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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 A. C. H. Durham, unpublished.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
`
`Mylan v. Genentech
`IPR2016-00710
`Merck Ex. 1103, Pg. 1
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`to structure repeating at intervals of
`about 100 angstroms along the length
`of the chromatin fiberJNeither histone
`nor DNA alone gives x-ray patterns
`with such bands.
`A Asuper-coil" model has been pro-
`to account for the x-ray
`posed (6)
`data on chromatin. It consists of a
`DNA double helix with "a coating of
`into a single
`coiled
`larger
`histone"
`helix of axial repeat distance 120 A
`and diameter 100 A. There are 340 A
`of DNA double helix or 100 DNA
`base pairs per turn of the larger helix,
`which is a major drawback of the
`model in view of the following discus-
`sion of the true size of repeating unit
`in chromatin.
`
`A Repeating Unit
`
`The ratios of histone to DNA and
`x-ray data mentioned above do not
`indicate how the five types of histone
`are distributed in chromatin. The sim-
`plest case would be that the histones
`act together and form a unique struc-
`ture that gives rise to the x-ray pattern;
`the other extreme would be the
`at
`case of different combinations of his-
`tones in different regions of chromatin,
`some one of which gives rise to the
`x-ray pattern. Evidence from the pre-
`ceding article (1) helps to distinguish
`among these and the many possible
`intermediate cases. It was shown that
`histones F2A1 and F3 of calf thymus
`occur entirely
`as an (F2A1 ),(F3)2
`tetramer. It was further shown that a
`complex of tetramers, F2A2- F2B olig-
`omers, and DNA gives the x-ray pat-
`tern of chromatin, and that tetramers
`and F2A2-F2B oligomers are both re-
`quired, but Fl is not. t~he following
`conclusions may be drawn: F2A1 and:
`F3 form a unique structure; F2A1, F3,
`F2A2, and F2B act together and form,
`with DNA the repeating structure re-
`sponsible for the x-ray pattern of chro-
`matin;,,and Fl is either added on or
`located
`chromatin. ' In
`elsewhere
`in
`sum, four of the histones and DNA
`form a unique repeating structure.f
`Now suppose that the (F2A1 )2(F3)2
`tetramer defines a repeating unit of this
`structure and that all the DNA in chro-
`in
`matin is
`involved
`the
`structure.
`Then, /is chromatin contains roughly
`one each of F2A1, F3, F2A2, and F2B
`per 100 base pairs of DNA, the repeat-
`ing unit may contain two of each of
`these histones and about 200 base pairs
`of DNA. This coincides in a rather
`strikinL.wai.ith the results of diees-
`
`tion of chromatin by certain nucleases,
`in which most of the DNA is cleaved
`to pieces of about 200 base pairs./fhe
`first such observation was made by
`Hewish and Burgoyne in work on di-
`gestion of chromatin in rat liver nuclei
`by an endogenous nuclease (7),//In this
`digestion more than 80 percent of the
`DNA is cleaQTed to multiples of from
`one to six times 200 base pairs1j/ihe
`multiples
`occurrence of
`rather than
`just 200 base pair pieces is presumably
`being
`to some cleavage
`sites
`due
`blocked by nonhistone proteins./A more
`clear-cut result has come from an ex-
`tension of the work of Hewish and
`staphylococcal
`which
`in
`Burgoyne,
`nuclease has been shown to cleave
`more than 90 percent of the DNA in
`rat liver nuclei to pieces of about 200
`base pairs (8). Both the endogenous
`and staphylococcal nucleases produce
`a slight heterogeneity in size of the
`DNA pieces, the dispersion being about
`± 10 percent.
