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Nematode chromosomal proteins—IV. The nonhistones of Caenorhabditis elegans
Comp. Biochem. Physiol. Vol. 81B, No. 2, pp. 377-383, 1985 Printed in Great Britain
0305-0491/85 $3.00 + 0.00 © 1985 Pergamon Press Ltd
N E M A T O D E CHROMOSOMAL PROTEINS--IV. THE NONHISTONES OF CAENORHABDITIS ELEGANS L. MEHEUS and J. R. VANFLETEREN* Laboratorium voor Morfologie en Systematiek der Dieren, Rijksuniversiteit Gent, Ledeganckstraat 35, B-9000 Gent, Belgium (Tel: 091-22-78-21)
(Received 8 October 1984) Abstract--1. A method has been developed which yields reasonably clean nuclei from the free-living nematode Caenorhabditis elegans. Nonhistones were prepared from these nuclei by gentle extraction with 2 M NaC1 and analyzed by one- and two-dimensional electrophoresis using a nonequilibrium pH gradient electrophoresis in the first dimension after removal of nucleic acids by phenol extraction. 2. The extraction procedure yielded nonhistone chromatin proteins and proteins associated with nuclear RNA and a background of polypeptides derived from the nuclear matrix and pore-lamina complex. 3. Over 40 polypeptides were distinguished on silver stained one-dimensional slab gels. 4. The presence of actin, tropomyosin and tubulin was demonstrated by immunoprobing after transfer of the polypeptides to nitrocellulose. 5. Bands co-migrating with myosin and ct-actinin gave no specific reaction when probed with antibodies raised against the respective proteins from vertebrate tissues. 6. Over 200 spots were visible on two-dimensional gels stained with silver nitrate.
INTRODUCTION There is still growing evidence that nonhistone proteins are important in organizing the higher order structure of chromatin and controlling gene expression. A nuclear matrix, sometimes called nuclear ghost or scaffold and composed of protein and small amounts of nucleic acid extends throughout the interphase nucleus and forms a framework to which loops of the D N A fibres are attached (Berezney and Coffey, 1977; Adolph, 1980; Agutter and Richardson, 1980; Capco et al., 1982; Lebkowski and Laemmli, 1982; Lewis and Laemmli, 1982). The compaction of interphase chromatin effected by this structure might well have a bearing on local genome activity. On the other hand it has been thought that specific gene regulators reside in the nonhistone fraction. Evidence in support of this idea has been taken from the time and species specificity of several of the nonhistones associated with chromatin (Adolph and Phelps, 1982; Cartwright et al., 1982; Kabisch et al., 1982; Weihe et al., 1982; Treadgill and Arnstein, 1984) and from the changes in the nonhistone protein pattern associated with changes in gene expression during both normal development and neoplastic transformation (Bliitmann and Illmensee, 1981; Kilianska et al., 1981; Bojanovic et al., 1981; Medvedev et al., 1981; Wielgat and Kleczkowski, 1981; Yancheva and Djondjurov, 1982; Einck and Bustin, 1983; T o d o r o v a et al., 1983; Burkhardt et al., 1984). A major characteristic of ageing is the steep fall of organismic and cellular activity. The underlying mechanism is still a matter of controversy but there is some agreement that there is a reduced level of R N A synthesis in old animals and alterations of the structure and function of chromatin have been implicated (Kanungo, 1980; Rothstein, 1982). *To whom all correspondence should be addressed.
The free-living nematode species Caenorhabditis elegans is a good model for fundamental ageing research. It offers the advantage of small size, short life cycle (12-18 days pending on the nutritional regime), ease of cultivation, a genetically manipulatable system and a strictly determined development (eutely). Since very few somatic cell divisions occur after hatching and with exclusion for the gonadal cells, aged organisms can be considered as being composed of aged cells. Since the nonhistone proteins of free-living nematodes have never been studied before, the present investigation aimed to describe the diversity of the nonhistones of C. elegans and to identify some major components. This knowledge is prejudicial to the further study of age-related changes at this level. MATERIALS AND METHODS
Nematode growth The nematode used in this study is the wild type of the Bristol strain of Caenorhabditis elegans, designated N2 by Brenner (1974). It was obtained from Dr Epstein. Nematodes were grown in axenic culture in a medium consisting of 3% dry yeast extract, 3% soy peptone and 500 pg/ml haemoglobin. The basal medium was autoclaved for 20 min at 120°C. Haemoglobin was aseptically added from a stock solution made up in 0.1 N KOH (5 g/100ml) and sterilized by Seitz filtration. Routine cultures were grown in Fernbach flasks containing 200 ml of medium with continuous shaking on a gyrotory shaker at 100-150 oscillations/min. Larger cultures were established in 101 vessels equipped with a magnetically driven large Teflon bar. The need for increased gas exchange was met by continuous aerating through an in line membrane filter. Axenic culture methods have been described in much more detail elsewhere (Vanfleteren, 1978, 1980). When the optimal density of about 40,000 worms/ml was reached, which usually occurred on days 8 12 pending on the inoculum size, the worms were collected by centrifugation at 5000 rpm for 10 min in a refrigerated centrifuge. The pellet was washed 377
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several times with S buffer (0.1 M NaCI in 0.05 M phosphate buffer, pH 6) at 3000 rpm for 5 rain. The nematode pellets were then suspended in 2 vol of a solution containing 1.7 M sucrose, 0.5~ Nonidet P-40, 5 m M CaC1 z and 50mM Tris-HC1 (pH 7.4), dripped into liquid nitrogen and stored at -196°C. The wet weight from one litre of culture was approx. 5 g of nematodes.
Preparation of nuclei The frozen nematode nuggets were homogenized in a Waring blender with an equal vol of acid washed glass beads (0.2 mm in dia.) at medium speed for 1 min. The glass beads were allowed to settle by gravitation and the supernatant fluid was further homogenized with 10 strokes of a motor driven Teflon pestle in a Potter-Elvehjem homogenizer operated at 2000rpm. Intact nematodes and large debris were sedimented at 3000 rpm for 5 min. The supernatant was then layered over two vol of 1.7 M sucrose, 0.5~ Nonidet P-40, 5 mM CaC12, 50 mM Tris-HCl (pH 7.4) and a crude gradient was formed using a Pasteur pipette. The gradients were centrifuged at 10,000rpm in a 4 × 50ml swing-out rotor for 30 min. The nuclear pellets were suspended in 2ml 0.14M NaC1, l m M PMSF, 50mM Tris-HCl (pH 7.4) and centrifuged for 5 min in an Eppendorf centrifuge. This wash was repeated five times. The Eppendorf tubes were carefully cooled in ice water in between to avoid any rise of the temperature much above 4°C. All other handlings were performed at 4:'C.
Isolation of chromosomal proteins The nuclear pellet was suspended in 2 ml of 2 M NaCI, 1 mM PMSF, 10 m M Tris-HC1 (pH 7.4) and left on ice for 90 min with occasional shaking. Particulate material was sedimented by centrifugation for 5 min in an Eppendorf centrifuge and discarded. The solubilized chromatin was cooled in ice and finally clarified by one more run for 5 min. The yield of chromatin from 1 g of wet nematode tissue was 4-6 A260units. In another set of experiments chromatin was extracted in low salt buffer after digestion with DNase I. The nuclear pellet was suspended in 10 m M MgC12, 1 mM CaC12, 1 mM PMSF, 10. m M Tris-HCl (pH 7.4) and DNase I (Worthington) was added at a final concn of 100 #g/ml. Incubation was at 0°C for 60 min with gentle stirring. The solubilized chromatin was recovered as described above. The purity of all chromatin preparations was assessed by measuring the A260/A280 ratio.
