on March 22, 2017
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`Downloaded from
`
`carbons fi om nltuiallyo11OCCii riing SOuli ces ina
`be ilmorc widespread than was generally be-
`BuLbble
`lieved.
`clustcrs occtur both in areas
`which cor relate with shiallow suLbbottomii struLc-
`ttires and also in places where stuch features
`are absent. It is presently unknown how Imtich
`liquid hydrocat bon is associatcd
`with
`these
`gas sccps, hut initi,il obscrsvations aire that a
`simiall amllouLnt of liquiid hxydirocarbon is pi-esent
`in somlie cases. The readet- shotuld see the fol-
`refcreniccs:
`lowing
`A.
`Tinkle,
`R.
`J. W.
`Antoine. R. Kuzela. Oceau
`ind. 8. 139 (1973);
`Mletall. Petrol.
`W. E. Sssect, Au,u.
`ltint.
`lt Nt.
`ELug. Ofsliore Tec/tnol. Cotif.. Ioulston (1973).
`p;aper No. OTC-1803. vol. 1. pp. 1667-1672.
`24. G. W. lIodgson, B. Hitchlon. K. TaguLchi, in
`tlhe Ficlds
`Recett ReNearclies
`ini
`of Hydro-
`sphlere, Attou sphere,
`Nuclear Geochtem-
`aui(t
`istrv,
`Y.
`Nlizake
`and
`Kozcinia,
`A.
`Eds.
`(Kenkytlsh.i, Tokyo, 1964), pp. 215-242.
`25. L. G. Weeks, Aml. Assoc. Petrol. Geol. Butll.
`49, 1680 (1965).
`Albers.
`P.
`26. J.
`D. C.artcr.
`A. L.
`Clark,
`NI.
`S. P. Schwcinfurtlh. U.S. Geol.
`A. B. Cout v,
`Prof. Pp. .517 (1973).
`.Sitri
`27. Sce NI. Gary, R. NicAfec. Jr., C. L. Wolf.
`G/lossar-
`Eds.,
`of Geolo,r (Amei can Geo-
`WX .ushitiniton, I).(., 1972), p. 194.
`lo-icil Inst..
`
`xsseight about 10. Such aggregates may
`be the cause of anomalous stoichiome-
`tries in complexes of the histones with
`DNA. For example, five to ten times
`as much F2AI will bind to a given
`weight of DNA as is bound to the
`same weight of DNA in chromatin.
`
`Oligoniers of the Histones in Solution
`
`The tenidency of histones
`to form
`larme a,-re,,ates may be a consequence
`denaturing
`the
`of
`conditions
`under
`which they are prepared. The histones
`are usually extracted from chromatin
`acid and fractionated by ethanol
`in
`and acetone precipitation, followed by
`gel filtration in acid or by ion-exchange
`chromatography in
`guLanidine hydro-
`chloride. We decided
`milder
`to
`try
`methods and, in particuLlar, the proce-
`dure of vaii der Westhuyzen and von
`Holt (9). which involves extracting the
`histones from chromatin in 2M sodium
`chloride-50 mM sodium acetate (pH 5)
`and fractionating the extract by Sepha-
`dex G-100 ,el filtration in 5OmM sodi-
`um acetate at pH 5. The Sephadex
`G-100 eluLtion
`profile consists of two
`peaks, both corming after the excluded
`volume and thus of molecular weight
`less than about 105. The first
`(higher
`molecular weight) peak contains Fl,
`F'AI. Land F3: and the second (lower
`molecuLlar weight) peak contains F2A2
`
`Dr. Koinbc-g is a Junior Fellow of the Societ)'
`of Fellows of Harvard Unisersity; he is woi-king
`the MRC Laboratory of MolecuLlar
`at
`Biology,
`Hills Road, Cambridge CB2 2QH, England. Dr.
`Thomiias is a lecturer in the Departmiient of Bio-
`chemistry, University of Camiibridge, Tennis Court
`Road, Canmbridge CB2 lQW, England.
`
`865
`
`Petrol.
`
`(,eol.
`
`Bull.
`
`11. K. K. Landes, Aml. Assoc. Petrol. Geol. Biill.
`57, 637 (1973).
`16, 739 (1932).
`12. E. DeGolyer, ibid.
`13. W. E. Pratt and D. Good, Eds., World Geog-
`raphy of Petroleutml
`(Princeton Univ. Press.
