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The isolation and properties of nucleohistone from the fission yeast, Schizosaccharomyces pombe
BIOCHIMICA ET BIOPHYSICAL ACTA
627
BBA 96726
T H E I S O L A T I O N AND P R O P E R T I E S OF N U C L E O H I S T O N E FROM T H E F I S S I O N YEAST, SCHIZOSACCHAROMYCES POMBE J. H. DUFFUS*
Department of Zoology, University of Edinburgh, Edinburgh, EH 9 3JT (Great Britain) (Received September 8th, 197 o)
SUMMARY
I. Nucleohistone has been prepared from the fission yeast, Schizosaccharomyces
pombe b y p H titration and partially characterised. 2. The histone appears to be a protein of molecular weight about 4 ° ooo. 3. Electrophoresis and immunological assay suggest that the histone is identical with a ribosomal protein. 4. The synthesis of the histone during the cell cycle and at different stages of growth is discussed. INTRODUCTION
The fission yeast, Schizosaccharomyces pombe, has been extensively used for studies on the biochemistry of the cell cycle 1. The inducible enzymes, sucrase, acid phosphatase and alkaline phosphatase follow a linear pattern of synthesis through the cell cycle, with a doubling in rate at a 'critical point' about one fifth of the way through the cycle 2. The following model has been suggested to explain this observation. The 'critical point' m a y be the time at which the chromatids separate in the nucleus and become available for transcription. I t is also the time at which the action spectrum for ultraviolet light sensitivity of the cells shifts from a peak at 260 nm to a peak at 280 nm. This suggests that the chromatids acquire a protein backbone at this point. If this is so, one would expect a related burst in the synthesis of this protein, presumably a histone. However, there is only one report of the presence of histones in yeast 3. Thus it is necessary to establish their presence in S. pombe before any role in the cell cycle can be investigated. Histones have been defined as basic proteins present in the nucleus and associated with DNA at some stage in the cell cycle 4. Ribosomal proteins m a y be similar in m a n y respects and so, initially, isolated chromatin or nuclei must be used to investigate the presence or absence of histones. This paper describes the isolation of a histone fraction from S. pombe nuclei and its subsequent extraction from whole cells. Some of the properties of the fraction are described. Its relationship to ribosomal protein and variation through the cell cycle and with growth phases of the yeast are discussed. EXPERIMENTAL
Except for spore production, the strain of S. pombe used was No. 132 from the British National Collection of Yeast Cultures. It was grown at 32 ° in Edinburgh * Present adress: Department of Brewing and Biological Sciences, Heriot-Wart University, Edinburgh, E H I I H X (Great Britain).
Biochim. Biophys. Acta, 228 (1971) 627-635
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J.H. DUFFUS
minimal medium: either in its original form (EMMI) or supplemented with 30 times the original amount of phosphate (EMM2). Synchronous cultures were prepared by the method of MITCHISONAND VINCENT5 or by transfer of cells from cultures 3 days stationary in EMMI to fresh medium 6. Spores were obtained by mating strains 972h- and 975 h÷ of the yeast on EMM2 agar plates and leaving the plates at 32 ° until spores were formed. Spores and unchanged cells were then washed off the plates with distilled water and pelleted by centrifuging at 2000 × g for 3 min. The pellets were suspended in about 20 ml distilled water each and 20 ml liquid paraffin added ~. The mixture was shaken thoroughly and centrifuged at 2000 × g for 5 rain. The paraffin layer was separated with a Pasteur pipette, together with a small amount of the water layer to ensure that all the spores which collected at the interface were obtained. The detergent, Triton N-Ioo, was added to the paraffin layer in the proportion 1:50 and the layer was centrifuged at 200o × g for 15-2o min. A pellet of spores was obtained. The pellet was washed three times by suspension in distilled water before being analysed. Spores obtained in this way were shown to be viable by inoculation on EMM2 agar.
Preparation of histone ]rom nuclei Nuclei were isolated from S. pombe in exponential growth as previously described s. They were homogenised in a glass homogeniser with 5 ml of a KC1-HC1 buffer 9, ionic strength o.I, pH 2.8, and the suspension left at 4 ° for 20 min before being centrifuged at 15oo × g for 20 min at the same temperature. The supernatant was discarded and the pellet washed once by a brief resuspension in the 2.8 buffer. The washed pellet was homogenised further in 5 ml of an identical buffer at pH 2.1 and the extraction procedure repeated. After centrifuging, the supernatant was decanted and 9 volumes of cold acetone added to it. The mixture was left at --20 ° overnight for complete precipitation to take place. The resultant precipitate was pelleted in a bench centrifuge, washed three times with pure acetone, and dried under vacuum at room temperature. This constituted the crude histone preparation. Crude histone was purified by dissolving it in cold 0.02 M H2SO 4, dialysing the solution at 4 ° against 4 1 of the solvent, and finally precipitating the histone with 4 vol. of ethanol at --20 °. This was then repeated to give the preparation which will be referred to as pure histone. The method described is based on the technique developed by MURRAY:° for the fractionation of chicken erythrocyte histones.
Preparation o/histone ]rom whole cells S. pombe cells were harvested by filtration on Whatman No. 50 paper, transferred to centrifuge tubes in suspension in distilled wate:, and pelleted. To the pellet was added approx. 3 times its volume of pH 2.8, o.I M phosphate-citrate buffer:. This buffer was used because it was found that the KCI-HC1 buffer used above would not maintain its nominal pH in the subsequent homogenate. The cells were suspended in the buffer and the mixture put through an Eaton :e press. The homogenate obtained was centrifuged for IO min at speed IO in an MSE bench centrifuge at 4 °. The pellet was suspended in pH 2.1 KC1-HC1 buffer using a glass homogeniser and left for 20 rain at 4 °. The suspension was centrifuged and the supernatant decanted. The crude histone was precipitated from the supernatant as described above for nuclear extracts and similarly purified.
Biochim. Biophys. Acta, 228 (1971) 627-635
NUCLEOHISTONE FROM S. pombe
629
Samples from synchronous cultures were treated in the same way save that the cells were collected on Millipore filters, pore size 3/~m, frozen rapidly and kept in the deep freeze until extraction. The amount of histone was determined b y measurement of the absorbance of the p H 2.1 extract at 215 and 225 nm ~8'x4. DNA was assayed b y the method of BURTON15.
