Am J Cardiol, 1987 Jan 23, 59(2), 56A - 58A The myocardial intracellular renin-angiotensin system; Re R; In recent years several tissues have been found to contain the components of the renin-angiotensin system . Locally synthesized renin could conceivably function in an endocrine, paracrine or autocrine manner . Isolated left ventricular cardiac myocytes of the rat were examined in the present study and renin and angiotensin II were detected in these cells . This observation suggests the existence of a functioning renin-angiotensin system in the heart and, more particularly, in the left ventricular cardiac myocyte . Additional evidence, derived from micrococcal nuclease and deoxyribonuclease I treatment of angiotensin II-exposed chromatin, suggests that angiotensin II could subserve an intracellular function in some of its cells of synthesis. Biochemistry, 1987 Jan 13, 26(1), 290 - 5 Selective solubilization of beta-globin oligonucleosomes at low ionic strength; Ridsdale JA et al.; We {Rocha, E., Davie, J.R., van Holde, K.E., & Weintraub, H . (1984) J . Biol . Chem . 259, 8558-8563} have previously reported that the transcriptionally competent beta-globin gene domain is selectively enriched in chromatin fractions eluted with solutions of approximately physiological ionic strength from micrococcal nuclease digested mature chicken erythrocyte nuclei . In this report, we demonstrate that beta-globin chromatin is eluted as oligonucleosomes while vitellogenin, a transcriptionally inactive gene, is eluted as mononucleosomes as is the bulk of sequences found in this fraction . Following removal of the salt, the eluted chromatin was made 100 mM KCl and separated into aggregation-prone and aggregation-resistant fractions . Globin sequences were present in both fractions and had the greatest enrichment in the aggregation-prone fraction which contained H1 and H5, H1 being more abundant . A procedure is presented in which H1 is selectively removed from the erythrocyte nuclei . Following the selective removal of H1 and subsequent fractionation, globin but not vitellogenin oligonucleosomes were present in the aggregation-resistant chromatin fraction . The results indicate the beta-globin domain is a mosaic of aggregation-resistant and aggregation-prone regions with the latter being associated with H1 and H5 . Vitellogenin sequences were associated principally with aggregation-prone regions complexed with H5. Nucleic Acids Res, 1987 Jan 12, 15(1), 277 - 92 Chromatin association and DNA binding properties of the c-fos proto-oncogene product; Renz M et al.; As a first step in the analysis of the molecular function of the nuclear c-fos proto-oncogene product we have studied its subnuclear localization in serum-stimulated mouse fibroblasts where it forms a non-covalent, apparently monodisperse complex with another nuclear protein, p39 . The c-fos/p39 complex is almost quantitatively released from intact nuclei by DNasel or micrococcus nuclease treatment under conditions where only a minor fraction of DNA and nuclear proteins is released . In gel filtration experiments, c-fos/p39 comigrates with chromatin and seems to be associated with regions of increased DNasel accessibility . c-fos/p39 is bound to chromatin by electrostatic forces of moderate strength since greater than 90% of the complex can be eluted from nuclei at 0.4 M NaCl . In vitro, the c-fos/p39 complex in nuclear extracts binds to double- and single-stranded calf thymus DNA, suggesting that the association of c-fos/p39 with chromatin is at least in part due to its interaction with DNA . In agreement with this conclusion, c-fos/p39 is released from nuclei by incubation with tRNA, presumably due to competition for binding sites . Our observations are compatible with the hypothesis that c-fos may play a role in the regulation of gene expression. FEBS Lett, 1987 Jan 5, 210(2), 173 - 6 Retention of actin synthesis in liver under conditions that inhibit synthesis of almost all other proteins; Felipo V et al.; As briefly reported {(1986) Fed . Proc . 45, 1771, Abstr . 1690}, rats fed a protein-free diet for a few days often show a marked inhibition of protein synthesis in liver cytosol . However the synthesis of a protein of molecular mass approximately 42 kDa is fully retained . We show here on the basis of its molecular mass, number of bands on isoelectric focusing, isoelectric point and immunological reactivity that this protein is actin and also that actin mRNA is not degraded by micrococcal nuclease under conditions which degrade the bulk of other mRNAs. J Steroid Biochem, 1987 Jan, 26(1), 35 - 40 Analysis of the nuclear estrogen receptor from MCF-7 cells by limited proteolysis; Geier A et al.; The proteolytic fragments of the nuclear estrogen receptor in the MCF-7 cell line were characterized following limited digestion with chymotrypsin and trypsin . Nuclei were isolated from cells previously exposed to 10 nM {3H}estradiol . The proteolytic digestion was performed either on the micrococcal nuclease hydrolysate or on intact nuclei . The molecular weights (Mr) were calculated from the sedimentation coefficients determined on a sucrose gradient and from the Stokes radii estimated by gel filtration . Digestion of the nuclei with micrococcal nuclease solubilized a receptor form of Mr = 151,000 . This receptor form was degraded by chymotrypsin to a receptor of Mr = 33,000 and by trypsin to a receptor of Mr = 60,000 . Digestion of intact nuclei with chymotrypsin solubilized a receptor form of Mr = 62,000 which dissociated in 0.4 M KCl to a receptor of Mr = 32,000 . Digestion of intact nuclei with trypsin followed by micrococcal nuclease solubilized a receptor form of Mr = 75,000 which was further dissociated by 0.4 M KCl to a receptor form of Mr = 60,000 . The ability of the receptor forms to bind DNA was tested using DNA-cellulose column chromatography . About 40% of the micrococcal nuclease solubilized receptor form, compared to about 7% of the chymotrypsin degraded receptor and to about 13% of the trypsin degraded receptor forms, all bound to the column and could be eluted by high salt concentrated buffer . We conclude that the nuclear estrogen receptor in the MCF-7 cell line can be partially degraded either in the micrococcal nuclease hydrolysate or in intact nuclei by chymotrypsin or trypsin generating protein moieties, probably receptor fragments of Mr = 33,000 and 60,000 respectively . Both fragments retain their estradiol binding domain and it may be hypothesized that the heavier fragment retains its chromatin binding domain. Microbiol Immunol, 1987, 31(5), 403 - 15 Characterization of mesosomes of Micrococcus luteus: isolation and properties of mesosomal ribosomes, and localization of penicillin-binding proteins in mesosomal membranes; Nakasone N et al.; Mesosomes were isolated and purified from Micrococcus luteus under hypertonic conditions throughout preparation processes . The purified mesosomal preparation was composed of closed tubules and vesicles . Electron-dense ribosome-like particles were observed within the isolated mesosomal vesicles by electron microscopy . The ribosome-like particles were isolated from the purified mesosomes by a procedure involving solubilization of the membranes with detergents followed by centrifugation on a linear density gradient of sucrose . The isolated particles have a sedimentation coefficient of 70S in the presence of 10 mM Mg2+, when Mg2+ concentration was lowered to 0.1 mM, the particles were dissociated into two sub-particles of 30S and 50S . The 70S particles had the same appearance as cytoplasmic 70S ribosome particles upon observations of negatively stained preparations . These findings indicate that mesosomal tubules contain ribosomes . The isolated mesosomal ribosomes had the ability for protein synthesis when polyuridylic acid-directed polyphenylalanine synthesis was assayed . The sensitivity of mesosomal ribosomes to inhibitors, chloramphenicol and streptomycin, for protein synthesis was significantly lower than that of both cytoplasmic and cytoplasmic membrane-bound ribosomes . In addition, three penicillin-binding proteins were detected in the mesosomal membranes . One of these was localized predominantly in the mesosomal membranes and the other two were distributed almost equally in both mesosomal and cytoplasmic membranes. Prostate, 1987, 10(3), 207 - 22 Chemical demonstration of nuclear androgen receptor following affinity chromatography with immobilized ligands; Bruchovsky N et al.; With increasing purification of the androgen receptor from nuclei of rat ventral prostate, a receptor-like protein could be demonstrated by chemical staining with silver nitrate . After sonication and digestion of nuclei with micrococcal nuclease, the solubilized receptor was applied to a column of Matrex Gel Green A and eluted with a linear gradient of 0-2 M NaCl . Characterized by specific binding of dihydrotestosterone, this form of the receptor was also androgen dependent and yielded an apparent Mr of 33,000 when analyzed by polyacrylamide gel electrophoresis and silver nitrate staining . To facilitate recovery following chromatography, the receptor was precipitated with 0-40% ammonium sulfate . Analysis of the 15-fold enriched fraction by sucrose density-gradient centrifugation confirmed the presence of a 3S androgen-binding protein . About 200 ng of the precipitated protein was applied to a column of dihydrotestosterone-17 beta-succinyl agarose (ligand concentration, 0.25 mumol/ml) . The fractions eluted with 50 microM dihydrotestosterone were electrophoresed and stained as before; again, the presence of a 33,000 Mr protein sensitive to castration was demonstrated . Alternatively, when the precipitated protein was fractionated by fast protein liquid chromatography utilizing a Superose 12 HR 10/30 column, the receptor coeluted with nuclear proteins in the 29,000-36,000 Mr range as determined both by retention time and electrophoresis . In combination, the above methods may be used to obtain a receptor protein purified to near homogeneity with a yield of 5-10% . The amount of receptor afforded by the purification sequence is small but nevertheless sufficient for chemical detection . We anticipate that with modification, the procedures may prove suitable for the recovery of nuclear androgen receptor on a preparative scale. Differentiation, 1987, 36(2), 125 - 9 The antitumor drug 3-nitrobenzothiazolo(3,2-a)quinolinium chloride (NBQ): effects on lens regeneration and interaction with DNA of Notophthalmus viridescens; Gonzalez FA et al.; We have studied the effect of 3-nitrobenzothiazolo(3,2-a)quinolinium (NBQ) on the regeneration of the lens in adult newt Notophthalmus viridescens . NBQ has marked cytotoxic effects in tumor cells, intercalates DNA, and was found to enhance lens regeneration . Newt liver DNA was isolated, and the thermal denaturation temperature (Tm) determined to be 76.6% +/- 0.8% . The G-C content was determined to be 44.0% +/- 0.4% and 45.0% +/- 0.1% . Parameters of NBQ binding to newt DNA were determined by spectrophotometric methods and compared with those obtained for calf thymus and Micrococcus lysodeikticus . The association constant, K(o), was found to be 1.1 x 10(+5) M-1 with a site-size parameter, n, of 8.7 nucleotides . No explanation is apparent for the paradoxical stimulation of lens regeneration. Braz J Med Biol Res, 1987, 20(5), 539 - 48 Characterization of inducible lysozyme activity in the hemolymph of Rhodnius prolixus; Azambuja P et al.; 1 . The characterization and partial purification of an induced lysozyme activity in the hemolymph of adult Rhodnius prolixus inoculated with Micrococcus lysodeikticus is described . 2 . Little or no activity against M . lysodeikticus appeared in the first hours after inoculation, but the activity increased reaching a maximum 4 days later, which was maintained to day 12 . 3 . The activity was characterized as lysozyme on the basis of the following considerations: 1) pH optimum and thermostability at acidic pH; 2) rate of lysis negatively dependent on ionic strength; 3) binding to SP-Sephadex at pH 5.5; 4) apparent molecular weight of 15 kDal . 4 . Crude or semi-purified enzyme preparations showed a high degree of stability during handling, freezing and thawing, and standing at 5 degrees C . 5 . Incubation of abdominal fat bodies from treated insects resulted in the release of activity into the medium . 