`1950).
`Princeton, N.J.,
`14. W. K. Link, Amii.
`Ass.
`36, 1505 (1952).
`15. These observations
`sccps can and do
`that
`soLurce beds and that
`originate directly fromii
`the presence of seeps is not directly coi re-
`coimmercial
`produIction
`latable
`oil
`with
`are
`commiionly known. One imiay choose to
`call
`noncommiiiiercial
`acculmUlations
`these
`ilmicro-
`reservoirs; there is a gradation between that
`commlinercial
`oil reservoired in
`accumllulations,
`that in suibcommiiiiercial accumultilations, that in
`miiicroreservoirs
`fracttures,
`that
`and
`in
`or
`isolated porosity or in transit
`after the gen-
`eration and imiigration processes. The "driving
`force" for scepage fromii fracttures and isolated
`porosity is probably, in pait, associated with
`miiigration processes;
`the sccpagc driive from
`reservoirs is the "reset-voir drivc" associated
`with that accumultilation.
`16. W. A. Ver Wiebe, Howv Oil Is Foltd (Ed-
`w.rds, Ann Arbor, Michigan,
`1951).
`Yackel,
`17. M. Steineke and M. P.
`in
`Worldl
`Geography of PetroleumZ. W. E.
`Pratt and
`
`D. Good, Eds. (Princeton Univ. Press, Princc-
`ton, N.J,, 1950), pp. 203-229.
`18. V. E. McKelvey and F. F. H. Wang, U.S.
`Surr.
`Geol.
`A1isc. Geol. Imiest. Map 1-632
`(1970), sheet 3 of 4.
`19. P. G. Mikolaj, A. A. Allen, R. S. Schlucter-,
`Am1. Ist. Alini. Aletall. Petrol. Enzg. OfJ.shoue
`Techlntol. Cotnf., Dallas (1972). paper No. OTC-
`1549, vol. 1. pp. 1365-1378.
`20. D. Straughan and B. C. Abbott, in Water
`l'olluitioni be Oil.
`P. Hepple, Ed. (Institute
`of Petroleulm., London, 1971), pp. 257-262.
`2I. The' tirnm API grasvity is a standard adopted
`by the American PetrolcLum
`InstitLute for de-
`n1oting the specific wcight of oils9 the lower the
`glavity,
`specific
`the
`higlher the API gravity:
`141.5
`specific grxsity of
`the liquid zit 1.5 C'
`22. T. C. Johnson. U.S. Coa.st Guatd Rep. Proj.
`No. 714141/002 (1971).
`geophysical
`23. Rccent
`advances
`the
`detection
`of gas hLhbblcs
`column.
`(ii)
`identification
`the
`shallow sedimiients,
`aind
`(iii)
`the
`of gas resetrvoil s
`froIml amplitude
`esvidence
`constittite
`that
`seepagec
`
`API gr.iits
`
`131.5
`
`perimiitting
`(i)
`in
`the water
`of
`gas-filled
`recognition
`atnomaizilics
`of
`hydro-
`
`Chromatin Structure:
`Oligomers of the Histones
`
`The histones comprise an (F2A 1 )(F3)L2 tetramer, a
`different oligomer of F2A2 and F2B, and monomer of Fl .
`
`Roger D. Kornberg and Jean 0. Thonm'as
`
`Biochemical and x-ray diffractioni re-
`sults concerning the oligomeric struc-
`ture of the histones are presented. The
`results show pairwise associations
`in
`solution, two types of histone forming
`a tetramer and two other types of his-
`tone forming a different oligomer. The
`same pairwise associations appear to
`occur in chromatin.
`
`Introduction to the Histones
`
`There are five maini types of histone
`in the chromatin of euk.aryotes. known
`.as Fl (or 1) with a mass of 21,000
`daltons; F2AI (or IV), of 11,300 dal-
`toIns; F2A2 (or IIbi), of 14,500 dal-
`tons; F2B (or Ilb2), of 13,700 daltons:
`and F3 (or III), of 15,300 daltons.
`There are
`usually
`equimolar
`nearly
`amounts of all the histones except for
`24 MAY 1974
`
`is about half as
`
`Fl, ol which ther-e
`much (M).
`The arrang>,emenit
`histones and
`of
`DNA in chromatin has been studied by
`x-ray ditfraiction (2. 3), leading to the
`proposal (4) of a "Super-coil" model.
