MATERIALS AND METHODS
Materials The preparation of herring sperm DNA and its sonicated specimen were described previously 6. Clupeine was isolated from the testis of Pacific herring and purified according to the method developed by ANDO et al?. The preparation of oligo-Llysine and oligo-L-arginine used as the model substances of clupeine was also described previouslyt The mature testis of trout was obtained at the end of February.
Formation o] DNA-clupeine and other complexes DNA-clupeine and its model complexes were prepared b y titrating, under constant stirring, a constant volume of DNA in 4 M ammonium acetate with the equal volume of the peptide dissolved in the same solvent. The final concentration of DNA was 5O-lOO/*moles P per 1 (A2~o n m = 0.3 -o.6) and the ratio of the basic amino acid residue in the peptide to DNA-phosphorus was I : I .
Extraction o/native nucleoprotamine All extraction procedures were carried out at about 4 °. Trout testis was suspended in o.14 M NaC1 and homogenized in a Waring blender. The homogenate was filtered through nylon gauze and the filtrate was centrifuged at 2000 ×g. The precipitate was washed with o.14 M NaC1 3 times. The washed nuclei were suspended in 4 M ammonium acetate and gently homogenized in a glass homogenizer with a few strokes b y hand. The homogenate was stirred for an hour using a magnetic stirrer and centrituged at IO ooo × g for IO rain. The opalescent supernatant was diluted so that the concentration of DNA was about 8o/~moles P per 1 (A260 n m approx. 0.5).
Electron microscopy Samples were prepared for electron microscopy according to the technique introduced b y KLEINSCHMIDTet al. TM. The solutions, containing the complexes (A260 nm----0.1--0.3) and o.oi % of cytochrome c in 4 or 2 M ammonium acetate, were spread onto the surface of double-distilled water. Portions of the monolayers were transferred onto grids coated with carbon films. The accompanying water was removed b y letting the grid touch the surface of ethanol. They were stained b y floating the grids on a 1 % solution of uranyl acetate in water-acetone (33 : 67, v/v) or shadow-casted rotationally with platinum-palladium (80:20, v/v) at an angle of tan -1 (I/IO). A J E O L JEM-7A electron microscope was used. The samples of native nucleoprotamines were similarly prepared either with or without adding cytochrome c. RESULTS
Electron microscopic analysis o/DNA--protamine complexes Fig. I is a typical electron micrograph of a well-dispersed sample of the complexes formed between intact DNA and clupeine. It shows a network in which all the fragments of DNA are associated. When this DNA specimen alone was examined,
Biochim. Biophys. Acta, 204 (197o) 296-3o 3
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s. INOUE, M. FUKE
Fig. I. A n e t w o r k of D N A clupeine complexes. T h e s a m p l e solution c o n t a i n i n g D N A - c l u p e i n e c o m p l e x e s (d2s 0 nm ~ o.3) a n d o.oi ~o c y t o c h r o m e c in 4 M a m m o n i u m a c e t a t e was spread o n t o t h e surface of water. T h e p r o t e i n m o n o l a y e r film was t r a n s f e r r e d onto a c a r b o n - c o a t e d grid a n d r o t a t i o n a l l y s h a d o w e d w i t h a p l a t i n u m - p a l l a d i u n l alloy.
fragments with lengths of o.5-15 ~ were seen in the field oI the electron microscope. Appaiently clupeine caused the association, probably by forming clupeine bridges between two adjacent DNA fibrils. Short segments of unit fibrils frequently occur either as loops along thick fibrils or as links between two independent thick fibrils. The diameter of the unit fibrils appears to be equal to that of DNA fibrils. However, it has not been determined whether the unit fibrils represent free DNA or isolated complexes in which clupeine is wrapped arouPd DNA double-helices. The thick fibrils consist of 2-4 (6 in some instances) unit fibrils associated side by side. The association appears to occur either between two independent fibrils or between parts of the same fibril which has folded on itself. A similar network is seen in the electron Inicrograph of the nucleoprotalnine extracted from the sperm nuclei of trout (Fig. 2a). The amino acid sequences of trout protamines have been found to be similar to those of clupeine 11. It should be noted that the sample for this electron micrograph was prepared in the absence of cytochrome c (see MATERIALS AND METHODS). Similar electron inicrographs were obtained when cytochrome c was added to the samples. Thus cytochrome c, although it is also a basic protein, does not affect the association of DNA and the protalnine. Fig. 2b shows an electron micrograph of the trout nucleoprotamine stained with Biochim. Biophys. Acta, 2o 4 (197 o) 296-3o3
Fig. z. A. Nucleoprotamine fibrils extracted from trout sperm nuclei with 4 M ammonium acetate. The sample solution was spread onto the surface of water without adding cytochrome C. Shadow-casting was with a platinum-palladium alloy. B. Same as A, but stained with I yO solution of uranyl acetate in a water-acetone mixture (33 : 67, v/v).
