An analysis of the phenol-soluble nuclear proteins in the ciliate Stylonychia mytilus

~ell Differentiation, 6 (1977) 17--24 © Elsevier/North-H~!!andScientific PublishersLtd.

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AN~ANALYSIS OF THE ~PHENOL-SOLUBLE NUCLEAR PROTEINS IN T H E C I L I A T E S T Y L O N Y C t t l A M Y T I L U $

Hans Joachim LIPPS Institut i:"~r]3iologie IH, Abt. Zellbiologie, A u f der Morgenstelle 28, 74 T~bingen, Federal Republic of Germany Accepted 19 January~ 1977

Phenol~oluble nuclear proteins were compared in the vegetative macronucleus and the macronuclear anlagen of the ciliate Stylonychia rnytflus. Characterization by means of isoelectric focua~ing on polyacrylamide gels and amino acid analysis showed a high similarity to those of other organisms. Electrophoretic comparison on SDS-polyacrylamide gels revealed only quantitative differences in some protein fractions between the two nuclear types. In the macronuclei of conjugating cells one protein with a molecular weight of about 51.000 accumulated. It seems now certain that histones are involved in the preservation o f a highly ordered chromatin structure and as such they may act as nonspecific repressors Of gene activity in eucaryotic cells (Hewish et al., 1973; Olins et al., 1974; Komberg, 1974). Whereas the histones are very similar in most higher organisms (DeLm~ge e t a l . , 1975) chromosomal nonhistonc proteins represent a very heterogenous group of p r o ~ i n s (MacGillivray et al., 1972; Elgin et al~, 1973). It has been supposed that some o f these proteins are involved in the specific regulation of gene activity (Paul et al., 1975); more~ler, recent observations made it very likely that certain of these proteins differ in t~eir affinities to different sequences of DNA (A]lfrey et al.~ 1975). The nuclear dualism of cfliates provides several advantages for studying the function of nuclear proteins.. As in other ciliates,the hypotrichous ciliate Stylonychi~ mytilss contains a DNA.rich macronucleus which controls the metabolim~ of the cell and several generative micronuclei which are trans. criptionaly ~n~tive. During sexual reproduction (conjugation) the macronucleus LreaI~s down, the micronuclei undergo meiosis leading to haploid gametic nlele~ which are exchanged between the partner cells. A diploid syncaryon is ~het. formed in each cell. After mitotic division of the syncaryon one of ~he daughter nuclei differentiates into a new macronucleus, the other becor,~es micronuclei. In the prospective rnacronucleus (macronuclear anla~'e) v rapid increase in D N A content takes place and polytene giant chromo~or~s are formed (Ammerrnann~ 1968). These giant chromosomes disinter,rate and more than 9 0 % of the D N A gets lost from the nucleus. The arJage elongates and reaches its final D N A content by progres-

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sire doublings of the quantity of DI~A. As in the micronucleus, 1;here is little or no RNA synthesis in the macronuclear anlage. While the DNA of the micronuclei and the macronuclear anlage is enriched in repetitive sequences, these sequences are ~bsent in the vegetative macronucleus (Ammermann et al., 1974). :~ It seemed therefor,:~ of reasonable interest to analyze in this cell.some of the nonhistone prot~i~ns. In this report the pattern of macronuclear phenolsoluble proteins under different growth conditions is described and a comparison of these proteins between the vegetative macronucleus and the macronuclear anhge h~ the giant chromosome stage was made. MATERIALAND METHODS

