Chromosomal proteins and chromatin structure.

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1975.

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CHROMOSOMAL PROTEINS AND

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Annu. Rev. Biochem. 1975.44:725-774. Downloaded from www.annualreviews.org by Kansas State University on 07/17/14. For personal use only.

CHROMATIN STRUCTURE Sarah C. R. Elgin Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138

Harold Weintraub Department of Biochemical Sciences, Princeton University, Princeton, New Jersey 08540

CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . THE HISTONES ................................................ '.' . .. ........ Primary Structure. . ..... .......... . . . . . . . ... . .. . .. . ..... .... ... ..... . . .. ...... . Tissue, Species, and Developmental Specificity. . ...... .. .. . . ... .. .. ...... .. . .. ........ The Histones of Primitive Eukaryotes and Animal Cell Viruses. . ........ ....... .. .... . THE HISTONE GENES. . ...... ........ . . .. .. . .... ... .. .... . . ...... . ... .. . .... Histone Genes are Repetitious and Clustered. Sequence Homology. ................. .. . .. . ........ ..... . . .. .... . . . . . ... . . . .... Evolution and Inheritance of the Histone Genes. ....... . . . . ..... ... .. . .... . . ...... .... TIJE NONHISTONE CHROMOSOMAL PROTEINS.. .. . . . ..... ...... . . .. ... . ..... Definition and I solation. . .......... . . . .. . .. . ..... .

726 726 728 732 733 734 734 735 736 737

. . ....... . . ......... .

737

Chemical Characterization. . .. .. ................. . . .. .. ... ..... ....... ....... . ... .

738

Tissue Specificity. . .. ....... ....... ..... ........ .. . . ............ ... ...

740

Biological Roles.

. . .. ... . . ..... . . .... ..... . . ............

THE METABOLISM OF CHROMOSOMAL PROTEINS. . . .... .. ... .. . .. . ........ . Histone Synthesis............. ... ....... .... .. ....... .. ... .. ..... ... .. ..... ... Nonhistone Chromosomal Protein Synthesis. . .......... .. . . . .. . . . . . . . . .... ... . .. . Replication of Chromatin. . . ........... .. ........ .... .. ... ..... . . . . . ...... .. ... .

738 743 743 745 745

Other Chromosome Forms...... . . . ...... ...... ... ... . ..... .. .. . . ......

745

Turnover and Dife f rential Synthesis of Chromosomal Proteins. ... . .......... ... ... .... .. . .

746

Modifications of Chromosomal Proteins. .................... . . ..... ...... .... ...... . .

747

CHROMATIN STRUCTURE.......... . ..... .. . .... . . ... .. ..... ....... . . ...... . Periodicity of Histone Along the DNA Fiber. . .. . . . ...... . . . .. . . . . . . . ..... . . . . ...... . Electron Microscopy. . ......................... .. .. ... . . .. .. .. .. ...... ... ....... . Nu Body Structure. . .. . ....... .. .. ...... . . . . .. .. ...... . . ......... .. ... ......... Histone: Histone Interactions.. . . . . . . . . . . . . . . . ....... .. . ........ . .... ...... .. .. ... . ... . ... .. . ...... .. .. . .. ... . ..... .......... . Histone Accessibility. . ......

749

Conclusions.

. . . . . . . .. . .

751 753 754 757 759 760

TE MPLATE ACTIVITY..

...................... ' ... ... ..................

Histones as General Repressors .

760

. ..... ... . . . ...... ... .

760 761

. . .. .. . .......... .

. . . . .. . .... ..... ... . . . . ..... . ... ..... Chromatin Fractionation. . ........ . Model Systems. . .... .. . . .... ....... .... ... .... .. . . ..... .... . ... . .. .... . . . .....

CONCLUSIONS. .. . .... ........ . ...... .... .. . . .. ...... .... . . . .... .. .. . ... ....

763 764

725

726

ELGIN & WEINTRAUB


INTRODUCTION One important aspect of gene regulation during cellular differentiation in eukaryotes occurs at the level of gene transcription (for examples, see 1-4; see 5 for review). Transcription of eukaryotic chromatin is tissue specific and represents a small fraction of the total number of nucleotide sequences in DNA. This has now been established by examination of the products of both repetitious and unique gene sequences ; further, this specificity is preserved in chromatin isolated and transcribed in vitro (6-8). Most convincing are the in vitro results demonstrating tissue-specific control of transcription of the hemoglobin gene (9-13). The control of template activity must be encoded in some way by particular nucleotide sequences in DNA. It is possible that these sequences are read by specific activator or repressor proteins, as has been shown in prokaryotic systems (14-16). However, in the eukaryotic genome the effects of primary and higher order chromosome structures must also be considered. There is no a priori reason that there should be structural differences in the packaging of active and inactive regions of the genome. Selective gene transcription o r translation can in principle be accomplished by only "transacting" solubl e factors which modify the acti vi ty of the transcribing or translating machinery. That this is unlikely for all forms of gene control in higher organisms is best illustrated by the classical observations dealing with the inactivation of the X chromosome ( 17). Here it is quite clear that some structural change, as visualized in the light microscope, occurs with the loss of function of an entire chromosome. Examples of structural effects with cis control have been observed in Drosophila and are termed "position effects" (18, 1 9). In this instance the activity of a particular gene depends on its proximity to inactive heterochromatin and presumably on the structural environ ment established by the heterochromatin. These position effects often vary in the different cells of the organism. Structural changes as an early step in gene activation have been observed in the giant polytene chromosomes of Drosophila (20, 21). Our main purpose in mentioning these phenomena (and there are other examples) is to illustrate that at least some, and perhaps all, types of transcriptional controls in higher organisms have a structural basis, which is best explained as an altered DNA packaging pattern. The packaging pattern appears to be a consequence of the population of associated protein molecules. Thus we wish to consider the problem of differential gene expression from the point of view of chromatin structure. We will consider the chemistry of chromosomal proteins, some parameters influencing their interaction with DNA, basic repeating units of chromatin structure, and possible structure/function relationships in transcriptional control. THE HISTONES The his tones, the small basic proteins found in association with DNA, were thc first chromosomal proteins identified (22). The histones are major general structural proteins of chromatin and can act as repressors of template activity (see the later


Table 1

("'J

Characterization of the histonesa

� o Lys/Arg Ratio

Total Residues

Molecular Weight

N terminal

C terminal

HI (I, fl, KAP)

22.0

�215

-21,500

Ac-Ser

Lys

H2a (IIbl, f2a2,

1.17

129

14,004

Ac-Ser

Lys

2.50

125

13,774

Pro

Lys

Fraction

Class

Very lysine rich Lysine rich

ALG) H2b (IIb2, f2b,

a

H3 (III, f3, ARE)

0.72

135

15,324

Ala

Ala

H4

0.79

102

11,282

Ac-Ser

Gly

(IV, f 2at, GRK)

:::

� ;g ::J ;!l z

'"

KSA) Arginine rich

�

All data for histones of calf thymus. Compiled from (112) and (118) and the references cited therein.

;l>

Z o ("'J :r: :;d o ::: ;l>

:l z

�c:

�:;d

m

-.) IV -.)


728

ELGIN & WEINTRAUB

section on template activity). They have now been isolated, cnaracterized, and sequenced; their salient chemical characteristics and some of the different nomen clatures in use are summarized in Table 1. (We will use the Ciba Symposium nomenclature, H 1, H2a, H2b, H3, and H4.) There are a small number of different histones, as defined by amino acid sequence criteria. Studies to date have indicated that the his tones are very highly conserved proteins, there being very little variation in the amino acid sequences of histones from widely differing creatures. The histones are among the most highly modified proteins ; the modifications include acetylation, methylation, and phosphorylation. We do not know the functional consequences of these variations ; however, since the amino acid sequences are so highly conserved, such changes are likely to have significant effects on chromatin structure (23). Because the histones are encoded in the genome by repetitious DNA sequences, an additional dimension is involved in the problem of histone variability.

Primary Structure The sequences of the calf thymus histones are presented in Figures 1-5. Amino acid positions where substitutions have been observed are underlined ; deletions are indicated by an overline, and insertions by *. Sources of partial sequence data are indicated on the figure legends by a superscript p. Only apparent genetic polymorphisms (within the species) are marked for histones 1 and 2b ; genetic and evolutionary polymorphisms (all known substitutions) are marked for histones 2a, 3, and 4. Unless otherwise discussed, the amino acid substitutions are conservative ones. Post-transcriptional modifications are not shown. The conserved nature of the histones is apparent from the data presented. The estimated mutation rate of histone 4 is 0.06 per 100 amino acid residues per 100 million years, clearly the lowest mutation rate yet observed (23). Histone 3 is Histone 4

20

10

Ac-Ser-Gly-Arg-Gly-Lys-Gly-Gly-Lys-Gly-Leu-Gly-Lys-Gly-Gly-Ala-Lys-Arg-His-Arg-Lys-

30

40

Val-Leu-Arg-Asp-Asn-Ile-Gln-Gly-Ile-Thr-Lys-Pro-Ala-Ile-Arg-Arg-Leu-Ala-Arg-Arg-

50

60

Gly-Gly-Val-Lys-Arg-Ile-Ser-Gly-Leu-Ile-Tyr-Glu-Glu-Thr-Arg-Gly-Val-Leu-Lys-Val-

70

80

Phe-Leu-Glu-Asn-val-Ile-Arg-Asp-Ala-Val-Thr-Tyr-Thr-GlU-His-Ala-Lys-Arg-Lys-Thr-

90

100

Val-Thr-Ala-Met-Asp-Val-Val-Tyr-Ala-Leu-Lys-Arg-Gln-Gly-Arg-Thr-Leu-Tyr-Gly-Phe-

Gly-Gly-COOH

Figure 1

Calf histone 4 (38 1 -383). Comparative amino acid sequence data have been

obtained for H4 of rat (384), pig (385), bovine lymphosarcomaP (386), Novikoff hepatomaP (386), troutP (58), sea urchinP (387), and pea (382).

729

CHROMOSOMAL PROTEINS AND CHROMATIN STRUCTURE Histone

20

10

H2N-Ala-Arg-Thr-Lys-Gln-Thr-Ala-Arg-Lys-Ser-Thr-Gly-Gly-Lys-Ala-Pro-Arg-Lys-Gln-Leu-

40

30

Ala-Thr-Lys-Ala-Ala-Arg-Lys-Ser-Ala-Pro-Ala-Thr-Gly-Gly-Val-Lys-Lys-Pro-His-Arg-�50

60

Arg-Pro-Gly-Thr-Val-Ala-Leu-Arg-Glu-Ile-Arg-�-Tyr-Gln-Lys-Ser-Thr-Glu-Leu-Leu-Ile 80

70


Arg-Lys-Leu-Pro-Phe-Gln-Arg-Leu-Val-Arg-Glu-Ile-Ala-Gln-Asp-Phe-Lys-Thr-Asp-Leu-Arg 100

90

P he- G ln- Ser - s e r-A1a -V a1 -�-A1 a- L e u-G 1 n � G 1 u- A 1a-Cys - G 1 u- A1a -Tyr- L eu-V a 1- G 1 y - Le u-P he -

120

110

Glu-Asp-Thr-Asn-Leu-Cys-Ala-Ile-His-Ala-Lys-Arg-Val -Thr-Ile-Met-Pro-Lys-Asp-Ile-Gln130 Leu-Ala-Arg-Arg-Ile-Arg-Gly-Glu-Arg-Ala-COOH

Figure 2

Calf histone

3 (388-390). Comparative amino acid sequence data have been (391-393), carp (394), troutP (279), shark (395), DrosophilaP (S. C. R. Elgin, R. Goodfleisch, and L. Hood, unpublished observations), sea urchinP (396), mollusc (Patella)P (396), pea (397), and cycadp (396). obtained for H3 of chicken

Histone 2b

10 Calf

HN-Pro-Glu-Pro-Ala-Lys-Ser-Ala-Pro-----Ala-Pro-Lys-Lys-----Gly-Ser-Lys-Lys-Ala----

Trout

HN-Pro-G l x-Pro-Ala-Lys-Ser-Ala-Pro-------------Lys-Lys-----Gly-Ser-Lys-Lys-A1a----

Drosophila

HN-Pro-----Pro- --- -Lys-Thr -Ala- Gl y-L ys -Ala-Ala-Lys- Lys -Ala-Gly--------- L ys-Ala -Glx 20

30

Calf

-------Val-Thr-Lys-----Ala-Gln-Lys-Lys-Asp-Gly-Lys-Lys-Arg-Lys-Arg-Ser-Arg-Lys-Glu-

Trout

-------Va1-Thr-Lys-Thr-A1a-Gly-Lys-

Drosophila

Lys-Asx- Ilu-Thr-Lys-Thr----Asx-Lys-L ys-

Calf

Ser-Tyr-Ser-Val-Tyr-Val-Tyr-Lys-Val-Leu-Lys-Gln-Val-His-Pro-Asp-Thr-Gly-Ile-Ser-Ser

calf

Lys-A1a-Met-G1y-I1e-Met-Asn-Ser-Phe-Va1-Asn-Asp-I1e-Phe-G1u-Arg-Ile-A1a-G1y-Glu-Ala

Calf

Ser-Arg-Leu-Ala-His-Tyr-Asn-Lys-Arg-Ser-Thr- Ile-Thr-Ser-Arg-Glu-I 1 e-Gln-Thr-Ala-Val

Calf

Arg-Leu -Leu-Leu-Pro-Gly-Glu-Leu-Ala-Lys -H is -Ala-Val- Ser-Glu-Gly-Thr-Lys-Ala-Val-Thr

40

50

60

70

80

90

100

120 Calf

Lys -

Figure 3

110

125

Tyr-Thr-Ser-Ser-Lys-C OOH

Calf, troutP, and DrosophiiaP histone 2b (279, 398; S. C. R. Elgin, R. Goodfleisch,

and L. Hood, unpublished observations).


730

ELGIN & WEINTRAUB

also highly conserved, whereas H2a and H2b have evolved at a more rapid rate. Comparative gel electrophoresis of the reduced and oxidized forms of H3 has shown that many mammals, including rodents and those more highly developed, have a major H3 component containing two cysteines, while all other eukaryotes have only H3 containing one cysteine (24-26). In all cases examined this alteration represents the replacement of cysteine at position 96 with serine (see references of Figure 2). Originally it was thought that this replacement was a significant change in the histone sequence. However, physical chemical evidence indicates that the cysteine at residue 96 is buried in the interior of the molecule ; consequently, replacement with serine is a conservative change (27). H istone 1 is the most divergent histone, both in terms of the number of sub fractions within any given tissue and species and in terms of its evolution. From one to eight subfractions of histone 1 (different amino acid sequences) have been observed in various species. Within a species the quantitative amounts of the sub fractions will vary in different tissues (28-34). Post-translational modifications also occur; see the section on metabolism of chromosomal proteins. The available data suggest that histone 1 may vary as much as 15% by amino acid substitutions among the subfractions of a given organism. The comparison between rabbit and trout HI (Figure 5) suggests a variation of 15-28% between species. In both instances the substitutions are not conservative; they frequently involve interchanges of lysine, alanine, proline, and serine. Some of the substitutions affect functional sites. For example an alanine/serine substitution is observed at position 37 ; this serine is known to be specifically phosphorylated by a cAMP-dependent enzyme following hormone stimulation in the rat liver (35, 36). In addition to simple substitutions, there is also considerable heterogeneity in the size of this histone, both from a given Histone 2a

20

10

....--. . Ac�Ser-GIY-Arg-GIY-Lys-Gln-Gly-�-Lys-Ala-�-Ala-LYs-Ala-Lys-Thr�Arg-Ser-Ser-Arg 30

40

Ala-GlY-LeU-Gln-Phe-Pro-Val-Gly-Arg-Val-His-Arg-�-Leu-Arg-Lys-Gly-Asn-Tyr-Ala50

,...--,

60

�-Arg-val-GlY-Ala-GlY-Ala-Pro-val-Tyr-Leu-Ala-Ala-Val-Leu-Glu-Tyr-Leu-Thr-Ala70 80 .---. Glu-Ile-Leu-Glu-Leu-Ala-Gly-Asn-Ala-Ala-AIg-Asp-Asn-Lys-Lys-Thr-AIg-Ile-Ile-Pro90

100

Arg-His-Leu-Gln-Leu-Ala-Ile-Arg-Asn-Asp-Glu-Glu-Leu-Asn-Lys-Leu-Leu-Gly-�-val-

110

120

Thr-Ile-Ala-Gln-Gly-Gly-Val-Leu-Pro-Asn-Ile-Gln-Ala-Val-Leu-Leu-Pro-Lys-Lys-Thr-

�

"" 129

Glu-'Ser-His-Hi -Lys-Ala-Lys-GlY-Lys-COOH

Figure 4 Calf histone 2a (399, 400). Comparative amino acid sequence dat� have been . obtained for H2a of rat (401) and trout (402).

731

CHROMOSOMAL PROTEINS AND CHROMATIN STRUCTURE

*

Ac-Ser-G I u-AI a-Pro-�-�-Thr-AIa-A Ia-Pro-AI a-Pro-AIa-ffiU'-Lys-Ser-Pro-'A I a-Lys bY2.llli-Lys-

Trout

____,unknown __ ______________________________

Rabbit-3

AIa-�-Lys-Lys-Pro-Q!...t-AI a-GI V-AIa-AI a-Lys-Arg-Lys-A r a-�-GI y-Pro-Pro-Va I-Ser-Gr u-Leu- r I u-

�

�

unknown__________ _________________

Trout


20

10

*

Rabblt-3

�

�

Rabb It-3

Thr-Lys-AI a-VaI-AI a-AI a-Ser-Lys-G I u-Arg-Asn....G . I y-Leu-Ser-Leu-A I a-A r a-Leu-Lys-Lys-AI a-Leu-AI a-

Trout

__________________________ -'Arg-Ser-GIv-VaI-Ser-Leu-A I a-AIa-Leu-Lys-Lys-Ser-Leu-AI a-

Rabb It-3

AIa-Q!y-GI y-Tyr-Asp-Va I-GI u-Lys-Asn-Asn-Ser-Arg-II e-Lys-Leu-GI y-Leu-Lys-Ser-Leu-VaI-Ser-Lys-

Trout

AIa-GI y-GI y-Tyr-Asp-VaI-GI u-Lys-Asn-Asn-Ser-Arg-VaI-Lys-II u-A I a-VaI-Lys-Ser-Leu-Va 1-Thr-Lys-

Rabblt-3

Gly-Thr-Leu-VaI-GI u-Thr-Lys-GI'1-Thr-GIV-Alo-Ser-GI y-Ser-Phe-Lys-Leu-Asp-Lys-Lys-AIa-AIa-Ser-

Trout

GI y-Thr-Leu-Vol-GI u-Thr-Lys-GIy-Th r-GIV-AIa-Ser-GI y-Ser-Phe-Lys-Leu-Asn-Lys-Lys-A 10---------

�

�

�

100

110

I�

I�

Rabblt-3

GI y-GI u-A t a-Lys-Pro-Lys-Pro-----lys-Lys-A I a-G I V-A Ia-AIa-Lys-Pro-Lys-Lys-Pro-AIa-GIV-AI a-A 1 a-

Trout

Va I-G I u-A I a-Lys-----Lys-Pro-AI a-Lys-Lys-AI a-----A I a-A I a-----Pro-Lys-At a-Lys-Lys-Va I-At a-At a-

Rabblt-3

Lys -Lys- Pro-At a-Gt v-A t a,At a, Lys,At a,Pro, (Thr ,Pro,Lys) (Va I -A I a-----LyS) (Lys-AI a-Va I -Lys) (At a-Lys

Trout

Lys-Lys-Pro-AIa-AI a-----AI a-Lys-AIa-Pro------Lys-Lys--Val-AIa-AI a-Lys--Lys-Ala-Vol-AI a--AI a-Lys-

Rabblt-3

I� r� Lys) (Ser-Pro-Lys) (Lys-AI a-Lys) (Lys-Pro-Lys) (AIa-Pro-Lys) (Ser-AI a-AI a-Lys) (Sar-Pro-AI a-Lys-

Trout

Lys--Ser-Pro- Lys--Lys-AI a-Lys--Lys-Pro-A 10--Thr-Pro-Lys-- Lys-Ala-A la-Lys--Ser-Pro-Lys-Lys-

Rabb It-3

Pro-----Lys) (AIa-AI a-Lys-Pro-Lys-AI a-Pro-Lys-Pro-Lys) (----AIa-A I a-Lys-) ILys) (AIa-AI a-Lys) (�-Pro-

Trout

AI a-Th r-Lys--AI a-AI a-Lys-Pro-Lys-AIa-A r a-Lys -Pro-Lys --Lys-AI a-A! a-Lys- (Lys--AIa-A I a-Lys)-Ser-Pro-

Rabb It-3

AI a-Lys) (AI a-VaI-Lys-Pro-Lys) (AI a-AIa-AIa-Lys-Pro-Lys) (AI a-AI a-Gly-AI a-Lys) (Lys-Lys-COOH

Trout

Lys-Lys------Va !-Lys-Lys-Pro--A Ia-AIa-AIa-----------------------------------Lys-Lys-COOH

140

Figure 5

150

180

190

210

Rabbit histone

I,

200

220

subfraction 3 (34, 403; M. Hsiang and R, D, Cole, personal

communication; M. Hsiang, C Largman, and R. D. Cole, personal communication) and trout histone I

(58; k

R, Macleod and G, R Dixon, personal communication), Where

overlaps are missing between tryptic peptides parentheses are shown; residues not yet sequenced are separated by commas,

organism and from different creatures (25, 37-39). The existence of subfractions in histone 1 makes it difficult to assign variability to polymorphisms per se, as opposed to varying expression of different subfractions represented in the genome, Some general conclusions may be drawn from considering the primary sequences of the histones. A prominent and early observation was that of a skewed distri bution of the basic residues in histones, In histones 2a, 2b, 3, and 4, a predominance of the basic residues occurs in the N-terminal region, with a secondary cluster of basic residues occurring again in the C-terminal region, the intermediate region being dominated by hydrophobic and acidic amino acids, The pattern is to some degree reversed in histone 1, with the dominant basic region at the C-terminal end, One may note a statistical dominance of a spacing of basic residues every fourth position rather than every third or fifth in the N-terminal region of the smaller


732

ELGIN & WEINTRAUB

histones. No characteristic spacing is noted for histone 1. The positioning of basic residues does not seem to be absolutely critical for the functioning of the histones; for example, a deletion of the peptide Scr-His-His has occurred in the C-terminal region of histone 2b of trout relative to that of calf. Some sequence homologies, generally of 4 to 10 residues, can be observed in direct comparisons of sequences from H2a, H3, and H4 and again for sequences in H2b and H l (23). There is little information available on the secondary and tertiary structure of the individual histones. Physical and conformational studies, including estimates of a-helices and fJ-structures, have been reviewed by Bradbury & Crane-Robinson (40). The considerable variability in post-synthetic modification may be contributing to our failure to obtain histone crystals for X-ray diffraction analysis. Perhaps the unit cell will have to be the chromatin subunit, discussed in the section on chromatin structure.

