Micrococci

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 these domains into micrococcal nuclease-resistant DNA were observed . Our results indicate that periodic changes of chromatin organization associated with rearrangement of repair patches accompany the process of excision repair in mammalian cells.

J Mol Biol, 1986 May 5, 189(1), 217 - 26
Fine analysis of the active H5 gene chromatin of chicken erythroid cells at different stages of differentiation; Renaud J et al.; We have analyzed the chromatin structure of a region that encompasses 14.4 X 10(3) base-pairs of the chicken histone H5 locus in adult erythroid cells at different stages of maturation . Seven of eight major lineage-specific DNase I-hypersensitive sites, some of which show complex substructure, were found in the flanking regions of the gene . The hypersensitivity of some of these sites is modulated during erythrocyte maturation in a way that parallels the transcriptional activity of the gene . DNase I, micrococcal nuclease, and S1 nuclease recognize the same regions, which differ from those cleaved by S1 on supercoiled plasmid DNA . This suggests that hypersensitivity of DNA in chromatin reflects a greater accessibility of the DNA rather than its altered conformation . The DNA sequence of some of the DNase I target sites contains repeated motifs, (T-C-C-C)2, (T-C-C)2, (T-G-G-G-G)2, which are found in the hypersensitive sites of other genes . Detailed analysis across sections of the H5 gene and flanking sequences revealed differences in the DNase I sensitivity of the different regions examined . Notably, the first one-third of the gene is more sensitive than the rest . The sequences downstream from the region where most RNA polymerases terminate transcription were found to be the most resistant.

Mol Biol (Mosk), 1986 May-Jun, 20(3), 853 - 60
{Structural transformations of oligonucleosomes from pigeon erythrocyte chromatin}; Osipova TN et al.; The methods of velocity sedimentation and circular dichroism have been used to investigate structural rearrangements of pigeon erythrocyte oligonucleosomes isolated after digestion with micrococcal nuclease (oligonucleosomes-M) or pancreatic DNase I (oligonucleosomes-D), in the wide range of ionic strength (mu from 0.005 to 0.5) . The electrophoretic analysis of DNA isolated from the oligonucleosomes has revealed internal cuts in the DNA chain of oligonucleosomes-D . In spite of this fact the conformational parameters of DNA in both types of oligonucleosomes are practically indistinguishable, and their optical and hydrodynamic properties vary in a similar way with increasing ionic strength of the solution . The specificity of DNase I action results in the ability of oligonucleosomes-D to form homogeneous associates at mu = 0.065, which seems to be due to the existence of elongated intact ends of linker DNA in oligonucleosomes-D . It has been shown that the integrity of oligonucleosomes-D in a wide range of ionic strength is maintained by histones H1 and H5, because after their dissociation the sedimentation coefficient sharply decreases . The results obtained reveal the multifunctional role of lysine-rich histones and intact linker in the processes of compaction and association of oligonucleosomes.

J Steroid Biochem, 1986 May, 24(5), 971 - 6
Physical-chemical properties of the estrogen receptor released by deoxyribonuclease I; Geier A et al.; The physical-chemical properties of the nuclear estrogen receptor released by DNase I were characterized . Nuclei were isolated from MCF-7 cells previously exposed to 10-nM-{3H}estradiol . The parameters determined were: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding ability to a DNA-cellulose column . The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values . The properties of the receptor released by DNase I obtained from Worthington were compared to the properties of the receptor released by DNase I obtained from Sigma . Digestion with DNase I (Worthington) excised a receptor form which could be solubilized from nuclei by EDTA . This form sedimented at 5.2S with a Rs = 7.08 nm and a calculated Mr = 152.000 . About 40% of this receptor form bound to a DNA-cellulose column . 0.4 M KCl dissociated this receptor form into a smaller form sedimenting at 4.2S with Rs = 4.64 nm and a calculated Mr = 80.000 . The properties of the receptor solubilized by micrococcal nuclease followed by DNase I (Worthington) digestion were identical to the properties of the DNase I (Worthington) released receptor . Digestion with DNase I (Sigma) released a 3.2S receptor form, which diffused through the nuclear membrane and a 4-5S form which could be extracted from nuclei by EDTA . The 3.2S receptor had a Rs = 2.41 nm, a calculated Mr = 32.000 and less than 5% of it bound to a DNA-cellulose column . Digestion with micrococcal nuclease followed by DNase I (Sigma) solubilized a receptor form with identical properties to the 3.2S receptor . These results suggest that DNase I (Worthington) released a receptor form still associated with some molecules, probably chromatin proteins, which complexed it to DNA, while DNase I (Sigma) released the estradiol binding fragment of the receptor (meroreceptor) as a result of a proteolytic activity present in this preparation.

Cell Biol Int Rep, 1986 May, 10(5), 339 - 45
Rat CNS neurons are not yet programmed to shorten their chromatin repeat length at the end of fetal neurogenesis; Savettieri G et al.; Neurons from rat fetal cerebral hemispheres were grown in a synthetic medium (Maat medium), as previously described, for different periods of time . The repeat length of their chromatin was determined by micrococcal nuclease digestion and compared with that of neurons isolated from postnatal rat brain of corresponding ages . In contrast to the in vivo situation, we found that neurons, dissociated at the 16th gestational day and cultured in vitro, did not undergo the shortening of their chromatin repeat, thus indicating that, at the end of their mitotic cycles, they are not yet programmed to this event.

Biochem Cell Biol, 1986 May, 64(5), 474 - 84
Protein-protein and protein-DNA interactions in calf thymus nuclear matrix using cross-linking by ultraviolet irradiation; Boulikas T; Nuclear matrices from calf thymus contained 30-50 protein species with one prominent band at 70 kilodaltons tentatively identified by its isoelectric point, apparent molecular weight, charge modification, and abundance as bovine lamin . The amount of DNA present in the matrix fraction was strongly dependent on the extent of digestion of the nuclei by micrococcal nuclease . The size of the DNA was higher than two kilobase pairs, although the chromatin DNA had been digested down to short oligonucleosomes . The lamin band was preferentially dissociated from isolated matrices during repeated treatment by 2 M NaCl or 5 M urea . Irradiation of calf thymus nuclear matrices at 313 nm induced protein-protein and protein-DNA cross-linking, as well as double-strand breaking of DNA, presumably at unprotected, protein-free regions . Lamin protein was more dramatically affected than other protein species by ultraviolet (UV) irradiation . In situ DNA hydrolysis, after the separation of the cross-linked matrix components on polyacrylamide-sodium dodecyl sulfate gels, followed by two-dimensional electrophoresis, showed lamin to be the major protein that was cross-linked to the DNA . Lamin molecules were also cross-linked by UV light to each other to form lamin homo-oligomers . A discrete size DNA fragment of approximately 450 base pairs is protected by lamin homo-oligomers from breakdown during UV irradiation . It is proposed that the direct contact between lamin and DNA found in this study is responsible for anchoring chromatin loops (domains) to a stable, immobile matrix structure.

Arch Biochem Biophys, 1986 May 1, 246(2), 690 - 8
Specific DNA alkylation damage and its repair in carcinogen-treated rat liver and brain; Wani AA et al.; The in vivo formation and repair of specific DNA lesions produced by alkylating agents of contrasting carcinogenic potencies were investigated . Male Sprague-Dawley rats were treated with direct-acting alkylating agents methylmethane sulfonate (MMS) or methylnitrosourea (MNU) . The amounts of N-3-methyladenine (3-meA), N-7-methylguanine (7-meG), and methylphosphotriesters (mePTE) in the DNA of liver and brain were determined following selective removal of the methylated bases by enzyme 3-meA N-glycosylase from Micrococcus luteus and thermal depurination at neutral pH . Both enzyme- and heat-induced alkali-labile apurinic sites were converted to single-strand breaks on incubation with 0.1 M NaOH . The number of such sites was quantitated following centrifugation of the DNA in alkaline sucrose gradients, fluorescent detection of unlabeled DNA, and estimation of number-average molecular weight . The results show a carcinogen dose-dependent initial linear increase in the number of enzyme- and heat-induced DNA strand breakage in both liver and brain DNA . With a half-life of approximately 3 h, 3-meA was removed from the tissues, whereas 45 to 55% of 7-meG remained unrepaired at 48 h . The study of the alkylation damage induced by MNU treatment of rats showed that the kinetics of repair for 3-meA and 7-meG was similar to the MMS-treated tissues and that mePTE persisted over a 7-day period . The technique developed does not require the use of radiolabeled reagents of DNA and allows for the selective quantitation of DNA alkylation lesions like 3-meA and 7-meG in the presence of nitrosourea-induced phosphotriesters.

Prikl Biokhim Mikrobiol, 1986 May-Jun, 22(3), 315 - 9
{Thermal inactivation and stabilization of lysozyme substrate-- Micrococcus lysodeicticus cells}; Tarun EI et al.; Heat inactivation of the acetonic powder of Micrococcus lysodeicticus cells suspended in phosphate buffer pH 6.2 was quantitatively characterized in the temperature range from 34 to 52 degrees . The total value of the rate constant for heat inactivation of the cells equals 2.88 X 10(8) exp(-18360/RT) sec-1 . The activation parameters of the process at 34 degrees are the following: delta H* = 17.7 kcal/mole; delta S* = 21.8 E . U.; delta F* = 24.4 kcal/mole . The effect of ethylene glycol, mannitol, dextran, polyvinyl alcohol (PVA) and polyethylene glycols with different molecular weights on the lysis rate and cell stability was studied . Polyvinyl alcohol was found to be the most effective stabilizer . At concentrations of about 10(-5) it enhances the thermostability of the cells threefold.

Virology, 1986 Apr 30, 150(2), 342 - 51
Adenovirus DNA synthesis in vitro is inhibited by the virus-coded major core protein; Korn R et al.; The effects of the adenovirus type 2 (Ad2) structural proteins on Ad DNA synthesis in vitro have been examined . Both of the viral core proteins, polypeptides V and VII were shown to inhibit Ad2 DNA synthesis in vitro; however, only the major core protein, polypeptide VII, inhibited DNA synthesis at a ratio of protein to DNA proportional to the number of polypeptide VII molecules associated with the Ad2 DNA in the mature virion . In addition, fractions containing the precursor to polypeptide VII, pVII, were capable of inhibiting Ad2 DNA replication in vitro to the same extent as polypeptide VII . Purified polypeptide VII bound to double-stranded DNA with no apparent sequence specificity . In addition, polypeptide VII protected Ad2 DNA from digestion with micrococcal nuclease . The binding of polypeptide VII was probably responsible for the inhibition of Ad2 DNA synthesis in vitro by virtue of rendering the DNA inaccessible to viral replication proteins . These results suggest that the core proteins must be removed from the Ad2 genome before the template can function in genome replication and that assembly of pVII on Ad2 DNA can terminate the replication process.

Biochemistry, 1986 Apr 22, 25(8), 2055 - 60
Assembly of active chromatin; Kumar S et al.; In eukaryotic cells, transcriptionally active chromatin and inactive chromatin exist in dissimilar configurations which correlate with their functional states . The present study asks whether the differences in structure and function of active and inactive chromatin are also reflected in the process of replication . We have used mild micrococcal nuclease digestion {Bloom, K.S., & Anderson, J.N . (1978) Cell (Cambridge, Mass.) 15, 141-150} to release selectively an active chromatin fraction, S1, from MSB cell nuclei . The S1 fraction is greater than 6-fold enriched in sequences complementary to polyadenylated RNA and thus consists of virtually pure active chromatin . As isolated, the S1 chromatin comprises mononucleosomes which contain approximately 160 base pairs of DNA bound by core histones but which are enriched in non-histones and free of histone H1 . Chemical cross-linking of S1 chromatin yields octamers containing the normal stoichiometry of core histones . When MSB cells are pulse labeled with isotopically dense amino acids and the recovered S1 chromatin is isolated and cross-linked, the labeled histone octamers are found in a single hybrid density octamer band on isopycnic gradients . These results indicate that, in contrast to the conservative assembly of transcriptionally inactive nucleosomes, the deposition of histones during the replication of active chromatin follows a nonrandom, semiconservative pathway.

Eur J Biochem, 1986 Apr 15, 156(2), 367 - 74
Different micrococcal nuclease cleavage patterns characterize transcriptionally active and inactive sea-urchin histone genes; Anello L et al.; The micrococcal nuclease cleavage sites have been mapped in the H2A coding and flanking regions of the sea-urchin histone DNA chromatin . A hypersensitive area, centered around - 100 base pairs from the H2A starting site, is found only in embryos actively transcribing the alpha-subtype histone genes . In mesenchyme blastula embryos, upon inactivation of the H2A gene, this region becomes protected while two other areas, near the transcription starting site and in the proximity of the 3' palindromic sequence, become preferential targets for the enzyme . Analysis of the pattern of micrococcal nuclease cleavage on the same region of the histone gene cluster in sperm and late blastula chromatin and on the corresponding segment of protein-free DNA indicates that distinct nucleosomal arrangements characterize the histone genes in the two cell populations.

FEBS Lett, 1986 Apr 7, 199(1), 89 - 91
Temperature-dependent cleavage of chromatin by micrococcal nuclease near the nucleosome center; Huang SY et al.; Digestion of nuclei at 4 degrees C with micrococcal nuclease results in significant intranucleosomal cleavage compared to digestion conducted at 37 degrees C . Employing nucleoprotein gel electrophoresis in one dimension followed by DNA electrophoresis in a second dimension, we demonstrate that such temperature-sensitive, internal cleavage predominantly occurs about 20 bp from the nucleosome center . We suggest that lower temperatures reduce the stability of hydrophobic interactions within the histone octamer and lead to a conformational alteration in nucleosomes that is detected by micrococcal nuclease.

Radiat Res, 1986 Apr, 106(1), 73 - 7
Repair of gamma-ray-induced DNA base damage in xeroderma pigmentosum cells; Fornace AJ Jr et al.; The repair of DNA damage produced by 137Cs gamma irradiation was measured with a preparation from Micrococcus luteus containing DNA damage-specific endonucleases in combination with alkaline elution . The frequency of these endonuclease sensitive sites (ESS) was determined after 54 or 110 Gy of oxic irradiation in normal and xeroderma pigmentosum (XP) fibroblasts from complementation groups A, C, D, and G . Repair was rapid in all cell strains with greater than 50% repair after 1.5 h of repair incubation . At later repair times, 12-17 h, more ESS remained in XP than in normal cells . The frequency of excess ESS in XP cells was approximately 0.04 per 10(9) Da of DNA per Gy which was equivalent to 10% of the initial ESS produced . The removal of ESS was comparable in XP cells with normal radiosensitivity and XP3BR cells which have been reported to be moderately radiosensitive.

Mol Cell Biol, 1986 Apr, 6(4), 1058 - 64
RNase P activity in the mitochondria of Saccharomyces cerevisiae depends on both mitochondrion and nucleus-encoded components; Hollingsworth MJ et al.; A requisite step in the biosynthesis of tRNA is the removal of 5' leader sequences from tRNA precursors . We have detected an RNase P activity in yeast mitochondrial extracts that can carry out this reaction on a homologous precursor tRNA . This mitochondrial RNase P was sensitive to both micrococcal nuclease and protease, demonstrating that it requires both a nucleic acid and protein for activity . The presence of RNase P activity in vitro directly correlated with the presence of a locus on yeast mitochondrial DNA previously shown by genetic and biochemical studies to be required for tRNA maturation . The product of the locus, the 9S RNA, and this newly described mitochondrial RNase P activity cofractionated, providing further evidence that the 9S RNA is the RNA component of yeast mitochondrial RNase P.

Proc Natl Acad Sci U S A, 1986 Apr, 83(8), 2387 - 91
Fractionation and characterization of a yeast mRNA splicing extract; Cheng SC et al.; We have fractionated a yeast whole cell extract that can accurately splice synthetic actin and CYH2 pre-mRNAs . Three fractions, designated I, II, and III, have been separated by use of ammonium sulfate fractionation and chromatography on heparin agarose . Each fraction alone has no splicing activity . Fractions I and II allow the first step of the splicing reaction to proceed, giving rise to the splicing intermediates, free exon 1, and intron-exon 2 . Addition of fraction III completes the reaction . Micrococcal nuclease treatment of the whole cell extract or of either fraction I or II abolished splicing activity, indicating that fractions I and II have RNA moieties that are required in the splicing reaction . The nature of the RNAs was examined using antibodies directed against the trimethylated cap structure unique to small nuclear RNAs . Preincubation of the whole cell extract with protein A-Sepharose coupled to trimethylated cap antibody abolished splicing activity . This indicates that at least one essential RNA component contains a trimethyl cap . Thus, in yeast as in mammalian systems, small nuclear RNAs are involved in mRNA splicing.

Proc Natl Acad Sci U S A, 1986 Apr, 83(8), 2315 - 9
Cell cycle-dependent expression of a stable episomal human histone gene in a mouse cell; Green L et al.; We have constructed a recombinant plasmid that includes a cell cycle-dependent human H4 histone gene with 650 base pairs of 5' and 900 base pairs of 3' flanking sequences and the 69% transforming fragment of bovine papilloma virus . When transfected into C127 mouse cells, this plasmid is maintained as a stable episome with approximately 20 copies per cell . Micrococcal nuclease digestion indicates that the episomal human histone gene is packaged as chromatin . The human H4 histone transcript is initiated at the correct 5' start site and undergoes selective destabilization when DNA synthesis is inhibited . When C127 cells containing the episomal H4 histone gene are synchronized, the human H4 histone mRNA levels are regulated coordinately with DNA replication and parallel those of transcripts from the murine chromosomal H4 histone genes . Our results suggest that the regulatory sequences and/or regulatory molecules associated with murine and human histone genes are compatible . The human histone gene-bovine papillomavirus episome is therefore a viable system for studying cell cycle-regulated histone gene expression under conditions where control is not influenced at the site of chromosomal integration by cis-acting elements of genes normally not contiguous.

Antimicrob Agents Chemother, 1986 Apr, 29(4), 598 - 601
Inhibition of Micrococcus luteus DNA gyrase by norfloxacin and 10 other quinolone carboxylic acids; Zweerink MM et al.; The ability of norfloxacin, amifloxacin, cinoxacin, ciprofloxacin, flumequine, nalidixic acid, ofloxacin (OFL), oxolinic acid, perfloxacin, pipemidic acid, and rosoxacin to inhibit the in vitro supercoiling activity of Micrococcus luteus DNA gyrase was compared with the ability of each drug to inhibit the growth of the M . luteus strain from which the gyrase was purified . The potency of the quinolones as DNA gyrase inhibitors did not always correlate with antimicrobial potency . For example, OFL was a less potent inhibitor of gyrase than rosoxacin, yet the MIC of OFL was 16-fold lower than that of rosoxacin . Similarly, the MICs of norfloxacin and ciprofloxacin (the most potent of the antibiotics tested in these assays) were several hundredfold lower than the MIC of nalidixic acid (the least potent of these antibiotics), but the inhibition of purified gyrase by these two quinolones was only 8- to 16-fold lower than that of nalidixic acid . These results suggest that factors in addition to inhibition of gyrase supercoiling activity are important in determining the potency of these drugs . Further studies indicated that the uptake of norfloxacin, OFL, and amifloxacin by M . luteus cells may not account for the large differences in MICs observed for these drugs (MICs of 0.8, 2.0, and 128 micrograms/ml, respectively).

Cancer Res, 1986 Apr, 46(4 Pt 1), 1703 - 6
Repair of ionizing radiation DNA base damage in ataxia-telangiectasia cells; Fornace AJ Jr et al.; Micrococcus luteus endonuclease sensitive sites were measured by alkaline elution in normal human and ataxia-telangiectasia (AT) fibroblasts after ionizing radiation . Due to the sensitivity of this assay, repair of base damage after 3 to 6 kilorads has been measured after oxic or hypoxic radiation . With 5.5 kilorads of oxic radiation, more than 50% of the base damage was removed after 1.5 h of repair incubation in all cells, including exr+ and exr- AT cells, and approximately 75% was removed by 4 h . After 3 or 4.5 kilorads of hypoxic X-irradiation, repair was equivalent in normal and exr- AT cells . This study included three exr- AT strains which have been reported to be deficient in the removal of gamma-ray base damage at higher doses . Since these strains repaired ionizing radiation base damage normally at lower doses, which are more relevant to survival, it is concluded that the X-ray hypersensitivity of AT cells is probably not related to the repair of base damage.

J Biol Chem, 1986 Mar 15, 261(8), 3838 - 45
Transcription termination and chromatin structure of the active immunoglobulin kappa gene locus; Xu M et al.; To investigate the chromatin surrounding an active gene, we have determined the distribution of RNA polymerase molecules, the intactness of nucleosomal structure, and the subnuclear compartmentalization along 15 kilobase pairs (kb) of the mouse kappa immunoglobulin locus of MPC-11 plasmacytoma cells . Hybridization of in vitro nuclear transcripts to probes specific for the template strand reveals that transcription terminates within the region between 1.1 and 2.3 kb downstream from the poly(A) addition site . Ten different short sequences (8-13 base pairs) reside within 460 base pairs of this termination region that exhibit homology with sequences found in the termination regions of mouse beta-globin and chicken ovalbumin genes . Transcription of the nontemplate strand occurs on either side of this termination region . We find that both within the transcription unit and 6.5 kb downstream of the termination region of the kappa gene, the canonical nucleosomal structure is perturbed, the chromatin exhibits pronounced insolubility, and the nucleosomes liberated by micrococcal nuclease appear to lack histone H1 . The insolubility is characterized by interactions that are disrupted by 0.3 to 0.6 M NaCl treatment . We conclude that the active chromatin phenotype spreads a considerable distance along the kappa locus, well beyond the region of transcription termination.

Nucleic Acids Res, 1986 Mar 11, 14(5), 2089 - 107
Methidiumpropyl-EDTA-iron(II) cleavage of ribosomal DNA chromatin from Dictyostelium discoideum; Parish RW et al.; We have used methidiumpropyl-EDTA-iron(II) {MPE.Fe(II)} in parallel with micrococcal nuclease to investigate the chromatin structure of the extrachromosomal palindrome ribosomal RNA genes of Dictyostelium . Confirming our earlier results with micrococcal nuclease (1,2), MPE.Fe(II) digested the coding region of rapidly transcribing rRNA genes as a smear, indicating the absence or severe disruption of nucleosomes, whereas in slowly transcribing rRNA genes, a nucleosomal ladder was produced . In the central non-transcribed spacer region of the palindrome, MPE.Fe(II) digestion resulted in a normal nucleosomal repeat, whereas micrococcal nuclease gave a complex banding pattern . The difference is attributed to the lower sequence specificity of MPE.Fe(II) compared to micrococcal nuclease . In the terminal region of the palindrome, however, both substances gave a complex chromatin digestion pattern . In this region the DNA appears to be packaged in structures strongly positioned with respect to the underlying DNA sequence.