Stylonychia mytiius, syngen I was grown in neugral Pringsheim medium (Ammermann et al., 1974). Logarithmic growth was achieved by feeding the ceils daily with the ~flgae Chlorogonium elongatum. Under these conditions cells divide twice per day. Nuclei from the stationary phase were obtained when feeding was stopped for 48 h before isolation of nuclei. In such cultures DNA and RNA synthesis are highly reduced and no divisions occur. Conjugation was achieved by mixing cells of different mating types. Usually up to 95% conjugat:~on was obtained. From these conjugating cells macronuclei were isolated about 5 h after pair formation. It has been suggested that at this time stable m R N A necessary for the further development are transcribed (Sapra oet al., 1973); very soon after that macronuclei break down. Isolation and pu]~fication of macronuclei was performed as described earlier ( A m m e n n a n n et al.,1974; Lipps et al.,1974; Lipps, 1975). Isolation of macronuclear anh~en in the giant chromosome stage was accomplished as described by Amme1.~nann et al. (1974). The quality of these preparations is shown in Figure 1. Fractionation of nuclear proteins followed the technique of Teng et al. (1971) as described by LeStourgeon et al. (1974). Following this procedure, in Stylonychia the ~'Lpproximate ratio of h~tones (acid-soluble proteins) to nonbJstone proteins was in the macronuclei 1 : 0 . 3 + 0.0~' ~ d in the macronuclear anlagen 1:{|.2 -+ 0.05. From these nonhistone proteins about 70% can be solubilized ir~ phenol. Proteins were prepared for SDS-polyacrylamide gel electrophoresis by dialysis in 0.01 M sodium phosphate buffer pH 7.2, 0.1% SDS, 0.14 M mercaptoethanol and 0.25 M sucrose. For isoelectric focussing on polyact~llamide gels the phenol phase was dialysed against 8 M urea. Protein concentrations were estimated according to the technique of Lowry et al. (1951) with bovine serum albumin as standard. SDS-polyacrylamide gel electrophosesis was performed using the system of Weber et al. (1969). Molecular weights were determined using rabbit aldolase (MW 147.000), bovine serum albumin (MW 67.000), chymotryp-

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Fig. 1. FeuJgen~tained preparations of 3tylonychia nuclei. × 170 a) Macronuclei. b) Macronucle;~r anlagen in the giant chromosome stage.

sinogen (MW 25.000) and myoglobin (MW 17.000) as standards. Gels were stained with 1% Coomassie Brilliant Blue i n 7% acetic acid and destained in the same solvent. Isoelect~c focussing was carried o u t on 5.5% acrylamide gels containing 5 M urea and 4% ampholyte pH 2--11 (Serva). Gels were stained as described by Vesterberg (1972). Stained gels were scanned with a J o y c e - Loebl Scan 400. For amino acid analysis, macronuclear phenol-soluble proteins were hydrolized in double distilled 6 N HCI for 18 h at 102°C. The hydrolysates were evaporated to dryness ~ d the residue dissolved in 0.2 N sodium citrate buffer pH 2.2. Aliquots were analyzed using the Unichrom amino acid analyzer (Beckmann Instruments, Mfinchen). RESULTS Macronuclear phenol-soluble proteins were characterized by means of araino acid analysis, isoelectric focussing and SDS-polyacrylamide gel electrophoresis. Table I gives the amino acid composition of these nuclear proteins. The most predominant amino acids are glutamic and aspartic acid, the ratio of acidic : basic amino acids is 1.75 ~ d only traces of cysteine and methionine are detectable in this preparation. Determination of the isoelectric points of macronuclear phenol-soluble proteins revealed isoelectric points between pH 8.5 and 3, but most of these proteins have isoelectric points between pH 5 and S (Fig. 2). On SDS-polyacry]arnide gels these proteins coul,t be resolved into at least 30 bands with molecular weight~ ranging from 13).000 to 20.000 daltons. Comparison of these pro~eins in the macronuclei of logarithmic growing

20 TABLE I Amino acid composition of m a c r o n u ~ a r phenol-solubl© proteinso~ S~ylonych~ mytilus (1 p/~ amino acid/100 g M totalamino ~de). Lysine Histidlne

8.52 1.11 5.81

Ar~inine

Aspic

12.45

Th~onine

5.41 10.31 13.73

Serine Glutarnic I~oline G|ycine Alanine Cysteine Valine Methionine ]soleucine

4.33 9.16 6.46

4.58 > 0.5

Leucine ~rosine

4.85 8.49 2.07

Phenylalanine

3.15

Acidic amL1o =cids : basic amino acids = 1.75

cultures (Fig. 8a), stationary cultures (Fig. 3b) and conjugating cells (Fig. 3c) indicated no significant differences in protein pattern of logarithmic growing cells and cells in the stationazy phase; however, in maczonuclei which have been obtained from conjugatL~g cells (see Mate~al and Methods) a fraction with molecular weight 51.000 dalt~ns was present in l a r g e r quantity. Very soon a~ter that the macronuclei break d o w n and all nuclear pro-

i 10

9

a

7

6 5 pH- gradient

Fig. 2. Densitometer tracings of macronuclea~ phenol~oiubP~ proteins separated on isoelectric f ocussed po] yacryl amid e - g e l~ . E ] e c ~ o p h o r c t i c v~eparation was performed o n 5.5% ac~lam]de, 5 M urea, 4%.a~pholyt~ pH 2~11 (8e~a) at a c o . r a n t v o l ~ e of 100 V for

14 h.