`The convergence of work on oligo-
`mers of histones and work on cleavage
`of DNA makes a /strong case for a
`repeating unit containing two each of
`F2A1, F3, F2A2, and F2B, and about
`200 base pairs of DNA.,'3oth kinds of
`work bear on how much repeating
`in chromatin, /6ne
`structure there is
`kind showing that most of the histone
`is involved (four of the five types of
`histone) and the other showing that
`most of the DNA is involved (more
`than 90 percent in rat liver) ./The gen-
`erality of the results can of course be
`tested by repeating the work on chro-
`matin from other sources. Short of
`that, it may be asked whether the rela-
`tive amounts of the histones and rela-
`tive amounts of total histone and DNA
`are independent of source. The relative
`histones have been
`amounts of the
`measured (9-12) by extraction from
`chromatin and fractionation by prepar-
`ative methods or
`in polyacrylamide
`gels. The measurements should be re-
`garded as only approximate, because
`of possible
`differential
`extractability,
`proteolysis, losses during fractionation,
`and overlaps
`of bands in
`the
`gels
`(especially
`bands
`the
`arising from
`F2A2 and F2B, and minor bands aris-
`ing from histone modification). The
`results, expressed as molar ratios of
`F3, F2A2, and F2B to F2A1, are 0.9,
`0.8, and 1.1
`in calf thymus (9) and
`nearly the same in other calf tissues
`(10), 0.7, 0.7, and 1.0 in Drosophila
`0.5, and 2.6 in pea
`(11 ), and 0.9,
`bud and other pea tissues (12).1F2Al
`and F3 may in fact be eQuimolar in all
`
`organisms, and F2A2 and F2B roughly
`equimolar with exceptions.
`'Despite the approximate nature of
`these measurements, it may be signifi-
`that F2Al and F3 are more
`cant
`nearly equimolar than F2A2 and F2B.
`F2A1 and F3, which occur as an
`(F2AI ) 2(F3).2 tetramer in calf thy-
`mus, would be expected, on the basis
`of the conservation of their amino acid
`sequences during evolution
`(13), to
`occur as a tetramer in all organisms,
`and might therefore be expected to
`occur in equimolar amounts in all or-
`ganisms. The oligomeric structure of
`F2A2 and F2B, on the other hand, has
`not been as well established as for
`F2AI and F3, and the amino acid se-
`quences of F2A2 and F2B appear to
`be less conserved than those of F2A1
`and F3 (1). The numbers of F2A2
`and F2B that I have taken to be in
`repeating
`unit
`based
`the
`are
`on
`the roughly equimolar amounts of all
`calf thymus. These
`histones
`in
`the
`numbers (two each of F2A2 and F2B)
`may not be exactly right (there may
`be two of F2A2 and three of F2B in
`calf thymus),
`in
`the repeating unit
`and they may vary from one organism
`is possible to envisage
`to another. It
`for F2A2 and F2B
`structural
`roles
`(see
`compatible with such variation
`below) .)
`ZMeasurements of relative amounts
`of total histone and DNA are more
`accurate than measurements of relative
`amounts of the various histones since
`amounts of total histone are less sensi-
`extractability, and
`tive to differential
`forth. The results,
`expressed
`as
`so
`weight ratios of total histone to DNA,
`are nearly 1.0 for chromatin from a
`wide range of sources, for example:
`1.15, 0.95,
`1.08, and 1.10 for
`1.17,
`liver,
`rat kidney, chicken liver,
`rat
`chicken
`erythrocytes,
`pea bud
`and
`(14); 1.02,
`1.04, and 0.86 at three
`in
`the
`development
`of
`stages
`sea
`urchin embryos (15); and 1.05 in the
`slime mold Physarum polycephalum
`(16). This invariance, together with
`the invariance of amino acid sequences
`of F2A1 and F3, is the strongest evi-
`dence for the generality of a repeating
`unit of two each of four of the histones
`and about 200 base pairs of DNA. ?
`, Fl is not involved in forming the
`repeating unit (see above), so it must
`either be added on to the unit or
`located in a different region of chro-
`matin. The amount of Fl relative to
`the other histones suggests that Fl is
`in fact associated with the unit: the
`molar ratio of Fl to F2Al is 0.54 in
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`nating with more extended DNA and
`associated protein, rather like beads on
`a string.>
`Some evidence for such a structure
`digestion
`the
`nuclease
`comes from
`work mentioned above. Endonucleases
`may produce 200 base pair pieces of
`DNA by cleaving the connecting strand
`between tetramers. And recent work
`(24) has shown that the 200 base pair
`piece and associated protein occurs as
`a discrete complex in solution.