Fig. 3. Complexes formed between sonicated DNA palladium alloy. B. Stained with uranyl acetate.
and clupeine.
A. Shadowed
with a platinum-
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MICROSCOPYOF
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Fig. 4. Aggregates formed by various DNA-peptide complexes. Complexes were prepared in z M ammonium acetate with sonicated DNA. Samples were shadowed with a platinum-pallaC. DNA-(Arg),,. D. DNA-clupeine. dium alloy. A. DNA-(Arg),. B. DNA-(Lys),,. Biochim.
Biqbhys.
Acta,
204
(1970)
296-303
302
S. INOUE, M. FUKE
uranyl acetate. The magnification of this photograph is higher than that of Fig. 2a. The fact that the fibrils seen in Fig. 2a can be stained well with uranyl acetate indicates that DNA is one of the main components of the fibrils. This photograph clearly shows the segments where two unit fibrils are combined into one thick fibril. Figs. 3 a and 3b show that complexes formed between sonicated DNA and clupeine also aggregate into large networks. The average length of tile sonicated DNA was found to be o.2/*. The mode of association of the fragments can be seen in these electron micrographs.
Comparison o/ DNA-clupeine with other complexes containing model peptides Fig. 4 compares the electron micrographs of DNA-(Arg)s', DNA-(Lys)~o ~, DNA-(Arg)~0 ~ and DNA-clupeine complexes formed under identical conditions. In this series of experiments, 2 M instead of 4 M ammonium acetate was used as a solvent. DNA had been subjected to the sonic treatment at 5o ~o power supply; the treatment was milder than for the sonicated DNA used to prepare complexes shown in Figs. 3a and 3b, and the specimen had an average length of 0.4/~. The electron micrograph of the DNA-(Arg)8 system shows only isolated fibrils which cannot be distinguished from those of free DNA. An essentially similar pattern was obtained for the DNA-(Lys)8 system. Both the DNA-(Arg)2 0 and DNA-clupeine aggregates shown in Fig. 4 are poorly dispersed. These patterns were obtained when the concentration of ammonium acetate was low. It is noted that the aggregates formed by DNA-clupeine complexes are larger than those formed by DNA-(Arg)2 0 complexes. The DNA-(Lys)~ 0 complexes show quite different features in the shape of the aggregate. The unit fibrils tend to form particles of rather uniform size and do not form long thick threads or networks.
DISCUSSION
On the basis of X-ray analysis, it has been established that the protamine is wrapped in the smaller groove of the DNA double helix 1~. Practically the nucleoprotamine, either native (the chromosome) or reconstituted, forms a gel which has a complex structure. It has been shown previously that the artificially formed DNAclupeine complexes can be dispersed into dilute "solutions". Sedimentation 6 and gel filtration n studies have shown that DNA-clupeine complexes are aggregated when the ratio of arginine to phosphorus is about unity. In the present study, an attempt has been made to observe the aggregates formed by the DNA-protamine complexes by use of the electron microscope. When the complexes were prepared in dilute salt solutions by the direct mixing or gradient dialysis method s and then placed on grids, clumps similar to those reported by MATSUO et al. s were observed under the electron microscope. In 4 M (or 2 M) ammonium acetate, DNA and the protamine (or basic polyamino acids) are apparently dissociated and the complexes as observed in the present study are formed during the process of spreading the sample on a monolayer. Thus although these DNA-protamine and other complexes were formed under limited conditions, the present electron microscope study may have visualized several phenomena associated with the complex formation. First, the DNA unit " The suffix indicates the degree of polymerization of oligo- and polyamino acids.
