Proc. NatL Acad. Sci. USA Vol. 79, pp. 1535-1539, March 1982 Cell Biology

Isopeptidase: A novel eukaryotic enzyme that cleaves isopeptide bonds (histone H2A//chromatin/carbon-nitrogen lyase/cell cycle) SEI-ICHI MATSUI*, AVERY A. SANDBERG*t, SHIGERU NEGORO*, BEN K. SEON*, AND GIDEON GOLDSTEIN: *Roswell Park Memorial Institute, Buffalo, New York 14263; and tOrtho Pharmaceutical Corporation, Raritan, New Jersey 08869 Communicated by David Harker, November 20, 1981 ABSTRACT In an attempt to clarify the regulatory mech- MATERIALS AND METHODS anism that accounts for the shift ofprotein A24 in the mitotic cycle, we demonstrated the existence ofan enzyme, provisionally termed Cultured cell lines of a Chinese hamster (DON) and a mouse isopeptidase, that cleaves A24 stoichiometrically into histone H2A (L929) were maintained at 37°C in RPMI 1640 medium/8% fetal and ubiquitin. Properties of this enzyme are (i) most eukaryotes, calf serum (20, 21). E. coli (strain K-12, wild type) and yeast including mammals, amphibia, chicken, and yeast, contain iso- were gifts ofAllen Leonard and Joel Huberman ofour institute. peptidase in the cytoplasm; (ii) a significant increase in enzyme Metaphase chromosomes, nuclei, acid-soluble chromatin pro- binding to chromatin occurs when cells enter mitosis; (iii) Esche- teins, and A24 were prepared as described (13, 21, 22). '25I richia coli does not contain isopeptidase; (iv) isopeptidase has a Labeling ofA24 was carried out by the chloramin T method (23). molecular weight of 38,000; (v) at an ionic strength that induces To protect A24 molecules from radiation-caused disintegration, globular conformation of H2A, isopeptidase activity is repressed; the iodinated sample was immediately adjusted to a concentra- (vi) a SH group is an essential cofactor; and (vii) most divalent cat- tion of 18.7 nmol of A24/ml and A24-free calf thymus histone ions (except Mg2+ and Ca2+) are inhibitory. In view of the stoi- at 1 was chiometric conversion of A24 into H2A and ubiquitin by isopep- mg/ml added as carrier. 125I Stoichiometry, as deter- tidase in vitro, A24 probably contains a Gly-Gly dipeptide in mined by the isopeptidase reaction (see Fig. 1), was 6:1 (H2A/ isopeptide linkage but no other intervening polypeptides. Since ubiquitin). To avoid extreme modification of side ubiquitin in various eukaryotes binds to proteins other than H2A, chains, labeling of A24 by reductive methylation of a- and e- and is proteolytically released, isopeptidase probably acts on iso- NH2 groups (24), as adapted by others (25), was not used. bonds in general and not uniquely on those of A24. In- For enzyme preparation, 0.5-2.0 g ofcells was washed once asmuch as isopeptidase is present throughout the cell cycle, the with 0.14 M NaCl and disrupted by using a glass/Teflon ho- level of A24 in chromatin appears to be controlled by a balance mogenizer in 2 to 3 vol of50 mM Na2HPO4/NaH2PO4, pH 7.5/ between isopeptidase and an as yet unestablished H2A-ubiquitin 10 mM MgCl2/2 mM EGTA/0.5 mM phenylmethylsulfonyl ligase. fluoride (BzlSO2F). The homogenate was centrifuged for 10 min at 45,000 X g in a Beckman 65 Ti rotor, and the supernatant Despite extensive advances in chromatin biochemistry (1-3), was used as an enzyme source. Intracellular localization of en- the molecular basis of the higher order organization of chro- zyme was determined as follows. The cells were homogenized matin remains unknown. Histone phosphorylation has been im- in buffer/0.25 M sucrose and centrifuged as above. The su- plicated in chromatin condensation in mitosis (4-9); however, pernatant was used as a cytoplasmic enzyme. The pellet was this idea has been questioned recently (10, 11). In attempts to suspended in 2.0 M sucrose and centrifuged over 