Nature, 1986 Mar 6-12, 320(6057), 81 - 4
Removal of the Alu structural domain from signal recognition particle leaves its protein translocation activity intact; Siegel V et al.; Alu-like elements comprise the most abundant family of interspersed repetitive sequences in primates and rodents, and contain many features of processed genes, suggesting that they were initially derived by reverse transcription of processed RNA transcripts . Transcripts containing Alu family members are represented in heterologous nuclear RNAs, cytoplasmic messenger RNAs and small RNAs, although nothing is known about their function . Evolutionary studies strongly suggest that the parent RNA for the Alu-like elements is the highly conserved 7SL RNA, which is an essential component of signal recognition particle (SRP), a small cytoplasmic ribonucleoprotein whose function is the targeting of nascent secretory and membrane proteins to the rough endoplasmic reticulum (for a review see ref . 6) . 7SL RNA is composed of both unique and Alu-like sequences . SRP is rod-shaped and, in addition to its RNA, contains four proteins (two monomers composed of a polypeptide of relative molecular mass (Mr) 19,000 (19K) and one of 54K, and two heterodimers, one composed of a 9K and a 14K polypeptide, and the other composed of a 68K and a 72K polypeptide, respectively) . The RNA moiety is required for SRP activity, as well as for structural integrity of the particle . To investigate whether the Alu-like segments of 7SL RNA have a specific role in SRP activity, we have now purified and analysed a SRP subparticle that is created upon extensive digestion with micrococcal nuclease and entirely lacks the Alu-like sequences . We find that it contains the 72/68K, 54K and 19K proteins tightly bound, but lacks the 9/14K protein . In vitro activity assays demonstrated that the subparticle could still promote secretory protein translocation across the microsomal membrane, but could no longer trigger an arrest of pre-secretory protein synthesis . Re-addition of the 9/14K protein did not restore the elongation arrest . We conclude that the region of SRP comprised of the Alu-like RNA and the 9/14K protein exists in a distinct structural domain which is not required for the protein translocation promoted by SRP but apparently confers elongation-arresting activity on the particle.

Carcinogenesis, 1986 Mar, 7(3), 423 - 9
n-Butyrate alters chromatin accessibility to DNA repair enzymes; Smith PJ; Current evidence suggests that the complex nature of mammalian chromatin can result in the concealment of DNA damage from repair enzymes and their co-factors . Recently it has been proposed that the acetylation of histone proteins in chromatin may provide a surveillance system whereby damaged regions of DNA become exposed due to changes in chromatin accessibility . This hypothesis has been tested by: (i) using n-butyrate to induce hyperacetylation in human adenocarcinoma (HT29) cells; (ii) monitoring the enzymatic accessibility of chromatin in permeabilised cells; (iii) measuring u.v . repair-associated nicking of DNA in intact cells and (iv) determining the effects of n-butyrate on cellular sensitivity to DNA damaging agents . The results indicate that the accessibility of chromatin to Micrococcus luteus u.v . endonuclease is enhanced by greater than 2-fold in n-butyrate-treated cells and that there is a corresponding increase in u.v . repair incision rates in intact cells exposed to the drug . Non-toxic levels of n-butyrate induce a block to G1 phase transit and there is a significant growth delay on removal of the drug . Resistance of HT29 cells to u.v.-radiation and adriamycin is enhanced in n-butyrate-treated cells whereas X-ray sensitivity is increased . Although changes in the responses of cells to DNA damaging agents must be considered in relation to the effects of n-butyrate on growth rate and cell-cycle distribution, the results are not inconsistent with the proposal that increased enzymatic-accessibility/repair is biologically favourable for the resistance of cells to u.v.-radiation damage . Overall the results support the suggested operation of a histone acetylation-based chromatin surveillance system in human cells.

J Biochem (Tokyo), 1986 Mar, 99(3), 971 - 9
Effect of ionic strength on chain elongation in ADP-ribosylation of various nucleases; Ohashi Y; With the use of a reconstituted poly(ADP-ribosyl)ating enzyme system and three purified nucleases, micrococcal nuclease (MN), bull seminal RNase (BS RNase) and Ca2+, Mg2+-dependent endonuclease (BS DNase), as model acceptor proteins for ADP-ribose, the effect of ionic strength on the modification reaction was examined in detail . When these three nucleases were extensively poly(ADP-ribosyl)ated in this system at a low ionic strength (5 mM Tris), they were all inhibited by about 80% and the chain length of the polymer covalently bound to the nucleases was 13 to 23 ADP-ribose units . The observed inhibition was markedly prevented by increasing the ionic strength in the reaction mixture with a concomitant decrease in the polymer size bound to the nucleases . The NaCl concentrations required for decreasing the extent of the inhibition to half of the maximum were calculated to be 20, 50, and 100 mM for MN, BS RNase, and BS DNase, respectively . These values are similar to the NaCl concentrations required for decreasing the average chain lengths of the polymer to half, suggesting that the length of polymer is closely correlated to the extent of inhibition of these nucleases . DNA-binding affinities of these nucleases, expressed in terms of the NaCl concentrations required for eluting the enzymes from DNA-cellulose, were 140, 280, and 340 mM for MN, BS RNase, and BS DNase, respectively . Considering that maintainance of a ternary complex of poly(ADP-ribose) synthetase, acceptor and DNA may be essential for the modification reaction, the relatively strong salt effect observed in the modification of MN may be explained by its low DNA-binding affinity.

J Dairy Sci, 1986 Mar, 69(3), 633 - 42
Purification and characterization of extracellular caseinolytic enzyme of Micrococcus sp . MCC-315 isolated from cheddar cheese; Prasad R et al.; Micrococcus sp . MCC-315, an organism isolated from Cheddar cheese, produced an extracellular calcium metalloenzyme . This protease was purified to homogeneity from culture supernatant by precipitation with ammonium sulfate (50 to 70% saturation) and gel filtration through Sephadex G-100, resulting in about 82 times increase of specific activity and 53% recovery of the enzyme . The protease exhibited a pH optimum at 10.6 for both whole casein and beta-casein . It had optimum activity for whole casein in the presence and absence of calcium++ at 60 and 50 degrees C, respectively, and at 37 to 40 degrees C for beta-casein with or without calcium++ . The enzyme was stable at 45 degrees C but lost activity at higher temperatures . It was inhibited by heavy metal ions but calcium++, cobalt++, manganese++, strontium++, and iron++ had a slight stimulatory effect . The enzyme was inhibited completely and irreversibly by metal chelating agents . Calcium ions were required for maintenance of an active conformation of the enzyme . The enzyme had molecular weight of 28,900 and Michaelis constants 6.66 and 5.00 mg/ml for whole casein and beta-casein . Amino acid analysis of the hydrolyzed enzyme revealed the absence of sulfhydryl groups as was indicated also by lack of inhibition by thiol reagents.

J Biochem Biophys Methods, 1986 Mar, 12(3), 147 - 59
Sensitive quantitation of endonuclease kinetics; Rories CC et al.; Described here is an assay permitting exclusive quantitation of the kinetics of endonucleolysis by a mixed-function nuclease at substrate DNA concentrations much lower than those necessary with other assay methods . It makes possible determination of Michaelis parameters Km and kcat for endonuclease activity of such enzymes . The sensitivity of the assay method to very low DNA concentrations is obtained through use of 32P-labeled DNA as substrate . Digested DNA is electrophoresed, and computerized analysis of an autoradiogram made from the gel gives the extent of digestion . The analysis produces a profile of weight fraction vs . DNA fragment size for each sample taken from a nuclease-DNA reaction mixture . Each experimental profile is compared to a family of theoretical profiles generated by computer using theory of polymer statistics and assuming random cleavage . Theoretical profiles are found whose shapes most closely match those of the experimental profiles . Associated with each best-fitting theoretical profile is a value of the number of cuts made in the DNA sample . These values, plotted against time, give initial reaction velocities . Initial velocities from experiments at different DNA concentrations have been used to obtain Km and kcat for micrococcal nuclease under specific reaction conditions.

Mech Ageing Dev, 1986 Mar, 34(1), 23 - 34
Nuclease digestion of DNA and RNA in nuclei from young adult and senescent Caenorhabditis elegans (Nematoda); Meheus L et al.; Nuclei prepared from young adult and senescent Caenorhabditis elegans (Nematoda) were subjected to digestion by micrococcal nuclease and DNaseI . The kinetics of digestion of nuclei by micrococcal nuclease showed no change with age . There was, however, an age-related increase of acid-soluble deoxyribonucleotides released by DNaseI, suggesting that subtle alterations of chromatin conformation occur in aged nematodes . The ratio of nuclear RNA to DNA decreased and the nuclear RNA became more susceptible to enzymatic degradation as the worms grew old . These findings appear to indicate that nuclear RNA is less protected by protein in old nematodes . The decline of the nuclear RNA/DNA ratio with age is in good agreement with the generally accepted idea that there is a reduced level of RNA and protein synthesis in old animals.

Cell, 1986 Feb 28, 44(4), 535 - 43
DNA methylation affects the formation of active chromatin; Keshet I et al.; To study the mechanism of gene repression by DNA methylation, M13 gene constructs were methylated to completion and inserted into mouse L cells by DNA-mediated gene transfer . All unmethylated sequences, regardless of their source, integrated into the DNA in a potentially active DNAase I-sensitive conformation . Total CpG methylation prevented the formation of this structure and rendered these sequences DNAase I-insensitive over the entire methylated domain . Whereas unmethylated DNA demonstrated additional conformational features of active genes, such as DNAase I hypersensitivity and restriction endonuclease-sensitive segments, these markers were not present when methylated DNA was used for transfection . The use of micrococcal nuclease to probe for active or inactive supranucleosome particles also showed that DNA methylation directs DNA into an inactive type of structure . The results suggest that DNA methylation may exert its effect on gene transcription by altering both specific and nonspecific interactions between DNA and nuclear proteins.

Nucleic Acids Res, 1986 Feb 25, 14(4), 1667 - 82
The beta-globin domain in immature chicken erythrocytes: enhanced solubility is coincident with histone hyperacetylation; Nelson DA et al.; A 60 minute exposure of chicken immature erythrocytes to n-butyrate shifts actively acetylated and deacetylated histones to hypermodified forms . Micrococcal nuclease digestion of nuclei from n-butyrate treated cells and subsequent fractionation of the chromatin releases 40-45% of the adult beta-globin (beta A) nucleohistone into a soluble fraction . This is an eleven fold enrichment over the soluble chromatin from untreated cells (Ferenz and Nelson (1985) Nucleic Acids Res . 13, 1977-1995) . The enhanced beta A chromatin solubility and induced histone hyperacetylation are coincident . Removal of n-butyrate from the cell incubation medium allows rapid histone deacetylation and a striking reduction in beta A chromatin solubility . Chromatin from cells incubated in the absence of n-butyrate, or in medium containing 10 mM NaCl or 2% dimethylsulfoxide, does not exhibit histone hyperacetylation, or the acquired solubility of beta A chromatin . We show that the H4 histone co-isolated with the beta A DNA is in a hyperacetylated state and present evidence that the n-butyrate incubation increases the solubility of both coding and noncoding chromatin regions in the beta-globin domain.

J Mol Biol, 1986 Feb 20, 187(4), 591 - 601
Roles of H1 domains in determining higher order chromatin structure and H1 location; Allan J et al.; Peptides derived from calf thymus H1 and rat liver H1, comprising only the globular and COOH-terminal domains of the intact molecule and therefore lacking NH2-terminal domains, have been shown by reconstitution to be as effective as the complete H1 molecule in inducing higher-order-chromatin structure . As the globular domain of H1 alone cannot induce chromatin folding, our results demonstrate that this function is primarily controlled by the COOH-terminal domain of the molecule . Surprisingly, these peptides do not locate correctly with respect to the nucleosome . This is demonstrated by their failure to confer upon reconstitutes the ability to protect DNA fragments of chromatosome length when digested with micrococcal nuclease . The precise placement of the H1 molecule (globular domain) with respect to the nucleosome is shown to be influenced by the "tail" domains of both H1 and the core histones.

Biochem Int, 1986 Feb, 12(2), 303 - 11
ADP-ribosylation induced changes in the conformation of the chromatin of the brain of developing rats; Das BR et al.; Conformational changes in the chromatin of the cerebral hemisphere of 3-, 14- and 30-day old developing rats were studied before and after its ADP-ribosylation using DNase I and micrococcal nuclease (MNase) . The rate and extent of digestion of chromatin by DNase I are the highest at 3-day and decline progressively thereafter . The rate and extent of digestion by MNase do not change during development . ADP-ribosylation of chromosomal proteins was carried out by incubating nuclei with NAD+ for 30 min and was followed by endonuclease digestion . Both the rate and extent of digestion by DNase I and MNase were enhanced after ADP-ribosylation which was the maximum for 3-day rats.

Mol Cell Biochem, 1986 Feb, 69(2), 169 - 78
Mannose incorporation and lectin recognition of pronase-sensitive components in Micrococcus lysodeikticus (M . luteus) membranes; Guerrero A et al.; Isolated cytoplasmic membranes from Micrococcus lysodeikticus were able to incorporate {14C}mannose from GDP-{14C}mannose . Labelled mannose remained in the membrane fraction after its repeated washing and lipid extraction . Sodium dodecyl sulfate gel electrophoresis in 12% acrylamide showed a set of bands with molecular weights ranging from 230 000 to 19 000 which stained for protein and carbohydrate, and incorporated {14C}mannose . Some of these bands reacted with different lectins (concanavalin A, wheat germ agglutinin and ricin) . Furthermore, the mannose was incorporated via a glycosylation pathway similar to that followed in eukaryotic system as shown by the preliminary identification of a lipid intermediate transferring the sugar to proteins and by the differential sensitivity to bacitracin and tunicamycin . These complex membrane components were sensitive to digestion with pronase . All the results presented suggest their glycoprotein nature.

EMBO J, 1986 Feb, 5(2), 293 - 300
Digestion of the chicken beta-globin gene chromatin with micrococcal nuclease reveals the presence of an altered nucleosomal array characterized by an atypical ladder of DNA fragments; Sun YL et al.; The structure of the chicken adult beta-globin gene chromatin in immature and mature erythrocyte nuclei has been analysed using micrococcal nuclease digestion . The resulting DNA fragments were blotted onto DBM-papers and probed with labelled DNA fragments spanning the adult beta-globin gene and its 5'- and 3'-flanking regions . The structure of the nucleosomes within and in the regions flanking the adult beta-globin gene appears to be altered in at least two ways in erythrocyte chromatin, when compared with either bulk or inactive ovalbumin gene chromatin . First, oligomeric DNA fragments containing the beta-globin gene are released faster than those of either bulk or ovalbumin gene chromatin . Second, although the difference in size of the liberated oligomeric DNA fragments is similar to the nucleosomal repeat length of bulk and ovalbumin gene chromatin, the individual oligomers are approximately 100 bp shorter than their bulk or ovalbumin gene counterparts, most noticeably when the nuclease digestion is performed at 37 degrees C . This results in an atypical ladder of approximately 300, 500, 700, 900 bp instead of the canonical chicken erythrocyte ladder which is an integral multiple of 207 bp . The same ladder was obtained from immature erythrocytes, in which the beta-globin gene is actively transcribed, and from mature erythrocytes, in which it is considered to be inactive with RNA polymerase molecules clustered in the 5' moiety of the gene . This indicates that the alteration of the nucleosomal structure is not due to transcription per se.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Genet, 1986 Feb, 24(1-2), 79 - 92
Satellite DNA-correlated nucleosomal proteins in Drosophila virilis; Viglianti GA et al.; Three major satellite DNAs comprise 40-45% of the genome of Drosophila virilis . Since these satellites are not substrates for most restriction enzymes, we were able to digest D . virilis nuclei with HaeIII and micrococcal nuclease and isolate chromatin fractions containing variable levels of satellite DNA . Electrophoretic analysis of these chromatin fractions revealed that the level of the acid-soluble chromosomal protein, cp17.3, was directly related to the percentage of satellite DNA in chromatin . The correlation between cp17.3 and satellite DNA abundance suggests that cp17.3 is involved in the heterochromatic condensation of satellite DNAs . cp17.3 occurs at a frequency of one molecule per 10-20 nucleosomes . It is detected in an electrophoretically distinguishable class of mononucleosomes, provisionally identified as MN1uH2A, which contains ubiquitinated histone H2A (uH2a) but lacks histone H1 . It is not detected in MN1, a second class of mononucleosomes, which lacks uH2A and H1 . Since cp17.3 is correlated with satellite DNAs and present in nucleosome cores, it might be a histone variant specifically associated with satellite DNAs.

Virology, 1986 Jan 30, 148(2), 375 - 80
Characterization of vaccinia virus transcripts involved in selective inhibition of host protein synthesis; Bablanian R et al.; Vaccinia virus transcripts synthesized in vitro inhibit protein synthesis directed by globin mRNA, EMC RNA, and total cytoplasmic RNA isolated from HeLa and CHO cells in micrococcal nuclease-treated reticulocyte lysates . In contrast, RNA isolated from vaccinia virus-infected cells is efficiently translated in the same system in the presence of the in vitro transcripts (G . Coppola and R . Bablanian (1983), Proc . Natl . Acad . Sci . USA 80, 75-79) . In this study, we have fractionated the in vitro transcripts by acid-urea-agarose gel electrophoresis and demonstrated that the smallest RNA fraction inhibited translation of HeLa cell mRNA in vitro but had no effect on vaccinia virus mRNA translation . On the other hand, the larger RNA species inhibited nonselectively . These results indicate that the selective inhibitory activity of vaccinia virus transcripts resides only in the small poly(A)-containing in vitro RNA fraction.

Biochemistry, 1986 Jan 28, 25(2), 495 - 502
Chromatin structure of the chicken lysozyme gene domain as determined by chromatin fractionation and micrococcal nuclease digestion; Stratling WH et al.; The chromatin structure encompassing the lysozyme gene domain in hen oviduct nuclei was studied by measuring the partitioning of coding and flanking sequences during chromatin fractionation and by analyzing the nucleosome repeat in response to micrococcal nuclease digestion . Following micrococcal nuclease digestion, nuclei were sedimented to obtain a chromatin fraction released during digestion (S1) and then lysed in tris(hydroxymethyl)aminomethane-(ethylenedinitrilo)tetraacetic acid-{ethylenebis(oxyethylenenitrilo)}tetraacetic acid and centrifuged again to yield a second solubilized chromatin fraction (S2) and a pelleted fraction (P2) . By dot-blot hybridization with 14 specific probes, it is found that the fractionation procedure defines three classes of sequences within the lysozyme gene domain . The coding sequences, which partition with fraction P2, are flanked by class I flanking sequences, which partition with fractions S1 and P2 and which extend over 11 kilobases (kb) on the 5'side and probably over about 4 kb on the 3' side . The partitioning of class II flanking sequences, which are located distal of class I flanking sequences, is different from that of class I flanking sequences . Coding sequences lack a canonical nucleosome repeat, class I flanking sequences possess a disturbed nucleosome repeat, and class II flanking sequences generate an extended nucleosomal ladder . Coding and class I flanking sequences are more readily digested by micrococcal nuclease than class II flanking sequences and the inactive beta A-globin gene . In hen liver, where the lysozyme gene is inactive, coding and class I flanking sequences fractionate into fractions S2 and P2.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1986 Jan 25, 261(3), 1445 - 52
Chromatin structure of the human dihydrofolate reductase gene promoter . Multiple protein-binding sites; Shimada T et al.; The chromatin structure of the promoter region of the human dihydrofolate reductase gene was determined using a variety of nucleases including DNase I, micrococcal nuclease, several restriction endonucleases, exonuclease III, and Bal31 . Two separate regions from -670 to -340 (the distal hypersensitive region) and from -170 to +150 (the proximal hypersensitive region) were shown to be essentially free of proteins as indicated by their accessibility to both endo- and exonucleases . Within the proximal hypersensitive region, one protein appears to be bound at the start site for transcription . A 170-base pair fragment between the two hypersensitive regions was highly resistant to all nucleases tested . Multiple barriers against exonuclease digestion and resistance to dissociation by high salt concentrations suggest that more than one protein is tightly bound to this region . The upstream sequence from -670 and the downstream sequence from +150 were shown to be packaged into nucleosomes . The selective accessibility of certain sites to micrococcal nuclease cutting indicates that the initial nucleosomes are phased upstream from the distal hypersensitive region . There appears to be a protein bound between the phased nucleosomes and the upstream boundary of the distal hypersensitive region . These results suggest that the normal nucleosome array is interrupted by about 900 base pairs of nucleosome-free DNA to which several nuclear proteins bind in a DNA sequence-specific manner.

Mol Biol Rep, 1986, 11(3), 143 - 7
Study of chromatin structure in ataxia-telangiectasia cells; Houldsworth J et al.; Micrococcal nuclease was used as a probe to study chromatin structure in control and ataxia-telangiectasia cells . The rate and extent of release of acid-soluble nucleotide was similar in both cell types . Production of mono- and oligonucleosomes by micrococcal nuclease as determined by gel electrophoresis also failed to reveal differences in chromatin structure between control and ataxia-telangiectasia cells . Radiation exposure did not significantly alter the kinetics of digestion . These results indicate that there are no gross alterations in chromatin structure in ataxia-telangiectasia cells.

J Invest Dermatol, 1986 Jan, 86(1), 34 - 6
Higher pyrimidine dimer yields in skin of normal humans with higher UVB sensitivity; Freeman SE et al.; We have measured UVB (280-320 nm)-induced DNA damage in skin of individuals with different sensitivities to UVB irradiation as measured by minimal erythema dose (MED) . The DNA damage was susceptible to cleavage by Micrococcus luteus UV endonuclease, which recognizes pyrimidine dimers in DNA . An alkaline agarose gel electrophoresis method was used to quantitate the number of M . luteus UV endonuclease-sensitive sites in nonradioactive DNA from skin biopsies of 7 individuals irradiated with UVB (0-180 mJ X cm-2) . The production of sites correlated well with MED (correlation coefficient = 0.78) . The slope of the dose response curve for the most UVB-sensitive individual (MED = 24 mJ X cm-2) and for the least UVB-sensitive individual (MED = 146 mJ X cm-2) were 11.5 X 10(-4) and 2.6 X 10(-4) sites per 1000 bases per mJ X cm-2, respectively . The UVB-induced DNA damage was determined to be pyrimidine dimers by its susceptibility to cleavage by M . luteus UV endonuclease and its photoreactivability by Escherichia coli photoreactivating enzyme.

Toxicon, 1986, 24(6), 622 - 5
Biological properties of Pyrularia thionin prepared from nuts of Pyrularia pubera; Evett GE et al.; Pyrularia thionin is a 47 amino acid basic peptide which resembles wheat purothionin and mistletoe viscotoxin . It is toxic to mice, with an LD50 of 1.5 mg/kg body weight and is cytotoxic to tumor and normal cells in culture, with ID50 values ranging from 0.62 to 17 micrograms/ml . Against seven bacterial species it was toxic only to Micrococcus luteus . The toxin did not protect mice against transplanted B16 melanoma, nor did it show any mitogenic activity with mouse spleen lymphocytes.

Mol Biol Rep, 1986, 11(2), 63 - 8
In vitro ADP-ribosylation of chromosomal proteins of the brain of developing rats; Das BR et al.; In vitro ADP-ribosylation of chromosomal proteins and its modulation by spermine, 3-aminobenzamide (3-AB) and benzamide were studied by incubating the nuclei of cerebral hemisphere of 3-, 14- and 30-day old rats with 32P-NAD+ . Histones get ADP-ribosylated more than the non-histone chromosomal (NHC) proteins . H1 is the major target for ADP-ribosylation . Among the nucleosomal histones, H2B is ADP-ribosylated most . The other core histones also get ADP-ribosylated to a lesser extent . ADP-ribosylation of both histones and NHC proteins decreases during development . Spermine stimulates, whereas 3-AB and benzamide inhibit, 32P-ADP-ribose incorporation into histones and NHC proteins . These effects decrease with development . Mild digestion of chromatin by micrococcal nuclease (MNase), EcoRI and AluI prior to ADP-ribosylation stimulates incorporation of 32P-ADP-ribose . The degree of stimulation decreases as development proceeds . Such alterations indicate progressive condensation of chromatin with development.

