G1- and S-phase syntheses of histones H1 and H1o in mitotically selected CHO cells: utilization of high-performance liquid chromatography

We have employed high-performance liquid chromatography (HPLC) to investigate the syntheses of histones H1 and H1o as synchronized cells traverse from mitosis to S phase. Chinese hamster (line CHO) cells were synchronized by mitotic selection, and, at appropriate times, they were pulse labeled for 1 h with [3H]lysine. Histones H1 and H1o were extracted by blending radiolabeled and carrier cells directly in 0.83 M HC1O4; the total HC1O4-soluble, Cl3CCO2H-precipitable proteins were then separated by a modification of an HPLC system employing three mu Bondapak reversed-phase columns [Gurley, L. R., D'Anna, J. A., Blumenfeld, M., Valdez, J. G., Sebring, R. J., Donahue, D. K., Prentice, D. A., & Spall, W. D. (1984) J. Chromatogr. 297, 147-165]. These procedures (1) produce minimally perturbed populations of synchronized proliferating cells and (2) maximize the recovery of radiolabeled histones during isolation and analysis. Measurements of rates of synthesis indicate that the rate of H1 synthesis increases (3.6 +/- 0.5)-fold as cells traverse from early to mid G1; as cells enter S phase, the rate of H1 synthesis increases an additional congruent to 22-fold and is proportional to the number of S-phase cells. In contrast to H1, the rate of H1o synthesis is nearly constant throughout G1. As cells progress into S phase, the rate of H1o synthesis increases (3.1 +/- 0.2)-fold so that it also appears to be proportional to the number of S-phase cells. Except for the first 1-2 h after mitotic selection, these results are similar to those obtained when cells are synchronized in G1 with the isoleucine deprivation procedure.     

Characterization of the six chicken histone H1 proteins and alignment with their respective genes

Six histone H1 subtypes and histone H5, isolated from chicken erythrocyte nuclei, were visualized on acid/urea polyacrylamide gels. Four of the H1 subtypes have been purified to homogeneity by fast protein liquid chromatography on a strong cation exchange column. The other two subtypes were obtained as enriched fractions from the same fast protein liquid chromatography experiments. Because six chicken H1 genes have been completely sequenced (Coles, L.S., Robins, A. J., Madley, L.K., and Wells, J. R. E. (1987) J. Biol. Chem. 262, 9656-9663), it was possible to assign each of the six H1 proteins to a specific gene after amino acid sequence analysis of peptides derived from the subtypes.     

Interleukin-1 receptor antagonist competitively inhibits the binding of interleukin-1 to the type II interleukin-1 receptor.

The interleukin-1 receptor antagonist (IL-1ra) inhibits the binding of interleukin-1 (IL-1) to T-cell lines possessing the type I IL-1 receptor; evidence has been published (Carter, D. B., Deibel, M. R. J., Dunn, C. J., Tomich, C. S., Laborde, A. L., Slightom, J. L., Berger, A. E., Bienkowski, M. J., Sun, F. F., McEwan, R. N., Harris, P. K. W., Yem, A. W., Waszak, G. A., Chosay, J. G., Sieu, L. C., Hardee, M. M., Zurcher-Neely, H. A., Reardon, I. M., Heinrickson, R. L., Truesdell, S. E., Shelly, J. A., Eessalu, T. E., Taylor, B. M., and Tracey, D. E. (1990) Nature 344, 633-638; Hannum, C. H., Wilcox, C. J., Arend, W. P., Joslin, F. G., Dripps, D. J., Heimdal, P. L., Armes, L. G., Sommer, A., Eisenberg, S. P., and Thompson, R. C. (1990) Nature 343, 336-340) that IL-Ira does not bind to the type II IL-1 receptor (IL-1RtII). In this study we examined the ability of human recombinant IL-1ra to block the binding of IL-1 to the IL-1RtII on human polymorphonuclear leukocytes (PMN) and Raji human B-lymphoma cells. The binding of 125I-IL-1 beta to PMN was competively inhibited by IL-1ra. IL-1 beta was more potent in inhibiting the binding of 125I-IL-1 beta than IL-1ra. Incubating PMN with 125I-IL-1ra in the presence of increasing concentrations of IL-1 beta or IL-1ra showed that IL-1 beta was an approximately 40-fold more potent inhibitor of binding of 125I-IL-1ra than unlabeled IL-1ra. The IL-1ra was approximately 500-fold less potent in inhibiting the binding of 125I-IL-1 alpha than IL-1 alpha. IL-1ra was also able to competitively inhibit binding of 125I-IL-1 beta to Raji cells. PMN or Raji cells were also incubated with 125I-IL-1 in the absence or presence of IL-1 or IL-1ra. After cross-linking of IL-1 to cells followed by specific immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a band at 85 kDa corresponding to the 68-kDa IL-1RtII. However, in the presence of an excess of either unlabeled IL-1 or IL-1ra, the 85-kDa IL-1.IL-1RtII complex was not present. These findings demonstrate that the IL-1ra recognizes and blocks IL-1 binding to the IL-1RtII.     