Tissue, Species, and Developmental Specificity Many years ago, Stedman & Stedman (41) proposed that tissue-specific histones would be observed and that the interaction of these proteins with the DNA in a specific manner would lead to differential repression of gene activity. This early prediction was based on the observation of specific histones in two terminally differentiated cell types, histone 5 in chick erythrocytes and protamines in sperm. However, these are the only two salient examples of tissue-specific histones that have been observed. Histone 5, found in the nucleated erythrocytes of birds, amphibians, and fish, has been partially sequenced (42, 43). The appearance of histone 5 per se does not correlate well with repression of RNA synthesis. However, recent data indicate that newly synthesized histone 5 is phosphorylated, ::tnd the subsequent appearance of dephosphorylated H5 does correlate well with the decrease in RNA synthesis in developing erythroid cells. Thus it seems likely that this histone plays a critical role in establishing and maintaining the highly repressed state of chromatin in erythrocytes (44-47, 457). It is not clear why a specific histone is used for this purpose when simpler mechanisms for inactivation would be adequate. Major his tones specific to meiosis have been reported (48-49a). During spermato genesis the male gametes undergo a condensation of the chromatin, frequently becoming associated with other small basic proteins instead of or in addition to the somatic histones. A number of these proteins have been isolated and characterized ; examples with up to 75% arginine (from fish) and 50% lysine (from mollusc) are known (50, 5 1 ; for review see 52-54). Some of these proteins from mammalian sperm contain a number of cysteine residues; disulfide linkages appear during the condensation process (55). The process of the transition from a cell with chromatin having associated somatic histones and nonhistone chromosomal (NHC) proteins to the mature sperm, with DNA associated with basic sperm proteins only, has been studied extensively in the trout by Dixon and his co-workers. The somatic histones are gradually removed from the DNA and replaced by the protamines. Histones 2a, 2b, 3, and 4 are removed from the DNA by a process that involves acetylation followed by limited proteolysis ; at the end of the process histone 1 is displaced by competition from the protamine, which is present in increasing con-


733


.

centration. The level of nonhistone chromosomal proteins is reduced to very low amounts (56-58). There is no information concerning removal of the protamines from the DNA following fertilization of an egg by the sperm. With these exceptions, virtually no qualitative changes in the histone fraction have been documented as events in tissue differentiation (e.g. 59). Recently, studies have been carried out with antibody techniques to examine the question of tissue and species specificity of the histones. In all instances studied the tissue specificity has been of lower order than the species specificity, as judged by the antibody-antigen interactions (60-63). Species-specific histones are noted only rarely in the higher organisms, always as minor components. A good example, HT from the trout, has been isolated and partially characterized by Dixon and his colleagues. HT is �O.5% of the trout histone fraction (64, 65). Some interesting patterns of quantitative alteration in the histone complement have been reported, particularly regarding the subfractions of histone 1. Regular somatic histones are synthesized very early in embryogenesis from both maternal and newly transcribed messenger (66-75). In the case of sea urchin this occurs as early as the first cleavage. Paternal, as well as maternal, genes are expressed (76). A specific histone I subfraction is synthesized in the early sea urchin embyro, and a different subfraction is observed (by polyacrylamide gel analysis) in the late or hatching blastula. Preliminary evidence suggests that this switch may be regulated at the translational as well as the transcriptional level (72, 74, 75, 77). A marked increase in the relative amount of H I during early embryogenesis has been reported for other systems (78, 79). Alterations in the pattern of synthesis of histone I sub fractions have also been observed as a consequence of hormonal stimulation of the mouse mammary gland (80-82). The functional consequences of these changes remain to be established.

T he Histones of Primitive Eukaryotes and Animal Cell Viruses Histones are not found in association with the DNA of prokaryotes in vivo (83, 84), although they are capable of forming regular complexes with bacterial and viral DNAs in vitro, as shown by X-ray diffraction and nuclease studies (85; T. Maniatis, personal communication). The histones of unicellular and other primitive eukaryotes have been studied to examine a possible role of histones in chromosome replication, mitosis, and meiosis, as contrasted to a role in cellular differentiation. Slime molds and fungi appear to have an incomplete complement of histones; commonly H2a and H2b are present, H I and H3 absent. However, negative evidence is not entirely convincing, particularly given the susceptibility of histones to proteases. Different but histone-like proteins are frequently observed in association with the DNA (86-91). Under the electron microscope yeast nucleohistone fibers appear to be organizcd as are those of higher creatures, despite the apparent absence of H I and perhaps other histones (92). Volvox and Euglena likewise appear to have only a partial complement of histones (93, 94), whereas Tetrahymena and Stylonychia appear to have all the normal histones of higher eukaryotes (95-97). Recently, information has been obtained on a protein associated with the DNA of Thermoplasma acidophilum, a mycoplasm that normally grows at 59°C, pH 2. The


734

ELGIN & WEINTRAUB

protein has the same mobility as histone 4 ; it contains 23 % basic amino acids, 20% acidic amino acids, and no cysteine, tryptophan, tyrosine, histidine, or methionine (D. Searcy, personal communication). It is possible that this protein, which is capable of stabilizing the DNA to heat denaturation, could be an evolutionary precursor of the histones or the lysine-rich nonhistone chromosomal proteins (see Table 3). The DNAs of the mammalian viruses SV40 and polyoma are associated with histones (98-100). It is thought that the basic proteins bound to the viral DNA in the capsid and in the nucleoprotein complex are his tones 2a, 2b, 3, and 4, on the basis of analyses reflecting amino acid labeling patterns, behavior on disc gel electrophoresis, column chromatography behavior, and tryptic peptide maps (101). It will be extremely interesting to see if these viral systems can be exploited to study the role of histones in repressing transcription of specific genes (see the section on template activity). THE HISTONE GENES

Histone Genes are Repetitious and Clustered One of the exciting discoveries of the past few years has been the finding that by repetitious genes in the eukaryotic DNA (102). This finding and the further accomplishment of isolation of the histone genes (103) have been made possible by the availability of histone mRNA in significant quantities, isolated primarily from early embryos during stages of rapid nuclear replication. Considerable evidence that the 7-12S mRNA so isolated does indeed code for the his tones has now been compiled; see the section on metabolism of chromosomal proteins. Recently it has been possible to obtain three purified subfractions of message, presumably coding for histones 1, 2a-2b--3 , and 4, respectively (104-106, 471). The histone mRNA subfraction designated C3 by Grunstein et al ( 1 06) is 370400 nucleotides long. This is an appropriate length to code for histone 4 assuming some processing or regulatory sequences. Data obtained from translation experi ments in cell-free systems indicate, and partial sequencing of this RNA is consistent with the hypothesis that this is the histone 4 messenger (106). Most studies on the histone genes have been carried out in the sea urchin. Studies of the histone genes of four different species of sea urchin indicated from 400 copies in Lytechinus pictus to 1200 copies in Psammechinus milaris of each histone gene (104, 105, 107). In the most extreme case, that of P. milaris, from 0.5 to 0.8% of the genome is thus required to encode the histone genes and their associated spacer DNA ( 103). While it is clear from cross species hybridization studies that all eukaryotic organisms examined to date contain multiple copies of the histone genes in their DNA (107), the numbers are generally lower: on the order of 50 to 1 00 copies for both Drosophila and mammals (M. L. Pardu e ; and M. Obinata and B. 1. McCarthy, personal communcations). The reiteration is characteristic of the portion of the messenger coding for the protein and not for some general processing sequence. In experiments examining the distribution of DNA sequences hybridizable with histone mRNA in cesium chloride gradients, the the histones are coded for



735

histone messages are all found to hybridize exclusively with a specific cryptic satellite DNA and not with the bulk of the DNA (102, 103, 106). Hybridization experiments utilizing purified messenger for individual histones indicate that about the same number of genes exists for each histone in a given haploid genome (104, 105). It seems likely that this number is also a constant for all tissues, including meiotic ones; this has been directly determined for sea urchin sperm and embryos (102). As noted above, the histone genes band together in cesium chloride and other density gradients. Hybridization assays with the individual messenger RNAs give the same distribution curves within thc dcnsity gradients (102-104). The GC com position of the individual messengers varies considerably, from 51 to 58% (105); thus the evidence suggests that the individual histone genes are interspersed among each other. Only one region of in situ hybridization, 2L 39D-E, is observed in experiments in which Drosophila melanogaster polytene chromosomes are hybridized with tritiated total histone mRNA (108). In experiments carried out with individual histone mRNA fractions, it appears that all the mRNAs hybridize throughout this region, which contains two large bands and possibly several small ones (M. L. Pardue, personal communication). Thus there is now some evidence that the genes for the ' smaller histones (2a, 2b, 3, and 4) are organized in a system of complex, tandem repeats in a fashion analogous to that of the ribosomal genes, although as yet there is no information on the regularity of the pattern. Questions concerning the

locus and distribution or interspersion of the H I genes are at present unresolved because of difficulty in obtaining pure HI mRNA. One might anticipate that special histones, such as H5 of reticulocytes or the histone of meiosis; will not be an integral part of the "locus" because of the differences in the regulation of their expression.

Sequence Homology Early studies on the hybrids formed by the annealing of DNA with homologous

histone mRNA indicated that the hybrids were of very high fidelity with little mismatch, as the RNA-DNA hybrids melted at the temperature anticipated for that GC composition (104). In general, it could be said that within a given species there was no indication of variance among the histone genes. Recently more sensitive assays have suggested that some variance is possible. The histone genes have now been isolated in the DNA form. Melting and re-annealing of these DNA sequences suggest from 1 to 3% mismatch (103). Evidence obtained in studying the histone mRNAs per se also suggests some variation. Fractionation of the histone messengers by polyacrylamide gel electrophoresis indicates several subfractions of each. For example, the messenger C (which codes for H4) includes three size subfractions. These subfractions show small differences in the oligonucleotide maps produced from them (106). However, it is not known whether this heterogeneity is a true indicator of gene heterogeneity, a consequence of genetic polymorphism within the population, an indicator of a precursor-product relationship, i.e. a processing of the mRNA through the three forms, or the consequence of artifactual degrada tion. Substantial random synonymous codon substitutions (t hird position base changes) have not occurred in these repetitious genes within a given species.

736

ELGIN & WEINTRAUB

Substitution at the theoretically possible level would lead to genes that hybridize as single copy rather than as multicopy genes (104). One may conclude that some preventive or corrective mechanism must intervene to conserve and maintain the coding sequence within a species.


Evolution and Inheritance of the Histone Genes Comparison of the histone genes at the nucleic acid level in closely and distantly related organisms indicates that, as in the case of the ribosomal genes, the histone genes of any given species are homologous whereas divergence has occurred during the evolution of species. The homology of the total histone genes of different organisms follows patterns anticipated in terms of relationships in evolutionary time established on other bases (109). It appears that the rates of divergence are greater for the AT-rich than for the GC-rich portions of the histone genes. It is thought that the AT-rich region represents spacer between the GC-rich histone messengers; thus the situation may be analogous to that of the ribosomal genes (103, 107). Note in addition that the evolution rate of the different histones at the amino acid level varies considerably. While estimates of the evolution rate are difficult to make from hybridization studies, the data at hand suggest that the rate

for the total histone genes is approximately one half that for total DNA (109, 1 10). The results support the notion that, while equivalent codon substitutions have certainly occurred, these changes have not accumulated at such a rate as to imply that they are entirely neutral. The histones represent a unique opportunity to study the inheritance and evolution of multicopy genes because they are the only proteins known to be so encoded. Amino acid sequence investigations are capable of much finer analysis than can be achieved with conventional DNA/DNA or DNA/RN A hybridization exper iments. Work is just now beginning on the study of histone mutants. Several have been discovered; these are presented in Table 2. In general, the genetics has not yet been examined. The h istone 5 variant appears to be inherited as a single allele; however, the investigation involvcd only one generation (42). In a second instance, where two subfractions of HI were studied, each variant showed independent segregation of Table 2

Histone

Known histone polymorphisms

Organism

Amino Acid Pos ition

Alternative Amino Acid

References

H3

Pea

96

alanine/serine

397

H3

Calf

96

cysteine/serine

H2a

R at

16

serine/threonine

H2a

Rat, calf

99

arginine/lysine

438; L. Patthy and E. L . Smith, person al communication 401 401; P. Sautiere, personal

H5

Chicken

15

arginine/glutamine

42

communication



737

the other (111). (Note that this observation supports the suspicion that the HI genes are not interspersed with the rest of the histone genes.) It is to be hoped that in the near future two types of experiments will provide further evidence on this question. In the first instance, it should soon be possible to generate data on the relative position of the specific histone messenger RNAs to each other on the DNA using electron microscopy techniques. In the second instance, searches are now under way in the laboratories of M. L. Pardue and others to find histone mutants (as temperature-sensitive lethals) in Drosophila. One can immediately envision genetic experiments that should shed further light on the inheritance of such mutations and on the speed with which these mutations could spread throughout the gene family. THE NONHISTONE CHROMOSOMAL PROTEINS Considerable progress has been made in the last few years in the isolation, the NHC proteins. These proteins play structural, enzymic, and regulatory roles in the chromatin complex. Here we will merely summarize the more salient conclusions of the last several years of work; for more detailed reviews see (23, 112-118). fractionation, and chemical and functional characterizations of

Definition and Isolation The nonhistone chromosomal proteins are defined as those proteins (excluding the histones) that isolate together with DNA in purified chromatin or chromosomes. This class of proteins probably overlaps, but is not identical with the classes of acidic nuclear proteins and nuclear phosphoproteins, which are also under intensive study (119-125). That most, if not all, of the NRC proteins isolated in chromatin are true constituents of the complex as it exists in vivo is now substantiated by considerable evidence (reviewed in 126). The major NHC proteins have proved to be difficult to isolate and study because of their tendency to aggregate with the histones and with each other. A number of protocols are in use or under develop ment but none have been found that are satisfactory in all aspects (total yield, recovery of all NRC proteins, complete separation of NRC proteins from DNA and histones, avoidance of harsh denaturing reagents, widespread applicability, possibility of further fractionation of the NRC proteins). The NRC proteins may be obtained in solution in 1 % sodium dodecylsulfate (SDS) from chromatin treated with dilute mineral acids to remove the histones (e.g. 127). Such preparations have been used extensively in comparative studies utilizing SDS disc gel electrophoresis. A frequently used, less denaturing method of preparation is to dissociate chromatin in 5 M urea-2 M NaCl, remove the DNA by hydroxylapatite chromatography or by centrifugation, and separate the histones and NHC proteins by hydroxylapatite or by ion exchange chromatography (e.g. 128). Such preparations are typically used in reconstitution experiments. See the reviews cited above for further discussion and references on isolation techniques. The class of NHC protein is considerably more complex than the histone class. The number of NHC proteins (individual polypeptide chains) observed on analysis

738

ELGIN & WEINTRAUB

by SDS polyacrylamide gel electrophoresis is usually on the order of 20-115, depending on the chromatin source and the resolving power of the analytical system. [The lower limit of ' detection at present using disc gel electrophoresis is 5 X 10 3 protein molecules per mammalian haploid genome (129}.J Fractionation and analysis suggest that about 1 5-20 major proteins comprise 50 to 70% of the NHC protein fraction on a quantitative basis (129, 130). �


Chemical Characterization The NHC proteins possess many chemical, physical, and biological properties that contrast sharply with those of the histones. The amino acid compositions of total NHC protein fractions generally indicate a ratio of acidic to basic residues of 1.2 to 1.6 (calculated without the consideration of amides) (121, 128, 131). The individual polypeptide chains range from 10, 000 to several hundred thousand daltons and from less than 3.7 to 9.0 in isoelectric point (e.g. 130, 132). Several subclasses and individual major NHC proteins have now been isolated and chemically character ized. These have been grouped accordingly and are presented in Table 3. In addition, several NHC proteins of rat liver have been purified by Douvas & Bonner (439). This listing is by no means exhaustive; as the work progresses it is anticipated that further subclasses will be· characterized. Class D, that of the lysine-rich NHC proteins, is particularly intriguing. Pre liminary sequence data indicate that the charge distribution within these proteins is irregular, as is the case for the histones, with a basic N-terminal region and an acidic region elsewhere in the molecule (133; G. H. Dixon and J. Walker, personal communication). The D proteins bind extensively to DNA with little effect on template activity ( 133, 134). Further studies of the chemistry of the NRC proteins are not only of intrinsic interest, particularly in studying peculiar proteins such as the lysine-rich NHC proteins, but also aid in devising further experiments and models of chromatin structure. Several methods that have been used extensively in the past few years to try to isolate and characterize specific nonhistone chromosomal proteins rely on their binding interactions with eukaryotic DNA. While the DNA column methodology holds promise for the future, there are many technical difficulties at present. Several recent studies indicate that some NHC proteins, particularly low molecular weight ones, and some nuclear phosphoproteins bind extensively and in species-specific fashion to DNA ( 125, 1 35-139). Membrane filter assays analogous to those developed to study the lac repressor also hold promise for studies of NHC protein/DNA interactions (140, 440).