Fast sedimenting fraction of chromatin Nuclei prepared from 20 g of nematode tissue were digested with 100#g/ml DNase I for 60 min as described above. An equal vol of 4 M NaC1, 20 m M EDTA, 20 mM Tris-HCl (pH 7.4) was added and extraction was allowed to continue for another 30 min. The suspension was centrifuged for 3 min in an Eppendorf centrifuge and filtered through a No. 4 glass filter. The filtrate was layered on 5~o sucrose in 2 M NaCI, 10mM Tris-HCl (pH 7.4) and centrifuged for 60 min at 8000 rpm in a 3 × 25 ml swing out rotor as described for the preparation of nuclear scaffolds (Lebkowski and Laemmli, 1982). The sediment was dissolved in TEM buffer (20 mM Tris-HCl, pH 8.2, 20mM EDTA, 0.1~o 2-mercaptoethanol) containing 2 ~ SDS and further processed for electrophoretic analysis.
sedimentation coefficients of 40 S and above. The sedimented material was then solubilized directly in sample buffer and analyzed by SDS polyacrylamide gel electrophoresis.
Sample preparation .for electrophoretic analysis The chromatin preparation was made 20°/0 in TCA. After standing for at least 60 min the precipitate was sedimented at 10,000 rpm for 5 min. The pellet was washed three times in a solution containing 70~o acetone, 20% ethanol and 10 mM Tri~HC1 (pH 7.4) and dissolved overnight in TEM buffer containing 2~o SDS. The sample was then boiled for 5 min, cooled again and mixed with an equal vol of phenol equilibrated with TEM buffer. After brief centrifugation (5min, 10,000rpm) to separate the phases, the nuclear proteins were precipitated from the phenol phase with 3 vol of methanol at - 1 8 ° C overnight. The precipitate was sedimented at 5000 rpm for 5 min, washed with ether to remove traces of phenol and air dried.
Electrophoresis Gel electrophoresis in the presence of SDS was performed according to Laemmli (1970) in 11~o polyacrylamide slab gels. The mol. wt of any given polypeptide was determined from a comparison of its mobility and that of standards run in a separate lane. The standards used were: myosin from rabbit muscle, 205,000 daltons; fl-galactosidase from E. coli, 116,000 daltons; phosphorylase B from rabbit muscle, 97,000 daltons; bovine serum albumin, 68,000 daltons; egg albumin, 45,000 daltons; carbonic anhydrase from bovine erythrocytes, 29,000 daltons (Sigma). For two-dimensional gel electrophoretic analysis the nonequilibrium pH gradient electrophoresis (NEPHGE) procedure of O'Farrell et al. (1979) was used. Only pH 3 10 ampholytes (LKB) were used. The gels were run at 400 V for 6 hr. For probing the pH gradient 5 mm sections of a control gel were put into separate vials and soaked with degassed water. After completion of the nonequilibrium gel electrophoresis the gels were fixed in 50~o methanol for 1 hr (with 4 changes) and equilibrated for 2 hr against the sample buffer for the second dimension. 2-Mercaptoethanol was omitted from this buffer because it produces a few heavy horizontal lines in the 60,000-70,000 mol. wt range in silver stained gels.
Silver stain detection of polypeptides Silver staining was performed essentially according to Morrissey (1981) with the following modifications. The slab gel was fixed in 50~o methanol-12~ TCA for at least 30 min, the secondary fixation with glutaraldehyde was omitted. The silver nitrate concentration was raised to 0.2~o. Some polypeptides (histones for example) produce a negative stain with a single cycle of this staining procedure, yet stain very well after recycling. When a highly sensitive silver stain was
Preparation of proteins associated with nuclear RNA Sucrose washed nuclei were suspended in 0.14 M NaCI, 1 mM MgCI z, 1 mM 2-mercaptoethanol, 0.1 m M PMSF, 50 mM Tris-HC1 (pH 7.4) containing 0.1 pg/ml pancreatic RNase (preheated for 15 min at 80°C) for 1 hr at 4°C to extract nuclear ribonucleoprotein. The nuclei were then pelleted at 10,000 rpm for 5 min. The supernatant portion was further centrifuged in a MSE 3 × 5 ml swing out rotor at 45,000 rpm for 3hr at 4°C to pellet particles with
Fig. 1. Isolated nuclei of C. elegans, stained with Giemsa. × 450.
Nonhistones of C. elegans
a
b
c
d
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e
Fig. 2. Comparison of the chromosomal proteins released from nematode nuclei by different methods. (a) Extraction with 2 M NaC1; (b) extraction with 2 M NaC1 following digestion with 100 #g/ml DNase I; (c) proteins released in 10mM Tris-HC1 (pH 7.4), 10.mM MgCI2, 1 mM CaC12 by digestion with 100#g/ml DNase I; (d) DNase I, 10/zg; (e) mol. wt markers.
desired gels were stained as above and destained in concentrated Agefix (Agfa Gevaert), thoroughly rinsed with distilled water and recycled as described above.
of numerous nuclei (Fig. 1). Occasional cuticular fragments and amorphous components are also present.