Preparation o/ribosomes Cells of S. pombe were homogenised in a solution containing 5o mM KC1, IO mM magnesium acetate, and IO mM Tris buffer, p H 7.4, b y passage through an EATON press. To the homogenate was added 0.03 ml 20 °/o Triton X - I o o per ml and it was spun at IOOOx g for IO min at o °. The supernatant from this was then spun at 25 ooo x g for IO rain at o ° and the resultant supernatant spun at lO5 ooo x g for 4 h at o ° after layering it on top of I ml of I M sucrose in the original homogenising medium. The pellet from this was resuspended in the homogenising medium (o.12 ml) and an aliquot containing about 200/,g applied to 5 ml of a IO to 34 % sucrose gradient. The gradient was centrifuged in a Spinco SW39 head in a Spinco Model L centrifuge at 37 ooo rev./min for 9 ° rain at 4 °. This separates the ribosomes from 4o-S and 6o-S subunits and from polysomes. The b o t t o m of the tube was pierced with a needle and the fractions collected dropwise. The ribosomal fraction was used directly as ribosomal protein in the immunological assay. (W. H. WAIN, personal communication).
Analytical procedures Polyacrylamide gel electrophoresis was carried out b y the method of JOHNS16 for histones and b y the method of SHAPIRO et al. 17 using sodium dodecyl sulphate for the determination of molecular weight. Amino acid analyses were performed on a Beckman Auto-Analyser using samples hydrolysed in 6 M HC1 for 24 h at lO5 °. Radioactivity was determined by scintillation counting in a Packard Tricarb liquid scintillation spectrometer. Each sample (o.I ml) was mixed with io ml of a solution of 5 g/1 CIBA scintillator butyl-I-phenyl-3,4-biphenylyloxadiazole in a mixture of A.R. toluene and 2-methoxyethanol (6:4, b y vol.). 14C was counted with an efficiency of 9 ° °/o and 85S with an efficiency of 7 ° °/o. Imnmnoelectrophoresis in agar followed the method described b y GRABARTM and samples were also analysed immunologically b y double diffusion in agar 19. To prepare antibody, one Half-lop rabbit was immunised b y subcutaneous injection of 5 mg of protein, partially dissolved in normal saline with Io l° Bacillus pertussis (Wellcome Vaccine) incorporated in Freund's complete adjuvant (Difco) and bled after 5 weeks. Phosphate content of the protein was measured b y the method of AMES2°.
RESULTS
The lowest p H at which appreciable amounts of protein are obtained from nuclei or whole cells b y the p H titration technique is 2.1. The protein obtained at this p H gives predominantly one band on electrophoresis b y the method of Jonr~s 16 (Fig. I) if the period in acid solution is kept to a minimum. Otherwise a variable
Biochim. Biophys. Acta, 228 (1971) 627-635
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j.H. DUFFUS
Fig. I. A c r y l a m i d e gel e l e c t r o p h e r o g r a m of (a) p H 2.8 f r a c t i o n from S. pombe, (b) calf t h y m u s h i s t o n e a n d (c) p H 2.1 f r a c t i o n f r o m S. pombe.
number of bands is obtained and this is the case too if the protein is further purified by reprecipitation from 0.02 M H~S04. However, the pure protein gives one main band on sodium dodecyl sulphate acrylamide gel electrophoresis with only one minor component. The main band corresponds to a protein of molecular weight 40 ooo, TABLE I AMINO
ACID
S. cerevisiae
COMPOSITION
OF
S. pombe
HISTONE
COMPARED
WITH
S. pombe
TOTAL
PROTEIN
AND
HISTONE 3
A m o u n t s of a m i n o acids are e x p r e s s e d as m o l e s / i o o of t o t a l r e c o v e r e d a m i n o acids; no correct i o n s are a p p l i e d for h y d r o l y t i c losses of a n y of t h e a m i n o acids.
Amino acid
S. pombe histone (crude)
S. pombe histone (pure)
S. pombe protein (total)
S. cerevisiae histone
I . ys His Arg Asp Thr Ser Glu Pro Gly Ala Cy~ Val Met I le Leu Tvr Piae Trp Lys/Arg
7.6 1.8 5.4 lO. 4 6.6 8.8 13. 5 4.4 8. 7 8. 5 0.8 4.7 1.6 4-9 6.2 2. 7 3.1 o. 4 1.4
7.4 2.2 5 .2 Io. 4 8. 7 lO. 5 11. 4 4.4 7.0 8. 9 o.o 5.5 1. 5 3.7 6. 4 3.0
8.2 2. 4 4.4 9.9 5.8 6.8 12.3 4 .1 7.9 8.9 2.1 6.2 i .7 5.1 7.2 3. I
lO.8 2.1 7.4 7.4 5-9 8. 4 lO.3 4 .1 7.4 IO.i o.o 5.7 0.6 5.8 8.5 2. 7 2.6 0. 5 1.5
3.3
3.4
0. 7 1.4
0. 5 1.9
Biochim. Biophys. Acta, 228 (1971) 627-635
NUCLEOHISTONE FROM S.
pombe
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which is also the minimum molecular weight calculated from the amino acid composition (Table I). A protein with the same electrophoretic properties is present in the pH 2.8 extract of whole cells. This extract contains largely ribosomal protein. The pH 2.8 extract of washed nuclei does not contain a similar protein. Immunoelectrophoresis also indicates that the purified pH 2.1 extractable protein consists of a major and a minor component. Both components have similar electrophoretic properties. Immunological analysis by double diffusion in agar gives the same results (Fig. 2). If the protein is compared with ribosomal protein, either in a pH 2.8 extract of whole cells or in purified ribosomes, it appears that the same protein is present in the ribosomes. Only the minor component is found in pH 2.8
Fig. 2. I m m u n o l o g i c a l a n a l y s i s b y double diffusion in a g a r of S. pombe h i s t o n e a n d p H 2.8 ext r a c t s of nuclei a n d whole cells. (a) p H 2.1 e x t r a c t of whole cells. (b) p H 2.1 e x t r a c t of whole cells after reprecipitation. (c) p H 2.1 e x t r a c t of 3 d a y E M M I s t a t i o n a r y cells. (d) p H 2.8 e x t r a c t of nuclei. (e) p H 2.8 e x t r a c t of whole cells (ribosomal protein).