Tissue Specificity In contrast to the histones, the nonhistone chromosomal protein fraction shows a limited tissue specificity. On comparative electrophoretic gel analysis most of the major nonhistone chromosomal proteins are observed in the NHC protein fraction from all tissues of an organism. A few tissue-specific proteins are observed, as' well as quantitative variations. Such analyses have been carried out using both one- and two-dimensional techniques, the latter in general utilizing charge electrophoresis or


Table 3

Major nonhistone chromosomal proteins Number of

Class Al

(Asx+Glx)/

Organism

Bands on

Molecular

and Tissue

SDS Gels

Weight

R a t liver

43,000

pI Z tl ("J ::c " 0

� > ::l z

f/l

1.1

rich)

S

;l

C

Sl

C

" m

-.l w .-.0


740

ELGIN & WEINTRAUB

isoelectric focusing in conjunction with SDS gel electrophoresis. The conclusion of limited tissue specificity appears valid for a number of nuclear protein prepara tions, including the nonhistone chromosomal proteins, the acidic nuclear proteins, the nuclear phosphoproteins, and the DNA binding proteins (125, 127, 128, 132, 141-149). For example, considerable similarities are seen in the gel pattern of the NHC protein fraction of rat liver and rat kidney, whereas the rat brain fraction shows a number of unique, high molecular weight proteins (128, 146, 150). Such limited tissue specificity is nonetheless compatible with this fraction having a role in the control of specific gene expression. This would be accomplished either if those elements involved in the specific regulatory mechanisms were present in small amounts, or if the controlling elements act in a combinatorial system, as suggested by Gierer (lSI). Studies of the nonhistone chromosomal protein fractions by im munological techniques often suggest a considerable amount of tissue specificity. This has been reported in particular for DNA-protein complexes of the low molecular weight NHC proteins which show high affinity for DNA (139, 152-155). Nonetheless, analyses indicate that the bulk of the nonhistone chromosomal proteins are common to all tissues, and thus imply that these proteins are involved in common structural and enzymatic roles. Biological Roles ENZYME ACTIVITIES

Many enzymes of chromosomal metabolism, including nucleic acid polymerases, nucIeases, and enzymes of histone metabolism, are integral com ponents of chromatin. For example, it is estimated that from 10 to 50% of the cell's RNA polymerase is bound to the chromatin in vivo (156, 157). A representative sample of enzyme activities that have been identified in isolated chromatin is given in Table 4. In addition, several proteins that bind to and affect the conformation of DNA have been isolated and characterized, including proteins that stabilize single stranded DNA (158-160) and that unwind superhelices (161, 162). Certain chromosomal structures, such as the meiotic synaptonemal complex, probably are made up of NHC protein elements. Since it has been observed that recombination frequencies in eukaryotic chromosomes can be both structurally dependent and genetically determined, it has been suggested that specific NHC proteins play a role in bringing about the chromosome pairing observed in meiosis, etc (163, 164); however, such proteins have not been isolated as yet. CHROMOSOME STRUCTURE AND TEMPLATE ACTIVITY

NHC proteins no doubt play general structural and enzymatic roles in the process of gene activation, and a subset may be involved in determining the specificity of transcription. A number of studies have been published in which the presence of nonhistone chromosomal proteins is observed to have a mitigating effect on the repression of template activity by the histones (e.g. 6 and 165; see 112 and 114 for a review of this question). Recent experiments in which chromatin is fractionated into DNA, histones, and NHC protein components and subsequently reassociated by salt-urea gradient dialysis implicate the NHC protein fraction as including positive regulators of gene


Table 4

Enzyme activities associated with chromatin Enzyme


1. RNA polymerase

Organism and Tissue

Reference

Rat liver

404, 405

Mouse myeloma

406

Hen oviduct

1 57

Coconut

407, 408

2. Poly(A) polymerase

Wheat

409

3. DNA polymerase

Rat liver

4 1 0-4 12

Rat ascites hepatoma

4 1 3, 4 1 4

Sea urchin

415

Calf thymus

416

4. DNA endonuclease 5. DNA l igase

HeLa cells

417

Rabbit bone marrow

418

6. DNase

Rat liver

419

7.

741

Terminal DNA nucleotidyltransferase -

8. Poly(adenosine diphosphate ribose)

Calf th ymu

Tobacco

s

420 42 1

Rat liver

422, 423

polymerase Rat liver

424

10. Histone acetyltransferase

Rat thymus

425-427

1 1 . Histone methylase

Calf thymus

43 1

1 2. Histone kinase

Rat Walker 256

433

9. Poly ADP-ribose glycohydrolase

(acid-labile phosphate) 1 3. Histone protease 14. N H C protein kinases

carcinosarcoma cells Rat liver

432

Rat liver

428, 44 1

Calf thymus

429

Rat liver

430

expression (12, 166). In all such work the test assay has been correct transcription of the hemoglobin gene as detected by hybridization of product RNA to a reverse transcript of hemoglobin mRNA. The NHC protein fractions used include small RNAs, which have also been suggested as positive regulators of gene expression (7, 167, 168). It is thought that the NHC proteins play an important role in the interaction of steroid hormones with target cell nuclei and the subsequent specific gene activation (e.g. 4, 169). However, a successful in vitro inductive system has yet to be established. [See (170) for a review of thi s field ] Hopeful ly, the use of mutant cells deficient in the activity of the cytoplasmic receptor, the ability to bind hormone in the nucleus, or in subsequent steps will help to resolve the many interesting questions. Such mutants of a lymphoma cell line, normally killed by glucocortoiGs, have been isolated by Sibley & Tomkins (171). The polytene chromosomes of Drosophila present a unique opportunity for COr,l bined biochemical and cytological analysis of gene activation. Specific bands puff either normally in development or in response to stimuli such as the hormone .

742

ELGIN & WEINTRAUB

ecdyson or heat shock. In puffing a band takes on a diffuse appearance and becomes the site of new RNA synthesis. A critical early event in the transition to the active state is a 100% increase in nonhistone chromosomal protein fit the puff locus ; no decrease in histone is observed (172-174). Early reports suggested that new, stimulus-specific nuclear proteins could be detected in this system (1 75, 176). Unfortunately, this could not be substantiated in a study of the polytene chromosomal proteins (459). This is not surprising considering the percentage of the genome involved, less than 1 %. Proteins as activators are implicated, however, by the finding that the induction of puffs occurring late in the hormone response Annu. Rev. Biochem. 1975.44:725-774. Downloaded from www.annualreviews.org by Kansas State University on 07/17/14. For personal use only.

can be blocked by inhibitors of protein synthesis (177). It is likely that the detailed

questions will remain unresolved until they can be studied with a more specific, restricted chromatin system, wherein a greater percentage of the genomic material undergoes the transition in question. See Berendes (20, 21) for a review of the

polytene chromosome system. Structural features are important clues to the mechanisms involved in chromo somal replication as well as transcriptional activity. Certain NHC proteins may play a major role in the shift from the inactive quiescent cell state (Go) to the active growing state (G]). Specific synthesis of certain NHC proteins is observed and may be required for this transition and the concomitant increase in template activity (178-182, 453). Comparative studies of the chromatins of Go and G] cells and of normal and transformed cells suggest that a fraction of NHC proteins dissociable in 0.25 M NaCI is responsible for the differences in the structural and functional properties of these chromatins ( 1 83, 1 84). See Baserga (455) for a review of this topic. Parallel observations on the quantity of certain NHC proteins in relation to the growth state have been made on a primitive eukaryote, Physarum. and cells from an advanced eukaryote, HeLa (181). LeStourgeon has recently suggested that myosin, actin, and a "tropomyosin" are major NHC proteins in Physarum poly cephalum ; an increase in the actin : "tropomyosin" ratio is observed to be character istic of both the metaphase and the quiescent nonproliferative Go states (185).

Actin and myosin-like filaments have previously been identified in the dividing cell and are thought to be important in movement of chromosomes (186, 466). LeStourgeon, on the basis pf th e quantitative observations, has suggested that these proteins play an additional role in chromosome condensation per se. NHC proteins with the molecular weights of actin and myosin have been observed by others as characteristic of mitosis (see the following section). Perhaps the use of fluorescent antibody techniques to study the distribution of chromosomal proteins in situ will resolve the question in the next few years. NUCLEAR MEMBRANE PROTEINS Chromatin as it is isolated contains some lipid, suggesting associated or contaminating nuclear membrane material (187-190). This is hardly surprising because much evidence indicates that the chromatin fibers are associated with the annuli of the nuclear membrane (e.g. 191, 192). Certain specific proteins may be involved in this membrane DNA interaction and as such may be legitimate chromosomal proteins as well as nuclear membrane proteins. The question remains to be resolved.


743

RNA TRANSPORT PARTICLE PROTEINS Since the early work of Georgiev and his associates, there has been a considerable effort to characterize the proteins of the RNA transport particles, or informosomes (see 193 for a more detailed review of early work). These particles are thought to assist in packaging, transport, and other wise processing of mRNA. The particles possess a major protein component of 40,000 daltons ; reports vary concerning the amount and characteristics of other proteins present (194-196, 454). Certain preparations, such as the lampbrush chromosomes of amphibian oocytes, are particularly enriched for these particles and it seems likely that such proteins will be major nonhistone chromosomal protein components in this instance (197-199). Fluorescent antibodies prepared to proteins from the Triturus oocyte nuclear ribonucleoprotein particles will specifically stain the loops of the lampbrush chromosomes ; in fact, an antibody preparation to a specific size protein selectively stains only 10 of the loop pairs ( 198, 2(0). The pro portion of such nonhistone proteins in chromatin may increase as a consequence of increased template activity in response to stimuli such as hormones (201). Such particles may turn out in some way to be locus or stimulus specific, as indicated by work in the polytene chromosomes (202). Studies concerning such proteins have been recently reviewed by Williamson (203).


�

THE METABOLISM OF CHROMOSOMAL PROTEINS

Histone Synthesis HISTONE mRNA TRANSCRIPTION It is now well established that the bulk of the histone synthesis is synchronized with DNA synthesis in the cell cycle. The relation ship has been tested in a number of cell systems synchronized by artificial or natural means, e.g. in Euplotes (204), synchronized tissue culture cells (205, 206), and the regenerating rat liver (207). This coupling of synthesis operates in both directions, in that an inhibition of DNA synthesis will depress histone synthesis, while an inhibition of histone synthesis will slow DNA synthesis to about half its former level (208-21 0 ; see below). There are also several reports of small amounts of histone synthesis at other stages of the cell cycle (e.g. 206, 2 1 1 ), and histone synthesis is known to be uncoupled from DNA synthesis in certain situations, e.g. erythropoeisis (21 2) and oogenesis (213). However, it is safe to conclude that the bulk of histone synthesis occurs in S phase during the replication of the chromatin. The observation of this fact has allowed experimenters to successfully isolate histone mRNA fractions from synchronized cells in S phase and from early embryos, which are undergoing rapid nuclear divisions. The histone mRNA has been separated from cellular RNA as a peak sedimenting between 7 and 12S on sucrose gradients (208, 214, 21 5). The mRNA fractions may be further separated for preparative as well as analytical purposes on polyacrylamide gels into three to five bands, as discussed above (106). That such RNA is indeed messenger for histones and is not significantly contaminated by mRNA for other proteins has been shown by its translation in cell-free systems. The presumptive messenger peak, isolated from synchronized HeLa cells in S phase, is able to direct the synthesis of all five histones in both the rabbit reticulocyte system and the


744

ELGIN & WEINTRAUB

mouse ascites cell-free system (2 14�216). Studies of tyrosine and tryptophan incor poration indicate that over 90% of the protein synthesized in the mouse ascites system is histone (214). The histone mRNA and the histone synthesizing system possess several interesting characteristics apparently oriented towards the production of a large amount of protein within a short time. At least 20,000 histone molecules are synthesized by the cell per minute in S phase (21 0). The fact that the histones are coded for by repetitious DNA sequences is obviously beneficial in producing a great deal of mRNA in a very short time. In addition, Schochetman & Perry (217) have demon strated that histone mRNA is processed and transported to the cytoplasm within several minutes of its synthesis, a very short time relative to that of other mRNAs. Histone synthesis is not inhibited by cordecypin (219) ; this is consistent with the observation that the mRNA appears not to contain the 3'-terminal poly(A) sequence characteristic of other mRNAs (2 1 8). The histone message is found in cytoplasmic polysomes that are free or loosely associated with membranes (220). Synthesis of the protein is initiated, as for other proteins, with the methionyl tRNAf(221). Current evidence suggests that this residue is cleaved from the nascent polypeptide chain, and the N-terminal serine is sub sequen tl y acetylated (222). The newly synthesized histones are transported quite rapidly (within 10 sec) into the nucleus (2 1 0). The extranuclear free histone pool is very low and is estimated to be 0.2% of the total (223). There appears to be no gross translational control exercised on the synthesis of histones. The histone messenger can be translated equally well in cell-free translation systems isolated from S phase cells and from G 1 cells ; the latter are not normally active in histone synthesis. Likewise, cells in which DNA synthesis has been inhibited-a condition that normally results in a loss of biologically active histone mRNA from polysomes-possess translational systems that can use histone mRNA with full efficiency (219, 224�226). HISTONE TRANSLATION

COUPLING OF HISTONE AND DNA SYNTHESIS The interacting controls of histone and DNA synthesis present a very intriguing system. The lifetime of histone mRNA in the cytoplasm is much shorter after inhibition or cessation of DNA synthesis, although histone synthesis is relatively resistant to inhibition by cycloheximide or actinomycin D (210, 225, 227, 228). The lifetime of histone mRNA in mouse L cells has been estimated to be roughly equal to S phase, in contrast to other mRNAs whose lifetime is estimated to be approximately equal to the cell generation time (229). The evidence suggests that a specific degradation of histone mRNA occurs in the absence of DNA synthesis (219, 226, 229, 467). Conversely, cycloheximide inhibition of protein synthesis during S phase causes an inhibition of 50% in the rate of DNA synthesis (210, 230, 23 1). It has been suggested that the histones play a role as chain elongation proteins in the eukaryotic chromatin (210, 232). The reduced rate of DNA synthesis would then be a consequence of the availability of only the parental, and no newly synthesized, histones. The simplest hypothesis to explain this control circuitry is that free histone (that not bound to DNA) inhibits further �


745

histone synthesis in a way that results in rapid histone mRNA degradation as well as inhibiting production of new histone mRNA (228, 233).

Nonhistone Chromosomal Protein Synthesis


The synthesis of NHC proteins stands in contrast to that of histones in that it generally occurs throughout the cell cycle (234-236), although some increase in the rate has been reported in G 1 in mammalian tissue culture cells (237, 238). More detailed studies have indicated that specific NHC proteins are synthesized at specific times in the cell cycle (239, 240).

Replication of Chromatin An intriguing problem which is just now beginning to be studied is chromatin replication. Whereas the transfer of the information in the DNA takes place by the well-known mechanisms indicated by the Watson-Crick structure of the DNA double helix, there is no obvious model for the replication of the information contained in the distribution of proteins on the DNA. The tight coupling of DNA and histone synthesis and the rapid binding of histone to newly synthesized DNA indicate that this replication of information takes place at the growing fork (233). However, the molecular mechanism remains obscure. It has recently been shown that following the inhibition of new histone synthesis with cycloheximide, the old histone becomes associated exclusively with only one of the daughter DNA double helices (233). Under conditions where protein synthesis is not inhibited, double label experiments indicate that new histone is associated with the newly made single strands of DNA. When cells are pulse-labeled con currently with H 3 -BUdR (5-bromodeoxyuridine) and C 14-leucine, the chromatin isolated and then denatured, the individual DNA strands separate but the histones are only partly removed. If the separated chromatin strands are irradiated to break the BUdR strand (which is then resolved from the parental strand by its lower sedimentation rate on sucrose gradients), one finds that newly made histone migrates together with the newly made BUdR strand of the DNA (24 1 ).

Other Chromusome Forms MITOSIS It is of interest to look for changes in the protein population that might be indicative of particular states of chromatin. The histones are extremely stable throughout the cell cycle and are synthesized mostly in tandem with the new DNA. It is not surprising, then, that the histones of mitotic chromosomes are essentially the same as those of the interphase chromatin (242). It has been suggested that histone 3 plays a role in condensing the chromosomes by forming intermolecular Cys-Cys disulfide bonds. However, as discussed above, one of the cysteines (position 96) is interior and nonreactive in the H3 of isolated chromatin (and can be replaced by serine), suggesting that at most only one cysteine (position 1 10) could be involved in such intermolecular linkages. Several studies of the question have found no evidence for an increase in the amount of histone 3 in a dimer or other oxidized form in metaphase chromosomes (243, 465 ; R. Chalkley, personal communication).

746

ELGIN & WEINTRAUB


A comparison of the NRC proteins of metaphase chromosomes with those of interphase chromatin shows that this protein fraction also remains much the same (1 26, 243, 244). Certain significant quantitative shifts in the NRC protein pattern . were found to be common to the metaphase chromosomes of ReLa and Chinese hamster ovary cells. These were a decrease in the amount of two 78,000--80,000 dalton proteins and an increase in proteins of 50,000 and 200,000 daltons in metaphase chromosomes. The latter NHC proteins thus could be involved in the metaphase chromosome condensation (see section on NRC proteins). A number of minor bands are observed in either G or M for each tissue (243). POLYTENY Polytene chromosomes are observed in certain tissues of Diptera, most notably Drosophila. By virtue of the differential underreplication of much of the repetitious DNA, they are greatly enriched in euchromatin (245). The chemical composition of the polytene chromosomes is similar to that of diploid chromatin, being reported as DNA : histone : NRCP : RNA of 1.0 : 0.97 : 1 .03 : 0.03 (246). The histones of the polytene chromosomes are identical with those of Drusophila diploid chromatin (38) ; R4 is relatively quantitatively deficient (459). Certain relative deficiencies are observed in the analytical gel pattern of the nonhistone chromo somal proteins. These deficiencies may indicate proteins normally associated with the highly repetitious and heterochromatic DNA, as this DNA is severely under represented in the polytene chromosomes ( 1 26).

Turnover and Differential Synthesis of Chromosomal Proteins The histones and NRC proteins stand in contrast to one another in their metabolic stability or estimated half-lives. Various labeling experiments indicate that the his tones have very long half-lives, on the order of that of the DNA (247-249), although exceptions to this are noted in particular circumstances (212, 250, 25 1 ). In general, the NRC proteins possess shorter half-lives, similar to those of the cytoplasmic proteins ; however, considerable variation within this fraction is observed (252-254). Alterations in the patterns of synthesis of the histones and the NRC proteins have been observed when new genes are being expressed. Such observations include (a) the new synthesis of specific NRC proteins in target tissues in response to hormones ( 124, 255, 256), (b) a general stimulation of the synthesis of NRC proteins and DNA binding proteins during the transition from a quiescent to a growing cell popUlation (see the previous section) (257, 258), and (c) changes in the NHC protein fraction during embryogenesis (79, 259-263). Alterations in the pattern of synthesis of histone 1 subfractions have also been observed and were discussed above. Unfortunately, most stimuli affect cell division rates as well as differentiation, so cause and effect relationships are difficult to sort out. An increase in the cell division rate will lead to an increase in the percentage of time a given cell spends in S and M ; thus one might expect to see an increase in proteins specifically associated with either of these states of the chromatin. Indeed, this is suggested in the case of alterations of the NHC protein pattern during early embryogenesis in Drosophila. In this instance the rate of nuclear division goes from once every 10 min to once \


747


every several hours or less during the 18 hr period covered by the study ; a con comitant decline in the amount of an NHC protein of 48,000 daltons is observed (79). Minor histones have been reported as associated with cells having higher or lower rates of cell division (264, 265). Rubio et al (266) have observed consistent changes in the DNA binding protein pattern for transformed and normal mouse cells, and such a difference for the NHC protein fraction has been confirmed using im munological techniques ( 1 55, 267). No such differences have been noted for the his tones (268-270). Much work remains to be done to sort out roles and cause and effect relationships in this field [see (53, 271, 272) for further review] .