`Electron micrographs of chromatin
`compatible with a jointed
`also
`are
`struLcture. Chromatin fibers observed
`after critical point drying have a gen-
`(25).
`"knobby"
`appearance
`erally
`Spray-mounted and shadow-cast speci-
`mens show "nodules" alternating with
`thin strands, although the nodules are
`often widely spaced and are absent
`from some preparations (26). Striking
`examples of micrographs showing alter-
`nate thick and thin regions were pub-
`lished (27) while this manuscript was
`in preparation. In these micrographs,
`which were obtained by formaldehyde
`fixation and positive or negative stain-
`ing, the thick regions are quite closely
`spaced and have a beadlike appear-
`ance. These regions were suggested to
`in contrast
`contain all
`five histones,
`with the arrangement of histones and
`DNA suggested above (28). Of course
`electron micrographs alone say nothing
`of the locations of particular molecules.
`But it may be possible, for example by
`selective extraction of histones
`(23)
`and nuclease digestion, to relate some
`features of the micrographs to par-
`ticular histones and to DNA.
`
`Summary
`
`Many lines of evidence on chro-
`matin structure have been discussed.
`The essential facts are:
`1) Chromatin contains roughly one
`of each type of histone per 100 base
`pairs of DNA, except for histone Fl.
`2) X-ray patterns reveal a structure
`repeating along the length of the chro-
`matin fiber. F2A1, F3, F2A2, and F2B
`are required in this structure, but Ft
`is not.
`3) Two each of F2A1 and F3 com-
`bine to form a tetramer.
`4) Certain nucleases cleave almost
`all the DNA in chromatin to pieces of
`about 200 base pairs.
`5) Chromatin fibers are often exten-
`sively coiled or folded.
`These facts lead to two proposals:
`1) Chromatin structure is.based on
`
`The full significance of the repeating
`unit of histones and DNA may lie in
`the relation of the units to base se-
`quences in the DNA. It may be asked,
`for example, whether there is a specific
`phase relation between the units and
`base sequences in the DNA. In other
`words, do the 200 base pair pieces
`arising from endonuclease digestion of
`chromatin form a unique set with re-
`spect to base sequence or do they over-
`lap in sequence (21)?
`
`A Flexibly Jointed Chain of
`Repeating Units
`
`My views on the arrangement of
`histones and DNA in the repeating unit
`are speculative and meant to be taken
`as a working hypothesis. The basic idea
`is that a chromatin fiber is a flexibly
`jointed chain of repeating units. The
`point is that a jointed structure may be
`the underlying DNA,
`flexible
`as
`as
`whereas a continuous structure, such
`as a helix, is not. The idea arises from
`the fact that a chromatin fiber is flexi-
`ble enough to be extensively coiled or
`folded. Such coiling or folding must
`occur, for example, in the bands of
`polytene chromosomes of Drosophila,
`where the ratio of length of DNA to
`length of DNA-containing structure is
`an order of magnitude greater than in
`a chromatin fiber (22).
`A possible arrangement of histones
`and DNA in the repeating unit, leading
`to a jointed structure, is as follows.
`The (F2A ) 2(F3) 2
`forms
`tetramer
`the core of the repeating unit [this is
`suggested by the essentially globular
`nature of the tetramer (1), the con-
`servation in amino acid sequence of
`F2Al and F3, and the fact that these
`histones are the last to be removed
`from chromatin by mild methods of
`(23) ]. F2A2 and F2B
`extraction
`spacing
`of
`determine
`the
`tetramers
`along the length of the chromatin fiber,
`perhaps as F2A2-F2B dimers, or as an
`F2A2-F2B polymer running alongside
`[suggested by x-ray experiments show-
`ing that tetramers and F2A2-F2B olig-
`omers act together to form a structure
`repeating at regular intervals along the
`length of the fiber (see above)]. Much
`of the 200 base pairs of DNA in a re-
`peating unit would follow some path
`on the tetramer, and the remainder of
`the DNA would connect
`tetramers
`along a path defined by F2A2 and
`F2B. In brief, I suggest that a chro-
`matin fiber consists of tightly packed
`protein
`DNA and associated
`alter-
`
`calf thymus (9), 0.40 in Drosophila
`(11), and 0.52 in pea bud (12); thus
`there is one Fl for two each of the
`other histones, or one Fl for every re-
`peating unit.