Biochim. Biophys. Acta, 204 (197o) 296-303
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fibrils can be linked together by the protamine. Secondly, poly-L-arginine (degree of polymerization = 20) has an ability to form DNA complexes similar to DNAclupeine. Such complexes are not formed by oligo-L-arginine (degree of polymerization = 8); the binding of this oligomer to DNA is weak. These findings are in accord with the results obtained from the thermal melting studies on the binding of DNA with clupeine, polyamino acids and oligoamino acids6,14. Thirdly, the present results show that there are differences in the shape and size of the aggregates formed by the complexes. The DNA-clupeine complexes form larger aggregates than the other complexes. The DNA-(Lys)20 complexes show somewhat different shape and their size is smaller than that of DNA-(Arg)~ 0 or DNA-clupeine. These observations are comparable with the results obtained through gel filtration studies1~. ACKNOWLEDGMENTS
The authors are grateful to Professors Toshio Ando and Akiyoshi Wada for their interest in this study. This work was supported in part by a grant from the ministry of education of Japan. REFERENCES i T. ANDO, I~. IWAI, S. ISHII, lV[. AZEGAMI AND C. NAKAHARA, Biochim. Biophys. Acta, 56 (1962) 628. 2 T. ANDO AND K. SUZUKI, Biochim. Biophys. Acta, i 2 I (1966) 427. 3 T. ANDO AND K. SUZUKI, Biochim. Biophys. Acta, 14o (1967) 375. 4 S. INOUE AND T. ANDO, Biochim. Biophys. Acta, 129 (1966) 649. 5 S. INOU]~ AND T. ANDO, Biochem. Biophys. Res. Commun., 32 (1968) 5Ol. 6 S. INOUE AND T. ANDO, Biochemistry, 9 (197 o) 388, 395. 7 K. SUZUKI AND T. ANDO, J. Biochem. Tokyo, 65 (1969) 831. 8 K. 1V[ATSUO, M. FUKE, iV[. TSUBOI AND A. WADA, Biochim. Biophys. Acta, 179 (1969) 39. 9 T. ANDO, S. ISHII, M. YAMASAKI, K. IWAI, C. HASHIMOTO AND F. SAWADA,J. Biochem. Tokyo, 44 (1957) 275. io A. K. KLEINSCHMIDT, D. LANG, D. JACHERTS AND R, I~. ZAHN, Biochim. Biophys. Acta, 61 (1962) 857. I I S. WATANABE AND T. ANDO, Abstr. z9th Syrup. Protein Structure, Tokyo, r968, p. 9. i2 M. H. F. WILKINS, Cold Spring Harbor Symp. Quant. Biol., 2I (1956) 75. 13 S. INOUm AND T. ANDO, to be published. 14 S. KAWASHIMA, S. INOUE AND T. ANDO, Biochim. Biophys. Acta, 186 (1969) 145.
Biochim. Biophys. Acta, 204 (197 o) 296-.303
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96462
AN ELECTRON MICROSCOPE STUDY OF DEOXYRIBONUCLEOPROTAMINES S A D A K O I N O U E * AND M O T O H I R O F U K E ' " , * * "
*Department o[ Biophysics and Biochemistry and ""Department of Physics, Faculty of Science, The University o[ Tokyo, Bunkyo-ku, Tokyo II3 (Japan) (Received October 27th , 1969)
SUMMARY
The nucleoprotamine extracted from the trout testis and the DNA-clupeine complexes reconstituted in solutions have been examined by electron microscopy. Samples were spread on an air-water interphase and transferred onto carbon-coated grids. They were visualized by staining with a uranyl acetate solution or by shadowcasting in vacuum. The electron micrographs showed that both the native nucleoprotamine and the DNA-clupeine complexes were dispersed in solution in the form of extended networks. DNA fibrils appeared to be linked side by side through protamine bridges. No isolated complex, consisting of one DNA double helix with protamine molecules wrapping around it, was observed in these systems. The shapes of DNA-oligo-L-lysine and DNA-oligo-L-arginine complexes were also observed by use of the electron microscope. It was found that the DNA-(Arg)20 complexes form aggregates similar to those formed by the DNA clupeine complexes. However, the former aggregates were smaller than the latter. The DNA-(Arg)8 complexes formed no aggregate. The shape of the aggregates formed by the DNA-(Lys)20 complexes was different from the shape shown by the DNA-clupeine complexes.