M odijications of Chromosomal Proteins HISTONES

Considerable postsynthetic modification occurs in the chromosomal proteins, particularly in the histones. Among the unusual amino acids observed in histones are e-N-methyllysine in the mono-, di-, and trimethyl forms, w-N methylarginine, 3-methylhistidine, a-N-acetylserine, e-N-acetyllysine, O-phospho serine, O-phosphothreonine, N-phospholysine, and N-phosphohistidine. We will review the subject only very briefly here ; for more extensive discussion one may refer to other reviews (23, 58, 1 12, 1 18, 273-275). Here we wish to emphasize two aspects of histone modification. First, it is important to note the nontriviality of these modifications : they occur at specific times in the cell cycle and at specific sites on the histones as a consequence of the action of specific enzymes. In no case are all the histone molecules modified in the same way at the same time ; the percentage of histone modified can range from very little to almost all. Second, such modifica tions significantly alter the DNA-protein interaction. (Note that there is no evidence for the notion that such modifications can promote sequence specificity in the histonejDNA interaction.) It is of interest to contrast the extremely conservative amino acid sequence of the histones with the variability of the postsynthetic modification. Clearly if a conservative amino acid substitution, e.g. from leucine to isoleucine, is a forbidden one, the introduction of a methyl group or change of charge within the sequence must have a profound effect on some aspect of histone interactions. Both considerations combine to convince one that these postsynthetic modifications are important in determining the activities of the histones (23, 1 18). As the best studied example of histone modification, we will discuss here the case of HI phosphorylation. The phosphorylation of H I has been observed as a biochemical event of S phase, mitosis, and certain hormone responses. Several investigators have reported a large increase in H I phosphorylation during S phase, the time of most histone synthesis. A detailed analysis of histone modification during and immediately after their synthesis during trout spermatogenesis by Dixon and his colleagues indicates that considerable modification, phosphorylation, and/or acetylation occurs for all the histones and for the protamines at this time. They suggest that these modifications may aid in the transport and correct association of the histones with the DNA. In particular, Louie & Dixon (277) have proposed that during chromosome assembly histone 4 is "fitted" into its proper configuration with the DNA by a mechanism involving primarily histone acetylation. They suggest that acetylation


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will decrease the charge interactions between DNA and protein, and as a consequence a particular type of interaction will take place. After this association is achieved, deacetylation (which is observed) would allow electrostatic interactions which could "lock in" that conformation. Similarly, the phosphorylation of protamines may inhibit tight binding to DNA at a time when the chromatin is still being transcribed. The protamine is subsequently dephosphorylated, and presumably it is this form that is responsible for turning off the sperm nuclei (57, 58, 276-279). A similar mechanism appears to apply to the inactivation of avian erythrocyte nuclei in terms of histone 5 phosphorylation and dephosphorylation (see the section on the histones). It has been established in other diverse systems that phosphorylation of H I also occurs during metaphase (280, 281). In most eukaryotic cells, two to four sites of H I phosphorylation are present in S and additional sites are phosphorylated in M. The degree of overlap between the S and M phase sites is not completely known at this time, but it appears most likely that the sites of phosphorylation are independent (282). These phosphorylation sites are threonine as well as serine residues ; they are clearly different from those sites involved in H I phosphorylation in hormone response (283) (see below). A significant amount, half or more of HI, is phosphorylated in rapidly dividing cells (270). The increased phosphorylation at M is most likely the consequence of a six- to tenfold elevation of a specific ATP : histone phosphotransferase in the mitotic mammalian cells (284, 285). A rapid de phosphorylation of H I occurs as cells move into early G 1> suggesting that the phosphorylation ofH 1 is necessary for the condensed metaphase state (270, 286, 287). The slime mold P. polycephalum is particularly advantageous for studying this role of phosphorylation of H I , in that in the slime mold S phase and M phase are clearly separated in time. Using Physarum, Bradbury and his colleagues (288-290) have obtained intriguing results suggesting that the phosphorylation of histone 1, in response to a steady increase in HI phosphokinase levels, constitutes the mitotic trigger and leads to the condensation of chromosomes in this system. That the specific phosphorylation of H I is an early response to glucagon stimulation in the rat liver is now well established. The response is mediated by the glucagon-induced increase in cellular cAMP, which in turn stimulates the rate of histone phosphorylation. The cAMP effect can be d�monstrated in vitro with the isolated enzyme as well as in the rat and the isolated perfused rat liver (29 1-295). The H I phosphorylation is a very early event in response to glucagon, occurring in about 1 5 min, whereas enzyme induction occurs between 30 and 240 min (35, 292, 296). The enzyme responsible for this phosphorylation is clearly different from those discussed above, which are not affected by the cAMP level. The cAMP dependent enzyme phosphorylates H I at position 37. Thus two enzymes are clearly different, and it is likely that there are other enzymes showing specificities for histone substrates (35, 291-293, 297). Rat liver contains a phosphatase specific for phosphorylated histones and protamine. This enzyme has been found in all eukaryotic tissues examined, but not in any prokaryotic cells (298, 299). The existence of this complete and specific H I phosphorylation/dephosphorylation system has led to speculation concerning a possible role in genc activation.



749

That such modifications do affect the interactions of histone with DNA has been shown by physical chemical studies. Phosphorylated H I is eluted from a DNA cellulose column at lower salt concentrations than nonphosphorylated H I (S. Fisher and U. Laemmli, personal communication). Circular dichroism techniques show that the modified histones are less able to produce the conformational distortions of DNA that histones normally cause. This is true both of histone phosphorylation at thc specific sites discussed and for histone acetylation. A random change in the charge of HI is not effective in this regard (300, 301 ). Recently Watson & Langan (302) have presented evidence suggesting that H I and phosphorylated HI differ in their ability to repress template activities of chromatin following selective reassociation of these histone fractions to H I-depleted chromatin. It should be noted that in in vivo hormone stimulation, only 1 % of the total H I is phosphorylated. This is in accordance with expectations in that only a very limited portion of the genome should be activated in response to hormones. However, this makes it difficult or impossible to detect the physical chemical change, if any, in the total chromatin. NONHISTONE CHROMOSOMAL PROTEINS The only postsynthetic modification of nuclear nonhistone proteins that has been studied in detail is phosphorylation. It has been shown that within the mammalian nucleus there exist a large number of enzymes specifically responsible for phosphorylation and dephosphorylation of nonhistone proteins (including RNA polymerase and associated factors) at serine and threonine residues. There is evidence that, in parallel with the observations on histones, their activity and response may be highly specific ( 1 1 6, 254, 275, 303-3 12). Unfortunately, it has not been possible to correlate the nuclear phosphoproteins with the known nonhistone chromosomal proteins. Interesting questions remaining to be studied concern the relatedness of these two fractions, the binding coefficient to DNA of the proteins in their phosphorylated and nonphosphorylated forms, as well as the interaction of these proteins and enzymes with the histone component. See Kleinsmith (3 1 2) for a review of recent progress in this complex field.

CHROMATIN STRUCTURE The essential features describing the gross structure of interphase eukaryotic chromatin will probably be elucidated within the next few years. The field is developing rapidly and we will therefore try to anticipate many of the probable conclusions that are emerging as well as enumerate some of the basic questions that will probably remain for quite some time. Several extensive reviews and discussions of the literature regarding chromosome structure and protein-DNA interactions have been presented recently (40, 3 1 3-3 1 5, 442, 450). Consequently we will concentrate on the more important observations of the last few years. These observations have changed our concept of chromatin from that of a smooth, linear fiber dominated by a regular (3 1 6) or irregular (3 1 7) supercoiling of the DNA double helix, to one where the DNA folds around histone complexes spaced fairly regularly along the chromosome fiber. This "beads on a string" model is visualized in the electron micrograph shown in Figure 6. We do not yet know how the DNA

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of each particle is packed. Some elements of the supercoil model may be applicable to the arrangement of the DNA in these particles or to the arrangement of the particles themselves.

o. t fLIT! , I

Figure 6

Chromatin fibers streaming out of a chicken erythrocyte nucleus. The spheroid

chromatin units (nu bodies) are about 70 A in diameter. Negative stain : 0.5 mM uranylacetate in methanol. Preparation as described in D. E. Olins, 1974.)

(333). 167,000 x . (Courtesy of A. L. Olins and


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Periodicity of Histone Along the DNA Fiber The most important recent advance has been the use of acrylamide gels to analyze in detail the products of nuclease digestion of chromatin. Hewisch & Burgoyne (3 18) showed that an endogenous nuclease from rat liver cuts nuclear DNA into a series of discrete bands that migrated ao if they were multiples of a basic repeat. The size of this repeat was later quoted as about 200 base pairs (3 1 5). One difficulty in interpreting these data comes from the fact that exhaustive treatment fails to digest all the nuclear DNA into pieces of 200 base pairs, much of the DNA being pre�ent in higher multiples of 200. The reason for this is still unclear ; however, subsequent experiments using staphylococcal nuclease have made this distinction somewhat less important. Low concentrations of staphylococcal nuclease (or DNase I) digest nuclear DNA into pieces consistent with a repeating unit(s) of between 140 and 200 base pairs (3 19-321 ; B. Shaw and K. Van Holde, personal communication). [A similar treatment of yeast nuclei yields an analogous pattern of resistant DNA fragments (B. Shaw and K. Van Holde, personal communication). This is particularly striking because yea�t� are purported to be missing some histones.] After the initial nuclease attack, less than 15% of the DNA is acid soluble. Upon additional digestion with the nuclease, there is a decrease in DNA size from 200 to 1 70 base pairs (3 19). Extensive digestion with staphylococcal nuclease yields a limit-digest of at least eight DNA bands that range in size between 45 and 145 base pairs (320, 321). Under these conditions, approximately half of the DNA is rendered acid soluble, while the remainder is present in these discrete DNA fragments. The acid soluble DNA reflects the so-called open regions in chromatin, while the eight limit digest DNA fragments reflect nuclease-resistant closed regions (322). At very low concentrations of nuclease, discrete chromatin fragments can be separated on sucrose gradients (3 19, 321 ; R. Axel and G. Felsenfeld, and B. Shaw and K. Van Holde, personal communications). These fragment� �ediment in a modal distribution consisting of monomer, dimer, trimer, etc. The DNA in each peak migrates on gels as if it were a multiple of a monomer repeat of 1 50-200 base pairs. When the monomer chromatin fragment is treated with nuclease, approximately 43% of the DNA is rendered acid soluble (320) and the resistant DNA consists of approximately eight DNA fragments between 45 and 1 45 base pairs which are characteristic of the staphylococcal nuclease limit-digest of whole chromatin. It has been proposed that extensive digestion with nuclease gives rise to artifacts due to the redistribution of histones during the digestion (3 1 5, 323, 324). While this is undoubtedly true for exhaustive digestion with pancreatic DNase (325), it seems extremely unlikely for staphylococcal nuclease for two major reasons : 1 . the staphylococcal nuclease limit-digest yields most of the DNA in extremely discrete fragments and 2. redigestion of the monomer chromatin fragment with nuclease results in the same DNA limit-digest as found with whole chromatin. The latter finding indicates that if transfer of protein occurs it must happen between particles ; yet, such a hypothetical transfer has not been detected by a number of sensitive assays. It is likely, therefore, that different degrees of nuclease digestion reveal different characteristics of chromatin structure (see 452 and references cited above).


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The clear advantage of staphylococcal nuclease is that it digests essentially all the chromosome into discrete units, whereas the liver enzyme, although giving a rather clear indication that chromatin is digestible into discrete units, senses additional structural information since it is unable to cleave all the DNA into a single unit. An explanation offered by Hewisch & Burgoyne (3 18) is an enticing and testable one, namely that nonhistone protein can protect certain open regions from the liver endonuclease ; however, as yet there is no evidence for this. (Note that there is no formal proof that staphylococcal nuclease and liver nuclease generate the same repeat.) Indeed, although it is becoming increasingly c1car that the histoncs arc organized into defined "repeats" along the chromosome and that the DNA itself is organized into repeats of reiterated and nonreiterated sequences (326), the organiza tion of nonhistone proteins in this general scheme is unknown. The repeat size obtained from nuclease digestion may be in very good agreement with the 1 10 A repeat observed from X-ray diffraction studies (3 1 5). Unfortunately, it is not clear how the two repeats are related. While the nuclease digestion data indicate that a nuclease-sensitive site, one that is presumably deficient in protein, exists at regular intervals along the chromosome, the X-ray data (at least in the most superficial analysis) indicate only a structural repeat about every 1 10 A. It is not clear whether it is the protein or DNA component that gives rise to this reflection, what portion of the chromosome structure gives rise to this repeat, or whether the lower angle reflections at 55, 37, 27, and 22 A are higher orders of the 1 10 A repeat or attributable to independent, repeating structures. Reconstitution with selected histones (327-329) demonstrates that H2a, H2b, H3, and H4 are all required for the generation of the normal X-ray pattern and that H I is not (328-330). In addition, selective histone removal by chymotrypsin indicates that only histone 4 is needed to maintain some of the X-ray structure (331). Clearly maintenance and generation of the structure are two separate phenomena. Much of the X-ray diffraction data can be better understood in terms of recent data obtained using neutron scattering (332). The basic method involves matching the scattering of the solvent with that of either DNA or histone. Because DNA and histone have different scattering properties and either can be theoretically eliminated by increasing the concentration of DzO in the solvent (scattering from DNA is matched by a solvent concentration of 63% DzO while that from histone is matched at 37.5% DzO), it is possible to determine which diffraction spacings arise from which macromolecule. By varying the D20-to-HzO ratio of the medium and analyzing the changes in the reflections at 1 10, 55, 37, and 27 A, Bradbury and his colleagues (332) have found that all the spacings do not change coordinately, and consequently all chromatin reflections do not come from higher orders of the same basic repeat. The data indicate that the 1 10 and 37 A reflections come from protein and that the 55 and 27 A reflections come from DNA. Their interpretation of these findings is in remarkable agreement with recent electron microscopic observations (333, 334) and with results from nuclease and trypsin digestions of chromatin, i.e. a "particles on a string" model. It is proposed that the repeat of 1 1 0 A corresponds to a histone core and that the folding of the DNA within the particle corresponds to the DNA rings at 55 and 27 A. The 37 A repeat may come from the way the histones are packaged within each particle.


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Electron Microscopy Recent electron microscopic data are strikingly consistent with the emerging physical and chemical picture of chromatin. OIins & OIins (333, 473) and Woodcock (334) have observed that chromatin, when prepared according to their methods, appears as a chain of beads on a string (see Figure 6). The beads ("nu" bodies) have a diameter of about 70 A (333) (approximate molecular weight 160,000) and their spacing is roughly consistent with a DNA repeat of about 200 base pairs (335), although this is only very approximate. Although it is difficult to prove that the "particles on a string" view of chromatin as visualized by electron microscopy is not an artifact, it is nevertheless very consistent with the suggestions from nuclease experiments. Indeed, it is most likely that this picture is a meaningful "vestige" (333) of the original structure within the nucleus. There are four major questions raised by the recent electron microscopic findings. The first is the composition of each nu body. Are they homogeneous? The second is the composition of the threadlike substances (about 1 5 A in diameter) between them. Is it sensitive to nuclease? If so, is it sensitive at a particular point or throughout its length? Does it contain histones? nonhistones ? The third and most difficult question is the natu re of the higher order structure that was probably destroyed in obtaining these pictures ; or equivalently, how do the nu bodies interact to produce the next order of packing? Do they interdigitate between fibers? Do they fold back on themselves ? And finally, what is the internal structure of the DNA and protein within the nu body? Clearly the electron microscopic findings are quite compatible with the original nuclease digestion experiments of Clark & Felsenfeld (322), which indicated that chromatin exists as a series of alternating segments of protein-covered and protein-depleted DNA, each about 75,000 daltons. Although not yet proven, we shall assume that staphylococcal nuclease is attacking sites between individual nu bodies. Senior et al (335) have shown that extensive sonication of formaldehyde-fixed,

chick erythrocyte chromatin yields a subpopulation consisting almost entirely of single nu bodies. An analysis of these particles indicates a DNA strand of about 200 base pairs and about an equal weight of protein. Interestingly, the authors report preliminary evidence indicating that the isolated nu bodies give the same X-ray reflections as intact chromatin. Since nu bodies are about 80 A in diameter, it seems unlikely that they themselves give rise to the 1 10 A repeat. An explanation offered by Senior et al (335) i s that the repeat is actually a conse quence of the packing of the nu bodies, in much the same way as the packing of spheres might give rise to a higher order reflection. In contrast to the features of chromosome structure detected by nucleases over rather short distances of DNA (50-200 base pairs), some structural properties present over longer distances appear to be quite variable. Georgiev and his colleagues (336, 337) have investigated the quantity of free DNA generated by shearing chromatin and banding according to density in CsCI after formaldehyde fixation. Their data cl ear l y show that as the ionic strength of the buffer increases, there is a corresponding increase in free DNA, from essentially zero at low ionic strength to approximately 25% in 2 mM MgCl2 or 225 mM NaC!. Interestingl y the digestion ,


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pattel n from staphylococcal nuclease is hardly changed in similar ionic conditions (H. \tleintraub, unpublished observations). As intimated by the authors, these findings may be relevant to the problems of differential gene activity ; thus, they are presently doing the appropriate hybridization experiments to determine whether the free DNA generated at high ionic strength is in any way related to transcriptional activity. In any event, the behavior of histones in chromatin is highly dependent on ionic conditions and it would not be surprising if subtle, ionic effects (from small or large molecules) occurring in particular regions of the nucleus were used to impose specific local chromosome conformations. One of the models that the authors propose to explain their data is that of a long DNA loop. One side of the loop is extended and not tightly bound to histone, whereas the other side is highly compacted by histones. It should be emphasized that this model represents a higher order of structure than that indicated by nuclease experiments and it is in no way in consi stent with that data.

Nu Body Structure Accepting the general model that seems to be emerging, that of a globular, protein rich structure associated with every 1 50-200 base pairs of the chromosome fiber and bordered by short protein-poor, nuclease-sensitive regions, it is possible to ask certain questions about the internal structure of these nu bodies. One insight into their substructure comes from the fact that the limit-digest with staphylococcal nuclease yields very small but discrete DNA bands on electrophoresis (320). The size of these bands is significantly less than the 1 50-200 base pair repeat for nu bodies. Consequently, exhaustive digestion with nuclease must be hydrolyzing less accessible sites within each nu body. If this is so, then since there are a minimum of eight limit-digest DNA fragments of 45--145 base pairs, there are likely to be some differences between individual nu bodies. These differences would determine the variable points at which nuclease is hydrolyzing in the limit-digestion conditions. Sahasrabuddhe & Van Holde (338) have shown that with mild nuclease digestion chromatin falls apart into fairly homogeneous particles that sedimented at 1 1-12S, and (by equilibrium sedimentation) have a molecular weight of about 1 76,000 (remarkably consistent with the 160,000 dalton estimate of the size of a nu body obtained from electron microscopy). These particles were therefore almost spherical and it is now likely that they correspond to nu bodies (456). When these particles were digested with trypsin, there was a rapid shift in sedimentation to about 5S. The shift was stable over long periods of trypsin digestion and the resulting particles had a molecular weight of about 158,000, indicating that trypsin did not remove very much protein. Thc rather meager loss of material with trypsin digestion allowed the interpretation that the altered sedimentation was due to an unfolding of the nu body. These observations have been extended by Weintraub & Van Lente (339), who have further analyzed the digestion of chromosomal histones by trypsin. They have

shown that exhaustive digestion of chick erythrocyte chromatin results in the complete digestion of. histones 1 and 5 and the cleavage of only about 20-30 amino acid residues from the positively charged N-terminal parts of histones 3, 4, 2b, and



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(possibly) 2a. The resistance of C-terminal histone fragments (from 80-1()O amino acids in length) seems to be due to their mutual interaction, since removal of the histones from the DNA by 2 M NaCl fails to alter the trypsin digestion pattern. The sensitivity of the basic N-terminal regions was interpreted in terms of a model in which these basic "arms" extend from a trypsin-resistant histone complex and define the binding sites for the folding of a DNA fiber about this complex. This type of structure is consistent with the nu bodies observed in the electron microscope. When trypsin-digested chromatin was treated with nuclease (339), the resistant DNA contained some, but not all of the eight DNA fragments characteristic of the staphylococcal nuclease limit-digest. The authors interprctcd this as indicating that histone N terminals protected the DNA corresponding to some DNA fragments, whereas histone C terminals protected the DNA corresponding to other DNA fragments. In addition, they postulated that histones could fold the chromosome by crosslinking the DNA regions that corresponded to the different DNA fragments. Subsequent experiments, repeating and extending the work of Sahasrabuddhe & Van Holde (338), showed that the transition from the l I S nuclease particles to the 5S trypsin-treated nuclease particle occurred when the N termini of histones 3 and 4 were being digested (340). If the l IS particles are generated by slightly higher nuclease concentrations than used by Van Holde (so that the chromatin is digested to the limit with nuclease) and then trypsinized and run on 6% acrylamide gels, pH 7.S, the resulting 5S particles are resolved by the gel into about eight extremely discrete subparticles which are visualized as bands when stained with ethidium bromide. In addition, five of these bands stained with Coomasie blue, while three or four did not. Clearly, trypsinization of the l 1 S particles generated by extensive nuclease treatment results in the production of protein-free and protein-associated DNA fragments of defined size. These observations have specific implications for the internal structure of the l I S nuclease particles, o r nu bodies. Since trypsinization results i n the cleavage o f 20-30 amino acids from the N-terminal ends of the arginine and slightly lysine-rich histones, it is likely that the protein-free DNA fragments released by trypsin treat ment of the nuclease particles were originally bound to amino-terminal parts of the histones. The protein associated with the DNA fragments released after these procedures were shown by peptide analysis to be the trypsin-resistant histone C-terminal segments. The model derived from this data is that the general structure of a nu body consists of a group of histones which fold the DNA in the nu body by binding to a stretch of DNA of about 80 base pairs with their N termini and another stretch of about 80 base pairs with their C termini, the intervening DNA defining a nuclease-sensitive site. To summarize, by cleaving histones at their N termini, trypsin in effect splits the l IS nuclear particles into two subparticles, one of which contains protein and the other does not. The evidence that histones crosslink the DNA supports previous work and speculations from a number of laboratories. Are the individual nu bodies homogeneous? The finding from �eptide analysis that most subchromosomal particles obtained by nuclease and trypsin treatment of chromatin contain trypsin-resistant fragments from all four major histones would


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argue that these particles are qualitatively homogeneous (340). Why then are so many DNA fragments generated by nuclease treatment, and why so many sub chromosomal particles? Several explanations are possible. One is that there are quantitative differences in the ratios of the different histones in each of the nu bodies. There is some evidence for this, and the finding of one subchromosomal particle (generated by nuclease and trypsin treatment of chromatin and isolated by gel electrophoresis) with only histones 3 and 4 further supports this idea (340). A second possibility is that the ratio of the histones is the same, but their conformations are different. This could be a consequence of 1. their association with particular DNA sequences, 2. their entrapment in specific metastable energy states, or 3. their selective modification. The first explanation is unlikely because reconstitution experi ments generate the same limit-digest DNA bands with staphylococcal nuclease whether the DNA is from calf thymus or phage ). (320 ; T. Maniatis, personal communication). Also, three of the eight DNA bands of the nuclease limit-digest (all that were examined) hybridize with tracer amounts of labeled cellular DNA with kinetics identical to those obtained when the reaction is driven by total cellular DNA, indicating that they contain a random collection of DNA sequences (H. Weintraub, unpublished observation). In addition, three of the remaining bands contain a pattern of pyrimidine tracts which are indistinguishable from total cellular DNA (H. Weintraub, unpublished observation). There is no evidence for or against the second two possibilities suggested to explain the number of subchromosomal particles generated by nuclease ; however, since reconstitution experiments appear to generate a structure very similar to the original one, both of these possibilities can be tested. What is the structure of the DNA in the nu bodies? Assuming that nu bodies are equivalent to nuclease particles, the work of Sahasrabuddhe & Van Holde (338) indicates that they have a circular dichroism (CD) spectra very similar to C-type DNA. In this context, it is worth discussing the proposals made by Crick (341). He suggested that histones crosslinked the DNA and that this produced a strain on the double helix conformation, resulting in the unpairing of double helice.s. The unpaired bases could serve as specific recognition signals. These predictions are consistent with much of the data emerging about chromosome structure. Thus, there is growing evidence that the histones fold the chromosome by crosslinking DNA within a given nu body, and the recent CD data (338) indicate that as a consequence of this crosslin king the conformation of the DNA is altered. This is not surprising from what is known about the physical chemistry of DNA because the type of bending needed to package about 1 50 base pairs of DNA into a particle the size of a nu body would probably require distortion of the normal base pairing schemes. Since we do not know the path taken by the DNA within or around the nu body, it is impossible to say just how much of the double helix should be distorted. As far as we are aware, no one has shown that nu bodies are sensitive to single stranded nucleases ; however, this is a difficult experiment because the single stranded regions may not be accessible and it is possible that the denatured DNA relaxes as soon as it is nicked. One prediction made by Crick (341) will clearly be very difficult to prove, i.e.