`1 The repeating structure formed by
`DNA and all the histones except Fl
`gives rise to the x-ray pattern of chro-
`matin (see above). It may be asked
`whether the quantities of histone and
`DNA in the repeating unit, inferred
`from biochemical evidence (see above),
`are compatible with the size of the
`repeating unit indicated by the x-ray
`pattern. The answer may be seen by
`taking the dimensions of the repeating
`unit from the x-ray pattern and elec-
`the
`with
`together
`microscopy,
`tron
`proportion of chromatin in the repeat-
`ing unit from additional x-ray data.
`The x-ray pattern, as mentioned above,
`shows bands corresponding to structure
`repeating along the length of the chro-
`matin fiber at intervals of about 100 A.
`Electron micrographs generally show
`fiber diameters of about 100 A (17).
`This suggests a repeating unit about
`100 A long in the fiber direction and
`about 100 A in diameter. The x-ray
`pattern disappears when the chromatin
`concentration is raised above about 45
`percent by weight (3, 5); this sug-
`are packed as
`that the fibers
`gests
`closely as the structure permits when
`the concentration is about 45 percent.
`A unit 100 A long and 100 A in diam-
`eter which is 45 percent by weight in
`chromatin upon close-packing contains
`2.8 x 105 daltons of chromatin (18).
`of
`each
`equivalent
`is
`This
`2.3
`to
`F2A1, F3, F2A2, and F2B and 230
`base pairs of DNA. Thus, the repeat-
`ing unit inferred from biochemical evi-
`dence and the repeating unit that gives
`rise to the x-ray pattern may be the
`same (19).)
`g Some indication of the unit of pack-
`aging of histones and DNA might be
`expected in studies of events requiring
`at least partial unpackaging, such as
`DNA replication. Kriegstein and Hog-
`ness (20) have suggested that the rate
`of movement of DNA replication forks
`in eukaryotes is limited by a process
`involving the histones. As discontinu-
`ous DNA synthesis in Drosophila pro-
`steps of about 200 bases
`ceeds in
`(Kriegstein and Hogness show that the
`single-stranded gaps at replication forks
`and the fragments of newly synthesized
`DNA in Drosophila are about 200 and
`150 bases), the rate-limiting process
`could well be unpackaging of units of
`two each of four of the histones and
`about 200 base pairs of DNA.
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`a repeating unit of two each of F2A1,
`F3, F2A2, and F2B and about 200
`base pairs of DNA.
`2) A chromatin fiber
`consists
`of
`many such units forming a flexibly
`jointed chain.
`
`References and Notes
`l. R. D. Kornberg and J. 0. Thomas, Scietnce
`184, 865 (1974).
`2. M. H. F. Wilkins, Cold Spring Harbor Synip.
`Quant. Biol. 21, 75 (1956);
`, G. Zubay,
`H. R. Wilson, J. Mol. Biol. 1, 179 (1959).
`3. V. Luzzati and A. Nicolaieff, J. Mol. Biol.
`7, 142 (1963).
`4. B. M. Richards and J. F. Pardon, Exp. Cell
`Res. 62, 184 (1970); E. M. Bradbury, H. V.
`Molgaard, R. M. Stephens,
`L. A. Bolund,
`E. W. Johns, Eur. J. Biochent. 31, 474 (1972);
`C. W. Carter and A. Klug, unpublished.
`5. R. D. Kornberg, A. Klug, F. H. C. Crick,
`preparation.
`in
`6. J. F. Pardon and M. H. F. Wilkins, J. Mol.