The enhancement of histone H4 and H2A serine 1 phosphorylation during mitosis and S-phase is evolutionarily conserved

Histone phosphorylation has long been associated with condensed mitotic chromatin; however, the functional roles of these modifications are not yet understood. Histones H1 and H3 are highly phosphorylated from late G2 through telophase in many organisms, and have been implicated in chromatin condensation and sister chromatid segregation. However, mutational analyses in yeast and biochemical experiments with Xenopus extracts have demonstrated that phosphorylation of H1 and H3 is not essential for such processes. In this study, we investigated additional histone phosphorylation events that may have redundant functions to H1 and H3 phosphorylation during mitosis. We developed an antibody to H4 and H2A that are phosphorylated at their respective serine 1 (S1) residues and found that H4S1/H2AS1 are highly phosphorylated in the mitotic chromatin of worm, fly, and mammals. Mitotic H4/H2A phosphorylation has similar timing and localization as H3 phosphorylation, and closely correlates with the chromatin condensation events during mitosis. We also detected a lower level of H4/H2A phosphorylation in 5-bromo-2-deoxyuridine-positive S-phase cells, which corroborates earlier studies that identified H4S1 phosphorylation on newly synthesized histones during S-phase. The evolutionarily conserved phosphorylation of H4/H2A during the cell cycle suggests that they may have a dual purpose in chromatin condensation during mitosis and histone deposition during S-phase.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00412-004-0281-9Communicated by G. Almouzni     

A 1H NMR study of a ternary peptide complex that mimics the interaction between troponin C and troponin I.

The troponin I peptide N alpha-acetyl TnI (104-115) amide (TnIp) represents the minimum sequence necessary for inhibition of actomyosin ATPase activity of skeletal muscle (Talbot, J.A. & Hodges, R.S. 1981, J. Biol. Chem. 256, 2798-3802; Van Eyk, J.E. & Hodges, R.S., 1988, J. Biol. Chem. 263, 1726-1732; Van Eyk, J.E., Kay, C.M., & Hodges, R.S., 1991, Biochemistry 30, 9974-9981). In this study, we have used 1H NMR spectroscopy to compare the binding of this inhibitory TnI peptide to a synthetic peptide heterodimer representing site III and site IV of the C-terminal domain of troponin C (TnC) and to calcium-saturated skeletal TnC. The residues whose 1H NMR chemical shifts are perturbed upon TnIp binding are the same in both the site III/site IV heterodimer and TnC. These residues include F102, I104, F112, I113, I121, I149, D150, F151, and F154, which are all found in the C-terminal domain hydrophobic pocket and antiparallel beta-sheet region of the synthetic site III/site IV heterodimer and of TnC. Further, the affinity of TnIp binding to the heterodimer (Kd = 192 +/- 37 microM) was found to be similar to TnIp binding to TnC (48 +/- 18 microM [Campbell, A.P., Cachia, P.J., & Sykes, B.D., 1991, Biochem. Cell Biol. 69, 674-681]). The results indicate that binding of the inhibitory region of TnI is primarily to the C-terminal domain of TnC. The results also indicate how well the synthetic peptide heterodimer mimics the C-terminal domain of TnC in structure and functional interactions.     

WAF1 retards S-phase progression primarily by inhibition of cyclin-dependent kinases.

The p21(WAF1/CIP1/sdi1) gene product (WAF1) inhibits DNA replication in vitro (J. Chen, P. Jackson, M. Kirschner, and A. Dutta, Nature 374:386-388, 1995; S. Waga, G. Hannon, D. Beach, and B. Stillman, Nature 369:574-578, 1994), but in vivo studies on the antiproliferative activity of WAF1 have not resolved G1-phase arrest from potential inhibition of S-phase progression. Here, we demonstrate that elevated WAF1 expression can retard replicative DNA synthesis in vivo. The WAF1-mediated inhibitory effect could be antagonized by cyclin A, cyclin E, or the simian virus 40 small-t antigen with no decrease in the levels of WAF1 protein in transfected cells. Proliferating-cell nuclear antigen (PCNA) overexpression was neither necessary nor sufficient to antagonize WAF1 action. Expression of the N-terminal domain of WAF1, responsible for cyclin-dependent kinase (CDK) interaction, had the same effect as full-length WAF1, while the PCNA binding C terminus exhibited modest activity. We conclude that S-phase progression in mammalian cells is dependent on continuing cyclin and CDK activity and that WAF1 affects S phase primarily through cyclin- and CDK-dependent pathways.     