`The x-ray data have not been sufficient,
`however, to prove the validity of the
`model. Varilous stuldies have been un-
`dertaken to stLpplement the x-ray data,
`such as studies of the association be-
`havior of the histones (5-7) and stud-
`ies of complexes of one or another
`type of histone with DNA (6,
`8). In
`calses. histonies purified to homo-
`Imlost
`geneity from calf thymus were used.
`histones, F2AI. F2A2.
`Four of the
`F2B, and F3. were observed to form
`large self-aggregates. For example, F2Al
`and F3, at pH 7 and ionic strength
`0.1, form self-aggregates of sedimenta-
`anid
`coefficient
`tion
`19S
`molecular
`
`Mylan v. Genentech
`IPR2016-00710
`Merck Ex. 1102, Pg. 1
`
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`

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`
`served (Fig. 2) a single sharp sedimen-
`tation boundary (sedimentation coeffi-
`cient 3S) and the behavior expected of
`a homogeneous species at sedimenta-
`tion equilibrium (alinear plot of log
`as a function of the
`concentration
`square of the distance from the axis of
`rotation).
`The
`sedimentation
`equi-
`librium results give a value of 53,900
`for the molecular weight (assuming a
`partial specific volume of 0.72 cm3 g-1).
`This is in good agreement with the
`53,200
`expected
`value
`of
`the
`for
`molecular weight of an (F2A1 )2(F3)2
`tetramer.
`When F2AI
`and F3 prepared by
`acid and solvent extraction (12) were
`mixed and analyzed in
`cross-linking
`and sedimentation experiments as de-
`scribed above, only a small amount of
`tetramer was observed. Most of the
`material was in the form of high molec-
`weight
`aggregates
`ular
`which were
`excluded from SDS gels after cross-
`linking and which sedimented rapidly
`(sedimentation coefficient about 13S).
`Since F2Al, F3, and Fl are eluted
`together from Sephadex G-100 in the
`van der Westhuyzen and von Holt pro-
`cedure, and since F2A1 and F3 form
`a tetramer, it seemed that Fl might
`form a dimer (13). However, previous
`work (5, 14) on Fl prepared by acid
`extraction has shown only monomers.
`
`2-5
`
`2-0
`
`5
`
`-
`
`1*0
`
`w
`
`/
`
`-
`
`-1
`37
`
`I
`
`I
`
`40
`
`5--
`.I
`I
`-1
`39
`38
`Radis Squared (CM2)
`experiments on
`Fig.
`2.
`Sedimentation
`F2A1 and F3. A mixture of F2A1 and F3
`was prepared as described in Fig. 1 and
`dialyzed against 0.15M NaCl-25 mM so-
`dium phosphate-lO mM sodium bisulfite
`(pH 7.0; ionic strength, 0.25). Sedimenta-
`velocity measurements were made
`tion
`with a protein concentration of 4 mg/ml,
`in a Beckman Spinco model E centrifuge
`at a speed of 60,000 rev/min, with a
`double sector cell, and with the schlieren
`phase plate at 60°. The photograph shown
`was taken 32 minutes after full speed
`was reached.
`Sedimentation equilibrium
`measurements were made with the model
`E at a speed of 12,000 rev/min and
`methods described by Durham (32). The
`temperature was 20°C throughout.
`SCIENCE, VOL. 184
`
`words, F2A1
`and
`and F2B. In other
`F3, which arealmos
`it the same as F2A2
`ular weight, behave
`and F2B in moleci
`nearly twice their
`like F1, which has
`molecular weight.I
`seemed to us that
`ht yield a dimer of
`this procedure migi
`monomers of F2A2
`F2A1
`and F3 and i
`of indirect evidence
`and F2B. In view 4
`nairwise associations
`(see below) forp
`i chromatin, F2A1
`of the histones in
`
`-
`
`6
`{
`
`C(F2A1)2(F3)2
`/ (F2A1)(F3)2
`(F2AI)2(F3)
`(F3)2
`(F2A1)(F3)
`(F2Al)2
`
`o/
`
`F3 -
`
`F2A1- /
`
`30
`
`40
`
`506C
`
`with F3, and F2A2 with F2B, it seemed
`that a dimer of F2Al and F3 could
`be significant. So we followed the pro-
`cedure of van der Westhuyzen and von
`Holt (9), including the final step of
`precipitating F2AI
`and F3 with am-
`monium sulfate to separate them from
`Fl, and then looked for dimers. Instead
`we found an (F2Al)2(F3)2 tetramer.