Chromosoma, 1986, 93(6), 515 - 20
Organization within the mammalian kinetochore; Rattner JB; The organization within the mammalian kinetochore was examined using whole-mount electron microscopic techniques on chromosomes digested with restriction enzymes or micrococcal nuclease . These preparations revealed that a portion of the kinetochore is highly resistant to nuclease digestion and can be visualized as a discrete structure . The relationship of this structure to the remainder of the chromosome suggests that it represents the outer kinetochore plate . The plate is composed of a series of fibrillar loops that are arranged in a parallel array along the plane of the plate . These fibers are 25-30 nm in diameter . The morphology, particulate substructure, and ultimate susceptibility to nuclease digestion suggest that these fibers contain DNA . A model is presented that suggests that the outer plate contains the apexes of chromatin loops that originate within the body of the primary constriction.

Anim Genet, 1986, 17(1), 47 - 59
Some properties of the lysozymes in serum and colostrum from cows with high and low lytic power against Micrococcus lysodeikticus; Lie O et al.; Previous work has uncovered a dominant gene for high bacteriolytic activity of bovine serum against the test bacterium Micrococcus lysodeikticus . This major gene effect is also fully expressed in colostrum . In the present study the lytic power of serum and colostral whey from high and low level cows was subjected to a degree of characterization . It was found that the enzyme activities studied exhibited properties in accordance with those defined for a lysozyme (EC 3.2.1.17), i.e . (1) lysis of a suspension of M . lysodeikticus, (2) basic protein (pI = 10.0 and pI = 10.3 for bovine serum lysozyme (BSL) and bovine colostrum lysozyme (BCL), respectively), (3) molecular weight (MW) approximately 16 000 for both BSL and BCL, (4) liberation of free reducing sugars during action on cell wall peptidoglycan (the kinetics of BSL and BCL differed strongly), and (5) fairly heat stable, especially at acidic pH and relative labile at alkaline pH (BCL was far more sensitive to heating at alkaline pH than was BSL) . The dramatic differences in activity between high and low level animals might be due to a major genetic mechanism influencing the amount of, or the activity of, circulating enzyme molecules, rather than a structural gene coding for a certain enzyme with a particular specific activity . This is also supported by the high correlation between the lytic capacity of BSL and BCL in spite of the different properties of these lysozymes (i.e . in respect of pI, enzyme kinetics and heat stability) reported in the present study.

Adv Enzyme Regul, 1986, 25, 87 - 97
Alkylating antitumor agents reduce histone acetyl-transferase activity; Grunicke H et al.; N-Mustard depresses the acetylation of histones in Ehrlich ascites and Walker carcinoma cells . It is demonstrated that this effect is not caused by an accelerated deacetylation but is due to an inhibition of the acetyl-transferase reaction . Employing 4-sulphonatoethylthio-cyclophosphamide it is demonstrated that the alkylating agent affects predominantly the acetylation of a chromatin fraction which is soluble in 0.1M NaCl after digestion with micrococcal nuclease . After removal of the alkylating agent, the recovery of histone acetylation is relatively slow and--in contrast to the repair of DNA cross-links--characterized by a 4-hr lag period . The reduction of histone acetylation by N-mustard is much less expressed in cells which are resistant to the drug than in the sensitive parental lines . This is in contrast to DNA-interstrand cross-links in Walker cells where both N-mustard sensitive and resistant cells inhibit the same cross-link frequency and identical repair rates . Based on these data it is concluded that the inhibition of histone acetylation may be an important part of the mechanism by which alkylating agents inhibit tumor growth.

Mol Biol Rep, 1986, 11(4), 231 - 6
Synthesis of somatomedin C/insulin-like growth factor I by human placenta; Mills NC et al.; We have reported the presence of insulin-related poly A+ RNA sequences in human placenta by RNA to DNA hybridization . In this study we have used a monoclonal antibody to somatomedin C/insulin-like growth factor I (Sm-C/IGF-I) to identify somatomedin-like proteins whose synthesis is directed by placental mRNA . Poly A+ RNA from first trimester and term placenta was translated in a cell-free system using micrococcal nuclease-treated reticulocyte-lysate and {35S}methionine as a label . From 2.0 X 10(6) cpm of specifically incorporated {35S}methionine labeled protein, an immunoprecipitate with an apparent molecular weight of 14,000 represented about 0.1% of total radioactivity in the translational products of poly A+ RNA of first trimester placenta . A less prominent band (0.006%) of the same apparent molecular weight was also evident from translational products of term placental mRNAs . This protein could be competed with either acromegalic serum or synthetic Sm-C/IGF-I when added prior to immunoprecipitation . Translational products synthesized from mRNA of term placenta showed a second labeled band of 24,000 daltons . This band was less effectively competed by acromegalic serum and not competed with either Sm-C/IGF-I or IGF-II and therefore its identity is uncertain . A protein similar to Sm-C/IGF-I is, therefore synthesized in first trimester placenta and to a lesser extent at term, suggesting developmental changes in Sm-C/IGF-I synthesis . Because Sm-C/IGF-I may act in a paracrine fashion, our findings suggest a role for Sm-C/IGF-I in growth of the placenta during early gestation.

Comp Biochem Physiol B, 1986, 83(4), 819 - 22
Bacteriolytic factor in the salivary glands of Aedes aegypti; Rossignol PA et al.; Salivary gland homogenates from adult Aedes aegypti lyse Micrococcus lysodeikticus cells . The bacteriolytic factor is present in a cell type common to both male and female mosquitoes, as well as in the crop of sugar-feeding mosquitoes . The bacteriolytic factor releases digestion products from sacculi of Escherichia coli that are different from those of hen egg white lysozyme.

Appl Environ Microbiol, 1986 Jan, 51(1), 88 - 90
Simple method to demonstrate radiation-inducible radiation resistance in microbial cells; Tan ST et al.; A simple method for detection of radiation-inducible radiation resistance was developed by irradiating aliquots (0.01 ml) of cell suspension on agar plates . Part of each experimental plate was subjected to an induction treatment, and subsequent radiation resistance was compared with that of untreated cells on the same plate . The UV radiation resistance of a Micrococcus sp . was increased approximately 1.6 times by an induction treatment . This simple procedure of irradiating cells in a "fixed" position on agar avoided washing, centrifugation, and cell enumeration required in traditional methods.

Curr Genet, 1986, 10(5), 411 - 20
Two dimensional polyacrylamide gel electrophoresis analysis of Tetrahymena mitochondrial tRNA; Suyama Y; Two dimensional (2D) urea-polyacrylamide gel electrophoresis of tRNA isolated from Tetrahymena mitochondria separated at least 36 spots, while more than 45 major and minor spots were resolved with cytosolic tRNA . Co-electrophoresis of mitochondrial and cytosolic tRNAs revealed that many spots co-migrate . When radioactive mitochondrial tRNA was hybridized to mtDNA under various conditions and tRNA melted from the hybrid was analyzed by 2D gel electrophoresis, only 10 tRNA spots were found . Identified as mtDNA-encoded were 2 spots for tRNA(leu), 2 for tRNA(met), and 1 each for tRNA(phe), tRNA(trp) and tRNA(tyr) . The remaining three were unidentified . Mitochondrial tRNA spots that correspond to the tRNAs for arg, gly, ile, lys, ser, and val do not hybridize with mtDNA, and in gel positions they correspond to the cytoplasmic tRNA spots for the same respective amino acids . These mitochondrial tRNAs isolated from the gel can be acylated either by the mitochondrial or cytosolic enzymes . Mitochondrial tRNA isolated from a Tetrahymena cell homogenate which was pretreated with RNase A and Micrococcus nuclease exhibited the same 2D gel pattern as a non-treated control . Mitochondrial tRNAs from old and young cells showed generally similar tRNA spots in 2D gels, though more variable spots were seen with old cells . 3H-labeled whole-cell tRNA added to the cell homogenate prior to the mitochondrial isolation procedure did not remain associated with the final mitochondrial tRNA preparation . The present studies also showed mitochondrial tRNAs bound to the mitochondrial 80S monosome and polysome fractions . Radioactive tRNA added to the mitochondrial lysate does not adhere to the ribosomes, suggesting that the ribosome-bound tRNAs are not contaminating cytoplasmic tRNAs . These results are generally in good agreement with our previous data showing that only a small number of tRNAs are coded for by the mitochondrial DNA, while the others are a selected set of imported cytoplasmic tRNAs.

J Biochem (Tokyo), 1986 Jan, 99(1), 91 - 8
Nucleosome core particles of calf thymus, Tetrahymena, and the reconstituted hybrid . Their structure reflects the nature of the histone octamer; Kasai K et al.; The circular dichroism spectra and the thermal denaturation profiles of the nucleosome core particles isolated by micrococcal nuclease digestion from nuclei of calf thymus and the protozoan Tetrahymena pyriformis were compared with those of the homogeneous and hybrid core particles reconstituted from calf core DNA and either calf or Tetrahymena histone octamer . The core DNA was obtained from the calf core particle, and both the histone octamers were reconstituted from the acid-extracted four core histones of calf thymus or Tetrahymena, whose amino acid sequences show the largest differences hitherto known . The reconstituted homogeneous core particle was identical in both the physical properties with the isolated calf core particle, showing that the correct reconstitution was achieved . The circular dichroism spectra of the calf and Tetrahymena core particles and the hybrid core particle showed no essential differences, indicating that the three core particles have the same overall structure . The derivative thermal-denaturation profiles, however, clearly differed; the calf core particle showed two melting transitions at 60 degrees C and 72 degrees C, while the Tetrahymena and hybrid core particles showed the same three transitions at 48-50 degrees C, 60-61 degrees C, and 72 degrees C . Thus, the thermal denaturation properties of nucleosome core particles do not reflect the nature of DNA, but rather that of the histone octamer bound to the DNA . We conclude that the Tetrahymena histones are more weakly bound to the DNA than the calf thymus histones in the same overall structure of nucleosomes.

Carcinogenesis, 1986 Jan, 7(1), 83 - 7
Quantitation of carcinogen-induced DNA damage and repair in human cells with the UVR ABC excision nuclease from Escherichia coli; Van Houten B et al.; Recent studies by others have shown that the endonuclease complex coded for by the uvrA, uvrB and uvrC genes of Escherichia coli (UVR ABC excision nuclease) can incise DNA containing a variety of 'bulk-type' lesions, such as those resulting from u.v . light, (+/-)-7 alpha,8 beta-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene (anti-BPDE), and N-acetoxy-2-acetylaminofluorene . Using partially purified UVR ABC excision nuclease, we have quantitated the number of endonuclease sensitive sites (ESS) in purified DNA isolated from human fibroblasts treated with u.v . light or BPDE . The number of ESS/10(8) daltons of DNA were calculated from the number average mol . wt . of the DNA as determined by sedimentation in alkaline sucrose gradients . The number of endonuclease sites increased linearly with increasing doses of either u.v . light or BPDE . The UVR ABC excision nuclease was able to incise a majority of the BPDE-DNA adducts . Xeroderma pigmentosum fibroblasts, complementation group A (XP12BE) had 20-25% more ESS at each dose than the BPDE-treated normal (HSBP) cells . Cells treated with 4 microM BPDE and allowed 12 h of incubation to perform excision repair showed removal of 60% of the initial number of ESS from HSBP DNA and 40% of the ESS from XP-A DNA . Beyond 12 h XP12BE cells lost no additional ESS while HSBP cells continued to lose ESS, although at a slower rate, until at 48 h only 22% of the initial ESS remained . In cells treated with 10 J/m2 of u.v . light, the UVR ABC excision nuclease detected 60% of the sites recognized by the pyridimine dimer specific Micrococcus luteus glycosylase/apyrimidinic endonuclease . These results demonstrate the potential use of the UVR ABC excision nuclease in a quantitative assay for determining the number of carcinogen-induced lesions in human DNA.

Chemotherapy, 1986, 32(6), 494 - 8
Discrepancy between the antibacterial activities and the inhibitory effects on Micrococcus luteus DNA gyrase of 13 quinolones; Fu KP et al.; Thirteen quinolone antibacterial agents were investigated as to their ability to inhibit Micrococcus luteus DNA gyrase and cell growth, and compared to those of novobiocin and coumermycin . Among the quinolones tested, the most active were found to be CI-934 and ciprofloxacin, which inhibited gyrase full supercoiling activity at concentrations of 100 and 200 micrograms/ml, respectively, while inhibiting cell growth at a concentration of 1 microgram/ml . However, both novobiocin and coumermycin inhibited gyrase full supercoiling activity at concentrations of 0.5 and 1.0 microgram/ml, respectively, which were comparable to those concentrations causing inhibition of cell growth.

Gene, 1986, 43(1-2), 139 - 46
Cloning of Micrococcus luteus 3-methyladenine-DNA glycosylase genes in Escherichia coli; Pierre J et al.; The 3-methyladenine-DNA glycosylase (m3ADG) excises 3-methyladenine (m3A) residues formed in DNA after treatment with alkylating agents . In Escherichia coli, the repair of this type of damage depends on the products of the genes tagA and/or alkA, which code for m3ADG I (20 kDa) and II (30 kDa), respectively . The tagA- and alkA--single mutants are sensitive to alkylating agents, the double mutant much more so . We have cloned two genes of Micrococcus luteus that can partly substitute the function of the E . coli tagA- and alkA- genes . An M . luteus genome bank was made by shotgun cloning of EcoRI + BamHI-digested DNA into pBR322 . Two hybrid plasmids were identified that confer methylmethane sulfonate (MMS) resistance to the tagA- ada+ mutant and a capacity to reactivate MMS-treated bacteriophage lambda . Each hybrid plasmid directed the synthesis of 21-kDa m3ADG in E . coli tagA- ada-, which were not inhibited by 4 mM m3A . However, the restriction maps of the two cloned genes were different, and they showed no sequence homology as judged by the lack of cross hybridization.

CRC Crit Rev Biochem, 1986, 21(1), 1 - 26
Structure of transcriptionally active chromatin; Yaniv M et al.; Transcriptionally active or potentially active genes can be distinguished by several criteria from inactive sequences . Active genes show both an increased general sensitivity to endonucleases like DNase I or micrococcal nuclease and the presence of nuclease hypersensitive sites . Frequently, the nuclease hypersensitive sites are present just upstream of the transcription initiation site covering sequences that are crucial for the promoter function . Viral or cellular transcription enhancer elements are also associated with DNase I hypersensitive sites . At least for the SV40 enhancer, it was shown by electronmicroscopic studies that the DNase I hypersensitive DNA segment is excluded from nucleosomes . It is highly plausible that the binding of regulatory proteins to enhancer or promoter sequences is responsible for the exclusion of these DNA segments from nucleosomes and for the formation of nuclease hypersensitive sites . We speculate that the binding of such proteins may switch on a change in the conformation and/or the protein composition of a chromatin segment or domain containing one to several genes . Biochemical analysis of fractionated nucleosome particles or of active and inactive chromatin fractions have revealed differences in the composition as well as in the degree of modification of histones in these two subfractions of the chromosome . However, until present it is impossible to define unambiguously what are the crucial structural elements that distinguish between particles present on active and inactive chromatin.

Boll Ist Sieroter Milan, 1986, 65(2), 112 - 7
{Bacterial lysis by lysozyme}; Gagliardi R et al.; We confirmed the previous reports of Fleming, Nakamura et al . on the inability of lysozyme to lyse Micrococcus lysodeikticus cells at an acidic pH (3.0 - 3.5) . In these conditions, lysozyme binds to the mucopolysaccharides of the cell wall without showing any lytic activity; lysis occurs at very high rate when pH is raised at neutral values by adding alkaline solutions . Our results were obtained by monitoring bacterial cultures both in aqueous solutions and on agar plates.

Mutat Res, 1986 Jan, 165(1), 9 - 14
Damages induced in lambda phage DNA by enzyme-generated triplet acetone; Menck CF et al.; Exposure of lambda phage to triplet acetone, generated via the oxidation of isobutanal by peroxidase, leads to genome lesions . The majority of these lesions are detected as DNA single-strand breaks only under alkaline conditions, and so true breaks do not occur . Also, no sites sensitive to UV-endonuclease from Micrococcus luteus were found in DNA from treated phage . The participation of triplet acetone in the generation of such DNA damage is discussed.

Prostate, 1986, 8(4), 363 - 80
Association states of androgen receptors in nuclei of human benign hypertrophic prostate; Kyprianou N et al.; Androgen receptors (AR) were quantified in nuclei purified from unfractionated benign hypertrophic prostate (bph) tissue and from separated epithelium and stroma from bph specimens . Both epithelial and stromal cell nuclei contained AR, although concentrations in epithelial cell nuclei were higher and more variable . Variations in AR levels in epithelial cell nuclei reflected variations in unfractionated-tissue nuclei . Nuclear AR were further characterized regarding extractability with or resistance to 0.6 mol/lKCl and micrococcal nuclease . Nuclei from unfractionated tissue, epithelium, and stroma contained populations of AR susceptible and refractory to solubilization with KC1 and nuclease . Nuclease- and salt-sensitive populations of AR were similar numerically . The observed variability in epithelial cell nuclear AR was attributable to a wide range of solubilizable AR . Nuclease-digestion profiles and sedimentation analyses revealed that this wide range was not due to AR associated with soluble chromatin oligomers but to AR not detectably associated with other nuclear components . In contrast, AR in stromal cell nuclei was predominantly resistant to KC1 and nuclease, and variability in total nuclear AR concentration was due to variation in the nonextractable population.

J Mol Biol, 1985 Dec 20, 186(4), 743 - 58
Different conformations of ribosomal DNA in active and inactive chromatin in Xenopus laevis; Spadafora C et al.; The chromatin structure of the ribosomal DNA in Xenopus laevis was studied by micrococcal nuclease digestions of blood, liver and embryonic cell nuclei . We have found that BglI-restricted DNA from micrococcal nuclease-digested blood cell nuclei has an increased electrophoretic mobility compared to the undigested control . Micrococcal nuclease digestion of liver cell nuclei causes a very slight shift in mobility, only in the region of the spacer containing the "Bam Islands" . In contrast, the mobility of ribosomal DNA in chromatin of embryonic cells, under identical digestion conditions, remains unaffected by the nuclease activity . Denaturing gels or ligase action on the nuclease-treated DNA abolishes the differences in the electrophoretic mobility . Ionic strength and ethidium bromide influence the relative electrophoretic migration of the two DNA fragment populations, suggesting that secondary structure may play an important role in the observed phenomena . In addition, restriction analysis under native electrophoretic conditions of DNA prepared from blood, liver and embryonic cells shows that blood cell DNA restriction fragments always have a faster mobility than the corresponding fragments of liver and embryo cell DNA . We therefore propose that nicking activity by micrococcal nuclease modifies the electrophoretic mobility of an unusual DNA conformation, present in blood cell, and to a lesser extent, in liver cell ribosomal chromatin . A possible function for these structures is discussed . The differences of the ribosomal chromatin structures in adult and embryonic tissues may reflect the potential of the genes to be expressed.

Science, 1985 Dec 20, 230(4732), 1344 - 9
The 3' splice site of pre-messenger RNA is recognized by a small nuclear ribonucleoprotein; Chabot B et al.; A component present in splicing extracts selectively binds the 3' splice site of a precursor messenger RNA (pre-mRNA) transcript of a human beta-globin gene . Since this component can be immunoprecipitated by either autoantibodies of the Sm class or antibodies specifically directed against trimethylguanosine, it is a small nuclear ribonucleoprotein (snRNP) . Its interaction with the 3' splice site occurs rapidly even at 0 degrees C, does not require adenosine triphosphate, and is altered by certain mutations in the 3' splice site region . Binding is surprisingly insensitive to treatment of the extract with micrococcal nuclease . The U5 particle is the only abundant Sm snRNP with a capped 5' end that is equally resistant to micrococcal nuclease . This suggests that, in addition to the U1 and U2 snRNP's, U5 snRNP's participate in pre-mRNA splicing.

EMBO J, 1985 Dec 16, 4(13A), 3571 - 81
Purification of a protein required for the splicing of pre-mRNA and its separation from the lariat debranching enzyme; Kramer A et al.; We have used a complementation assay to test for activities required for the splicing of pre-mRNA in vitro . During the hypotonic lysis of HeLa cells, two components are released from the nuclei that specifically stimulate splicing in an extract prepared from washed nuclei . The two activities separate during chromatography on DEAE-Sepharose . One of these activities {splicing factor (SF)2} co-purified through several steps with the lariat debranching enzyme and with a nuclease which degrades the linear portion of lariat RNAs . These enzymes could, however, be separated from SF2 by chromatography on heparin-Sepharose . SF2 fractionates as a single protein with an apparent mol . wt . of 50 000 . SF2 is resistant to mild heat treatment and to treatment with micrococcal nuclease, but it is inactivated by N-ethylmaleimide, suggesting that it is a protein which is not associated with an essential RNA component . When SF2 is absent in a complementation assay, the generation of both intermediates and final products of the splicing reaction is completely abolished . Thus, SF2 functions in an early step of the splicing process.

Cancer Lett, 1985 Dec, 29(3), 245 - 54
An endodeoxyribonuclease activity in nuclei from rat-ascites hepatoma; Hibino Y et al.; The DNA in nuclei from rat-ascites hepatoma (AH) was rather resistant to endogenous endonucleolytic attack (autodigestion), compared with that in nuclei from normal rat liver (RL) . In contrast, by micrococcal nuclease, the DNA in AH nuclei was cleaved in the same manner as in RL nuclei . A 0.6 M NaCl extract was prepared from RL or AH nuclei and subjected to Sephadex G-100 filtration . The resulting-nuclease fraction was separated further into two nuclease fractions, I and II, by CM-Sephadex column chromatography . The activity ratio of II to I was 7.1 for the RL and 2.0 for the AH nuclei . Moreover, the activity of fraction II from the AH nuclei was rather low, compared with that from the RL nuclei . Regenerating-liver nuclei from the normal rat were also assayed in the same way . The results obtained were very similar to those from the AH nuclei . In addition, each of fractions, I and II, cleaved pBR322 DNA of superhelical form; in other words, each had endonucleolytic ability.

J Gen Virol, 1985 Dec, 66 ( Pt 12), 2671 - 84
The structure of adenovirus chromatin in infected cells; Dery CV et al.; The structure of adenovirus chromatin in infected cells was studied by micrococcal nuclease digestion and hybridization with virus-specific probes . In the early phase of infection (5 h) a significant proportion of viral molecules was organized like actively transcribed cellular chromatin . As expected for a transcriptionally active population of molecules, even at high multiplicity of infection the nucleosomal repeating pattern was less distinct than in a transformed cell which contained the corresponding but less active genomic region . The observed repeating pattern in infected cells was unlikely to be due to integrated molecules since less than 0.07% of input genomes became associated with cellular DNA . After the onset of viral DNA replication, the pool of viral chromatin organized like cellular chromatin rapidly increased . In addition, newly replicated molecules also maintained the cellular chromatin-like organization as measured by {3H}thymidine incorporation after the cessation of cellular DNA synthesis . These data suggest that newly replicated viral molecules are organized by histones into cell-like chromatin throughout the infection cycle . Coincident with the peak of viral DNA and core protein synthesis, and the decline of histone synthesis, the late, core-like non-repeating viral chromatin became dominant, increasingly obscuring the underlying repeating pattern . Experiments suggest that this late chromatin is destined for encapsidation, that the early chromatin persists and that viral core proteins do not displace histones on viral DNA . A model is proposed suggesting that transcription and type I replication occur on histone-condensed templates, while type II replication products late in infection are condensed by core proteins and are destined for encapsidation.