Immunoprobing
Isolation of chromosomal proteins
Chromosomal proteins were run on 11% SDS polyacrylamide gels. The resolved polypeptides were transferred to nitrocellulose in 25mM Tris-HC1 (pH 8.2), 150mM glycine, 20% methanol at 0.5 A for 4 hr. Residual protein binding sites were blocked by incubation in 20raM Tris-HC1 (pH 8.2), 150mM NaCI, 5% BSA for 30min at 37°C. The blot was then extensively washed in 20mM Tris-HC1 (pH 8.2), 150 mM NaC1, 0.1% BSA and cut into strips. These were incubated with approx. 1 #g/ml antibody in wash buffer. Bound antibodies were then visualized by immunogold staining and silver enhancement of the latter (Moeremans et al., 1984, and in press). RESULTS
Preparation of nuclei The preparation of nuclei from nematode tissue is complicated by their widely different dimensions and the co-sedimentation of cuticular debris during sucrose density gradient centrifugation. This problem was largely met by a two-step density gradient centrifugation. At low speed (3000 rpm) intact and broken nematodes and the bulk of cuticle fragments sedimented whereas the nuclei remained in the supernatant solution. They were pelleted at higher speed (10,000rpm) and freed of cytoplasm. Microscopic analysis of a typical preparation reveals the presence
Extraction of sheared chromatin with low salt buffer such as 10 mM Tris-HC1 (pH 8.0), generally known as the Bonner method (Bonner et al., 1968), has yielded low recoveries and less pure preparations. Instead, gentle extraction with 2 M NaCI has proved to be a fast and reliable method. The ratio of the absorbancies measured at 260 and 280nm was 1.6 + 0.02, which is comparable to published values (Bonner et al., 1968; Andrews and Roberts, 1974). As a second test for purity we have also compared the electrophoretic pattern of the polypeptides that were solubilized upon digestion with DNase I with those extracted with high salt after digestion with DNase I and those extracted with high salt solely (Fig. 2). Some interesting features emerge from this experiment. First, no qualitative differences are observed in the electrophoretic pattern of the polypeptides extracted with high salt and those extracted with high salt after digestion with DNase. Thus no polypeptides are selectively lost by a simple extraction with 2 M NaC1 at this level of resolution. Secondly, there is no enrichment of polypeptides as a result of extraction with high salt (lanes a and b vs c) suggesting that no detectable amounts of contaminating protein are preferentially solubilized with 2 M NaCI. Thirdly, a few polypeptides are preferen-
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L. MEHEUSand J. R, VANFLETEREN chromatin is cleared by centrifugation (see Materials and Methods). Proteins associated with nuclear RNA will be released in 2 M NaC1 and they may contribute considerably to the mass of the chromatin proteins (Peters and Comings, 1980; Weihe et al., 1982). The complexity of the proteins that co-sediment with RNA after digestion of nematode nuclei with RNase are shown in Fig. 4. A set of 4 major proteins migrates in the 42,000-50,000 daltons mol. wt range. A second cluster of about 6 abundant proteins occurs between 29,000 and 36,000 daltons. These proteins are probably associated with hnRNA (Beyer et al., 1977; Karn et al., 1977; Brunel and Lelay, 1979; Peters and Comings, 1980; Weihe et al., 1982). Some of these and perhaps most proteins in the lower molecular weight range might be derived from nucleolar RNP. Preliminary characterization of chromosomal nonhistone proteins As can be seen from Fig. 5 over 40 polypeptides are separated by SDS polyacrylamide gel electrophoresis. We have tried to identify some of them using antibodies raised against filamin, myosin, ~-actinin, tubulin, F-actin and tropomyosin. A precipitin reaction
a
13
Fig. 3. Proteins recovered from the pellet and from the supernatant fluid when chromatin was layered on 5~o sucrose and centrifuged at 8000rpm for 60 min. Chromatin was extracted with 2 M NaC1 after digesting nuclei with 100/~g/ml DNase I. (a) Proteins from the pellet; (b) proteins from the supernatant fluid.
tially liberated upon digestion with nuclease, e.g. the doublet band seen at 80,000 daltons in lane c. Actin is also enriched in this fraction probably due to the very high affinity of DNase I for actin (Lazarides and Lindberg, 1974). It should be noticed that the amount of chromatin solubilized with nuclease solely was consistently low, usually not exceeding 35~o of the chromatin present. Because of the foregoing and since the enzyme itself masks a considerable part of silver stained gels the high salt extraction procedure was chosen for further experiments. In order to examine to what extent other proteins of the nucleus, e.g. those of the nuclear (and nucleolar) matrix and the pore-lamina complex were present in our preparations, nematode nuclei were extracted with 2 M NaCI after treatment with DNase I and centrifuged through 5~o sucrose as described by Lebkowski and Laemmli (1982). As little as 1-2~o of the chromatin was recovered from the pellet, a first indication that the bulk of matrix proteins were not present in our chromatin preparations. Neither did a b the electrophoretic pattern show a typical preponFig. 4. Occurrence of proteins associated with nuclear RNA derance of a polypeptide set at 60,000-75,000 daltons among the chromatin proteins, analyzed by SDS poly(polypeptides of the pore-lamina) though a general acrylamide gel electrophoresis. (a) Nuclear proteins extracincrease of polypeptides with molecular weights ted with 2 M NaC1, (b) RNP particles prepared by digestion above 50,000 was effected (Fig. 3, lane a and Fig. 5). of nuclei with 0.1/~g/ml pancreatic ribonuclease (60min, Presumably intact matrices are largely removed along 4°C) and sedimented by ultracentrifugation as described under 'Materials and Methods'. with any particulate material when the solubilized
Nonhistones of C. elegans
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and produced a vertical streak in the second dimension. In contrast, the nonequilibrium procedure was found to be very useful in separating the chromosomal nonhistone proteins. Over 200 spots could be detected on silver stained two-dimensional gels (Fig. 6). Further research will be needed to characterize important chromatin constituents on such gels, however. At present we can only identify actin with reasonable certainty.
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11697-
66-
DISCUSSION TUBULIN
45ACTIN TROPOMYOSIN
29-
381
H1
Fig. 5. The SDS-polyacrylamide gel electrophoresis of the chromosomal proteins prepared from C. elegans. Tubulin, actin and tropomyosm were identified by their binding with the respective antibodies.
was obtained with tubulin, F-actin and tropomyosin. The respective bands are assigned in Fig. 5. The absence of a specific reaction when probing myosin, a-actinin and filamin does not exclude their presence among the nonhistone proteins since the antibodies used were raised against the respective proteins extracted from vertebrate tissue. A band at 200,000 daltons co-migrates with muscle myosin. We have found variable amounts of this polypeptide which could either result from some contamination of the nonhistones with muscle myosin or due to incomplete dissolution. No attempts were made to remove the histones so as to avoid loss of co-extracting nonhistones. The prominent band having an apparent mol. wt of approx. 30,000 daltons was shown to represent HI. This was derived from a comparison of its relative mobility and that of the H1 polypeptides of purified histones. A better estimate of their real mol. wt is 18,500-20,000. It has been known for long that histones behave anomalously in SDS polyacrylamide gel electrophoresis. The core histones are not resolved m 11~ polyacrylamide gels (Vanfleteren and Van Beeumen, 1983). We have also used two-dimensional electrophoretic methods as more appropriate tools to the study of the tremendous complexity of the nonhistones. Poor results were obtained when the proteins were focused at their isoelectric points in the first dimension, mainly because a major portion did not enter the gel
Nonhistone chromosomal proteins are generally defined as the proteins remaining after the five histones have been removed from chromatin, the extended chromosomes of the interphase nucleus. Besides chromatin the nucleus contains a fibrillar framework or matrix to which the chromatin is attached (Berezney and Coffey, 1977; Adolph, 1980; Agutter and Richardson, 1980; Capco et al., 1982; Lebkowski and Laemmli, 1982; Lewis and Laemmli, 1982). This framework is embedded in the nucleoplasm and surrounded by the nuclear envelope. One or several nucleoli are involved in r R N A synthesis and ribosome assembly. Newly synthetized hnRNA is packaged as ribonucleoprotein and attached to the inner side of the nuclear envelope and the nuclear matrix. It is a tremendous task to discriminate between these constituents. A first problem is to distinguish genuine constituents of the chromatin from contaminating proteins derived from the cytoplasm. Extensive purification of nuclei will considerably reduce cytoplasmic contamination. Chromatin is usually prepared from lysed nuclei by thoroughly washing of the particulate fraction with saline-EDTA and dilute Tris buffer and sedimentation across 1.7 M sucrose (Bonner et aL, 1968). The washed sediment is then resuspended in Tris buffer and sheared in a Virtis homogenizer to obtain soluble chromatin. Up to 80% of the mass of the nonhistone chromosomal proteins prepared by this method has been shown to be contributed by nuclear matrix and hnRNPs (Peters and Comings, 1980). For that reason and because more impurities were solubilized under the harsh conditions of the Bonnet method we have routinely extracted the nuclear pellet with 2 M NaC1. Neither the nuclear (and nucleolar) matrix nor the pore-lamina complex (i.e. the inner membrane of the nuclear envelope) are readily soluble in 2 M NaC1 (Dwyer and Blobel, 1976; Berezney and Coffey, 1977; Lebkowski and Laemmli, 1982; Miiller et al., 1983). Accordingly as little as 1-2% of the protein mass was recovered in the pellet when high salt extracted chromatin was sedimented through 5% sucrose. Since the protein content of the matrix and the pore-lamina complex accounts for about 10-20~ of the total nuclear protein (Agutter and Richardson, 1980; Lebkowski and Laemmli, 1982) these results suggest that they were not preferentially extracted. On the other hand we should not be surprised to find small but detectable amounts of matrix and lamina proteins among the chromatin proteins. Certainly present among the nonhistones are the proteins that are normally associated with nuclear RNA. Our chromatin preparations contain at least
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®
®
TUBULIN
,ACTIN 'TROPOMYOSIN
Fig. 6. Two-dimensional gel electrophoresis of the chromosomal proteins of C. elegans. Nonequilibrium pH gradient electrophoresis (NEPHGE) was used in the first dimension. The assignment of tubulin and tropomyosin is tentative.