T A B L E II RATIO
OF
VARIOUS
CRUDE GROWTH
pH
2.1
EXTRACTABLE
PROT]EIN
TO
DNA
IN WHOLE
CELLS
AND
NUCLEI
UNDER
CONDITIONS
Each result is an average quoted with the standard deviation. The number of experiments is given in parentheses.
Medium
E M M I a n d EMM2 EMM2 EMMI
Growth conditions
Exponential growth 3 days stationary 3 days stationary"
Ratio o[ protein to D N A Cells
Nu61ei
7 : I~.~_2(II) 9 : 1 4- 3 (3) i o o : 1-4-1o(3 )
6 : I±1(3 ) -20 : 14-3(3)
I n t h e s e cells t h e r e is o n l y o.o155 p g D N A / c e l l c o m p a r e d to o.o399 pg/cell in t h e o t h e r s s.
Biochim. Biophys. Acta, 228 (1971) 627-635
632
j.H. DUFFUS -$ 16 14 .°= "s 12 ~.. 1C
o ..~ 300
~
•-
x
20o
¢
1(3(2
P
~. ~ 16: •o~ g 3 1 2
I
=E~ 8 -~
0
7- E I O 0
~_ ~
200
%
C.P.
~
;
4
5
6
-;
Time (h)
Time (rain)
Fig. 3. Bulk change in histone in a synchronous culture prepared by the method of MITCHISON AND V I N C E N T s.
Fig. 4. Bulk change in histone and incorporation of [14C!amino acids following inoculation of 3day-old stationary phase cells of S. pombe from EMMI into fresh EM1Vf2 at 320 containing 50 #C of 14C-labelled protein hydrolysate per 1. C.P. = cell plate peak.
64 ~E
o~ c O
40
-'~
32
-~.
24
~ o
16 8 0
4o o 71 32 ~S 24 t
f
_
Time (rain)
Fig. 5- Bulk change in histone and incorporation of 8sS from 35SO4~- following inoculation of 3-day-old stationary phase cells of S. pombe from EMMI into fresh EMM2 at 32°. The fresh medium contained 2oo/zC 8sSO42- per 1. C.P. = cell plate peak.
Biochim. Biophys. Acta, 228 (1971) 627-635
NUCLEOHISTONE FROM S.
pombe
633
extracts of washed nuclei. The major component is absent from these extracts. Calf thymus histone does not react with antibody to the purified protein. Considering the crude fraction, the ratio of protein to DNA by weight is about 7:1 in preparations from both cells and nuclei but one reprecipitation reduces this to approx. I : I (Table II). Extensive dialysis of the crude extract against water for 24 h at 4 ° results in considerable precipitation of protein and again the protein left in solution shows a nearly I : I ratio to the DNA in the original cells. These results apply to cells in exponential growth and early stationary phase in EMM2. It should be noted that the analytical results apply only to the soluble protein except in the case of the amino acid analysis of the crude preparation. The amino acid analysis (Table I) shows that the purified material is appreciably different from the crude preparation. The content of threonine and serine is clearly higher while that of glutamic acid, glycine and isoleucine has dropped. Cysteine, present in the crude material, is completely absent after purification. The pure protein differs from total cell protein in having more serine and threonine, less isoleucine and no cysteine. N-terminal analysis shows that the N-terminal amino group of the pure protein is not free and hence may well be acetylated or formylated. The protein is not phosphorylated either in the crude preparation or in the purified form. The change in total amount of the pH 2.I extractable protein during the normal cell cycle shown in Fig. 3. It doubles in quantity at approximately the time of cell division. In cultures synchronised by growth into stationary phase in the phosphate deficient medium, EMMI, a different picture is obtained (Figs. 4 and 5)- The crude pH 2.1 extractable protein from EMMI three day stationary cells is 7 times more by weight per cell than from exponentially growing cells in EMMI or EMM2 and considerably enriched in the major component (Fig. 2). On inoculation of such cells into fresh medium it rapidly drops to the exponential phase value within one hour or less. In fact, a considerable drop occurs during the resuspension of the cells even before the first 'zero time' sample is taken. Following the intial drop, the total amount of pH 2.1 extractable protein remains constant till cell division takes place when it increases. Total incorporation of 35S from sulphate and 14C from a 14C-labelled protein hydrolysate is not appreciable for two hours but is virtually linear up to 6 h once it has begun. At 6 h there is a 3-fold increase in incorporation of 85S. Incorporation of the two labels into the pH 2.1 fraction does not occur till 3 h after inoculation. It levels off about an hour before the cell plate peak and then resumes immediately following it. The great increase in amount of the pH 2.1 protein in EMMI three day stationary cells is not reflected in the amount present in nuclei from such cells (Table II). Hence the extra protein must be cytoplasmic in origin. Further the solubility of the protein from these cells is different from that of EMM2 cells to the extent that the amount obtainable after one reprecipitation is nearly half of the crude total. Incorporation of 35S shows that there is a very slow turnover of the pH 2.1 fraction in stationary phase. A pH 2.1 extractable protein was also detected in spores of S. pombe. Unfortunately the amount of material available was insufficient to characterise it further.
Biochim. Biophys. Acta, 228 (i97 I) 627-635
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J.H.