6 . The relationship between induced lysozyme activity and its role as an insect defense mechanism is discussed. Acta Biochim Pol, 1987, 34(4), 461 - 76 Repair of UV-irradiated plasmid DNA in mutants of Saccharomyces cerevisiae and Escherichia coli deficient in repair of pyrimidine dimers; Dominski Z et al.; The repair of in vitro UV-irradiated DNA of plasmid pBB29 was studied in excision defective yeast mutants rad1, rad2, rad3, rad4, rad10 and in Escherichia coli mutants uvr- and recA-, by measuring the cell transformation frequency . Rad2, rad3, rad4, and rad10 mutants could repair plasmid DNA despite their inability to repair nuclear DNA, whereas the reduced ability of rad1 mutant for plasmid DNA repair demonstrated alone the same dependence on the host functions that are needed for nuclear DNA repair . In E . coli the repair of UV-irradiated plasmid DNA is carried out only by the excision-repair system dependent on uvr genes . Treatment of UV-irradiated plasmid DNA with UV endonuclease from Micrococcus luteus greatly enhances the efficiency of transformation of E . coli uvr- mutants . Similar treatment with cell-free extracts of yeast rad1 mutant or wild-type strains as well as with nuclease BaL31, despite their ability for preferential cutting of UV damaged DNA, showed no influence on cell transformation. Mol Cell Biol, 1987 Jan, 7(1), 495 - 503 Requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation of a simian virus 40 early pre-RNA in vitro; Ryner LC et al.; Using a pre-RNA containing the simian virus 40 early introns and poly(A) addition site, we investigated several possible requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation in a HeLa cell nuclear extract . Splicing and 3' end formation occurred under the same conditions but did not appear to be coupled in any way in vitro . Like splicing, 3' end cleavage and polyadenylation each required Mg2+, although spermidine could substitute in the cleavage reaction . Additionally, cleavage of this pre-RNA, but not others, was totally blocked by EDTA, indicating that structural features of pre-RNA may affect the ionic requirements of 3' end formation . The ATP analog 3' dATP inhibited both cleavage and polyadenylation even in the presence of ATP, possibly reflecting the coupled nature of these activities . A 5' cap structure appears not to be required for mRNA 3' end processing in vitro because neither the presence or absence of a 5' cap on the pre-RNA nor the addition of cap analogs to reaction mixtures had any effect on the efficiency of 3' end processing . Micrococcal nuclease pretreatment of the nuclear extract inhibited cleavage and polyadenylation . However, restoration of activity was achieved by addition of purified Escherichia coli RNA, suggesting that the inhibition caused by such a nuclease treatment was due to a general requirement for mass of RNA rather than to destruction of a particular nucleic acid-containing component such as a small nuclear ribonucleoprotein. Mol Cell Biol, 1987 Jan, 7(1), 111 - 20 Pre-mRNA splicing and the nuclear matrix; Zeitlin S et al.; We examined the relationship between pre-mRNA splicing and the nuclear matrix by using an in vivo system that we have developed . Plasmids containing the inducible herpesvirus tk gene promoter linked to an intron-containing segment of the rabbit beta-globin gene were transfected into HeLa cells, and then the promoter was transactivated by infection with a TK- virus . Northern analysis revealed that the globin pre-mRNA and all its splicing intermediates and products are associated with the nuclear matrix prepared from such transfected cells . When the nuclear matrix was incubated with a HeLa cell in vitro splicing extract in the presence of ATP, the amount of matrix-associated precursor progressively decreased without a temporal lag in the reaction, with a corresponding increase in free intron lariat . Thus, most of the events of the splicing process (endonucleolytic cuts and branching) occur in this in vitro complementation reaction . However, ligation of exons cannot be monitored in this system because of the abundance of preexisting mature mRNA . Since the matrix is not a self-splicing entity, whereas the in vitro splicing system cannot process efficiently deproteinized matrix RNA, we conclude from our in vitro complementation results (which can be reproduced by using micrococcal nuclease-treated splicing extract) that the nuclear matrix preparation retains parts of preassembled ribonucleoprotein complexes that have the potential to function when supplemented with soluble factors (presumably other than most of the small nuclear ribonucleoproteins known to participate in splicing) present in the HeLa cell extract. Eur Biophys J, 1987, 15(3), 133 - 40 The superstructure of chromatin and its condensation mechanism . IV . Enzymatic digestion, thermal denaturation, effect of netropsin and distamycin; Koch MH et al.; Changes in the structure of chicken erythrocyte chromatin fibres at low ionic strength resulting from enzymatic digestion, thermal denaturation and binding of Netropsin and Distamycin were monitored by synchrotron X-ray solution scattering . Digestion with micrococcal nuclease confirms the previous assignment of the 0.05 nm-1 band to an interference between nucleosomes with an average distance of 23 nm . The results of thermal denaturation indicate that above 40 degrees C there is a progressive increase of the internucleosomal distance and that above 60 degrees C the characteristic structure of the chromatin fibre is destroyed . Binding of Netropsin and Distamycin also results in an increase of the internucleosomal distance which can be estimated to correspond to about 0.2 nm/mol. J Cell Sci Suppl, 1987, 6, 111 - 25 Characterization of genes and proteins involved in excision repair of human cells; Hoeijmakers JH; To extend our knowledge of the excision repair system in mammalian cells we have focussed on the isolation of genes and proteins involved in this process . For the purification and characterization of human repair proteins the microneedle injection assay technique is utilized . This system is based on the transient correction of the excision repair defect of xeroderma pigmentosum (XP) fibroblasts (scored as increase of ultraviolet (u.v.)-induced unscheduled DNA synthesis (UDS) upon microinjection of crude extracts from complementing XP or normal cells . Specific correction is observed in fibroblasts of all (9) excision-deficient XP complementation groups . The XP-A and G correcting factors were found to be proteins and several purification steps (including (NH4)2SO4 fractionation, chromatography of phosphocellulose, heparin and u.v.-irradiated DNA-cellulose) have been worked out for the XP-A correcting protein . The microinjection system was also used for the introduction of (partially) purified repair enzymes of lower organisms . Micrococcus luteus endonuclease and bacteriophage T4 endonuclease V were able to correct all XP complementation groups tested, in marked contrast to the more sophisticated Escherichia coli uvrABC complex injected with uvrD . Photoreversal of dimers could be registered after introduction of the yeast photoreactivating enzyme in repair-competent, XP-variant, XP-C and XP-I fibroblasts (monitored as decrease of (residual) UDS) . Remarkably, no effect was noticed in XP-A, D, E and H, suggesting that something prevents dimers in these cells from being monomerized by the injected enzyme . Using DNA-mediated gene transfer we have cloned a human gene (designated ERCC-1) that compensates for the excision defect of the u.v . and mitomycin C-sensitive Chinese hamster ovary cell (CHO) mutant 43-3B (complementation group 2) . Characterization of this gene and its cDNA revealed the following features: (1) ERCC-1 corrects the full spectrum of repair deficiencies in mutants of complementation group 2 . No correction is observed in mutants of the other CHO complementation groups . (2) The ERCC-1 gene has a size of 15 X 10(3) base-pairs (bp) and consists of 10 exons, one of which appears to be differentially spliced . (3) It encodes two largely identical mRNAs, which differ in the presence or absence of a 72 bp coding exon, situated in the 3' half of the mRNA . Only the cDNA of the large transcript is able to confer repair proficiency to 43-3B cells . No effect of u.v . treatment is found at the level of ERCC-1 transcription in HeLa cells.(ABSTRACT TRUNCATED AT 400 WORDS) Urol Int, 1987, 42(2), 115 - 9 Intranuclear androgen receptor deployment and protooncogene expression in human diseased prostate; Phillips ME et al.; Androgen receptors were quantified in nuclei from human prostate tissue and in nuclear fractions derived by exhaustive digestion with micrococcal nuclease . In nuclei from benign hypertrophic prostate (BPH), the population of androgen receptors solubilized during nucleolysis predominated whereas in carcinoma nuclei the nuclease-resistant population was in excess . This phenomenon was restricted to intranuclear deployment and could not be attributed to recompartmentalization within the cell . Receptor content could not be correlated to the expression of the cellular protooncogenes myc, H-ras, K-ras or sis, in either BPH or carcinoma . However, in both BPH and carcinoma, significant correlation was observed between nuclear androgen receptor content and expression of c-fos . Expression of c-fos was not elevated in carcinoma compared to BPH, whereas expression of c-myc was elevated in carcinoma specimens of all grades of glandular differentiation, and expression of H-ras became increasingly elevated as differentiation was lost. J Biochem (Tokyo), 1987 Jan, 101(1), 153 - 61 Isolation and characterization of an activator for Azotobacter vinelandii nicotinamide mononucleotide glycohydrolase; Imai T; Azotobacter vinelandii NMN glycohydrolase {EC 3.2.2.14} has been shown to require absolutely GTP or a high-molecular-weight and heat-stable component for its function . The intracellular activator could be purified from its sonicate by heat treatment, acetone precipitation, phenol extraction, and acid precipitation in a good yield . The purified activator showed high affinity and effectiveness for NMN glycohydrolase (KA = 0.012 optical density unit at 257 nm/ml; Vmax standardized by the activity at 1 mM GTP = 88%) . Negative cooperativity of the enzyme activation with the activator was also shown . On treatment with either micrococcal nuclease or pancreatic RNase, the activator activity was completely abolished, whereas pronase and trypsin had no effect . The activator could be replaced by yeast RNA as well as calf liver RNA, whereas DNAs purified from Micrococcus lysodeikticus, T 7 and calf thymus had no effect on the enzyme . Furthermore, poly(G) and poly(I) could function as activators with the same effectiveness as the purified activator, and the enzyme activation with these RNA homopolymers was inhibited by poly(C), suggesting that the activation mechanism is specific with respect to base composition . Based on a kinetic analysis of the enzyme activation with commercial RNAs, together with the results from enzymatic digestion, specific inhibition of the enzyme by spermine, and its chemical properties, the activator was identified as an RNA . A model is described for NMN glycohydrolase regulation in which the RNA activator plays an important role in the NMN salvage cycles. J Endocrinol, 1987 Jan, 112(1), 161 - 9 Intranuclear distribution of androgen receptors in human prostate carcinoma; Kyprianou N et al.; Androgen receptors in nuclei from human prostate carcinomas were characterized on the basis of their solubilization by, or resistance to, micrococcal nuclease . By this means, androgen receptors were assigned to three nuclear categories: those associated with nuclease-resistant structures, those associated with chromatin and those apparently uncommitted by association with either of these . Prostate carcinoma nuclei contained high concentrations (57-82% of total nuclear content) of nuclease-resistant androgen receptors . This was a different pattern from that observed previously for benign hypertrophic prostate epithelial nuclei which contained a variable high proportion of uncommitted androgen receptors . The differences could not be attributed to differential losses to cytosol, or to loss of functionality, as determined in vitro . The differences in distribution could reflect different responses of diseased cells to androgens, or the intervention of other factors more relevant to the disease process. Science, 1986 Dec 12, 234(4782), 1417 - 9 The Fos protein complex is associated with DNA in isolated nuclei and binds to DNA cellulose; Sambucetti LC et al.; The properties of the viral and cellular fos proteins (Fos) were investigated as a first step toward understanding the function of the fos gene . Treatment of nuclei with salt and nonionic detergents solubilized a complex that contained Fos together with several other cellular proteins . The majority of the Fos protein complex was released from isolated nuclei incubated in the presence of deoxyribonuclease I or micrococcal nuclease but not with ribonuclease A, suggesting that Fos is associated with chromatin . This hypothesis is supported by the finding that Fos protein from native or denatured nuclear extracts exhibited DNA-binding activity in vitro . These results suggest that Fos is involved in the regulation of gene expression. Nucleic Acids Res, 1986 Dec 9, 14(23), 9291 - 309 (A-T)n tracts embedded in random sequence DNA--formation of a structure which is chemically reactive and torsionally deformable; McClellan JA et al.; Alternating d(A-T)n sequences which are contiguous with DNA of effectively random sequence have an abnormal conformation in linear DNA molecules . These regions are strongly reactive towards chemical modification by osmium tetroxide, and are preferentially cleaved by micrococcal nuclease . Both the chemical modification and the enzymic cutting occur uniformly through the alternating tract, and there is no evidence for enzyme or chemical sensitivity in the interfaces between the tract and DNA of normal conformation . These reactivities have a requirement for an alternating sequence . In addition to chemical reactivity, alternating (A-T)n sequences exhibit anomalously small twist changes on cruciform formation, suggesting that the pre-extruded DNA is underwound . We propose that the alternating sequences adopt an altered conformation which is subject to easy torsional deformation. J Mol Biol, 1986 Dec 5, 192(3), 577 - 91 Mapping of the sites of protection on a 5 S RNA gene by the Xenopus transcription factor IIIA . A model for the interaction; Fairall L et al.; The contact points of transcription factor IIIA with the internal control region of the 5 S RNA gene of Xenopus have been investigated by probing the accessibility of the DNA in the protein-DNA complex to dimethylsulphate and to micrococcal nuclease . The results of quantitative measurements, combined with those from earlier DNase I and DNase II protection studies, are consistent with a series of multiple contacts about five base-pairs apart, or half a double-helical turn, along the whole length of the internal control region . The nine patches of contact we have mapped could correspond to nine DNA-binding fingers in the protein . A model for the overall geometry of the interaction is presented in which the protein lies on one face of the DNA double helix. Exp Cell Res, 1986 Dec, 167(2), 517 - 30 UV-induced DNA excision repair in rat fibroblasts during immortalization and terminal differentiation in vitro; Vijg J et al.; UV-induced DNA excision repair was studied as DNA repair synthesis and dimer removal in rat fibroblast cultures, initiated from either dense or sparse inocula of primary cells grown from skin biopsies . During passaging in vitro an initial increase in DNA repair synthesis, determined both autoradiographically as unscheduled DNA synthesis (UDS) and by means of the BrdU photolysis assay as the number and average size of repair patches, was found to be associated with a morphological shift from small spindle-shaped to large pleiomorphic cells observed