- Email: [email protected]
Isolation and characterization of chromosomal proteins from the mosquito Anopheles albimanus weidemann
In/. J. Bkxhem. Vol. 20. No.
Printed in Great Britain
Il. pp. 1247-1253, 1988
0020-71 I X/88 $3.00 + 0.00 Pergamon Press plc
ISOLATION CHROMOSOMAL
AND CHARACTERIZATION OF PROTEINS FROM THE MOSQUITO ANOPHELES ALBIMANUS WEIDEMANN STEVE MILLER
Control Technology
Branch,
Division of Parasitic Diseases, Center for Infectious Disease Control, Atlanta, GA 30333, U.S.A.
Disease,
Centers
for
(Received 26 February 1988) Abstract-l. Nuclei were isolated from adult anopheline mosquitoes and fractionated into nucleolar chromatin, nucleoplasmic chromatin and ribonucleoprotein particles by sucrose density gradients. 2. Histones and nonhistone proteins were selectively dissociated from chromatin by treatment with sodium chloride, urea and guanidine HCl. 3. A special class of nonhistone proteins (tight proteins) were extracted from chromatin with Na,P,O,.
4. The electrophoretic properties of the histones, nonhistone proteins and ribonucleoprotein particles were examined by isoelectric focusing and SDS multiphase polyacrylamide gel electrophoresis. 5. By contrast to the histones, the nonhistone proteins displayed considerable heterogeneity. 6. Possible functional implications of the chromosomal proteins are discussed.
INTRODUCTION
DNA of eukaryotes is associated with a specific level of histones, a variable amount of nonhistone chromosomal proteins (NHCP) and RNA to form the chromatin complex. There is sufficient evidence that the chromatin proteins play an important role in activation and regulation of genes. Histones are considered as nonspecific repressors of transcription of chromatin DNA as well as structural components of nucleosomes (Olins and Olins, 1974; Van Holde et al., 1974; Hnilica, 1972). They are conserved from an evolutionary standpoint (Isenberg, 1979). The nonhistone proteins are a more heterogenous group and are not only involved in the stabilization of DNA strands during strand separation but also may function as derepressors (Elgin and Weintraub, 1975; Dastugue and Crepin, 1979; Stein and Stein, 1976; Frenster, 1965). In this communication, a procedure is described for preparing nuclei from adult anopheline mosquitoes and resolving the nuclei into nucleoplasmic and nucleolar fractions and ribonucleoprotein particles. The histones and nonhistone proteins were extracted from the two fractions under mild conditions taking advantage of the differential DNA binding properties of the chromosomal proteins and characterized by SDS multiphase polyacrylamide electrophoresis and isoelectric focusing. MATERIALS AND METHODS
Experimental organism The experimental organism used in this study was Anopheles albimanus originating from Panama. Larvae were reared on essential larvae nutrients at 26°C (Miller, 1979). Pupae were placed in distilled water for emergence. Pharate adults were selected for the study (Hinton, 1968).
Sample preparation Anopheles albimanus pharate adults were suspended in ice cold ‘Buffer A (IOmM KCl, 1OmM MgCI,, 20mM Tris-HCl. DH 7.6. 3.0 mM CaCl,. 1 mM spermine sulfate, I mM phenylmethylsulfonyl Buo>de, 2 mM dithiothreitol) and homogenized 5 strokes with a tight fitting motor driven homogenizer. The homogenate was filtered through cheesecloth and the pellet was homogenized and filtered an additional two times, The combined filtrates were centrifuged at 800 g for 10 min. The nuclear pellet was washed three times with Buffer A to remove the cytoplasmic contaminants. Sucrose
gradient centrifugation
The nuclei were fractionated by the following procedure. Nucleoli were sedimented by low speed centrifugation (45OOg, 20 min) through 30% sucrose while nucleoplasmic chromatin and ribonucleoprotein particles (RNP) remained on top of the sucrose. Nucleoplasmic chromatin and RNP particles were removed, layered on a l5-30% linear sucrose gradient and centrifuged at 40,OOOg for I7 hr. Under these conditions, the nucleoplasmic chromatin sediments as a pellet at the bottom of the tube and most of the RNP particles remain in the gradient where they are resolved into a heterogeneous population due to their overlapping range of sizes. The sucrose was removed from the fractions by dialysis against 20 mM TrissHCl, pH 7.6 containing 1 mM PMSF and 50 mM NaHSO,. Extraction of chromosomal proteins Histones were extracted from nucleolar and nucleoplasmic chromatin with 2% NaCl-75% ethanol and 2.0 M NaCI. After removal of histones, the nonhistone proteins were extracted from the resulting chromatin with 2.0M NaCl-5.0 M urea and 2.0 M NaC14.0 M guanidine HCl. The tight bound proteins were then removed from the pellet with 0.07 M Na,P,O,. The combined extracts containing dissociated histones and non-histone proteins were dialyzed, lyophilized, and recovered for SDS multiphase polyacrylamide electrophoresis and isoelectric focusing. The extraction effectiveness was determined by protein analysis. 1247
STEVEMILLER
1248 Polyacrylamide gel electrophoresis
Proteins were separated by combining the techniques of sodium dodecylsulfate (SDS) gel electrophoresis and electrophoresis in a discontinuous buffer system (multiphase) as described by Neville (1971). A 3.5% acrylamide gel served as a stacking gel and a 10% acrylamide gel as a resolving gel. The runs were at 5 ‘C for 665 V hr. After electrophoresis. the gels were fixed in 10% (w/v) trichloroacetic acid for I hr at room temperature and stained overnight by immersion in a solution of 0.05% Page Blue 83 m ethanol acetic acid-water (30: IO:60 by vol). The gels were destained in ethanol&acetic acid-water (25: 8:65 by vol) and were photographed. Isoelecrric .focusing Thin layer isoelectric focusing was performed with a LKB*-2117 unit. Gels were prepared with LKB carrier ampholytes pH range 3.5510. The gels were prerun for 1 hr at 10 Watt constant power to establish a pH gradient before the protein samples were applied. The samples (25 pl) were applied at the cathodic end of the gel and isoelectric focusing was carried out for 1860 Volt hr. The fixation and staining of protein bands were as described previously.