DUFFUS
DISCUSSION
There is no doubt that S. pombe contains none of the well characterised histones isolated from calf thymus and elsewhere. Comparison of amino acid contents ~ of histone fractions shows this clearly, confirming the immunological assay. The p H 2.1 extractable protein is also different from the histone fractions obtained b y TONINO AND ROZlJN 3 from Saccharomyces cerevisiae (Table I). It has less arginine and lysine, more aspartic acid and more serine and threonine. The most striking similarity is the absence of cysteine but, b y comparison with other organisms, both these preparations have in common low contents of basic amino acids for presumed histone fractions. The differences m a y be due to the method of fractionation but attempts to obtain chromatin from S. pombe b y the method of TONINO AND R o z u ~ 3 were not successful. The prime reasons for suggesting that the p H 2.I extractable protein represents a histone according to MURRAY's4 definition are as follows. Firstly its extraction properties. All cytoplasmic basic proteins are reported to be extractable at p H 2.8 but no histones are soluble at this p H 1°,23. The only nucleoprotein so far reported to be extracted at p H 2.1 following thorough extraction at p H 2.8 is F I histone 1°. However, it must be noted that the results above indicate that under extreme conditions (phosphate deficiency) at least some cytoplasmic basic protein comes out in the pH 2.1 fraction. Secondly, there is the constant ratio by weight of the p H 2.1 extractable protein to DNA whether extracted from a nuclear preparation or from whole cells under conditions of normal growth. This at least indicates that it is a component of the nucleus and if we consider also the fact that it doubles in quantity in the normal cell cycle at about the time of cell division, immediately following DNA synthesis 6, the correlation with DNA becomes quite strong. It m a y be noted that the time of doubling does not correspond to the 'critical point' of MITCHISON AND CREANOR 2.
Accepting that the p H 2. I extractable protein is a histone, its identity with at least part of the ribosomal protein indicated by electrophoresis and immunological analysis becomes of considerable interest. It m a y be suggested that this is the primitive condition in eucaryotes. The accumulation of p H 2.1 extractable protein in cells grown into stationary phase in EMMI appears to reflect a change in properties of some cytoplasmic component, presumably a ribosomal protein. This m a y correlate with the drop in total cell RNA from 3.7 to 0.66 pg under the same conditions 2~. The rapid decrease in the p H 2.1 fraction following inoculation into fresh medium could reflect the almost immediate resumption of production of ribosomes and RNA. Perhaps tlle newly synthesised ribosomes under these conditions make use of the pre-existing protein. With regard to the synthesis of the p H 2.1 fraction in cultures of EMMI stationary phase cells inoculated into fresh medium, incorporation of 35S and 14C indicates a short burst between 3 and 4.5 h after inoculation followed by another after the cell plate peak at 5.5 h. The amount synthesised during the first burst must represent turnover since it is not reflected in the total extracted. The beginning of the first burst is about 2.5 h before the cell plate peak. Since the normal time for a cell cycle under these conditions is also about 2.5 h (ref. I) it m a y be that the first burst coincides with the resumption of normality within the cells. The second burst
Biochim. Biophys. Acta, 228 (1971) 627-635
NUCLEOHISTONE FROM S.
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is reflected, with a short delay, in the increase in the total amount of the pH 2.1 fraction. As in cells growing normally in EMM2, this correlates with cell division and follows DNA synthesis 6. Finally, if the pH 2.1 extractable protein is a histone and if it is also a component of the ribosomes, the concept of histone synthesis requires re-examination. It is likely that ribosomal protein is synthesised throughout the cell cycle. If this is so, the apparent step in 'histone' synthesis merely reflects a redistribution of the protein, more being taken up by the nucleus and binding to the newly synthesised DNA. This possibility will require to be carefully studied. ACKNOWLEDGMENTS
I thank Professor J. M. Mitchison for all his help and encouragement and Mr. C. J. Mitchell and Miss J. I. Clancy for their excellent technical assistance. My thanks to Mr. W. H. Wain for the pure ribosomes used in the immunological assays. I am grateful to Dr. K. Murray, Dr. H. J. Cruft and Mr. P. Greenaway for their advice about histones and to Dr. R. P. Ambler and Mr. P. Greenaway for the amino-acid analyses. The immunological assays were kindly carried out by Dr. H. S. Micklem and Mrs. J. Riddaway. This work was supported by an S.R.C. Grant. Fig. I was photographed by Mr. D. Cremer. REFERENCES I J. M. MITCHISON, in D. M. PRESCOTT, Methods in Cell Physiology, Vol. 4, Academic Press, N e w York, 197 o, p. I3I. 2 J. M. MITCHISON AND J. CREANOR, J. Cell Sci., 5 (1969) 3733 G. J. M. TONINO AND TH. H. ROZlJN, Biochim. Biophys. Acta, 124 (i966) 427 . 4 K. MURRAY, in J. BONNER AND P. T s ' o , The Nucleohistones, H o l d e n - D a y , S a n Francisco, 1964, p. 15. 5 J. M. MITCHISON AND W . S. VINCENT, Nature, 205 (1965) 987 . 6 C. J. BOSTOCK, Exptl. Cell Res., 6o (197 o) 16. 7 C. C. EMEIS AND H. GUTZ, Z. Natur[orsch., 15b (1958) 702. 8 J. H. DUFFUS, Biochim. Biophys. Acta, 195 (1969) 23 o. 9 V. E. BOWER AND R. Cx. BATES, J. Res. Natl. Bur. Std., Set..4, 55 (1955) 197. io K. NfORRAY, C-. VIDALI AND J. M. NEELIN, Biochem. J., lO 7 (1968) 207. t i T. C. MCILVAINE, J. Biol. Chem., 49 (1921) 183. 12 ]N. R. EATON, J. Bacteriol., 83 (1962) 1359. 13 W. J. WADDELL, .]. Lab. Clin. Med., 48 (1956) 311. 14 J. ]3. MURPHY AND W. M. KIES, Biochim. Biophys. Acta, 45 (196o) 382. 15 K. BURTON, Biochem. J., 62 (1956) 315 . 16 E. W . JOHNS, Biochem. J., lO 4 (1967) 78. 17 A. L. SHAPIRO, E. V1NUELA AND J. V. MAIZEL, Biochem. Biophys. Res. Commun., 28 (1967) 815 . 18 P. GRABAR, in P. GRABAR AND P. ]3URTIN, Immuno-electrophoretic Analysis, Elsevier, A m s t e r d a m , 1964, p. 3. 19 O. OUCHTERLONY, Progr. Allergy, 6 (t962) 3 o. 20 B. N. AMES, in S. P. COLOWICK AND N. O. I~APLAN, Methods in Enzymology, Vol. 8., A c a d e m i c Press, N e w York, 1966, p. 115. 2i K. MURRAY, in J. BONNER AND P. Ts'O, The Nucleohistones, H o l d e n - D a y , S a n Francisco, 1964, p. 21. 22 K. MURRAY, J. Mol. Biol., 15 (1966) 409. 23 N. STEBBING, P h . D. Thesis, U n i v e r s i t y of E d i n b u r g h , 1969.