Mol Immunol, 1985 Dec, 22(12), 1415 - 22
Binding of monoclonal anti-native DNA autoantibodies to DNA of varying size and conformation; Ali R et al.; A microchemical assay for phosphorus was applied to the measurement of DNA in immune complexes formed with monoclonal or serum anti-DNA autoantibodies and DNA of varying size and conformation . Two monoclonal antibodies were produced by hybridomas derived from spleen cells of autoimmune MRL-lpr/lpr mice and were purified from culture fluid by affinity chromatography on columns of goat anti-mouse Ig-Sepharose . Double-helical DNA fragments were prepared by brief digestion of calf thymus DNA with micrococcal and S1 nucleases and fractionation on Sepharose 4B; their double-stranded structures was confirmed by measurement of thermal denaturation . Immune complexes were formed with monoclonal or serum antibodies and native DNA or DNA fragments or denatured DNA; the complexes were precipitated with goat anti-mouse IgG and washed, and DNA phosphorus content of the precipitates was measured . With one monoclonal autoantibody (H241), there were discontinuous increases in the amount of DNA that could be bound (and decreases in the antigen concn required for half-maximal binding) as the DNA size increased . There were especially marked increases in binding efficiency as fragment size increased from an average of 100 (range 85-105) to an average of 150 (range 105-170) base pairs, and again between 450 (range 360-620) and 600 (range 425-825) base pairs . A second monoclonal antibody (H143) did not show significant variation in binding with DNA fragments larger than 300 base pairs . With smaller fragments, the amount of DNA bound by H143 was reduced, but the DNA concn required for half-maximal binding was not . Affinities of these monoclonal antibodies were within the spectrum of human systemic lupus erythematosus serum IgG anti-DNA autoantibodies . The dependence of binding on mol . wt is important in the evaluation of these monoclonal antibodies as biochemical reagents and as potential participants in formation of immune complexes in vivo.

Virology, 1985 Dec, 147(2), 373 - 81
Template-dependent RNA polymerase from black beetle virus-infected Drosophila melanogaster cells; Saunders K et al.; Infection of cultured cells of Drosophila melanogaster with black beetle virus (BBV) induces an RNA polymerase that is bound to cellular particulate material in a complex with a template RNA . We have solubilized the polymerase by treatment of the relevant particulates with detergents such as dodecyl-beta-D-maltoside . The polymerase activity was made dependent upon exogenous RNA by destruction of the endogenous template RNA with micrococcal nuclease . Addition of BBV RNA1 or RNA2 induced synthesis of full-length negative-strand RNA isolated as a double-stranded complex with the added RNA . Newly synthesized plus strands were also detected in the RNA2 complexes . Certain other viral RNAs also induced synthesis of their negative strands.

Biochim Biophys Acta, 1985 Nov 22, 843(1-2), 58 - 67
Specificity of the histone lysine methyltransferases from rat brain chromatin; Duerre JA et al.; The histone lysine methyltransferases catalyze the transfer of methyl groups from S-adenosylmethionine to specific epsilon-N-lysine residues in the N-terminal regions of histones H3 and H4 . These enzymes are located exclusively within the nucleus and are firmly bound to chromatin . The chromosomal bound enzymes do not methylate free or nonspecifically associated histones, while histones H3 and H4 within newly synthesized chromatin are methylated . These enzymes can be solubilized by limited digestion (10-16%) of chromosomal DNA from rapidly proliferating rat brain chromatin with micrococcal nuclease . Histone H3 lysine methyltransferase remained associated with a short DNA fragment throughout purification . Dissociation of the enzyme from the DNA fragment with DNAase digestion resulted in complete loss of enzyme activity; however, when this enzyme remained associated with DNA it was quite stable . Activity of the dissociated enzyme could not be restored upon the addition of sheared calf thymus or Escherichia coli DNA . Histone H3 lysine methyltransferase was found to methylate lysine residues in chromosomal bound or soluble histone H3, while H3 associated with mature nucleosomes was not methylated . The histone H4 lysine methyltransferase which was detectable in the crude nuclease digest was extremely labile, losing all activity upon further purification . We isolated a methyltransferase by DEAE-cellulose chromatography, which would transfer methyl groups to arginine residues in soluble histone H4 . However, this enzyme would not methylate nucleosomal or chromosomal bound histone H4, nor were methylated arginine nucleosomal or chromosomal bound histone H4, nor were methylated arginine residues detectable upon incubating intact nuclei or chromatin with S-adenosylmethionine.

J Steroid Biochem, 1985 Nov, 23(5A), 547 - 51
Characterization of the 4-hydroxytamoxifen (4-OHTAM) bound estrogen receptor of MCF-7 cells solubilized by micrococcal nuclease; Geier A et al.; In order to get an insight into the molecular mechanism of antiestrogen action at the chromatin level, we characterized the physical-chemical properties of the chromatin fragments released by micrococcal nuclease digestion of nuclei isolated from MCF-7 cells previously exposed to {3H}4-OHTAM . The {3H}4-OHTAM bound solubilized fragments were characterized in a low ionic strength buffer and in a high ionic strength buffer without and with urea . The following parameters were determined: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding ability to a DNA-cellulose column . The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values . Following mild nuclease digestion, the solubilized {3H}4-OHTAM bound receptor sedimented as an abundant 6-7 S form and a less abundant approximately 12 S species . Increasing the extensiveness of digestion resulted in one receptor form sedimenting at 5.2 S, Rs = 7.25 nm and Mr = 155,000 . About 45% of the applied receptor bound to a DNA-cellulose column could be eluted by a high salt concentrated buffer . Dissociation of the micrococcal nuclease solubilized receptor in 0.4 M KCl resulted in a smaller receptor form with a 4.9 S, Rs = 5.87 nm and Mr = 119,000 . Further dissociation in the presence of 3 M urea resulted in a receptor with a 3.5 S, Rs = 5.78 nm and Mr = 83,000 . These results suggested that the antiestrogen bound estrogen receptor in chromatin, is associated with a tightly bound protein component and with an additional less tightly bound protein, complexes with DNA.

Infection, 1985 Nov-Dec, 13(6), 280 - 1
Meningitis due to Micrococcus luteus; Fosse T et al.; We are presenting a new case of meningitis due to the Micrococcus luteus species . This germ was isolated twice in eight days from the CSF of a 57-year old woman . The patient had a ventriculoperitoneal shunt implanted for hydrocephalus following a meningeal haemorrhage . Antibiotic therapy was efficient but the patient died of a recurrent haemorrhage.

J Bacteriol, 1985 Nov, 164(2), 684 - 8
High levels of glycolipid and low levels of phospholipid in a marine caulobacter; De Siervo AJ; Studies of the lipid composition of the marine bacterium Caulobacter halobacteroides revealed the presence of glycolipid as the predominant lipid constituent . The presence of minor amounts of phospholipid was confirmed with the incorporation of 14C- and 32P-labeled compounds . Other marine caulobacters had similar lipid compositions . Five chromatographically separable glycolipids were detected, two of which were identified as mono- and diglycosyldiglycerides . Glycolipid constituted 90 to 99% of the total extractable lipid based on 14C-acetate incorporation into six marine caulobacter strains . In addition, comparisons were made with the lipid extracts of the nonmarine Caulobacter crescentus and Micrococcus lysodeikticus, which contain substantial amounts of phospholipid . Studies of lipid composition during growth showed the maximum amount of phospholipid during early logarithmic growth (2.9%) with a decrease to 0.3% in the early stationary phase . The finding of a group of organisms in which phospholipid is not a major constituent of the lipid fraction is unique and generates many questions about the lipid requirements for membrane structure and function.

Mol Cell Biol, 1985 Nov, 5(11), 3124 - 30
Alternative model for chromatin organization of the Saccharomyces cerevisiae chromosomal DNA plasmid TRP1 RI circle (YARp1); Long CM et al.; TRP1 RI circle (now designated YARp1, yeast acentric ring plasmid 1) is a 1,453-base-pair artificial plasmid composed exclusively of Saccharomyces cerevisiae chromosomal DNA . It contains both the TRP1 gene and ARS1 (a DNA sequence that permits extrachromosomal maintenance of recombinant plasmids) . This high-copy-number, relatively stable plasmid was shown to be organized into nucleosomes comparable to typical yeast chromatin, containing a possible maximum of nine nucleosomes per circle . Therefore, YARp1 can be used to examine the structure of chromatin of both a chromosomally derived replicator and a functional gene . By mapping regions of micrococcal nuclease cleavage in chromatin versus purified DNA, we located the positions of protected regions on the circle with reference to six unique restriction sites . Measurements made on patterns of early digestion products indicated that a region of approximately 300 base pairs in the vicinity of ARS1 was strongly resistant to micrococcal nuclease . The remainder of the plasmid appeared to be associated with five positioned nucleosomes and two nonnucleosomal, partially protected regions on the bulk of the molecules . After similar extents of digestion, naked DNA did not exhibit an equivalent pattern, although some hypersensitive cleavage sites matched sites found in the chromatin . These results are consistent with the interpretation that the protected domains are aligned with respect to a specific site or sites on the small circular chromatin.

J Nutr, 1985 Nov, 115(11), 1504 - 14
Enhancement of RNA synthesis in chick liver by food intake: possible role of high mobility group nonhistone proteins; Oka T et al.; RNA synthesis in the nuclei of liver from newly hatched (4-d-old) chicks is enhanced by intake of food . The enhanced synthesis was ascribed not to an increase in the activity of solubilized DNA-dependent RNA polymerase but to an increase in the initiation of RNA synthesis . Enhanced RNA synthesis in fed chicks was accompanied by greater susceptibility of nuclei to digestion by micrococcal nuclease . Salt extraction abolished the difference in nuclease sensitivity between the fed and fasted groups . Reconstitution with either 0.35 M NaCl extracts or high mobility group (HMG) nonhistone proteins restored digestion susceptibility, but changing the source of extracted proteins did not equalize the extent of digestion in nuclei from livers of fed and fasted chicks . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of HMG proteins revealed the presence of HMGs 1 and 2 as well as a 38,000-dalton protein . The nuclear HMG protein content in fed chicks was greater than that of fasted chicks (121 +/- 17 micrograms/mg DNA vs . 31 +/- 12 micrograms/mg DNA) . The electron microscopic examination of hepatocyte nuclei revealed the enlargment of nucleoli and scarcity of aggregated heterochromatin structures in the fed chicks as compared with the fasted chicks . These morphological features are compatible with the high transcriptional activity in liver of fed chicks.

Tumori, 1985 Oct 31, 71(5), 407 - 18
Polycyclic aromatic hydrocarbon binding macromolecules . Identification, characterization and temperature activation of a 4.5 S binding nucleoprotein; Sica V et al.; A macromolecule binding 3H-methylcholanthrene (3H-MCA) and 3H-benzo(a)pyrene (3H-BaP) and sedimenting in the 4-5 S region of sucrose gradient (4.5 S) was identified in rat liver cytosol . The binding was displaced by 100-fold molar excess unlabeled ligands whereas 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) was ineffective . The dissociation constant for both polycyclic aromatic hydrocarbons (PAHs) was of the order of 10(-8) M or lower . Both 3H-MCA and 3H-BaP bound to 4.5 S in a non covalent manner, since 92% of the bound radioactivity was extractable with ethyl ether . Furthermore the binding was strongly reduced by urea 8 M and by guanidine . HCl 4 M (99 and 70% respectively) . Thin layer chromatography of the ethyl ether-solubilized radioactivity showed a peak comigrating with PAHs used as standards . When chromatographed on Sephadex G-200, 4.5 S was eluted as a sharp peak with an apparent molecular weight of 50-60,000 daltons . Enzyme treatment of liver cytosol showed that the 4.5 S binding sites were destroyed by micrococcal nuclease (92% of inhibition) . Papain and phosphodiesterase I and II reduced the binding to 50%, whereas DNase I, DNase II, RNase, phospholipase A2 and C and trypsin were ineffective . These data suggest that the PAHs binding macromolecule of rat liver cytosol is a protein associated with a polynucleotide . The binding of both PAHs was enhanced by increasing the incubation temperature, the maximum being reached after 20-30 min at 37 degrees C . After 2.5 min at 65 degrees C, binding sites were completely destroyed . The same temperature-induced "activation" was obtained also by prewarming the cytosol at 37 degrees C in the absence of ligands.

Eur J Biochem, 1985 Oct 15, 152(2), 279 - 86
Attachment of the 5'-terminal portion of globin mRNAs to 5S-RNA X L5-protein in the 80S initiation complex; Takahashi Y et al.; An 80S initiation complex was formed by incubating a heterologous cell-free system with 125I-labeled globin mRNAs in the presence of sparsomycin . The 80S initiation complex was then digested with micrococcal nuclease . The ribosomal 5S-RNA X L5-protein (5S RNP) fraction, released by EDTA treatment, contained 125I-labeled mRNA fragments . The attachment of labeled mRNA fragments to 5S RNP was shown by (a) CsCl isopycnic centrifugation, (b) recentrifugation through a sucrose density gradient and (c) acrylamide gel electrophoresis of 5S RNP purified by (b) . Labeled fragments were released from 5S RNP by treatment with sodium dodecyl sulfate or pronase, indicating the participation of protein L5 in the attachment . The attached mRNA fragments were 23-25 nucleotides in length . Hybridization experiments, using restriction fragments of cDNA for rabbit beta globin mRNA, showed that the attached mRNA fragments were derived from the 5' portion of globin mRNAs . The attachment of 125I-labeled mRNA fragments to 5S RNP was also observed in the 80S initiation complex formed by incubation of reticulocyte lysate with 125I-labeled globin mRNA, but not in labeled polysomal fractions . These findings may indicate that 5S RNP interacted with the 5' portion of globin mRNA, containing the translation initiation codon of globin mRNA in the 80S initiation complex . The biochemical significance of these results is discussed.

J Biol Chem, 1985 Oct 15, 260(23), 12622 - 8
Nonrandom location of H1-H1 degree histones on chromatin of mouse liver and brain; Delabar JM; The electrophoretic behavior of nucleosome dimers reconstituted with H1 or H1 degrees and the features of the digestion of those reconstituted dimers with micrococcal nuclease were first investigated . Both criteria appear to support the notion that H1 degrees can replace H1 on the nucleosome on a one-to-one basis and that both proteins fulfill a similar structural role in chromatin . H1/H1 degrees ratios in different chromatin subfractions from mouse liver were then indirectly measured by means of an electrophoretic purification of H1 degrees- from H1-containing nucleosome monomers, followed by a titration of different specific nucleotide sequences in the corresponding DNAs . Satellite and globin containing chromatin subfractions were found to contain only about half the amount of H1 degrees which is normally encountered in bulk chromatin, indicating a nonrandom location of H1-H1 degrees variants on untranscribed sequences; in contrast, titrations with cDNA from poly(A+) RNA and albumin cDNA show an approximately 2-fold enrichment in H1 degrees for the corresponding chromatin when compared to the same bulk chromatin . In the brain, inactive albumin chromatin contains a relative amount of both H1 variants similar to that found in satellite or globin chromatin in liver . Amounts of H1 degrees can, therefore, be correlated with different states of chromatin: an inactive state with a lower amount of H1 degrees and a potentially active state with an enrichment in H1 degrees.

Mol Biol Rep, 1985 Oct, 10(4), 199 - 203
Distribution of histone variants in the sea urchin chromatin fractions obtained by selective micrococcal nuclease digestion; Jasinskiene NE et al.; Chromatin fractions differing in their transcriptional activity were isolated by selective micrococcal nuclease digestion of nuclei from sea urchin embryos (Strongylocentrotus droebachiensis) at the gastrula and pluteus stage . The electrophoretic analysis of the chromatin proteins at the gastrula stage showed that a soluble, transcriptionally active fraction of chromatin was enriched with early variants of histones H1 and H2A . The early and late variants of histone H2A at the pluteus stage were distributed randomly between chromatin fractions . However, the content of both variants of histone H1 was essentially decreased in the soluble transcriptionally active fraction of chromatin.

J Inorg Biochem, 1985 Oct, 25(2), 109 - 20
Circular dichroism of micrococcal nuclease-treated calf thymus chromatin (soluble chromatin) in presence of CH3HgOH; Gruenwedel DW; Exposing (soluble) calf thymus chromatin and, as reference, protein-free native calf thymus DNA (both in 0.01 M Na+, pH 6.8, 25 degrees C) to increasing concentrations of CH3HgOh produces cooperative transitions in their CD spectra . In the case of chromatin, and there especially at low concentrations of methylmercury, they are due to reactions affecting the relative orientation of the bases in the constituent DNA, without disrupting base-pairing . In the case of protein-free DNA, and with chromatin at higher methylmercury concentrations, the CD changes signal collapse of the DNA secondary structure . Primary data (molar ellipticities {theta}, zero-ellipticity points, and rotational strengths R) are presented as a function of methylmercury concentration and wavelength . The results are discussed in relation to previous findings of this laboratory regarding methylmercury-DNA and methylmercury-chromatin interactions, and it is pointed out that the structural alterations observed with chromatin at low levels of methylmercury may very well be the primary events in a chain that is responsible for the teratogenic and clastogenic damages caused by organic mercury.

Mutat Res, 1985 Oct, 152(1), 85 - 96
Formation and persistence of sterigmatocystin--DNA adducts in rat liver determined via 32P-postlabeling analysis; Reddy MV et al.; A 32P-postlabeling method has been employed to detect the in vitro and in vivo modification of DNA by the mycotoxin sterigmatocystin (ST) . ST-modified DNA was initially incubated under buffered alkaline conditions to convert unstable ST-N7-guanine moieties to stable, putative ST-formamidopyrimidine derivatives . DNA was subsequently digested with micrococcal nuclease and spleen phosphodiesterase, and the resulting ST-modified nucleotides, purified by reverse-phase thin-layer chromatography (TLC), were labeled at the 5' position via incubation with {gamma-32P}ATP and T4 polynucleotide kinase . 32P-labeled ST-nucleotides were separated by reverse-phase and anion-exchange TLC . Cerenkov quantitation of excised TLC fractions indicated that ST-DNA moieties could be detected with a sensitivity of 1 ST adduct in 3-5 X 10(7) nucleotides . Initial enzymatic digestion of ST-modified DNA was found to yield ST-modified di- and trinucleotides which, upon 32P-labeling followed by incubation with nuclease P1, liberated unmodified 5'-terminal nucleotides suggesting that ST-formamidopyrimidine-modified DNA was a poor substrate for micrococcal nuclease and spleen phosphodiesterase . Dose-dependent ST-DNA adduct formation was detected in the liver of male Fischer 344 rats over a 27-fold range of ST administered (0.33-9 mg/kg) . In addition, ST-DNA adducts, formed in rats given a 9 mg/kg dose, were found to persist up to 105 days after treatment at a level of 0.5% of the 2-h value . Loss of these adducts from liver DNA was observed to exhibit a triphasic profile: rapid loss during the first 24 h (t 1/2 = 12 h) followed by a slower decline from 1 to 14 days post dosing (t 1/2 = 7 days) and an extremely slow decline from days 14 to 105 post treatment (t 1/2 = 109 days) . This experimental approach to the study of mycotoxin-DNA interactions permits the quantitative description of DNA modification in ST-treated animals . Further refinement of this approach may be useful in defining the precise relationship between ST exposure and tumorigenesis in ST-exposed human populations.

Eur J Biochem, 1985 Oct 1, 152(1), 143 - 50
The structure of sub-nucleosomal particles . The octameric (H3/H4)4--125-base-pair-DNA complex; Read CM et al.; Chicken erythrocyte chromatin was depleted of histones H1, H5, H2A and H2B . The resulting (H3/H4)-containing chromatin was digested with micrococcal nuclease to yield monomer, dimer, trimer etc . units, irregularly spaced on the DNA, with even-number multimers being more prominent . Sucrose density gradient centrifugation separated monomers and dimers (7.7 S and 10.5 S) . Sodium dodecyl sulphate gel electrophoresis and cross-linking indicated: the monomer contains 50-base-pair (bp), 60-bp and 70-bp DNA and the dimer 125-bp DNA; the monomer contains a tetramer and the dimer an octamer of H3 and H4 . Partial association of monomer units to dimers inhibits structural studies of monomers . The internal structure of the dimer, i.e . and (H3/H4)4-125-bp-DNA particle, was studied using circular dichroism, thermal denaturation and nuclease digestion . Both micrococcal nuclease and DNase I digestion indicate that, unlike core particles, accessible sites occur in the centre of the particle and it is concluded that the (H3/H4)4-125-bp-DNA particle is not a 'pseudo-core particle' in which the 'extra' H3 and H4 replace H2A and H2B . It is proposed that the octamer particle is formed by the sliding together of two 'monomer' units, each containing the (H3/H4)2 tetramer and 70 bp of DNA . Excision of this dimer unit with micrococcal nuclease results in the loss of 10 readily digestible base pairs at each end, leaving 125 bp.

J Neurochem, 1985 Oct, 45(4), 1006 - 12
A low repeat length in oligodendrocyte chromatin; Di Liegro I et al.; The behavior of oligodendrocyte chromatin after micrococcal nuclease digestion of nuclei was assayed in brains of rats of four different ages . During oligodendrocyte differentiation, a decreasing sensitivity of the chromatin to enzymatic attack was observed . On the other hand, the nucleosomal repeat length showed a slight tendency to increase during development . It is worth noting that even the highest values reported here for "oligodendrocyte" chromatin repeat lengths are significantly lower than 200 base pairs, the value previously reported by others for "non-astrocytic glia."

Cell, 1985 Oct, 42(3), 725 - 36
Multiple factors including the small nuclear ribonucleoproteins U1 and U2 are necessary for pre-mRNA splicing in vitro; Krainer AR et al.; We have identified six distinct factors necessary for pre-mRNA splicing in vitro by selective inactivation and complementation studies, and by fractionation procedures . Splicing factor 1 (SF1) is sensitive to micrococcal nuclease, and appears to consist of at least U1 and U2 snRNPs, since splicing is inhibited when the 5' termini of U1 and U2 snRNAs are removed by site-directed cleavage with RNAase H . SF2 is a micrococcal nuclease-resistant factor present in the nuclear extract but absent from an S100 extract . SF3 is a factor that can be preferentially inactivated by moderate heat treatment . Two additional factors (SF4A and SF4B) were identified by fractionation of the nuclear extract using spermine-agarose and CM-sepharose chromatography . SF1, SF2, and SF4B appear to be required for cleavage of the pre-mRNA at the 5' splice site and lariat formation, whereas SF3 and SF4A are only required for cleavage at the 3' splice site and exon ligation.

J Virol, 1985 Oct, 56(1), 161 - 71
Evidence for the presence of an inhibitor on ribosomes in mouse L cells infected with mengovirus; Pensiero MN et al.; After infection of mouse L cells with mengovirus, there is a rapid inhibition of protein synthesis, a concurrent disaggregation of polysomes, and an accumulation of 80S ribosomes . These 80S ribosomes could not be chased back into polysomes under an elongation block . The infected-cell 80S-ribosome fraction contained twice as much initiator methionyl-tRNA and mRNA as the analogous fraction from uninfected cells . Since the proportion of 80S ribosomes that were resistant to pronase digestion also increased after infection, these data suggest that the accumulated 80S ribosomes may be in the form of initiation complexes . The specific protein synthetic activity of polysomal ribosomes also decreased with time of infection . However, the transit times in mock-infected and infected cells remained the same . Cell-free translation systems from infected cells reflected the decreased protein synthetic activity of intact cells . The addition of reticulocyte initiation factors to such systems failed to relieve the inhibition . Fractionation of the infected-cell lysate revealed that the ribosomes were the predominant target affected . Washing the infected-cell ribosomes with 0.5 M KCI restored their translational activity . In turn, the salt wash from infected-cell ribosomes inhibited translation in lysates from mock-infected cells . The inhibitor in the ribosomal salt wash was temperature sensitive and micrococcal nuclease resistant . A model is proposed wherein virus infection activates (or induces the synthesis of) an inhibitor that binds to ribosomes and stops translation after the formation of the 80S-ribosome initiation complex but before elongation . The presence of such an inhibitor on ribosomes could prevent them from being remobilized into polysomes in the presence of an inhibitor of polypeptide elongation.