NaHSO,. The histones are very susceptible to proteolytic degradation and extraction in the presence of inhibitors in no way changed the isoelectric focusing and multiphase polyacrylamide electrophoretic patterns (unpublished results). The resulting pellet containing deoxyribonucleoproteins was then extracted with 2.0 M NaCl. This step removed 60% of the DNA and contained the remaining histones. After removal of the histones. chromatin was extracted with 2.0 M NaCILS.0 M urea and 2.0 M NaCI4.0 M guanidine HCI. It was necessary to use urea and guanidine in the dissociation procedure because of the limited solubility and strong interaction of the chromatin components. Guanidine further disrupted hydrogen bonds. The extractions with each solvent system resulted in removal of a particular protein fraction from the chromatin complex. The tight bound proteins were removed with Na,P20;. The extraction series solubilized 95% or greater all proteins present in chromatin as shown by a final
Analytical methods Protein concentrations were determined, after separation of histones, nonhistone proteins and RNP particles. by the method of Lowry et al. (1951). Bovine serum albumin was used as a standard. The concentration of DNA in chromatin was estimated by the diphenylamine method of Burton (1968) using calf thymus DNA as a standard. RNA was determined by the orcinol method of Schneider (1957) using purified yeast RNA standard.
000
0%
RESULTS Studies establishing chromosomal proteins in mosquito populations require a method for the preparation of nuclei and a method for the separation, dissociation, and analysis of chromatins and ribonucleoprotein particles. In the preparation of nuclei, mosquito tissue were disrupted by homogenization and tissue fragments removed by filtration through cheesecloth. The nuclei were selectively sedimented by low speed centrifugation and washed by repeated suspension and sedimentation. Figure I outlines the procedure for the fractionation of nuclei. The largest structure, the nucleoli were removed by low speed 30% sucrose centrifugation. Nucleoplasmic chromatin and RNP particles were separated on a 15-30% sucrose gradient as first described by Pederson (1981). The RNP particles contained no detectable DNA and had a protein to RNA ratio of 4: 1. In dissociating the chromatin complex and separating histones from nonhistone chromosomal proteins (NHCP), the use of acid, strongly alkaline and phenol extractions were avoided because of the variable solubility of the histones and because of the potentially irreversible effect on the structure of NHCP. Figure 2 outlines the procedure for dissociating mosquito nucleoplasmic and nucleolar chromatin under mild conditions. The histones were extracted from chromatin with 2% NaClL75% ethanol containing the proteolytic inhibitors PMSF and *Use of trade names is for identification purposes only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.
(4,500 X a, 20
mid
RNP and chromatin
nucleoli
I
centrifuge (40,000 x g, 17 hrs)
RNP particles
chromatln Fig. 1. Fractionation
of mosquito sucrose.
nuclear
components
on
Anopheline chromosomal Nuclear
proteins
component (nucleoplasmic chromatin or nucleolar
1249 chromatin)
homogenization with 2% NaCl 75% ethanol
Centrifuge (12.100 x g, 20 min)
Suparnatant
= 2% NaCl 75% ethanol.soluble fraction
homogenization with 2 M NaCl
Canirifuge (12,100 x g, 20 min)
Supernatant
= 2 M NaCl soluble fraction
homogenization with 2 M NaCI/ 5 M urea
Centrifuge 112,100 x g. 20 min)
Supernatant
= 2 M NaCl/!iM urea soluble fraction
pellet P
homogamzation with 2.0 M NaCI/ 4 M guanidine HCI
Centrifuge (12,100 x g. 20 min)
Suparnatant
= 2 M NaCll4 M guanidine HCI soluble fraction
residual fraction solubilized in 0.07 M Na4P207 (pH 9.0)
Fig. 2. Scheme of nucleolar and nucleoplasmic chromatin dissociation. extraction with 1.0 M NaOH and analysis of the alkaline extract. Protein composition of the extracts was determined by isoelectric focusing and SDS multiphase polyacrylamide electrophoresis. IEF patterns are shown in Fig. 3 and the number of protein bands of each extract with the isoelectric point range is presented in Table 1. IEF of the 2% NaCl-75% ethanol extract from nucleoplasmic chromatin showed two protein bands at a pH of 5.8 and 5.9 and showed 16 bands from nucleolar chromatin in the pH region of the gel 3.8-6.1. The difference in the number of protein bands from the two extracts indicates that nucleolar chromatin contains unmodified as well as modified histones and high mobility group proteins. The proteins from the 2.0 M NaCl extracts focused into 7 bands from nucleoplasmic chromatin and 8 bands from nucleolar chromatin covering a region of 0.9 unit with a pI range of 5.4-6.3. Unlike the DNA histones, the nonhistone proteins are not dissociated from chromatin with 2.0 M NaCl. In order to determine the charge characteristics of the nonhistones chromosomal proteins, chromatin, after removal of histones, was first extracted with 2.0 M NaCl-5.0M urea and the extract analyzed by IEF. The banding patterns were complex with at least 11 protein bands from nucleoplasmic chromatin and 18 bands from nucleolar chromatin focusing in the acidic p1 range of 4.1-5.9. Non-histone proteins were then further extracted from chromatin with
2.0 NaC14.0 M guanidine HCl. IEF of the proteins from nucleoplasmic and nucleolar chromatin showed similar banding patterns. The proteins from the two types of chromatin focused moderately acidic with a p1 range 4.1-7.4 differing in range from the proteins extracted with sodium chloride-urea. There is a class of NHCP (tight bound proteins) which fail to dissociate from DNA at a very high ionic strength (2.5 M NaCI-5.0 M urea) (Pederson and Bhorjee, 1975). The tight bound proteins were extracted from the two types of chromatin with Na,P,O,. The protein extract from nucleoplasmic chromatin displayed ~1’s 4.5-5.9 and from nucleolar chromatin 4.3-7.0. IEF of tight bound proteins from HeLa cells revealed a p1 range of 5.0-6.0 (Pederson and Bhorjee, 1975). Isoelectric focusing of ribonucleoprotein particles showed that the proteins are moderately acidic with a p1 range of 4.3-6.7. The RNP particles had a protein-RNA ratio of 4: 1. Extensive characterization of RNP particles from various sources revealed a protein-RNA ratio of 4-5: 1 (Wilt et al., 1973; Wilks and Knowler, 1981). The major proteins of RNP particles from rat liver displayed PI’S between 4.9-6.5. (Pederson, 1974). Table 2 lists the approximate molecular weight range of protein extracts from the two types of chromatin. Figure 4 illustrates SDS multiphase polyacrylamide electrophoresis of chromosomal proteins. The 2% NaCl-75% ethanol extract from nucleo-