over the first twenty generations . In cell populations in growth crisis, a situation exclusively associated with thin-inoculum cultures in which the population predominantly consisted of large pleiomorphic cells, UDS was found to occur at a low level . After development of secondary cultures into immortal cell lines, both repair synthesis and morphology appeared to be the same as in the original primary spindle-shaped cells . At all passages the capacity to remove UV-induced pyrimidine dimers was found to be low, as indicated by the persistence of Micrococcus luteus UV endonuclease-sensitive sites . These results are discussed in the context of terminal differentiation and immortalization of rat fibroblasts upon establishment in vitro. Biochim Biophys Acta, 1986 Nov 28, 889(2), 128 - 35 Surface charge measurements on Micrococcus lysodeikticus and the catalytic implications for lysozyme; Price JA et al.; Electrophoresis measurements on Micrococcus lysodeikticus have shown that the net surface charge density on the cell wall is constant at around -1.5 microC/cm2 for the pH range 4-8 . This result has enabled a quantitative analysis to be made of how the electrostatic field associated with the negatively charged cell wall influences the ionic strength and pH dependency of the lytic activity of lysozyme towards M . lysodeikticus . A dominant effect is the creation of a local pH gradient at the cell wall, and at high ionic strengths the lytic activity is found to be controlled by an electrostatic force of attraction between the lysozyme molecule and the cell wall . As the ionic strength of the supporting electrolyte is decreased, however, an electrostatic force of repulsion becomes dominant and is associated with a negative charge carried by the lysozyme molecule, which could possibly be the ionized Asp-52 residue at the active site . This is considered to arise from the fact that at low ionic strengths the fine details of the heterogeneous charge distribution on the cell wall and lysozyme molecule are only partially screened by counter ions. Biochem Biophys Res Commun, 1986 Nov 26, 141(1), 213 - 21 Reassembly of c-myc and relaxation of c-fos nucleosomes during differentiation of human leukemic (HL-60) cells; Chou RH et al.; Human promyelocytic leukemic (HL-60) cells have amplified c-myc protooncogene sequences which lead to an elevated level of c-myc gene expression . Induction of HL-60 cells by phorbol esters to undergo monocytic differentiation results in the suppression of c-myc, but the activation of c-fos gene transcription . Chromatin structures of c-myc and c-fos were compared by measuring their sequences in nucleosome-associated DNA fragments . These nucleosomal particles were released from chromatin by micrococcal nuclease digestion and subsequently analyzed with two dimensional gel electrophoresis . C-myc related sequences were detected in nucleosomal DNA fragments of differentiated cells only, while the c-fos related sequences were found in nucleosomal DNAs of noninduced HL-60 cells . Since the enzyme preferentially digests relaxed DNAs, these results suggest that nucleosomal subunits of c-myc and c-fos chromatin are relaxed during the state of active transcription, and reassembled once their transcription is repressed. Nucleic Acids Res, 1986 Nov 25, 14(22), 8703 - 22 Hypersensitive sites in the 5' and 3' flanking regions of the cysteine proteinase I gene of Dictyostelium discoideum; Pavlovic J et al.; The cysteine proteinase I gene of Dictyostelium discoideum is a developmentally regulated single copy gene . Specific sites in the 5' and the 3' flanking regions of the gene were cleaved by an endogenous nuclease when the gene was being transcribed . The majority of these sites were not cut when the gene was inactive . A dramatic change in the pattern of micrococcal nuclease and DNase I hypersensitive sites occurred in the 5' flanking region when transcription commenced at the 8 h stage of development . The major sites, doublets at -220/-300 bp and -670/-770 bp upstream of the transcription start site, corresponded to those cut by the endogenous nuclease . When transcription subsequently ceased the hypersensitive sites did not significantly change, indicating the gene remained in an activated state . The micrococcal nuclease hypersensitive sites in the 3' flanking region did not change significantly during development. Biochemistry, 1986 Nov 18, 25(23), 7736 - 44 Nucleosome core particle self-assembly kinetics and stability at physiological ionic strength; Diaz P et al.; Micrococcal nuclease, DNase I, and trypsin have been employed to study the kinetics of core particle self-assembly by salt jump from 2.0 to 0.2 M NaCl . A few seconds after the initiation of the reassociation reaction, the bulk of core particle DNA becomes protected from digestion by micrococcal nuclease, whereas free DNA, under the same conditions, is completely hydrolyzed . The central and C-terminal regions of core histones are also protected from trypsin digestion immediately after the 2.0-0.2 M NaCl salt jump . Moreover, the extent of degradation produced by trypsin is the same for samples digested a few seconds after the salt jump and for samples digested 20 min after the salt jump . With DNase I, minor structural differences have been detected between samples obtained at different times during the reaction . However, even in this case our results indicate that many of the characteristic histone-DNA contacts within the core particle are made a few seconds after the initiation of the self-assembly reaction . Furthermore, core particles have been labeled with the fluorescent reagent N-(1-pyrenyl)maleimide (NPM), which was previously used as a sensitive probe for nucleosome conformation . Extensive DNase I or trypsin digestion of NPM-labeled core particles in 0.2 M NaCl does not produce significant changes in excimer fluorescence . This allows us to conclude that the covalent continuity of DNA is not required for the maintenance of the folded conformation of the core particle and that the trypsin-resistant domains of core histones play a fundamental role in the stabilization of this structure. Biochemistry, 1986 Nov 18, 25(23), 7440 - 5 Raman spectra of the model B-DNA oligomer d(CGCGAATTCGCG)2 and of the DNA in living salmon sperm show that both have very similar B-type conformations; Kubasek WL et al.; Raman spectra were obtained from aqueous solutions of the deoxyoligonucleotide d(CGCGAATTCGCG)2 (I), which has been suggested as a model for B-type DNA conformation . These spectra were compared with the Raman spectra of the aqueous solutions of several DNAs of natural origin taken under identical solution conditions . Since the model sequence has a high percent GC (66%), the Raman spectrum was compared with the Raman spectrum of the DNA from Micrococcus lysodeikticus (72% GC), and the spectra of the two different DNAs were found to be rather similar in both 50 mM salt and 6 M salt solutions . Computer-aided band-shape analysis of the backbone vibrational region of the Raman spectra shows the existence of several bands corresponding to different furanose ring puckers . This appears to indicate a heterogeneity of furanose ring pucker in both the model dodecamer and the native DNA . Significant differences were found in the intensity of the conformational marker band at 810 cm-1, which indicates corresponding differences in furanose ring pucker heterogeneities in these two high GC content DNAs . The Raman spectrum of the dodecamer (I) was used to analyze the Raman spectrum of the DNA inside the head of living intact salmon sperm . Sperm spectra were taken with both our conventional Raman spectrograph and a newly developed intracavity laser Raman microscope system . Although the DNA in the sperm head is required by packing considerations to be in a highly compact and condensed state, the Raman spectra of the intact sperm are almost identical with that of the model dodecamer (I) if the difference in base composition is taken into account.(ABSTRACT TRUNCATED AT 250 WORDS) Anal Biochem, 1986 Nov 15, 159(1), 157 - 62 A one-step technique for the subcellular fractionation of total cell homogenates; Morand JN et al.; A procedure was developed for the rapid, analytical subcellular fractionation of entire homogenates from the Chinese hamster ovary and HeLa cell lines . The procedure avoids a nuclear sedimentation step and the losses that accompany such a step . A key to the development of this procedure was the addition to homogenates of either micrococcal nuclease or DNase I . Nuclease-treated homogenates were fractionated on self-forming Percoll gradients . The entire procedure from cell harvesting through collecting gradient fractions took only 2.5 h . The position of marker enzymes in the gradient fractions indicated clear resolution of plasma membranes, Golgi apparatus, endoplasmic reticulum, and lysosomes . This procedure should facilitate many studies requiring subcellular fractionation of cultured cells. Biochemistry, 1986 Nov 4, 25(22), 7062 - 8 Chromatin structure of the cytochrome P-450c gene changes following induction; Einck L et al.; The chromatin structure of cytochrome P-450c and P-450d genes, which in the liver are highly inducible by 3-methylcholanthrene, was studied in normal and carcinogen-treated rats by using a cDNA probe specific for P-450c and a genomic probe that recognizes both genes . Digestion with micrococcal nuclease revealed that the active genes are not present in the typical 200 base pair nucleosomal structure . Gene induction is associated with a rearrangement of the nuclear organization of the genes . By use of indirect end-label hybridization, three DNase I hypersensitive sites were mapped, one in the 5'-terminal region and two in the 3' region of the P-450c gene . Gene induction, by treatment with 3-methylcholanthrene, changes the location of the DNase I site present in the 5' region without affecting the sites present in the 3' region . Rat thymus chromatin does not contain these DNase I hypersensitive sites, suggesting that, in the liver, the chromatin structure is altered so as to allow tissue-specific expression of the P-450c gene . The chromatin structure of the highly inducible P-450c gene is compared to that of the P-450m gene, which is induced to a significantly smaller extent and is constitutively expressed. Can J Neurol Sci, 1986 Nov, 13(4 Suppl), 427 - 31 Chromatin structure in scrapie and Alzheimer's disease; McLachlan DR et al.; Scrapie affected brains exhibit a number of pathological features in common with the human neurodegenerative condition, Alzheimer's disease . The present report describes studies on chromatin structure seen in these two disease processes . Chromatin associated proteins influence transcriptional activity of DNA through an effect upon chromatin structure . We examined chromatin structure by: measuring the capacity of the enzyme micrococcal nuclease to release mono- and dinucleosomes from isolated nuclei and measuring DNA-histone interactions by examining the effect of ambient tonicity upon the release of chromatin proteins . In two strains of mice infected with two strains of scrapie agent there was reduced accessibility to micrococcal nuclease and an increased content on dinucleosomes of the histone H1 and H1(0) types . These changes precede clinical signs of scrapie and resemble those found in the human conditions of Alzheimer's and Pick's disease . Scrapie mouse brain differs from Alzheimer brain in that scrapie does not alter histone-DNA interactions as monitored by ionically induced histone release from chromatin . Despite similarities, the scrapie agent appears to operate upon different molecular mechanisms than those found in Alzheimer's disease. J Invest Dermatol, 1986 Nov, 87(5), 585 - 7 De novo synthesis of lysozyme by human epidermal cells; Chen VL et al.; The presence of lysozyme in human epidermis was determined in extracts from the surface of human skin and in sonicates of human epidermal cell preparations with the use of a bacteriolytic assay employing Micrococcus lysodeikticus cell walls as substrate . Preincubation of the extracts with the Fab portion of the IgG fraction of an antiserum to human lysozyme abolished the lytic activity of the extracts showing the specificity of the assay . De novo synthesis of lysozyme by human epidermal cells was demonstrated by radiolabeling studies . Human epidermal cells cultured in serum-free medium and pulsed with {3H}leucine for 24 h were sonicated and fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . A major band at Mr 14,500 exhibited both immunoreactivity with a rabbit antihuman lysozyme antibody and radioactivity . Gel filtration of the cell sonicate revealed bacteriolytic activity in the fractions containing radioactive and immunoreactive proteins . These findings suggest that lysozyme is newly synthesized by human epidermal cells. Endocrinology, 1986 Nov, 119(5), 2163 - 7 Identification and characterization of L-triiodothyronine receptors in cells of glial and neuronal origin; Ortiz-Caro J et al.; High affinity-low capacity binding sites for thyroid hormone have been identified in the nuclei of glial (C6) and neuronal (Neuro 2A) cultured cells . Equilibrium dissociation constants, determined by Scatchard analysis, were very similar in both types of cells (0.2-0.3 nM) . The relative affinity of hormonal analogs was also similar: the affinity for T3 was lower than for triiodothyroacetic acid and higher than for T4 or tetraiodothyroacetic acid . The sedimentation coefficients obtained by gradient centrifugation of nuclear receptor extracted with 0.4 M KCl or excised by micrococcal nuclease digestion were 3.5 S and 6.5 S, respectively . These results suggest that the thyroid hormone receptor is not restricted to neuronal cells, but also appears in cells of glial origin. Biochem Int, 1986 Nov, 13(5), 773 - 9 Endonucleolytic activities of nuclei from rat ascites hepatoma; Hibino Y et al.; By autodigestion (endogenous endonucleolysis) of rat liver (RL) or rat-ascites hepatoma (AH) nuclei, the nucleosomes were released from the RL, but not from the AH, nuclei . In contrast, by micrococcal nuclease digestion (exogenous endonucleolysis), the nucleosomes were released more rapidly from the AH than from the RL nuclei . A 