J t 10

1 5

2.5 × 104 Daltons

4

10

5

2 5 x 104 Dalton~

Fig. S. Densitometer tracings of phenol-soluble nuclear proteins separated ,on SDS-polyaerylamide gel~ of a) macronuelei from logarlthmlc growing cells, b) macronuclei from cells in the stationary phase, e) macronuelei from conjugating cells, d) maeronuelear anlagen in the giant chromosome stage.

reins are degraded. To rule o u t t h e possibility that this protein becomes more soluble in phenol by some modification {i.e. phosphorylation), the residual proteins n o t solubilized with phenol were extracted with h o t 3% SDS--0.14 M mercapt0ethanol. In this fraction, which consists mainly of high molecular weight proteins, no difference between macronuclei of logarithmic growing cultures and conjugating cells could be found. This observation makes it likely that this protein becomes synthesized and incorporated into the macronucleus during conjugat!ion. Since it has been previously reported that durivg the interaction of the plant lectin Concanavalin A with Sty$~nychh~ mytilus a protein with molecular weight 52.000 becoraes Lncorporated into l;he reacronucleus (Lipps et al., submitted for publication), a preparation of phenol-soluble reacronuclear proteins after 30 rain Con. A treatment was compared with the proteins of conjugating cells. Figures 4a and b show that the proteL~s which become incorporated into the reacronucleus after Con. A treatment and during conjugation are not identical.

a

/

-

~

~, Daltons

Fig. 4. Comparison of macronuclfar phenol-soluble proteins of conjugatin~ cells ~nd cells treated for 30 rain with 4/~g/ml Concanavalin A. Proteins were separatec o~ ~,DS~olyacrylamide gels for 10 h at a constant current of 8 mA per gel. Densitorner~r t~einp of a) macronuclei of Con A treated cells, b) macronuclei of conjugating cei~. Figure 3d shows the densitometer tracings of the phenol-solub.'e proteins of the maeronuclear anlage in the giant chromosome stage. The qualitative pattern o f these proteins is very shnilar to that of the vegetatiw ~ ~acronucleus, but a significant quantitativ~ increase in high molecular weight proteins c~..-,,be observed in the macronuclear anlage. DISCUSSION Since it has been suggested that n o n h ~ t o n e chromosomal proteins are involved in the specific regulation of gone activity in the eukaryotic chromosome (Dingman e t a l . , 1964; Frenst~r, 1965; Gilmour et al., 1969) a number of techniques for the isolation and fractionation of these proteins from different sources has been d e a c r i b ~ (Pate], 1975). In the course of these investigations a limited tissue specificity of these proteins was demonstrated (Elgin e t a l . , 1975) as well a~ alterations in protein pattern during gone activation (Allfrey et al., 1975).

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In this study °one fraction of chromatin bound nonhistone proteins w ~ characterized. Phenol-soluble proteins represent a well defined class ot nuclear proteins in many organisms (LeStourgeon et al., 1975). In Stylonyehia about 70% of nonhi~tone proteins can be solubflized in this solvent. Characterization by a~nino acid analysis and isoelectrie focussing revealed a high similarity to those in other organisms (LeStourgeon et al., 1975). Eleetrophoretie comparisc,n of maeronuclear phenol-soluble proteins of logarithhnic growing cells and cel~s in the stationary phase in which DNA and RNA synthes~ are h . ~ y ~educed did n o t show differences in protein pattem~ sugges~ng that qualitative or quantitative alterations in some of these proteins can not be related to an overall increase in DNA or RNA synthesis. The quantitative increase in a fraction with molecular weight 51.000 in conjugating cells is difficult to ex~Jain. Comparison of proteins solubiiized with h o t SDS suggest~ that this fraction becomes newly synthesized and incorporated into the maeronueleu~ during conjugation. Only vague speculations about it~ possible function are possible at present: it has been shown in 8tylonychia that after the :interaction with Con. A the incorporation oi some nonhistone proteins into the macronucleus is essential for the transcription of RNA nece,~sary for the regeneration of the ceils (Lipps et al., submitted for publication). In conjugating cells about 5 to 6 h after pair formatior~ lon:g life m R N A is transcribed (Sapra et al., 1973). This protein could also represent DNases or proteases necessary for the degeneration of the macronucleus which occurs ~ery soon after the appearance of this protein. It has been reported that certain of the nonhistone proteins vary in their affinities to difIe~.e,,t DNA sequences (Allfrey e~ al., 1975). It was therefore of interest to compare nonhistone proteins from vegetative maeronuelei and maeronuclear anlagen in the giant ehrome~ome stage which differ considerably in their content of repetitive seql,.enees (Ammermann et al., 1974). As shown in Figure 3d, on SDS-polyacrylmuide gels the qualitative pattern of phenol-soluble nuclear proteins is very similar in both nuclear types. This result does not exclude possible qualitative differences not detectable with the methods used here. Howeve:, significant quantitative differences could be observed in the proteins of these two nuclei: in the vegetative macronucleus the most predominant fractions are in the molecular weight range of 50.000 to 60.000 daltons but the .,r,acmnuci~ar an!vge is enriched in higher molecular weight fractions. The structure and function of these two nuclear types is very different: Whereas the macronuclear anlage contains banded polytene chromosomes and has no RNA synthetic acff.vity, the macronucleus is very active in RNA synthesis, contains hundreds of nucleoli and has probably fragmented chromosomes. It seems likely that some of these quantitative alteraticns can be related to some of these differences between the r~uclei. The resu!ts presented here are a first attempt and further work, including other fractionatlo~ procedures for nuclear proteins, will be necessary to correlate the pattern of chr,~matin bc md nonhistone proteins with the differentiation of the ciliate nuclei