1250
STEVEMJLLER Table
1. Isoelectric
point range (PI’S) of protein extracts from nucleoplasm~c chromatm, chromatin and proteins from ribonucieourotein oartick iRNPl
Extract 2% N&-75% ethanol 2.0 M NaCl 2.0 M N&I-5.0 M urea 2.0M NaCI-4.0M GuHCl 0.07 M Na,P,O, RNP
Table 2. Molecular
Nucl~pl~ic Number of bands
Chromntin
2 7 II 25 17 21
5.x--5.9 5.4-6.3 4.5-5.x 4.4-7.4 4.5.-5.9 4.3-6.7
pi range
weight range of protein extracts from nucleoplasmic from ribonucieoprotein particles NueieopIasmk Number of bands
Extract 2% N&-75% ethanol 2.0 M NaCl 2.0 M NaCI-5.0 M urea 2.0 M NaCik4.0 M GuHCl 0.07 M NalP20, RNP
3 6 I6 22 16 9
plasmic chromatin showed 3 protein bands with molecular weights of lO,OO@-14,000and the extract from nucleolar chromatin showed 12 bands with a molecular weight range of 14,00~76,0~. The size of the proteins from nucleoplasmic chromatin are in the range of unmodified calf thymus and C~orist~~eura ~~~z~~u~~ histones (Hnilica, 1972; Pitel and Durzan, 1978). The remaining histones bound by electrostatic and Van der Waals forces were extracted with 2.0 M NaCl as described by Spelsberg and Hnihca (1970). The electrophoretic results of the 2.0 M NaCl extract revealed 6 protein bands distributed through the lO,OOO-72,000 molecular weight range from nucleoplasmic chromatin and revealed 6 bands from nucleolar chromatin in the range of 12,OOt&60,000. The nonhistone chromosomal protein banding patterns were complex as shown by the number of proteins separated by SDS multiphase electrophoresis. The 2.0 M N&I-5.0 M urea extract from nucleoplasmic and from nucleolar ~hromatin revealed quahtative similar but quantitatively very different banding patterns (Fig. 4). The electrophoretic patterns of the 2.0 M NaCI-4.0 M guanidine HCl extract from the two types of chromatin showed many similar bands with different intensities. The remaining NHCP were extracted with Na,P,O, under slightly basic conditions and were referred to as tight bound proteins. The tight bound proteins were enriched in the 14,000-30,000 molecular weight components. This is a general property of this class of chromatin proteins. SDS multiphase electrophoresis of RNP particles showed a population of proteins with a molecular weight range from 14,~94,~. SDS polyacrylamide gel electrophoresis of RNP from different cells revealed a distinctive pattern for each ceil type (Pederson, 1974). DISCUSSION
Transcription of genes in eukaroytic cells is intimately associated with chromosomal proteins (Tata
Ndeolar Number of bands
chromatin. (RNP)
Chromstin Molecular weight range 10,~14,0~ lO.ooo-72,000 14,00@120,000 14,000-120.000 14,ocu-92,000 14,000-94,000
16 X IX 23 IY
nucleolar
nucleola~
#mmatin pI range 3.X4.1 5.66.3 4.1~ s.9 4.1~7.3 4.3 7 0
chromatin
and proteins
Nucleolat Chromatin Number MOkCUkLr of bands weight range I2 6 17 I8 13
i4,0~76.~0 I2,00@60,000 l4,000- I20,oOtl 14,00& 120,000 14.000-92.000
and Baker, 1974). Most of the transcription restriction of DNA is undoubtedly due to histones. On the other hand, the nonhistone chromosomal proteins (NHCP) are complex and capable of activating transcription in ttitrn indicating a potential role of NHCP in gene expression. A search for biologically active chromosomal proteins is not possible without a suitable method for fractionation of nuclei and extraction of the chromatin complex. In this communication, a procedure is described for the fractionation of mosquito nuclei using differential centrifugation strategies as first reported by Pederson (1981). Two types of chromatin and ribonucleoprotein particles were separated by this procedure. In addition, a method was developed for the extraction of chromosomal proteins under mild conditions. The method is unique in that it does not employ strong acids (H,SO, or HCI) for extraction of histones and basic conditions for removal of NHCP. Such treatment affects the structural properties of histones and nonhistone proteins. The use of ionic detergents was also avoided due to avid binding to proteins and the consequent difficulty in removal of denaturants in allowing separation other than size. The solutions employed not only prevented protein aggregation with other proteins but also had no adverse affect on protein stability. The extractions yielded essentially a quantitative recovery of chromosomal proteins as shown by analytical methods. The protein extracts from the chromatin complex were analyzed by isoelectric focusing and SDS multiphase polyacrylamide electrophoresis. The ionic bonds in chromatin which link histones to DNA were first disrupted by 2% NaCl-75% ethanol. IEF and poly-acrylamide electrophoretic profiles of the sahethanol extract showed a greater number of proteins present in nucleolar chromatin than in nucleoplasmic chromatin. This indicates that more putative transcriptionally active fractions exist in nucleolar chromatin in acetylated forms. Modification of the electrical charge of the molecule can increase transcription, The remaining ionic bound histones
1 Fig. 3. Isoelectric focusing pattern of extracts from nucleoplasmic chromatin: I. 2% NaCl 75% ethanol; M guanidine HCI; 5. 0.07 M Na, P,O,; 2. 2.0 M NaCl; 3. 2.0 M NaCl -5.0 M urea; 4. 2.0 M NaCl4.0 chromatin: 7. 2% NaCl 75% ethanol; 8. 2.0 M NaCl; 9. 2.0 M NaCl 5.0 M urea; 6. RNP. Nucleolar HCI; 1 I. 0.07 M Na,P,O,. 10. 2.0 M NaC14.0 M guanidine
1
2
3
4
5
6
7
a
9
10
11
12
Fig. 4. SDS multiphase polyacrylamide electrophoresis of extracts from nucleoplasmic chromatin: 1. 2% NaCl-75% ethanol; 2. 2.0 M NaCl; 3. 2.0 M NaClkS.0 M urea; 4. 2.0 M NaCI4.0 M guanidine HCI 5. 0.07 M Na,P,O,; Nucleolar chromatin: 6. 2% NaClk75% ethanol; 7. 2.0 M NaCl; 8. 2.0 M NaCl-5.0 M urea. 9. 2.0 M NaCl4.0 M guanidine HCI; 10. 0.07 M Na,P,O,; I I. RNP; 12. BRL protein molecular weight standard mixture with numbers on far right corresponding to protein bands and representing: I. myosin (H chain) (200,000); 2. phosphorylase b (92,500); 3. bovine serum albumin (68,000); 4. ovalbumin (43,000); 5. chymotrypsinogen (25,000); 6. B-lactoglobulin (18,400); 7. cytochrome c (12,300). Sigma standard mixture is not numbered.