`The evidence for this tetramer comes
`from cross-linking and sedimentation
`experiments. Cross-linking was carried
`out by the method of Davies and
`OX
`Stark (10) which involves treating the
`70protein with dimethyl suberimidate, a
`bifunctional amino group reagent, and
`determining the molecular weights of
`the products by sodium dodecyl sul-
`fate (SDS)-polyacrylamide gel eleotro-
`phoresis. A tetramer of identical sub-
`units gives four bands on the gel, cor-
`responding to monomer, dimer, trimer,
`20
`and tetramer. In a case of nonidentical
`)ht(x10-3)
`Molecular Weig
`subunits a more complex pattern
`of
`of F2A1
`and F3. An
`Fig. 1. Cross-linking
`bands would be expected. The mixture
`precipitate of F2A1
`ammonium sulfate
`of F2A1
`and F3 prepared according to
`d by the method of
`and F3 was prepare
`l and von Holt (9)
`van der Westhuyzen and von Holt
`van der Westhuyzen
`romatin [prepared by
`from calf thymus chi
`gives eight bands (Fig. 1). The pOSi-
`)ay and Doty (25),
`the method of Zub
`of the
`intensities
`tions and relative
`)dium bisulfite as a
`including 50mM sc
`26)]. The precipitate
`bands were the same in experiments
`protease inhibitor(;
`performed at protein concentrations of
`protein concentration
`was dissolved at a
`[determined by the
`2 mth/dl if 50 mM 0.1, 1, and 2 mg/ml, showing that all
`et al. (27)] of about
`M sodium bisulfite,
`the bands derive from intramolecular
`sodium acetate-50 n
`against 70 mM reaction. The monomer bands were
`Eed
`pH 5.0, and dialyz
`identified by comparison with a control
`) mM sodium bisulfite,
`sodium phosphate-10
`in which dimethyl suberimidate was
`th, 0.25). The protein
`pH 8.0 (ionic strengi
`concentration was a
`omitted. The other bands were identi-
`dialysis buffer, and
`by dilution with the
`fied by finding combinations of F2A1
`te
`(freshly dissolved
`dimethyl suberimida
`e same buffer) was
`and F3 whose molecular weights, when
`in th
`at 20 mg/ml
`plotted
`against
`the
`entration of 1 mg/ml.
`of the
`positions
`added to a final conc
`t for 3 hours at room
`The mixture was kep
`bands in the gel, gave a straight line
`against bands In the gel,
`weightscae
`dialyzed
`then
`temperature,
`[Fig. 1; the molecular weight scale was
`thyl sulfonyl fluoride
`0.25 mM phenylme
`not calibrated with other proteins be-
`freeze-dried. The resi-
`(PMSF) at4°C, and
`cause the mobilities of histones in SDS
`SDS sample buffer,
`due was dissolved iI
`the
`All
`proteolysis (28),ald
`(11)].
`are anomalous
`containing 0.1 nM
`gels
`boiling to prevent
`bands expected for an (F2A1)2(F3)2
`cent SDS-polyacryla-
`analyzed in 7.5 per
`tetramer are present, but none of the
`to Weber and Osborn
`mide gels according
`xed, stained with Coo-
`additional bands that would arise from
`(29). The gels were fi:
`tetramers of other compositions. The
`e, and destained
`as
`blu
`massie brilliant
`positions and relative
`described (30). The
`intensities of the bands are
`relative
`are
`were the same in ex- roly
`epetedor an
`intensities of bands
`onic strength (40 mM roughly as expected for an (F2A1)2
`periments at lower ii
`(F3)2 tetramer (1: 2: 1 for the dimer
`rochloride-10 mM so-
`triethanolamine hydr
`bands and 1: 1
`D; ionic strength, 0.05)
`for the trimers), but
`dium bisulfite, pH 8.(
`not exactly as expected, presumably
`rcross-linking reagent,
`and when the shorter
`dimethyl pimelimidE
`because probabilities of forming cross-
`and Schroeder (31)]
`method of McElvair
`ons of the bands were
`links depend on proximities and re-
`was used. The positi
`ents at pH 7 (90 mM activities of amino groups.