0.6 M NaCl extract of the RL or AH nuclei was filtered through a Sephadex G-100 column . The resulting topoisomerase fraction was subjected to DNA relaxation and catenation assays with pBR322 DNA as a substrate . Consequently, the relaxation activity was almost the same between the RL and AH fractions, whereas the catenation activity was much higher in the AH fraction. J Antibiot (Tokyo), 1986 Nov, 39(11), 1565 - 73 Direct evaluation of phenylacetyl-CoA: 6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum by bioassay; Luengo JM et al.; The enzyme phenylacetyl-CoA: 6-Aminopenicillanic acid acyltransferase of Penicillium chrysogenum was evaluated by direct bioassay against Micrococcus luteus ATCC 9341 . The enzyme required dithiothreitol, was inactivated by 0.2 mM Hg2+ (100%), Zn2+ (80%), Cu2+ (60%), 1 mM N-ethylmaleimide (80%), and showed maximal catalytic activity at pH 8.4 and 20 degrees C . The V50 values for phenylacetyl-CoA and 6-aminopenicillanic acid were 0.55 mM and 1 microM, respectively . When octanoyl-CoA was employed as substrate similar results were obtained . In both cases the product generated showed strong antibacterial activity which was quickly lost when incubation was carried out with beta-lactamase . Reactions performed in the presence of Escherichia coli penicillin acylase did not generated active products when phenylacetyl-CoA was the substrate; they did with octanoyl-CoA . Time-course experiments revealed that the highest enzyme levels are found in 36 hours mycelium and remained almost constant from 48 to 96 hours; thereafter the level of the enzyme slowly decreased. FEBS Lett, 1986 Oct 20, 207(1), 75 - 8 Preferential binding of DNA methyltransferase and increased de novo methylation of deoxyinosine containing DNA; Pfeifer GP et al.; Mammalian DNA-cytosine 5-methyltransferases methylate cytosines in deoxyinosine containing DNA polymers more rapidly than in other synthetic or naturally occurring DNAs . The initial methylation rate of poly(dI-dC) X poly(dI-dC) is about 10-times higher than that of poly-(dG-dC) X poly(dG-dC) or of the native Micrococcus luteus DNA . In competitive binding experiments, DNA methyltransferase has about 10-fold higher affinity for the dI-containing alternating DNA polymer than for poly(dG-dC) X poly(dG-dC) . The observed high methyl accepting capacity of poly(dI-dC) X poly(dI-dC) may be a useful methodological advance to determine de novo DNA methyltransferase activity in extracts of mammalian cells. Biochim Biophys Acta, 1986 Oct 16, 868(1), 9 - 16 Mouse ascites DNA methylase: characterisation of size, proteolytic breakdown and nucleotide recognition; Adams RL et al.; We have purified the DNA methylase from mouse ascites tumour cells to a specific activity of 11,500 units per mg protein using denatured Micrococcus luteus DNA as methyl acceptor . Methyl groups are transferred to cytosines almost exclusively in CpG dinucleotides . The purified enzyme contains two polypeptides of molecular mass 185 and 160 kDa, and an antiserum raised in a rabbit to the purified enzyme specifically reacts with these two proteins in crude extracts . The two proteins can be partially separated by affinity chromatography when activity is associated with the 185 kDa protein which can be proteolytically degraded to give polypeptides of 170 and later 100 and 50 kDa . Only the 185 kDa methylase is lost when cells are treated with azadeoxycytidine and this is the predominant form firmly bound in the nucleus of dividing cells . Antibody bound to the 185 kDa band in protein blots will itself bind native DNA methylase, which can be detected by its binding 14C-labelled, azacytosine-containing DNA. Biochemistry, 1986 Oct 7, 25(20), 5910 - 4 Solid-phase processing of U2 snRNA precursors; Rohleder A et al.; HeLa cell cytoplasmic extracts contain both precursors to small nuclear RNA (snRNA) U2 and an activity that is capable of trimming these snRNA precursors to the size of mature U2 . The substrate for this RNA processing reaction is the ribonucleoprotein complex containing pre-U2 RNA . To circumvent the difficulty of biochemically isolating pre-U2 ribonucleoprotein (pre-U2 RNP) complexes for use as substrate for the analysis of the processing activity, we have developed a procedure for the processing of pre-U2 RNP complexes that have been immobilized on anti-Sm antibody/protein A-Sepharose columns . When the immobilized {3H}uridine-labeled substrate RNP complexes are incubated at 37 degrees C with unlabeled cytoplasmic extracts from HeLa cells, labeled molecules the size of mature U2 are produced in a linear fashion for up to 3 h . Similar results are obtained when substrate pre-U2 RNPs are immobilized with an anti-2,2,7-trimethylguanosine antibody . Thus, accurate processing of the 3' termini of U2 precursors occurs on the antibody columns . Incubation with buffer alone does not result in the production of mature-sized U2, indicating that the processing activity is not intrinsic to the pre-U2 RNP . Using this assay procedure, we have demonstrated that the processing activity is destroyed by trypsin or by preincubation at 65 degrees C but is resistant to treatment with micrococcal nuclease . These results are compatible with the conclusion that the processing activity is a classical enzyme that does not contain a nuclease-sensitive essential RNA component. Anal Biochem, 1986 Oct, 158(1), 119 - 29 Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis: application to pyrimidine dimers; Freeman SE et al.; We have developed an alkaline agarose gel method for quantitating single strand breaks in nanogram quantities of nonradioactive DNA . After electrophoresis together with molecular length standards, the DNA is neutralized, stained with ethidium bromide, photographed, and the density profiles recorded with a computer controlled scanner . The median lengths, number average molecular lengths, and length average molecular lengths of the DNAs can be computed by using the mobilities of the molecular length standards . The frequency of single strand breaks can then be determined by comparison of the corresponding average molecular lengths of DNAs treated and not treated with single strand break-inducing agents (radiation, chemicals, or lesion-specific endonuclease) . Single strand break yields (induced at pyrimidine dimer sites in uv-irradiated human fibroblasts DNA by the dimer-specific endonuclease from Micrococcus luteus) from our method agree with values obtained for the same DNAs from alkaline sucrose gradient analysis . The method has been used to determine pyrimidine dimer yields in DNA from biopsies of human skin irradiated in situ . It will be especially useful in determining the frequency of single strand breaks (or lesions convertible to single strand breaks by specific cleaving reagents or enzymes) in small quantities of DNA from cells or tissues not amenable to radioactive labeling. Biochem Pharmacol, 1986 Oct 1, 35(19), 3283 - 91 Interaction of cis-diamminedichloroplatinum (II) to chromatin . Specificity of the drug distribution; Foka M et al.; We have studied the interaction of the antitumoral drug, cis-diamminedichloroplatinum (II), cis-DDP, to chromatin . Degradation of chromatin-platinum complexes with micrococcal nuclease releases the platinum bound to the linker DNA . By comparing the percentage of platinum released throughout the digestion to the percentage of acid-soluble DNA we suggest that the linker DNA is the preferential target for this drug . This is mainly the case when the amount of bound platinum is low (r less than 0.03) and is less at higher drug concentrations . By comparing the rate constants corresponding to the reaction of cis-DDP to chromatin, DNA or core particle it appears that these constants are the same . This indicates that the bound platinum is located mainly at the DNA level . Our results are discussed with respect to the structure of chromatin and we conclude that this structure should play a role in the in vivo association of cis-DDP to DNA. Proc Natl Acad Sci U S A, 1986 Oct, 83(19), 7206 - 10 Isolation of an episomal yeast gene and replication origin as chromatin; Pederson DS et al.; A multicopy yeast plasmid containing the TRP1 gene (coding for N-5'-phosphoribosylanthranilate isomerase) and ARS1 (autonomously replicating sequence 1) has been purified as chromatin . Electrophoretic analysis of nucleic acid and proteins and electron microscopy show that the plasmid chromatin is largely free of contaminants . Electron-microscopic and linking-number analyses indicate that the plasmid chromatin contains seven nucleosomes, as predicted by the indirect end-label analyses of Thoma, Bergman, and Simpson {J . Mol . Biol . (1984) 177, 715-733} . Indirect end label mapping of micrococcal nuclease cuts demonstrates that nucleosome positions and nuclease-sensitive regions are not altered by the purification . The plasmid chromatin behaves homogeneously with respect to its elution from nuclei, template activity, and intrinsic buoyant density . Taken together, these observations suggest that different copies of the TRP1ARS1 plasmid do not differ from each other grossly in chromatin structure . We discuss the potential for understanding eukaryotic gene regulation offered by the ability to isolate unique genes as chromatin. Proc Natl Acad Sci U S A, 1986 Oct, 83(20), 7573 - 7 Tightly bound nuclear progesterone receptor is not phosphorylated in primary chick oviduct cultures; Garcia T et al.; Oviduct cells from estradiol-treated chicks were grown in primary culture . After 3-5 days of culture in medium containing estradiol, 90% of the cellular progesterone binding sites were detected in the cytosol . After exposure to {3H}progesterone at 37 degrees C, 80% of the progesterone binding sites were found in nuclear fractions . Progesterone receptor phosphorylation was assessed after incubating the cells with {32P}orthophosphate . Receptor components were immunoprecipitated with a specific polyclonal antibody (IgG-G3) and analyzed by NaDodSO4/PAGE and autoradiography . In the cytosol, constant amounts of 32P-labeled 110-kDa subunit (the B subunit, one of the progesterone-binding components of the receptor) and of the non-steroid-binding heat shock protein hsp90 were found, whether cells had been exposed to progesterone or not . No 32P-labeled 79-kDa subunit (the A subunit, another progesterone-binding subunit) was detected . Various procedures were used to solubilize nuclear progesterone receptor (0.5 M KCl, micrococcal nuclease, NaDodSO4), and in no case was 32P-labeled B subunit detected in the extracts . However, nonradioactive B subunit was detected by immunoblot in a nuclear KCl extract of progesterone-treated cells . These results suggest that the fraction of the B subunit that becomes strongly attached to nuclear structures is not phosphorylated upon exposure of cells to progesterone. Biochimie, 1986 Oct-Nov, 68(10-11), 1201 - 9 Fatty acids and carbohydrate-containing lipids in four Micrococcaceae strains; Melin AM et al.; Fatty acid composition and lipidic carbohydrate to lipidic phosphorus molar ratio of yellow pigmented micrococci are compared to red pigmented ones and may be summarized by three indexes . These bacteria show wide differences in their fatty acid composition: three strains possess saturated branched chain fatty acids and one has unsaturated straight chain ones . A significant increase in 'anteiso/iso indexes' is observed between pink (M . roseus) and yellow colored bacteria (M . lysodeikticus, S . lutea) . There is no significant difference (P greater than 0.05) between the 'unsaturation indexes' of the red pigmented parental D . radiodurans strain and its colorless mutant . Radioresistant strains exhibit a higher 'carbohydrate/phosphorus index' than other strains . There seems to be a relationship between a high carbohydrate-containing lipid content and a high resistance to physical and chemical agents, in particular to radiations . These differences observed in the lipid composition have implications in taxonomy and in establishing an evolutionary scheme. EMBO J, 1986 Oct, 5(10), 2681 - 7 Nuclease hypersensitive regions with adjacent positioned nucleosomes mark the gene boundaries of the PHO5/PHO3 locus in yeast; Almer A et al.; The chromatin structure of two tandemly linked acid phosphatase genes (PHO5 and PHO3) from Saccharomyces cerevisiae was analyzed under conditions at which the strongly regulated PHO5 gene is repressed . Digestion experiments with DNase I, DNase II, micrococcal nuclease and restriction nucleases reveal the presence of five hypersensitive sites at the PHO5/PHO3 locus, two of them upstream of PHO5 at distances of 920 and 370 bp, one in between the two genes and two downstream of PHO3 . Specifically positioned nucleosomes are located next to these hypersensitive sites as shown by indirect end-labeling experiments . The positions deduced from these experiments could be verified by monitoring the accessibility of various restriction sites to the respective nucleases . Sites within putative linker regions were about 50-60% susceptible, whereas sites located within nucleosome cores were resistant . Hybridizing micrococcal nuclease digests to a probe from in between the two upstream hypersensitive sites leads to an interruption of an otherwise regular nucleosomal DNA pattern . This shows directly that these hypersensitive sites represent gaps within ordered nucleosomal arrays. J Med Chem, 1986 Oct, 29(10), 2044 - 7 Chiral DNA gyrase inhibitors . 