INTRODUCTION
In the sperm nuclei of fish, DNA is associated with strongly basic proteins called the protamine. ANDO and co-workers 1-3 have recently determined the amino acid sequences of all three components of clupeine (YI, YII and Z), a typical protamine isolated from the testis of Pacific herring. A clupeine molecule, consisting of a total 3o-3I amino acids, has 20-2I arginine residues and no other basic amino acids. Arginine occurs singly or in sequences comprising a maximum 4 consecutive residues. Between arginine residues, 1-3 neutral amino acid residues are present. A great similarity has been found between the sequences of the three components. At present, clupeine is the only nuclear basic protein of which the amino acid sequence is known and, therefore, is one of the best materials for studying the properties and function of DNA-protein complexes in nuclei. INOUE AND ANDO4 6 have already reported some of the physicochemical properties of DNA-clupeine complexes which *"~ P r e s e n t address: D e p a r t m e n t of Biological Chemistry, H a r v a r d Medical School, Boston. Mass.
Biochim. Biophys. Acta, 204 (197 o) 296"--303
ELECTRON MICROSCOPY OF DEOXYRIBONUCLEOPROTAMINES
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are reconstituted in solutions, and SUZUKI AND ANDO~ showed an inhibitory effect of clupeine on a DNA-dependent RNA-synthesizing system. This paper reports an electron microscope study of the nucleoprotamine complexes. MATSUO et al. s also studied b y electron microscopy the shapes of the complexes formed between poly-Llysine and nucleic acids. MATERIALS AND METHODS
Materials The preparation of herring sperm DNA and its sonicated specimen were described previously 6. Clupeine was isolated from the testis of Pacific herring and purified according to the method developed by ANDO et al?. The preparation of oligo-Llysine and oligo-L-arginine used as the model substances of clupeine was also described previouslyt The mature testis of trout was obtained at the end of February.
Formation o] DNA-clupeine and other complexes DNA-clupeine and its model complexes were prepared b y titrating, under constant stirring, a constant volume of DNA in 4 M ammonium acetate with the equal volume of the peptide dissolved in the same solvent. The final concentration of DNA was 5O-lOO/*moles P per 1 (A2~o n m = 0.3 -o.6) and the ratio of the basic amino acid residue in the peptide to DNA-phosphorus was I : I .
Extraction o/native nucleoprotamine All extraction procedures were carried out at about 4 °. Trout testis was suspended in o.14 M NaC1 and homogenized in a Waring blender. The homogenate was filtered through nylon gauze and the filtrate was centrifuged at 2000 ×g. The precipitate was washed with o.14 M NaC1 3 times. The washed nuclei were suspended in 4 M ammonium acetate and gently homogenized in a glass homogenizer with a few strokes b y hand. The homogenate was stirred for an hour using a magnetic stirrer and centrituged at IO ooo × g for IO rain. The opalescent supernatant was diluted so that the concentration of DNA was about 8o/~moles P per 1 (A260 n m approx. 0.5).
Electron microscopy Samples were prepared for electron microscopy according to the technique introduced b y KLEINSCHMIDTet al. TM. The solutions, containing the complexes (A260 nm----0.1--0.3) and o.oi % of cytochrome c in 4 or 2 M ammonium acetate, were spread onto the surface of double-distilled water. Portions of the monolayers were transferred onto grids coated with carbon films. The accompanying water was removed b y letting the grid touch the surface of ethanol. They were stained b y floating the grids on a 1 % solution of uranyl acetate in water-acetone (33 : 67, v/v) or shadow-casted rotationally with platinum-palladium (80:20, v/v) at an angle of tan -1 (I/IO). A J E O L JEM-7A electron microscope was used. The samples of native nucleoprotamines were similarly prepared either with or without adding cytochrome c. RESULTS
Electron microscopic analysis o/DNA--protamine complexes Fig. I is a typical electron micrograph of a well-dispersed sample of the complexes formed between intact DNA and clupeine. It shows a network in which all the fragments of DNA are associated. When this DNA specimen alone was examined,
Biochim. Biophys. Acta, 204 (197o) 296-3o 3
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s. INOUE, M. FUKE
Fig. I. A n e t w o r k of D N A clupeine complexes. T h e s a m p l e solution c o n t a i n i n g D N A - c l u p e i n e c o m p l e x e s (d2s 0 nm ~ o.3) a n d o.oi ~o c y t o c h r o m e c in 4 M a m m o n i u m a c e t a t e was spread o n t o t h e surface of water. T h e p r o t e i n m o n o l a y e r film was t r a n s f e r r e d onto a c a r b o n - c o a t e d grid a n d r o t a t i o n a l l y s h a d o w e d w i t h a p l a t i n u m - p a l l a d i u n l alloy.