2.2 M sucrose seek feasible mechanisms, we found another mitosis-specific at 40,000 X g for 60 min in a Beckman SW 41 rotor. This pellet event that involves a reversible stoichiometric shift in one ofthe was washed twice with 0.25 M sucrose, and then the nuclei were structural chromatin proteins, A24 (12-14), a conjugate of his- extracted at 40C with 0.35 M NaCV0.5 mM BzlSO2F. More tone H2A and the nonhistone protein ubiquitin (15-17). A24 than 75% of the nuclear nonhistone chromatin protein re- virtually disappears as a structural component of chromatin in covered in 0.35 M NaCl extracts was dialyzed against phosphate mitosis (12-14, 18) and, at the same time, a 10% increase in the buffer and used as a nuclear enzyme. The size of the enzyme H2A/DNA mass ratio occurs (12, 13). When cells revert to the molecule was estimated by 5-20% linear sucrose gradient cen- G, phase from mitosis and the chromatin becomes decon- trifugation and by Sephadex G-100 chromatography (26, 27). densed, A24 is reformed by conjugation ofthe core histone H2A The and preformed ubiquitin, apparently without new protein syn- standard assay mixture for the isopeptidase reaction con- thesis (13). tained (total vol, 20,ul) 100,umol ofTris-HCl (pH 8.5), 37.3 pmol In view of the reversible stoichiometric shift of A24 in the ofA24, 2 ,umol of2-mercaptoethanol (or dithiothreitol), 1 ,umol mitotic cycle ofchromatin organization, we predicted the pres- of BzlSO2F, and 0.5-10 ,Ag of enzyme. The reaction was ter- ence ofnovel enzymes that catalyze the interaction of H2A and minated by the addition of5 ,ul of0.5% NaDodSOJpolyacryl- ubiquitin (13, 19). In this article, we present evidence that eu- amide gel electrophoresis buffer. Under these conditions, the karyotes, but not prokaryotes such as Escherichia coli, contain reaction was linear for up to 15 min and proportional to the a cytoplasmic enzyme, termed isopeptidase, that cleaves A24 amount of enzyme. The reaction products were analyzed by into H2A and ubiquitin stoichiometrically in vitro. 15% NaDodSO4 or two-dimensional polyacrylamide gel elec- trophoresis (26, 28). The gels were sliced, the proteins were The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviation: BzlSO2F, phenylmethylsulfonyl fluoride. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. t To whom reprint requests should be addressed. 1535 Downloaded by guest on September 26, 2021 1536 Cell Biology: Matsui et al. Proc. Natd Acad. Sci. USA 79 (1982) quantitatively eluted in 50 mM phosphate buffer, pH 7.5/ strength (Fig. 7). (v) Free SH groups are essential cofactors 0.1% NaDodSO4, and the radioactivity was determined. For (Table 2). (vi) Except for Ca2+ and Mg2+, divalent cations are autoradiography, the stained gels were exposed to a Kodak X- potent inhibitors (Table 2). (vii) Core histones and H1 are not Omat AR film for 16-72 hr. cleaved by isopeptidase (Fig. 3). (viii) The optimal pH is 8.0-8.5 (Fig. 7). RESULTS Primary Structure of A24 Reassessed by Isopeptidase Re- action. Polyacrylamide gel electrophoresis of the isopeptidase Existence of Isopeptidase in Cell Extracts. Our prediction reaction products confirmed that the only components of A24 that the interaction ofH2A and ubiquitin is areversible dynamic are H2A and ubiquitin (Figs. 1 and 3). Because no component process requiring involvement of specific enzymes (18, 19, 29) corresponding to [3H]glycine or [3H]Gly-Gly was produced was tested by addressing three major areas-identification of with [3H]glycine-labeled A24 as substrate (Fig. 3 A and B), the enzyme reaction, intracellular localization, and enzyme level question arises as to whether a Gly-Gly dipeptide is an integral during the cell cycle. Initial attempts were focused on an en- element in the , as initially proposed (30). This zyme(s) that cleaves A24 into H2A and ubiquitin. The results controversial point was studied. A24was purifiedfrom cells that obtained are summarized as follows. (i) A wide variety of eu- incorporated [3H]glycine for 6 generations to uniformly label karyotic cells from mammals, amphibia, birds, and yeast con- the entire molecule and then incubated with interphase cyto- tains an isopeptidase that cleaves A24 into H2A and ubiquitin plasmic proteins (Fig. 3C). Again, there was no radioactivity at in vitro (Fig. 1A). (ii) A prokaryote such as E. coli does not con- the ion front at which glycine and Gly-Gly would be located tain an isopeptidase (Fig. 1B). This was surprising, because (Fig. 3C). However, the H2A/ubiquitin radioactivity ratio was ubiquitin is present in E. coli (17), and its conjugate may well 2.0, in good agreeement with the ratio between the number of require isopeptidase for turnover. (iii) Isopeptidase attacks iso- glycine residues in H2A and ubiquitin terminating in Gly-Gly peptide bonds ofisolated A24, as well as those integrated in vivo (12:6) (13). as structural nucleosomal components (Figs. 2 and 3); therefore, the enzyme appears to have a very specific recognition site for DISCUSSION isopeptide bonds. (iv) Isopeptidase is localized mostly in the In this article, we report that various eukaryotic species contain cytoplasm (>99%) (Table 1) but it is unclear whether the re- an enzyme, tentatively referred to as isopeptidase, that cleaves sidual activity in nuclei reflects a real localization or merely an protein A24 into its components in vitro stoichiometrically. The absorbed portion during subcellular fractionation. (v) Isopep- failure ofA24 cleavage by E. coli extracts may indicate that iso- tidase is present not only in mitosis but also in interphase (Table peptidase is eukaryote specific. This enzyme selectively attacks 1). (vi) Cleavage ofA24 is stoichiometric; that is, neither inter- on isopeptide bonds between the COOH-terminal Gly-Gly di- vening or Gly-Gly dipeptides, initially proposed to be peptides attached to ubiquitin and the e-NH2 residue oflysine- an integral component between H2A and ubiquitin (30), are 119 on A24 (Figs. 1 and 3) and, hence, it probably belongs to detected in the isopeptidase products (Figs. 1 and 4). (vii) Iso- the carbon-nitrogen lyase group (E.C. 4.3). Since ubiquitin peptidase binds to chromatin in mitosis (Table 1). binds to many kinds ofproteins through isopeptidase bonding, Specificity of the Isopeptidase Reaction. Novel properties forming various ubiquitin adducts (32, 33), the same enzyme ofisopeptidase are as follows. (i) Isopeptidase differs from serine should have a wide substrate specificity. In fact, ubiquitin is hydrolase-type polypeptidases (including histone ) in known to be released, in an ATP-dependent of the that neither BzlSO2F nor trypsin inhibitor blocked the reaction rabbit reticulocyte system, from its adducts other than A24 (32, (Table 2). (ii) Isopeptidase differs from other endo- and exo- 33). Moreover, Wu et al. (18, 29) showed that uH2B is also con- peptidases, such as trypsin, chymotrypsin, carboxpeptidase, verted to H2B and ubiquitin during mitosis. Thus, the term Pronase, and H2A (31). These enzymes either failed isopeptidase rather than "A24 lyase" undoubtedly applies more to cleave A24 into H2A and ubiquitin (Fig. 5) or showed a dif- generally to the nature of the enzyme reaction. ferent reaction site on H2A (31). (iii) Isopeptidase has an ap- Interestingly, the isopeptidase reaction, at least its rate, was parent molecular weight of 38,000 as determined on Sephadex restricted at a higher ionic strength (Fig. 7B). A NMR study G-100 (Fig. 6), and its sedimentation coefficient is estimated showed that the presence ofions such as Na+, Ca2+, and Mg2e as 1-3. (iv) The isopeptidase reaction is depressed at high ionic induced a helical interaction ofapolar domain within H2A mol- A B C D / 1 2 3 4 5 6 7 8 9 1011 12 1314 15 1 2 3 4 5 6 7 8 9 101112 13 14 15 Hl4 I1 J!- 2 A24 w -A24' .,-. ll~T ~~~~~.06 4 * * .-"* ^ -t H4 H -4 q H2A ^ H2A -'W_ -W 4_ ._mwveo_.ww -MU 4 u