1 . Synthesis and antimicrobial activity of the enantiomers of 6-fluoro-7-(1-piperazinyl)-1-(2'-trans-phenyl-1'-cyclopropyl)-1, 4-dihydro-4-oxoquinoline-3-carboxylic acid; Mitscher LA et al.; New quinolone antimicrobial agents (racemic, (1'S,2'R)- and (1'R,2'S)-6-fluoro-7-(1-piperazinyl)-1-(2'-trans-phenyl-1'-cyclopropyl)- 1, 4-dihydro-4-oxoquinoline-3-carboxylic acids) were synthesized, and their in vitro antimicrobial potencies and spectra were determined . As compared to their conceptual parents, these agents retained a considerable amount of the antimicrobial potency and spectra of ciprofloxacin and of 6-fluoro-1-phenyl-7-(1-piperazinyl)-1,4-dihydro-4-oxoquinoline-3-carboxy lic acid against Gram-positives . Gram-negatives were considerably less sensitive . The (-)-(1'S,2'R) analogue was the more potent of the enantiomers, but the degree of chiral discrimination by most bacteria was only 4-fold . The 4-fold chiral discrimination was observed also using purified DNA gyrase obtained from Micrococcus luteus, whereas the two enantiomers were essentially equiactive against the enzyme derived from Escherichia coli . These results confirm that there is a substantial degree of bulk tolerance available at N-1 of quinolone antimicrobial agents and suggest that electronic factors controlled by substitution at that site are of considerable importance . On the other hand, chiral recognition brought about by attachment of optically active groups to the N-1 position in these derivatives is relatively small. Biochim Biophys Acta, 1986 Oct 1, 883(3), 389 - 95 Deacylation of penicilloylated penicillin-binding proteins from Micrococcus luteus: kinetic study of thiol-promoted release of the bound penicilloyl moiety; Pellon G; The stability of covalent complexes obtained by labelling penicillin-binding proteins 1-6 from Micrococcus luteus with a radioactive derivative of ampicillin has been examined in the presence of thiols . When the incubation medium contained only 1 mM 2-mercaptoethanol, the complexes were almost unaffected for at least 1 h . If 20 mM dithiothreitol was also included in the medium, the amount of bound radioactivity decreased throughout the incubation period . The breakdown of the complexes derived from penicillin-binding proteins 4 and 5 proceeded very slowly, following an apparent first-order kinetics, whereas the kinetics of deacylation of other penicillin-binding proteins exhibited a biphasic pattern with an initial fast phase followed by a slow one, each of which could be approximated by an apparent first-order reaction . This behavior is explained adequately by a two-step mechanism: the penicilloylated penicillin-binding proteins are first deacylated in a reversible exchange with the added thiol, giving rise to an intermediate thioester; once formed, this intermediate is hydrolysed irreversibly . A simple graphical method has been devised to deduce rate constants from the time course of the reaction . Theoretical curves have been constructed, and they fit experimental data satisfactorily . The results point out that added thiols may effectively interfere with the quantitation of penicillin-binding proteins; therefore, the stability of penicilloylated penicillin-binding proteins should be checked carefully when these protecting agents are included in membrane extracts or incubation media. Nucleic Acids Res, 1986 Sep 25, 14(18), 7361 - 78 Chromatin structure of the promoter region of the human c-K-ras gene; Jordano J et al.; The chromatin structure of the human c-K-ras gene has been investigated in various cultured normal and tumor human cells and in a rat cell line transformed with the human oncogene . The promoter region is hypersensitive to DNAse I, micrococcal nuclease, endogenous nucleases and to S1 nuclease in supercoiled plasmids . This hypersensitive region is present in the different cell types analyzed and both normal and mutant alleles exhibit similar general sensitivity to DNAse I digestion in the same tumor cells . However, the 5' more distal DNAse I hypersensitive site, which is coincident with a region of the gene containing sequence homologies with known enhancers, exhibits variable sensitivity which appears to be higher in the tumor than in the normal and in the human than in the rat cells which we have analyzed . These data suggest the presence of specific factors interacting with the promoter sequences and delimits the transcription unit of the c-K-ras locus. Biochemistry, 1986 Sep 23, 25(19), 5364 - 70 Reversible changes in the nucleosomal organization of a human H4 histone gene during the cell cycle; Moreno ML et al.; The organization of nucleosomes associated with a cell cycle regulated human H4 histone gene was examined in synchronized HeLa S3 cells . At various times during the cell cycle, nuclei were digested with micrococcal nuclease, and the nucleosomal pattern of the gene was obtained by Southern blot analysis using radiolabeled human histone H4 gene probes . We have detected reversible changes during the cell cycle in the chromatin structure of this gene, as reflected by the shortening of the nucleosomal spacing after replication and the peak of transcription . This variation is also observed when DNA and protein syntheses are inhibited . By using a probe that comprises 250 base pairs (bp) of the coding region and 240 bp of the 5' end of the gene, containing the promoter and DNase I sensitive sequences, we also have observed a general disruption of the nucleosomal organization, which is reflected by a degeneration of the characteristic nucleosomal ladder produced by micrococcal nuclease digestion . This modification coincides with the replication and active transcription of the gene (early S phase), which recovers its regular nucleosomal appearance when both processes have been completed, although the nucleosome linker length is shortened . When the probe utilized comprises the distal 3' end of the gene, there is no disruption of the nucleosomal pattern, but the linker region also exhibits a shortened length . A non-cell cycle regulated gene (beta-globin) does not exhibit such modifications in any of the situations analyzed.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1986 Sep 23, 25(19), 5388 - 91 Euglena gracilis chromatin: comparison of effects of zinc, iron, magnesium, or manganese deficiency and cold shock; Falchuk KH et al.; The effects induced by Fe, Mn, or Mg deficiency or cold shock on the DNA content and histones of Euglena gracilis have been examined and compared to those produced by Zn deficiency . The DNA content of the stationary-phase organisms used as controls is 2.1 micrograms/10(6) cells . The DNA of stationary-phase iron-deficient (-Fe), magnesium-deficient (-Mg), manganese-deficient (-Mn), zinc-deficient (-Zn), and cold-shocked (CS) cells is increased to 3.0, 4.6, 6.2, 3.8, and 3.8 micrograms/10(6) cells, respectively . The electrophoretic mobilities of proteins solubilized with 0.4 N H2SO4 from CS, -Fe, -Mg, and -Mn cells are nearly identical and are characteristic of the five histone classes, H1, H2A, H2B, H3, and H4 . In contrast, no histones are found in the equivalent acid extract from -Zn cells . The effect of micrococcal nuclease on chromatin from control, CS, and -Zn cells was examined . The chromatin of CS cells is 1.2-fold while that from -Zn cells is 10-30-fold more resistant to micrococcal nuclease digestion than is the chromatin of control cells . Thus, the chromatin of cells grown in Zn-deficient conditions differs markedly from that of organisms cultured in media deficient in Fe, Mn, or Mg or exposed to cold shock. Biochemistry, 1986 Sep 9, 25(18), 5057 - 63 A 10S particle released from deoxyribonuclease-sensitive regions of HeLa cell nuclei contains the 86-kilodalton-70-kilodalton protein complex; Yaneva M et al.; Digestion of HeLa cell nuclei with micrococcal nuclease or deoxyribonuclease I (DNase I) released the 86-kilodalton-70-kilodalton (kDa) protein complex in particles sedimenting at approximately 10 S in sucrose density gradients . Immunoaffinity-purified 32P-labeled complexes contained 86- and 70-kDa polypeptides with phosphorylated serine residues and DNA fragments, of which the largest was 110 base pairs long . Digestion of nick-translated nuclei with micrococcal nuclease released 32P-labeled 10S particles that were immunoaffinity-purified; they contained labeled 110-base-pair DNA fragments . The micrococcal nuclease digests were analyzed by two-dimensional electrophoresis, which separated nucleosomes in the first dimension and the associated proteins in the second . Western blots of the separated proteins showed that the 86-kDa-70-kDa complex was associated with the mono-, di-, and trinucleosomes . A more extensive electrophoretic separation revealed that the 10S particle from nick-translated nuclei migrated with a subfraction of the mononucleosomes that lacked H1 histones . These results suggest that the 10S particle which contains the 86-kDa-70-kDa complex is associated with an unfolded nucleosome that is present in DNase I sensitive regions. Anal Biochem, 1986 Sep, 157(2), 367 - 74 Determination of lysozyme activity at low levels with emphasis on the milk enzyme; McKenzie HA et al.; A method is described for the determination of lysozyme (muramidase) activity, whereby sensitivity is maximized by incubation of the reaction mixture (sample, buffer, and substrate (Micrococcus luteus} over an extended period . This approach is made feasible by exploiting our observation that the lytic reaction follows simple kinetic order during this time (e.g., 700 min for bovine lysozyme and 960 min for the eggwhite enzyme at low concentrations) . After this period, the reaction rates diminish, indicating biphasic behavior, and eventually become negligible . The kinetic order may vary with both the type of lysozyme and the buffer system used . The limit of detection for bovine milk lysozyme is 100 pg/ml reaction mixture, equivalent to 6 ng/ml milk, for a 50-microliters sample (with reference to hen eggwhite lysozyme) . With these limits, the method has proven valuable in our comparative studies, particularly for low levels of activity in bovine milk, but also in secretions and tissue extracts from various other eutherian, metatherian, and prototherian mammals . The method may also be applied to investigation of structure and function in modified forms of the enzyme. Carcinogenesis, 1986 Sep, 7(9), 1497 - 503 Selective repair of methylated purines in regions of chromatin DNA; Ryan AJ et al.; The distribution of methylated purines in different regions of liver chromatin DNA has been examined after treating rats with {14C}dimethylnitrosamine (2 mg/kg) . At different times after administration of the carcinogen, liver nuclei were isolated and fractionated by micrococcal nuclease digestion and low and high salt extractions into an active chromatin fraction, two fractions comprising the bulk of the genome, and a nuclear matrix fraction . Regions of active chromatin and nuclear matrix tended to be methylated more readily than bulk chromatin, with respect to formation of both O6-methylguanine and N-methyl purines . Removal of both 7-methylguanine and 3-methyladenine (by repair and depurination reactions) occurred at a relatively uniform rate in all chromatin fractions . In contrast, repair of O6-methylguanine proceeded more rapidly from active chromatin than from bulk chromatin, whereas repair of this lesion from nuclear matrix DNA was much slower than for bulk DNA . Pretreatment of rats for 4 weeks with non-radioactive dimethylnitrosamine before the administration of {14C}dimethylnitrosamine enhanced the rate of repair of radioactive O6-methylguanine from all chromatin fractions . Nevertheless the rate of loss of the adduct was still faster from active chromatin and slower from matrix DNA than from the bulk of the genome . Since pretreatment also elevated the rate of liver DNA synthesis especially in the nuclear matrix fraction, there is an increased probability of the fixation of mutations due to the presence of O6-methylguanine in this selected region of the genome . The implications of this persistent O-alkylation of matrix DNA, and rapid repair of O6-alkylguanine in active chromatin for the toxicity and carcinogenicity of alkylating agents are discussed. J Gen Microbiol, 1986 Sep, 132 ( Pt 9), 2577 - 81 Pyrimidine dimer excision repair of DNA in Bacteroides fragilis wild-type and mitomycin C-sensitive/UV-sensitive mutants; Abratt VR et al.; An enzyme preparation purified from Micrococcus luteus was shown to be specific for UV-induced pyrimidine dimers and was suitable for the detection of DNA excision repair systems . The wild-type Bacteroides fragilis Bf-2 strain and a mitomycin C-sensitive mutant (MTC25) had constitutive dimer excision systems which functioned efficiently under anaerobic and aerobic conditions . A UV-sensitive mutant (UVS9) had markedly reduced levels of the constitutive dimer excision systems under anaerobic and aerobic conditions . Since liquid holding recovery under aerobic conditions was inhibited by chloramphenicol whereas the final level of excision repair in B . fragilis Bf-2 was not affected, it is concluded that pyrimidine dimer removal is not the process responsible for increased physiological aerobic liquid holding recovery. Metabolism, 1986 Sep, 35(9), 861 - 8 Nuclear thyroid hormone receptors in cultured human fibroblasts: improved method of isolation, partial characterization, and interaction with chromatin; Ichikawa K et al.; In order to characterize the nuclear thyroid hormone receptors in human tissue, an improved method for isolation of nuclei from cultured human fibroblasts was developed . This method provided nuclei with a protein/DNA ratio of 2.8 and recovery of 42% . The purity of nuclei was verified by phase contrast and electron microscopy, which showed normal appearance of chromatin structure . Nuclear binding assay was performed by incubation of whole cells at 37 degrees C or isolated nuclei at 22 degrees C with L-triiodothyronine (T3) . In both cases, an affinity constant (Ka) of 2.0-3.0 X 10(10) M-1 and an average binding capacity of 41 femtomoles of T3/100 micrograms DNA (3,100 binding sites/nucleus) were obtained . During incubation of the nuclei, 13% to 16% of receptors that had an identical Ka was released into the medium . Salt extraction recovered 85% to 90% of the receptors, which had a Ka of 4.5 X 10(10) M-1 and the capacity of 0.13 pmol of T3/mg protein . The Ka fo . L-thyroxine (T4) was seven to 18 times lower than that for T3, but the capacity was the same in isolated nuclei, receptors released during incubation of nuclei, and in salt-extracted receptors . Of the iodothyronines examined, affinity for triiodothyroacetic acid was the highest, followed by L-T3, D-T3, L-T4 . Isokinetic glycerol gradient analysis revealed that salt-extracted receptors had a sedimentation coefficient of 3.4 S, whereas micrococcal nuclease digested receptors showed two major (6.0 to 6.5 and 12.5 S), and two minor (17 and 19 S) peaks . These results were virtually identical to those obtained with rat liver nuclei analyzed in parallel studies.