`the same in experimi
`>H 7.0; ionic strength,
`Strictly
`speaking,
`the
`of
`pattern
`sodium phosphate, p
`0.2) and at pH 9
`(0 15odiumNaCbiufitM
`cross-linked products is consistent with
`sodium borate-10
`h 0.25); but the rela-
`of monomers,
`either some mixture
`th0different, th'e rlowr dmr,orahmgnou ouino
`pH 9.0; ionic strengt
`dimers, or a homogeneous solution of
`different, the lower
`tive intensities were
`(F2A1)2(F3)2 tetramer. The possibil-
`lands being relatively
`molecular weight b
`ity of a .mixture was ruled out and the
`H 7 and the higher
`more intense at p]
`ands more intense at
`molecular weight b
`tetramer shown to be homogeneous by
`pH dependence of the
`pH 9, reflecting the
`experiments. We ob-
`sedimentation
`reactivity of e-aminc
`igroups.
`866
`
`Merck Ex. 1102, Pg. 2
`
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`

` on March 22, 2017
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`
`Downloaded from
`
`In fact, Fl prepared by the van der
`Westhuyzen and von Holt procedure
`is
`also a monomer, as shown by
`cross-linking
`experiments
`(in which
`only a monomer band was observed
`on the gel) and sedimentation equilib-
`rium experiments
`(which showed a
`homogeneous species with a molecular
`weight of 21,500 under the conditions
`given in Fig.
`2,
`in good agreement
`with the expected molecular weight of
`about 21,000). This leaves open the
`question of why F2A1, F3, and Fl are
`eluted together from Sephadex G-100.
`A possible explanation is that the tet-
`ramer of F2A1 and F3 is relatively
`compact and the monomer of Fl ex-
`tended [the sedimentation coefficient of
`Fl is known (5, 15) to be anomalously
`low].
`The association behavior of F2A2
`and F2B in the mixture that arises from
`the van der Westhuyzen and von Holt
`procedure has been difficult to analyze.
`Cross-linking experiments give the pat-
`tern of bands shown in Fig. 3. The
`most rapidly
`migrating
`species was
`found, by comparison with a control
`in which dimethyl suberimidate was
`omitted, to comprise both monomers.
`The other species were identified
`as
`dimers, trimers, tetramers, pentamers,
`and hexamers by the linearity of a
`semilog plot of the sort shown in Fig.
`1. (The plot was constructed using the
`average of the molecular weights of
`F2A2 and F2B. Plots constructed in
`this way and for the extreme cases of
`all the bands arising from F2A2 or all
`from F2B gave the same straight line.
`In other words, the resolution of the
`gel was not sufficient to distinguish the
`various possible combinations of F2A2
`and F2B.)
`In cross-linking experiments
`a
`at
`tenfold
`lower
`protein
`concentra-
`tion,
`the
`positions
`of
`the
`bands
`were the same,
`the
`but
`intensities
`of the dimer and the higher molec-
`ular
`weight
`bands
`relative
`the
`to
`monomer were greatly diminished. This
`that F2A2 and F2B form
`suggests
`oligomers in a reversible way. The sim-
`plest possibility is that they form short
`chains,
`such
`(-F2A2-F2B-)n
`as
`or
`(-F2A2-F2A2-F2B-F2B-),n, by reversi-
`ble polymerization. We have not so far
`established this or excluded other pos-
`sibilities, such as association behavior
`like that of a mixture of F2A2 and
`F2B prepared by acid extraction, which
`Kelley has shown (15)
`to comprise
`dimers of the two histones and higher
`aggregates of only F2B.
`We have also carried out a cross-
`24 MAY 1974
`
`.- Migration
`Fig. 3. Clross-linking of F2A2 and F2B.
`The mixture of F2A2 and F2B resulting
`from the van der Westhuyzen and von
`Holt (9) procedure was treated at a pro-
`concentration
`tein
`0.5 mg/ml with
`of
`dimethyl suberimidate and analyzed in
`SDS gels as for the experiment shown in
`Fig. 1.
`
`linking experiment on a mixture of the
`tetramer of F2A1 and F3 and oligo-
`mers of F2A2 and F2B. Equal weights
`of the pairs of histones described in
`Fig.
`1 (F2A1 and F3) and, Fig.