17 polypeptides derived from ribonucleoprotein particles, but their contribution to the total protein mass is less important than e.g. in rat liver and H e L a cell chromatin (Peters and Comings, 1980; Karn et al., 1977; Weihe et al., 1982). We are not surprised to find that actin, tropomyosin and tubulin and perhaps myosin as well are major constituents of nematode nuclei. Their occurrence among the nonhistone proteins of rat liver chromatin has already been documented (Douvas et al., 1975; Peters and Comings, 1980; Capco et al., 1982). However, the question remains whether they are genuine components of chromatin or contaminants of cytoplasmic origin as suggested by Lebkowski and Laemmli (1982). To date only actin has been detected in situ in nuclei of Dictyostelium and of H e L a cells (Fukui and Katsumaru, 1979) and a distinct species of nuclear actin, which was different from muscle and cytoplasmic actin has been prepared from Novikoff hepatoma ascites cells (Bremer et al., 1981). W o r k is now in progress to further characterize the nonhistone chromosomal proteins of C. elegans on two-dimensional gels and to study age related alterations of the electrophoretic pattern. Acknowledgements--The authors are much indebted to Dr J. De Mey and G. Danneels, Janssen Pharmaceutica (Beerse, Belgium) for providing the antibodies and for laboratory facilities. The first author gratefully acknowl-
edges a scholarship from the "lnstituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw'. J.R.V. is a senior research associate of the 'Nationaal Fonds voor Wetenschappelijk Onderzoek'. REFERENCES Adolph K. W. (1980) Organization of chromosomes in HeLa cells: isolation of histone-depleted nuclei and nuclear scaffolds. J. Cell Sci. 42, 291-304. Adolph K. W. and Phelps J. P. (1982) Role of nonhistones in chromosome structure. Cell cycle variations in protein synthesis. J. biol. Chem. 257, 9086-9092. Agutter P. S. and Richardson J. C. W. (1980) Nuclear non-chromatin proteinaceous structures: their role in the organization and function of the interphase nucleus. J. Cell Sci. 44, 395-435. Andrews P. and Roberts D. (1974) The preparation and characterization of chromatin from third instar larvae of Drosophila melanogaster. Nucleic Acids' Res. 1, 979-997. Berezney R. and Coffey D. S. (1977) Nuclear matrix. Isolation and characterization of a framework structure from rat liver nuclei. J. Cell Biol. 73, 616-637. Beyer A. L., Christensen M. E., Walker B. W. and LeStourgeon W. M. (1977) Identification and characterization of the packaging proteins of core 40 S hnRNP particles. Cell 11, 12~138. Blfithmann H. and Illmensee K. (1981) Nuclear nonhistone proteins in mouse teratocarcinomas. I. Cell lineage specificity. Roux Arch. Devl Biol. 190, 347-378. Bojanovic J. J., Drazic A. M. and Vujovic Z. R. (1981) Non-histone nuclear proteins in active and involuted rat thymuses. Biochimie 63, 791-794.
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occurring inhibitor of deoxyribonuclease I. Proc. natn. Acad. Sci., U.S.A. 71, 4742-4746. Lebkowski J. S. and Laemmli U. K. (1982) Non-histone proteins and long-range organisation of HeLa interphase DNA. J. molec. Biol. 156, 325-344. Lewis C. D. and Laemmli U. K. (1982) Higher order metaphase chromosome structure: evidence of metalloprotein interactions. Cell 29, 176-181. Medvedev Z. A., Medvedeva M. N. and Crowne H. M. (1981) Sharp increase of the content of liver specific histone satellite chromatin protein in spontaneous mouse hepatomas. IRCS Med. Sci. 9, 860-861. Moeremans M. , Danneels G. and De Mey J. Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes (in press). Moeremans M., Danneels G., Van Dijck A., Langanger G. and De Mey J. (1984) Sensitive visualisation of antigenantibody reactions in dot and blot immuneoverlay assays with immunogold and immunogold silverstaining. J. Immun. Meth. 74, 353 360. Morrissey J. H. (1981) Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Analyt. Biochem. 117, 307-310. Mfiller M., Spiess E. and Werner D. (1983) Fragmentation of 'nuclear matrix' on a mica target. Eur. J. Cell Biol. 31, 158-166. O'Farrell P. Z., Goodman M. M. and O'Farrell P. H. (1979) High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12, 1133-1142. Peters K. E. and Comings D. E. (1980) Two-dimensional gel electrophoresis of rat liver nuclear washes, nuclear matrix and hnRNA proteins. J. Cell Biol. 86, 135-155. Rothstein M. (1982) Biochemical Approaehes to Aging. Academic Press, New York. Todorova M., Anachkova B. and Russev G. (1983) Comparative study of the tightly bound nonhistone chromosomal proteins of rat liver and Ehrlich ascites tumor cells. Cell Diff. 13, 57-61. Treadgill G. J. and Arnstein H. R. V. (1984) The nonhistone proteins of the developing mammalian erythroid cells. Cell Differentiation 14, 7-17. Vanfleteren J. R. (1978) Axenic culture of free-living, plantparasitic and insect-parasitic nematodes. A. Rev. Phytopathol. 16, 131-157. Vanfleteren J. R. (1980) Nematodes as nutritional models. In Nematodes as Biological Models, Vol. 2 (Edited by Zuckerman B. M.), pp. 47-79. Academic Press, New York. Vanfleteren J. R. and Van Beeumen J. (1983) Nematode chromosomal proteins--III. Some structural properties of the histones of Caenorhabditis elegans. Comp. Biochem. Physiol. 76B, 179-184. Weihe A., Schmidt G., v. Mickwitz C.-U. and Lindigkeit R. (1982) Discrimination of several classes of nonhistone proteins in chromatin fractions from liver and thymus nuclei of rats. Acta biol. med. germ. 41, 609-624. Wielgat B. and Kleczkowski K. (1981) Nonhistone chromosomal proteins from gibberellic acid treated maize and pea plants and their effect on transcription in vitro. Int. J. Biochem. 13, 1201-1203. Yancheva N. and Djondjurov L. (1982) The growth inhibition in Friend erythroleukemia cells induces cycling of preexisting nonhistone proteins. Eur. J. Biochem. 121, 309 316.