Books

Book reviews in this article:
H andbookof M arine M ammals . V olume 6. the S econd B ookof D olphinsand P orpoises . S. H. Ridgway and Sir R. Harrison, eds.
T he B ottlenose D olphin : B iologyand C onservation . J. E. Reynolds, R. S. Wells and S. D. Eide, eds.     

Minor groove orientation for the (1S,2R,3S,4R)-N2- [1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyguanosyl adduct in the N-ras codon 12 sequence

The conformation of the trans-anti-(1S,2R,3S,4R)-N(2)-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyguanosyl adduct in d(G(1)G(2)C(3)A(4)G(5)X(6)T(7)G(8)G(9)T(10)G(11)).d(C(12)A(13)C(14)C(15)A(16)C(17)C(18)T(19)G(20)C(21)C(22)), bearing codon 12 of the human N-ras protooncogene (underlined), was determined. This adduct had S stereochemistry at the benzylic carbon. Its occurrence in DNA is a consequence of trans opening by the deoxyguanosine amino group of (1R,2S,3S,4R)-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthracenyl-3,4-diol. The resonance frequencies, relative to the unmodified DNA, of the X(6) H1' and H6 protons were shifted downfield, whereas those of the C(18) and T(19) H1', H2', H2' ', and H3' deoxyribose protons were shifted upfield. The imino and amino resonances exhibited the expected sequential connectivities, suggesting no interruption of Watson-Crick pairing. A total of 426 interproton distances, including nine uniquely assigned BA-DNA distances, were used in the restrained molecular dynamics calculations. The refined structure showed that the benz[a]anthracene moiety bound in the minor groove, in the 5'-direction from the modified site. This was similar to the (+)-trans-anti-benzo[a]pyrene-N(2)-dG adduct having S stereochemistry at the benzylic carbon [Cosman, M., De Los Santos, C., Fiala, R., Hingerty, B. E., Singh, S. B., Ibanez, V., Margulis, L. A., Live, D., Geacintov, N. E., Broyde, S., and Patel, D. J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1914-1918]. It differed from the (-)-trans-anti-benzo[c]phenanthrene-N(2)-dG adduct having S stereochemistry at the benzylic carbon, which intercalated in the 5'-direction [Lin, C. H., Huang, X., Kolbanovskii, A., Hingerty, B. E., Amin, S., Broyde, S., Geacintov, N. E., and Patel, D. J. (2001) J. Mol. Biol. 306, 1059-1080]. The results provided insight into how PAH molecular topology modulates adduct structure in duplex DNA.     

Identification of a uniquely immunodominant, cross-reacting site in the human immunodeficiency virus endonuclease protein.

One of the features of the life cycle of retroviruses is insertion of the proviral DNA into host chromosomes. A protein encoded by the 3' end of the pol gene of the virus genome has been shown to possess endonuclease activity (D. P. Grandgenett, A. C. Vora, and R. D. Schiff, Virology 89:119-132, 1978), which is necessary for DNA integration. Sera from the majority of human immunodeficiency virus (HIV)-infected individuals react with endonuclease protein p31 in serological tests (J. S. Allan, J. E. Coligan, T.-H. Lee, F. Barin, P. J. Kanki, S. M'Boup, M. F. McLane, J. E. Groopman, and M. Essex, Blood 69:331-333, 1987; E. F. Lillehoj, F. H. R. Salazar, R. J. Mervis, M. G. Raum, H. W. Chan, N. Ahmad, and S. Venkatesan, J. Virol. 62:3053-3058, 1988; K. S. Steimer, K. W. Higgins, M. A. Powers, J. C. Stephans, A. Gyenes, G. George-Nascimento, P. A. Liciw, P. J. Barr, R. A. Hallewell, and R. Sanchez-Pescador, J. Virol. 58:9-16, 1986). It is not known, however, which part of the protein represents the target(s) for antibody response. To study this, we synthesized peptides and used them in an enzyme-linked immunosorbent assay system to map the reactivity of human immunodeficiency virus type 1 (HIV-1) antibody-positive sera to the different regions of the HIV endonuclease. A uniquely antigenic, HIV-1- and HIV-2-cross-reacting site was identified in the central part of this protein from Phe-663 to Trp-670.     