FIG. 1. (A andB) Two-dimensional electrophoresis (acid urealNaDoSO4) of isopeptidase reaction products. Twenty micrograms of interphase cytoplasm protein was incubated for 10 min with 74.6 pmol 1 I-labeled A24 under standard assay conditions. The entire reaction mixture was subjected to electrophoresis with 50 ,Zg of unlabeled calf thymus histones and 5 j.g of ubiquitin (U). (C and D) NaDodSO4/polyacrylamide gel elec- trophoresis of the reaction products of cell extracts (10 and 20 ,ug as protein) from various species. Lanes: 1, control (buffer alone); 2 and 3, yeast; 4 and 5, E. coli; 6 and 7, mouse L929; 8 and 9, Xenopus laevis kidney; 10 and 11, chicken erythrocyte; 12 and 13, calf thymus; 14 and 15, X. laevis liver. Note the absence of isopeptidase activity in theE. coli extracts. The frogtissue extracts showed a uniquedigestionprofile, probablycompounded by other endopeptidases, but the presence of 'wI-labeled H2A and ubiquitin in the reaction products (lanes 14 and 15) points to the presence of isopeptidase. (A and C) Protein staining. (B and D) Autoradiograms. Downloaded by guest on September 26, 2021 Cell Biology: Matsui et al. Proc. NatL Acad. Sci. USA 79 (1982) 1537 A B Table 1. Intracellular localization of isopeptidase 1 2 3 4 5 1 2 3 4 5 Total Enzyme activity proteins, Units/mg Exp. Fraction mg of protein Total W. I-- 1 Interphase cytoplasm 66.5 6.4 425.6 :M. V Interphase* nuclei 1.11 0.89 1.0 w- 10- 2 Interphase cytoplasm - 7.58 - Metaphase cytoplasm - 12.32 - 3 Metaphase cytoplasm 38.0 12.11 460 Metaphase chromosomes* 0.82 8.10 6.6 4, One unit of enzyme activity is defined as the amount of enzyme that cleaves 1 nmol of A24 in 10 min under standard assay conditions. * Chromosomes were isolated as described in refs. 21 and 22, and 0.35 FIG. 2. NaDodSO/polyacrylamide gel electrophoresis of isopep- M NaCl soluble proteins were- used as an enzyme source. tidase reaction products. Ten micrograms of mitotic cytoplasmic pro- tein was incubated in the standard assay mixture at 10TC (lane 2) or the isopeptidase products at lower and higher ionic strengths 3700 (lane 3). The entire mixture and calf thymus histones were sub- are identical (that is, H2A and ubiquitin), recognition of the jected to 12.5% polyacrylamide gelelectrophoresis. Lanes: 1, unlabeled cleavage site on A24 molecules by isopeptidase should not be A24; 4, blank control (enzyme); 5, ubiquitin. {A) Protein staining. (B) determined by substrate conformation. Autoradiograms. , , (top to bottom), A24, H2A, ubiquitin. The A24 molecules located in chromatin were as accessible to isopeptidase as free A24 molecules; no appreciable degra- ecules (34). Furthermore, it has been reported that ubiquitin dation occurred in other core histones (Fig. 2). In accordance generally tends to display a globular trypsin-resistant structure with a previous prediction (13), as well as the findings of Bohm (35, 36). Thus, the conformation of the A24 molecule seems to et al. (37), this result further strengthens the hypothesis that be partly an enzyme reaction rate-limiting factor. Inasmuch as a COOH-terminal domain of H2A to which ubiquitin binds is probably exposed on the nucleosome surface but not embedded A B within a globular space. Such an exposed domain could be in- volved in specific interactions other than those among globular 1 2 3 l 2 3 domains ofcore histones. It is conceivable that the conjugation ofubiquitin to H2A would not significantly alter the interaction ofcore histones through globular domains. This interpretation appears to be supported by the. findings of others as well (38, 39). Using carbodiimide crosslinking as a probe for histone- histone interactions, Bonner and Stedman (38) showed that H2A and the H2A portion ofA24 were equally positioned within nucleosomes. Martinson et al. (39)- also showed that the attach-