`Biol. 68, 115 (1972).
`7. D. R. Hewish and L. A. Burgoyne, Biochein.
`Biophys. Res. Commun. 52, 504 (1973); the
`extent of digestion and size of the pieces are
`from D. R. Hewish, personal conmmiiunication
`(the size was determined by velocity sedimenta-
`tion in alkali and should be regarded as only
`approximate).
`8. Such cleavage (M. Noll, of this laboratory,
`conflict with
`unpublished) would appear to
`the report [R. J. Clark and G. Felsenfeld,
`Nat. New Biol. 229, 101 (1971)] that about
`half the DNA in chromatin is converted by
`staphylococcal nuclease to acid-soluble form
`while the remaining half is converted to pieces
`of about 175 base pairs. I suggest the follow-
`ing way of accounting for all the staphylo-
`results. There may be two
`coccal nuclease
`classes of sites of nuclease action in chro-
`matin: sites between 200 base pair repeating
`
`units where nuclease
`rapid, and
`is
`action
`sites within the repeating units where nu-
`clease action is slow. Brief digestion would
`be expected to convert most of the chro-
`matin to pieces of about 200 base pairs of
`(the
`result
`DNA with
`associated
`protein
`quoted in the text). Further digestion would
`be expected to involve breakdown of some
`of the 200 base pair pieces and binding of
`the histones that are released to the remain-
`ing pieces. The digestion should continue until
`the binding of extra histone completely pro-
`tects the pieces that remain. This linmit should
`pieces
`be reached when about half of the
`remain (the result of Clark and Felsenfeld)
`since roughly twice the amount of histone
`naturally occurring in chromatin is required
`to neutralize all the negative charge on the
`DNA (on the basis of the amino acid com-
`positions of the histones and relative amounts
`in chromatin of histones and DNA).
`9. E. W. Johns, Biochem. J. 104, 78 (1967).
`10. S. Panyim and R. Chalkley, Biochenmistry 8.
`3972 (1969).
`11. D. Oliver and R. Chalkley, Exp. Cell Res.
`73, 295 (1972).
`12. D. M. Fanmbrough, F. Fujimura, J. Bonner,
`Biochemistry 7, 575 (1968).
`13. R. J. DeLange, D. M. Fambrough, E. L.
`Smith, J. Bonner, J. Biol. Chemn. 244, 5669
`(1969); L. Patthy, E. L. Smith, J. Johnson,
`ibid. 248, 6834 (1973).
`14. S. C. R. Elgin and J. Bonner, Biochemistry
`9, 4440 (1970).
`15. R. J. Hill, D. L. Poccia, P. Doty, J. Mol.
`Biol. 61, 445 (1971).
`16. J. Mohberg and H. P. Rusch, Arch. Biochem.
`Biophys. 134, 577 (1969).
`17. H. Ris and D. F. Kubai, Annuz. Rev. Genet.
`4, 263 (1970).
`hexagonal
`close-
`calculation
`18. The
`assumes
`packing of chromatin fibers, and densities of
`Fl-depleted chromatin [the material used in
`recent x-ray work (5)] and solvent of 1.50 and
`1.00 g/cm3.
`the repeating
`19. The close
`correspondence of
`inferred from biochemical and x-ray
`units
`evidence does not necessarily mean they are
`
`identical (there may, for example, be 11 of
`one unit for every 10 of the other), and
`further work is needed to determine the exact
`relation between them.
`20. H. J. Kriegstein and D. S. Hogness, Proc.
`Natl. Acad. Sci. U.S.A. 71, 135 (1974).
`21. The matter is complicated by the occurrence
`of
`repeated
`sequences
`in
`most
`eukaryote
`DNA. The most convenient choice for testing
`would be the genomne of a small virus, such
`as polyoma or SV40. The histone-associated
`forms of these genomes should be cleaved
`pair
`by
`about
`25
`base
`pieces
`200
`into
`It should then be
`staphylococcal
`nuclease.
`possible-for example, by the use of restriction
`enzymes-to determine whether these pieces
`are of only 25 or else a very large number
`of types with respect to base sequence.