`3
`combined,
`(F2A2 and F2B)
`were
`treated at a total protein concentration
`of 1 mg/ml with dimethyl suberimi-
`date, and analyzed in SDS gels, as for
`the experiment shown in Fig. 1. The
`pattern of bands appeared to be the
`sum of the patterns shown in Figs. 1
`and 3, but many of the bands over-
`lapped. The dimers could be com-
`pletely resolved in a 17.5 percent SDS-
`polyacrylamide gel run according to
`Laemmli (16); there were four bands,
`corresponding exactly in position and
`intensity to the three dimer bands given
`by the tetramer and the one dimer band
`of the F2A2-F2B oligomers. Evidently
`no additional oligomers are formed.
`
`Oligomers of the Histones
`in Chromatin
`
`The histones thus appear to be as-
`sociated in pairs in solution, F2A1 with
`F3 and F2A2 with F2B. Many lines of
`evidence seem to us to suggest that the
`same pairs of histones are associated
`in chromatin. The strongest evidence
`comes from the work of Ilyin et al.
`(17) on the exchange of histones be-
`tween chromatin and free polynucleo-
`tides. In 5 mM tris-HCl, pH 8 at 0°C,
`there is no exchange of F2A1 and F3
`(no detectable exchange in 24 hours),
`slow exchange of F2A2 and F2B (a
`half-time for exchange of about 24
`hours), and rapid exchange of Fl
`(complete exchange in 15 minutes).
`An example of very different evidence
`which seems to us to
`the
`suggest
`same pairing of histones comes from
`amino acid sequence work. F2A1 and
`F3 have been conserved in sequence
`during evolution [there are two differ-
`ences in F2A1 (18) between pea seed-
`ling and calf thymus and four differ-
`ences in F3 (19)], F2A2 and F2B ap-
`pear less conserved [eight differences in
`F2A2 (20) between trout testis and
`calf thymus and three differences in the
`first 22 residues of F2,B (21)], and Fl
`appears variable [eight differences in
`the first 34 residues between rabbit
`thymus and calf thymus (22)].
`
`Chromatin
`
`A FB D
`
`(F2A1)2(F3)2+DNA
`(F2A2, F2B)+DNA
`
`Fig. 4. X-ray patterns of chromatin and
`of complexes of oligomers of histones
`with DNA. Chromatin was isolated as de-
`scribed in
`1, treated with ion-ex-
`Fig.
`change resin in 0.65M NaCl-50 mM so-
`dium phosphate-O1 mM sodium bisulfite,
`to remove Fl
`pH 7.0,
`(33), dialyzed
`against 0. 15M NaCl-25 mM sodium phos-
`phate-10 mM sodium bisulfite (pH 7.0;
`ionic strength, 0.25) and centrifuged in a
`Spinco 60 Ti rotor at 50,000 rev/min for
`at 40C. Complexes of
`all
`20 hours,
`oligomers of histones with DNA were
`(F2AI)(F3)2formed from 2 mg of one of the pairs of
`histones described in Figs.
`1 and 3, or
`+CF2A2 F2B)+DNA
`2 mg of each of the pairs, and 5 mg of
`DNA [isolated from calf thymus (34)
`and sheared by passage through a French
`press (35) to pieces of about 2500 base
`pairs, as measured by analytical velocity
`sedimentation in alkali (36)]. The histones
`and DNA were mixed in 2M NaCl-0.1M
`Jsodium phosphate, pH 7.0, in a total vol-
`ume of 25 ml, and dialyzed and centri-
`A-1
`fuged as described above for chromatin.
`The pellets of chromatin, (F2Al )2(F3)2 + (F2A2, F2B) + DNA, (F2Al)2(F3)2 +
`DNA, and (F2A2, F2B) + DNA contained 17, 19, 14, and 11 percent by weight of
`chromatin or histone + DNA. The pellets were sealed in 1-mm path length specimen
`holders and photographed with the use of a fine-focus rotating anode x-ray tube and
`mirror-morochromator focusing camera (37). Exposures were for about 20 hours at
`room temperature. Films were analyzed usinig an Optronics Photoscan model P-1000
`digital microdensitometer interfaced
`to a PDP 11/10 computer, with a program
`devised by Dr. R. A. Crowther; the optical densities were radially integrated to obtain
`the traces shown above (38).