1251
Anopheline chromosomal were extracted with 2.0 M NaCI. IEF of the 2.0 M NaCl extract from the two types of chromatin revealed no greater heterogeneity than was apparent from multiphase polyacrylamide electrophoresis. The presence of nonhistone proteins in chromatin
which bind strongly to DNA has been well established (Allfrey et al., 1978). Some proteins were found to bind selectively to unique or repetitive DNA sequences while others preferred native over denaturated DNA. In mosquito chromatin, nonhistone proteins were also tenaciously bound to DNA and required not only 2.0 M NaCI-5.0 M urea but also 2.0 M NaCIL4.0 M guanidine HCl for removal from DNA. The use of urea in the extraction procedure prevented protein aggregation and is particularly suitable for isoelectric focusing since it reduces the influence of protein conformation and noncovalent bound factors. The difference between IEF and SDS multiphase electrophoretic patterns of nonhistone proteins extracted with salt urea suggests that there is present a group of unrelated proteins rather than a mixture of protein components. The nonhistone proteins were further released from the two types of chromatin by guanidine HCI. IEF of the nonhistone proteins from the guanidine-HCl extracts showed a greater pI range than found with the sodium chloride-urea extracts indicating a release of different proteins held together by the same molecular force. The tightly bound proteins are a particular subclass of NHCP which are distributed asymmetrically between transcriptionally active and inactive chromatin regions. These proteins were extracted from mosquito chromatin with Na,P,O,. The proteins focused moderately acidic and the molecular weight range was 14,000-92,000. The tight bound proteins generally have molecular weights between 14,000 and 85,000 and display PI’S from 5.0 to 6.0. Heterogeneous nuclear RNA is associated with a set of specialized RNA binding proteins (RNP). Ribonucleoprotein particles were separated from chromatin components under conditions which minimize adventitious binding. The IEF protein pattern of RNP particles and the protein-RNA ratio were similar to that found in rat liver (Wilks and Knowler, 1981; Pederson, 1974). The method presented for the separation of RNP particles together with metrazimide density gradients would be useful in determining the relationship between nuclear RNP and the polyribosome associated particles containing mRNA. In summary, a method is presented for the preparation of mosquito nuclei, resolution of nuclear components, extraction and characterization of chromosomal proteins. This overall procedure seems adequate for further nuclear component studies with mosquito populations. established whether exposure in changes in composition somal proteins accompanying
expression.
It remains to be to insecticides results of insect chromomodification of gene
proteins
1253 REFERENCES
Allfrey V. G. Inoue A., Karn J., Johnson E. M. and Vidali G. (1978) Cold Spring Harbor Symp. Quant. Biol. 30, 785-80
I.
Burton K. (1968) A study of the conditions and mechanisms of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323.
Dastugue B. and Crepin M. (1979) Interaction of nonhistone protein with DNA and chromatin from Drosophilia and mouse cells. Eur. J. Biochem. 99, 488491.
Elgin S. C. R. and Weintraub H. (1975) Chromosomal proteins and chromatin structure. A. Reo. Biochem. 44, 125-744.
Frenster J. H. (1965) Nuclear polyanions as derepressors of synthesis of ribonucleic acid. Nature, Lond. 206.68@683. Hi&on H. E. (1968) Definition of stages during metamorphosis. Adv. Insect Physiol. 5, 68-71. Hnilica L. S. (1972) The Structure and BioloPical Function . of Histones. CRC Press, West Palm Beach, Florida. Isenberg I. (1979) Histones. A. Rev. Biochem. 48, 159%191. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Miller S. (1979) RNA synthesis in two strains of anopheline mosquitoes. Insect Biochem. 9, 509-5 15. Neville D. M. (1971) Molecular weight determination of protein dodecylsulfate complexes by gel electrophoresis in a discontinuous buffer system. J. biol. Chem. 246, I
63286338.
Olins A. L. and Olins D. E. (1974) Spheroid chromatin units. Science, N.Y. 183, 33(t332. Pederson T. (1974) Proteins associated with heterogeneous nuclear RNA in eukaroytic cells. J. molec. Biol. 83, 163-183.
Pederson T. (1981) Messenger RNA biosynthesis and nuclear structure. Am. Sci. 69, 7684. Pederson T. and Bhorjee J. S. (1975) A special class of nonhistone proteins tightly complexed with template inactive DNA in chromatin. Biochemistry 14, 3238-3242. Pitel J. A. and Durzan D. J. (1978) Chromosomal proteins from the spruce budworm Choristoneura Jiimiferana. Insect Biochem. 8, 197-201.
Schneider W. C. (1957) Determination of nucleic acids in tissue by pentose analysis. In Methods of Enzymology (Edited by Colowick S. P. and Kaplan N. O.), Vol. 3, pp. 68&684. Academic Press, New York. $&berg T. C. and Hnilica L. S. (1970) Deoxyribonucleoproteins and the tissue: specific restrictions of the deoxyribonucleic acid in chromatin. Biochem. J. 120, 435437.
Stein J. L. and Stein G. S. (1976) Chromosomal proteins: their role in the regulation of gene expression. Bioscience 26, 488498.
Tata J. A. and Baker B. (1974) Subnuclear fractionation. Intranuclear compartmentation of transcription in citio and in vitro. Expl Cell Res. 83, 125-138. Van Holde K. E., Scharsrabuddhi C. G. and Shaw B. R. (1974) A model for particulate structure of chromatin. Nucleic Acid Res. 1, 1575-1579. Wilks A. F. and Knowler J. T. (1981) The phosphorylation of the proteins of rat liver heterogenous ribonucleoprotein particles by an endogeneous kinase activity. Biochem. biophys. Acta 652, 228-233. Wilt F. H., Anderson M. and Ekenberg E. (1973) Centrifugation of nuclear ribonucleoprotein particles of sea urchin embryos in caesium sulfate. Biochemistry 12, 959-966.