Nucleic Acids Res, 1985 Sep 11, 13(17), 6185 - 203
Transient alterations of the chromatin structure of sea urchin early histone genes during embryogenesis; Wu TC et al.; We describe features of the chromatin structure of the early histone gene family of Strongylocentrotus purpuratus during development . Before and after the histone genes are transcriptionally active, chromatin structure is quite similar with well-defined spaced nucleosomes and no major 5'-flanking sites hypersensitive to nucleases . During the period when the genes are active, marked changes in chromatin structure occur . Micrococcal nuclease digestion generates monomer nucleosomes and only trace amounts of higher multimers . Regions hypersensitive to an endogenous nuclease and DNAase I appear in the 5'-flanking regions of genes for H2A, H2B and H3 . Each region consists of four sites spanning a DNA length of 200-250 base pairs . In each case, one major cutting site is near the TATA box; the bulk of the sensitive region is in the nontranscribed spacer . Other sites, in 3'-flanking regions of the genes, are sensitive to nucleases only when the histone genes are no longer transcribed.

Biochemistry, 1985 Sep 10, 24(19), 5269 - 75
Chromatin structure of a 3-methylcholanthrene-induced cytochrome P-450 gene; Einck L et al.; Plasmids carrying fragments of a cytochrome P-450 gene, inducible by 3-methylcholanthrene, were used to study the chromatin structure of this gene in the liver of normal and carcinogen-treated rats . Digestion with micrococcal nuclease revealed that the gene is not present in the typical 200 base pair nucleosomal structure . By use of indirect end-label hybridization, four DNase I hypersensitive sites were mapped in the 5'-terminal region of the gene . An S1 nuclease sensitive site is located close to a DNase I site . Gene induction by treatment with 3-methylcholanthrene does not result in detectable changes in the DNase I hypersensitive sites . Rat thymus chromatin does not contain DNase I hypersensitive sites in the P-450 gene, suggesting that in the liver the chromatin structure is altered so as to allow tissue-specific expression of the gene . This paper is the first study on the chromatin structure of a gene coding for a member of the cytochrome P-450 family of enzymes . The implications of our results to the understanding of gene regulation of the P-450 genes are discussed.

Mol Biol (Mosk), 1985 Sep-Oct, 19(5), 1231 - 41
{Analysis of cellular nucleoproteins by the nucleoprotein-celite chromatography method . III . Two types of DNA-nuclear matrix interaction}; S'iakste NI et al.; There are two types of DNA-nuclear matrix interactions in animal cells as revealed by the release of DNA from isolated nuclei by three successive gradients: NaCl, LiCl-urea and temperature . Nuclei were treated with dissociating agents while being adsorbed on the Celite columns . "Weak" DNA-matrix interactions which dissociate in 1.5 M LiCl-3 M urea at 2 degrees appear to be sensitive to ethidium bromide and resistant to exogeneous nucleases (DNAase I, DNAase II and micrococcal nuclease), to DNA-damaging agents, including alkylators and gamma-irradiation, and also to psoralen-induced cross-links . "Strong" DNA-matrix interactions proved to be very different . They dissociate in 4 M LiCl-8 M urea at approximately 90 degrees, are very sensitive to DNAase I and other nucleases, slightly sensitive to chemicals and irradiation at doses stimulating single-stranded DNA breaks, but resistant to ethidium bromide . DNA strand separation seems to be necessary prerequisite for DNA release from its "strong" complex with nuclear matrix . A model for the topological link between DNA and the nuclear matrix involved in DNA replication complex is discussed.

Mol Cell Biochem, 1985 Sep, 68(1), 49 - 57
A new nucleosomal protein in normal liver related to the cytoplasmic polypeptide target of a carcinogen; Bassuk JA et al.; Normal adult rat liver contains a nucleosomal protein that is related to the principal target polypeptide of a carcinogen in cytoplasm . Normal rat liver was found previously to contain a 14 000-dalton polypeptide that is the principal cytosolic target of the carcinogen, N-2-fluorenylacetamide (2-acetylaminofluorene; FAA), early during hepatocarcinogenesis . Elevated levels of immunohistochemically detectable target polypeptide in cytoplasm are associated with normal mitosis and carcinogen-induced hyperplasias in adult hepatocytes . A putatively related 17 500-dalton polypeptide was shown to be tightly bound to chromatin of normal liver nuclei . We report here that purified nucleosomes from normal rat liver contain the bound 17 500-dalton protein . Nuclei were digested with micrococcal nuclease, and the resultant nucleosomes were resolved into size classes by density gradient sedimentation . The monomers, dimers, and trimers of nucleosomes possessed bound 17 500-dalton polypeptide, as determined by SDS gel electrophoresis followed by immunoelectroblot analyses . Alterations in the levels of the two polypeptides were shown previously to occur during liver carcinogenesis by FAA and 3'-methyl-4-dimethylaminoazobenzene . The findings support the possibility that the 17 500-dalton polypeptide may function normally in a role related to the replication or expression of the hepatic genome, and may be connected with changes in hepatic genic activity brought about by the carcinogens.

J Cell Biol, 1985 Sep, 101(3), 1124 - 34
Fractionation and initial characterization of the kinetochore from mammalian metaphase chromosomes; Valdivia MM et al.; We have partially isolated the kinetochore and associated centromeric structures from mammalian metaphase chromosomes . Human autoantibodies from scleroderma CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) patients were used as immunofluorescent probes to monitor fractionation . The procedure includes digestion of total chromosomal DNA with micrococcal nuclease, dehistonization with heparin, and dissociation of the remaining material with detergent and urea . We used a density gradient (metrizamide) to obtain an enriched fraction of stained material (kinetochore) . When examined by electron microscopy, the kinetochore fraction is seen to contain numerous small immunoperoxidase-positive masses which are morphologically similar to the centromere/kinetochore region of intact metaphase chromosomes . The particulate fraction that contains kinetochore components represents less than 5% of total chromosomal proteins and contains less than 1% of total DNA . Two polypeptides of 18 and 80 kD were identified as kinetochore antigens by immunoblotting with CREST antiserum . In this paper we discuss the distribution of these kinetochore polypeptides with the associated centromeric chromatin.

Int J Radiat Biol Relat Stud Phys Chem Med, 1985 Sep, 48(3), 389 - 95
Changes in nuclease sensitivity of mammalian cells after irradiation with 60Co gamma-rays; Takahashi K et al.; Changes in sensitivity of mouse BALB/c 3T3 cells in the plateau phase to digestion with micrococcal nuclease were examined following gamma-irradiation . Immediately after irradiation, cell nuclei were more sensitive to micrococcal nuclease compared to unirradiated nuclei . However, there were no detectable changes in length of basic repeating subunits of 182 base pairs of DNA, which include the nucleosome cores consisting of approximately 140 base pairs of DNA, which When the cells were incubated at 37 degrees following irradiation, the sensitivity of cell nuclei to the nuclease first increased then decreased, reaching a similar level to unirradiated nuclei 6 h after irradiation . Both the initial increase and the subsequent decrease in sensitivity of nuclei to micrococcal nuclease were prevented when 15 microM novobiocin was present during the post-irradiation incubation, suggesting a possible involvement of type II DNA topoisomerase in repair of DNA lesions induced by gamma-rays.

Br J Cancer, 1985 Sep, 52(3), 377 - 82
DNA repeat length in chromatin from murine bone marrow and L1210 leukaemia cells; Dean SW et al.; Previous studies have suggested that 1-(4-amino-2-methylpyrimidine-5-yl)-methyl-3-(2-chloroethyl) -3-nitrosoureahydrochloride (ACNU) and 1,(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) bind specifically to the nucleosomal DNA of murine bone marrow and L1210 leukaemia cells whereas the glucose nitrosoureas, 2-(3-(2-chloroethyl)-3-nitrosoureido)-2-deoxy-D-glucopyranose, (chlorozotocin, CLZ) and 1-(2-chloroethyl)-3-(-D-glucopyranosyl)-1-nitrosourea (GANU), bind preferentially to the linker DNA of bone marrow but not tumour cell chromatin . In order to provide an explanation for this differential, the DNA repeat and linker lengths in murine bone marrow and L1210 leukaemia cells were measured using electrophoresis of micrococcal nuclease-digested DNA . The linker length of bone marrow chromatin was approximately 22% longer than that in L1210 leukaemia cells from mouse ascites . The linker length of L1210 cells maintained in suspension culture was 27% less than in those from ascites fluid . The tissue-specific toxicity of sugar nitrosoureas and the differential binding of these drugs to chromatin does not appear to correlate quantitatively with differences in DNA linker length.

Nucleic Acids Res, 1985 Aug 26, 13(16), 5727 - 46
Investigation of restriction-modification enzymes from M . varians RFL19 with a new type of specificity toward modification of substrate; Butkus V et al.; The characterization of MvaI restriction-modification enzymes, isolated from Micrococcus varians RFL19, is reported . Both enzymes recognize the 5'CC decreases (A/T)GG nucleotide sequence . The endonuclease cleaves the sequence at the position indicated by the arrow, whereas the methylase modifies the internal cytosine, yielding N4-methylcytosine . This type of modification protects the substrate from R.MvaI cleavage . 5-Methylcytosine in the same position of the recognition sequence does not protect the substrate from R.MvaI cleavage . R.MvaI proved to be the first example of a restriction endonuclease differentiating the position of the methyl group in the heterocyclic ring of cytosine, located in the same site of the recognition sequence . M.MvaI modifies DNA dcm+ in vitro yielding N4,5-dimethylcytosine . N4-methylcytosine cannot be differentiated from cytosine using the Maxam-Gilbert DNA sequencing procedure.

J Biol Chem, 1985 Aug 25, 260(18), 9981 - 7
The phosphate diester linkage of the peptidoglycan polysaccharide moieties of Micrococcus lysodeikticus cell wall; Nasir-ud-Din et al.; The external polysaccharide is a major component of Micrococcus lysodeikticus cell wall and displays distinct composition . The complete structure of the external polysaccharide had been elucidated as a basis for investigation of the cell wall structure-function relation . However, the mode of attachment of the polysaccharide to the peptidoglycan through a phosphodiester was not clear due to limitations in structural and biosynthetic studies . The present study describes purification of a lysozyme-resistant nondialyzable high-molecular-weight fragment of cell wall and identifies the sugar, D-glucose, as the point of external polysaccharide attachment to the peptidoglycan through a phosphate diester . Kinetic studies for the acid-catalyzed release of external polysaccharide from the peptidoglycan were performed in parallel with synthetic {methyl-2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-alpha-D- glucopyranoside-6-yl}-alpha-D-glucopyranosyl phosphate and alpha-D-glucopyranosyl phosphate and showed the presence of a phosphodiester linkage between external polysaccharide and peptidoglycan . In addition, type of phosphate residue and cross-linking between muramic acid and protein part have been determined.

J Biol Chem, 1985 Aug 25, 260(18), 10093 - 8
In situ cross-linking of androgen receptors to nuclear acceptor sites of rat prostate with formaldehyde; Foekens JA et al.; Androgen receptors were attached covalently in situ to their nuclear acceptor sites with the contact site cross-linker, formaldehyde . Chromatin, prepared from sonicated nuclei of rat prostate, was labeled by isotope exchange with {3H}dihydrotestosterone and found to contain 19,000 +/- 900 (mean +/- S.E.) salt-extractable androgen receptors/nucleus which sedimented in the 3-4 S region of 7.6-76% (v/v) glycerol gradients and at a density of approximately 1.28-1.35 g/ml in CsCl gradients . After incubation of the chromatin with 0.5% (w/v) formaldehyde for 1 h at 4 degrees C, there was a 90% reduction in the concentration of free androgen receptors and an increase in the density of the androgen binding sites recovered from CsCl gradients . Extensive digestion of the cross-linked chromatin with micrococcal nuclease liberated 18% of the androgen receptors as 3-4 S entities and caused an overall decrease in the density of the receptor-acceptor complexes . Ribonuclease digestions had no effect on the androgen receptors cross-linked to chromatin . Mild digestion of the cross-linked preparations with trypsin, alone or in combination with micrococcal nuclease, resulted in the release of 74% and 97% of the androgen receptors, respectively . Together, these findings imply that two classes of receptor-acceptor complexes are present in prostatic chromatin--one, containing about 20% of the androgen receptors in which the receptors are in direct contact with DNA but not with proteins and the other, containing most of the androgen receptors in which the receptors are adjacent to acceptor proteins but not to DNA.

J Biol Chem, 1985 Aug 5, 260(16), 9336 - 45
Monoclonal antibodies as probes for the complexity, phylogeny, and chromatin distribution of high mobility group chromosomal proteins 1 and 2; Vanderbilt JN et al.; Monoclonal antibodies were prepared against the high mobility group (HMG) proteins 1, 2a, and 2b from hen erythrocyte chromatin . One antibody that recognized multiple sites along HMG-1, -2a, and -2b reacted strongly with HMG proteins from all vertebrates tested . In contrast, five antibodies that detected unique epitopes on chicken HMG-1 and -2a recognized antigenic sites that exhibited restricted phylogenic distributions . The differential reactivity of these antibodies on vertebrate proteins was in agreement with traditional taxonomy in that the avian HMGs were most closely related to those from reptiles and less related to those from mammals, amphibians, bonyfish, and especially the jawless fish . Mononucleosomes generated by mild digestion of erythrocyte chromatin with micrococcal nuclease were highly enriched in HMG-2a . One antigenic determinant located within the N-terminal domain of HMG-2a was freely accessible to its antibody when the protein was bound to these mononucleosomes . In contrast, two antibodies that recognized determinants in the central region of HMG-2a exhibited little chromatin binding activity . The masking of the central domain by DNA binding was presumably not responsible for these results because all three determinants were available for antibody binding when HMG-2a was bound to DNA in vitro . Therefore, the central region of HMG-2a may be masked from antibody binding by protein-protein interactions in chromatin.

J Steroid Biochem, 1985 Aug, 23(2), 137 - 43
Physical-chemical properties of the estrogen receptor solubilized by micrococcal nuclease; Geier A et al.; The physical-chemical properties of the nuclear estrogen receptor from MCF-7 cells were determined . The receptor was solubilized by micrococcal nuclease . Nuclei were isolated from cells previously exposed to 10 nM {3H}estradiol . The amount of receptor released was parallel to the extent of chromatin solubilized, which suggested that the receptor is homogeneously distributed on the chromatin . Following mild nuclease digestion the excised receptor sedimented as an abundant 6-7 S form and as a less abundant approximately 12 S species . The 6-7 S form represented the receptor excised in association with linker DNA, while the approximately 12 S may represent receptor bound to linker DNA which remained associated with the nucleosome . Increasing the extensiveness of digestion resulted in one receptor form sedimenting at 5.6 S . Additional digestion with DNase I did not affect the sedimentation coefficient of the receptor . Sedimentation of the micrococcal nuclease hydrolysate in a 0.4 M KCl sucrose gradient resulted in a 4.2 S receptor form . The same receptor form was extracted from undigested nuclei with 0.4 M KCl . Using Sephadex G-200 column chromatography we have determined the Stokes radii (Rs), molecular weight (Mr) and frictional ratio (f/fo) for the 5.6 S and 4.2 S receptor forms . For the 5.6 S form: Rs = 7.04 nm, Mr = 163,000 and (f/fo) = 1.80 . For the 4.2 S receptor, Rs = 4.45 nm, Mr = 77,000 and (f/fo) = 1.46 . The ability of the nuclease solubilized 5.6 S receptor to bind DNA was tested using DNA-cellulose column and highly polymerized DNA . About 40% of the applied receptor bound to the column and could be eluted by high salt concentrated buffer . The 5.6 S receptor form was sedimented on sucrose gradient by the highly polymerized DNA . These results suggested that the receptor is bound in chromatin as a dimer or as a monomer in association with other protein(s) which complexed it with DNA.

Cell Biol Int Rep, 1985 Aug, 9(8), 689 - 98
Chromatin organization in Paracentrotus lividus eggs; Di Liegro I et al.; After purification by buoyant density centrifugation in ethidium bromide - CsCl gradient and electrophoretic fractionation, the DNA fragments isolated from P . lividus egg nuclei incubated with micrococcal nuclease exhibit a typical oligomeric pattern . Analysis of chromatin samples digested to an increasing extent by micrococcal nuclease reveals that the structural organization of egg chromatin is heterogeneous, both in terms of repeat size and degree of sensitivity to nuclease attack . The nucleosomal repeats of P . lividus sperms and embryos up to the mesenchyme blastula stage have also been determined, for comparison.

Eur J Biochem, 1985 Aug 1, 150(3), 441 - 6
Assembly of oligonucleosomes into a limit series of multimeric higher-order chromatin structures; Muyldermans S et al.; Chicken erythrocyte chromatin, obtained after fragmentation with micrococcal nuclease, appears to remain folded in a stable distribution of supranucleosomal structures in buffers containing 80 mM NaCl . These supranucleosomal particles are composed of on average 25 nucleosomes . However, the integrity of the linker DNA within these particles is not required . The supranucleosomal particles have been interpreted by others as superbeads cut out of a preexisting granular nominal 30-nm chromatin fibre . We show that the same distribution of supranucleosomal structures (even those containing internal DNA scissions) can be reconstituted from unfolded nuclear chromatin extracts as present in 10 mM or 600 mM NaCl . Moreover, fractions of oligonucleosomes with mean lengths between 6 and 15 nucleosomes reassemble or aggregate into a limit series of multimeric species . The existence of an assembly barrier could be inferred as we were unable to observe a stable and soluble assembly product containing more than about 25 nucleosomes . We propose an alternative explanation for the generation and observation of a constant distribution of supranucleosomal structures in nuclear extracts, based on the assembly or aggregation property of oligonucleosomes and on the existence of an assembly barrier.

Mol Cell Biol, 1985 Aug, 5(8), 1901 - 9
Suppressors of Saccharomyces cerevisiae his3 promoter mutations lacking the upstream element; Oettinger MA et al.; Transcription of the Saccharomyces cerevisiae his3 gene requires an upstream promoter element and a TATA element . A strain containing his3-delta 13, an allele which deletes the upstream promoter element but contains the TATA box and intact structural gene, fails to express the gene and consequently is unable to grow in medium lacking histidine . In this paper we characterize His+ revertants of his3-delta 13 which are due to unlinked suppressor mutations . Recessive suppressors in three different ope genes allow his3-delta 13 to be expressed at wild-type levels . In all cases, the suppression is due to increased his3 transcription . However, unlike the wild-type his3 gene, whose transcripts are initiated about equally from two different sites (+1 and +12), transcription due to the ope mutations is initiated only from the +12 site, ope-mediated transcription is regulated in a novel manner; it is observed in minimal medium, but not in rich broth . Although ope mutations restore wild-type levels of transcription, his3 chromatin structure, as assayed by micrococcal nuclease sensitivity of the TATA box, resembles that found in the his3-delta 13 parent rather than in the wild-type strain . This provides further evidence that TATA box sensitivity is not correlated with transcriptional activation . ope mutations are pleiotropic in that cells have a crunchy colony morphology and lyse at 37 degrees C in conditions of normal osmolarity . ope mutations are allele specific because they fail to suppress five other his3 promoter mutations . We discuss implications concerning upstream promoter elements and propose some models for ope suppression.

Biochemistry, 1985 Jul 30, 24(16), 4435 - 50
Structure of subnucleosomal particles . Tetrameric (H3/H4)2 146 base pair DNA and hexameric (H3/H4)2(H2A/H2B)1 146 base pair DNA complexes; Read CM et al.; The tetrameric (H3/H4)2 146 base pair (bp) DNA and hexameric (H3/H4)2(H2A/H2B)1 146 bp DNA subnucleosomal particles have been prepared by depletion of chicken erythrocyte core particles using 3 or 4 M urea, 250 mM sodium chloride, and a cation-exchange resin . The particles have been characterized by cross-linking and sedimentation equilibrium . The structures of the particles, particularly the tetrameric, have been studied by sedimentation velocity, low-angle neutron scattering, circular dichroism, optical melting, and nuclease digestion with DNase I, micrococcal nuclease, and exonuclease III . It is concluded that since the radius of gyration of the DNA in the tetramer particle and its maximum dimension are very close to those of the core particle, no expansion occurs on removal of all the H2A and H2B . Nuclease digestion results indicate that histones H3/H4 in the tetramer particle protect a total of 70 bp of DNA that are centrally located within the 146 bp . Within the 70 bp DNA length, the two terminal regions of 10 bp are, however, not strongly protected from digestion . The optical melting profile of both particles can be resolved into three components and is consistent with the model of histone protection of DNA proposed from nuclease digestion . The structure proposed for the tetrameric histone complex bound to DNA is that of a compact particle containing 1.75 superhelical turns of DNA, in which the H3 and H4 histone location is the same as found for the core particle in chromatin by histone/DNA cross-linking {Shick, V . V., Belyavsky, A . V., Bavykin, S . G., & Mirzabekov, A . D . (1980) J . Mol . Biol . 139, 491-517} . Optical melting of the hexamer particle shows that each (H2A/H2B)1 dimer of the core particle protects about 22 base pairs of DNA.

Biochem Biophys Res Commun, 1985 Jul 16, 130(1), 364 - 71
Presence of nucleosomal repeat in the transcribed alpha globin gene of induced murine erythroleukemia cells; Kirov N et al.; The sensitivity of the mouse alpha-globin gene to micrococcal nuclease and its nucleosomal repeat were studied in three different functional states of the gene: inactive in EAT cells, potentially active in uninduced MEL cells and active in induced MEL cells . The results show that: 1 . The nuclease sensitivity of the gene differs in the three different functional states . 2 . Both the coding and the 5'-flanking regions of the induced actively transcribed gene show a typical nucleosomal repeat pattern . 3 . Hypersensitive sites for micrococcal nuclease and for an endogenous nuclease appear upstream of the gene after induction of differentiation.

J Biochem (Tokyo), 1985 Jul, 98(1), 105 - 16
Biosynthesis of UDP-N-acetyl-D-glucosaminuronic acid and UDP-N-acetyl-D-mannosaminuronic acid in Micrococcus luteus; Kawamura T et al.; The occurrence and formation of UDP-N-acetyl-D-glucosaminuronic acid (UDP-GlcNAcA) and UDP-N-acetyl-D-mannosaminuronic acid (UDP-ManNAcA) were studied in Micrococcus luteus ATCC 4698 . UDP-N-acetylhexosaminuronic acid separated from D-cycloserine-inhibited cells was shown to be a mixture of UDP-GlcNAcA and UDP-ManNAcA in the ratio of 87:13, whereas that obtained from untreated cells was a 96:4 mixture of these two nucleotides . Crude enzyme preparations obtained from the supernatant fraction of cells catalyzed the NAD+-dependent conversion of UDP-GlcNAc into UDP-GlcNAcA and UDP-ManNAcA . Studies on the partial separation and properties of enzymes revealed that UDP-GlcNAcA is synthesized directly from UDP-GlcNAc by the action of UDP-GlcNAc dehydrogenase and that UDP-ManNAcA is synthesized from UDP-GlcNAc through the successive actions of UDP-GlcNAc 2-epimerase and UDP-ManNAc dehydrogenase . However, enzymatic conversion of UDP-GlcNAcA to UDP-ManNAcA was not detected . Ammonium sulfate protects both dehydrogenases from inactivation during storage and incubation . Partially purified UDP-GlcNAc dehydrogenase required dithiothreitol and the particulate fraction for its full activity . The apparent Km values of UDP-GlcNAc dehydrogenase for UDP-GlcNAc and NAD+ were 0.28 and 1.43 mM, respectively . The optimum pH of this enzyme was higher than 9 in Tris-HCl buffer . p-Chloromercuribenzoate at 27 microM as well as 10 mM ethanol almost completely inhibited the UDP-GlcNAc dehydrogenase reaction.