H2A A24 H2A A A24 I ,B I C 4

r;. 0 - ***I.0< - '-3 x ,1 E a 2 I

CV) s0 ,t . _~~~~~iiS > > uL At 20 30 40 50 20 30 40 50 20 30 40 50 FIG. 3. A24 lyase activity in interphase cells. Interphase chro- Slice matin (100 ,g as DNA) was incubated without (lanes 1) or with 100 jig of interphase cytoplasmic protein in the absence (lanes 2) or pres- FIG. 4. Isopeptidase reaction of 3H-labeled A24. (A) A24 alone. ence (lanes 3) of 10 mM 2-mercaptoethanol for 20 min at 3.700. After (B and C) Isopeptidase products of A24 prepared from cells labeled for dilution with 10 vol of incubation buffer, chromatin was isolated by 1.5 generations (20 hr) with [3Hlysine, [5Hlleucine, [5H]arginine, and centrifugation at 20,000 x g for 10 min and extracted with 0.2 M [3H]glycine (B) or for 6 generations (72 hr) with [3Hlgl]cine alone (C). H2SO4. The proteins were subjected to NaDodSO4polyacrylamide gel There is no radioactivity at the ion front at which [ Higlycine and electrophoresis at 5 (A) and 10 (B) pg per lane. Note decrease of A24 [3H]Gly-Gly would be expected to- migrate (I). In C, the H2A/ubi- chromatin incubated with cytoplasmic proteins. Apparently, no sig- quitin (U) radioactivity ratio is2.00 (16,355:8150), whichis compatible nificant degradation of other histones occurs. Bars, cytoplasmic pro- with the ratio of the number of glycine residues in H2A and ubiquitin teins adventitiously bound to chromatin during the incubation. that has Gly-Gly attached (12 and 6 residues per molecule) (13). Downloaded by guest on September 26, 2021 1538 Cell Biology: Matsui et al. Proc. Natd Acad. Sci. USA 79 (1982) Table 2. Effects of cofactors Ak Enzyme activity, Exp. Addition units x 102 1.0 . 1 None (control) 6.050 (100) 5 mM DTNB 0.007 (1.1) 5 mM DTNB/10 mM dithiothreitol 5.016 (82.9) 5 mM p-CMB 0 (0) I 5 mM p-CMB/10 mM dithiothreitol 5.116 (84.6) 10 20 30 50 70 90 110 130 150 170 190 210 5 mM N-ethylmaleimide 0 (0) FRACTION NUMBER 2 None (control) 7.500 (100) 91 I 123 5 mM Mg2- 6.405 (85.4) 91 123 10 mM Mg2+ 5.012 (66.8)

10 mM Ca2+ 7.525 (100.3) , 424 - 10 mM Mn2+ 0.006 (0.08) _ _ 10 mM Cd2+ 0 (0) eB_, m m - IH2AV 10 mM Cu2+ 0(0) C 0.1% Triton X-100 6.57 (87.6) B u Heat (1000C, 5 min) 0 (0) FIG. 6. Sephadex G-100 chromatography of isopeptidase. Twenty Assays were carried out at370C for 10 min understandard conditions milligrams of interphase cytoplasmic protein was applied to a 1.5 X using 10 ,g of interphase cytoplasmic protein and cofactors as indi- 90 cm column equilibrated with 50 mM Tris-HCl, pH 8.5 /1 mM di- cated. Values in parentheses are % control. DTNB, 5,5'-dithiobis-(2- thiothreitol/0.5 mM BzlSO2F, and 1-ml fractions were collected at a nitrobenzoic acid); p-CMB, p-chloromercuribenzoate. flow rate of 12 ml/hr. Assays were carried out on 10 u.l each of every other fraction. Only the isopeptidase-containing regions are shown (fractions 91-123). (A) Protein elution pattern at 280 nm. (B) Protein ment of ubiquitin to H2A has no significant effect on its ability staining pattern. (C) Autoradiogram of B. +-, peak activity (fraction to interact with H2B. Using a DNase I digestion probe and chro- 101). Note the appearance of a polypeptide of Mr 38,000 throughout matin reconstitution, Martinson and Kleinschmidt (40) further the fractions with isopeptidase activity but