`
`100
`
`50
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`This evidence suggests pairwise asso-
`ciations of the histones in chromatin
`but says nothing of details, such as
`whether the F2A1 and F3 pair, which
`occurs as an (F2Al)2(F3)2 tetramer
`in solution, also occurs as a tetramer
`in chromatin. The most direct evidence
`in
`(F2Al)2(F3)2 tetramer
`for
`an
`chromatin is that a complex formed
`from tetramers, F2A2-F2B oligomers,
`and DNA gives the same x-ray pattern
`as chromatin (Fig. 4, upper two traces).
`Tetramers and F2A2-F2B oligomers
`are both required to give the x-ray pat-
`tern (Fig. 4, lower two traces), but Fl
`is not-in keeping with previous obser-
`vations (3, 23 ) that removing Fl from
`chromatin does not affect the x-ray
`pattern. Further implications of these
`results are discussed in the accompany-
`ing article (24).
`We are currently studying associa-
`tions of the histones in chromatin by
`cross-linking. There are two difficulties
`that do not arise in experiments on the
`histones in solution: the amino side
`salt linkages
`chains are involved in
`with the phosphate groups of DNA
`and are thus less available for chemical
`modification; and the presence of five
`rather than two histones complicates
`identification of products from molec-
`ular weights. -Preliminary results do
`show less cross-linking of histones in
`
`chromatin than in solution, but cross-
`linked products up to pentamers are
`readily observed and call for further
`investigation.
`
`References and Notes
`1. Molecular weights are from R. J. DeLange
`and E. L. Smith, Accounts Chem. Res. 5,
`368 (1972). Relative amounts of the histones
`are discussed in the accompanying article (24).
`2. M. H. F. Wilkins, Cold Spring Harbor Symp.
`Quant. Biol. 21, 75 (1956); - , G. Zubay,
`H. R. Wilson, J. Mol. Biol. 1, 179 (1959);
`ibid.
`V. Luzzati and A. Nicolaieff,
`7, 142
`(1963).
`3. B. M. Richards and J. F. Pardon, Exp. Cell
`Res. 62, 184 (1970).
`4. J. F. Pardon and M. H. F. Wilkins, J. Mol.
`Biol. 68, 115 (1972).
`5. P. A. Edwards and K. V. Shooter, Biochem.
`114, 227 (1969).
`J.
`6. R. Ziccardi and V. Schumaker, Biochemistry
`12, 3231 (1973).
`7. A. C. H. Durham, unpublished.
`8. A. B. Barclay and R. Eason, Biochim. Bio-
`phys. Acta 269, 37 (1972).
`9. D. R. van der Westhuyzen and C. von Holt,
`FEBS (Fed. Eur. Biochem. Soc.) Lett. 14, 333
`(1971).
`10. G. E. Davies and G. R. Stark, Proc. Natl.
`Acad. Sci. U.S.A. 66, 651 (1970).
`11. S. Panyim and R. Chalkley, J. Biol. Chem.
`246, 7557 (1971).
`12. Electrophoretically pure F2AI and F3 were
`gifts of Dr. E. W. Johns.
`13. Preliminary work shows that the sedimenta-
`coefficient of the van der Westhuyzen
`tion
`and von Holt (9) preparation of F2AI and F3
`is the same at pH 5 as at pH 7, so the two
`histones most likely occur as a tetramer in
`the Sephadex G-100 gel filtration (pH 5) jusi
`as they do in the cross-linking (pH 8) and
`sedimentation (pH 7) experiments described
`in Figs. 1 and 2.
`14. A. J. Haydon and A. R. Peacocke, Chem.
`Soc. Spec. Publ. No. 23 (1968), p. 315.
`15. R. I. Kelley, Biochem. Biophys. Res. Conmniun.
`54, 1588 (1973).
`16. U. K. Laemmli, Natuire
`(Lond.)
`227,
`680
`(1970).
`
`1
`
`Smith, J. Johnson, ibid.
`
`17. Y. V. Ilyin, A. Ya. Varshavsky, U. N. Mick-
`elsaar, G. P. Georgiev, Eur. J. Blochem. 22,
`235 (1971).
`18. R. J. DeLange, D. M. Fambrough, E. L.
`Smith, J. Bonner, J. Biol. Chem. 244, 5669
`(1969).
`19. L. Patthy, E. L.
`248, 6834 (1973).
`20. G. S. Bailey and G. H. Dixon, ibid. p. 5463.