Biokhimiia, 1985 Jul, 50(7), 1132 - 40
{Aggregation of fragmented chromatin associated with the synthesis of products of its treatment with nuclease}; Lobanenkov VV et al.; Isolated cell nuclei were incubated with nucleases followed by extraction of chromatin with a low salt buffer . With an increase of nuclear chromatin degradation with DNAse I or micrococcal nuclease, solubilization of deoxyribonucleoprotein (DNP) by a low salt buffer increases, reaching a maximum upon hydrolysis with 2-4% nuclear DNA and then decreases appreciably after extensive treatment with nucleases . Soluble fragmented chromatin aggregates in the course of treatment with DNAase . I . Addition to gel chromatin preparations of exogenous products of nuclease treatment of isolated nuclei leads to its aggregation . Pretreatment of nuclear chromatin with RNAase prevents solubilization of DNP by low ionic strength solutions . Some experimental data obtained with the use of severe nuclease treatment are discussed; for a correct interpretation of these data the aggregation of fragmented chromatin by products of its nuclease degradation should be taken into consideration.

Arch Biochem Biophys, 1985 Jul, 240(1), 464 - 9
Formation of single-stranded regions in the course of digestion of DNA with DNAase II and micrococcal nuclease; Galcheva-Gargova Z et al.; In the course of digestion of DNA with DNAase II or micrococcal nuclease, considerable amounts of single-stranded (ss) regions are formed, as determined by a second digestion with ss-specific nucleases, hyperchromicity measurements, and electron microscopy . Most of the ss stretches are located internally in the DNA molecules . The effect appears to be related to regions of decreased stability arising around single-stranded cuts in the double helix.

Proc Natl Acad Sci U S A, 1985 Jul, 82(13), 4351 - 5
Isolation and characterization of two fractions from HeLa cells required for mRNA splicing in vitro; Furneaux HM et al.; A nuclear extract from HeLa cells has been separated by DEAE-cellulose chromatography into two fractions, both of which are required for mRNA splicing in vitro . Both fractions are heat labile and sensitive to N-ethylmaleimide . The activity of one of the fractions was abolished by preincubation with micrococcal nuclease, while the other fraction was unaffected by this treatment . This abolition indicates an essential nucleic acid component . Fractions I and II are required for the in vitro splicing of human beta-globin and adenovirus transcripts.

Mol Biol (Mosk), 1985 Jul-Aug, 19(4), 926 - 35
{Structuro-functional organization of the SV40 virus chromosome . IV . Specific organization of regulatory sequences of the SV40 genome in the mini-chromosome}; Shakhov AN et al.; Using previously described technique of hybridization end-labeling, we analysed nucleosomal organization of the regulatory region of SV40 minichromosome . We showed that DNAase II, in spite of certain specificity observed on the naked DNA, cut the minichromosome in a highly specific manner with the major hypersensitive site inside the enhancer . This hypersensitivity and that to micrococcal nuclease were not found when the chromosome of mature SV40 virions was tested.

Biochem Biophys Res Commun, 1985 Jun 28, 129(3), 645 - 50
Structural studies on Allium cepa L . chromatin: enhanced stability of internucleosome interactions in plant chromatin; Moreno ML et al.; The pattern of micrococcal nuclease digestion of chromatin from different organs of Allium cepa has been studied . The DNA from small oligonucleosomes appears to be highly degraded and heterogeneous . In solutions of intermediate ionic strength (0.15 M NaCl) histones H2A and H2B form dimers, however at high salt concentrations (2 M NaCl) they tend to form complexes of higher order, such as tetramers . It is proposed that a correlation exists between the ability of these proteins to form tetramers and the particular stability of internucleosome interactions.

Biochim Biophys Acta, 1985 Jun 18, 840(2), 280 - 6
Purification and kinetic properties of polynucleotide kinase from rat testes; Bosdal T et al.; Polynucleotide kinase (EC 2.7.1.78) has been purified from rat testes, and an approximately 2000-fold purification was obtained . The purified enzyme had an Mr of 38000 +/- 3800 . The enzyme phosphorylated micrococcal nuclease-treated calf thymus DNA and (dT)10 while 5'-HO-tRNA was a very poor substrate . A certain degree of specificity towards purine-containing 5'-HO-nucleotides was observed . The polynucleotide kinase had an absolute requirement for a divalent cation . Both Mg2+ and Mn2+ could be used, but 10 mM MgCl2 gave optimal activity . The monovalent cations Na+, K+ and NH4+ all stimulated enzyme activity, and the optimal concentration was 0.1 M . The enzyme was inhibited by inorganic phosphate, pyrophosphate and sulphate . A 50% inhibition was obtained with 20, 0.3 and 2 mM, respectively . At 2 mM MgCl2, 1 mM spermine enhanced the enzyme activity 3-times . The apparent KATP was estimated to be 36 microM and KHO-DNA was found to be 2 microM.

Mol Cell Biol, 1985 Jun, 5(6), 1287 - 94
Localization of specific rDNA spacer sequences to the mouse L-cell nucleolar matrix; Bolla RI et al.; Mouse L-cell nucleoli were isolated from sonicated nuclei by centrifugation and extensively treated with pancreatic DNase or micrococcal nuclease to obtain "core nucleoli." Core nucleoli still contained the precursors to rRNA and about 1% of the total nuclear DNA, which remained tightly bound even after the removal of some chromatin proteins with 2 M NaCl . The core nucleolar DNA electrophoresed in a series of discrete bands, 20 to about 200 base pairs in length . Hybridization tests with specific DNA probes showed that the DNA was devoid of sequences complementary to mouse satellite, mouse Alu-like, and 5S RNA sequences . It also lacked sequences coding for cytoplasmic rRNA species, since it did not hybridize to the 18S to 28S portion of rDNA in Northern blot analyses and none of it was protected by hybridization to a 100-fold excess of total cytoplasmic RNA in S1 nuclease assays . However, the core nucleolar DNA did hybridize to nontranscribed and external transcribed spacer rDNA sequences . We infer that specific portions of rDNA are protected from DNase action by a tight association with nucleolar structural proteins.

Mol Cell Biol, 1985 Jun, 5(6), 1220 - 8
Specific regions of beta-globin RNA are resistant to nuclease digestion in RNA-protein complexes in chicken reticulocyte nuclei; Patton JR et al.; The interaction between beta-globin RNA and proteins in chicken reticulocyte nuclei was studied by determining the sequence of nuclease-resistant beta-globin RNA . Two types of nuclease-resistant RNAs were isolated for this study: endogenous nuclease-resistant RNA from 50S heterogeneous nuclear RNA-protein complexes and micrococcal nuclease-resistant nuclear RNA from whole nuclei . The nuclease-resistant regions were identified with the use of a RNA mapping method we recently developed (J.R . Patton and C.-B . Chae, J . Biol . Chem . 258:3991-3995, 1983) . We found that beta-globin RNA is assembled into heterogeneous nuclear RNA-protein complexes in a specific manner . There are several regions of nuclease resistance in the first and third exons interrupted at regular intervals by sensitive regions . The second exon has only one nuclease-resistant region . The resistant regions range in size from 20 to 50 nucleotides . This organization may reflect a specific mode of assembly for heterogeneous nuclear RNA-protein complexes.

Cell Differ, 1985 Jun, 16(4), 235 - 40
DNA repeat size in chick embryonic lens epithelium, lens fiber, brain and liver cell nuclei; Muel AS et al.; The DNA repeat size is determined by micrococcal nuclease digestion kinetics and subsequent electrophoresis of the products among various chick embryonic tissues . The repeat size is found to be not significantly different from 193 to 197 bp, for brain and liver at 11 days and for lens epithelium and fiber at different embryonic stages . However, the pattern of micrococcal digestion seems to reveal an overall chromatin modification as a function of development in the lens fibers.

Br J Surg, 1985 Jun, 72(6), 440 - 2
Significance of positive bacterial cultures from aortic aneurysm contents; Buckels JA et al.; Aneurysm contents were cultured in 275 patients out of a series of 546 cases undergoing infrarenal aortic aneurysm repair between 1961 and 1981 . The incidence of positive cultures was 8 per cent . Cultures were more likely to be positive if taken from ruptured (16.7 per cent) and acute (9.1 per cent) aneurysms than from elective (4.2 per cent) cases (X2 = 6.69, P less than 0.01) . Gram-positive organisms predominated with Micrococcus being the commonest isolate . Positive cultures were seen at an annual rate of 1-3 cases up to 1976 since which time all have been negative and we believe this may be due to prophylactic antibiotics being given preoperatively rather than postoperatively . The incidence of subsequent graft sepsis was greater in patients with positive aneurysm contents cultures (7 out of 22) than in those with negative cultures (6 out of 253) (X2 = 32.7, P less than 0.001) . We recommend the routine culture of aneurysm contents to identify patients who are at high risk of developing graft sepsis and suggest that those cases with positive cultures receive prolonged organism-specific antibiotic therapy . In addition, there is evidence that pre-operative antibiotics may eliminate organisms from aneurysms, thus reducing the subsequent risk of graft sepsis.

Mol Cell Endocrinol, 1985 Jun, 41(1), 45 - 59
Binding of the glucocorticoid receptor complex to the nucleosomal core in the P1798 mouse lymphosarcoma; Ip MM et al.; Binding of the glucocorticoid receptor complex to nucleosomes has been studied using the mouse P1798 lymphosarcoma . Cells were incubated with {3H}triamcinolone acetonide (TA), and nuclei prepared and digested with 3 different concentrations of micrococcal nuclease . After fractionation with EDTA and NaCl, it was observed that {3H}TA bound with similar specific radioactivity to mononucleosomes containing both core and linker DNA, of 183 +/- 5, and 168 +/- 4 base pair lengths, respectively, as well as to core size DNA, of 148 +/- 3 base pair length, suggesting that the glucocorticoid receptor bound to the core portion of the nucleosome . Steroid binding was found to be associated with regions of the nucleosome that were depleted in histone H1 and enriched in high mobility group (HMG) proteins 1 and 2; only negligible binding was noted in nucleosomes enriched in histone H1 and depleted in HMG proteins . In addition to binding to core nucleosomes, the glucocorticoid receptor complex was also shown to bind to a fraction sedimenting at 5-6 S on sucrose gradients characterized by subnucleosome and mononucleosome size DNA, as well as by core histones . While binding of the steroid receptor complex to linker regions of the nucleosome cannot be ruled out, this data would appear to present the first concrete evidence that glucocorticoid binding, at least in the P1798 lymphosarcoma, is to core nucleosomes . Some caution in interpretation of the results is indicated, however, on 2 points: (1) receptor redistribution during nuclease digestion cannot be ruled out; (2) only the binding of a small proportion of the steroid receptor complex may be physiologically relevant.

J Neurochem, 1985 Jun, 44(6), 1799 - 808
Experimental methyl mercury neurotoxicity: locus of mercurial inhibition of brain protein synthesis in vivo and in vitro; Cheung MK et al.; Brain cell-free protein synthesis is inhibited by methyl mercury chloride (MeHg) following in vivo or in vitro administration . In this report, we have identified the locus of mercurial inhibition of translation . Intraperitoneal injection of MeHg (40 nmol/g body wt) induced variable inhibition of amino acid incorporation into the post-mitochondrial supernatant (PMS) harvested from the brain of young (10-20-day-old) rats . No mercurial-induced disaggregation of brain polyribosomes nor change in the proportion of 80S monoribosomes was detected on sucrose density gradients . No difference in total RNA was found in the PMS . Initiation complex formation was stimulated by MeHg, as detected by radiolabelled methionine binding to 80S monoribosomes following continuous sucrose density gradient centrifugation . After micrococcal nuclease digestion of endogenous mRNA, both in vivo and in vitro MeHg inhibited polyuridylic acid-directed incorporation of {3H}phenylalanine . However, the in vivo inhibition was no longer observed when {3H}phenylalanyl-tRNAPhe replaced free {3H}phenylalanine in the incorporation assay . The formation of peptidyl{3H}puromycin revealed no difference from controls . There was significant mercurial inhibition of phenylalanyl-tRNA Phe synthetase activity in pH 5 enzyme fractions derived from brain PMS of MeHg-poisoned rats . These experiments revealed that the apparent MeHg inhibition of brain translation in vivo and in vitro is due primarily to perturbation in the aminoacylation of tRNA and is not associated with defective initiation, elongation, or ribosomal function.

J Biol Chem, 1985 May 25, 260(10), 5942 - 9
Characterization of an RNase P activity from HeLa cell mitochondria . Comparison with the cytosol RNase P activity; Doersen CJ et al.; A ribonuclease P-like activity was partially purified from HeLa cell mitochondria by DEAE-cellulose and octyl-Sepharose chromatography . RNase P-like activity can be quantitatively recovered from intact mitochondrial preparations treated with micrococcal nuclease, strongly suggesting that the enzyme is localized within the organelles . Mitochondrial RNase P (mtRNase P) cleaves the precursor to Escherichia coli suppressor tRNATyr at the same site as E . coli RNase P, producing the mature 5'-end of tRNATyr . The sensitivity of mtRNase P to pretreatment with nucleases or Pronase indicates that the enzyme has essential RNA and protein components . Although the ionic requirements of mtRNase P are similar to those of the RNase P activity isolated from the post-mitochondrial cytosol fraction, the chromatographic properties of mtRNase P are distinct . Mitochondrial RNase P is probably a part of the mitochondrial RNA processing machinery of mammalian mitochondria, being responsible for the endonucleolytic cleavage of the RNA transcripts at the 5'-side of the tRNA sequences.

Biochim Biophys Acta, 1985 May 24, 825(1), 80 - 8
IgG binding enhances DNAase I sensitivity of N-acetoxy-N-2-acetylaminofluorene-modified phi X-174 RF DNA; Bases R et al.; DNA restriction fragments of phi X-174 RF were modified with the carcinogen, N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) . Immune complexes of 5'-32P-labeled AAF-modified DNA and rabbit immunoglobulin (IgG) against AAF-guanosine were specifically bound by surface membranes of Cowan I strain micrococci whose protein A binds the Fc portion of IgG . DNAase I sensitivity of the bound DNA was 20-fold greater than in solution, but the normal pattern of hydrolysis was not altered, as determined in sequencing gels . Nonadducted DNA ligated to AAF-modified DNA acquired the enhanced sensitivity to DNAase I hydrolysis when the ligation hybrid was immunobound.

Eur J Biochem, 1985 May 2, 148(3), 529 - 32
Anomalous nuclease digestion of Holothuria sperm chromatin containing histone H1 variants; Azorin F et al.; We have investigated the micrococcal nuclease digestion of chromatin from the spermatozoa of the sea cucumber Holothuria tubulosa . This chromatin contains minor protein variants related to histone H1 with a high proportion of basic amino acids . One of these variants, protein phi 0, represents about 4% of the total histones . It is 78 amino acids long and its amino acid composition and sequence are related to the very basic C-terminal region of histone H1 . The presence of these proteins induces an unusual digestion pattern . Oligonucleosomal particles which are soluble at 150 mM NaCl are depleted of protein phi 0 and they are also defective in histone H1 . A low percentage of the insoluble material can be solubilized at lower NaCl concentrations (50 mM) . These oligonucleosomal particles show a very peculiar protein content, since at early digestion times, they contain histone H1 and protein phi 0 exclusively . We conclude that these particles arise from a cooperative displacement of core histones by protein phi 0 and histone H1 . These results show that minor changes in histone H1 complement can result in the formation of artifactual particles upon microccocal nuclease digestion . These observations may be of interest in other systems which contain H1 variants.

Proc Natl Acad Sci U S A, 1985 May, 82(9), 2769 - 73
Preferential binding of benzo{a}pyrene diol epoxide to the linker DNA of human foreskin fibroblasts in S phase in the presence of benzamide; Kurian P et al.; Addition of benzamide (BZ) at the onset of S phase inhibited expression of the neoplastic phenotype in human foreskin fibroblasts treated in vitro with (+/-)-7 alpha,8 beta-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene (B{a}P diol epoxide) in early S phase . Analysis of the specific B{a}P diol epoxide-DNA adducts revealed that ca . 65% of the total adducts in BZ and non-BZ carcinogen-treated cells was the B{a}P diol epoxide-deoxyguanine adduct . Limited micrococcal nuclease digestion of the early S phase nuclei from cells treated with B{a}P diol epoxide indicated that the carcinogen binds equally to linker and core DNA . However, when the cells were predominantly in S phase, in the presence of BZ, there was ca . three times more binding of B{a}P diol epoxide to the linker DNA compared to the core region . The confluent cells in G1 cell arrest treated only with B{a}P diol epoxide also bound the carcinogen preferentially to the linker region . These data indicate that pretreatment of the cells with BZ at the onset of S phase established a preferential binding pattern in the linker DNA similar to that observed in the cells treated with B{alpha}P diol epoxide in G1 arrest.

Proc Natl Acad Sci U S A, 1985 May, 82(9), 2642 - 6
Differential scanning calorimetry of nuclei reveals the loss of major structural features in chromatin by brief nuclease treatment; Touchette NA et al.; Differential scanning calorimetry revealed that chromatin melts in four distinct transitions in intact HeLa nuclei at 60 degrees C, 76 degrees C, 88 degrees C, and 105 degrees C . Calorimetry of whole cells was characterized by the same four transitions along with another at 65 degrees C, which was probably RNA . Isolated chromatin, however, melted in only two transitions at 72 degrees C and 85 degrees C . Very brief digestion of HeLa nuclei with either micrococcal nuclease or DNase I resulted in the conversion of a structure that melted at 105 degrees C to one that melted at 88 degrees C . Further digestion with micrococcal nuclease to the level of the mononucleosome did not result in any further substantial changes in either enthalpy or melting temperatures . In contrast, further DNase I digestion that resulted in cleavage within the nucleosome produced a pronounced shift in melting temperatures to broad transitions at 62 degrees C and 78 degrees C.

J Histochem Cytochem, 1985 May, 33(5), 389 - 99
Silver staining of the nucleolar organizer regions (NORs) on Lowicryl and cryo-ultrathin sections; Moreno FJ et al.; Nucleolar organizer region (NOR) silver staining was applied to sections of fixed material . A positive reaction on cryo-ultrathin sections was found as well as on semithin and ultrathin Lowicryl sections . Repeatable staining that was easy to control was obtained by a one-step procedure after aldehyde-Carnoy fixation . Fixation of the material by formaldehyde and glutaraldehyde alone in cacodylate buffer also maintained reaction selectivity when ammonium chloride was used after fixation . Enzymatic digestion by pronase, RNase A, DNase I, or micrococcal nuclease was applied to ultrathin Lowicryl sections . Pronase digestion removed the silver-stained proteins, whereas digestion by the nucleases did not . A routine procedure is proposed for easy NOR silver staining of sections that preserves a good tissue ultrastructure and is also compatible with cytochemical and immunological investigations.

Nucleic Acids Res, 1985 Apr 25, 13(8), 2843 - 53
The involvement of histone H1{0} in chromatin structure; Roche J et al.; Micrococcal nuclease digestion and light scattering are used to compare native chromatins with various histone H1{0} contents . The experimental data show that the higher the H1{0} content, the greater the ability to form compact structures with increasing ionic strength, and the lower the DNA accessibility to micrococcal nuclease . On the contrary, reconstituted samples from H1-depleted chromatin and pure individual H1 fractions behave in such a way that samples reconstituted with pure H1 degree give rise to a looser structure, more accessible to nuclease than samples reconstituted with H1-1 . This contradiction suggests that the effect of H1o on chromatin structure must originate from the interaction of this histone with other components in native chromatin among which other histone H1 subfractions are good candidates.

J Biol Chem, 1985 Apr 25, 260(8), 4558 - 60
Studies on nuclease digestion of chromatin phosphorylated in vivo; West MH et al.; We have previously shown that, by culturing cells in hypertonic media, histone 2A becomes hyperphosphorylated (Pantazis, P., West, M . H . P., and Bonner, W . M . (1984) Mol . Cell . Biol . 4, 1186-1188) . In the present study we have probed the effect of this histone modification on the overall chromatin structure by micrococcal nuclease and DNase I digestion . Although no significant quantitative differences in the extent of hydrolysis were observed between control and hyperphosphorylated chromatin by micrococcal nuclease, DNase I digested hyperphosphorylated chromatin at a 3- to 4-fold higher rate than unmodified chromatin.

Biochem Biophys Res Commun, 1985 Apr 16, 128(1), 226 - 32
The association of human c-Ha-ras sequences with chromatin and nuclear proteins; Kasid UN et al.; As a step towards the understanding of possible relationship between chromatin organization and regulation of the oncogene expression, we have investigated the chromatin structure of one of the more frequently activated oncogenes, c-Ha-ras, in HeLa-S3 cells . This was accomplished by isolation of the chromatin fractions (soluble and insoluble) after micrococcal nuclease digestion of purified nuclei and probing for the distribution of ras sequences . The polynucleosomal fraction was further resolved by sucrose gradient sedimentation . Southern-blot hybridization of the DNA isolated from various fractions yielded following results: (1) c-Ha-ras sequences segregated predominantly in the lysate fraction . (2) Unlike the B-globin (transcriptionally inactive) sequences, ras-H associated chromatin lacked typical nucleosomal packaging . Furthermore, since post-translational modifications of nuclear proteins have been suggested to modulate the nucleosome structure during DNA transcription and replication, ras sequences, in polynucleosomes immunofractionated on anti-poly (ADP-Ribose) Sepharose were also examined . The data suggested that the major class of this oncogene sequence exists in chromatin more distal to the sites of this particular chromatin modification.

Nucleic Acids Res, 1985 Apr 11, 13(7), 2569 - 82
Efficient modification of E . coli RNA polymerase in vitro by the N gene transcription antitermination protein of bacteriophage lambda; Goda Y et al.; The N gene protein of bacteriophage lambda prevents termination of transcription by E . coli RNA polymerase . We describe here the conditions of a cell-free reaction system in which pure N stimulates net transcription up to tenfold and therefore nearly stoichiometrically modifies transcribing RNA polymerase molecules . The reaction contains micrococcal nuclease-treated S100 extract derived from E . coli and a plasmid template DNA containing the lambda early promoter PL, the N utilization site nutL, and the Rho-dependent terminator tL1 . Stimulation by N in this system is specific and biologically relevant since it is absent with vector pBR322 DNA and with extracts derived from E . coli strains bearing the nusA1 and nusE71 mutations known to block N function in vivo . We use the system to provide further evidence that ribosomes are not necessary for N function and to demonstrate the direct involvement in N function of the NusA protein of E . coli.

Radiat Res, 1985 Apr, 102(1), 119 - 29
Sequence specificity of DNA cleavage by Micrococcus luteus gamma endonuclease; Hentosh P et al.; DNA fragments of defined sequence have been used to determine the sites of cleavage by gamma-endonuclease activity in extracts prepared from Micrococcus luteus . End-labeled DNA restriction fragments of pBR322 DNA that had been irradiated under nitrogen in the presence of potassium iodide or t-butanol were treated with M . luteus gamma endonuclease and analyzed on high resolution, denaturing, polyacrylamide gels . Gamma endonuclease was found to cleave irradiated DNA preferentially at the positions of cytosines and thymines . DNA cleavage occurred immediately to the 3' side of pyrimidines in irradiated DNA and resulted in fragments that terminate in a 5'-phosphoryl group . These studies indicate that both altered cytosines and thymines may be important DNA lesions requiring repair after exposure to gamma radiation.