Biochim. Biophys. Acta, 228 (1971) 627-635
627
BBA 96726
T H E I S O L A T I O N AND P R O P E R T I E S OF N U C L E O H I S T O N E FROM T H E F I S S I O N YEAST, SCHIZOSACCHAROMYCES POMBE J. H. DUFFUS*
Department of Zoology, University of Edinburgh, Edinburgh, EH 9 3JT (Great Britain) (Received September 8th, 197 o)
SUMMARY
I. Nucleohistone has been prepared from the fission yeast, Schizosaccharomyces
pombe b y p H titration and partially characterised. 2. The histone appears to be a protein of molecular weight about 4 ° ooo. 3. Electrophoresis and immunological assay suggest that the histone is identical with a ribosomal protein. 4. The synthesis of the histone during the cell cycle and at different stages of growth is discussed. INTRODUCTION
The fission yeast, Schizosaccharomyces pombe, has been extensively used for studies on the biochemistry of the cell cycle 1. The inducible enzymes, sucrase, acid phosphatase and alkaline phosphatase follow a linear pattern of synthesis through the cell cycle, with a doubling in rate at a 'critical point' about one fifth of the way through the cycle 2. The following model has been suggested to explain this observation. The 'critical point' m a y be the time at which the chromatids separate in the nucleus and become available for transcription. I t is also the time at which the action spectrum for ultraviolet light sensitivity of the cells shifts from a peak at 260 nm to a peak at 280 nm. This suggests that the chromatids acquire a protein backbone at this point. If this is so, one would expect a related burst in the synthesis of this protein, presumably a histone. However, there is only one report of the presence of histones in yeast 3. Thus it is necessary to establish their presence in S. pombe before any role in the cell cycle can be investigated. Histones have been defined as basic proteins present in the nucleus and associated with DNA at some stage in the cell cycle 4. Ribosomal proteins m a y be similar in m a n y respects and so, initially, isolated chromatin or nuclei must be used to investigate the presence or absence of histones. This paper describes the isolation of a histone fraction from S. pombe nuclei and its subsequent extraction from whole cells. Some of the properties of the fraction are described. Its relationship to ribosomal protein and variation through the cell cycle and with growth phases of the yeast are discussed. EXPERIMENTAL
Except for spore production, the strain of S. pombe used was No. 132 from the British National Collection of Yeast Cultures. It was grown at 32 ° in Edinburgh * Present adress: Department of Brewing and Biological Sciences, Heriot-Wart University, Edinburgh, E H I I H X (Great Britain).
Biochim. Biophys. Acta, 228 (1971) 627-635
628
J.H. DUFFUS
minimal medium: either in its original form (EMMI) or supplemented with 30 times the original amount of phosphate (EMM2). Synchronous cultures were prepared by the method of MITCHISONAND VINCENT5 or by transfer of cells from cultures 3 days stationary in EMMI to fresh medium 6. Spores were obtained by mating strains 972h- and 975 h÷ of the yeast on EMM2 agar plates and leaving the plates at 32 ° until spores were formed. Spores and unchanged cells were then washed off the plates with distilled water and pelleted by centrifuging at 2000 × g for 3 min. The pellets were suspended in about 20 ml distilled water each and 20 ml liquid paraffin added ~. The mixture was shaken thoroughly and centrifuged at 2000 × g for 5 rain. The paraffin layer was separated with a Pasteur pipette, together with a small amount of the water layer to ensure that all the spores which collected at the interface were obtained. The detergent, Triton N-Ioo, was added to the paraffin layer in the proportion 1:50 and the layer was centrifuged at 200o × g for 15-2o min. A pellet of spores was obtained. The pellet was washed three times by suspension in distilled water before being analysed. Spores obtained in this way were shown to be viable by inoculation on EMM2 agar.
Preparation of histone ]rom nuclei Nuclei were isolated from S. pombe in exponential growth as previously described s. They were homogenised in a glass homogeniser with 5 ml of a KC1-HC1 buffer 9, ionic strength o.I, pH 2.8, and the suspension left at 4 ° for 20 min before being centrifuged at 15oo × g for 20 min at the same temperature. The supernatant was discarded and the pellet washed once by a brief resuspension in the 2.8 buffer. The washed pellet was homogenised further in 5 ml of an identical buffer at pH 2.1 and the extraction procedure repeated. After centrifuging, the supernatant was decanted and 9 volumes of cold acetone added to it. The mixture was left at --20 ° overnight for complete precipitation to take place. The resultant precipitate was pelleted in a bench centrifuge, washed three times with pure acetone, and dried under vacuum at room temperature. This constituted the crude histone preparation. Crude histone was purified by dissolving it in cold 0.02 M H2SO 4, dialysing the solution at 4 ° against 4 1 of the solvent, and finally precipitating the histone with 4 vol. of ethanol at --20 °. This was then repeated to give the preparation which will be referred to as pure histone. The method described is based on the technique developed by MURRAY:° for the fractionation of chicken erythrocyte histones.
Preparation o/histone ]rom whole cells S. pombe cells were harvested by filtration on Whatman No. 50 paper, transferred to centrifuge tubes in suspension in distilled wate:, and pelleted. To the pellet was added approx. 3 times its volume of pH 2.8, o.I M phosphate-citrate buffer:. This buffer was used because it was found that the KCI-HC1 buffer used above would not maintain its nominal pH in the subsequent homogenate. The cells were suspended in the buffer and the mixture put through an Eaton :e press. The homogenate obtained was centrifuged for IO min at speed IO in an MSE bench centrifuge at 4 °. The pellet was suspended in pH 2.1 KC1-HC1 buffer using a glass homogeniser and left for 20 rain at 4 °. The suspension was centrifuged and the supernatant decanted. The crude histone was precipitated from the supernatant as described above for nuclear extracts and similarly purified.
Biochim. Biophys. Acta, 228 (1971) 627-635
NUCLEOHISTONE FROM S. pombe
629
Samples from synchronous cultures were treated in the same way save that the cells were collected on Millipore filters, pore size 3/~m, frozen rapidly and kept in the deep freeze until extraction. The amount of histone was determined b y measurement of the absorbance of the p H 2.1 extract at 215 and 225 nm ~8'x4. DNA was assayed b y the method of BURTON15.