(ABSTRACT TRUNCATED AT 250 WORDS) Dev Biol, 1986 Sep, 117(1), 109 - 13 Site and stage specific action of endogenous nuclease and micrococcal nuclease on histone genes of sea urchin embryos; Anderson OD et al.; The early histone genes of sea urchin embryos are expressed exclusively during cleavage stages of embryogenesis . The chromatin containing these genes was examined by nuclease sensitivity . An endogenous nuclease active during cleavage, produces 1300-bp segments containing early histone genes . The cutting sites have been mapped; there are very sensitive sites close to the cap site for H1, H2A, H2B, and H4 . Chromatin obtained from embryos of later stages, when the genes are not expressed, do not display this pattern of nuclease sensitivity . Micrococcal nuclease produces nucleosomes that contain histone genes when used with nuclei from later stages, but not with nuclei from cleavage stages. J Virol, 1986 Sep, 59(3), 764 - 7 Chromosomal organization of the herpes simplex virus genome during acute infection of the mouse central nervous system; Muggeridge MI et al.; After corneal inoculation, herpes simplex virus type 1 replicates in the mouse eye, trigeminal ganglia, and brainstem, producing first an acute and then a latent infection . Previous work from this laboratory focused on the structure of the viral DNA in this system . We have now examined the structure of the viral genome at the chromosome level by using micrococcal nuclease digestion . Studies with disaggregated cell preparations made from the brainstems of acutely infected mice show that the majority of the viral DNA is in a nonnucleosomal form; however, a nucleosomelike fraction was also consistently detected . A similar result was obtained for viral DNA in herpes simplex virus type 1-infected C1300 (clone NA) neuroblastoma cells (a neuronal cell line). Biochim Biophys Acta, 1986 Aug 29, 888(1), 49 - 61 Tubulin-chromatin interactions: evidence for tubulin-binding sites on chromatin and isolated oligonucleosomes; Mithieux G et al.; The interaction of tubulin with chromatin has been studied using a radiolabeled tubulin binding assay and velocity sedimentation analysis on isokinetic sucrose gradients . Soluble chromatin was prepared by mild micrococcal nuclease digestion of rat liver nuclei and tubulin was purified from rat brain by temperature-dependent assembly-disassembly and phosphocellulose chromatography . The tubulin-binding assay is based on the ability of chromatin to precipitate quantitatively at physiological ionic strength allowing separation of free tubulin from chromatin-bound tubulin . The binding of tubulin to unfractionated soluble chromatin was rapid, reversible and saturable . Saturation of binding sites was obtained using tubulin concentrations ranging from 0.5 to 400 micrograms/ml, in the presence of a high concentration (2.5 mg/ml) of another acidic protein, bovine serum albumin . The Scatchard and Hill plots showed that tubulin bound to a single class of non-interacting sites and yielded values of (0.5-0.6) X 10(7) M-1 for an apparent Ka and a maximal binding capacity of 0.8 nmol tubulin/mg DNA, i.e . about 1 molecule of tubulin/10 nucleosomes . Similar binding parameters were obtained when binding experiments were performed with insoluble chromatin in 0.15 M NaCl . Velocity sedimentation analysis of tubulin-chromatin complexes revealed that tubulin bound to all classes of chromatin oligomers, irrespective of the length of the nucleosomal chain . Tubulin-trinucleosome complexes formed from isolated trinucleosome in the presence of an excess of tubulin were separated from free reactants . It was found that 10-15% of the starting oligonucleosomal species reacted with tubulin, in a stoichiometry of about 0.8 molecule of tubulin/nucleosome . Given the characteristics of the binding and the expected cellular free tubulin concentration, the tubulin-chromatin interaction could possibly take place in vivo, when the nuclear membrane breaks down during the first steps of mitosis. J Biol Chem, 1986 Aug 25, 261(24), 11393 - 7 Study of barley endonucleases and alpha-amylase genes; Kalinski A et al.; We have identified an endonuclease(s) that preferentially cleaves the internucleosomal linker regions in the aleurone chromatin producing mono- and oligonucleosomes . This enzyme(s) has been designated as a "linker"-specific nuclease(s) . This nuclease does not require divalent cations for activity, and therefore it is not the "Ca2+-Mg2+-DNase" found in mammalian cells . The linker-specific nuclease activity is not detectable in the dry aleurone tissue and in the tissue treated with 0.5 mM cordycepin . The endonuclease activity of the aleurone tissue incubated with gibberellic acid is higher than the level of this endonuclease in tissue treated with abscisic acid or water alone . Nuclei isolated from embryos have lower levels of endonuclease activities compared to those from aleurone tissue . Digestion of the nuclei from embryos with micrococcal nuclease revealed the subunit structure of chromatin . In Southern blots of the HindIII digests of DNA from embryos, five DNA bands hybridized to a nick-translated alpha-amylase cDNA clone . In similar autoradiograms with aleurone DNA, particular bands are less visible, notably in the DNA isolated from the tissue treated with gibberellic acid . This is the first report of the presence of a linker-specific nuclease activity in plant cells. J Mol Biol, 1986 Aug 20, 190(4), 619 - 33 Micrococcal nuclease as a DNA structural probe: its recognition sequences, their genomic distribution and correlation with DNA structure determinants; Flick JT et al.; We have analyzed micrococcal nuclease (MNase) DNA cleavage patterns at the sequence level by examining 2.3 X 10(3) base-pairs of data derived from the Drosophila melanogaster 44D larval cuticle locus . Within this region, MNase preferentially cleaved 140 sites . Clusters of these sites appear to generate the preferential MNase eukaryotic DNA cleavage sites seen on agarose gels at roughly 100 to 300 base-pair intervals . These clusters of preferential cleavage sites rarely occur within gene coding regions . The analysis revealed that duplex DNA sequences preferentially cleaved by MNase are generally determined by a single strand sequence: d(A-T)n, where n greater than or equal to 1, flanked by a 5' dC or dG . Cleavage of the other strand is generally staggered 5' by several nucleotides and occurs even if such sequences are absent on that strand . An empirical predictive DNA cleavage model derived from a statistical analysis of the sequence level data was applied to seven eukaryotic gene loci of known sequence . The predicted patterns were in good general agreement with the previously observed eukaryotic gene/spacer cleavage pattern . Statistical analysis also revealed that sites of predicted preferential DNA cleavage occur less frequently in protein coding regions than for randomized sequences of the same length and nucleotide content . Comparison of the MNase cleavage patterns to the sequence-dependent pattern of binding energies between duplex DNA strands indicates that MNase preferentially cleaves sequences with low helix stability. Mol Cell Biochem, 1986 Aug, 71(2), 167 - 75 Molecular and functional diversity of non-histone protein fraction NHCP1 from hamster Kirkman-Robbins hepatoma and liver; Kilianska Z et al.; Non-histone protein fraction NHCP1 of micrococcal nuclease-sensitive and nuclease-resistant chromatin from Kirkman-Robbins hepatoma and hamster liver was studied by two-dimensional electrophoresis followed by Coomassie and silver staining and by microcomplement fixation technique in the presence of antibodies elicited against NHCP1 of both tissues . Apart from many common spots several tissue specific components associated with either nuclease-sensitive or nuclease-resistant chromatin were found . The presence of tissue specific components among NHCP1 from hepatoma and liver was confirmed by immunological analysis . It was stated that these components are exclusively localized in nuclease-resistant part of chromatin from neoplastic and normal tissues thus suggesting their structural function. J Steroid Biochem, 1986 Aug, 25(2), 271 - 6 Characterization of the progesterone receptor solubilized by micrococcal nuclease and DNase I digestion; Geier A et al.; In order to investigate the functional organization of the progesterone receptor in chromatin we characterized the physical-chemical properties of the receptor bound chromatin fragments released by micrococcal nuclease and DNase I digestion . The crude nuclear fraction was isolated from T 47 D cells, previously exposed to 0.1 microM {3H}ORG 2058 . The parameters determined in low and high salt concentrated buffers were: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding abilities to a DNA-cellulose column . The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values . Micrococcal nuclease digestion solubilized a receptor form sedimenting as a single peak at 4.4 S with a Rs = 7.78 nm and an estimated Mr = 144,000 . About 53% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer . 0.4 M KCl dissociated this receptor form into a smaller receptor sedimenting at 3.3 S with Rs = 5.53 nm and a calculated Mr = 76,000 . A similar receptor form was extracted by 0.6 M KCl from the undigested crude nuclear fraction . DNase I digestion solubilized a receptor form sedimenting at 3.3 S with a Rs = 6.87 nm and a calculated Mr = 94,000 . About 26% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer . Dissociation of this receptor form by 0.4 M KCl resulted in a receptor sedimenting at 2.8 S with a Rs = 6.53 nm and an estimated Mr = 76,000 . These results suggest: The progesterone receptor in chromatin is associated with several molecules probably proteins which complexed it to DNA . Some of these molecules still associated with the progesterone receptor could be released by nucleases digestion . Micrococcal nuclease releases a larger portion of these molecules than those release by DNase I. Biophys J, 1986 Aug, 50(2), 307 - 17 Bifilar enzyme-sensitive sites in ultraviolet-irradiated DNA are indicative of closely opposed cyclobutyl pyrimidine dimers; Lam LH et al.; Incubation of UV-irradiated DNA with pyrimidine dimer-DNA glycosylase in cell-free lysates prepared from Micrococcus luteus results in the appearance of double-strand breaks . It has previously been assumed that such double-strand breaks result from cleavage at closely opposed dimers . We have used hybrid molecules of bacteriophage T7 DNA comprised of two unirradiated strands, two UV-irradiated strands, or one unirradiated and one UV-irradiated strand to test this hypothesis . Bifilar cleavage was observed only with molecules consisting of two irradiated strands and no bifilar cleavage was observed after the monomerization of pyrimidine dimers by enzymatic photoreactivation . Our results indicate that at least 80% of the double-strand breaks result from cleavage at closely opposed dimers and that the induction of dimers in one strand does not influence the induction of dimers at closely opposed positions in the complementary strand of a DNA double helix. Fed Proc, 1986 Aug, 45(9), 2394 - 8 Diet-mediated alteration of chromatin structure; Castro CE et al.; Higher-order chromatin structure and the process of transcription are related . The significance of a nutritional state's altering chromatin structure lies in the potential role of that nutritional state in the regulation of gene expression . In rats short-term feeding of semisynthetic diets varying in the proportion of carbohydrate, protein, or fat alters the configuration of liver chromatin as measured by sensitivity to micrococcal nuclease (EC 3.1.31.1) . A carbohydrate-rich, fat-free diet increases the sensitivity of rat liver chromatin to micrococcal nuclease and decreases the nucleosome repeat length . In contrast, a protein-free diet or a diet deficient in magnesium or zinc decreases the sensitivity of liver nuclear chromatin to micrococcal nuclease . Diet-mediated mechanisms that alter chromatin structure are now unknown, but the continued study of nutritional interaction with the genome should identify the responsible features as well as their significance to gene function. J Biomol Struct Dyn, 1986 Aug, 4(1), 23 - 39 A study of phi X-174 DNA torus and lambda DNA torus tertiary structure and the implications for DNA self-assembly; Marx KA et al.; Hydrated torus shaped complexes were examined by transmission electron microscopy in both spermidine-condensed linear and nicked circular phi X-174 DNA and lambda DNA preparations . Freeze-etch replicas of both these torus samples, produced with very low Pt metal deposition levels (9APt/C), were found to have circumferentially wound single DNA double helix size surface fibers in the range of 30A width . Measurements of torus inner and outer circumference as well as ring thickness were performed . Observed differences in the torus dimension distributions from circular phi X-174 DNA and linear phi X-174 DNA may be related to the different topological constraints on DNA folding in these two samples (1) . On the basis of annulus thickness measurements phi X-174 DNA toruses, in contrast to lambda DNA toruses, were observed to fall into two classes identified as being formed from monomer DNA condensation and multimer DNA condensation . All of the torus substructure and population dimensions observed here are consistent with the continuous circumferential DNA winding model of torus organization proposed by Marx and Reynolds (1) to explain the micrococcal nuclease cleavage properties of the toruses . End-on view measurements of the torus thickness were made from micrographs obtained by extensive tilting of the object replica . These direct measurements confirmed quaternary structure interpretations made from simple strand packing models . We compared the measured torus properties in this linear DNA size series (5386-48000 bp) . With increasing DNA length the pattern of DNA strand self-assembly was found to be more varied producing lambda DNA toruses of varying shape . The relevance of our study to the problem of lambda bacteriophage DNA head packaging was discussed. Biochem Biophys Res Commun, 1986 Jul 