fragments with lengths of o.5-15 ~ were seen in the field oI the electron microscope. Appaiently clupeine caused the association, probably by forming clupeine bridges between two adjacent DNA fibrils. Short segments of unit fibrils frequently occur either as loops along thick fibrils or as links between two independent thick fibrils. The diameter of the unit fibrils appears to be equal to that of DNA fibrils. However, it has not been determined whether the unit fibrils represent free DNA or isolated complexes in which clupeine is wrapped arouPd DNA double-helices. The thick fibrils consist of 2-4 (6 in some instances) unit fibrils associated side by side. The association appears to occur either between two independent fibrils or between parts of the same fibril which has folded on itself. A similar network is seen in the electron Inicrograph of the nucleoprotalnine extracted from the sperm nuclei of trout (Fig. 2a). The amino acid sequences of trout protamines have been found to be similar to those of clupeine 11. It should be noted that the sample for this electron micrograph was prepared in the absence of cytochrome c (see MATERIALS AND METHODS). Similar electron inicrographs were obtained when cytochrome c was added to the samples. Thus cytochrome c, although it is also a basic protein, does not affect the association of DNA and the protalnine. Fig. 2b shows an electron micrograph of the trout nucleoprotamine stained with Biochim. Biophys. Acta, 2o 4 (197 o) 296-3o3
Fig. z. A. Nucleoprotamine fibrils extracted from trout sperm nuclei with 4 M ammonium acetate. The sample solution was spread onto the surface of water without adding cytochrome C. Shadow-casting was with a platinum-palladium alloy. B. Same as A, but stained with I yO solution of uranyl acetate in a water-acetone mixture (33 : 67, v/v).
Fig. 3. Complexes formed between sonicated DNA palladium alloy. B. Stained with uranyl acetate.
and clupeine.
A. Shadowed
with a platinum-
ELECTRON
MICROSCOPYOF
301
DEOXYRIBONUCLEOPROTAMINES
Fig. 4. Aggregates formed by various DNA-peptide complexes. Complexes were prepared in z M ammonium acetate with sonicated DNA. Samples were shadowed with a platinum-pallaC. DNA-(Arg),,. D. DNA-clupeine. dium alloy. A. DNA-(Arg),. B. DNA-(Lys),,. Biochim.
Biqbhys.
Acta,
204
(1970)
296-303
302
S. INOUE, M. FUKE
uranyl acetate. The magnification of this photograph is higher than that of Fig. 2a. The fact that the fibrils seen in Fig. 2a can be stained well with uranyl acetate indicates that DNA is one of the main components of the fibrils. This photograph clearly shows the segments where two unit fibrils are combined into one thick fibril. Figs. 3 a and 3b show that complexes formed between sonicated DNA and clupeine also aggregate into large networks. The average length of tile sonicated DNA was found to be o.2/*. The mode of association of the fragments can be seen in these electron micrographs.
Comparison o/ DNA-clupeine with other complexes containing model peptides Fig. 4 compares the electron micrographs of DNA-(Arg)s', DNA-(Lys)~o ~, DNA-(Arg)~0 ~ and DNA-clupeine complexes formed under identical conditions. In this series of experiments, 2 M instead of 4 M ammonium acetate was used as a solvent. DNA had been subjected to the sonic treatment at 5o ~o power supply; the treatment was milder than for the sonicated DNA used to prepare complexes shown in Figs. 3a and 3b, and the specimen had an average length of 0.4/~. The electron micrograph of the DNA-(Arg)8 system shows only isolated fibrils which cannot be distinguished from those of free DNA. An essentially similar pattern was obtained for the DNA-(Lys)8 system. Both the DNA-(Arg)2 0 and DNA-clupeine aggregates shown in Fig. 4 are poorly dispersed. These patterns were obtained when the concentration of ammonium acetate was low. It is noted that the aggregates formed by DNA-clupeine complexes are larger than those formed by DNA-(Arg)2 0 complexes. The DNA-(Lys)~ 0 complexes show quite different features in the shape of the aggregate. The unit fibrils tend to form particles of rather uniform size and do not form long thick threads or networks.