Printed in Great Britain
Il. pp. 1247-1253, 1988
0020-71 I X/88 $3.00 + 0.00 Pergamon Press plc
ISOLATION CHROMOSOMAL
AND CHARACTERIZATION OF PROTEINS FROM THE MOSQUITO ANOPHELES ALBIMANUS WEIDEMANN STEVE MILLER
Control Technology
Branch,
Division of Parasitic Diseases, Center for Infectious Disease Control, Atlanta, GA 30333, U.S.A.
Disease,
Centers
for
(Received 26 February 1988) Abstract-l. Nuclei were isolated from adult anopheline mosquitoes and fractionated into nucleolar chromatin, nucleoplasmic chromatin and ribonucleoprotein particles by sucrose density gradients. 2. Histones and nonhistone proteins were selectively dissociated from chromatin by treatment with sodium chloride, urea and guanidine HCl. 3. A special class of nonhistone proteins (tight proteins) were extracted from chromatin with Na,P,O,.
4. The electrophoretic properties of the histones, nonhistone proteins and ribonucleoprotein particles were examined by isoelectric focusing and SDS multiphase polyacrylamide gel electrophoresis. 5. By contrast to the histones, the nonhistone proteins displayed considerable heterogeneity. 6. Possible functional implications of the chromosomal proteins are discussed.
INTRODUCTION
DNA of eukaryotes is associated with a specific level of histones, a variable amount of nonhistone chromosomal proteins (NHCP) and RNA to form the chromatin complex. There is sufficient evidence that the chromatin proteins play an important role in activation and regulation of genes. Histones are considered as nonspecific repressors of transcription of chromatin DNA as well as structural components of nucleosomes (Olins and Olins, 1974; Van Holde et al., 1974; Hnilica, 1972). They are conserved from an evolutionary standpoint (Isenberg, 1979). The nonhistone proteins are a more heterogenous group and are not only involved in the stabilization of DNA strands during strand separation but also may function as derepressors (Elgin and Weintraub, 1975; Dastugue and Crepin, 1979; Stein and Stein, 1976; Frenster, 1965). In this communication, a procedure is described for preparing nuclei from adult anopheline mosquitoes and resolving the nuclei into nucleoplasmic and nucleolar fractions and ribonucleoprotein particles. The histones and nonhistone proteins were extracted from the two fractions under mild conditions taking advantage of the differential DNA binding properties of the chromosomal proteins and characterized by SDS multiphase polyacrylamide electrophoresis and isoelectric focusing. MATERIALS AND METHODS
Experimental organism The experimental organism used in this study was Anopheles albimanus originating from Panama. Larvae were reared on essential larvae nutrients at 26°C (Miller, 1979). Pupae were placed in distilled water for emergence. Pharate adults were selected for the study (Hinton, 1968).
Sample preparation Anopheles albimanus pharate adults were suspended in ice cold ‘Buffer A (IOmM KCl, 1OmM MgCI,, 20mM Tris-HCl. DH 7.6. 3.0 mM CaCl,. 1 mM spermine sulfate, I mM phenylmethylsulfonyl Buo>de, 2 mM dithiothreitol) and homogenized 5 strokes with a tight fitting motor driven homogenizer. The homogenate was filtered through cheesecloth and the pellet was homogenized and filtered an additional two times, The combined filtrates were centrifuged at 800 g for 10 min. The nuclear pellet was washed three times with Buffer A to remove the cytoplasmic contaminants. Sucrose
gradient centrifugation
The nuclei were fractionated by the following procedure. Nucleoli were sedimented by low speed centrifugation (45OOg, 20 min) through 30% sucrose while nucleoplasmic chromatin and ribonucleoprotein particles (RNP) remained on top of the sucrose. Nucleoplasmic chromatin and RNP particles were removed, layered on a l5-30% linear sucrose gradient and centrifuged at 40,OOOg for I7 hr. Under these conditions, the nucleoplasmic chromatin sediments as a pellet at the bottom of the tube and most of the RNP particles remain in the gradient where they are resolved into a heterogeneous population due to their overlapping range of sizes. The sucrose was removed from the fractions by dialysis against 20 mM TrissHCl, pH 7.6 containing 1 mM PMSF and 50 mM NaHSO,. Extraction of chromosomal proteins Histones were extracted from nucleolar and nucleoplasmic chromatin with 2% NaCl-75% ethanol and 2.0 M NaCI. After removal of histones, the nonhistone proteins were extracted from the resulting chromatin with 2.0M NaCl-5.0 M urea and 2.0 M NaC14.0 M guanidine HCl. The tight bound proteins were then removed from the pellet with 0.07 M Na,P,O,. The combined extracts containing dissociated histones and non-histone proteins were dialyzed, lyophilized, and recovered for SDS multiphase polyacrylamide electrophoresis and isoelectric focusing. The extraction effectiveness was determined by protein analysis. 1247
STEVEMILLER
1248 Polyacrylamide gel electrophoresis
Proteins were separated by combining the techniques of sodium dodecylsulfate (SDS) gel electrophoresis and electrophoresis in a discontinuous buffer system (multiphase) as described by Neville (1971). A 3.5% acrylamide gel served as a stacking gel and a 10% acrylamide gel as a resolving gel. The runs were at 5 ‘C for 665 V hr. After electrophoresis. the gels were fixed in 10% (w/v) trichloroacetic acid for I hr at room temperature and stained overnight by immersion in a solution of 0.05% Page Blue 83 m ethanol acetic acid-water (30: IO:60 by vol). The gels were destained in ethanol&acetic acid-water (25: 8:65 by vol) and were photographed. Isoelecrric .focusing Thin layer isoelectric focusing was performed with a LKB*-2117 unit. Gels were prepared with LKB carrier ampholytes pH range 3.5510. The gels were prerun for 1 hr at 10 Watt constant power to establish a pH gradient before the protein samples were applied. The samples (25 pl) were applied at the cathodic end of the gel and isoelectric focusing was carried out for 1860 Volt hr. The fixation and staining of protein bands were as described previously.