`22. The ratio of length of DNA to length of struc-
`salivary X chromosome
`of
`the
`in
`ture
`Drosophila
`melanogaster
`is about
`80 [W.
`Beermann, in Resuilts and Problemns in Cell
`Diflerentiation, W. Beermann, Ed. (Springer-
`1]. The
`Berlin,
`1972),
`vol.
`Verlag,
`4,
`p.
`ratio in a chromatin fiber is about 6.8 (based
`on 200 base pairs or 680 A length of DNA
`in about 100 A length of fiber).
`Ilyin,
`Varshavsky, U. N.
`Ya.
`23. Y. V.
`A.
`Mickelsaar, G. P. Georgiev, Eur. J. Biochem.
`22, 235 (1971).
`24. M. Noll, unpublished result.
`25. S. Bram and H. Ris, J. Mol. Biol. 55, 325
`(1971).
`26. H. S. Slayter, T. Y. Shih, A. J. Adler, G. D.
`Fasman. Biochemnistry 11. 3044 (1972).
`27. A. L. Olins and D. E. Olins, Science 183, 330
`similar micrographs have been ob-
`(1974);
`tained by Dr. J. T. Finch of this laboratory.
`28. The beadlike thick regions are observed to
`be about 70 A in diameter. This is compatible
`with these regions consisting of a globular
`(F2Al)j(F3), tetramer (diameter 40 to 50 A)
`covered by DNA (double helix diameter about
`20 A).
`29. I thank Drs. A. Klug, F. H. C. Crick. and
`M. S. Bretscher for helpful discussions and
`Drs. F. H. C. Crick, A. Klug, and S. Brenner
`for criticism of the manuscript.
`
`Budget and the National Cancer
`Program
`
`National Cancer Institute funding through grants and
`contracts for 1972 to 1974 is presented and discussed.
`
`Frank J. Rauscher, Jr.
`
`Since its creation in 1937, the Na-
`tional Cancer Institute (NCI) has been
`the primary agency through which the
`federal
`supported
`has
`government
`cancer research. The National Cancer
`Act of 1971 gave the NCI responsi-
`bility for conducting a much broader
`National Cancer Program with the goal
`of bringing cancer under control. The
`act specifically directs the director of
`24 MAY 1974
`
`the institute to "plan and develop an
`expanded, intensified, and coordinated
`cancer research program encompassing
`the programs of the National Cancer
`Institute, related programs of the re-
`search institutes, and other Federal and
`non-Federal programs." This mandate
`includes support for cancer research in
`industry and in countries outside the
`United States.
`
`The purpose of this article is to pre-
`sent accurate information on funding
`from the NCI for fiscal years 1972,
`1973, and 1974, and, in particular, to
`compare dollars allocated through the
`grant and contract mechanisms for
`these years. All figures for 1972 and
`1973 are actual obligations, whereas
`those for 1974 are estimates. Because
`of the lateness of the present fiscal
`year and the concomitant firmness of
`spending plans these estimated 1974
`figures, with the possible exception of
`those for training grants, will vary only
`slightly.
`The National Cancer Act was signed
`into law by the President in December
`1971. In fiscal year 1971, prior to this
`enactment, the total budget available
`to the NCI was $233 million. In 1972
`this was increased by $145 million to
`$378 million. In 1973 the Congress
`authorized $492 million but the NCI
`was permitted to spend no more than
`$432 million in accordance with the
`Administration's overall spending plan.
`Recently, the President decided to spend
`
`The author is director of the National Cancer
`Program, National Cancer Institute, at the Na-
`tional Institutes of Health, U.S. Public Health
`Service, Department of Health, Education, and
`Welfare, Bethesda, Maryland 20014.
`
`871
`
`Merck Ex. 1103, Pg. 4
`
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`Chromatin Structure: A Repeating Unit of Histones and
`DNA
`Roger D. Kornberg (May 24, 1974)
`184
` (4139), 868-871. [doi:
`Science 
`10.1126/science.184.4139.868]
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