Exp Cell Res, 1985 Apr, 157(2), 504 - 10
Influence of histone H5 on mononucleosome structure during differentiation in the avian erythroid series; Haye KR et al.; Changes in nucleosome repeat length during avian erythroid development have been previously correlated with changes in H5 content . In order to determine the effects of H5 on the length of DNA in mononucleosomal particles as a function of differentiation, a two-dimensional electrophoretic system was used to analyse DNA and histones of particles generated by micrococcal nuclease digestion of nuclei from several stages of erythroid development . Although the relative proportions of H5- to H1-containing mononucleosomes increased during development, only in mature erythrocytes did H5 protect a greater length of linker DNA from micrococcal nuclease digestion than did H1 . These results suggest that changes in average nucleosome repeat length during erythroid development can be attributed only partially to an increase in the proportion of H5-containing nucleosomes which contribute to this average.

Cell, 1985 Apr, 40(4), 923 - 32
Structure of the two distinct types of minichromosomes that are assembled on DNA injected in Xenopus oocytes; Ryoji M et al.; DNA injected into germinal vesicles of Xenopus oocytes is assembled into two distinct types of minichromosomes . One type is soluble and behaves like conventional nucleosomal chromatin . The other type is insoluble, is sensitive to DNAase I and to micrococcal nuclease, lacks a canonical nucleosome repeat, and generates a half-nucleosome size limit digest with micrococcal nuclease . We suggest that these peculiar minichromosomes may be the ones that display the unconstrained, "dynamic" DNA supercoils in the living oocyte.

Exp Cell Res, 1985 Apr, 157(2), 307 - 14
Reverse differential staining of sister chromatids induced by Hoechst plus black light and endonuclease; Jan KY et al.; A differential Giemsa staining between sister chromatids was obtained by treating chromosomes replicated twice in medium containing 5-bromodeoxyuridine (BrdU) with Hoechst 33258 plus black light at 55 degrees C (HB pretreatment) and deoxyribonuclease (DNase) I, II, or micrococcal nuclease . In this staining pattern the BrdU bifilarly substituted chromatids were darkly and the unifilarly substituted chromatids lightly stained . This staining pattern was obtained only by staining the HB-DNase I-treated chromosomes with Giemsa and methylene blue, not by several other dyes tested . Relatively more DNA labelling was removed from the non-BrdU-substituted than the BrdU-substituted chromosomes, when the HB-pretreated chromosomes were digested with DNase I . But the protein labelling was not removed appreciably in the same treatment . The differential DNase I sensitivity between the non-BrdU-substituted and BrdU-substituted chromosomes disappeared when the HB-pretreated chromosomes were incubated with proteinase K before The DNase I digestion . Moreover, no differential DNase I sensitivity was found between the HB-pretreated isolated DNA containing and not containing BrdU . We propose that during the HB pretreatment, more DNA-protein cross-linkings are induced in BrdU bifilarly substituted than the unifilarly substituted chromatids . This structure protects the chromosomal DNA against the DNase I digestion . Thus, a reverse differential Giemsa staining between sister chromatids is obtained by the HB-DNase I treatment.

Nucleic Acids Res, 1985 Mar 25, 13(6), 1977 - 95
N-Butyrate incubation of immature chicken erythrocytes preferentially enhances the solubility of beta A chromatin; Ferenz CR et al.; The solubility of adult beta-globin chromatin (beta A chromatin) from immature chicken red blood cells can be controlled by the presence or absence of n-butyrate in a cell incubation medium . In the absence of n-butyrate, only a small percentage (approximately 4%) of the total beta A chromatin is in a soluble chromatin fraction following micrococcal nuclease digestion and centrifugation . This percentage increases to approximately 40-45% of the beta A chromatin if cells are incubated 1 hour in the presence of 10 mM sodium n-butyrate . The highest yield and enrichment of solubilized beta A chromatin is attained when 1-4% of the DNA is rendered acid soluble, and in buffers containing 1.5 - 5 mM MgCl2 . The soluble beta A nucleohistone is nucleosome oligomer size (contains DNA 250-600 bases in length) and can be separated from soluble, transcriptionally inert mononucleosomes by agarose A-5m exclusion chromatography . The enhanced solubility appears to be specific for transcriptionally active chromatin . Whereas 40-45% of the beta A chromatin is recovered in the supernatant fraction from n-butyrate incubated immature erythrocytes, nucleohistone containing ovalbumin DNA sequences remains insoluble.

Eur J Biochem, 1985 Mar 15, 147(3), 575 - 80
Lack of nucleosomal structure in a DNase-I-solubilized transcriptionally active chromatin fraction of Physarum polycephalum; Czupryn M et al.; Light treatment of nuclei of Physarum polycephalum microplasmodia with DNase I, at low MgCl2 concentration (less than or equal to 3% DNA acid solubility, 0.1 mM MgCl2) selectively solubilizes a defined fraction of chromatin, in the form of a macromolecular complex . This fraction (up to 15% of the total chromatin) contains a full complement of the core histones and a reduced amount of histone H1, and is enriched in the high-mobility-group type of proteins . It is preferentially associated with nascent RNA and RNA polymerase B actively engaged in transcription . Digestion of DNAase-I-solubilized chromatin by micrococcal nuclease releases a size-heterogeneous population of cleavage products, indicative of lack of a typical nucleosomal packaging . It is concluded that the procedure used allows the isolation of structurally and functionally distinct regions of Physarum chromatin.

J Biol Chem, 1985 Mar 10, 260(5), 3035 - 40
Sequential alterations in globin gene chromatin structure during erythroleukemia cell differentiation; Yu J et al.; During differentiation of murine erythroleukemia cells, adult beta-globin gene chromatin acquires site-specific, DNase I hypersensitivity and an increased sensitivity in the globin gene region toward micrococcal nuclease (MNase) digestion . The relationship of these changes in chromatin structure to globin gene activation and to cellular commitment events has been studied . Imidazole, which blocks globin gene transcription during induction does not affect the terminal differentiation of the cells nor does it prevent the acquisition of DNAse I hypersensitivity . The formation of the inducible DNase I-hypersensitive site near the globin gene accompanies the developmental events which lead to cellular differentiation independent of the transcription process . The increased MNase sensitivity of the adult beta-globin gene region, normally preceded by the acquisition of 5' DNase I hypersensitivity, was blocked by the addition of imidazole prior to but not after globin gene activation . The enhanced MNase sensitivity was not abolished by the addition of actinomycin D and, thus, reflects a part of chromatin alterations that define potential for transcription . Therefore, there is a sequential series of chromatin alterations in the globin gene region associated with murine erythroleukemia cell differentiation . The appearance of the inducible 5' DNase I-hypersensitive site precedes the onset of globin gene transcription and is strongly correlated with commitment events . The enhanced MNase sensitivity is closely related to globin gene transcription, but it is not a consequence of the transcription process . In addition, the commitment of cells to terminal differentiation is dissociable from the stimulation of globin gene transcription.

J Mol Biol, 1985 Mar 5, 182(1), 109 - 29
Nucleosome disruption precedes transcription and is largely limited to the transcribed domain of globin genes in murine erythroleukemia cells; Cohen RB et al.; We used micrococcal nuclease to separate murine erythroleukemia cell (MELC) chromatin into soluble and insoluble fractions which differ in gene content and chromatin structure . Genes that are not expressed in the erythroid lineage, such as the Ig alpha and albumin genes, distribute preferentially into the soluble rather than the insoluble fraction, and are organized into nucleosomes in both fractions . Both alpha 1- and beta maj-globin genes are enriched in the insoluble fraction and are organized into structures that are partially devoid of nucleosomes in uninduced MELC, when the genes are transcriptionally inactive . Following chemical induction of MELC and the onset of globin gene transcription, globin gene enrichment and nucleosome disruption in the insoluble chromatin fraction increase . Using seven DNA subclones that span the beta maj-globin gene we show that insolubility and nucleosome disruption are largely limited to DNA sequences lying within the transcribed domain . Non-transcribed, flanking sequences are soluble and organized into nucleosomes . In addition, the globin genes found in insoluble, non-nucleosomal chromatin contain previously engaged RNA polymerases which can elongate globin RNA chains in vitro in a pattern qualitatively and quantitatively similar to intact nuclei . These results are discussed in terms of a model for globin gene activation during erythropoeisis.

Eur J Cell Biol, 1985 Mar, 36(2), 315 - 22
The effects of sodium and magnesium-ion interactions on chromatin structure and solubility; Dixon DK et al.; The effects of sodium and magnesium-ion interactions on chromatin structure and solubility were examined in isolated mouse liver nuclei . To facilitate this study, a simple assay of chromatin structure was developed, based on the absorbances at 260 nm (A260) and 320 nm (A320) of nuclei in test solutions . By subtracting the A320 from the A260, a single "spectral index" was obtained which served as a useful, but not absolute, indicator of chromatin structure . Electron microscopy verified the validity of this approach . The results indicate that either 200 mM NaCl or 0.5 mM MgCl2 were capable of preserving the native 20 to 30 nm chromatin fiber structure . Below 200 mM NaCl, the native fiber progressively uncoiled to the 10 nm unit fiber . The presence of 0.5 mM MgCl2 inhibited this uncoiling . Only divalent cations stabilized condensed chromatin (heterochromatin) within the nucleus . Monovalent and divalent cations interacted with one another at critical concentrations and modified their individual effects on chromatin structure; e.g., 10 to 25 mM NaCl interfered with the action of 0.5 to 1.5 mM MgCl2, causing a complete loss of condensed chromatin . Maximum solubility of micrococcal nuclease-digested chromatin occurred at 10 mM NaCl, which treatment allowed the chromatin to unfold to the 10 nm fiber . However, ionic conditions that disrupted condensed chromatin but maintained the native chromatin fiber morphology still resulted in relatively high yields of soluble chromatin . Minimum solubility occurred under conditions which preserved the structure of condensed chromatin.

Ann Inst Pasteur Microbiol, 1985 Mar-Apr, 136A(2), 213 - 26
{Stomatococcus mucilaginosus: cultural and biochemical properties of 100 strains isolated from the ORL area}; Baldellon C et al.; A study of the growth characteristics and 80 biochemical tests using API System micromethods (API-50CH, API-20E, API-Zym) was carried out on 100 strains of Stomatococcus mucilaginosus isolated from the human oral cavity . Seventy-seven other enzymatic tests from the API system (AP-arylamidases, osidases and esterases) were also performed on 10 of these strains . The results of these tests were compared to those of 24 type strains of Micrococcaceae and Aerococcus viridans . A simple identification scheme for S . mucilaginosus is proposed.

Arch Biochem Biophys, 1985 Mar, 237(2), 354 - 60
Partial characterization of nuclear binding sites for retinol delivered by cellular retinol binding protein; Liau G et al.; Retinol (vitamin A alcohol), which plays an important role in the differentiation of epithelia, can be transferred to chromatin in vitro . Rat liver chromatin can accept retinol in a specific and saturable manner only when the retinol is presented as a complex with cellular retinol-binding protein (CRBP) . A partial characterization of the nuclear components responsible for accepting retinol is reported here . A preparation of solubilized chromatin isolated from liver nuclei was able to accept retinol from its complex with CRBP as described previously for nuclei and chromatin . The binding of retinol to chromatin was noncovalent . However, chromatin prepared from nuclei which were incubated with DNase I or micrococcal nuclease did not accept retinol specifically . Chromatin in the form of mono and dinucleosomes also did not accept retinol . However, treatment of nuclei with RNase did not affect the specific binding of retinol . Furthermore, it has been found that retinol was not transferred to purified double or single stranded DNA . These results are interpreted to indicate that the transfer of retinol to specific nuclear binding sites requires a higher order of chromatin structure than that occurring in nucleosome preparations.

Exp Cell Res, 1985 Mar, 157(1), 227 - 41
Isolation and characterization of nuclear particles containing rapidly labelled hnRNA and snRNA in combination with a distinct set of polypeptides of Mr 74000 and 72000; Hatzoglou M et al.; A heterogeneous RNP structure has been isolated from rat liver nuclei by a method previously used for the isolation of 30S RNP complexes carrying heterogeneous RNA (hnRNA) {1} . The RNP sediments in sucrose gradients with s-values of 70-110S . Formaldehyde-fixed preparations band at Q = 1.40 in isopycnic CsCl gradients . The RNP structure is composed of a heterogeneous population of polypeptides, prominent among which are two proteins with Mr 74000 and 72000 . It contains both rapidly labelled RNA as well as several species of snRNA, as demonstrated by double-labelling experiments and gel electrophoresis . Treatment of rats with alpha-amanitin leads to a significant decrease in the amount of recovered RNP . In the presence of 0.7 M NaCl the s-value of the complex changes from 70-110S to 40-80S . The RNP structure is stable to mild RNase A or micrococcal nuclease digestion . Transmission electron microscopy reveals the presence of a heterogeneous population of particles with a mean diameter of 300-360 A . The isolated RNP structure differs completely from the well-known monoparticle or polyparticle hnRNP complexes and from the 30S or smaller snRNP particles but could be similar to or identical with the heterogeneous complex described by Jacob et al . {29}.

Biochimie, 1985 Mar-Apr, 67(3-4), 371 - 6
Template properties of ultraviolet-irradiated poly(dC) replicated by E . coli DNA polymerase I: indication for a role of apyrimidinic-sites in UV-induced mutagenesis; Boiteux S et al.; Ultraviolet irradiation alters the template properties of poly(dC) when replicated by Escherichia coli DNA polymerase I . These effects are due to base modifications . Some of them are identified as apurinic/apyrimidinic sites (AP-sites) by their sensitivity to AP-endonuclease B purified from Micrococcus luteus, and their template properties . The rate of formation of AP-sites in poly(dC) is estimated at 3 X 10(-7) site per nucleotide per J.m-2 . Exposure of supercoiled or relaxed pBR322 DNA to UV light results also in the formation of sites sensitive to AP-endonuclease B . In this case, the rate of formation of AP-sites is the same in relaxed or supercoiled DNA: 0.3 X 10(-7) site per nucleotide per J.m-2 . The apyrimidinic sites are generated through the processing of an ultraviolet induced primary lesion . We suggest that this lesion is cytosine hydrate by its rate of decay and preferential formation in single stranded DNA . Our results suggest that AP-sites might be a minor pathway leading to UV-induced mutagenesis.

Biochemistry, 1985 Feb 26, 24(5), 1186 - 93
Solubility and structure of domains of chicken erythrocyte chromatin containing transcriptionally competent and inactive genes; Komaiko W et al.; Chromatin generated by micrococcal nuclease digestion of erythrocyte nuclei can be fractionated into two pools of differing solubility in solvents containing 0.15-0.25 M NaCl . A fixed percentage of the chromatin is soluble under these conditions, independent of the average size of the DNA in the unfractionated chromatin . Chromatin containing particular gene sequences is also distributed between soluble and insoluble fractions in a way that is independent of the average size of the starting material . However, the actual percentage of gene copies present in each fraction is not necessarily the same as for bulk chromatin . The transcriptionally active chicken erythrocyte adult beta-globin gene is more soluble than the bulk, while the ovalbumin gene in the same tissue is less soluble . These differences do not appear to be related to variations in content of RNA, core histones, or two classes of non-histone proteins . Instead, we find that the soluble chromatin pool is somewhat depleted in histones H1 and H5 and contains lower molecular weight DNA than precipitable chromatin . The soluble fraction can be made insoluble by addition of H1 . If the precipitable chromatin fraction is redigested to reduce its size and then recombined with the soluble fraction and reprecipitated, the distribution of globin gene is randomized . The results suggest that the partitioning of chromatin into soluble and insoluble pools in 0.15-0.25 M NaCl arises from redistribution of a limiting amount of histones H1 and H5 to the chromatin fractions containing the longest DNA.

Anal Biochem, 1985 Feb 15, 145(1), 137 - 43
A method for determining oligo- and poly(ADP-ribosyl)ated enzymes and proteins in vitro; Tanaka Y et al.; A new method to determine oligo- and poly(ADP-ribosyl)ated enzymes and proteins in vitro has been developed . This method is based on the facts that in Mg2+-depleted condition automodification of poly(ADP-ribose)polymerase is minimized and exogenously added acceptor protein is oligo(ADP-ribosyl)ated predominantly, and in Mg2+-fortified conditions the exogenous acceptor can be poly(ADP-ribosyl)ated . When 13 proteins, including several enzymes, were subjected to this system, dimeric bovine seminal RNase and micrococcal nuclease were found to be oligo(ADP-ribosyl)ated under Mg2+-depleted conditions but their activity was unchanged . Under Mg2+-fortified conditions however, the RNase was deactivated concomitantly with its extensive poly(ADP-ribosyl)ation . When dimeric bovine seminal RNase was monomerized in advance by treatment with dithiothreitol and urea, the enzyme lost ADP-ribose-accepting ability in spite of a significant residual enzyme activity . As used here successfully, the Mg2+-depleted and Mg2+-fortified ADP-ribosylation and subsequent chromatographic analysis of various proteins and enzymes might be an useful method for proving their oligo- and poly(ADP-ribosyl)ation.

J Mol Biol, 1985 Feb 5, 181(3), 333 - 49
Chromatin-specific hypersensitive sites are assembled on a Xenopus histone gene injected into Xenopus oocytes; Gargiulo G et al.; A cloned histone H4 gene of Xenopus laevis is efficiently transcribed after injection into germinal vesicles of X . laevis oocytes . Deletion analyses indicate that less than 140 base-pairs of 5' flanking sequences and 50 base-pairs of 3' flanking sequences are required for efficient transcription of this gene in Xenopus oocytes . Chromatin footprint analysis by a direct end-label technique reveals discrete DNase I-hypersensitive and micrococcal nuclease-hypersensitive sites at the 5' and 3' boundaries of the gene, which bracket the transcribed region of this minichromosome . The specific chromatin structure assembled around this homologous gene, together with the finding that histone genes of Drosophila melanogaster are not assembled into specific nucleoprotein structures within Xenopus oocytes, strongly suggest that sequence-specific and species-specific factors may be responsible for generating the chromatin-specific hypersensitive sites at the boundaries of active genes.

Acta Pathol Microbiol Immunol Scand {B}, 1985 Feb, 93(1), 1 - 6
Etiologic importance of coagulase-negative Micrococcaceae isolated from blood cultures; Hansen BG; Coagulase-negative Micrococcaceae from blood cultures were classified biochemically according to Baird-Parker and to Kloos & Schleifer and by means of antibiotic susceptibility testing, in an attempt to distinguish between bacterial growth due to contamination and growth due to bacteremia . S . epidermidis biotype 1 (according to Baird-Parker) accounted for c . 60% of the isolates and for c . 85% of the isolates considered to be of clinical importance . The more time- and resource-demanding classification of Kloos & Schleifer provided no further clinically useful information, as the predominant number of isolates turned out to be S . epidermidis . Isolates other than S . epidermidis (according to Kloos & Schleifer) accounted for c . 45% of the total number of isolates but for only 20% of the isolates considered of clinical importance . Resistance to multiple antibiotics was recorded more often in isolates from patients with positive clinical information than in isolates thought to be contaminators . The frequency of blood cultures contaminated with coagulase-negative Micrococcaceae was estimated at c . 1.5% . Records from 91 patients were reviewed . By correlating the clinical findings to biochemical classification, resistance types, and the massiveness of growth it was suggested that the quantitative rather than the qualitative findings are suitable for determining clinical significance of isolates of coagulase-negative Micrococcaceae from blood cultures.

Mol Cell Biol, 1985 Feb, 5(2), 342 - 51
Reconstitution of functional mRNA-protein complexes in a rabbit reticulocyte cell-free translation system; Greenberg JR et al.; A variety of evidence suggests that the cytoplasmic mRNA-associated proteins of eucaryotic cells are derived from the cytoplasm and function there, most likely in protein synthesis or some related process . Furthermore, the evidence suggests that protein-free mRNA added to a cell-free translation system should become associated with a set of proteins similar to those associated with mRNA in native polyribosomes . To test this hypothesis, we added deproteinized rabbit reticulocyte mRNA to a homologous cell-free translation system made dependent on exogenous mRNA by treatment with micrococcal nuclease . The resulting reconstituted complexes were irradiated with UV light to cross-link the proteins to mRNA, and the proteins were analyzed by gel electrophoresis . The proteins associated with polyribosomal mRNA in the reconstituted complexes were indistinguishable from those associated with polyribosomal mRNA in intact reticulocytes . Furthermore, reticulocyte mRNA-associated proteins were very similar to those of cultured mammalian cells . The composition of the complexes varied with the translational state of the mRNA; that is, certain proteins present in polyribosomal mRNA-protein complexes were absent or reduced in amount in 40S to 80S complexes and in complexes formed in the absence of translation . However, other proteins, including a 78-kilodalton protein associated with polyadenylate, were present irrespective of translational state, or else they were preferentially associated with untranslated mRNA . These findings are in agreement with previous data suggesting that proteins associated with cytoplasmic mRNA are derived from the cytoplasm and that they function in translation or some other cytoplasmic process, rather than transcription, RNA processing, or transport from the nucleus to the cytoplasm.

Exp Cell Res, 1985 Feb, 156(2), 563 - 9
Fractionation of micrococcal nuclease-digested chromatin solubilized at physiologic ionic strength; Dixon DK et al.; When mouse brain nuclei are optimally digested with micrococcal nuclease, most of the chromatin is soluble in a 180 mM salt/1 mM EDTA buffer {1} . At this ionic concentration, chromatin maintains its native structure {2} . In an attempt to selectively extract different fractions of chromatin from digested nuclei, we have examined the differential solubility of chromatin in the 180 mM salt buffer containing concentrations of MgCl2 ranging from 2 to 0 mM . The results suggest that digested chromatin may be fractionated into specific soluble chromatin fractions which correspond to nuclease-sensitive chromatin, bulk chromatin, and heterochromatin . These soluble fractions have a high molecular weight (up to 20 kbp), and contain a full complement of histones as well as a complex assortment of non-histone proteins . The residual insoluble fraction may be equivalent to a native, nuclear matrix-bound chromatin fraction.

J Bacteriol, 1985 Feb, 161(2), 602 - 8
Repair of nonreplicating UV-irradiated DNA: cooperative dark repair by Escherichia coli uvr and phr functions; Hays JB et al.; The system previously used to study recombination of nonreplicating UV-irradiated phage lambda DNA was adapted to study UV repair . Irradiated phages infected undamaged homoimmune lysogens . Pyrimidine dimer content (by treatment with Micrococcus luteus UV endonuclease and alkaline sucrose sedimentation) and a biological activity endpoint (infectivity in transfection of uvrB recA recB spheroplasts) were followed . Unless room light was excluded during DNA extraction procedures, photoreactivation (Phr function) was significant . In uvr delta phr bacteria, repair, by both assays, was very low but not zero . Even when light was totally excluded, Phr function appeared to play a role in Uvr-mediated excision repair: both dimer removal and restoration of infectivity were two to five times as efficient in uvr+ phr+ bacteria as in uvr+ delta phr bacteria . Similarly, UV-irradiated phages plated with higher efficiencies on phr+ than delta phr bacteria even under totally dark conditions . In uvr phr+ repressed infections, removal of dimers from nonreplicating DNA did not increase infectivity as much as in uvr+ infections, suggesting a requirement for repair of nondimer photoproducts by the uvrABC system.