NaHSO,. The histones are very susceptible to proteolytic degradation and extraction in the presence of inhibitors in no way changed the isoelectric focusing and multiphase polyacrylamide electrophoretic patterns (unpublished results). The resulting pellet containing deoxyribonucleoproteins was then extracted with 2.0 M NaCl. This step removed 60% of the DNA and contained the remaining histones. After removal of the histones. chromatin was extracted with 2.0 M NaCILS.0 M urea and 2.0 M NaCI4.0 M guanidine HCI. It was necessary to use urea and guanidine in the dissociation procedure because of the limited solubility and strong interaction of the chromatin components. Guanidine further disrupted hydrogen bonds. The extractions with each solvent system resulted in removal of a particular protein fraction from the chromatin complex. The tight bound proteins were removed with Na,P20;. The extraction series solubilized 95% or greater all proteins present in chromatin as shown by a final
Analytical methods Protein concentrations were determined, after separation of histones, nonhistone proteins and RNP particles. by the method of Lowry et al. (1951). Bovine serum albumin was used as a standard. The concentration of DNA in chromatin was estimated by the diphenylamine method of Burton (1968) using calf thymus DNA as a standard. RNA was determined by the orcinol method of Schneider (1957) using purified yeast RNA standard.
000
0%
RESULTS Studies establishing chromosomal proteins in mosquito populations require a method for the preparation of nuclei and a method for the separation, dissociation, and analysis of chromatins and ribonucleoprotein particles. In the preparation of nuclei, mosquito tissue were disrupted by homogenization and tissue fragments removed by filtration through cheesecloth. The nuclei were selectively sedimented by low speed centrifugation and washed by repeated suspension and sedimentation. Figure I outlines the procedure for the fractionation of nuclei. The largest structure, the nucleoli were removed by low speed 30% sucrose centrifugation. Nucleoplasmic chromatin and RNP particles were separated on a 15-30% sucrose gradient as first described by Pederson (1981). The RNP particles contained no detectable DNA and had a protein to RNA ratio of 4: 1. In dissociating the chromatin complex and separating histones from nonhistone chromosomal proteins (NHCP), the use of acid, strongly alkaline and phenol extractions were avoided because of the variable solubility of the histones and because of the potentially irreversible effect on the structure of NHCP. Figure 2 outlines the procedure for dissociating mosquito nucleoplasmic and nucleolar chromatin under mild conditions. The histones were extracted from chromatin with 2% NaClL75% ethanol containing the proteolytic inhibitors PMSF and *Use of trade names is for identification purposes only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.
(4,500 X a, 20
mid
RNP and chromatin
nucleoli
I
centrifuge (40,000 x g, 17 hrs)
RNP particles
chromatln Fig. 1. Fractionation
of mosquito sucrose.
nuclear
components
on
Anopheline chromosomal Nuclear
proteins
component (nucleoplasmic chromatin or nucleolar
1249 chromatin)
homogenization with 2% NaCl 75% ethanol
Centrifuge (12.100 x g, 20 min)
Suparnatant
= 2% NaCl 75% ethanol.soluble fraction
homogenization with 2 M NaCl
Canirifuge (12,100 x g, 20 min)
Supernatant
= 2 M NaCl soluble fraction
homogenization with 2 M NaCI/ 5 M urea
Centrifuge 112,100 x g. 20 min)
Supernatant
= 2 M NaCl/!iM urea soluble fraction
pellet P
homogamzation with 2.0 M NaCI/ 4 M guanidine HCI
Centrifuge (12,100 x g. 20 min)
Suparnatant
= 2 M NaCll4 M guanidine HCI soluble fraction
residual fraction solubilized in 0.07 M Na4P207 (pH 9.0)
Fig. 2. Scheme of nucleolar and nucleoplasmic chromatin dissociation. extraction with 1.0 M NaOH and analysis of the alkaline extract. Protein composition of the extracts was determined by isoelectric focusing and SDS multiphase polyacrylamide electrophoresis. IEF patterns are shown in Fig. 3 and the number of protein bands of each extract with the isoelectric point range is presented in Table 1. IEF of the 2% NaCl-75% ethanol extract from nucleoplasmic chromatin showed two protein bands at a pH of 5.8 and 5.9 and showed 16 bands from nucleolar chromatin in the pH region of the gel 3.8-6.1. The difference in the number of protein bands from the two extracts indicates that nucleolar chromatin contains unmodified as well as modified histones and high mobility group proteins. The proteins from the 2.0 M NaCl extracts focused into 7 bands from nucleoplasmic chromatin and 8 bands from nucleolar chromatin covering a region of 0.9 unit with a pI range of 5.4-6.3. Unlike the DNA histones, the nonhistone proteins are not dissociated from chromatin with 2.0 M NaCl. In order to determine the charge characteristics of the nonhistones chromosomal proteins, chromatin, after removal of histones, was first extracted with 2.0 M NaCl-5.0M urea and the extract analyzed by IEF. The banding patterns were complex with at least 11 protein bands from nucleoplasmic chromatin and 18 bands from nucleolar chromatin focusing in the acidic p1 range of 4.1-5.9. Non-histone proteins were then further extracted from chromatin with
2.0 NaC14.0 M guanidine HCl. IEF of the proteins from nucleoplasmic and nucleolar chromatin showed similar banding patterns. The proteins from the two types of chromatin focused moderately acidic with a p1 range 4.1-7.4 differing in range from the proteins extracted with sodium chloride-urea. There is a class of NHCP (tight bound proteins) which fail to dissociate from DNA at a very high ionic strength (2.5 M NaCI-5.0 M urea) (Pederson and Bhorjee, 1975). The tight bound proteins were extracted from the two types of chromatin with Na,P,O,. The protein extract from nucleoplasmic chromatin displayed ~1’s 4.5-5.9 and from nucleolar chromatin 4.3-7.0. IEF of tight bound proteins from HeLa cells revealed a p1 range of 5.0-6.0 (Pederson and Bhorjee, 1975). Isoelectric focusing of ribonucleoprotein particles showed that the proteins are moderately acidic with a p1 range of 4.3-6.7. The RNP particles had a protein-RNA ratio of 4: 1. Extensive characterization of RNP particles from various sources revealed a protein-RNA ratio of 4-5: 1 (Wilt et al., 1973; Wilks and Knowler, 1981). The major proteins of RNP particles from rat liver displayed PI’S between 4.9-6.5. (Pederson, 1974). Table 2 lists the approximate molecular weight range of protein extracts from the two types of chromatin. Figure 4 illustrates SDS multiphase polyacrylamide electrophoresis of chromosomal proteins. The 2% NaCl-75% ethanol extract from nucleo-