not in thefractions without activity. Mr standards: a, bovine serum albumin; b, ovalbumin; c, confirmed that A24 has little influence on structure at the level RNase. ubiquitin. of individual nucleosomes; U, Even though the role of A24 is controversial (12-15, 18, 41, is that, when ubiquitin binds to the core histone H2A, the chro- 42), a general implication we formulate from available evidence matin-becomes generally relaxed and, hence, transcriptionally active (13, 43). In fact, the A24 content is high in interphase chromatin (compared with metaphase) (13), nucleolar euchro- matin (compared with whole chromatin) (26), active immature chicken erythrocytes (compared with inactive matured cells) (42), andphytohemagglutinin-stimulatedhumanTlymphocytes m (unpublished results). It is tempting to assume that the attach- 0 10 x ment ofubiquitin to core histones (H2A and H2B) (18, 29) leads E to repulsion of adjacent chromatin fibers by blocking certain Q 0 electrostatic interactions that involve the charged COOH do- mainofH2A and that this relaxation ofchromatin fibers, in 4; turn, ._ 0.Q Q o I-r0 B 0 0 x ° 12 -z X x E X co 10 E Q a 10 0. -o0 o0 0 . -C o -o 0 -D0. 0 0 I J[ J _ z .n 0 6 10 20 10 20 10 20 CL0I0 L..!.0 Fraction 0 , - .L .0io FIG. 5. Digestion of '25I-labeled A24 with known proteolytic en- 10 zymes. A24 was digested for 30 min at 370C in 0.125 M Tris-HCl, pH -J 7.5/10 mM 2-mercaptoethanol, and the whole digests and calf thymus histones were subjected to NaDodSO/polyacrylamide gel electropho- resis. Gels were fixed in 50% methanol/10% acetic acid, destained, 5 6 7 8 9 1011 11.0 1.5 2.0 2.5 sliced, and extracted with 50 mM phosphate, pH 7.5/0.1% NaDodS04 pH log ionic strength for radioactivity assay. (A) Control (buffer alone). (B) Carboxypepti- dase A (2.63 units). (C) Carboxypeptidase B (0.28 unit). (D) Trypsin FIG. 7. (A) Effect of pH on isopeptidase. Assays were carried out (2.62 units). (E) Chymotrypsin (2.4 units). (F) Pronase (0.24 unit; Wor- in standard mixture buffered with 50 mM acetate (pH 5.0), phosphate thington). I (left to right): A24, H2A, and ubiquitin. Pronase com- (pH 6-7.5), Tris-HCl (pH 7.0-pH 9.0), or borate (pH 9.0-11.0). At pH pletely digested A24. After digestion with carboxypeptidase A or B, 7.0 and 7.5, there was no difference in the activity between phosphate A24 migrated slightly fasterthanthe undigested peptide, probably due and Tris-HCl buffers. Activity was determined,by H2A (0) and ubi- to removal of the COOH-terminal region (from threonine-120 to ly- quitin (o) produced. (B) Effects of ionic strength on isopeptidase ac- sine-129) in the H2A portion. tivity. Ionic strength was adjusted with Na+ (10-500 mM). Downloaded by guest on September 26, 2021 Cell Biology: Matsui et al. Proc. Natl. Acad. Sci. .USA 79 (1982) 1539

.enables nonhistone chromatin proteins such as regulatory pro- 20. Matsui, S., Weinfeld, H. & Sandberg, A. A. (1972)J. Cell Biol teins and RNA polymerases to bind to the accessible genes (43). 54, 120-132. The present results point to promise for the use ofpurified iso- 21. Matsui, S., Weinfeld, H. & Sandberg, A.A. (1979) J. Cell Biol peptidase in removing Since 80, 451-464. gently ubiquitin. conjugation of 22. Goyanes, V., Matsui, S. & Sandberg, A. A. (1980) Chromosoma ubiquitin to the e-NH2 groups oflysines on H2A should be pos- 78, 123-135. sible with rabbit reticulocyte enzymes (32, 33), the use ofboth 23. Hunter, W. M. (1973) in Handbook of Experimental Immunol- enzyme systems will make it possible to study ubiquitin mod- ogy, ed. Weir, D. M. (Blackwell, Oxford), p. 1000. .ification of structural proteins. 