31, 138(2), 758 - 63 A high melting cis-{Pt(NH3)2{d(GpG)}}adduct of a decanucleotide duplex; van Hemelryck B et al.; The {cis-Pt(NH3)2(d(GCCGGATCGC)-N7(4), N7(5))}-d(GCGATCCGGC) duplex has been prepared with Tm = 49 degrees C (vs 58 degrees C for the unplatinated form) . NMR of the ten observable imino protons supports a kinked structure with intact base pairing of the duplex on the 3'-side of the d(GpG).cis-Pt chelate (relative to the platinated strand) The modification of the B-DNA type CD spectrum, due to the platinum chelate, is comparable to that observed for the platination (at a 0.05 Pt:base ratio) of the Micrococcus Lysodeikticus DNA (72% GC). Eur J Biochem, 1986 Jul 15, 158(2), 393 - 401 Chromatin structure and induction-dependent conformational changes of human interferon-beta genes in a mouse host cell; Bode J et al.; Multiple copies of a human interferon-beta gene introduced into a mouse host cell line can be activated by induction with double-stranded RNA . Several induction-dependent changes of the chromatin structure could be traced by mapping techniques using four different agents {DNase I, micrococcus nuclease, bromoacetaldehyde and methidiumpropyl-EDTA X iron(II)} . Our data show that all copies of the interferon gene have adopted a very similar conformation in the host cell and respond to the inducing stimulus in a highly synchronous fashion . Detailed induction-specific changes were observed best with the chemical reagents which disclose a specific hypersensitive site within a sequence that has been shown to be required for the induction process (around position -80) and three other regions which, in addition to the transcribed region itself, gain single-strand character by an auxiliary process which can be mimicked by the addition of butyrate to the medium and may therefore involve histone hyperacetylation . Six discrete 'phased' nucleosomes are present upstream from the gene and are modulated by induction . At least four nucleosomes are located downstream . The interferon genes are largely protected from micrococcus nuclease in the inactive state . Gene activation increases access to micrococcus nuclease and DNase I indicating gross conformational changes on a higher level of chromatin structure. J Immunol, 1986 Jul 15, 137(2), 651 - 5 Down-regulation of macrophage lysozyme by lipopolysaccharide and interferon; Warfel AH et al.; Lipopolysaccharide (LPS) treatment of resident mouse peritoneal macrophages (M phi) was found to suppress intracellular as well as secreted lysozyme (LZM) . Interferon (IFN) had a similar effect . LZM was identified by the capacity of cell lysates or medium to lyse Micrococcus lysodeikticus, and by the presence of a 14.5 Kd protein band which co-migrated with human LZM in SDS-PAGE and which reacted positively in Western blots with antiserum to human LZM . The size of the 14.5 Kd band decreased sequentially with increasing concentrations of LPS to which the cells were exposed . Although the LPS influence on LZM levels was dose-dependent, the intracellular LZM pool responded more readily than secreted LZM . Maximal intracellular LZM suppression of 80% was obtained with 10 micrograms LPS, whereas secreted LZM was reduced by only 66% . An IFN concentration of 100 U reduced secreted LZM by 24%, whereas 10,000 U of IFN decreased the amount of LZM secreted by 71% . Thioglycolate-elicited M phi had 75% less intracellular LZM than untreated resident M phi . Moreover, thioglycolate-elicited M phi were hyporesponsive to the suppressive effects of LPS added in vitro . Because both LPS and IFN have been shown to stimulate numerous M phi functions, the data are of interest because they support the concept, based on other studies, that agents which are capable of enhancing some M phi activities may concomitantly down-regulate other functions. Nucleic Acids Res, 1986 Jul 11, 14(13), 5513 - 29 The effect of preincubation of HeLa cell nuclei with ATP on the degradation of mononucleosomal DNA by micrococcal nuclease; Pentz M et al.; HeLa cell nuclei with DNA labeled with {3H} thymidine have been preincubated under varying conditions and then incubated with micrococcal nuclease . Aliquots, removed at increasing times, were analyzed for mononucleosomal size DNA and for acid-soluble DNA, the ratios were plotted and a slope was determined . Preincubation with ATP and a regenerating system increased the slope 2 fold . Optimum ATP concentrations were above 0.25 mM . The ATP effect was reversed by novobiocin . No inhibition of the ATP effect was observed with nalidixic acid, coumermycin, oxolinic acid, VM-26, aphidicolin, or 3 amino-benzamide . NAD or cAMP or cGMP had no effect with or without ATP . Other nucleoside triphosphates could substitute to varying degrees for ATP as could ATP analogues . Nuclei from log phase cells showed no ATP effect, but log phase cells, partially depleted of ATP by incubation with deoxyglucose, showed the effect . The effect was lost in nuclei on long-term storage . No evidence was found for differential degradation of core histones, histone H-1 or DNA, and there was no evidence of nucleosome sliding. EMBO J, 1986 Jul, 5(7), 1633 - 44 Adenoviral protein VII packages intracellular viral DNA throughout the early phase of infection; Chatterjee PK et al.; The proteins associated with parental, adenoviral DNA in productively-infected HeLa cells have been examined both directly and indirectly . HeLa cells infected with 32P-labelled Ad2 were irradiated with u.v . light at various points in the infectious cycle . Following degradation of the DNA, nuclear proteins carrying cross-linked nucleotides, or oligonucleotides, were distinguished from virion phosphoproteins by the resistance of their 32P radioactivity to 1 M NaOH . The major core protein of the virion, protein VII, was found to be associated with viral DNA throughout infection, even when cells were infected at a multiplicity of 0.14 . Micrococcal nuclease digestion of intranuclear viral DNA 4 h after infection liberated two nucleoprotein particles containing viral DNA, neither of which co-migrated with HeLa cell mononucleosomes . These results indicate that core protein VII remains associated with parental adenoviral DNA during productive infections . The observation that protein VII can be cross-linked to DNA in cells infected at very low multiplicity, together with the results of a comparison of proteins cross-linkable to viral DNA in cells infected by wild-type virus and a non-infectious mutant containing the precursor to protein VII, suggest that nucleoproteins comprising viral DNA and protein VII must be the templates for expression of pre-early and early viral genes. Mech Ageing Dev, 1986 Jul, 35(2), 199 - 208 Changes in the higher order organization of DNA during aging of human fibroblast-like cells; Dell'Orco RT et al.; Nucleosome spacing (DNA repeat length) was determined in human diploid fibroblast-like cells (HDF) of different in vitro ages following the electrophoretic separation of micrococcal nuclease digestion products . The results indicate that a heterogeneity of DNA repeat lengths is present in HDF of all in vitro ages . In older cells the organization of part of the DNA is conserved, but a greater proportion of shorter repeats is evident . The shorter repeat lengths are not due to nucleosome sliding, but result from the presence of shorter linker regions which are reduced by as much as 25% in part of the chromatin of high PDL cells. Immunology, 1986 Jul, 58(3), 489 - 94 Functional effects of CRP binding to nuclei; Shephard EG et al.; Binding of human CRP and rat CRP to their respective autologous liver nuclei has been demonstrated . The binding was shown to consist of both a calcium- and non-calcium-dependent component . The co-precipitation of human CRP with histones in physiological calcium concentrations suggests that the calcium-independent binding to chromatin is mediated by the CRP-polycation site . Complement-dependent chromatin solubilization by human CRP bound to nuclei was confirmed and show that the solubilized material is intact chromatin with a large proportion of small monosome structures of 180 base pair repeat . The binding of human and rat CRP to nuclei enhanced micrococcal nuclease digestion, suggesting that CRP binding in both these species alters chromatin structure by increasing linker DNA exposure . The suppressed transcription of DNA, after saturating CRP binding to nuclei, could limit aberrant transcription of damaged chromatin. Biokhimiia, 1986 Jul, 51(7), 1100 - 7 {Localization of cysteine residues in the alpha-chain of histidine decarboxylase from Micrococcus sp . n.}; Grebenshchikova OG et al.; It was shown that the alpha-chain of histidine decarboxylase of Micrococcus sp . n . is split off by 2-nitro-5-thiocyanobenzoic acid at only one of the two cysteine residues . Determination of the C-terminal sequences, amino acid composition, molecular weight of the fragments obtained demonstrated that these fragments constitute a complete alpha-chain whose cleavage occurs at the cysteine residue which is readily modified by SH-reagents . the Ile-Cys peptide bond appeared to be resistant to cleavage under these conditions . This cleavage permitted to identify the amino acid environment of the cysteine residue active center and its localization in the alpha-chain of histidine decarboxylase. Biochemistry, 1986 Jul 1, 25(13), 3839 - 45 Modulation of the sensitivity of chromatin to exogenous nucleases: implications for the apparent increased sensitivity of transcriptionally active genes; Walker PR et al.; We have examined the effects of changing the ionic composition of the buffers in which nuclei are isolated on the sensitivity of chromatin to micrococcal nuclease and deoxyribonuclease I . Unless nuclei are isolated in buffers containing physiological levels of monovalent (150 mM KCl) and divalent (2-5 mM MgCl2) cations, there is a substantial loss of higher order structure . The ionic composition of the buffer in which the digestion is carried out also affects the amount of material digested both by modulating higher order structure and by determining the solubility of the released material . Magnesium ion concentrations greater than 2 mM and calcium ions at virtually any concentration precipitate substantial amounts of the released chromatin fragments . These observations can be interpreted in light of the known effects of the ions on 10- and 30-nm fiber structure and used as a basis for improvements in techniques for isolating chromatin and for studying its structure and function using exogenous nuclease probes . The apparent nuclease sensitivity of transcriptionally active chromatin was reexamined and shown to be more likely a reflection of differential solubility rather than an overall increase in nuclease sensitivity. J Inorg Biochem, 1986 Jul, 27(3), 179 - 89 Enhancement of in vitro ribonucleic acid synthesis on chromium(III)-bound chromatin; Ohba H et al.; Z chromatin-chromium (Cr) complex, prepared from mouse liver chromatin and CrCl3, showed a significantly enhanced template activity for in vitro RNA synthesis . Digestion experiments with this complex using micrococcal nuclease and DNase I suggested that Cr(III) preferentially binds to linker regions rather than core regions of chromatin . Further, it was found that Cr(III) binds to DNA and nonhistone proteins (NHP), but hardly to histones . Moreover, the template activity of an NHP-Cr complex, when added to a DNA-histones complex, was inhibited remarkably . The template activity of the chromatin-Cr complex was not significantly altered by proteinase K digestion . Furthermore, experiments using rifampicin and {gamma-32P}guanosine 5'-triphosphate (GTP) demonstrated an increase in the number of initiation sites in the chromatin-Cr complex . These results suggest that, in this in vitro system, Cr(III) preferentially binds to DNA in chromatin and causes an increase in the number of initiation sites, thus enhancing RNA synthesis. Proc Natl Acad Sci U S A, 1986 Jul, 83(13), 4612 - 6 Simian virus 40 DNA replication in vitro: study of events preceding elongation of chains; Wobbe CR et al.; We have evidence for the formation of a stable preelongation complex during the replication of simian virus 40 (SV40) origin containing DNA (ori+ DNA) in vitro . Preincubation of ori+ DNA with HeLa cytosolic extracts and SV40-encoded large tumor antigen (T antigen) in the absence of deoxynucleoside triphosphates eliminates a lag that normally precedes replication . This effect requires ATP and is inhibited by RNase A; subsequent elongation is inhibited by aphidicolin but not by RNase A . A T antigen and SV40 origin-dependent complex can be isolated by gel-filtration chromatography of preincubation reaction mixtures . In both cases, the products formed by replication after complex formation resemble those formed during in vitro replication reactions described previously . HeLa cytosolic extract was separated into two ammonium sulfate fractions: a 0-40% fraction (AS 40) that shows low levels of DNA synthesis and a 40-65% fraction (AS 65) that is inactive by itself but stimulates synthesis when added to the AS 40 fraction . DNA synthesis by these combined fractions has the same requirements as crude extract, occurs in two stages as described above, and is sensitive to RNase A . Pretreatment of both fractions with micrococcal nuclease eliminated replication activity, whereas the combination of a pretreated fraction (either AS 40 or 65) with an untreated fraction was active . A heat-inactivated (55 degrees C, 5 min) AS 65 fraction restored replication activity to the combination of micrococcal nuclease-treated AS 40 and AS 65 fractions. Biochim Biophys Acta, 1986 Jun 20, 867(3), 124 - 34 Selective insolubility of active hsp70 gene chromatin; Hanks SK et al.; A gentle chromatin fractionation procedure was used to investigate solubility properties of Drosophila hsp70 heat-shock genes . After a brief digestion of isolated nuclei with micrococcal nuclease, most DNA is readily solubilized under low-ionic-strength conditions that maintain native nucleosomal organization . Actively transcribing hsp70 genes, however, are found to be enriched in the insoluble nuclear residue . Inactive genes are not resistant to solubilization, showing a fractionation pattern