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that the single stranded regions are specific recognition signals. The evidence, previously mentioned, indicating that histones are bound to chromosomal DNA independent of base sequence, argues against the possibility that histones fold the DNA to expose specific signals. On the other hand, most of this work was done with bulk chromosomal DNA and it is clear that one must look at particular genes. In this respect, it is extremely interesting that Axel, Cedar & Felsenfeld (10, 446) have shown that a reverse transcriptase probe to globin mRNA hybridized only partially to DNA obtained from nuclease-sensitive parts of the genome. Addition of nuclease-insensitive sequences led to a protection of the entire probe. These experiments are extremely important and clearly indicate that along the globin gene, some portions of the globin sequence in every reticulocyte are covered and other portions are exposed. Whether the same results would be obtained with other cell types, whether the exposed sequences in the globin gene are single stranded, whether they are involved in control (they may not be because the probe is detecting largely those sequences common to the globin structural gene), and whether they are generated by a base sequence specificity peculiar to histones, certain combinations of histones, or combinations of histones and nonhistone chromosomal proteins remain to be determined.

Histone: Histone Interactions In general, the experiments described thus far indicate that specific histone clusters are periodically located along the chromosome fiber and that these clusters are intimately involved in folding chromosomal DNA. The nature of the histone inter actions that may be responsible for generating these clusters has been extensively studied in solution by Isenberg and his colleagues over the past few years (342, 343, 447, 448). Using fluorescence anisotropy (to detect the rotational mobility of the transition moments of tyrosine residues) and circular dichroism, they have shown that specific interactions occur between the histones. Very strong associations exist

between histones 3 and 4, 2b and 2a, and 2b and 4. For many of these interactions, there is an increased amount of a-helix produced upon complex formation. Weaker associations also exist between 2a and 4, and it would not be surprising if many other types of interactions could occur in different types of environments. This work extends earlier studies (344, 449) showing that histone 2b undergoes a pro gressive association with increasing salt concentration. The nuclear magnetic resonance (NMR) studies of Bradbury and colleagues (40, 345), which demonstrated salt-induced changes in resonance due to the altered mobility of amino acids in particular regions of histones 4, 1, and 2b, have recently been reviewed and we will mention only that they support the growing consensus that histone : histone interactions are important for establishing the folding parameters of chromosomal DNA. Additional evidence comes from the work of Kelley (346), who showed that histones 2b and 2a can form a 1 : 1 complex. In contrast, using the crosslinking reagent dimethylsuberimidate, Kornberg & Thomas (329) have shown that H2b and H2a can form polymers of the form (2b-2a)n. In addition, they have demon strated by both sedimentation analysis and crosslinking that histones 3 and 4 can


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form a specific association in solution, probably as a tetramer. The same general conclusions from similar types of studies come from the work of Rouark et al (347), who have additionally presented data indicating that at low concentrations the 3-4 complex may be a dimer. Both groups have questioned the validity of information obtained by using histones that have been extracted and purified with acid ; however, other types of experiments indicate that the acid denaturation of histone is reversible (e.g. 458). To summarize, studies with isolated histones in solution show that a great number of interactions are possible ; given the appropriate ionic condition and methods of histone preparation, a great many more will no doubt be observed. The primary question of course is which of these interactions occur in the chromosome? With respect to the types of histone : histone interactions occurring in chromatin, several observations are of importance. It has been shown that most particles obtained after nuclcase and trypsin treatment of chromatin contain the C-terminal fragments from all four major histones (340). The stoichiometry was such that depending on the particle, anywhere from 6-10 histones were bound to DNA fragments between 60 and 140 base pairs. One particle, however, appeared to contain only histones 3 and 4. A second type of observation involved the use of crosslinking reagents to produce high molecular weight protein products from chromatin. Kornberg & Thomas (329) have reported the generation of oligomers up to pen tamers using dimethylsuberimidate, but have not yet identified the specific crosslinked products. In addition, Olins & Wright (348) have used glutaraldehyde to crosslink chick erythrocyte chromatin. They found that at low concentrations of glutaraldehyde several polymeric bands appeared on their SDS gels. By their amino acid com position, the authors were able to infer that these crosslinked products were probably derived from the red cell-specific histone 5. From their apparent molecular weight, a minimum of up to eight molecules could be crosslinkcd and consequcntly, it was suggested that histone 5 appeared in clusters either because of its contiguous alignment along a particular stretch of DNA or because of the tertiary folding of the chromosome fiber. What is disturbing about the glutaraldehyde data and what appears to be emerging as a general phenomenon with reagents that crosslink chromosomal histones is the fact that only a small percentage (5-10%) of a particular histone ever gets crosslinked into a resolvable product. When attempts are made to increase this percentage by increasing the concentration of reagent, then essentially all the material fails to enter the gel. Thus, it may prove difficult to show that a crosslinked product representing about 10% of the protein is indeed representative of a general structure and not a specific type of variant. Recently, Martinson & McCarthy (349), using the tyrosine-specific crosslinking agent, tetranitromethane, have presented evidence consistent with the notion that all four major histones are intimately associated in chromatin. They demonstrated that the protein product of tetranitromethane treatment of chromatin was a dimer. By reconstituting DNA with specific histones, they showed that only histones 2b, 4, and 2a were required to produce a crosslinked product with the same electro phoretic properties as that obtained from chromatin. After reconstituting DNA with specifically labeled histone species, they were able to identify the crosslinked



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product as a dimer of 2b and 4 and argued that the crosslinked product from native chromatin had the same composition. Since 2a was required for the proper reconstitution of 2b and 4, it was proposed that this histone was also in the complex, but because of the rigid specificity of the crosslinking reagent (tetra nitromethane requires intimate contact between the activated tyrosine and a neigh boring molecule) 2a was not crosslinked into the product. Relying on the strong evidence from the solution chemistry which indicated that 3 and 4 were tightly complexed, they interpret their studies in terms of a model where all four histones interact to produce a basic chromosomal unit. As with the other crosslinking experiments, only about 10% of the histones appear in the product. J. F. Jackson, F. Van Lente, and H. Weintraub (unpublished observations) have observed dimer products, H4 + H2b and H2a + H2b, on crosslinking with formaldehyde or glutaraldehyde. To summarize, the X-ray evidence, the crosslinking data, and the analysis of the particles released after digestion with nuclease and trypsin support the notion that all four major histones are involved as a group in generating the final chromosomal structure.

Histone Accessibility Specific labels have been used to map the accessibility of specific histone residues. This approach is extremely valuable with histones because their sequences have been so well characterized. One of the first attempts in this direction was by Simpson (350), who showed that at very high concentrations of acetic anhydride, only about 25% of the potential lysine residues in chromatin become acetylated. By analyzing the electrophoretic mobility of the histones after treatment, he was able to show that all histone species were accessible to the pwbe. No attempt was made, however, to localize the sites where acetylation was occurring. In vivo, of course, the acetylating enzymes in chromatin appear to be very specific for histone N-terminal regions. This work was extended by Ma\Chy & Kaplan (35 1 ), who used an internal standard to compare the acetylation of particular his tones. In essence, they were able to show that at rather low concentrations of acetic anhydride their marker was much more reactive and they concluded that all the potential lysine residues in chromatin were essentially inaccessible to acetylation. This is in contrast to the pH titration studies (451) which reveal that about 25% of the lysines in chromatin are freely titratable. A second type of label available to measure accessibility is iodination. Using 1 2 5 1 to label histone tyro sines, F. Van Lente (unpublished observations) has shown that in chromatin no tyrosine in H2a is accessible, even at very high concentrations of 1 2 51. When the chromatin is treated with increasing concentrations of urea, H2a readily becomes iodinated. Van Lente has also shown that histone 3, although iodinated in chromatin, is much more available when free in solution. Histone 3 is the only histone with tyrosine in its N-terminal region and hence these tyro sines are intimately associated with basic residues. By mapping all the iodinatable peptides from all the histones, Van Lente has demonstrated that the histone 3 peptides become relatively more accessible to 1 2 5 1 with increasing ionic strength. The data are used to support the notion that histone 3 N-terminal segments are involved in DNA binding.

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Potentially the most useful histone probes will be antibodies of different specificity. Bustin (352) has used antibodies made against specific his tones and has observed that antigenic determinants of H I and H2b are more available in chromatin to interact with homologous antibody than those in H3 and H4, and that determinants of H2a are the least available. Most striking was the additional observation that antibodies bound to any one histone class failed to interfere with subsequent antibody binding to a second histone class. The implication of these findings is that antibody determinants from one histone class are spatially separated from those of another. Hopefully, it will be possible to show where the antibody determinants reside in the individual histonc molecules.

Conclusions It is increasingly clear that histone : histone interactions are determining the folding parameters of chromosomal DNA. The basic unit involved in these interactions is probably the nu body. It is likely that specific histone complexes form the core of this structure and that positively charged N-terminal arms extend from this core, defining the path taken by the folding DNA fiber, although it is also clear that othcr regions toward the C termini can bind DNA very tightly. We do not really know how homogeneous nu bodies are, we do not know the conformation and path of the DNA within (or around) these structures, and we know nothing about the structures between these nu bodies or what determines their spacing. Most important, we do not even know whether it is possible to expect solutions of histones to behave in the same manner as histones in chromatin. What role does DNA have in forcing histones to form the "proper" associations? What function might histone modifica tion play in generating these conformations? Finally, what function, if any, might be served by nonequilibrium associations between the various histones? Clearly, there are many questions to be answered, but perhaps the ones that are most outstanding involve the possible differences in structure between active and inactive chromatin. There is a great deal of evidence indicating that differences do occur ; however, further characterization of these differences and, most importantly, an explanation for how these differences are generated are required. Finally, we should emphasize that our attempts in this section have been focused on events of the past few years. Consequently, we have not discussed the less recent work involving X-ray diffraction, cation binding to DNA, flow dichroism, circular dichroism, viscosity, and sedimentation. All these data are documented in the reviews cited earlier in this section. TEMPLATE ACTIVITY

H istones as General Repressors If RNA polymerase is specifically restricted by the conformation of DNA in chromatin, this implies that tissue-specific gcnes are accessible in the chromatin of one tissue and inaccessible in that of another. Transcription from isolated duck chromatin from reticulocytes and from liver has been examined. The chromatin was transcribed with RNA polymerase from Escherichia coli and the RNA isolated and hybridized to a labeled reverse transcript probe for globin sequences. The



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results showed that the addition of RNA polymerase and nucleoside triphosphates to reticulocyte chromatin led to the production of RNA that significantly increased protection from a single stranded nuclease for the labeled probe. In contrast, addition of RNA polymerase and nucleoside triphosphates to the liver chromatin did not lead to an increase in RNA sequences providing probe protection (9, 10). The interpretation of these findings is that the structure of the globin DNA sequences allowed transcription in reticulocyte chromatin but not in liver chromatin. The same results have been obtained in studies of erythropoietic and brain chromatin of mouse ( 1 1, 12). How these differences are generated during development and what other factors may amplify this differential signal in the reticulocyte are far from being worked out. Nevertheless, a basic difference between cells that make hemo globin and cells that do not is that the "accessibility" of the globin sequences is different. This basic approach has recentlybeen extended to the transcription of chro matin from viral-infected myeloblasts (353) and SV40-transformed cells (468, 469). The limited transcription potential of eukaryotic chromatin with RNA polymerase appears to be largely a consequence of the association of chromosomal DNA with histones. The removal of these proteins from chromatin with any of several reagents leads to an increase in template activity (354-356). There is some indication that histone 1 plays a special role in this generalized repression because its removal has been reported in some instances to result in a large increase in transcription (357, 358). It is not at all clear how the histones are blocking transcription. Recent work by Cedar & Felsenfeld (359) shows that E. coli RNA polymerase binds to about 10% the number of sites in chromatin as in DNA. The E. coli enzyme elongates RNA chains using a chromatin template at about 33% the rate observed with purified DNA. Assuming the endogenous enzyme works at least approximately in the same way as the E. coli enzyme, these experiments demonstrate that the major restriction imposed on the DNA by the histones involves the blocking of sites for RNA chain initiation. There is no indication from any published work that the polymerase, once properly initiated, cannot transcribe histone-associated DNA. Whether the histones actually come off or slide during this process remains to be determined. There appears to be no a priori reason to assume that the histones must sterically block the polymerase, although they certainly could do so, for example, by actually crosslinking the individual strands of the double helix. Again, there is no evidence for or against this possibility.

Chromatin Fractionation Biochemical attempts to isolate so-called active chromatin presuppose that such a fraction is structurally different from the bulk of the chromatin. Such an "active" subfraction can readily be evaluated : it should contain a unique subset of DNA sequences ; it should contain sequences for genes that are known to be active, for example, rRNA, tRNA, globin (if the chromatin is from erythroblasts), and histone genes (if the chromatin is from S phase cells). Somewhat less definitive assays include co purification of endogenous RNA polymerase or nascent RNA chains, assays of template activity with exogenous RNA polymerase, etc. The earliest fractionation attempts used diffcrcntial centrifugation of sheared chromatin on sucrose gradients (360, 361). A similar approach has been adopted


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by McCarthy et al (362, 443, 444) and others (363) who have separated active fractions on the basis of their hydrodynamic properties, either as being preferentially exeluded by agarose columns or as slower sedimenting in sucrose gradients. Both fractionations succeed in isolating a subfraction of sheared chromatin that is more extended than the bulk chromatin. A second type of fractionation takes advantage of a possible difference in charge between active and inactive chromatin. The fractionation involves chromatography on epichlorohydrin-coupled tris(hydroxymethyl)amino methane (ECTHAM) cel lulose (364) where the putative active fraction is the last to be eluted; indicating that it is probably more negatively charged than the bulk chromatin. Active chromatin isolated in this manner sediments more slowly than bulk chromatin and is enriched in low melting (relatively histone-free) sequences. Moreover, it is highly enriched in E. coli RNA polymerase binding sites, containing approximately half as many as protein-free DNA (365). A third type of fractionation involves very limited cutting of chromatin with DNase II followed by a differential precipitation in 2 mM MgCl2 (366-368). The resulting soluble material, the active fraction, contains about 1 1 % of the total chromatin. It fractionates together with nascent RNA chains and has a high relative template activity with exogenous polymerase. The DNA of the active fraction hybridizes with a kinetic complexity that is about one tenth that of total DNA, which indicates that this fraction contains a unique subset of single copy DNA sequences (and probably a unique subset of middle repetitive DNA as well). The observation that bulk cellular RNA preferentially hybridizes with DNA from this fraction further supports the notion that this fraction is indeed active in in vivo transcription. The bulk of the work on chromatin fractionation has indicated that active chromatin is relatively enriched in nonhistone chromosomal proteins and deficient in histones, particularly H I . Specific major NHC proteins have been suggested to be present exclusively in the euchromatin (362, 368, 369). Active chromatin is consistently found to melt at lower temperatures, implying less stabilization of the DNA double helix by proteins, presumably because of the depletion of histone. McCarthy and his colleagues have fractionated chromatin by thermal chromatog raphy on hydroxyapatite. Here the lowest melting chromatin fraction has been shown to be enriched for DNA sequences that hybridized to homologous cellular RNA (370). This reinforces the idea of a structural basis of gene activity that can be exploited for chromatin fractionation. It will be important to determine whether or not thc chromatin of the active fraction is in the nu body form, and if so, whether any differences in frequency, spacing, or substructure exist. Much of the present data are consistent with the idea that the histones can block the accessibility of the polymerase to initiation sites, but only marginally affect the accessibility of the polymerase to the DNA bases once the chain is initiated. Thus it may be not the accessibility of the polymerase to the bases that is being hampered, but some other type of block preventing initiation. What immediately comes to mind is the conformation of the DNA, especially since it is increasingly clear that proper initiation with both the E. coli RNA polymerase and mammalian RNA polymerase requires a particular promoter configuration (37 1-374, 445). It is possible



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that by folding the DNA, the histones alter the structure of potential promoter sites and hence initiation fails to occur. Assuming that all histones participate in the folding of the DNA chain, then the removal of any one would presumably destroy the structure and allow the polymerase to initiate properly. Indeed, experiments involving selective histone removal have led Smart & Bonner (355, 356) to precisely this conclusion. It remains to be discovered how desired promoters are identified and protected. The nonhistone chromosomal proteins may play such a role. In some instances histones or similar basic proteins have been reported to have a stimulatory effect on transcriptional activity, perhaps implying the removal of nonpromoter binding sites in competition for the RNA polymerase (375, 376). An indication of broad levels of structural organization related to activity has been obtained from studies of metaphase chromosomes. Unique and reproducible chromosome banding patterns can be obtained by treating metaphase chromosomes with a number of reagents and dyes, e.g. quinacrine (377, 378). The banding patterns appear to reflect the state and accessibility of the DNA, and thus to be a consequence of differential protein-DNA interactions along the chromatids. Experiments by Gottesfeld et al (379) show that the fluorescence of quinacrine associated with DNA in active chromatin (euchromatin) is quenched very effectively relative to that in inactive chromatin (heterochromatin), and suggest that this difference is due to the difference in DNA conformation caused by the associated proteins. Thus the banding pattern may correlate roughly with those structural features that help to define heterochromatin and euchromatin. It has further been suggested that certain types of heterochromatin that stain differently, such as the N bands, may do so as a consequence of a specific association of NHC proteins with DNA (380).