0305-0491/85 $3.00 + 0.00 © 1985 Pergamon Press Ltd
N E M A T O D E CHROMOSOMAL PROTEINS--IV. THE NONHISTONES OF CAENORHABDITIS ELEGANS L. MEHEUS and J. R. VANFLETEREN* Laboratorium voor Morfologie en Systematiek der Dieren, Rijksuniversiteit Gent, Ledeganckstraat 35, B-9000 Gent, Belgium (Tel: 091-22-78-21)
(Received 8 October 1984) Abstract--1. A method has been developed which yields reasonably clean nuclei from the free-living nematode Caenorhabditis elegans. Nonhistones were prepared from these nuclei by gentle extraction with 2 M NaC1 and analyzed by one- and two-dimensional electrophoresis using a nonequilibrium pH gradient electrophoresis in the first dimension after removal of nucleic acids by phenol extraction. 2. The extraction procedure yielded nonhistone chromatin proteins and proteins associated with nuclear RNA and a background of polypeptides derived from the nuclear matrix and pore-lamina complex. 3. Over 40 polypeptides were distinguished on silver stained one-dimensional slab gels. 4. The presence of actin, tropomyosin and tubulin was demonstrated by immunoprobing after transfer of the polypeptides to nitrocellulose. 5. Bands co-migrating with myosin and ct-actinin gave no specific reaction when probed with antibodies raised against the respective proteins from vertebrate tissues. 6. Over 200 spots were visible on two-dimensional gels stained with silver nitrate.
INTRODUCTION There is still growing evidence that nonhistone proteins are important in organizing the higher order structure of chromatin and controlling gene expression. A nuclear matrix, sometimes called nuclear ghost or scaffold and composed of protein and small amounts of nucleic acid extends throughout the interphase nucleus and forms a framework to which loops of the D N A fibres are attached (Berezney and Coffey, 1977; Adolph, 1980; Agutter and Richardson, 1980; Capco et al., 1982; Lebkowski and Laemmli, 1982; Lewis and Laemmli, 1982). The compaction of interphase chromatin effected by this structure might well have a bearing on local genome activity. On the other hand it has been thought that specific gene regulators reside in the nonhistone fraction. Evidence in support of this idea has been taken from the time and species specificity of several of the nonhistones associated with chromatin (Adolph and Phelps, 1982; Cartwright et al., 1982; Kabisch et al., 1982; Weihe et al., 1982; Treadgill and Arnstein, 1984) and from the changes in the nonhistone protein pattern associated with changes in gene expression during both normal development and neoplastic transformation (Bliitmann and Illmensee, 1981; Kilianska et al., 1981; Bojanovic et al., 1981; Medvedev et al., 1981; Wielgat and Kleczkowski, 1981; Yancheva and Djondjurov, 1982; Einck and Bustin, 1983; T o d o r o v a et al., 1983; Burkhardt et al., 1984). A major characteristic of ageing is the steep fall of organismic and cellular activity. The underlying mechanism is still a matter of controversy but there is some agreement that there is a reduced level of R N A synthesis in old animals and alterations of the structure and function of chromatin have been implicated (Kanungo, 1980; Rothstein, 1982). *To whom all correspondence should be addressed.
The free-living nematode species Caenorhabditis elegans is a good model for fundamental ageing research. It offers the advantage of small size, short life cycle (12-18 days pending on the nutritional regime), ease of cultivation, a genetically manipulatable system and a strictly determined development (eutely). Since very few somatic cell divisions occur after hatching and with exclusion for the gonadal cells, aged organisms can be considered as being composed of aged cells. Since the nonhistone proteins of free-living nematodes have never been studied before, the present investigation aimed to describe the diversity of the nonhistones of C. elegans and to identify some major components. This knowledge is prejudicial to the further study of age-related changes at this level. MATERIALS AND METHODS
Nematode growth The nematode used in this study is the wild type of the Bristol strain of Caenorhabditis elegans, designated N2 by Brenner (1974). It was obtained from Dr Epstein. Nematodes were grown in axenic culture in a medium consisting of 3% dry yeast extract, 3% soy peptone and 500 pg/ml haemoglobin. The basal medium was autoclaved for 20 min at 120°C. Haemoglobin was aseptically added from a stock solution made up in 0.1 N KOH (5 g/100ml) and sterilized by Seitz filtration. Routine cultures were grown in Fernbach flasks containing 200 ml of medium with continuous shaking on a gyrotory shaker at 100-150 oscillations/min. Larger cultures were established in 101 vessels equipped with a magnetically driven large Teflon bar. The need for increased gas exchange was met by continuous aerating through an in line membrane filter. Axenic culture methods have been described in much more detail elsewhere (Vanfleteren, 1978, 1980). When the optimal density of about 40,000 worms/ml was reached, which usually occurred on days 8 12 pending on the inoculum size, the worms were collected by centrifugation at 5000 rpm for 10 min in a refrigerated centrifuge. The pellet was washed 377
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several times with S buffer (0.1 M NaCI in 0.05 M phosphate buffer, pH 6) at 3000 rpm for 5 rain. The nematode pellets were then suspended in 2 vol of a solution containing 1.7 M sucrose, 0.5~ Nonidet P-40, 5 m M CaC1 z and 50mM Tris-HC1 (pH 7.4), dripped into liquid nitrogen and stored at -196°C. The wet weight from one litre of culture was approx. 5 g of nematodes.
Preparation of nuclei The frozen nematode nuggets were homogenized in a Waring blender with an equal vol of acid washed glass beads (0.2 mm in dia.) at medium speed for 1 min. The glass beads were allowed to settle by gravitation and the supernatant fluid was further homogenized with 10 strokes of a motor driven Teflon pestle in a Potter-Elvehjem homogenizer operated at 2000rpm. Intact nematodes and large debris were sedimented at 3000 rpm for 5 min. The supernatant was then layered over two vol of 1.7 M sucrose, 0.5~ Nonidet P-40, 5 mM CaC12, 50 mM Tris-HCl (pH 7.4) and a crude gradient was formed using a Pasteur pipette. The gradients were centrifuged at 10,000rpm in a 4 × 50ml swing-out rotor for 30 min. The nuclear pellets were suspended in 2ml 0.14M NaC1, l m M PMSF, 50mM Tris-HCl (pH 7.4) and centrifuged for 5 min in an Eppendorf centrifuge. This wash was repeated five times. The Eppendorf tubes were carefully cooled in ice water in between to avoid any rise of the temperature much above 4°C. All other handlings were performed at 4:'C.
Isolation of chromosomal proteins The nuclear pellet was suspended in 2 ml of 2 M NaCI, 1 mM PMSF, 10 m M Tris-HC1 (pH 7.4) and left on ice for 90 min with occasional shaking. Particulate material was sedimented by centrifugation for 5 min in an Eppendorf centrifuge and discarded. The solubilized chromatin was cooled in ice and finally clarified by one more run for 5 min. The yield of chromatin from 1 g of wet nematode tissue was 4-6 A260units. In another set of experiments chromatin was extracted in low salt buffer after digestion with DNase I. The nuclear pellet was suspended in 10 m M MgC12, 1 mM CaC12, 1 mM PMSF, 10. m M Tris-HCl (pH 7.4) and DNase I (Worthington) was added at a final concn of 100 #g/ml. Incubation was at 0°C for 60 min with gentle stirring. The solubilized chromatin was recovered as described above. The purity of all chromatin preparations was assessed by measuring the A260/A280 ratio.