1250
STEVEMJLLER Table
1. Isoelectric
point range (PI’S) of protein extracts from nucleoplasm~c chromatm, chromatin and proteins from ribonucieourotein oartick iRNPl
Extract 2% N&-75% ethanol 2.0 M NaCl 2.0 M N&I-5.0 M urea 2.0M NaCI-4.0M GuHCl 0.07 M Na,P,O, RNP
Table 2. Molecular
Nucl~pl~ic Number of bands
Chromntin
2 7 II 25 17 21
5.x--5.9 5.4-6.3 4.5-5.x 4.4-7.4 4.5.-5.9 4.3-6.7
pi range
weight range of protein extracts from nucleoplasmic from ribonucieoprotein particles NueieopIasmk Number of bands
Extract 2% N&-75% ethanol 2.0 M NaCl 2.0 M NaCI-5.0 M urea 2.0 M NaCik4.0 M GuHCl 0.07 M NalP20, RNP
3 6 I6 22 16 9
plasmic chromatin showed 3 protein bands with molecular weights of lO,OO@-14,000and the extract from nucleolar chromatin showed 12 bands with a molecular weight range of 14,00~76,0~. The size of the proteins from nucleoplasmic chromatin are in the range of unmodified calf thymus and C~orist~~eura ~~~z~~u~~ histones (Hnilica, 1972; Pitel and Durzan, 1978). The remaining histones bound by electrostatic and Van der Waals forces were extracted with 2.0 M NaCl as described by Spelsberg and Hnihca (1970). The electrophoretic results of the 2.0 M NaCl extract revealed 6 protein bands distributed through the lO,OOO-72,000 molecular weight range from nucleoplasmic chromatin and revealed 6 bands from nucleolar chromatin in the range of 12,OOt&60,000. The nonhistone chromosomal protein banding patterns were complex as shown by the number of proteins separated by SDS multiphase electrophoresis. The 2.0 M N&I-5.0 M urea extract from nucleoplasmic and from nucleolar ~hromatin revealed quahtative similar but quantitatively very different banding patterns (Fig. 4). The electrophoretic patterns of the 2.0 M NaCI-4.0 M guanidine HCl extract from the two types of chromatin showed many similar bands with different intensities. The remaining NHCP were extracted with Na,P,O, under slightly basic conditions and were referred to as tight bound proteins. The tight bound proteins were enriched in the 14,000-30,000 molecular weight components. This is a general property of this class of chromatin proteins. SDS multiphase electrophoresis of RNP particles showed a population of proteins with a molecular weight range from 14,~94,~. SDS polyacrylamide gel electrophoresis of RNP from different cells revealed a distinctive pattern for each ceil type (Pederson, 1974). DISCUSSION
Transcription of genes in eukaroytic cells is intimately associated with chromosomal proteins (Tata
Ndeolar Number of bands
chromatin. (RNP)
Chromstin Molecular weight range 10,~14,0~ lO.ooo-72,000 14,00@120,000 14,000-120.000 14,ocu-92,000 14,000-94,000
16 X IX 23 IY
nucleolar
nucleola~
#mmatin pI range 3.X4.1 5.66.3 4.1~ s.9 4.1~7.3 4.3 7 0
chromatin
and proteins
Nucleolat Chromatin Number MOkCUkLr of bands weight range I2 6 17 I8 13
i4,0~76.~0 I2,00@60,000 l4,000- I20,oOtl 14,00& 120,000 14.000-92.000
and Baker, 1974). Most of the transcription restriction of DNA is undoubtedly due to histones. On the other hand, the nonhistone chromosomal proteins (NHCP) are complex and capable of activating transcription in ttitrn indicating a potential role of NHCP in gene expression. A search for biologically active chromosomal proteins is not possible without a suitable method for fractionation of nuclei and extraction of the chromatin complex. In this communication, a procedure is described for the fractionation of mosquito nuclei using differential centrifugation strategies as first reported by Pederson (1981). Two types of chromatin and ribonucleoprotein particles were separated by this procedure. In addition, a method was developed for the extraction of chromosomal proteins under mild conditions. The method is unique in that it does not employ strong acids (H,SO, or HCI) for extraction of histones and basic conditions for removal of NHCP. Such treatment affects the structural properties of histones and nonhistone proteins. The use of ionic detergents was also avoided due to avid binding to proteins and the consequent difficulty in removal of denaturants in allowing separation other than size. The solutions employed not only prevented protein aggregation with other proteins but also had no adverse affect on protein stability. The extractions yielded essentially a quantitative recovery of chromosomal proteins as shown by analytical methods. The protein extracts from the chromatin complex were analyzed by isoelectric focusing and SDS multiphase polyacrylamide electrophoresis. The ionic bonds in chromatin which link histones to DNA were first disrupted by 2% NaCl-75% ethanol. IEF and poly-acrylamide electrophoretic profiles of the sahethanol extract showed a greater number of proteins present in nucleolar chromatin than in nucleoplasmic chromatin. This indicates that more putative transcriptionally active fractions exist in nucleolar chromatin in acetylated forms. Modification of the electrical charge of the molecule can increase transcription, The remaining ionic bound histones
1 Fig. 3. Isoelectric focusing pattern of extracts from nucleoplasmic chromatin: I. 2% NaCl 75% ethanol; M guanidine HCI; 5. 0.07 M Na, P,O,; 2. 2.0 M NaCl; 3. 2.0 M NaCl -5.0 M urea; 4. 2.0 M NaCl4.0 chromatin: 7. 2% NaCl 75% ethanol; 8. 2.0 M NaCl; 9. 2.0 M NaCl 5.0 M urea; 6. RNP. Nucleolar HCI; 1 I. 0.07 M Na,P,O,. 10. 2.0 M NaC14.0 M guanidine
1
2
3
4
5
6
7
a
9
10
11
12
Fig. 4. SDS multiphase polyacrylamide electrophoresis of extracts from nucleoplasmic chromatin: 1. 2% NaCl-75% ethanol; 2. 2.0 M NaCl; 3. 2.0 M NaClkS.0 M urea; 4. 2.0 M NaCI4.0 M guanidine HCI 5. 0.07 M Na,P,O,; Nucleolar chromatin: 6. 2% NaClk75% ethanol; 7. 2.0 M NaCl; 8. 2.0 M NaCl-5.0 M urea. 9. 2.0 M NaCl4.0 M guanidine HCI; 10. 0.07 M Na,P,O,; I I. RNP; 12. BRL protein molecular weight standard mixture with numbers on far right corresponding to protein bands and representing: I. myosin (H chain) (200,000); 2. phosphorylase b (92,500); 3. bovine serum albumin (68,000); 4. ovalbumin (43,000); 5. chymotrypsinogen (25,000); 6. B-lactoglobulin (18,400); 7. cytochrome c (12,300). Sigma standard mixture is not numbered.