Model Systems It is clear that the animal viruses are going to be helpful in future analyses of chromatin structure and activity. As mentioned previously, SV40 viruses contain all histones with the possible exception of histone 1 . The SV40 complex can exist in two major forms, as the packaged virion released from the cell and as the replicating and transcribing nucleoprotein complex found in the nucleus. The former may be a prototype for inactive chromatin and the latter a prototype for active chromatin. Preliminary studies by B. Polisky and B. J. McCarthy (personal com munication), using the packaged particle, have shown that all six DNA fragments produced by the Hin 3 restriction endonuclease appear when the particle is treated with the enzyme ; however, only about 30% of the total DNA is in these fragments, the remaining DNA being only partially digested. The data imply that, as in chromatin, there is a random arrangement of "open" and "closed" or protected regions of approximately equal lengths along the SV40 DNA. A given Hin digestion site has a particular probability of being either open or closed. This probability depends on a number of factors ; the position of the Hin sites relative to each other, the average distance between closed regions, and the average length of a closed and an open region, to mention only a few. The actual analysis of the data from the Hin cutting will no doubt yield a great deal of information about a number of packaging parameters. Similar conclusions have been reached from studies with the R l restriction enzyme. Finally, it has been observed that approximately 50% of the

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nucleoprotein complex (the replicating form of SV40) is sensitive to staphylococcal nuclease, a nonspecific enzyme (1. Beltz, A. 1. Levine, and H. Weintraub, unpublished observations). Moreover, the same limit-digest sized DNA bands are obtained as were found from eukaryotic chromatin. These experiments indicate that the nucleo protein complex and virion are good structural models of chromatin. Many aspects of SV40 replication are also quite similar to those of eukaryotic chromatin. In addition, the in vivo transcription behavior of the virus is very well worked out. Taken as a whole, it is likely that SV40 will be a very useful prototype for active genes in higher cells. CONCLUSIONS It is hoped that just as the solving of the DNA structure yielded insights into the way the genetic information was replicated and transcribed, so the solving of the structure of higher chromosomes will be equally informative about the way chromo somes are replicated and information transfer is controlled during eukaryotic growth and differentiation. Our present knowledge of chromatin structure is derived from the work on primary structure and chemical physical characterization of the histones. This information is being used to determine the way in which the histones interact with each other and fold the DNA ; in the future, additional work on the histones will undoubtedly provide valuable insights into the evolutionary changes in chromo some structure and control processes. Biological evidence implies that the nonhistone chromosomal proteins play an equally important role. It will be necessary to achieve an equivalent chemical analysis of these proteins if fruitful experiments are to be designed. The ultimate goal, that of understanding gene expression in terms of chromatin structure, appears attainable, but much work remains to be done. NOTE ADDED IN PROOF D'Anna & Isenberg (460) have extended and summarized their results showing that specific interactions between all pairs of his tones (excluding histone 1) can occur in solution. The energies of some of these interactions are enormous (for example, the binding coefficient of H3 to H4 is on the order of 1021 M - 3), while some of these interactions are comparatively weak (for example, the binding coefficient of H3 to H2a is about 1 0 - 6 M). Several very important papers concerning nuclease digestion of chromatin have also appeared recently. Burgoyne, Hewish & Mobbs (461 ) have shown that the endogenous nuclease of rat liver cuts chromatin into single stranded multiples of a regular repeat of about 55 to 60 bases. In addition, Noll (462) has shown that when nuclei are digcsted with pancreatic DNase and the resistent DNA fragments are analyzed under denaturing conditions, a series of bands is observed between about 40 and 200 bases. Each successive band occurs as an integer multiple of 10 base pairs. These very striking results were interpreted in terms of a model where pancreatic DNase nieks exposed DNA regions, which occur every 10 base pairs (or one turn of a double helix) on the outside of the nu body. Finally, the experiments of Honda, Baillie & Candido (463) have shown that during the development of trout sperm there is an inverse relationship between the percentage of chromatin digested into l I S particles by staphylococcal nuclease and the amount of DNA associated with protamine. The



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nuclease particles obtained contain only histones and negligible quantities of protamine. In a very novel approach, Mirzabekov & Melnikova (464) have uscd H3_ dimethylsulfate as a probe of DNA accessibility in chromatin. This reagent methylates the N-7 atom of guanine (located in the major DNA groove), the N-3 atom of adenine (in the minor groove), and the N- l atom of adenine (available only in single stranded regions). The results suggest that 1. the minor groove of DNA is freely available to methylation in chromatin, 2. the major groove is only marginally protected by histones, and 3. less than 0.5% of the DNA is in a single stranded conformation. The surprising level of accessibility of both DNA grooves in chromatin is consistent with the idea that the DNA may be on the outside of the nu body (332, 462). All these recent data support the numerous and similar "beads on a string" models of chromatin structure that have recently been proposed. Wilson et al (470) have recently reported that the human genome contains 1 0-20 copies of each histone gene. This is the lowest reiteration frequency reported to date. The characteristic electron microscope pictures of chromatin fibers showing nu body structure have recently been obtained from preparations of Drosophila polytene chromosomes (c. L. F. Woodcock, J. P. Safer, and J. E. Stanchfield, personal communication). This is of particular interest in that the polytene chromosomes are almost totally euchromatin, the heterochromatin having been severely under replicated during polytenization. The nucleoplasmic form of SV40 has similarly been visualized as a "minichromosome" with one nu body per 200 base pairs of DNA (472). Using a filter binding assay, Renz has shown that histone 1 prefers to bind to purified native mammalian DNA relative to E. coli DNA. The data indicate that there are preferential binding sites on the mammalian DNA which appear to be distributed at intervals of approximately 0.5 to 2 x 1 06 daltons of DNA (474). 1. Thomas and R. D. Kornberg (personal communication) have recently shown that dimethylsuberimidate can produce crosslinked products up to octamers from histones in solution. In chromatin at pH 9, a similar pattern of crosslinked histones is produced, while at lower pH, higher order crosslinked products appear. In addition, using a reversible crosslinking reagent, they have been able to identify the his tones present in several different dimer products. These data support the proposal of a repeating chromosomal unit of eight of the smaller histones, H2a, H2b, H3, and H4. ACKNOWLEDGMENTS

We would like to thank the many scientists who have provided us with copies of work in press and allowed us to quote their unpublished observations, as well as those who have helped by criticizing the manuscript. Work in our laboratories is supported by the Jane Coffin Childs Memorial Fund for Medical Research and USPHS Grant ROJ GM 20779-01 from the National Institutes of Health (S.C.R.E.) and by the American Cancer Society and Grant GB 40148 from the National Science Foundation (H.W.). We thank L. Silver and N. Niles for technical assistance.

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Literature Cited 1. Grouse, L., Chilton, M. D., M cCarthy, B. J. 1972. Biochemistry 1 1 : 798-805 2. Brown, I. R., Church, R. B. 1 972. Del'. Bioi. 29 : 73-84 3. Palmiter, R. D. 1 973. J. Bioi. Chan. 248 : 8260--70 4. Chan, L., M eans, A. R., O'Malley, B. W.

1973. Proc. Nat. A cad. Sci. USA 70 :

1 870-74 Davidson, E. H., Britten, R. 1. 1 973. Quart. Rev. Bioi. 48 : 565-6 1 3 Paul, J., Gilmour, R. S. 1 968. J. Mol. Bioi. 34 : 305-1 6 Bekhor, I., Kung, G. M., Bonner, J. 1969. J. Mol. Bioi. 39 : 3 5 1 --64 Smith, K. D . , Church, R. B . , M cCarthy, B. J. 1 969. Biochemistry 8 : 4271-77 Axel, R., Cedar, H., Felsenfeld, G. 1973. Proc. Nat. A cad. Sci. USA 70 : 2029-32 Axel, R., Cedar, H., Felsenfeld, G. 1973. Cold Spring Harbor Symp. Quant. Bioi. 38 : 773-83 Gilmour, R. S., Paul, 1. 1973. Proc. Nat. A cad. Sci. USA 70 : 3 440-42 Pau l, J., Gil mo u r, R. S., A ffara, N ., Birnie, G., Harrison, P., Hell, A., Humphries, S . , W indass, J., Young, B . 1973. Cold Spring Harbor Symp. Quant. Bio/. 3 8 : 885-90 Steggles, A. W., Wilson, G. N., Kantor, 1. A. 1974. Proc. Nat. A cad. Sci. USA 7 1 : 1 2 19-23 Gilbert, W., Maizels, N., Maxam, A.

35. Langan, T. A. 1 9 7 1 . Ann. N Y A cad. Sci. 1 8 5 : 1 66--8 0 36. Langan, T. A., Rail, S. C, Cole, R. D. 1 97 1 . J. Bioi. Chern. 246 : 1942---44 37. Subirana, J. A., Palau, J., Cozcolluela, C Ruiz-Carrillo, A. 1970. Nature 228 : 992-93 38. Cohen, L. H., Gotchel, B. V. 1 9 7 1 . J. Bioi. Chern. 246 : 1 8 4 1---4 8 39. Sherod, D., Johnson, G., Chalkley, R. 1 974. J. BioI. Chern. 249 : 3923-3 1 40. Bradbury, E. M., Crane-Robinson, C. 1 97 1 . Histones and Nucleohistones, 85134. New York : Plenum 4 1 . Stedman, E., Stedman, E. 1 950. Nature 1 66 : 780-81 42. Greenaway, P. 1., Murray, K. 1 9 7 1 .

Bioi. 38 : 845-56 1 5 . M aniatis, T., Ptashne, M., Maurer, R. 1973. Cold Spring H arbor Symp. Quant. Bioi. 3 8 : 857-68 16. Maniatis, T., Ptashne, M ., Barrell, B. G., Donelson, J. 1 974. Nature 250 : 394-97 17. Lyon, M. F. 1 96 1 . Nature 190 : 372-73 18 . . Lewis, E. B. 1 950. Advan Genet. 3 : 73115 19. Baker, W. K . 1968. Advan. Genet. 1 4 : 133-69 20. B eren des, H. D. 1 9 7 1 . Symp. Soc. Exp. Bioi. 25 : 145-61 2 1 . Berendes, H . D. 1973. Int. Rev. Cyto. 35 : 6 1 - 1 1 6 22. Kassel, A . 1884. Z . Physiol. Chern. 8 : S J l-IS 23. DeLange, R. J . , S mit h, E. L. 1 975. Ciba Found. Symp. 28 : 59-70 24. Panyim, S., Chalkley, R., Spiker, S., Oliver, D. 1970. Biochim. Biophys. Acta 2 1 4 : 2 1 6---2 1 25. Panyim, S., Bilek, D., Chalkley,. R. 197 1 . J . Bioi. Chern. 246 : 4206--- 1 5 26. Panyim, S., Sommer, K. R., Chalkley, R . 1 9 7 1 . Biochemistry 1 0 : 39 1 1- 1 7

Garel, A. et al 1975. FEBS Lett. 50 : 1 95-99 44. Bradbury, E. M., Crane-Robinson C, Johns, E. W. 1972. Nature New Bioi. 238 : 262-64 45. Billett, M. A., H indley, J. 1 97 2. Hur. J. Biochern. 28 : 45 1-62 46. Moss, B. A., Joyce, W. G., Ing ram, V. M . 1 973. J. BioI. Chern. 248 : 1025--3 1 47. Brasch, K . , Adams, G . H . M . , Neelin, 1. M. 1974. J. Cell Sci. 1 5 : 659-77 48. Sheridan, W. F., Stern, H. 1967. Exp. Cell Res. 45 : 323-35 49. Strokov, A.; Bogdanov, Yu. F., Reznikova, S.A. 1 973. C hrornosorna 43 : 247-60 49a. Liapunovo, N. A., Babadjanian, D. P. 1973. Chrornosoma 40 : 387-99 50. A n do, T., Wa tanabe, S. 1 969. Int. J. Protein Res. 1 : 221-24 5 1 . Subirana, J. A., Cuzcolluela, C., Palau, J., Unzeta, M . 1 973. Biochim. Biophys. . Acta 3 1 7 : 364-79 52. Bloch, D. P. 1 969. Genet. Suppl. 6 1 : 93 - 1 1 1 53. Hnilica, L. S., McClure, M. E., Spelsberg,

5.


27. Palau, J., Padros, E . 1972. FEBS Lett. 27 : 1 57-60 28. Kinkade, J. M. Jr., Cole, R. D. 1 966. J. Bioi. Chern. 241 : 5790--97 29. Kin kade, 1. M. Jr. 1968. Fed. Proc. 27 : 773 30. Bustin, M., Cole, R. D. 1968. J. Bioi. Chern. 243 : 4500--5 3 1 . Bu stin, M., Cole, R. D. 1969. 1. Bioi. Chern. 244 : 5286--90 32. Evan s, K., Hohmann, P., Cole, R. D. 1970. Biochirn. Biophys. Acta 221 : 1 28-31 33. Stellwagen, R. H., Cole, R. D. 1 969. Ann. Rev. Biochern. 38 : 9 5 1 -90 34. Rail, S. C, Cole, R . D. 1 9 7 1 . J. Bioi.

6.

7. 8. 9. 10. 1 1. 12.

13. 14.

1973. Cold Spring Harbor Syrnp. Quant.

Chern. 246 : 7 1 75-90

43.

Nature New Bioi. 229 : 233-38



T. C. See Ref. 40, 1 87-240 54. Phillips, D. M. P. See Ref. 40, 47-83 5 5 . Marushige, Y., Marushige, K. 1974. Biochim. Biophy'. Acta 340 : 498-508 56. Marushige, K., Dixon, G. H. 1 97 1 . J. BioI. Chem. 246 : 5799-5805 57. Dixon, G. H. 1972. Karolinska Symp. 5 : 1 30-54 58. Dixon, G. H. et al 1 975. Ciba Found. Symp. 28 : 229-50 59. Kischer, C. W., Hnilica, L. S. 1969. Exp. Cell Res. 48 : 424-30 60. Stollar, B. D., Ward, M. 1 970. J. BioI. Chem. 245 : 1 26 1 -66 6 1 . Bustin, M., Stol l ar, B. D. 1973. J. BioI. Chem. 248 : 3506-10 62. Hekman, A., Sluyser, M. 1 973. Biochirn. Biophys. Acta 295 : 6 1 3-20 63. Sluyser, M., Bustin, M. 1 9 74. J. Bioi. Chern. 249: 2507- 1 1 64. Wigle, D. T., Dixon, G. H. 1 97 1 . J. Bioi. Chern. 246 : 5636-44 65. Huntley, G. H., Dixon, G. H. 1 972. 1. Bioi. Chern. 247 : 49 1 6-1 9 66. Thaler, M. M., Cox, M. C. L., Vil1ee, C. A. 1 970. J. Bioi. Chem. 245 : 1479-83 67. Byrd, E. W. Jr., Kasinsky, H. E. 1973. Biochirn. Biophys. Acta 3 3 1 : 430-41 68. Farquhar, M. N., McCarthy, B. J. 1 973. Biochem. Biophys. Res. Cornrnun. 53 : 5 1 5-22 69. Seale, R. L., Aronson, A. I. 1 973. J. Mol. Bioi. 75 : 633-45 70. Skoultchi, A., Gross, P. R. 1 973. Proc. Nat. A cad. Sci. USA 70 : 284()...44 7 1 . Gross, K. W., Jacobs-Lorena, M., Baglioni, c., Gross, P. R. 1 973. Proc. Nat. A cad. Sci. USA 70 : 2614- 1 8 72. Gross, P . R., Gross, K . W . 1973. The Role of R NA in Reproduction and Development, 4-10. New York : Elsevier 73. Destree, O. H. 1., d'Adelhart Toorop, H. A., Charles, R. 1 973. Cell Differ. 2 : 229-42 74. Ruderman, 1. V., Baglioni, c., Gross, P. R. 1974. Nature 247 : 36-38 75. Ruderman, J. V., Gross, P. R. 1974. Dev. Bioi. 36 : 286-98 76. Easton, D. P., Chamberlain, J. P., Whiteley, A. II., Whiteley, H. R. 1 974. Biochem. Biophys. Res. Cornmun. 57 : 5 1 3- 1 9 77. Ruderman, 1 . V., Gross, P. R . 1 973. J. Cell B ioi. 59 : 29 6a 78. Fambrough, D. M., Fujimura, F., Bonner, J. 1968. Biochemistry 7 : 575-84 79. Elgin, S. C. R., Hood, L. E. 1973. Biochemistry 1 2 : 4984-91 80. Stellwagen, R. H., Cole, R. D. 1968. J. BioI. Chern. 243 : 4456-62 8 1 . Stellwagen, R. H., Cole, R. D. 1969. J. Bioi. Chem. 244 : 4878-87

767

82. Hohmann, P., Cole, R. D. 1 97 1 . J. Mol. Bioi. 58 : 533-40 83. Raaf, J., Bonner, J. 1968. Arch. Biochem. Biophy,. 1 2 5 : 567-79 84. Makino, F., Tsuzuki, 1. 197 1 . Nature 2 3 1 : 446-47 85. Baldwin, 1. P., Bradbury, E. M., Butler Browne, G. S., Stephens, R. M. 1 97 3. FEBS Lett. 34 : 1 3 3-36 86. Mohberg, J., Rusch, H. P. 1 969. Arch. Biochem. Biophys. 134 : 5 7 7-89 ll7. Mohberg, 1., Rusch, H. P. 1 9 70. Arch. Biochern. Biophys. 1 38 : 418-32 88. Coukell, M . B., Walker, 1 O. 1973. Cell Differ. 2 : 8 7-95 89. Hsiang, M. W., Cole, R. D. 1 973. J. Bioi. Chem. 248 : 2007-1 3 90. Wintersberger, U., Smith, P., Letnansky, K. 1 973. Eur. J. Biochem. 33 : 1 2 3-30 9 1 . Franco, L., Johns, E. W., Navlet, 1. M. 1 974. Eur. J. Biochem. 45 : 83-89 92. Gray, R. H., Peterson, J. B., Ris, H . 1973. J. Cell Bioi. 58 : 244-47 93. Bradley, D. M., Goldin, H. H., Clay brook, J. R. 1 9 74. FEBS Lett. 4 1 : 2 1 922 94. Netrawali, M . S. 1 970. Exp. Cell Res.

63 : 422-26

95. Iwai, K., Hamana, K., Yabuki, H. 1 970. J. B io ch em. 68 : 597-601 96. Gorovsky, M. A., Pleger, G. L., Keevert, J. B., Johmann, C. A. 1 973. J. Cell Bioi. 57 : 773-8 1 97. Lipps, H. J., Sapra, G. R., Ammermann, D. 1 974. Chromosoma 45 : 273-80 98. Rob lin, R., Harle, E., Dulbecco, R. 1 97 1 . Virology 45 : 555-66 99. Frearson, P. M., Crawford, L. V. 1 972. J. Gen. Vi m l. 1 4 : 1 4 1-55 100. Lake, R. S., Barban, S., Salzman, N. P. 1 973. Biochem. Biophys. Res. Cornmun. 54 : 64()...4 7 1 0 1 . Cold Spring Harbor Symp. Quant. Bioi. 1 974. Vol. 39 102. Kedes, L. H., Birnstiel, M. L. 1 97 1 . Nature New Bioi. 230 : 165-69 103. Birnsticl, M. L., Telford, J., Weinberg, E., Stafford, D. 1 974. Proc. Nat. A cad. Sci. USA 71 : 2900--4 104. Weinberg, E. S., Birnstiel, M. L., Purdom, I. F., Williamson, R. 1 972. Nature New Bioi. 240 : 225-28 105. Grunstein, M., Schedl, P., Kedes, L. 1 973. Symposium on Molecular Cyto genetics, 1 1 5-23. New York : Plenum 106. G runstein, M., Levy, S., Schedl, P., Kedes, L. 1973. Cold Spring Harbor Syrnp. Quant. Bioi. 38 : 7 1 7-24 107. Farquhar, M. N., McCarthy, B. J. 1973. Biochernistrv 1 2 : 4 1 1 3-22 108. Pardue, M., Weinberg, E. , Kedes, L., Birnstiel, M. L., 1 972. J. Cell Bioi. 55 : 1 99a

768

ELGIN & WEINTRAUB

1 09. McCarthy, R J., Farquhar, M. N. 1 972. Brookhaven Symp. BioI. 23 : 1-43 1 10. Birnstiel, M . L., Weinberg, E., Pardue, M. L. See Ref. 105, 75-93 1 1 1 . Stout, J. T., Phillips, R. L. 1973. Proc. Nat. Acad. Sci. USA 70 : 3043-47 1 12. Elgin, S. C R., Froehner, S. C, Smart, 1. E., Bonner, J. 1 9 7 1 . Advan. Cell Mol. BioI. 1 : 1-57 1 1 3. Spelsberg, T. c., Wilhelm, J. A., Hnilica, L. S. 1972. Subcel/. Biochem.


1 : 107-45

1 14. M acGillivray, A. J, Paul, J., ThrelfaJl, G. 1 972. Advan. Cancer R es. 1 5 : 931 62 1 1 5. Elgin, S. C. R., Bonner, J. 1 973. The Biochemistry of Gene Expression in Higher Organisms, 1 42-63. Sydney : Aust. New Zealand Book Co. 1 1 6. Johnson, J. D., Douvas, A., Bonner, J. 1 974. Int. Rev. Cytol. Supp\. 4 1 17. Elgin, S. C R., Stumpf, W. 1 975. Ciba Found. Symp. 28 : 1 1 3-23 1 1 8. DeLange, R. J., Smith, E. L. 1974. Proteins 4 : Chap 2, 3rd ed. 1 19. Langan, T. A. 1967. Biochim. Biophys. Acta Libr. Ser. 1 0 : 233-50 1 20. Holoubek, V., Crocker, T. T. 1968. Biochim. Biophys. Acta 1 57 : 352-6 1 1 2 1 . Benjamin, w., Gellhorn, A. 1968. Froc. Nat. A cad. Sci. USA 59 : 262-68 1 22. Kleinsmith, L. J., Allfrey, V. G. 1969. Biochim. Biophys. Acta 1 75 : 123-35 1 23. Gershey, E. L., Kleinsmith, L. J. 1969. Biochim. Biophys. Acta 1 94 : 33 1-34 1 24. Shelton, K. R., Allfrey, V. G. 1 970. Nature 228 : 1 3 2-34 125. Teng, C. S., Teng, C. T., Allfrey, V. G. 1 97 1 . J. BioI. Chem. 246 : 3597-3609 126. Elgin, S. C. R., Boyd, J. R, Hood, L. E., Wray, W., Wu, F. C. 1973. Cold Spring

Harbor Symp.