Preparation o/ribosomes Cells of S. pombe were homogenised in a solution containing 5o mM KC1, IO mM magnesium acetate, and IO mM Tris buffer, p H 7.4, b y passage through an EATON press. To the homogenate was added 0.03 ml 20 °/o Triton X - I o o per ml and it was spun at IOOOx g for IO min at o °. The supernatant from this was then spun at 25 ooo x g for IO rain at o ° and the resultant supernatant spun at lO5 ooo x g for 4 h at o ° after layering it on top of I ml of I M sucrose in the original homogenising medium. The pellet from this was resuspended in the homogenising medium (o.12 ml) and an aliquot containing about 200/,g applied to 5 ml of a IO to 34 % sucrose gradient. The gradient was centrifuged in a Spinco SW39 head in a Spinco Model L centrifuge at 37 ooo rev./min for 9 ° rain at 4 °. This separates the ribosomes from 4o-S and 6o-S subunits and from polysomes. The b o t t o m of the tube was pierced with a needle and the fractions collected dropwise. The ribosomal fraction was used directly as ribosomal protein in the immunological assay. (W. H. WAIN, personal communication).
Analytical procedures Polyacrylamide gel electrophoresis was carried out b y the method of JOHNS16 for histones and b y the method of SHAPIRO et al. 17 using sodium dodecyl sulphate for the determination of molecular weight. Amino acid analyses were performed on a Beckman Auto-Analyser using samples hydrolysed in 6 M HC1 for 24 h at lO5 °. Radioactivity was determined by scintillation counting in a Packard Tricarb liquid scintillation spectrometer. Each sample (o.I ml) was mixed with io ml of a solution of 5 g/1 CIBA scintillator butyl-I-phenyl-3,4-biphenylyloxadiazole in a mixture of A.R. toluene and 2-methoxyethanol (6:4, b y vol.). 14C was counted with an efficiency of 9 ° °/o and 85S with an efficiency of 7 ° °/o. Imnmnoelectrophoresis in agar followed the method described b y GRABARTM and samples were also analysed immunologically b y double diffusion in agar 19. To prepare antibody, one Half-lop rabbit was immunised b y subcutaneous injection of 5 mg of protein, partially dissolved in normal saline with Io l° Bacillus pertussis (Wellcome Vaccine) incorporated in Freund's complete adjuvant (Difco) and bled after 5 weeks. Phosphate content of the protein was measured b y the method of AMES2°.
RESULTS
The lowest p H at which appreciable amounts of protein are obtained from nuclei or whole cells b y the p H titration technique is 2.1. The protein obtained at this p H gives predominantly one band on electrophoresis b y the method of Jonr~s 16 (Fig. I) if the period in acid solution is kept to a minimum. Otherwise a variable
Biochim. Biophys. Acta, 228 (1971) 627-635
630
j.H. DUFFUS
Fig. I. A c r y l a m i d e gel e l e c t r o p h e r o g r a m of (a) p H 2.8 f r a c t i o n from S. pombe, (b) calf t h y m u s h i s t o n e a n d (c) p H 2.1 f r a c t i o n f r o m S. pombe.
number of bands is obtained and this is the case too if the protein is further purified by reprecipitation from 0.02 M H~S04. However, the pure protein gives one main band on sodium dodecyl sulphate acrylamide gel electrophoresis with only one minor component. The main band corresponds to a protein of molecular weight 40 ooo, TABLE I AMINO
ACID
S. cerevisiae
COMPOSITION
OF
S. pombe
HISTONE
COMPARED
WITH
S. pombe
TOTAL
PROTEIN
AND
HISTONE 3
A m o u n t s of a m i n o acids are e x p r e s s e d as m o l e s / i o o of t o t a l r e c o v e r e d a m i n o acids; no correct i o n s are a p p l i e d for h y d r o l y t i c losses of a n y of t h e a m i n o acids.
Amino acid
S. pombe histone (crude)
S. pombe histone (pure)
S. pombe protein (total)
S. cerevisiae histone
I . ys His Arg Asp Thr Ser Glu Pro Gly Ala Cy~ Val Met I le Leu Tvr Piae Trp Lys/Arg
7.6 1.8 5.4 lO. 4 6.6 8.8 13. 5 4.4 8. 7 8. 5 0.8 4.7 1.6 4-9 6.2 2. 7 3.1 o. 4 1.4
7.4 2.2 5 .2 Io. 4 8. 7 lO. 5 11. 4 4.4 7.0 8. 9 o.o 5.5 1. 5 3.7 6. 4 3.0
8.2 2. 4 4.4 9.9 5.8 6.8 12.3 4 .1 7.9 8.9 2.1 6.2 i .7 5.1 7.2 3. I
lO.8 2.1 7.4 7.4 5-9 8. 4 lO.3 4 .1 7.4 IO.i o.o 5.7 0.6 5.8 8.5 2. 7 2.6 0. 5 1.5
3.3
3.4
0. 7 1.4
0. 5 1.9
Biochim. Biophys. Acta, 228 (1971) 627-635
NUCLEOHISTONE FROM S.
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which is also the minimum molecular weight calculated from the amino acid composition (Table I). A protein with the same electrophoretic properties is present in the pH 2.8 extract of whole cells. This extract contains largely ribosomal protein. The pH 2.8 extract of washed nuclei does not contain a similar protein. Immunoelectrophoresis also indicates that the purified pH 2.1 extractable protein consists of a major and a minor component. Both components have similar electrophoretic properties. Immunological analysis by double diffusion in agar gives the same results (Fig. 2). If the protein is compared with ribosomal protein, either in a pH 2.8 extract of whole cells or in purified ribosomes, it appears that the same protein is present in the ribosomes. Only the minor component is found in pH 2.8
Fig. 2. I m m u n o l o g i c a l a n a l y s i s b y double diffusion in a g a r of S. pombe h i s t o n e a n d p H 2.8 ext r a c t s of nuclei a n d whole cells. (a) p H 2.1 e x t r a c t of whole cells. (b) p H 2.1 e x t r a c t of whole cells after reprecipitation. (c) p H 2.1 e x t r a c t of 3 d a y E M M I s t a t i o n a r y cells. (d) p H 2.8 e x t r a c t of nuclei. (e) p H 2.8 e x t r a c t of whole cells (ribosomal protein).