DISCUSSION
On the basis of X-ray analysis, it has been established that the protamine is wrapped in the smaller groove of the DNA double helix 1~. Practically the nucleoprotamine, either native (the chromosome) or reconstituted, forms a gel which has a complex structure. It has been shown previously that the artificially formed DNAclupeine complexes can be dispersed into dilute "solutions". Sedimentation 6 and gel filtration n studies have shown that DNA-clupeine complexes are aggregated when the ratio of arginine to phosphorus is about unity. In the present study, an attempt has been made to observe the aggregates formed by the DNA-protamine complexes by use of the electron microscope. When the complexes were prepared in dilute salt solutions by the direct mixing or gradient dialysis method s and then placed on grids, clumps similar to those reported by MATSUO et al. s were observed under the electron microscope. In 4 M (or 2 M) ammonium acetate, DNA and the protamine (or basic polyamino acids) are apparently dissociated and the complexes as observed in the present study are formed during the process of spreading the sample on a monolayer. Thus although these DNA-protamine and other complexes were formed under limited conditions, the present electron microscope study may have visualized several phenomena associated with the complex formation. First, the DNA unit " The suffix indicates the degree of polymerization of oligo- and polyamino acids.
Biochim. Biophys. Acta, 204 (197o) 296-303
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303
fibrils can be linked together by the protamine. Secondly, poly-L-arginine (degree of polymerization = 20) has an ability to form DNA complexes similar to DNAclupeine. Such complexes are not formed by oligo-L-arginine (degree of polymerization = 8); the binding of this oligomer to DNA is weak. These findings are in accord with the results obtained from the thermal melting studies on the binding of DNA with clupeine, polyamino acids and oligoamino acids6,14. Thirdly, the present results show that there are differences in the shape and size of the aggregates formed by the complexes. The DNA-clupeine complexes form larger aggregates than the other complexes. The DNA-(Lys)20 complexes show somewhat different shape and their size is smaller than that of DNA-(Arg)~ 0 or DNA-clupeine. These observations are comparable with the results obtained through gel filtration studies1~. ACKNOWLEDGMENTS
The authors are grateful to Professors Toshio Ando and Akiyoshi Wada for their interest in this study. This work was supported in part by a grant from the ministry of education of Japan. REFERENCES i T. ANDO, I~. IWAI, S. ISHII, lV[. AZEGAMI AND C. NAKAHARA, Biochim. Biophys. Acta, 56 (1962) 628. 2 T. ANDO AND K. SUZUKI, Biochim. Biophys. Acta, i 2 I (1966) 427. 3 T. ANDO AND K. SUZUKI, Biochim. Biophys. Acta, 14o (1967) 375. 4 S. INOUE AND T. ANDO, Biochim. Biophys. Acta, 129 (1966) 649. 5 S. INOU]~ AND T. ANDO, Biochem. Biophys. Res. Commun., 32 (1968) 5Ol. 6 S. INOUE AND T. ANDO, Biochemistry, 9 (197 o) 388, 395. 7 K. SUZUKI AND T. ANDO, J. Biochem. Tokyo, 65 (1969) 831. 8 K. 1V[ATSUO, M. FUKE, iV[. TSUBOI AND A. WADA, Biochim. Biophys. Acta, 179 (1969) 39. 9 T. ANDO, S. ISHII, M. YAMASAKI, K. IWAI, C. HASHIMOTO AND F. SAWADA,J. Biochem. Tokyo, 44 (1957) 275. io A. K. KLEINSCHMIDT, D. LANG, D. JACHERTS AND R, I~. ZAHN, Biochim. Biophys. Acta, 61 (1962) 857. I I S. WATANABE AND T. ANDO, Abstr. z9th Syrup. Protein Structure, Tokyo, r968, p. 9. i2 M. H. F. WILKINS, Cold Spring Harbor Symp. Quant. Biol., 2I (1956) 75. 13 S. INOUm AND T. ANDO, to be published. 14 S. KAWASHIMA, S. INOUE AND T. ANDO, Biochim. Biophys. Acta, 186 (1969) 145.
Biochim. Biophys. Acta, 204 (197 o) 296-.303