similar to that of bulk DNA . The insolubility characteristic correlates well with two other structural features of active hsp70 chromatin: increased sensitivity to endonuclease attack and disruption of the nucleosomal repeat pattern . The 5'-flanking regulatory region of active hsp70 genes is particularly resistant to solubilization, suggesting a role for binding of transcription factors in mediating this effect. Biochem Biophys Res Commun, 1986 Jun 13, 137(2), 716 - 21 The conversion of native adenylylated glutamine synthetase into phosphotyrosine enzyme by micrococcal nuclease; Kimura K et al.; Micrococcal nuclease treatment of the native adenylylated glutamine synthetase from M . smegmatis yielded adenosine and phosphotyrosyl enzyme . The rate of the deadenosylation reaction was monitored by the appearance of the adenosine in HPLC analysis . The o-phosphotyrosyl enzyme had catalytic activity comparable to that of the adenylylated enzyme suggesting that the adenosine part in AMP was not essential to the regulation of the enzyme activity . Further, upon treatment of the phosphotyrosyl enzyme with alkaline phosphatase, the glutamine synthetase activity was increased . This means that the regulation site of glutamine synthetase by covalent modification simply requires the phosphorylation of the tyrosine residue. J Mol Biol, 1986 Jun 5, 189(3), 457 - 76 Mechanism of chromatin assembly in Xenopus oocytes; Ruberti I et al.; We have analyzed the chromatin assembly reaction catalyzed by the Xenopus oocyte extract (S-150) . A 50 S complex is formed upon mixing the 17 S pUC DNA and the S-150 . Mature histones are not detected in this complex, which contains relaxed DNA and protein, and generates subnucleosomal 7 S particles upon digestion with micrococcal nuclease . The relaxed nucleoprotein is gradually supercoiled into nucleosomal chromatin in the S-150, via a pathway that requires ATP and is blocked by novobiocin, and this process is accompanied by the appearance of mature histones H3 and H4 . Isolated complexes also supercoil in vitro, which implies the complex is a kit that contains histone precursors, as well as topoisomerases and other enzymes required for assembly . We discuss the biological implications of these findings. Virology, 1986 Jun, 151(2), 315 - 28 Changes in the nucleoprotein complexes of a baculovirus DNA during infection; Wilson ME et al.; The nature of the DNA-protein complexes assumed by Autographa californica nuclear polyhedrosis virus (AcNPV) DNA during infection of Spodoptera frugiperda cells was investigated by micrococcal nuclease digestion of infected nuclei . Both parental viral DNA and progeny viral DNA assumed a chromatin-like structure early in infection . By late times (24 hr) p.i., the viral DNA acquired a unique nucleoprotein structure . In addition to fragments of mononucleosome size (185 bp), two subnucleosomal bands of 120 and 90 bp were observed . The subnucleosomal bands contained exclusively viral DNA . No alteration in the nature of the host chromatin structure following AcNPV infection was observed . An examination of the basic chromatin-associated proteins revealed two major viral-induced proteins having molecular weights of 15K and 39K . The induction of the basic 15K protein between 10 and 24 hr p.i . coincided with the appearance of the altered nucleoprotein structure observed by 24 hr p.i . and the cessation of histone synthesis. Carcinogenesis, 1986 Jun, 7(6), 907 - 13 Preferential binding of the carcinogen benzo{a}pyrene to DNA in active chromatin and the nuclear matrix; Obi FO et al.; Rat liver nuclei or hepatocytes were incubated with the proximate carcinogen, benzo{a}pyrene (BP) and its ultimate carcinogen, anti-benzo{a}pyrene-7,8-diol-9,10-epoxide (BPDE) . Following carcinogen exposure, nuclei were fractionated by micrococcal nuclease digestion and stepwise extraction to yield an active chromatin fraction enriched in transcribed versus non-transcribed genes, a bulk chromatin fraction, a high-salt-extracted chromatin fraction and a nuclear matrix fraction containing elevated concentrations of transcribed and nontranscribed genes . BP binds more readily to DNA of active chromatin and nuclear matrix than to bulk chromatin . Since low concentrations of BPDE also selectively damage active chromatin and matrix DNA, selectivity is not due to the subnuclear location of enzymes which activate BP to BPDE . Higher BPDE concentrations cause more uniform DNA damage . Selective carcinogen attack may result from an accessible DNA conformation in active chromatin and matrix or from partitioning of carcinogen in the nuclear membrane. Exp Cell Res, 1986 Jun, 164(2), 379 - 87 Endonuclease banding of isolated mammalian metaphase chromosomes; Burkholder GD et al.; Evidence is presented that endonuclease digestion of isolated, unfixed chromosomes results in the production of banding patterns similar to those produced by digestion of fixed, air-dried chromosomes . Mouse L cell chromosomes were isolated under acidic or relatively neutral pH conditions, exposed in situ (as wet mounts on glass slides) or in vitro (in suspension) to micrococcal nuclease, Alu I or Eco RI, treated with a buffered salt solution, and stained with Giemsa . After any of these endonuclease treatments in situ, the centromeric regions of the chromosomes were intensely stained, characteristic of the C-banding observed in fixed chromosomes exposed to the same treatments . Although the fixed chromosomes were morphologically well-preserved after endonuclease digestion, the morphology of chromosomes digested in situ was variable, ranging from normal to swollen to highly distorted chromosomes . In the latter, the endonucleases induced dispersion of non-C-band chromatin; however, C-bands were still apparent as condensed, differentially-stained regions . Exposure of isolated chromosomes to Alu I in vitro also resulted in well-defined C-banding and led to the extraction of about 70% of the chromosomal DNA . From these results, the mechanism of endonuclease-induced C-banding appears to involve the dispersion and extraction of digested chromatin. Cell Biophys, 1986 Jun, 8(3), 177 - 88 DNA topology in a chromatin model system; Calascibetta FG et al.; The synthetic copolypeptide (Lys33, Leu67)100-Orn20, modeled on some general features of the histone sequences, has been found to supercoil the DNA double helix, wrapping it into a micelle, as a result of cohesive interactions between the polypeptide hydrophobic moieties . X-ray low-angle diffraction of complexes between the polypeptide and DNA is characterized by maxima at 50, 32, and 23 A, reminiscent of the chromatin pattern . The existence of a nucleosome-like structure along the DNA is suggested by gel electrophoresis analysis of DNA fragments after micrococcal nuclease digestion, showing the presence of a fragment of about 100 basepairs (bp) long . Topological experiments on the complexes with supercoiled as well as relaxed circular DNA by two-dimensional gel electrophoresis show the presence of left-handed superhelical turns . The results are in agreement with an intrinsic propensity of B-DNA to writhe into left-handed supercoils. Arch Pathol Lab Med, 1986 Jun, 110(6), 497 - 501 An unusual central nervous system infection in a young immunocompromised host; Ambler MW et al.; A 13-year-old girl with a ten-year history of lymphoblastic leukemia and several central nervous system (CNS) relapses developed a bone marrow relapse and accelerated CNS leukemia . Following treatment with CNS radiation and intravenous chemotherapy, she developed fever, pancytopenia, headache, and vomiting . Her neurological function deteriorated and she died on the 20th hospital day . Multiple CSF examinations failed to disclose either leukemic cells or organisms . Blood cultures obtained from a Broviac catheter yielded Micrococcus species . Postmortem examination showed meningoependymitis with intracellular coccal organisms . The pathology of this infection resembles intracranial Whipple's disease . Intracranial intracellular bacterial infection should be excluded in the infectious complications in the immunocompromised host. Biochim Biophys Acta, 1986 May 27, 867(1-2), 1 - 8 Helix-destabilization of deoxyribonucleic acid and poly{d(A-T).d(A-T)} by bovine seminal ribonuclease; Pandit MW et al.; A ribonuclease isolated earlier from bovine seminal plasma by DNA-affinity chromatography (Ramakrishnamurti, T . and Pandit, M.W . (1983) J . Chromatogr . 260, 216-222) has now been shown by thermal denaturation studies to destabilize the double-helical structure of DNA and poly{d(A-T).d(A-T)} . Thermal denaturation profiles of DNA in the presence of the protein are much more complicated due to the denaturation of protein itself in the temperature range over which DNA predominantly melts . The protein shows relatively stronger affinity towards denatured DNA as compared to native DNA . The action of micrococcal nuclease on DNA and its complexes with ribonuclease A and bovine seminal ribonuclease indicates that both of these proteins destabilize the double-helical structure of native DNA and thereby render the DNA more sensitive to the micrococcal nuclease. J Biol Chem, 1986 May 25, 261(15), 7044 - 51 Chromatin structure . Nuclease digestion profiles reflect intermediate stages in the folding of the 30-nm fiber rather than the existence of subunit beads; Walker PR et al.; Conditions have been found for the isolation of rat liver nuclei which maintain chromatin in its native state and suppresses endogenous nuclease activity . Chromatin prepared in this way can be dispersed into buffers containing various concentrations of monovalent or divalent cations so that the 30-nm fiber is either totally or partially decondensed . When probed with micrococcal nuclease, the digestion profiles show that although the 30-nm fiber can be cleaved periodically to generate superbead-like particles this only occurs under certain ionic conditions when the fiber is partially decondensed . It is likely that this cleavage pattern reflects the transient exposure of specific nuclease sensitive sites as the 30-nm fiber condenses, rather than the existence of a specific subunit of a beaded 30-nm fiber . The periodicity of these nuclease-sensitive sites appear to be related to the asymmetric distribution of histone H1 molecules along the length of the fiber. Cell, 1986 May 23, 45(4), 555 - 65 Chromatin assembly during SV40 DNA replication in vitro; Stillman B; A cytosol extract from human 293 cells supports efficient replication of SV40 origin-containing plasmid DNA in the presence of the SV40 T antigen . Addition of a nuclear extract from the same cells promotes negative supercoiling of the replicated DNA but not the bulk of the unreplicated DNA . The level of superhelicity is affected by the concentrations of T antigen and nuclear extract factors and by the time of addition of the nuclear extract . The replicated DNA in isolated DNA-protein complexes resists relaxation by purified HeLa cell topoisomerase I . Micrococcal nuclease digestion, sucrose gradient sedimentation, and electron microscopy demonstrate that the negative supercoils result from assembly of the replicating DNA into a chromatin structure . These results suggest that, during DNA replication, the core histones can be assembled on both sides of the replication fork by an active, replication-linked mechanism that does not require a template of preexisting nucleosomes. Cell, 1986 May 23, 45(4), 581 - 91 A small nuclear ribonucleoprotein associates with the AAUAAA polyadenylation signal in vitro; Hashimoto C et al.; RNAs containing the polyadenylation sites for adenovirus L3 or E2a mRNA or for SV40 early or late mRNA are substrates for cleavage and poly(A) addition in an extract of HeLa cell nuclei . When polyadenylation reactions are probed with ribonuclease T1 and antibodies directed against either the Sm protein determinant or the trimethylguanosine cap structure at the 5' end of U RNAs in small nuclear ribonucleoproteins, RNA fragments containing the AAUAAA polyadenylation signal are immunoprecipitated . The RNA cleavage step that occurs prior to poly(A) addition is inhibited by micrococcal nuclease digestion of the nuclear extract . The immunoprecipitation of fragments containing the AAUAAA sequence can be altered, but not always abolished, by pretreatment with micrococcal nuclease . We discuss the involvement of small nuclear ribonucleoproteins in the cleavage and poly(A) addition reactions that form the 3' ends of most eukaryotic mRNAs. Biochim Biophys Acta, 1986 May 5, 866(4), 233 - 41 Methylation of chromatin in vitro; Davis T et al.; The endogenous DNA methylase in nuclei isolated from growing mouse cells preferentially methylates DNA in micrococcal nuclease-resistant regions probably as a result of the location in these regions of the preponderance of hemimethylated sites . Added mouse ascites cell DNA methylase catalyses the methylation of exposed, nuclease-sensitive DNA in chromatin from growing or non-growing mouse or insect cells . The poor acceptor ability of nuclease-resistant regions in this situation is due to the presence of histone proteins which block de novo methylation . Transcriptionally active regions of chromatin are selectively methylated in vitro by either endogenous or added DNA methylase. J Biol Chem, 1986 May 5, 261(13), 5758 - 65 Periodic changes of chromatin organization associated with rearrangement of repair patches accompany DNA excision repair of mammalian cells; Mathis G et al.; We have used 8-methoxypsoralen to probe the chromatin structure of mammalian cells in situ while they repair pyrimidine dimers or bulky lesions in DNA . We observed that excision repair of these DNA lesions is accompanied by periodic alterations of chromatin organization . In parallel, fluctuations of the rates of repair patch synthesis accompanied these structural changes . Taking advantage of the accessibility of free DNA domains for psoralen intercalation, we have developed a technique to quantitatively isolate the micrococcal nuclease-sensitive, free DNA fraction of native bulk chromatin . We have determined the location of newly synthesized repair patches relative to free DNA domains as a function of repair time . Extensive rearrangements of repair patches from