T A B L E II RATIO
OF
VARIOUS
CRUDE GROWTH
pH
2.1
EXTRACTABLE
PROT]EIN
TO
DNA
IN WHOLE
CELLS
AND
NUCLEI
UNDER
CONDITIONS
Each result is an average quoted with the standard deviation. The number of experiments is given in parentheses.
Medium
E M M I a n d EMM2 EMM2 EMMI
Growth conditions
Exponential growth 3 days stationary 3 days stationary"
Ratio o[ protein to D N A Cells
Nu61ei
7 : I~.~_2(II) 9 : 1 4- 3 (3) i o o : 1-4-1o(3 )
6 : I±1(3 ) -20 : 14-3(3)
I n t h e s e cells t h e r e is o n l y o.o155 p g D N A / c e l l c o m p a r e d to o.o399 pg/cell in t h e o t h e r s s.
Biochim. Biophys. Acta, 228 (1971) 627-635
632
j.H. DUFFUS -$ 16 14 .°= "s 12 ~.. 1C
o ..~ 300
~
•-
x
20o
¢
1(3(2
P
~. ~ 16: •o~ g 3 1 2
I
=E~ 8 -~
0
7- E I O 0
~_ ~
200
%
C.P.
~
;
4
5
6
-;
Time (h)
Time (rain)
Fig. 3. Bulk change in histone in a synchronous culture prepared by the method of MITCHISON AND V I N C E N T s.
Fig. 4. Bulk change in histone and incorporation of [14C!amino acids following inoculation of 3day-old stationary phase cells of S. pombe from EMMI into fresh EM1Vf2 at 320 containing 50 #C of 14C-labelled protein hydrolysate per 1. C.P. = cell plate peak.
64 ~E
o~ c O
40
-'~
32
-~.
24
~ o
16 8 0
4o o 71 32 ~S 24 t
f
_
Time (rain)
Fig. 5- Bulk change in histone and incorporation of 8sS from 35SO4~- following inoculation of 3-day-old stationary phase cells of S. pombe from EMMI into fresh EMM2 at 32°. The fresh medium contained 2oo/zC 8sSO42- per 1. C.P. = cell plate peak.
Biochim. Biophys. Acta, 228 (1971) 627-635
NUCLEOHISTONE FROM S.
pombe
633
extracts of washed nuclei. The major component is absent from these extracts. Calf thymus histone does not react with antibody to the purified protein. Considering the crude fraction, the ratio of protein to DNA by weight is about 7:1 in preparations from both cells and nuclei but one reprecipitation reduces this to approx. I : I (Table II). Extensive dialysis of the crude extract against water for 24 h at 4 ° results in considerable precipitation of protein and again the protein left in solution shows a nearly I : I ratio to the DNA in the original cells. These results apply to cells in exponential growth and early stationary phase in EMM2. It should be noted that the analytical results apply only to the soluble protein except in the case of the amino acid analysis of the crude preparation. The amino acid analysis (Table I) shows that the purified material is appreciably different from the crude preparation. The content of threonine and serine is clearly higher while that of glutamic acid, glycine and isoleucine has dropped. Cysteine, present in the crude material, is completely absent after purification. The pure protein differs from total cell protein in having more serine and threonine, less isoleucine and no cysteine. N-terminal analysis shows that the N-terminal amino group of the pure protein is not free and hence may well be acetylated or formylated. The protein is not phosphorylated either in the crude preparation or in the purified form. The change in total amount of the pH 2.I extractable protein during the normal cell cycle shown in Fig. 3. It doubles in quantity at approximately the time of cell division. In cultures synchronised by growth into stationary phase in the phosphate deficient medium, EMMI, a different picture is obtained (Figs. 4 and 5)- The crude pH 2.1 extractable protein from EMMI three day stationary cells is 7 times more by weight per cell than from exponentially growing cells in EMMI or EMM2 and considerably enriched in the major component (Fig. 2). On inoculation of such cells into fresh medium it rapidly drops to the exponential phase value within one hour or less. In fact, a considerable drop occurs during the resuspension of the cells even before the first 'zero time' sample is taken. Following the intial drop, the total amount of pH 2.1 extractable protein remains constant till cell division takes place when it increases. Total incorporation of 35S from sulphate and 14C from a 14C-labelled protein hydrolysate is not appreciable for two hours but is virtually linear up to 6 h once it has begun. At 6 h there is a 3-fold increase in incorporation of 85S. Incorporation of the two labels into the pH 2.1 fraction does not occur till 3 h after inoculation. It levels off about an hour before the cell plate peak and then resumes immediately following it. The great increase in amount of the pH 2.1 protein in EMMI three day stationary cells is not reflected in the amount present in nuclei from such cells (Table II). Hence the extra protein must be cytoplasmic in origin. Further the solubility of the protein from these cells is different from that of EMM2 cells to the extent that the amount obtainable after one reprecipitation is nearly half of the crude total. Incorporation of 35S shows that there is a very slow turnover of the pH 2.1 fraction in stationary phase. A pH 2.1 extractable protein was also detected in spores of S. pombe. Unfortunately the amount of material available was insufficient to characterise it further.
Biochim. Biophys. Acta, 228 (i97 I) 627-635
634
J.H.
DUFFUS
DISCUSSION
There is no doubt that S. pombe contains none of the well characterised histones isolated from calf thymus and elsewhere. Comparison of amino acid contents ~ of histone fractions shows this clearly, confirming the immunological assay. The p H 2.1 extractable protein is also different from the histone fractions obtained b y TONINO AND ROZlJN 3 from Saccharomyces cerevisiae (Table I). It has less arginine and lysine, more aspartic acid and more serine and threonine. The most striking similarity is the absence of cysteine but, b y comparison with other organisms, both these preparations have in common low contents of basic amino acids for presumed histone fractions. The differences m a y be due to the method of fractionation but attempts to obtain chromatin from S. pombe b y the method of TONINO AND R o z u ~ 3 were not successful. The prime reasons for suggesting that the p H 2.I extractable protein represents a histone according to MURRAY's4 definition are as follows. Firstly its extraction properties. All cytoplasmic basic proteins are reported to be extractable at p H 2.8 but no histones are soluble at this p H 1°,23. The only nucleoprotein so far reported to be extracted at p H 2.1 following thorough extraction at p H 2.8 is F I histone 1°. However, it must be noted that the results above indicate that under extreme conditions (phosphate deficiency) at least some cytoplasmic basic protein comes out in the pH 2.1 fraction. Secondly, there is the constant ratio by weight of the p H 2.1 extractable protein to DNA whether extracted from a nuclear preparation or from whole cells under conditions of normal growth. This at least indicates that it is a component of the nucleus and if we consider also the fact that it doubles in quantity in the normal cell cycle at about the time of cell division, immediately following DNA synthesis 6, the correlation with DNA becomes quite strong. It m a y be noted that the time of doubling does not correspond to the 'critical point' of MITCHISON AND CREANOR 2.