24. Means, G. E. & Feeney, R. E. (1971) Chemical Modification of Proteins (Holden-Day, San Francisco), p. 130. We thank Miss Anne Marie Conti, Miss Elena Greco, and Mr. 25. Andersen, M. W., Ballal, N. R., Goldknopf, I. L. & Busch, H. George Fox for their help in the preparation of this manuscript. This (1981) Biochemistry 20, 1100-1104. study was aided in part by American Cancer Society Institutional Re- 26. Matsui, S. & Busch, H. (1977) Exp. Cell Res. 109, 151-161. search Grant IN-54T-17 and National Cancer Institute Grant CA-14555. 27. Matsui, S., Fuke, M. & Busch, H. (1977) Biochemistry 16, 39-45. 1. Kornberg, R. & Thomas, J. O. (1974) Science 184, 865-871. 28. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 2. Cold Spring Harbor Symp. Quant. Biol. (1978) 42. 29. Wu, R. S., Nishioka, D., Pantazis, P., West, M. H. P. & Bonner, 3. Olins, D. E. & Olins, A. L. (1978) Am. Sci. 66, 704-711. W. M. (1980) J. Cell Biol 87, 46a. 4. Lake, R. S., Goidl, J. A. & Salzman, N. P. (1972) Exp. Cell Res. 30. Goldknopf, I. L. & Busch, H. (1977) Proc. Natl Acad. Sci. USA 73, 113-121. 74, 864-868. 5. Lake, R. S. (1973) Nature (London) New Biol 242, 145-146. 31. Eickbush, T. H., Watson, D. K. & Moudrianakis, E. N. (1976) 6. Marks, D. B., Park, W. K. & Borun, T. W. (1974)J. Biol Chem. Cell 9, 785-792. 248, 5660-5667. 32. Ciechanover, A., Heller, H., Elias, S., Haas, A. L. & Hershko, 7. Bradbury, E. M., Inglis, R. J. & Matthews, H. R. (1974) Nature A. (1980) Proc. Natl Acad. Sci. USA 177, 1365-1368. (London) 247, 257-261. 33. Hershko, A., Ciechanover, A., Heller, H., Haas, A. L. & Rose, 8. Gurley, L. R., Walters, R. A. & Tobey, R. A. (1974)J. Cell Biol I. A. (1980) Proc. Nati Acad. Sci. USA 77, 1783-1786. 60, 356-364. 34. Bradbury, E. M. & The Biophysics Group (1975) in The Struc- 9. Gurley, L. R., D'Anna, J. A., Barham, S. S., Deaven, L. L. & ture and Function ofChromatin, ed. Bradbury, E. M. (Elsevier/ Tobey, R. A. (1978) Eur. J. Biochem. 84, 1-15. North-Holland, Amsterdam), p. 138. 10. Gorovsky, M. A. & Keevert, J. B. (1975) Proc. Natt Acad. Sci. 35. Schlesinger, D. H., Goldstein, G. & Niall, H. D. (1975) Bio- USA 72, 2672-2676. chemistry 14, 2214-2218. 11. Tanphaichitr, N., Moore, K. C., Granner, D. K. & Chalkley, R. 36. Cary, P. D., King, D. S., Crane-Robinson, C., Bradbury, E. M., (1976) J. Cell Biol 69, 43-50. Rabbani, A., Goodwin, G. H. & Johns, E. W. (1980) Eur. J. 12. Matsui, S. & Sandberg, A. A. (1975) J. Cell Biol 83, 145a. Biochem. 112, 577-580. 13. Matsui, S., Seon, B. K. & Sandberg, A. A. (1979) Proc. Natl 37. Bohm, L., Crane-Robinson, C. & Sautiere, P. (1980) Eur. 1. Acad. Sci. USA 76, 6386-6390. Biochem. 106, 525-530. 14. Matsui, S. & Sandberg, A. A. (1980) Eur. J. Cell Biol 22, 83. 38. Bonner, W. M. & Stedman, J. D. (1979) Proc. Natl Acad. Sci. 15. Goldknopf, I. L., Taylor, C. W., Baum, R. M., Yeoman, L. C., USA 76, 2190-2194. Olson, M. 0. J., Prestayko, A. W. & Busch, H. (1975) J. Biol 39. Martinson, H. G., True, R., Busch, J. B. E. & Kunkel, G. (1979) Chem. 250, 7182-7187. Proc. Natl Acad. Sci. USA 76, 1030-1034. 16. Hunt, L. T. & Dayhoff, M 0. (1977) Biochem. Biophys. Res. 40. Kleinschmidt, A. M. & Martinson, H. G. (1981) Nucleic Acids Commun. 74, 650-655. Res. 9, 2423-2431. 17. Goldstein, G., Schneid, M., Hammerling, U., Boyse, E. A., 41. Levinger, L. & Varshavsky, A. (1980) Proc. Nati Acad. Sci. USA Schlesinger, D. H. & Nial, H. D. (1975) Proc. Natl Acad. Sci. 77, 3244-3248. USA 72, 11-15. 42. Goldknopf, I.L., Wilson, G., Ballal, N. R. & Busch, H. (1980)J. 18. Wu, R. S., Kohn, K. W. & Bonner, W. (1981)J. Biol Chem. 256, Biol Chem. 255, 10555-10558. 5916-5920. 43. Matsui, S., Weinfeld, H. & Sandberg, A. A. (1982) in Premature 19. Matsui, S., Nicotera, T. & Sandberg, A. A. (1980)J. Cell Biol 87, Chromosome Condensation, eds. Rao, P., Johnson, R. & Sper- 53a ling, K. (Academic, New York), in press. Downloaded by guest on September 26, 2021