Quant. BioI. 38 : 82 1-33

127. Elgin, S. C. R., Bonner, J. 1 970. Bio chemistry 9 : 4440-47 128. MacGillivray, A. J., Cameron, A., Krauze, R. J., Rickwood, D., Paul, J. 1972. Biochim. Biophys. Acta 227 : 384402 1 29. Garrard, W. T., Pearson, W. R., Wake, S. K., Bonner, J 1974. Biochem. Biophys. Res. Commun. 58 : 50-57 1 30. Elgin, S. C. R., Bonner, J. 1972. Bio chemistry 1 1 : 772-8 1 1 3 1 . Marushige, K., Brutlag, D., Bonner, .T. 1 968. Biochemistry 7 : 3 149-55 1 32. MacGillivray, A. 1., Rickwood, D. 1974. Eur. J. Biochem. 4 1 : 1 8 1-90 1 3 3 . .Tohns, E. W., Goodwin, G. H . , Walker, J. M., Sanders, C 1975. Ciba Found. Symp. 28 : 95- 1 07 1 34. Shooter, K. V., Goodwin, G. H.,

Johns, E. W. 1 974. Eur. J. Biochem. 47 : 263-70 1 3 5 . Kleinsmith, L. J., H eidema, 1., Carroll, A. 1970. Nature 226 : 1025--26 136. van den Broek, H. W. 1., Nooden, L. D., Sevall, J. S., Bonner, J. 1973. Biochemistry 1 2 : 229-36 1 37. Kleinsmith, L. J. 1 973. J. BioI. Chem. 248 : 5648-53 1 3 8 . Patel, G. H., Thomas, T. L. 1973. Proc. Nat. A cad. Sci. USA 70 : 2524-28 1 39. Wakabayashi, K., Wang, S., Hord, G., Hnilica, L. S. 1973. FEBS Lett. 32 : 46-48 1 40. Sheehan, D. M., OlillS, D. E. 1 9 74. Biochim. Biophys. Acta 353 : 438-46 1 4 1 . Platz, R. D., Kish, V. M., Kleinsmith, L. J. 1970. FEBS Lett. 1 2 : 38-40 142. M acGillivray, A. J., Carroll, D., Paul, J. 1 97 1. FEBS Lett. 1 3 : 204-8 143. Wang, 1. C. 1 9 7 1 . J. Mol. Bioi. 55 : 523-33 1 44. Wu, F. c., Elgin, S. C. R., Hood, L. 1975. J. Mol. Evol. In press 145. Gronow, M., Thackrah, T. 1 973. Arch. Biochem. Biophys. 1 58 : 377-86 146. Wu, F. C, Elgin, S. C. R., Hood, L. E. 1973. Biochemistry 1 2 : 2792-97 147. Yeoman, L. C, Taylor, C. W., Jordan, 1. J., Busch, H. 1 973. Biochem. Biophys. Res. Commun. 53 : 1 067-76 148. Barrett, T., Gould, H. J. 1 973. Biochim. Biophys. A cta 294 : 1 65-70 149. Fujitani, H . , Holoubek, V. 1974. Experientia 30 : 474-76 1 50. Tashiro, T., Mizobe, F., Kurokawa, M . 1974. FEBS Lett. 38 : 1 2 1 -24 1 5 1. Gierer, A. 1973. Cold Spring Harbor Symp. Quant. BioI. 38 : 95 1-61 1 52. Chytil, F., S pelsberg, T. C. 1 9 7 1 . Nature New Bioi. 233 : 2 1 5- 1 8 1 53. Wakabayashi, K., Wang, S., Hnilica, L. S. 1 9 74. Biochemistry 1 3 : 1 027-32 154. Wakabayashi, K., Hnilica, L. S. 1 973. Nature New Bioi. 242 : 1 53-55 1 55. Chiu, 1. F., Craddock, c., Morris, H. P., Hnilica, L. S. 1974. FEBS Lett. 42 : 94-97 1 56. Liao, S., Sagher, D., Fang, S. M. 1 968. Nature 220 : 1 336-37 1 57. Cox, R. F. 1 973. Eur. J. Biochem. 39 : 49-61 1 58. Hotta, Y., Stern, H. 1 9 7 1 . Dev. Bioi. 26 : 87-99 1 59. HOlla, Y., Stern, H. 1 97 1 . Nature New Bioi. 234 : 83-86 1 60. Herrick, G., Alberts, B. 1 973. Fed. Proc. 32 : 497 1 6 1 . Champoux, J. J., Dulbecco, R. 1972. Proc. Nat. Acad. Sci. USA 69 : 1 43-46 1 62. Basse, W. A., Wang, J. C. 1 974. Bio-



chemistry 13 : 4299--4303 1 63. Putrament, A. 1 97 1 . Genet. Res. 1 8 : 85-95 1 64. Comings, D. E., Riggs, A. D. 1 97 1 . Nature 233 : 48-50 165. Shea, M., Kleinsmith, L. J. 1 973. Biochem. Biophys. Res. Commun. 50 : 473-77 1 66. Barrett, T, M aryanka, D., Hamlyn, P. H., Go uld, H . J. 1 974. Proc. Nat. A cad. Sci. USA 7 1 : 5057-61 1 67. Britten, R. J., Davidson, E. H. 1969. Science 165 : 349-57 1 68. Steggles, A. W., Spelsberg, T C, O'Malley, B. W. 1 97 1 . Biochem. Biophys. Res. Commun. 43 : 20-27 1 69. Holmes, D. S., Mayfield, 1. E., Sander, G., Bonner, J. 1 972. Science 1 77 : 72-74 1 70. O'Malley, B. W., Means, A. R. 1 974. The Cell Nucleus, ed. H. Busch, 3 : 379--4 16. New York : Academic 1 7 1 . Sibley, C H., Tomkins, G. M. 1974. Cell 2 : 2 1 3-27 1 72. Gorovsky, M., Woodard, J. 1967. J. Cell. BioI. 33 : 723-28 1 73. Berendes, H. D. 1 968. Chromosoma 24 : 4 1 8-37 1 74. Holt, Th. K. H. 1 97 1 . Chromosoma 32 : 428-35 1 75. Helmsing, P. J., Berendes, H. D. 1 9 7 1 . J. Cell Bioi. 50 : 893-96 1 76. H elmsing, P. 1. 1972. Cell Differ. 1 : 1 9-24 1 77. Ashburner, M . 1974. Dev. Bioi. 39 : 14157 1 78 . Rovera, G., Farber, J., Baserga, R. 1 9 7 1 . Proc. Nat. A cad. Sci. USA 68 : 1 72 5-29 1 79. Rovera, G., Baserga, R. 1973. Exp. Cell Res. 7 8 : 1 1 8-26 1 80. LeStourgeon, W. M . , Rusch, H . P. 1 9 7 1 . Science 1 74 : 1 233-35 1 8 1 . LeStourgeon, W. M., Wray, W., Rusch, H. P. 1973. EXp. Cell Res. 79 : 487-92 1 82. LeStourgeon, W. M., Goodman, E. M., Rusch, H. P. 1 973. Biochim. Biophys. Acta 3 1 7 : 524-28 1 83. Baserga, R., Bombik, B., Nicolini, C 1 975. Ciba Found. Symp. 28 : 269-79 1 84. Lin, J. C, Nicolini, C, Baserga, R. 1974. Biochemistry 13 : 4 1 27-33 1 8 5. LeStourgeon, W. M., Forer, A., Yang, Y. Z., Bertram, J. S., Rusch, H. P. 1975. Biochim. Biophys. Acta 379 : 53952 1 86. Hinkley, R., Telser, A. 1 974. Exp. Cell Res. 86 : 1 61-64 1 8 7. Jackson, V., Earnhardt, J., Chalkley, R. 1968. Biochem. Biophys. Res. Commun. 33 : 253-59 1 88 . H arlow, R., Tolstoshev, P., Wells, J. R. E. 1972. Cell Differ. 2 : 341--49

769

189. Tata, J. R., Hamilton, M. J., Cole, R. D. 1 972. J. Mol. BioI. 67 : 2 3 1 -46 190. Shelton, K. R. 1 973. Can. J. Biochem. 5 1 : 1442--47 1 9 1 . Comings, D. E . , Okada, T. A. 1 970. Exp. Cell Res. 62 : 293-302 192. Aaronson, R. P., Blobel, G. 1 974. .J. Cell BioI. 62 : 746-54 193. Georgiev, G. P., Samarina, O. P. 1 97 1 . Advan. Cell Bioi. 2 : 47- 1 1 0 194. Samarina, O . P., Lukanidi, E. M., Molnar, J., Georgiev, G. P. 1 968. J. Mol. Bioi. 33 : 2 5 1 -63 1 95. Krichevskaya, A. A., Georgiev, G. P. 1 969. Biochim. Biophys. Acta 1 94 : 6 1 921 1 96. Martin, T . E., Swift, H . 1 973. Cold Spriny Harbor Symp. Quant. BioI. 3 8 : 921-32 1 97. Hill, R. J., Maundrell, K. G., Callan, H. G. 1 973. Molecular Cytogenetics,

147-53. New York : Plenum

1 98. Sommerville, J. 1 973. J. Mol. Bioi. 78 : 487-503 1 99. Sommerville, J., Hill, R. J. 1 973. Nature New Bioi. 245 : 1 04-6 200. Scott, S. E. M., Sommerville, J. 1 974. Nature 250 : 680-82 201 . Pederson, T 1974. Fro c. Nat. A cad. Sci. USA 7 1 : 6 1 7-21 202. Derksen, J., Berendes, H. D., Willart, E. 1 973. J. Cell Bioi. 59 : 661-68 203. Williamson, R. 1 973. FEBS Lett. 3 7 : 1-6 204. Prescott, D. M. 1 966. J. Cell BioI. 3 1 : 1-9 205. Robbins, E., Borun, T. W. 1 967. Proc. Nat. A cad. Sci. USA 57 : 409-1 6 206. Sadgopal, A., Bonner, J. 1 969. Biochim. Biophys. A ctu 1 86 : 349-57 207. Takai, S., Borun, T W., M uchmore, J., Lieberman, I. 1 968. Nature 2 1 9 : 860-61 208. Kedes, L. H., Gross, P. R. 1 969. Nature 223 : 1 335-39 209. Yarbro, J. W. 1 967. Bioc/Jim. Biophys. Acta 1 45 : 53 1 -34 2 1 0. Weintraub, H. 1 972. Nattlre 240 : 44953 2 1 1 . Gurley, L. R., Walters, R. A., Tobey, R. A. 1 972. Arch. Biochem. Biophys. 148 : 633--41 2 12. Appels, R., Wells, J. R. E. 1 972. J. Mol. Bioi. 70 : 425-34 2 1 3. Adamson, E. D., Woodland, H. R. 1 974. J. Mol. BioI. 88 : 263-8 5 2 14. Jacobs-Lorena, M., Baglioni, C, Borun, T W. 1 972. Froc. Nat. A cad. Sci. USA 69 : 2095-99 2 1 5. BreindI, M., Gallwitz, D. 1 973. Eur. J. Biochem. 32 : 38 1-9 1 2 1 6. Gallwit z, D., Breindl, M. 1 972. Biochem.


770

ELGIN & WEINTRAUB

Biophys. Res. Commun. 47 : 1 106- 1 1 2 1 7. Schochetman, G., Perry, R. P. 1 972. J. Mol. Bioi. 63 : 577-90 2 1 8. Adesnik, M., Darnell, J. E. 1 972. J. Mol. Bioi. 67 : 397-406 2 19. Breindl, M., Gallwitz, D. 1 974. Mol. BioI. Rep. 1 : 263-68 220. Zauderer, M., Liberti, P., Baglioni, e. 1973. J. Mol. Bioi. 79 : 5 57-86 22 1 . Jacobs-Lorena, M., Baglioni, e. 1973. Mol. Bioi. Rep. 1 : 1 1 3-1 7 222. Pestana, A., Pitot, H . e. 1 9 74. Nature 247 : 200-2 223. Oliver, D., Granner, D., Chalkley, R. 1 9 74. Biochemistry 1 3 : 746-49 224. Gallwitz, D., M ueller, G. C. 1 969. J. Bioi. Chern. 244 : 5947-52 225. Jacobs-Lorena, M., Gabrielli, F., Borun, T. W., Baglioni, e. 1973. Biochirn. Biophys. Acta 324 : 275-8 1 226. Breindl, M., Gallwitz, D. 1 974. Eur. J. Biochern. 45 : 9 1-97 227. Borun, T. W., Scharff, M. D., Robbins, E. 1967. Proc. Nat. Acad. Sci. USA 58 : 1 977-83 228. Butler, W. B., M ueller, G. e. 1973. Riochim. Biophys.

Acta 294 : 48 1 -96

229. Perry, R. P., Kelley, D. E. 1 973. J. Mol. Bioi. 7 1 : 6 8 1 -96 230. Hershey, H., Stieber, 1., Mueller, G. e. 1973. Biochirn. Biophys. Acta 3 1 2 : 50917 2 3 1 . Balhorn, R., Tanphaichitr, N., Chalkley, R., Granner, D. K. 1 973. Biochemistry 1 2 : 5 1 46-50 232. Woese, e. R. 1 973. J. Mol. Evol. 2 : 205-8 233. Weintraub, H. 1 973. Cold Spring Harbor Symp. Quant. Bioi. 38 : 247-56 234. Cave, M. D. 1968. Chrornosorna 25 : 392-401 235. Stein, G., Baserga, R. 1 970. Biochern. Biophys. R es. Commun. 4 1 : 7 1 5-22 236. Cross, M. E. 1 972. Biochern. J. 1 28 : 1 2 1 3- 1 9 237. Stein, G . S., Borun, T . W. 1 972. J . Cell Res. 52 : 292-307 238. Gerner, E. W., Humphrey, R. M. 1973. Biochirn. Biophys. Acta 331 : 1 1 7-27 239. Bhorjee, 1. S., Pederson, T. 1973. Biochemistry 1 2 : 2766-73 240. Borun, T. W., Stein, G. S. 1972. J. Cell Bioi. 52 : 308-1 5 24 1 . Tsanev, R., Russev, G. 1 974. Eur. J. Biochem. 43 : 257-63 242. Sadgopal, A., Bonner, 1. 1970. Biochirn. Biophys. Acta 207 : 227-39 243. Wray, W., Elgin, S. e. R. 1 975. J. Cell Bioi. In press 244. Comings, D. E., Tack, L. O. 1 973. Exp. Cell Res. 82 : 1 75-9 1

245. Gall, J. G. 1973. Symposium on Molecular Cytology, 59-74. New York : Plenum 24,6. Helmsing, P. J., van Eupen, O. 1 973. 'Biochirn. Biophys. Acta 308 : 1 54-60 247. Bloch, D. P., Macquigg, R. A., Brack, S D., Wu, 1. R. 1967. J. Cell Bioi. 33 : 451-67 248. Byvoet, P. 1 966. J. Mol. Bioi. 1 7 : 3 1 1 18 249. Hancock, R . 1 969. J. Mol. Bioi. 40 : 457-66 250. Appels, R., Ringertz, N. R. 1 974. Cell Differ. 3 : 1-8 251. Garrard, W. T., Bonner, J. 1 974. J. Bioi. Chern. 249 : 5570-79 252. Bondy, S. e. 1 97 1 . Biochern. J. 1 23 : 465-69 253. Dice, 1. F., Schimke, R. T. 1 973. Arch. Biochern. Biophys. 1 58 : 97-105 254. Karn, 1., Johnson, E. M . Vidali, G., Allfrey, V . G. 1 9 74. J. Bioi. Chern. 249 : 667-77 255. Teng, C. S., Hamilton, T. H. 1 970. Biochern. Biophys. Res. Commun. 40 : 1 23 1-38 256. Enea, V., Allfrey, V. G. 1 97 3 . Nature 242 : 265-67 257. Stein, G., Baserga, R. 1 970. J. Bioi. Chern. 245 : 6097-6 1 05 258. Choe, B. K., Rose; N. R. 1 974. Exp. o Cell Res. 83 : 26 1-80 259. Cognetti, G., Settineri, D., Spinelli, G. 1 972. Exp. Cell Res. 71 : 465-68 260. Hill, R. 1., Poccia, D. L., Doty, P. 1 97 1 . J . Mol. Bioi. 6 1 : 445-62 26 1 . Sevaljevic, L. 1973. Dev. Bioi. 34 : 26773 262. Pipkin, 1. L. Jr., Larson, D. A. 1 973. Exp. Cell Res. 79 : 28-42 263. Chytil, F., Glasser, S. R., Spelsberg, T. e. 1 9 14. Dev. Bioi. 37 : 295-305 264. Panyim, S., Chalkley, R. 1 969. Biochem. Biophys. Res. Cornrnun. 37 : 1 042-49 265. Lea, M. A., Youngworth, L. A., Morris, H. P. 1 9 74. Biochern. Biophys. Res. Commun. 58 : 862-67 266. Rubio, V., Tsai, W. P., Rand, K., Long, e. 1973. Int. J. Cancer 1 2 : 545--5 0 267. Zardi, L., Lin, 1. e., Baserga, R. 1 973. Nature New BioI. 245 : 2 1 1 - 1 4 268. Johns, E. W . , Davies, A. J. S . , Barton, M.E. 1 970. Biochirn. Biophys. Acta 2 1 3 : 537-38 269. Hohmann, P., Cole, R. D., Bern, H. A. 1 9 7 1 . J. Nat. Cancer Inst. 47 : 337-41 270. Balhorn, R., Chalkley, R., Granner, D. 1972. Biochemistrv 1 1 : 1 094-98 27 1 . McClure, M. E., Hnilica, L. S. 1 972. Subcell Biochem. 1 : 3 1 1-32 272. Stein, G., Baserga, R. 1 972. Advan.



Cancer Res. 1 5 : 287-330 273. DeLange, R. 1., Smith, E. L. 1971. Ann. Rev. Biochem. 40 : 279-3 14 274. Shepherd, G. R., Noland, B. 1., Hardin, J. M., Byvoet, P. See Ref. 1 1 5, 1 64-76 275. Johnson, E. M., Karn, J., Allfrey, V. G. 1974. J. Bioi. Chem. 249 : 4990-99 276. Ingles, C. J., Dixon, G. H. 1 967. Proc. Nat. A cad. Sci. USA 58 : 101 1-18 277. Louie, A. J., Dixon, G. H. 1972. Proc. Nat. A cad. Sci. USA 69 : 1 975-79 278. Louie, A. J., Dixon, G. H. 1973. Nature New Riol. 243 : 1 64-6R 279. Candido, E. P. M., Dixon, G. H. 1972. Proc. Nat. Acad. Sci. USA 69 : 2015-19 280. Lake, R. S., Goidl, 1. A., Salzman, N. P. 1972. Exp. Cell Res. 73 : 1 1 3-21 28 1 . Gurley, L. R., Walters, R. A., Tobey, R. A. 1973. Biochem. Biophys. Res. Commun. 50 : 744-50 282. Balhorn, R., Jackson, V., Granner, D., Chalkley, R. 1975. Biochemistry. In press 283. Langan, T. A., Hohmann, P. 1974. Fed. Proc. 33 : 1 597 284. Lake, R. S., Salzman, N . P., 1972. Biochemistry 1 1 : 48 1 7-26 285. Lake, R. S. 1 9 73. Nature New Bioi. 242 : 145-46 286. Marks, D. B., Park, W. K., Borun, T. W. 1973. J. Bioi. Chem. 248 : 566067 287. Gurley, L. A., Walters, R. A., Tobey, R. A. 1974. J. Cell Bioi. 60 : 356-64 288. Bradbury, E. M., Inglis, R. 1., M atthews, H. R., Sarner, N. 1973. Eur. J. Biochem. 33 : 1 3 1 -39 289. Bradbury, E. M., Inglis, R. 1., M atthews, H. R. 1 974. Nature 247 : 257-61 290. Bradbury, E. M., Inglis, R. 1., Matthews, H. R., Langan, T: A. 1 974. Nature 249 : 553-56 29 1 . Langan, T. A. 1 968. Science 1 62 : 57980 292. Langan, T. A. 196 1. Fed. Proc. 28 : 600 293. Langan, T. A. 1 969. J. Bioi. Chem. 244 : 5763-65 294. Langan, T. A. 1 969. Proc. Nat. A cad. Sci. USA 64 : 1 276-83 295. Mallette, L. E., Neblett, M., Exton, J. H., Langan, T. A. 1973. J. Bioi. Chem. 248 : 6289-9 1 296. Takeda, M., Ohga, Y. 1973. J. Biochem. 73 : 62 1 -29 297. Langan, T. A., Smith, L. 1967. Fed. Proc. 2 6 : 603 298. Meisler, M. H., Langan, T. A. 1967. J. Cell Bioi. 3 5 : 9 1 a 299. Meisler, M . H . , Langan, T . A. 1969. J. BioI. Chern. 244 : 4961--68 300. Adler, A. 1., Langan, T. A., Fasman,

30 1 .