Accepting that the p H 2. I extractable protein is a histone, its identity with at least part of the ribosomal protein indicated by electrophoresis and immunological analysis becomes of considerable interest. It m a y be suggested that this is the primitive condition in eucaryotes. The accumulation of p H 2.1 extractable protein in cells grown into stationary phase in EMMI appears to reflect a change in properties of some cytoplasmic component, presumably a ribosomal protein. This m a y correlate with the drop in total cell RNA from 3.7 to 0.66 pg under the same conditions 2~. The rapid decrease in the p H 2.1 fraction following inoculation into fresh medium could reflect the almost immediate resumption of production of ribosomes and RNA. Perhaps tlle newly synthesised ribosomes under these conditions make use of the pre-existing protein. With regard to the synthesis of the p H 2.1 fraction in cultures of EMMI stationary phase cells inoculated into fresh medium, incorporation of 35S and 14C indicates a short burst between 3 and 4.5 h after inoculation followed by another after the cell plate peak at 5.5 h. The amount synthesised during the first burst must represent turnover since it is not reflected in the total extracted. The beginning of the first burst is about 2.5 h before the cell plate peak. Since the normal time for a cell cycle under these conditions is also about 2.5 h (ref. I) it m a y be that the first burst coincides with the resumption of normality within the cells. The second burst
Biochim. Biophys. Acta, 228 (1971) 627-635
NUCLEOHISTONE FROM S.
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635
is reflected, with a short delay, in the increase in the total amount of the pH 2.1 fraction. As in cells growing normally in EMM2, this correlates with cell division and follows DNA synthesis 6. Finally, if the pH 2.1 extractable protein is a histone and if it is also a component of the ribosomes, the concept of histone synthesis requires re-examination. It is likely that ribosomal protein is synthesised throughout the cell cycle. If this is so, the apparent step in 'histone' synthesis merely reflects a redistribution of the protein, more being taken up by the nucleus and binding to the newly synthesised DNA. This possibility will require to be carefully studied. ACKNOWLEDGMENTS
I thank Professor J. M. Mitchison for all his help and encouragement and Mr. C. J. Mitchell and Miss J. I. Clancy for their excellent technical assistance. My thanks to Mr. W. H. Wain for the pure ribosomes used in the immunological assays. I am grateful to Dr. K. Murray, Dr. H. J. Cruft and Mr. P. Greenaway for their advice about histones and to Dr. R. P. Ambler and Mr. P. Greenaway for the amino-acid analyses. The immunological assays were kindly carried out by Dr. H. S. Micklem and Mrs. J. Riddaway. This work was supported by an S.R.C. Grant. Fig. I was photographed by Mr. D. Cremer. REFERENCES I J. M. MITCHISON, in D. M. PRESCOTT, Methods in Cell Physiology, Vol. 4, Academic Press, N e w York, 197 o, p. I3I. 2 J. M. MITCHISON AND J. CREANOR, J. Cell Sci., 5 (1969) 3733 G. J. M. TONINO AND TH. H. ROZlJN, Biochim. Biophys. Acta, 124 (i966) 427 . 4 K. MURRAY, in J. BONNER AND P. T s ' o , The Nucleohistones, H o l d e n - D a y , S a n Francisco, 1964, p. 15. 5 J. M. MITCHISON AND W . S. VINCENT, Nature, 205 (1965) 987 . 6 C. J. BOSTOCK, Exptl. Cell Res., 6o (197 o) 16. 7 C. C. EMEIS AND H. GUTZ, Z. Natur[orsch., 15b (1958) 702. 8 J. H. DUFFUS, Biochim. Biophys. Acta, 195 (1969) 23 o. 9 V. E. BOWER AND R. Cx. BATES, J. Res. Natl. Bur. Std., Set..4, 55 (1955) 197. io K. NfORRAY, C-. VIDALI AND J. M. NEELIN, Biochem. J., lO 7 (1968) 207. t i T. C. MCILVAINE, J. Biol. Chem., 49 (1921) 183. 12 ]N. R. EATON, J. Bacteriol., 83 (1962) 1359. 13 W. J. WADDELL, .]. Lab. Clin. Med., 48 (1956) 311. 14 J. ]3. MURPHY AND W. M. KIES, Biochim. Biophys. Acta, 45 (196o) 382. 15 K. BURTON, Biochem. J., 62 (1956) 315 . 16 E. W . JOHNS, Biochem. J., lO 4 (1967) 78. 17 A. L. SHAPIRO, E. V1NUELA AND J. V. MAIZEL, Biochem. Biophys. Res. Commun., 28 (1967) 815 . 18 P. GRABAR, in P. GRABAR AND P. ]3URTIN, Immuno-electrophoretic Analysis, Elsevier, A m s t e r d a m , 1964, p. 3. 19 O. OUCHTERLONY, Progr. Allergy, 6 (t962) 3 o. 20 B. N. AMES, in S. P. COLOWICK AND N. O. I~APLAN, Methods in Enzymology, Vol. 8., A c a d e m i c Press, N e w York, 1966, p. 115. 2i K. MURRAY, in J. BONNER AND P. Ts'O, The Nucleohistones, H o l d e n - D a y , S a n Francisco, 1964, p. 21. 22 K. MURRAY, J. Mol. Biol., 15 (1966) 409. 23 N. STEBBING, P h . D. Thesis, U n i v e r s i t y of E d i n b u r g h , 1969.
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