Fast sedimenting fraction of chromatin Nuclei prepared from 20 g of nematode tissue were digested with 100#g/ml DNase I for 60 min as described above. An equal vol of 4 M NaC1, 20 m M EDTA, 20 mM Tris-HCl (pH 7.4) was added and extraction was allowed to continue for another 30 min. The suspension was centrifuged for 3 min in an Eppendorf centrifuge and filtered through a No. 4 glass filter. The filtrate was layered on 5~o sucrose in 2 M NaCI, 10mM Tris-HCl (pH 7.4) and centrifuged for 60 min at 8000 rpm in a 3 × 25 ml swing out rotor as described for the preparation of nuclear scaffolds (Lebkowski and Laemmli, 1982). The sediment was dissolved in TEM buffer (20 mM Tris-HCl, pH 8.2, 20mM EDTA, 0.1~o 2-mercaptoethanol) containing 2 ~ SDS and further processed for electrophoretic analysis.
sedimentation coefficients of 40 S and above. The sedimented material was then solubilized directly in sample buffer and analyzed by SDS polyacrylamide gel electrophoresis.
Sample preparation .for electrophoretic analysis The chromatin preparation was made 20°/0 in TCA. After standing for at least 60 min the precipitate was sedimented at 10,000 rpm for 5 min. The pellet was washed three times in a solution containing 70~o acetone, 20% ethanol and 10 mM Tri~HC1 (pH 7.4) and dissolved overnight in TEM buffer containing 2~o SDS. The sample was then boiled for 5 min, cooled again and mixed with an equal vol of phenol equilibrated with TEM buffer. After brief centrifugation (5min, 10,000rpm) to separate the phases, the nuclear proteins were precipitated from the phenol phase with 3 vol of methanol at - 1 8 ° C overnight. The precipitate was sedimented at 5000 rpm for 5 min, washed with ether to remove traces of phenol and air dried.
Electrophoresis Gel electrophoresis in the presence of SDS was performed according to Laemmli (1970) in 11~o polyacrylamide slab gels. The mol. wt of any given polypeptide was determined from a comparison of its mobility and that of standards run in a separate lane. The standards used were: myosin from rabbit muscle, 205,000 daltons; fl-galactosidase from E. coli, 116,000 daltons; phosphorylase B from rabbit muscle, 97,000 daltons; bovine serum albumin, 68,000 daltons; egg albumin, 45,000 daltons; carbonic anhydrase from bovine erythrocytes, 29,000 daltons (Sigma). For two-dimensional gel electrophoretic analysis the nonequilibrium pH gradient electrophoresis (NEPHGE) procedure of O'Farrell et al. (1979) was used. Only pH 3 10 ampholytes (LKB) were used. The gels were run at 400 V for 6 hr. For probing the pH gradient 5 mm sections of a control gel were put into separate vials and soaked with degassed water. After completion of the nonequilibrium gel electrophoresis the gels were fixed in 50~o methanol for 1 hr (with 4 changes) and equilibrated for 2 hr against the sample buffer for the second dimension. 2-Mercaptoethanol was omitted from this buffer because it produces a few heavy horizontal lines in the 60,000-70,000 mol. wt range in silver stained gels.
Silver stain detection of polypeptides Silver staining was performed essentially according to Morrissey (1981) with the following modifications. The slab gel was fixed in 50~o methanol-12~ TCA for at least 30 min, the secondary fixation with glutaraldehyde was omitted. The silver nitrate concentration was raised to 0.2~o. Some polypeptides (histones for example) produce a negative stain with a single cycle of this staining procedure, yet stain very well after recycling. When a highly sensitive silver stain was
Preparation of proteins associated with nuclear RNA Sucrose washed nuclei were suspended in 0.14 M NaCI, 1 mM MgCI z, 1 mM 2-mercaptoethanol, 0.1 m M PMSF, 50 mM Tris-HC1 (pH 7.4) containing 0.1 pg/ml pancreatic RNase (preheated for 15 min at 80°C) for 1 hr at 4°C to extract nuclear ribonucleoprotein. The nuclei were then pelleted at 10,000 rpm for 5 min. The supernatant portion was further centrifuged in a MSE 3 × 5 ml swing out rotor at 45,000 rpm for 3hr at 4°C to pellet particles with
Fig. 1. Isolated nuclei of C. elegans, stained with Giemsa. × 450.
Nonhistones of C. elegans
a
b
c
d
379
e
Fig. 2. Comparison of the chromosomal proteins released from nematode nuclei by different methods. (a) Extraction with 2 M NaC1; (b) extraction with 2 M NaC1 following digestion with 100 #g/ml DNase I; (c) proteins released in 10mM Tris-HC1 (pH 7.4), 10.mM MgCI2, 1 mM CaC12 by digestion with 100#g/ml DNase I; (d) DNase I, 10/zg; (e) mol. wt markers.
desired gels were stained as above and destained in concentrated Agefix (Agfa Gevaert), thoroughly rinsed with distilled water and recycled as described above.
of numerous nuclei (Fig. 1). Occasional cuticular fragments and amorphous components are also present.
Immunoprobing
Isolation of chromosomal proteins
Chromosomal proteins were run on 11% SDS polyacrylamide gels. The resolved polypeptides were transferred to nitrocellulose in 25mM Tris-HC1 (pH 8.2), 150mM glycine, 20% methanol at 0.5 A for 4 hr. Residual protein binding sites were blocked by incubation in 20raM Tris-HC1 (pH 8.2), 150mM NaCI, 5% BSA for 30min at 37°C. The blot was then extensively washed in 20mM Tris-HC1 (pH 8.2), 150 mM NaC1, 0.1% BSA and cut into strips. These were incubated with approx. 1 #g/ml antibody in wash buffer. Bound antibodies were then visualized by immunogold staining and silver enhancement of the latter (Moeremans et al., 1984, and in press). RESULTS
Preparation of nuclei The preparation of nuclei from nematode tissue is complicated by their widely different dimensions and the co-sedimentation of cuticular debris during sucrose density gradient centrifugation. This problem was largely met by a two-step density gradient centrifugation. At low speed (3000 rpm) intact and broken nematodes and the bulk of cuticle fragments sedimented whereas the nuclei remained in the supernatant solution. They were pelleted at higher speed (10,000rpm) and freed of cytoplasm. Microscopic analysis of a typical preparation reveals the presence
Extraction of sheared chromatin with low salt buffer such as 10 mM Tris-HC1 (pH 8.0), generally known as the Bonner method (Bonner et al., 1968), has yielded low recoveries and less pure preparations. Instead, gentle extraction with 2 M NaCI has proved to be a fast and reliable method. The ratio of the absorbancies measured at 260 and 280nm was 1.6 + 0.02, which is comparable to published values (Bonner et al., 1968; Andrews and Roberts, 1974). As a second test for purity we have also compared the electrophoretic pattern of the polypeptides that were solubilized upon digestion with DNase I with those extracted with high salt after digestion with DNase I and those extracted with high salt solely (Fig. 2). Some interesting features emerge from this experiment. First, no qualitative differences are observed in the electrophoretic pattern of the polypeptides extracted with high salt and those extracted with high salt after digestion with DNase. Thus no polypeptides are selectively lost by a simple extraction with 2 M NaC1 at this level of resolution. Secondly, there is no enrichment of polypeptides as a result of extraction with high salt (lanes a and b vs c) suggesting that no detectable amounts of contaminating protein are preferentially solubilized with 2 M NaCI. Thirdly, a few polypeptides are preferen-
380
L. MEHEUSand J. R, VANFLETEREN chromatin is cleared by centrifugation (see Materials and Methods). Proteins associated with nuclear RNA will be released in 2 M NaC1 and they may contribute considerably to the mass of the chromatin proteins (Peters and Comings, 1980; Weihe et al., 1982). The complexity of the proteins that co-sediment with RNA after digestion of nematode nuclei with RNase are shown in Fig. 4. A set of 4 major proteins migrates in the 42,000-50,000 daltons mol. wt range. A second cluster of about 6 abundant proteins occurs between 29,000 and 36,000 daltons. These proteins are probably associated with hnRNA (Beyer et al., 1977; Karn et al., 1977; Brunel and Lelay, 1979; Peters and Comings, 1980; Weihe et al., 1982). Some of these and perhaps most proteins in the lower molecular weight range might be derived from nucleolar RNP. Preliminary characterization of chromosomal nonhistone proteins As can be seen from Fig. 5 over 40 polypeptides are separated by SDS polyacrylamide gel electrophoresis. We have tried to identify some of them using antibodies raised against filamin, myosin, ~-actinin, tubulin, F-actin and tropomyosin. A precipitin reaction
a
13
Fig. 3. Proteins recovered from the pellet and from the supernatant fluid when chromatin was layered on 5~o sucrose and centrifuged at 8000rpm for 60 min. Chromatin was extracted with 2 M NaC1 after digesting nuclei with 100/~g/ml DNase I. (a) Proteins from the pellet; (b) proteins from the supernatant fluid.