Spatial and temporal regulation of RACK1 function and N-methyl-D-aspartate receptor activity through WD40 motif-mediated dimerization

Efficient signaling requires accurate spatial and temporal compartmentalization of proteins. RACK1 is a scaffolding protein that fulfils this role through interaction of binding partners with one of its seven WD40 domains. We recently identified the kinase Fyn and the NR2B subunit of the N-methyl-D-Aspartate receptor (NMDAR) as binding partners of RACK1. Scaffolding of Fyn near its substrate NR2B by RACK1 inhibits Fyn phosphorylation of NR2B and thereby negatively regulates channel function. We found that Fyn and NR2B share the same binding site on RACK1; however, their binding to RACK1 is not mutually exclusive (Yaka, R., Thornton, C., Vagts, A. J., Phamluong, K., Bonci, A., and Ron, D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5710-5715). We therefore tested the hypothesis that RACK1 forms a homodimer that allows the simultaneous binding of Fyn and NR2B. We found that RACK1 binds to itself both in vitro and in the brain. Deletion analyses identified a RACK1-RACK1 dimer-binding site within the 4th WD40 repeat, and application of the 4th WD40 repeat or a peptide derivative to hippocampal slices inhibited NMDAR activity. We further found that in hippocampal slices, both RACK1 and NR2B associated with another WD40 protein, the beta-subunit of G protein (Gbeta), previously shown to heterodimerize with RACK1 in vitro (Dell, E. J., Connor, J., Chen, S., Stebbins, E. G., Skiba, N. P., Mochly-Rosen, D., and Hamm, H. E. (2002) J. Biol. Chem. 277, 49888-49895). However, activation of the pituitary adenylate cyclase polypeptide (1-38) G protein-coupled receptor, previously found to induce the dissociation of RACK1 from the NMDAR complex (Yaka, R., He, D. Y., Phamluong, K., and Ron, D. (2003) J. Biol. Chem. 278, 9630-9638), attenuated the association of Gbeta with RACK1 and NR2B. Based on these results, we propose that WD40-mediated homo- and heterodimerization of RACK1 mediate the formation of a transient signaling complex that includes the NMDAR, a G protein and Fyn.     

Role of conserved transmembrane cationic amino acids in the prostaglandin transporter PGT

The prostaglandin transporter "PGT" interacts electrostatically with its anionic substrate, based on inhibition by the disulfonic stilbenes [Chan, B. S. (1998) J. Biol. Chem. 273, 6689-6697], inhibition by the thiol-reactive anion sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) [Chan, B. S. (1999) J. Biol. Chem. 274, 25564-25570], and the requirement for a negatively charged 1-position carboxyl on the substrate [Itoh, S. (1996) Mol. Pharm. 50, 736-742]. Here we found that modification of positively charged residues on wild-type PGT by arginine- and lysine-specific reagents significantly inhibited transport. We previously found that the binding site of PGT is formed, at least in part, by its membrane-spanning segments [Chan, B. S. (1999) J. Biol. Chem. 274, 25564-25570]. Three charged residues within predicted transmembrane spans (E78, R560, and K613) are conserved in PGT and in related transporters. Substitution of the anionic residue E78 (E78D and E78C) produced an essentially functional transporter, whereas substitution of the cationic residues with neutral residues (R560N and K613Q) resulted in poorly functional transporters. Immunoblotting revealed similar expression levels of wild-type and mutant transporters, and immunostaining indicated correct targeting. Conservative charge substitutions (R560K, K613R, and K613H) resulted in generally functional transporters. In contrast, R560N was nonfunctional, whereas the substrate affinity of K613G decreased greater than 50-fold. Conservative substitutions retaining the charge at position 613 (K613R and K613H) restored the substrate affinity, suggesting a direct role of K613 in substrate binding. Double-neutral mutants E78G/R560C and E78G/K613C were inactive, indicating that these residues are not simply charge-paired. Our results suggest that an arginine at position 560 is critical for maximal substrate translocation, and that a positively charged side chain at position 613 contributes to electrostatic binding of the anionic substrate.     