`21. E. P. M. Candido and G. H. Dilon, Proc.
`Nail. Acad. Sci. U.S.A. 69, 2015 %1972).
`22. S. C. Rail and R. D. Cole, J. Biot. Chem.
`246, 7175 (1971).
`23. K. Murray, E. M. Bradbury, C. Crane-Rob-
`inson, R. M. Stephens, A. J. Haydon, A. R.
`Peacocke, Biochem. 1. 120, 859 (1970).
`24. R. D. Kornberg, Science 184, 868 (1974).
`25. G. Zubay and P. Doty, J. Mol. Biol. 1,
`(1959).
`26. S. Panyim, R. H. Jensen, R. Chalkley, Bio-
`chim. Biophys. Acta 160, 252 (1968).
`27. 0. H. Lowry, N. J. Rosebrough, A. L. Farr,
`Biol. Chem. 193,
`265
`Randall,
`J.
`R. J.
`(1951).
`28. J. R. Pringle, Biochem. Biophys. Res. Com-
`mun. 39, 46 (1970).
`29. K. Weber and M. Osborn, J.
`Biol. Chem.
`244, 4406 (1969).
`30. R. N. Perham and J. 0. Thomas, FEBS (Fed.
`Eur. Biochem. Soc.) Lett. 15, 8 (1971).
`31. S. M. McElvain and J. P. Schroeder, J. Am.
`Chem. Soc. 71, 40 (1949).
`67, 289
`32. A. C. H. Durham, J. Mol. Biol.
`(1972).
`33. E. M. Bradbury, H. V. Molgaard, R. M.
`Stephens, L. A. Bolund, E. W. Johns, Eur.
`J. Biochem. 31, 474 (1972).
`34. E. R. M. Kay, N. S. Simmons, A. L. Dounce,
`J. Am. Chem. Soc. 74, 1724 (1952).
`35. C. D. Laird, Chromosoma 32, 378 (1971).
`36. F. W. Studier, J. Mol. Biol. 11, 373 (1965).
`37. H. E. Huxley and W. Brown, ibid. 30, 383
`(1967).
`38. R. D. Kornberg, A. Klug, F. H. C. Crick,
`in preparation.
`39. We thank Drs. A. Klug and F. H. C. Crick
`for helpful discussions and criticism of the
`manuscript. We thank Janet Francis for ex-
`thanks
`R.D.K.
`assistance.
`technical
`pert
`the National Cystic Fibrosis Research Foun-
`dation for support during the early part ot
`this work.
`
`Chromatin Structure: A Repeating
`Unit of Histones and DNA
`
`Chromatin structure is based on a repeating unit of eight
`histone molecules and about 200 DNA base pairs.
`
`Roger D. Kornberg
`
`Evidence is given in the preceding
`article (1) for oligomers of the his-
`tones, both in solution and in chro-
`matin. Here I wish to discuss this and
`other evidence in relation to the ar-
`rangement of histones and DNA in
`chromatin. In particular, I propose that
`f868J
`
`the structure of chromatin is based
`on a repeating unit of two each of
`the four main types of histone and
`of DNA. A
`about 200 base pairs
`chromatin fiber may consist of many
`such units forming a flexibly jointed
`chain.
`
`Introduction to Chomatin Structure
`
`( Chromatin of eukaryotes contains
`nearly equal weights of histone and
`DNA. This corresponds, on the basis
`of the molecular weights and relative
`amounts of the five main types of his-
`tone, F1, F2A1, F2A2, F2B, and F3,
`to roughly one of each type of histone
`per 100 base pairs of DNA with the
`exception of Fl, of which there is half
`as much. The arrangement of histones
`and DNA involves repeats of structurej
`The first evidence of this comes from
`the work of Wilkins and co-workers
`(2) who obtained x-ray diffraction
`patterns from whole nuclei of cells
`showing relatively sharp bands.( Chro-
`matin isolated from the nucli as a
`nearly pure complex of histone and
`DNA/ gives x-ray patterns w
`the
`same bands. Further x-ray work (3-5)
`has shown thatthese bands corropond
`
`The author is a Junior Fellow of the Society
`of Fellows of Harvard UniversIty; he is working
`at the MRC Laboratory of Molecular Biology,
`Hills Road, Cambridge CB2 2QH, England.
`SCIENCE, VOL. 184
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