tially liberated upon digestion with nuclease, e.g. the doublet band seen at 80,000 daltons in lane c. Actin is also enriched in this fraction probably due to the very high affinity of DNase I for actin (Lazarides and Lindberg, 1974). It should be noticed that the amount of chromatin solubilized with nuclease solely was consistently low, usually not exceeding 35~o of the chromatin present. Because of the foregoing and since the enzyme itself masks a considerable part of silver stained gels the high salt extraction procedure was chosen for further experiments. In order to examine to what extent other proteins of the nucleus, e.g. those of the nuclear (and nucleolar) matrix and the pore-lamina complex were present in our preparations, nematode nuclei were extracted with 2 M NaCI after treatment with DNase I and centrifuged through 5~o sucrose as described by Lebkowski and Laemmli (1982). As little as 1-2~o of the chromatin was recovered from the pellet, a first indication that the bulk of matrix proteins were not present in our chromatin preparations. Neither did a b the electrophoretic pattern show a typical preponFig. 4. Occurrence of proteins associated with nuclear RNA derance of a polypeptide set at 60,000-75,000 daltons among the chromatin proteins, analyzed by SDS poly(polypeptides of the pore-lamina) though a general acrylamide gel electrophoresis. (a) Nuclear proteins extracincrease of polypeptides with molecular weights ted with 2 M NaC1, (b) RNP particles prepared by digestion above 50,000 was effected (Fig. 3, lane a and Fig. 5). of nuclei with 0.1/~g/ml pancreatic ribonuclease (60min, Presumably intact matrices are largely removed along 4°C) and sedimented by ultracentrifugation as described under 'Materials and Methods'. with any particulate material when the solubilized
Nonhistones of C. elegans
205
and produced a vertical streak in the second dimension. In contrast, the nonequilibrium procedure was found to be very useful in separating the chromosomal nonhistone proteins. Over 200 spots could be detected on silver stained two-dimensional gels (Fig. 6). Further research will be needed to characterize important chromatin constituents on such gels, however. At present we can only identify actin with reasonable certainty.
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11697-
66-
DISCUSSION TUBULIN
45ACTIN TROPOMYOSIN
29-
381
H1
Fig. 5. The SDS-polyacrylamide gel electrophoresis of the chromosomal proteins prepared from C. elegans. Tubulin, actin and tropomyosm were identified by their binding with the respective antibodies.
was obtained with tubulin, F-actin and tropomyosin. The respective bands are assigned in Fig. 5. The absence of a specific reaction when probing myosin, a-actinin and filamin does not exclude their presence among the nonhistone proteins since the antibodies used were raised against the respective proteins extracted from vertebrate tissue. A band at 200,000 daltons co-migrates with muscle myosin. We have found variable amounts of this polypeptide which could either result from some contamination of the nonhistones with muscle myosin or due to incomplete dissolution. No attempts were made to remove the histones so as to avoid loss of co-extracting nonhistones. The prominent band having an apparent mol. wt of approx. 30,000 daltons was shown to represent HI. This was derived from a comparison of its relative mobility and that of the H1 polypeptides of purified histones. A better estimate of their real mol. wt is 18,500-20,000. It has been known for long that histones behave anomalously in SDS polyacrylamide gel electrophoresis. The core histones are not resolved m 11~ polyacrylamide gels (Vanfleteren and Van Beeumen, 1983). We have also used two-dimensional electrophoretic methods as more appropriate tools to the study of the tremendous complexity of the nonhistones. Poor results were obtained when the proteins were focused at their isoelectric points in the first dimension, mainly because a major portion did not enter the gel
Nonhistone chromosomal proteins are generally defined as the proteins remaining after the five histones have been removed from chromatin, the extended chromosomes of the interphase nucleus. Besides chromatin the nucleus contains a fibrillar framework or matrix to which the chromatin is attached (Berezney and Coffey, 1977; Adolph, 1980; Agutter and Richardson, 1980; Capco et al., 1982; Lebkowski and Laemmli, 1982; Lewis and Laemmli, 1982). This framework is embedded in the nucleoplasm and surrounded by the nuclear envelope. One or several nucleoli are involved in r R N A synthesis and ribosome assembly. Newly synthetized hnRNA is packaged as ribonucleoprotein and attached to the inner side of the nuclear envelope and the nuclear matrix. It is a tremendous task to discriminate between these constituents. A first problem is to distinguish genuine constituents of the chromatin from contaminating proteins derived from the cytoplasm. Extensive purification of nuclei will considerably reduce cytoplasmic contamination. Chromatin is usually prepared from lysed nuclei by thoroughly washing of the particulate fraction with saline-EDTA and dilute Tris buffer and sedimentation across 1.7 M sucrose (Bonner et aL, 1968). The washed sediment is then resuspended in Tris buffer and sheared in a Virtis homogenizer to obtain soluble chromatin. Up to 80% of the mass of the nonhistone chromosomal proteins prepared by this method has been shown to be contributed by nuclear matrix and hnRNPs (Peters and Comings, 1980). For that reason and because more impurities were solubilized under the harsh conditions of the Bonnet method we have routinely extracted the nuclear pellet with 2 M NaC1. Neither the nuclear (and nucleolar) matrix nor the pore-lamina complex (i.e. the inner membrane of the nuclear envelope) are readily soluble in 2 M NaC1 (Dwyer and Blobel, 1976; Berezney and Coffey, 1977; Lebkowski and Laemmli, 1982; Miiller et al., 1983). Accordingly as little as 1-2% of the protein mass was recovered in the pellet when high salt extracted chromatin was sedimented through 5% sucrose. Since the protein content of the matrix and the pore-lamina complex accounts for about 10-20~ of the total nuclear protein (Agutter and Richardson, 1980; Lebkowski and Laemmli, 1982) these results suggest that they were not preferentially extracted. On the other hand we should not be surprised to find small but detectable amounts of matrix and lamina proteins among the chromatin proteins. Certainly present among the nonhistones are the proteins that are normally associated with nuclear RNA. Our chromatin preparations contain at least
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L. MEHEUSand J. R. VANFLETEREN
®
®
TUBULIN
,ACTIN 'TROPOMYOSIN
Fig. 6. Two-dimensional gel electrophoresis of the chromosomal proteins of C. elegans. Nonequilibrium pH gradient electrophoresis (NEPHGE) was used in the first dimension. The assignment of tubulin and tropomyosin is tentative.