In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model

Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP2- and for AMP2- in human cytosolic adenylate kinase (EC 2.7.4.3, hAK1), we have investigated the enzymic effects of replacement of the arginine residues (R44, R132, R138, and R149), which had been assumed by Pai et al. [Pai, E. F., Sachsenheimer, W., Schirmer, R. H., & Schulz, G. E. (1977) J. Mol. Biol. 114, 37-45] to interact with the phosphoryl groups of AMP2- and MgATP2-. With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 [Kim, H. J., Nishikawa, S., Tanaka, T., Uesugi, S., Takenaka, H., Hamada, M., & Kuby, S. A. (1989) Protein Eng. 2, 379-386] to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the Km,app values for AMP2- of the mutant enzymes, the relatively small increases in the Km,app values for MgATP2-, and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. (1977) have been reversed and that their ATP-binding site may be assigned as the AMP site.     

Isolation of BamHI variants with reduced cleavage activities

Derivation of the bamhIR sequence (Brooks, J. E., Nathan, P.D., Landry, D., Sznyter, L.A., Waite-Rees, P., Ives, C. C., Mazzola, L. M., Slatko, B. E., and Benner, J. S. (1991) Nucleic Acids Res., in press), the gene coding for BamHI endonuclease, has facilitated construction of an Escherichia coli strain that overproduces BamHI endonuclease (W. E. Jack, L. Greenough, L. F. Dorner, S. Y. Xu, T. Strezelecka, A. K. Aggarwal, and I. Schildkraut, submitted for publication). As expected, low-level constitutive expression of the bamhIR gene in E. coli from the Ptac promotor construct is lethal to the host unless the bamHIM gene, which encodes the BamHI methylase, is also expressed within the cell. We identified four classes of BamHI endonuclease variants deficient in catalysis by selecting for survival of a host deficient for bamHIM gene, transformed with mutagenized copies of the bamhIR gene, and then screening the surviving cell extracts for DNA cleavage and binding activities. Class I variants (G56S, G91S/T153I, T114I, G130R, E135K, T153I, T157I, G194D) displayed 0.1-1% of the wild-type cleavage activity; class II variant (D94N) lacked cleavage activity but retained wild-type DNA binding specificity; class III variants (E77K, E113K) lacked cleavage activity but bound DNA more tightly; class IV variants (G56D, G90D, G91S, R122H, R155H) lacked both binding and cleavage activities. Variants with residual cleavage activities induced the E. coli SOS response and thus are presumed to cleave chromosomal DNA in vivo. We conclude that Glu77, Asp94, and Glu113 residues are essential for BamHI catalytic function.     

Complete sequence-specific 1H NMR assignments for human insulin

Solvent conditions where human insulin could be studied by high-resolution NMR were determined. Both low pH and addition of acetonitrile were required to overcome the protein's self-association and to obtain useful spectra. Two hundred eighty-six 1H resonances were located and assigned to specific sites on the protein by using two-dimensional NMR methods. The presence and position of numerous dNN sequential NOE's indicate that the insulin conformation seen in crystallographic studies is largely retained under these solution conditions. Slowly exchanging protons were observed for seven backbone amide protons and were assigned to positions A15 and A16 and to positions B15-B19. These amides all occur within helical regions of the protein [Chawdhury, S.A., Dodson, E.J., Dodson, G.G., Reynolds, C.D., Tolley, S.P., Blundell, T.L., Cleasby, A., Pitts, J.E., Tickle, I.J., & Wood, S.P. (1983) Diabetologia 25, 460-464].     

Dynamic properties of nuclear lamins: lamin B is associated with sites of DNA replication

The nuclear lamins form a fibrous structure, the nuclear lamina, at the periphery of the nucleus. Recent results suggest that lamins are also present as foci or spots in the nucleoplasm at various times during interphase of the cell cycle (Goldman, A. E., R. D. Moir, M. Montag- Lowy, M. Stewart, and R. D. Goldman. 1992. J. Cell Biol. 104:725-732; Bridger, J. M., I. R. Kill, M. O'Farrell, and C. J. Hutchison. 1993. J. Cell Sci. 104:297-306). In this report we demonstrate that during mid- late S-phase, nuclear foci detected with lamin B antibodies are coincident with sites of DNA replication as detected by the colocalization of sites of incorporation of bromodeoxyuridine (BrDU) or proliferating cell nuclear antigen (PCNA). The relationship between lamin B and BrDU is not maintained in the following G1 stage of the cell cycle. Furthermore, the nuclear staining patterns seen with antibodies directed against lamins A and C in mid-late S-phase do not coalign with the lamin B/BrDU-containing structures. These results imply that there is a role for lamin B in the organization of replicating chromatin during S phase.