1251
Anopheline chromosomal were extracted with 2.0 M NaCI. IEF of the 2.0 M NaCl extract from the two types of chromatin revealed no greater heterogeneity than was apparent from multiphase polyacrylamide electrophoresis. The presence of nonhistone proteins in chromatin
which bind strongly to DNA has been well established (Allfrey et al., 1978). Some proteins were found to bind selectively to unique or repetitive DNA sequences while others preferred native over denaturated DNA. In mosquito chromatin, nonhistone proteins were also tenaciously bound to DNA and required not only 2.0 M NaCI-5.0 M urea but also 2.0 M NaCIL4.0 M guanidine HCl for removal from DNA. The use of urea in the extraction procedure prevented protein aggregation and is particularly suitable for isoelectric focusing since it reduces the influence of protein conformation and noncovalent bound factors. The difference between IEF and SDS multiphase electrophoretic patterns of nonhistone proteins extracted with salt urea suggests that there is present a group of unrelated proteins rather than a mixture of protein components. The nonhistone proteins were further released from the two types of chromatin by guanidine HCI. IEF of the nonhistone proteins from the guanidine-HCl extracts showed a greater pI range than found with the sodium chloride-urea extracts indicating a release of different proteins held together by the same molecular force. The tightly bound proteins are a particular subclass of NHCP which are distributed asymmetrically between transcriptionally active and inactive chromatin regions. These proteins were extracted from mosquito chromatin with Na,P,O,. The proteins focused moderately acidic and the molecular weight range was 14,000-92,000. The tight bound proteins generally have molecular weights between 14,000 and 85,000 and display PI’S from 5.0 to 6.0. Heterogeneous nuclear RNA is associated with a set of specialized RNA binding proteins (RNP). Ribonucleoprotein particles were separated from chromatin components under conditions which minimize adventitious binding. The IEF protein pattern of RNP particles and the protein-RNA ratio were similar to that found in rat liver (Wilks and Knowler, 1981; Pederson, 1974). The method presented for the separation of RNP particles together with metrazimide density gradients would be useful in determining the relationship between nuclear RNP and the polyribosome associated particles containing mRNA. In summary, a method is presented for the preparation of mosquito nuclei, resolution of nuclear components, extraction and characterization of chromosomal proteins. This overall procedure seems adequate for further nuclear component studies with mosquito populations. established whether exposure in changes in composition somal proteins accompanying
expression.
It remains to be to insecticides results of insect chromomodification of gene
proteins
1253 REFERENCES
Allfrey V. G. Inoue A., Karn J., Johnson E. M. and Vidali G. (1978) Cold Spring Harbor Symp. Quant. Biol. 30, 785-80
I.
Burton K. (1968) A study of the conditions and mechanisms of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323.
Dastugue B. and Crepin M. (1979) Interaction of nonhistone protein with DNA and chromatin from Drosophilia and mouse cells. Eur. J. Biochem. 99, 488491.
Elgin S. C. R. and Weintraub H. (1975) Chromosomal proteins and chromatin structure. A. Reo. Biochem. 44, 125-744.
Frenster J. H. (1965) Nuclear polyanions as derepressors of synthesis of ribonucleic acid. Nature, Lond. 206.68@683. Hi&on H. E. (1968) Definition of stages during metamorphosis. Adv. Insect Physiol. 5, 68-71. Hnilica L. S. (1972) The Structure and BioloPical Function . of Histones. CRC Press, West Palm Beach, Florida. Isenberg I. (1979) Histones. A. Rev. Biochem. 48, 159%191. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Miller S. (1979) RNA synthesis in two strains of anopheline mosquitoes. Insect Biochem. 9, 509-5 15. Neville D. M. (1971) Molecular weight determination of protein dodecylsulfate complexes by gel electrophoresis in a discontinuous buffer system. J. biol. Chem. 246, I
63286338.
Olins A. L. and Olins D. E. (1974) Spheroid chromatin units. Science, N.Y. 183, 33(t332. Pederson T. (1974) Proteins associated with heterogeneous nuclear RNA in eukaroytic cells. J. molec. Biol. 83, 163-183.
Pederson T. (1981) Messenger RNA biosynthesis and nuclear structure. Am. Sci. 69, 7684. Pederson T. and Bhorjee J. S. (1975) A special class of nonhistone proteins tightly complexed with template inactive DNA in chromatin. Biochemistry 14, 3238-3242. Pitel J. A. and Durzan D. J. (1978) Chromosomal proteins from the spruce budworm Choristoneura Jiimiferana. Insect Biochem. 8, 197-201.
Schneider W. C. (1957) Determination of nucleic acids in tissue by pentose analysis. In Methods of Enzymology (Edited by Colowick S. P. and Kaplan N. O.), Vol. 3, pp. 68&684. Academic Press, New York. $&berg T. C. and Hnilica L. S. (1970) Deoxyribonucleoproteins and the tissue: specific restrictions of the deoxyribonucleic acid in chromatin. Biochem. J. 120, 435437.
Stein J. L. and Stein G. S. (1976) Chromosomal proteins: their role in the regulation of gene expression. Bioscience 26, 488498.
Tata J. A. and Baker B. (1974) Subnuclear fractionation. Intranuclear compartmentation of transcription in citio and in vitro. Expl Cell Res. 83, 125-138. Van Holde K. E., Scharsrabuddhi C. G. and Shaw B. R. (1974) A model for particulate structure of chromatin. Nucleic Acid Res. 1, 1575-1579. Wilks A. F. and Knowler J. T. (1981) The phosphorylation of the proteins of rat liver heterogenous ribonucleoprotein particles by an endogeneous kinase activity. Biochem. biophys. Acta 652, 228-233. Wilt F. H., Anderson M. and Ekenberg E. (1973) Centrifugation of nuclear ribonucleoprotein particles of sea urchin embryos in caesium sulfate. Biochemistry 12, 959-966.