24 ACKNOWLEDGMENT This work was supposed by the VW-foundation ,rod by the Deutsche Forsch~ngsgemeinschaf~. REFERENCES Allfrey, V.G., A L~ouc, J. Karn, E.M. Johnson, R.A. Good and J.W. Hadden: L~: The Structure and Function of Chromatin; Ciba Foundation Symposium 28 (Elsevier/ Excerpta Medica, North-Holland, Amsterdam) pp0 199--228 (1975). Arnmermann, D.: Chromosoma (Bed.) 25,197--120 (1968). Ammennann, D., G. Ste|nbriick, L. v. Berger and W. Hennig: Chromosoma (Berl.) 45, 401--429 (1974). DeLange, R.J. and E.L. Smith: In: The Structure and Function of Chromatin; Ciba Foundation Symposium 28 (Elsevier, Excerpta Medica, North-Holland, Amsterdam) pp, 59--.76 (1975) Dingman, C.W. and M.B. Sporn: J. Biol. Chem. 239, 3483L-3492 (1964). Elgin, S.C.R., J.B. Boyd, L.E, Hood, W. Wray and F.C. Wu: Cold Spring Harbor Syrup. Quant. Biol. 3 8 , 8 2 1 - 8 3 3 (1973). Elgin, S.C.R. and H. Weintraub: In: Annual Review in Biochemistly, ed. E.S. Shell (Annual Reviews Inc., Palo Alto) pp. 725--774 (1975). Frer~ter, J.H.: Nature 206, 680--683 (1965). 6 Gilmour, R.S. and J. Paul: J. Mol° Biol..~0, 137--139 (1969). Hewlsh, D.R. and L.A. Burgoyne: Biochem. Biophys. Res. Commun. 52, 504--510 (1973). Komberg, R.D.: Science 184,868--871 (1974). LeStourgeon, W.M. and W. Wray: In: Acidic Pro~iws of the Nucleus; eds. LL. Cameron and J.R. Jctcr (Academic Press, New York) pp. 59--102 (1974). Lip~, H.J.: Cell Differentiation 4,123--129 (1975). Lipid, H.J.,'G.R. Sapra and D. Ammerrnann: Chromo$oma (]~rl.) 45,273--280 (1974). Lowry, O.H., N.J. R ~ b r o u g h , A.L. Parr and R.J. Randall: J. Biol. Chem. 193,265--275 (1951). MacGilllvray, A.J., J. Paul and G. Threlfall: Adv. Cancer Res. 15, 93--162 (1972). Olins, A.L. and D.E. Olin: Science 183,330--332 (1974). Patel, G.L.: Xn. Acidic Fro~ir~ of the Nucleus; eds. I.L. Cameron and J.R. J e e r (Academic ~ , New York) PP. 3 ~ 7 (1974). Paul, J. and R.S. G~|mour: in: The Structure and Function of Chromattn; Ctba Four|dation S y m ~ u m 28 ~E~vier, ~x~rp~a Medica, North-Holland, A m s ~ r d ~ ) pp. IBI-198 (1975). Sapr~, G.R. and D. ~ c ~ a n n : E~p. Ce~l Res. 78, 168--174 (1973). Teng, C.T., C°~. T~ng and V.G. A]lfr~y: J. Biol. Chem. 2~6, 3597--3609 (1971). Ves~r~rg, Oo: ~i~ch|~. Biophy~. Ac~ 257, 11--19 (1972).