17 polypeptides derived from ribonucleoprotein particles, but their contribution to the total protein mass is less important than e.g. in rat liver and H e L a cell chromatin (Peters and Comings, 1980; Karn et al., 1977; Weihe et al., 1982). We are not surprised to find that actin, tropomyosin and tubulin and perhaps myosin as well are major constituents of nematode nuclei. Their occurrence among the nonhistone proteins of rat liver chromatin has already been documented (Douvas et al., 1975; Peters and Comings, 1980; Capco et al., 1982). However, the question remains whether they are genuine components of chromatin or contaminants of cytoplasmic origin as suggested by Lebkowski and Laemmli (1982). To date only actin has been detected in situ in nuclei of Dictyostelium and of H e L a cells (Fukui and Katsumaru, 1979) and a distinct species of nuclear actin, which was different from muscle and cytoplasmic actin has been prepared from Novikoff hepatoma ascites cells (Bremer et al., 1981). W o r k is now in progress to further characterize the nonhistone chromosomal proteins of C. elegans on two-dimensional gels and to study age related alterations of the electrophoretic pattern. Acknowledgements--The authors are much indebted to Dr J. De Mey and G. Danneels, Janssen Pharmaceutica (Beerse, Belgium) for providing the antibodies and for laboratory facilities. The first author gratefully acknowl-
edges a scholarship from the "lnstituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw'. J.R.V. is a senior research associate of the 'Nationaal Fonds voor Wetenschappelijk Onderzoek'. REFERENCES Adolph K. W. (1980) Organization of chromosomes in HeLa cells: isolation of histone-depleted nuclei and nuclear scaffolds. J. Cell Sci. 42, 291-304. Adolph K. W. and Phelps J. P. (1982) Role of nonhistones in chromosome structure. Cell cycle variations in protein synthesis. J. biol. Chem. 257, 9086-9092. Agutter P. S. and Richardson J. C. W. (1980) Nuclear non-chromatin proteinaceous structures: their role in the organization and function of the interphase nucleus. J. Cell Sci. 44, 395-435. Andrews P. and Roberts D. (1974) The preparation and characterization of chromatin from third instar larvae of Drosophila melanogaster. Nucleic Acids' Res. 1, 979-997. Berezney R. and Coffey D. S. (1977) Nuclear matrix. Isolation and characterization of a framework structure from rat liver nuclei. J. Cell Biol. 73, 616-637. Beyer A. L., Christensen M. E., Walker B. W. and LeStourgeon W. M. (1977) Identification and characterization of the packaging proteins of core 40 S hnRNP particles. Cell 11, 12~138. Blfithmann H. and Illmensee K. (1981) Nuclear nonhistone proteins in mouse teratocarcinomas. I. Cell lineage specificity. Roux Arch. Devl Biol. 190, 347-378. Bojanovic J. J., Drazic A. M. and Vujovic Z. R. (1981) Non-histone nuclear proteins in active and involuted rat thymuses. Biochimie 63, 791-794.
Nonhistones of C. elegans Bonner J., Chalkley G. R., Dahmus M., Fambrough D., Fujimura F. Huang R-C. C., Huberman J., Jensen R., Marushige K., Ohlenbusch H., Olivera B. and Widholm J. (1968) Isolation and characterization of chromosomal nucleoproteins. In Methods in Enzymology Vol. XIIB (Edited by Colowick S. P. and Kaplan N. O.), pp. 3-65. Academic Press, New York. Bremer J. W., Busch H. and Yeoman U C. (1981) Evidence for a species of nuclear actin distinct from cytoplasmic and muscle actins. Biochemistry, N.Y. 20, 2013-2017. Brenner S. (1974) The genetics of Caenorhabditis elegans. Genetics 77, 71-94. Brunel C. and Lelay M.-N. (1979) Two-dimensional analysis of proteins associated with heterogeneous nuclear RNA in various animal cell lines. Eur. J. Biochem. 99, 273 283. Burkhardt A. L., Huang D. P. and Chiu J. F. (1984) Hepatoma-associated nonhistone chromosomal proteins. Biochim. biophys. Acta 781, 165-172. Capco D. G., Wan K. M. and Penman S. (1982) The nuclear matrix: three-dimensional architecture and protein composition. Cell 29, 847-858. Cartwright I. U, Abmayr S. M., Fleischmann G., Lowenhaupt K., Elgin S. C. R., Keene M. A. and Howard G. C. (1982) Chromatin structure and gene activity: the role of nonhistone chromosomal protiens. CRC Critical Rev. Biochem. 13, 1-86. Douvas A. S., Harrington C. A. and Bonner J. (1975) Major nonhistone proteins of rat liver chromatin: preliminary identification of myosin, actin, tubulin and tropomyosin. Proc. natn. Acad. Sci. U.S.A. 72, 3902-3906. Dwyer N. and Blobel G. (1976) A modified procedure for the isolation of a pore complex-lamina fraction from rat liver nuclei. J. Cell Biol. 70, 581-591. Einck L. and Bustin M. (1983) Inhibition of transcription in somatic cells by microinjection of antibodies to chromosomal proteins. Proc. natn. Acad. Sci. U.S.A. 80, 6735-6739. Fukui Y. and Katsumaru H. (1979) Nuclear actin bundles in Amoeba, Dictyostelium and human HeLa cells induced by dimethyl sulfoxide. Expl Cell Res. 120, 451-455. Kabisch R., Krause J. and Bautz E. K. F. (1982) Evolutionary changes in non-histone chromosomal proteins within the Drosophila melanogaster group revealed by monoclonal antibodies. Chromosoma 85, 531-538. Kanungo M. S. (1980) Biochemistry of Ageing. Academic Press, London. Karn J., Vidali G., Boffa L. C. and Allfrey V. G. (1977) Characterization of the non-histone nuclear proteins associated with rapidly labeled heterogeneous nuclear RNA. J. biol. Chem. 252, 7307-7322. Kilianska Z., Lipinska A., Krajewska W. M. and KlyszejkoStefanowicz L. (1982) Distribution of chromatin proteins between fractions of hamster liver chromatin differing in their susceptibility to micrococcal nuclease. Molec. Biol. Rep. g, 203-211. Krajewska W. M. and Klyszejko-Stefanowicz L. (1982) Immunospecificity of nonhistone chromatin proteins tightly bound to DNA from chicken thrombocytes and erythrocytes. Molec. Biol. Rep. 8, 199-202. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature, Lond. 227, 680-685. Lazarides E. and Lindberg V. (1974) Actin is the naturally
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