302. 303. 304. 305. 306.

307. 308. 309. 3 10. 311. 3 12. 3 [3. 3 14. 31 5. 3 1 6. 3 1 7. 3 1 8. 3 1 9. 320. 321. 322. 323. 324. 325. 326.

771

G. D. 1972. Arch. Biochem. Biophys. 1 5 3 : 769-77 Adler, A. 1., Fasman, G. D., Wangh, w., Allfn;y, V. G. 1974. J. Bioi. Chem. 249 : 29 1 1- [ 4 Watson, G., Langan, T . A . 1 973. Fed. Proc. 32 : 588 Kleinsmith, L. 1., Allfrey, V. G., Mirsky, A. E. 1966. Science 1 54 : 780-8 1 Gershey, E. L., Kleinsmith, L. 1. 1969. Biochim. Biophys. Acta 194 : 5 1 9-25 Johnson, E. M., Allfrey, V. G. 1972. Arch. Biochem. Biophys. 1 5 2 : 786-94 Platz, R. D., Stein, G. S., Kleinsmith, L. 1. 1 973. Biochem. Biophys. Res. Commun. 51 : 735-40 Platz, R. D., Hnilica, L. S. 1 973. Biochem. Biophys. Res. Commun. 54 : 222-27 Rickwood, D., Riches, P. G., Mac Gillivray, A. J. 1973. Biochim. Biophys. A cta 299 : 1 62-7 1 Kish, V. M., Kleinsmith, L. J. 1974. J. Bioi. Chem. 249 : 750-60 Trevithick, J. R. 1 974. Can. J. Biochem. 52 : 406-1 3 Fleischer-Lambropoulos, H . , Sark ander, H. I., Brade, W. P. 1974. FEBS Lett. 45 : 329-32 Kleinsmith, L. J. 1975. J. Cell Physiol. In press Huberman, 1. A. 1973. Ann. Rev. Biochem. 42 : 355-78 Simpson, R. T. 1973. Advan. Enzymol. 38 : 41-108 Kornberg, R. D. 1 974. Science 1 8 4 : 868-71 Pardon, 1. F., Wilkins, M. H. F. 1972. J. Mol. Bioi. 68 :. 1 1 5-24 Bram, S., Ris, H. 1971. J. Mol. Bioi. 55 : 325-36 Hewisch, D., Burgoyne, L. 1973. Biochem. Biophys. Res. Commun. 52 : 504-10 Noll, M . 1974. Nature 2 5 1 : 249 52 Axel, R., Melchior, W., Sollner-Webb, B., Felsenfe[d, G. 1974. Proc. Nat. Acad. Sci. USA 71 : 4 101-5 Weintraub, H., Van Lente, F. 1975. Ciba Found. Symp. 28 : 291 -307 Clark, R. J., Felsenfeld, G. [ 9 7 1 . Nature New BioI. 229 : 1 0 1-6 Itzhaki, R. F. 1971. Biochem J. 125 : 22 1 -24 Itzhaki, R. F. 1 974. Eur. J. Biochem. 47 : 27-33 Mirsky, A. E. 1 97 1 . Proc. Nat. Acad. Sci. USA 68 : 2945-48 Davidson, E. H., Graham, D. E., Neufie[d, B. R., Chamberlin, M . E., Amenson, C. S., Hough, B. R., Britten,

772

327. 328.

329. 330.


33 1 .

332.

333. 334. 335. 336.

ELGIN & WEINTRAUB

R. 1. 1973. Cold Spring Harbor Symp. Quant. Bioi. 38 : 295�302 Garrett, R. A. 1 960. J. Mol. Bioi. 3 8 : 249�50 plus plate Bradbury, E. M., Molgaard, H. V., Stephens, R. M. 1972. Eur. J. Biochem. 3 1 ; 474�82 Kornberg, R. D., Thomas, 1. O. 1 974 . Science 1 84 : 865--68 Richards, B. M., Pardon, J. F. 1970. Exp. Cell Res. 62 : 184�96 SkIdmore, c., Walker, I. 0., Pardon, J. F., Richards, B. M. 1973. FEBS Lett. 32 : 1 75�78 Baldwin,J. P., Boseley, P. G., Bradbury, E. M., Ibel, K. 1975. Nature 253 : 245� 49 Olins, A. 1., Olins, D. E. 1974. Science 1 8 3 : 330-32 Woodcock, C. 1. F. 1973. J. Cell Bioi. 59 : 368a Senior, M. B., Olins, A. 1., Olins, D. E. 1975. Science 1 8 7 : 1 73�75 Georgiev, G. P., Varshavsky, A. J., Church, R. B., Ryskov, A. P. 1973. Cold Spring Harbor Symp. Quant. BioI. 38 : 869�84

337. Varshavsky, A. J., Ilyin, Y. V., Georgiev, G. P. 1 974. Nature 250 : 602�6 338. Sahasrabuddhe, C. G., Van Holde, K. E. 1974. J. Bioi. Chem. 249 : 1 52�56 339. Weintraub, H., Van Lente, F. 1974. Proc. Nat. A cad. Sci. USA 71 : 4249�53 340. Weintraub, H. 1 975. Proc. Nat. A cad. Sci. USA. In press 34 1. Crick, F. H. C. 1 9 7 1 . Nature 234 : 25�27

342. D'Anna, J. A. Jr., Isenberg, I. 1972. Biochemistry 1 1 : 401 7�25 343. Smerdon, M. 1., Isenberg, 1 . 1974. Biochemistry 1 3 : 4046-49 344. Ed wards, P. A., Shooter, K. V. 1969. Biochem. J. 1 1 4 : 53 345. Bradbury, E. M., Rattle, H . W. E. 1 972. Eur. J. Biochem. 27 ; 270-81 346. Kelley, R. I. 1973. Biochem. Biophys. Res. Commun. 54 : 1 588�94 347. Roark, D. E., Geoghegan, T. E., Keller, G. H. 1974. Biochem. Biophys. Res. Commun. 59 ; 542-47 34K Olins, D. E., Wright, E. B. 1 973. J. Cell Bioi. 59 : 304� 1 7 349. Martinson, H., McCarthy, B . 1 . 1975. Biochemiwy 14; 1 07 3�78 350. Simpson, R. T. 1 9 7 1 . Biochemistry 1 0 : 4466-70 3 5 1 . Malchy, B., Kaplan, H. 1 974. J. Mol. Bioi. 82 ; 537-45 352. Bustin, M. 1 973. Nature New BioI. 245 : 207�9 353. Jacquet, M., Groner, Y., Monroy, G.,

354. 355. 356. 357. 358. 359. 360.

361. 362.

363.

364.

Hurwitz, 1. 1974. Proc. Nat. Acad. Sci. USA 7 1 : 3045-49 Shih, T. Y., Bonner, J. 1 970. J. Mol. Bioi. 48 : 469�87 Smart, J. E., Bonner, J. 1 97 1 . J. Mol. Bioi. 58 : 661�74 Smart, 1. E., Bonner, J. 1 9 7 1 . J. Mol. BioI. 58 : 675�8 4 Koslov, Y. V., Georgiev, G. P. 1 970. Nature 228 : 245-47 Limborska, S. A., Georgiev, G. P. 1 972. Cell Dijjer. 1 : 245�5 1 Cedar, H., Felsenfeld, G. 1 973. J. Mol. Bioi. 77 : 237�54 Frenster, J. H., Allfrey, V. G., Mirsky, A. E. 1 963. Proc. Nat. A cad. Sci. USA 50 ; 1026-32 Frenster, J. H . 1 965. Nature 206 : 68083 McCarthy, B. 1., Nishiura, J. T., Doenecke, D., Nasser, D. S., Johnson, C. B. 1 973. Cold Spring Harbor Symp. Quant. Riol. 38 : 763�7 1 Murphy, E . C . Jr., Hall, S . H . , Shepherd, J. H., Weiser, R. S. 1973. Biochemistry 1 2 : 3843�53 Reeck, G. R., Simpson, R. T., Sober, H. A. 1 9 72. Proc. Nat. A cad. Sci. USA 69 : 23 1 7�2 1

365. Simpson, R. T. 1 974. Proc. Nat. A cad. Sci. USA 7 1 : 2740-43 366. Marushige, K., Bonner, J. 1 9 7 1 . Proc. Nat. Acad. Sci. USA 68 : 2941-44 367. Billing, R. 1., Bonner, 1. 1972. Biochim. Biophys. Acta 28 1 : 453�62 368. Gottesfeld, 1. M., Garrard, W. T., Bagi, G., Wilson, R. F., Bonner, J. 1 9 74. Proc. Nat. Acad. Sci. USA 7 1 : 2 1 93� 97 369. Simpson, R. T., Reeck, G. R. 1973. Biochemistry 1 2 : 3853 58 370. McConaughy, B. L., McCarthy, B. 1. 1972.

Biochemistry 1 1 : 998� 1 003

371. Saucier, 1. M., Wang, J. C. 1972. Nature New Bioi. 23 9 : 167�70 372. Travers, A., Baillie, D. 1., Pederson, S. 1 972. Nature New BioI. 243 : 1 6 1 �63 373. Travers, A. 1974. Cell. 3 : 97�104 374. Hossenlopp, P., audet, P., Chambon, P. 1974. Eur. J. Biochem. 4 1 : 367�78 375. Ghosh, S., Echols, H. 1 972. Proc. Nat. A cad. Sci. USA 69 : 3660-64 376. Hall, B. D., Brzezinska, M., Hollenberg, C. P., Schultz, 1. D. See Ref. 105, 2 1 7� 26

377. Hsu, T. C. 1972. Methods Cell Physiol.

5 : 1�36

378. Hsu, T. C. 1973. Ann. Rev. Genet. 7 : 1 53�76 379. Gottesfeld, J. M., Bonner, J., Radda, G. K., Walker, 1. O. 1 9 74. Biochemistry


CHROMOSOMAL PROTEINS AND CHROMA TIN STRUCTURE

1 3 : 2937-45 380. Faust, 1., Vogel, W. 1 974. Nature 249 : 352-53 3 8 1 . DeLange, R. 1., Fambrough, D. M., Smith, E. L., Bonner, J. 1 969. J. BioI. Chem. 244 : 3 19-34 382. DeLange, R. J., Fambrough, D. M., Smith, E., Bonner, 1. 1 969. J. Bioi. Chem. 244 : 5669-79 383. Ogawa, Y. et al 1969. J. Bioi. Chem. 244 : 4387-92 384. Sautiere, P., Tyrou, D., Moschetto, Y., Biserte, G. 1 97 1 . Biochimie 53 : 479-83 385. Sautiere, P., Lambelin-Breynaert, M. D., Mos�hetto, Y., B i serte, G. 1 97 1 . Biochimie 53 : 7 1 1 - 1 5 386. Desai, L . , Ogawa, Y . , Mauritzen, C. M., Taylor, C. W., Starbuck, W. C. 1 969. Biochim. Biophys. Acta 1 8 1 : 1 4653 387. Strickland, M., Strickland, W. N., Brandt, W. F. 1974. FEBS Lett. 40 : 346 48 388. DeLange, R. J., Hooper, J . A., Smith, E. L. 1 972. Proc. Nat. A cad. Sci. USA 69 : 882-84 389. DeLange, R. J., Hooper, 1. A., Smith, E. L. 1973. J. Bioi. Chem. 248 : 3261-74 390. Olson, M. O. 1., Jordan, 1., Busch, H . 1 972. Biochem. Biophys. Res. Commun. 46 : 50-55 39 1 . Brandt, W. F., van Holt, C. 1972. FEBS Lett. 23 : 357-60 392. Brandt, W. F., van Holt, C. 1 974. Eur. J. Biochem. 46 : 407- 1 7 393. Brandt, W . F., van Holt, C . 1 9 74. Eur. J. Biochem. 46 : 4 1 9-29 394. Hooper, J. A., Smith, E. L., Sommer, K. R., Chalkley, R. 1973. J. Bioi. Chem. 248 : 3275-79 395. Brandt, W. F., Strickland, W. N., von Holt, C. 1974. FEBS Lett. 40 : 349-52 396. Brandt, W. F., Strickland, W. N., Morgan, M . , von Holt, C. 1 9 74. FEBS Lett. 40 : 1 67-72 397. Patthy, L., Smith, E. L., Johnson, J. 1 973. J. Bioi. Chem. 248 : 6834-40 398. lwai, K., Hayashi, H., Ishikawa, K. 1972. 1. Rinchem. 72 : 3 57-67 399. Yeoman, L. C. et aI 1972. J. Bioi. Chem. 247 : 60 1 8-23 400. Sautiere, P., Tyrou, D., Laine, B., Mizon, J., Ruffin, P., Biserte, G. 1974. Eur. J. Biochem. 4 1 : 563-76 40 1 . Sautiere, P. 1975. Ciba Found. Symp. 28 : 77-'138 402. Bailey, G. S., Dixon, G. H. 1973. J. BioI. Chem. 248 : 5463-72 403. Jones, G. M., Rail, S. c., Cole, R. D. 1 974. J. Bioi. Chem. 249 : 2548-53 404. W ei ss, S. B. 1 960. Proc. Nat. Acad. Sci.

773

USA 46 : 1 020-30 405. Tata, 1. R., Baker, B. 1974. Exp. Cell Res. 1)3 : 1 1 1 -24, 125-3!\ 406. Lentfer, D., Lezi us, A. G. 1 972. Eur. J. Biochem. 30 : 278-84 407. Mondal, H., Ganguly, A., Das, A., Mandai, R. K, Biswas, B. B. 1 972. Eur. J. Biochem. 28 : 1 43-50 408. Ganguly, A., Das, A., Mondal, H . , Mand ai, R. K, Biswas, B. B. 1973. FEBS Lett. 34 : 27-30 409. Sasaki, K., Tazawa, T. 1 973. Biochem. Biophys. Res. Commun. 52 : 1440-49 4 10. Patel, G., Howk, R., Wang, T. Y. 1 967. Nature 2 1 5 : 1488-89 4 1 1 . Howk, R., Wang, T. Y. 1 969. Arch. Biochem. Biophys. 1 33 : 238-46 4 1 2. Howk, R., Wang, T. Y. 1 970. Eur. J. Biochem. 1 3 : 455-60 413. Tsuruo, T., Tomita, Y., Satoh, H., U kita, T. 1 972. Biochem. Biophys. Res. Commun. 48 : 776-82 4 1 4. Tsuruo, T., Ukita, T. 1 974. Biochim. Biophys. Acta 353 : 1 46-59 4 1 5. Loeb, L. A. 1970. Nature 226 : 448-49 4 1 6. Chang, L. M. S. 1 973. J. Bioi. Chem. 248 : 3789-95 4 1 7. Urbanczyk, J., Studzinski, G. P. 1974. Biochem. Biophys. Res. Commun. 59 : 6 1 6-22 4 1 8. Gaziev, A. I., Kuzin, A. M. 1 973. Eur. J. Biochem. 37 : 7- 1 1 419. O'Connor, P . 1 . 1 969. Biochem. Biophys. Res. Commun. 35 : 805-10 420. Wang, T. Y. 1 968. Arch. Biochem. Biophys. 1 2 7 : 23 5-40 42 1 . Srivastava, B. I. S. 1972. Biochem. Biophys. Res. Commun. 48 : 270-73 422. Yamada, M.. Sugimura. T. 1 973. Biochemistry 1 2 : 3303-8 423. Sugimura, T. 1 973. Progr. Nucl. Acid Res. Mol. BioI. 1 3 : 1 27-5 1 424. Miyakawa, N., Veda, K, Hayaishi, O. 1 972. Biochem. Biophys. Res. Commun. 49 : 239-45 425. Racey, L. A., Byvoct, P. 1 97 1 . Exp. Cell Res. 64 : 366-70 426. Racey, L. A., Byvoet, P. 1972. Exp. Cell Res. 73 : 329-34 427. Gallwitz, D., Sures, I. 1 972. Biochim. Biophys. Acta 263 : 3 1 5-328 428. Garrels, 1. I., Elgin, S. C. R., Bonner, 1. 1972. Biochem. Biophys. Res. Commun. 46 : 545-5 1 429. Bartley, J., Chalkley, R. 1 970. J. Bioi. Chem. 245 : 4286-92 430. Takeda, M., Yamamura, H., Ohga, Y. 197 1 . Biochem. Biophys. Res. Commun. 42 : 1 03-1 0 43 1 . Comb, D . G., Sarkar, N., Pinzino, C . J. 1 966. J. Bioi. Chem. 24 1 : 1 857-62


774

ELGIN & WEiNTRAUB

432. Chen, C, Smith, D. L., Bruegger, B., Halpern, R. M., Smith, R. A. 1974. Biochemistry 1 3 : 378 5-89 433. Smith, D. L., Chen, C C, Bruegger; B. B. 1974. Biochemistry 1 3 : 3780-85 434. Smith, J. A., Stocken, L. A. 1973. Biochem. J. 1 3 1 : �59-6 1 435. Goodwin, G. H., Johns, E. W. 1 973. Eur. J. Biochem. 40 : 2 1 5-- 19 436. Goodwin, G. H., Sanders, C, Johns, E. W. 1973. Eur. J. Biochem. 38 : 14-19 437. Patel, N. T., Holoubek, V. 1974. FEBS Lett. 46 : 1 54-57 438. Miuzluff, W. F. Jr., Sanders, L. A., Miller, D. M., McCarty, K. S. 19 72. J. Bioi. Chern. 247 : 2026-33 439. Douvas, A., Bonner, 1. 1 974. J. Cell Bioi. 63 : 89a 440. Sevall, J. S., Cockburn, A., Savage, M., Bonner, 1. 1975. Biochemistry 14 : 78289 441 . Chong, M . T., Garrard, W. T., Bonner, J. 1 9 74. Biochemistry 1 3 : 5 1 28-34 442. Simpson, R. T. 1974. Curro Top. Biochem. 1973 : 1 3 5-85 443. Duer!

Chromatin-bound protease: degradation of chromosomal proteins under chromatin dissociation conditions.

Effect of chromosomal proteins extractable with low concentrations of NaCl on chromatin structure of resting and proliferating cells.

HMGA proteins as modulators of chromatin structure during transcriptional activation.

Analysis of chromosomal proteins of fractionated chromatin from rat liver and transplantable hepatocellular carcinomas.

Role of LET and chromatin structure on chromosomal inversion in CHO10B2 cells.

Increase in initiation sites for chromatin directed RNA synthesis by acetylation of chromosomal proteins.

DNA-binding activity of tightly-bound nonhistone chromosomal proteins in chicken liver chromatin.

Selective release of chromosomal proteins during limited DNAase 1 digestion of avian erythrocyte chromatin.

Effect of gamma irradiation on chromosomal proteins from Yoshida ascites tumour chromatin.

Role of nonhistone chromosomal proteins in determining circular dichroism spectra of chromatin.

Enhanced template activity in chromatin from adrenal medulla after phosphorylation of chromosomal proteins.

Interaction of chromosomal proteins with BrdU substituted DNA as determined by chromatin-DNA competition.

Chromatin structure and transcription.

Chromatin and nucleosome structure.

Chromatin: Histone influences on chromosomal translocations.

Differentiation-dependent alteration in the chromatin structure of chromosomal protein HMG-17 gene during erythropoiesis.

The chromatin architectural proteins HMGD1 and H1 bind reciprocally and have opposite effects on chromatin structure and gene regulation.

Chromosomal proteins and gene regulation.

Chromatin structure and gene expression.

Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins.

DNA methylation and chromatin structure.

Chromatin structure and dynamics in hot environments: architectural proteins and DNA topoisomerases of thermophilic archaea.

Quaternary structure of chromatin.

Yeast chromatin subunit structure.