J Appl Biochem, 1985 Feb, 7(1), 25 - 32
Topography in relation to activity of the F1-ATPase of Micrococcus lysodeikticus (M . luteus): a study using trypsin digestion and hydrophobic interaction chromatography; Pivel JP et al.; Micrococcus lysodeikticus (M . luteus) ATPase digested in a controlled manner with trypsin behaves like the native protein when chromatographed on alkyl agarose supports . The enzyme immobilized on the supports through noncovalent interaction is able to hydrolyze ATP with a specific activity similar to that of native membrane-bound ATPase . However, the response of M . lysodeikticus ATPase to the interaction with the hydrophobic columns can be modified by changing the protein-ligand ratio . These results support the notion that the catalytic site of M . lysodeikticus ATPase is not involved in the interaction with alkyl agarose, but rather that binding of the ATPase to the hydrophobic columns takes place through polypeptide or protein domains other than those which mediate binding to the native membranes, since they are very easily modified by trypsin . It is proposed that the alpha subunit plays a role in the interaction of the bacterial ATPase with hydrophobic ligands . These results are discussed in relation to the topography of the enzyme as established previously.

Nucleic Acids Res, 1985 Jan 25, 13(2), 319 - 35
Excision of uracil residues in DNA: mechanism of action of Escherichia coli and Micrococcus luteus uracil-DNA glycosylases; Delort AM et al.; Various octadeoxynucleotides containing uracil at different positions were synthesized and submitted to the action of Escherichia coli and Micrococcus luteus uracil-DNA glycosylases . A uracil residue situated at the 5'-end was excised by the M.luteus enzyme but not by the E.coli one . Uracil residues located at the ultimate and penultimate positions at the 3'-end were not cleaved by either enzymes . At the other central positions, uracil was eliminated with different initial velocities . Single stranded phi X 174 DNA fragments were used to study the influence of the sequence . Cytosine bases were deaminated to give uracil by bisulfite treatment . It was shown that the initial excision velocity of two vicinal uracil residues was decreased . The same observation was made for two uracils separated by one base . A hypothetical scheme is suggested to explain the mechanism of action of uracil-DNA glycosylases.

Nucleic Acids Res, 1985 Jan 11, 13(1), 15 - 30
A lysine-rich protein functions as an H1 histone in Dictyostelium discoideum chromatin; Parish RW et al.; Mononucleosomes released from Dictyostelium discoideum chromatin by micrococcal nuclease contained two distinctive DNA sizes (166-180 and 146 bp) . Two dimensional gel electrophoresis suggested a lysine-rich protein protected the larger mononucleosomes from nuclease digestion . This was confirmed by stripping the protein from chromatin with Dowex resin . Subsequently, only the 146 bp mononucleosome was produced by nuclease digestion . Reconstitution of the stripped chromatin with the purified lysine-rich protein resulted in the reappearance of the larger mononucleosomes . Two-dimensional gel electrophoresis showed the protein was associated with mononucleosomes . Hence, the protein functions as an H1 histone in bringing the two DNA strands together at their exit point from the nucleosome . Trypsin digestion of the lysine-rich protein in nuclei resulted in a limiting peptide of approx . 10 kilodaltons . Trypsin concentrations which degraded the protein to peptides of 12-14 kilodaltons and partially degraded the core histones did not change the DNA digestion patterns obtained with micrococcal nuclease . Thus, the trypsin-resistant domain of the lysine-rich protein is able to maintain chromatosome structure.

Neoplasma, 1985, 32(5), 521 - 8
Drug action and chromatin structure . I . Adriamycin binding to core particle of nucleosomes and subsequent enhanced DNA fragmentation induced by micrococcal nuclease; Gyapay G et al.; The relevance of the subunit structure of chromatin to the mode of action of Adriamycin in Novikoff hepatoma had been investigated . To elucidate whether drug binding takes place at random along the chromatin fiber or not Novikoff hepatoma nuclei treated with Adriamycin in vivo were digested with Micrococcal nuclease and were subsequently separated into three fractions, S1, S2 and P2 . In these chromatin fraction Adriamycin and the DNA content was determined . DNA were isolated from the above fractions and were analyzed by polyacrylamide gel electrophoresis, furthermore the S2 fraction was sedimented on sucrose density gradient . The results indicate that binding of Adriamycin takes place preferentially on the core particle inducing structural alterations . Adriamycin binding not only enhanced DNA nuclease digestion but altered the size of the fragments.

Chromosoma, 1985, 92(4), 304 - 12
Interactions of nuclear proteins with DNA, during sperm differentiation in the ram; Loir M et al.; Ram spermatid nuclei and caput epididymal sperm nuclei were prepared and treated with DTT under conditions avoiding proteolysis . Whole-mount preparations for the electron microscope were made in the presence or absence of the detergent Joy . The chromatin of the less mature, non-round spermatid nuclei displayed a nucleosomal organization that gradually disappeared at the time the histones leave the nuclei (elongating spermatids) . Digestion with micrococcal nuclease suggests that polynucleosome arrays are scarcer and more accessible to nuclease in the elongating than in the round nuclei, with increasing amounts of DNA becoming devoid of nucleosomes . In the protamine-containing nuclei (elongated spermatids), only smooth filaments were observed, which formed thick fibers by parallel aggregation . The change from a nucleosomal organization to bundles of smooth filaments appeared to result from a complex process involving the transitory presence of conspicuous "knobby fibers" that suggest a periodicity in the organization of the spermatidal proteins along the DNA molecules . X-ray diffraction patterns obtained with protamine-containing spermatid nuclei and with sperm nuclei confirm that the DNA is arranged in smoothly bent bundles of parallel molecules . No higher-order reflections that might correspond to nucleosome structures were detected in the 30-200 A region.

Prog Neuropsychopharmacol Biol Psychiatry, 1985, 9(3), 251 - 8
Effects of trichothecenes (T-2 toxin) on protein synthesis in vitro by brain polysomes and messenger RNA; Murthy MR et al.; The effects of T-2 toxin on protein synthesis were tested in two reticulocyte lysate in vitro systems pretreated with micrococcal nuclease . One of the test systems contained purified globin mRNA and was initiation dependent . The other contained rat brain polysomes and incorporated amino acids by an elongation dependent process . T-2 toxin inhibited the translation of globin mRNA at all concentrations tested, from 10(-8) M to 10(-4) M . Rat brain polysomes were much less sensitive to T-2 toxin than globin mRNA . While high concentrations of the toxin (10(-4) M) led to partial inhibition of protein synthesis by polysomes, low concentrations (10(-8) M and 10(-6) M) stimulated protein synthesis . Comparison of the above results with those obtained by other workers suggest that the T-2 toxin may inhibit not only the initiation step of translation, but also elongation and termination, depending upon the concentration of the toxin and the nature of the translation system . A similar mechanism may operate for all the trichothecene toxins that exert their effect through binding to ribosomal peptidyl transferase.

Int J Biochem, 1985, 17(3), 325 - 30
RNA polymerase activities and chromatin protein composition of rat liver during methionine deprivation and refeeding; Norell M et al.; The reversible effect of dietary methionine deficiency was studied in young adult rats . The sensitivity of nuclear chromatin to micrococcal nuclease (EC3.1.4.7) digestion and the composition of the chromatin proteins were unaffected by the dietary regimens . The specific chromatin-bound RNA polymerase II activity decreased during methionine deficiency . Refeeding of methionine for 2 days restored the activity in the nuclease-released chromatin . RNA polymerase I plus III activity remained unchanged . Total RNA polymerase activity changed with the liver wet weight which was reduced during methionine deficiency and was not restored to control level after 2 days of methionine refeeding . RNA polymerase activity was altered by methionine deficiency . The recovery was independent of major modifications of the chromatin structure and protein composition.

Radiat Environ Biophys, 1985, 24(3), 161 - 74
Cell lethality after selective irradiation of the DNA replication fork; Hofer KG et al.; It has been suggested that nascent DNA located at the DNA replication fork may exhibit enhanced sensitivity to radiation damage . To evaluate this hypothesis, Chinese hamster ovary cells (CHO) were labeled with 125I-iododeoxyuridine (125IUdR) either in the presence or absence of aphidicolin . Aphidicolin (5 micrograms/ml) reduced cellular 125IUdR incorporation to 3-5% of the control value . The residual 125I incorporation appeared to be restricted to low molecular weight (sub-replicon sized) fragments of DNA which were more sensitive to micrococcal nuclease attack and less sensitive to high salt DNase I digestion than randomly labeled DNA . These findings suggest that DNA replicated in the presence of aphidicolin remains localized at the replication fork adjacent to the nuclear matrix . Based on these observations an attempt was made to compare the lethal consequences of 125I decays at the replication fork to that of 125I decays randomly distributed over the entire genome . Regardless of the distribution of decay events, all treatment groups exhibited identical dose-response curves (D0: 101 125I decays/cell) . Since differential irradiation of the replication complex did not result in enhanced cell lethality, it can be concluded that neither the nascent DNA nor the protein components (replicative enzymes, nuclear protein matrix) associated with the DNA replication site constitute key radiosensitive targets within the cellular genome.

Microbios, 1985, 44(178), 95 - 105
Isolation and characterization of a succinylated polysaccharide from the cell wall of Micrococcus agilis; Lim S et al.; A polysaccharide consisting of rhamnose, galactose, glucosamine and ester-linked succinic acid was extracted from the isolated cell walls of Micrococcus agilis by the hot water-phenol and 5% trichloroacetic acid (TCA) extraction methods . The hot water-phenol extractable polysaccharide accounted for 30% of the weight of the wall, with 23% by the TCA method . Phosphorus contents were less than 0.01% of the polysaccharide . Succinyl residues released by alkali treatment (0.1 N NaOH, 30 min, 37 degrees C) were identified by gas-liquid chromatography, and accounted for 6.3% and 5.1% of the polysaccharide purified from the hot water-phenol and TCA extracts, respectively . The polysaccharide was not bound when chromatography on Concanavalin A-Sepharose 4B (Con A/Sepharose 4B) columns was performed and it could thus be separated from any residual membrane lipomannan . The purified polysaccharide behaved as a negatively-charged polymer on electrophoresis in 1% agarose (at pH 8.6) . A strong cross-reaction, unaffected by removal of the succinyl groups, was observed with type XXIII pneumococcal polysaccharide antiserum indicating the presence of L-rhamnose, linked through non-reducing, lateral end groups.

Braz J Med Biol Res, 1985, 18(4), 455 - 64
Rearrangement of mammalian chromatin structure following excision repair: absence of an effect of inhibitors of poly (ADP-ribose) polymerase and topoisomerase; Ortega JM et al.; Newly-repaired DNA in chromatin is more sensitive to micrococcal nuclease than bulk DNA, but tends to become equally sensitive with time . This rearrangement of chromatin, which had previously been observed following repair of lesions produced by UV-light and of some bulky adducts, has now been shown to occur after repair of lesions induced by hydrogen peroxide and dimethylsulfate . In both cases there was an enhanced sensitivity to nuclease digestion of newly repaired DNA followed by a rearrangement whose kinetics was very similar to that observed in UV irradiated cells . Benzamide and 3-aminobenzamide, inhibitors of the synthesis of poly (ADP-ribose) and novobiocin, an inhibitor of topoisomerase, had no effect on the initially enhanced digestibility of repaired regions or on the chromatin rearrangement that followed . Poly (ADP-ribose) polymerase and topoisomerase are known to play some role in excision repair and have also been shown to cause alterations in the chromatin structure . However, the present results show that these alterations are not involved in this kind of chromatin rearrangement.

J Interferon Res, 1985 Fall, 5(4), 597 - 604
Increased nuclease sensitivity of the human interferon-alpha 1-related genes and the interferon-beta 1 gene during induction by virus; Sagar AD et al.; The chromatin structure of the human interferon (IFN) genes was evaluated during induction of human lymphoblastoid (Namalwa) cells by Sendai virus . Namalwa cells were treated with bromodeoxyuridine (BrdUrd) for 36-48 h and induced with Sendai virus for 7 h; the nuclear fraction was isolated and treated with low levels of either micrococcal nuclease or DNAse I . DNA was extracted from the nuclease-treated chromatin, restricted with Eco RI and analyzed by Southern blotting using IFN-alpha 1 and -beta 1 cDNA probes . An increase in the digestibility of the IFN-alpha 1-related genes and the IFN-beta 1 gene was observed in chromatin prepared from BrdUrd-treated, Sendai virus-induced Namalwa cells as compared with chromatin from uninduced Namalwa cells . Our results indicate that, during IFN induction in Namalwa cells by Sendai virus, the IFN genes assume a more open conformation consistent with increased transcriptional activity across these genes.

Microbios, 1985, 44(177), 21 - 32
Subunit analysis of latent and non-latent F1-ATPase from Micrococcus lysodeikticus (luteus); Urban C et al.; Limited trypsin and chymotrypsin digestions were performed on the latent F1-ATPase from M . lysodeikticus, and subsequent analysis on SDS-polyacrylamide gels revealed subunit profiles with degraded alpha and delta subunits similar to those of ATPase preparations with spontaneously occurring lower degrees of latency . The ATPase obtained from M . lysodeikticus membranes after n-butanol extraction was also non-latent with similar SDS-gel patterns to the aforementioned ATPases . In addition, the sensitive technique of crossed immunoelectrophoresis was used to show that all of the above ATPases contained alpha, beta, gamma, delta and epsilon subunits but some of them were in degraded forms . Although the delta subunit was the first to be cleaved, the loss of latency can be attributed to the degradation of the alpha subunit.

Mol Cell Biol, 1985 Jan, 5(1), 173 - 86
Kinetochore components recognized by human autoantibodies are present on mononucleosomes; Palmer DK et al.; We have developed a competitive enzyme-linked immunosorbent assay for solubilized kinetochore components, using human CREST (calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly, telangiectasia) scleroderma autoimmune antibodies specific for these kinetochore elements . Using this quantitative assay, we found interphase persistent or "pre-kinetochore" components in low- and moderately high-salt (375 mM salt) extracts of micrococcal nuclease-digested rat liver and chicken erythrocyte nuclei . The release of antigen activity from nuclei under these conditions has been correlated with loss of pre-kinetochore foci as determined by immunofluorescence microscopy . Combined biochemical and competition assay analysis of chicken erythrocyte nuclear extracts indicates that pre-kinetochore components are tightly bound to chromatin of mononucleosome size . The conclusions based on competition assay data are supported by a direct binding assay, which confirms that antigens recognized by CREST sera are present on chromatin . These results raise the possibility that the kinetochore-specific chromosomal antigen(s) we have detected substitutes for "standard" mononucleosome components, such as histone H1 . Furthermore, they suggest approaches to the isolation of kinetochore-specific DNA sequences from higher eucaryotes.

Comp Biochem Physiol C, 1985, 81(1), 19 - 23
In vivo distribution of dimethylnitrosamine metabolites in mouse liver and their binding to micrococcal nuclease sensitive and resistant chromatin; Klaude M et al.; Mice were injected i.p . with a single dose (5 mg/kg body wt) of {14C}dimethylnitrosamine and killed at time intervals between 15 and 120 min . Isolated nuclei were incubated with micrococcal nuclease and the chromatin separated into a 1100 g pellet P1 and supernatant fraction S1 . The incorporation of 14C from {14C}dimethylnitrosamine into chromatin was significantly higher in the P1 than the S1 fraction . The purine bases of the P1 DNA showed a lower methylation than those of the S1 DNA . In contrast, radioactivity of the proteins was higher in the P1 than the S1 fraction . It is concluded that the open structures of the S1 chromatin were preferentially attacked by the hepatotoxin leading to a high 14C-labelling of the DNA relative to that of the proteins.

J Biol Chem, 1984 Dec 25, 259(24), 15301 - 6
Photoaffinity cross-linking of the coupling factor 1 from Micrococcus luteus by 3'-arylazido-8-azido-ATP; Schafer HJ et al.; The vicinity of nucleotide binding sites and the mechanism of ATP synthesis/hydrolysis have been studied with the bifunctional photosensitive ATP analog 3'-arylazido-8-azido-ATP . 3'-Arylazido-8-azido-ATP is hydrolyzed by the F1-ATPase from Micrococcus luteus in the absence of ultraviolet light . Irradiation, by ultraviolet light, of F1-ATPase in the presence of 3'-arylazido-8-azido-ATP results in the specific formation of cross-links between alpha and beta subunits . The results suggest that a hydrolytic nucleotide binding site is located on a beta subunit at or near an alpha subunit, probably at the interface between these subunits . Such a constellation would permit direct subunit-subunit interactions during ATP synthesis/hydrolysis.

Biochim Biophys Acta, 1984 Dec 14, 783(3), 283 - 92
High-affinity microtubule protein-higher organism DNA complexes . Many-fold enrichment in repetitive mouse DNA sequences comprised of satellite DNAs; Marx KA et al.; We have examined aspects of the interaction of cycled microtubule protein preparations with 35S-labeled mouse DNA tracer in a competition system with unlabelled competitor E . coli or mouse DNA . The nitrocellulose filter binding assay was used to measure interaction by scintillation counting . DNA molecular weight affected the levels of filter retained 35S-labelled mouse tracer DNA . Filter retention levels increased if 35S-labelled mouse DNA tracer size was increased, and the filter binding level decreased if competitor DNA size was increased . There was a sizeable, reproducible difference in the 35S-labelled mouse DNA tracer binding level of about 1% when E . coli or mouse DNA competitors were compared . Mouse DNA more effectively competed with 35S-labelled mouse DNA for microtubule protein binding than did E . coli DNA, suggesting that a small class of higher-organism DNA sequences interacts very strongly with microtubule protein . From other studies we know this to be the MAP fraction (Marx, K.A . and Denial, T . (1984) in The Molecular Basis of Cancer (Rein, R., ed.), Alan R . Liss, New York, in the press; and Villasante, E., Corces, V.G., Manso-Martinez, R . and Avila, J . (1981) Nucleic Acids Res . 9, 895-908) . We find that this difference in competitor DNA strength is qualitatively similar under high-stringency conditions (0.5 M NaCl, high competitor {DNA}) we developed for examining high-affinity complexes . Under high-stringency conditions we isolated 1.2% and 0.6% of 35S-labelled mouse DNA at 4200 and 350 bp respective sizes as nitrocellulose filter bound DNA-protein complexes . At both molecular weights these high-affinity DNA sequences, isolated from the filters, were shown to be significantly enriched in repetitive DNA sequences by S1 nuclease solution reassociation kinetics . The kinetics are consistent with about a 4-fold mouse satellite DNA enrichment as well as enrichment in other repetitious DNA sequence classes . The high molecular weight filter-bound DNA samples were sedimented to equilibrium in CsCl buoyant density gradients and found to contain primarily mouse satellite DNA density sequences (1.691 g/cm3) with some minor fractions at other density positions (1.670, 1.682, 1.705, 1.740, 1.760 g/cm3) similar to those observed by our laboratory in previous investigations of micrococcal nuclease-resistant chromatin (Marx, K.A . (1977) Biochem . Biophys . Res . Commun . 78, 777-784) . That the high-affinity microtubule-bound DNA was some 3-5-fold enriched in mouse satellite sequences was demonstrated by its characteristic BstNI restriction enzyme cleavage pattern.

Nucleic Acids Res, 1984 Dec 11, 12(23), 9191 - 204
The chromatin structure of the human epsilon globin gene: nuclease hypersensitive sites correlate with multiple initiation sites of transcription; Zhu J et al.; We have mapped sites in chromatin flanking the epsilon-globin gene in the K562 cell which are hypersensitive to digestion with DNAseI, micrococcal nuclease and S1 nuclease . Many of those in the 5' flanking region correspond to minor upstream transcriptional starts . However, one prominent site occurs upstream of the boundary of transcription; it maps to a region with an unusual DNA sequence . In baby hamster kidney cells stably transformed with recombinant DNA containing the human epsilon-globin gene and in Cos 7 cells transiently transfected with DNA containing the epsilon-globin gene, hypersensitive sites can be demonstrated.

Nucleic Acids Res, 1984 Dec 11, 12(23), 9025 - 38
Chromatin structure at the 44D larval cuticle gene locus in Drosophila: the effect of a transposable element insertion; Eissenberg JC et al.; The chromatin structure of the larval cuticle gene cluster at 44D was characterized in embryos from wild-type (Oregon R) and a variant line (2/3) of Drosophila melanogaster . A major DNase I hypersensitive (DH) site was found between genes II and III in the chromatin, in a position 5' to the transcriptional start of the genes in the cluster . The introduction of a 7.3 kilobase transposable element into the cluster in the 2/3 variant enhanced the sensitivity of the major site in 2/3 chromatin but had no other effect upon the pattern of DH sites associated with the wild-type sequences . The wild-type sequences were packaged into an ordered nucleosome-like array in embryos, as revealed by digestion with the chemical cleavage reagent (methidiumpropyl-EDTA) iron (II) {MPE . Fe(II)} . Nucleolytic cleavage within the transposable element chromatin shows it to be organized in an ordered array punctuated by several DH sites . While the patterns of DNase I hypersensitivity are similar in the vicinity of the direct terminal repeats, the patterns revealed by micrococcal nuclease and MPE . Fe(II) are not, indicating a different chromatin organization of these two identical sequences.

Eur J Biochem, 1984 Dec 3, 145(2), 339 - 44
Increased ribosomal affinity for mRNA causes resistance to edeine in a mutant of Saccharomyces cerevisiae; Herrera F et al.; The effect of edeine on the translation of mRNA or poly(U)-directed polyphenylalanine synthesis has been studied in an edeine-resistant mutant of Saccharomyces cerevisiae under three different experimental conditions: in the whole lysate system, in a micrococcal-nuclease-treated lysate, and in a high-salt-treated lysate . The results indicate that translation of messenger is more resistant to edeine in the whole lysate than in the depleted lysates; these observations suggest that resistance to edeine is associated with the presence of endogenous mRNA . It is shown that 40S mutant subunits have a higher affinity for polysomal RNA than 40S wild-type subunits . Since the mRNA binding is inhibited by 7-methylguanosine 5'-monophosphate, the interaction between polysomal RNA and 40S ribosomes is specific for mRNA . The data demonstrate that in each of the depleted lysates, with edeine initially present, the formation of the 80S initiation complex is inhibited . However, edeine inhibition of {3H}methionine binding to 80S ribosomes is overcome completely in the mutant extract by preincubation of this lysate with polysomal RNA . The results indicate that the mutant may carry a specific change in a messenger-binding factor or in a ribosomal protein thereby permitting an increased stability of the messenger-ribosome complex which consequently results in an increased resistance of the mutant lysate to edeine.

Arch Biochem Biophys, 1984 Dec, 235(2), 679 - 91
Characterization of three intermediates in the biosynthesis of teichuronic acid of Micrococcus luteus; Johnson GL et al.; Teichuronic acid, the Micrococcus luteus cell wall polysaccharide which consists of D-glucose and N-acetyl-D-mannosaminuronic acid, is synthesized in vitro from uridine diphosphate N-acetyl-D-glucosamine, uridine diphosphate N-acetyl-D-mannosaminuronic acid, and uridine diphosphate D-glucose in a series of reactions catalyzed by a particulate enzyme preparation . Several lipid-linked intermediates are formed, of which the first three are called components A, B, and C . The formation of these intermediates is inhibited by tunicamycin . The lipid moiety of the intermediates is approximately 95% undecaprenol and 5% dodecaprenol as determined by mass spectrometry . The oligosaccharide moieties of components B and C are the disaccharide, N-acetyl-D-mannosaminuronyl-(1,3)-N-acetyl-D-glucosamine, and the trisaccharide, N-acetyl-D-mannosaminuronyl-(1,4)-N-acetyl-D-mannosaminuronyl++ +-(1, 3)-N-acetyl-D-glucosamine, respectively, as determined by the complete degradation of the former and partial degradation of the latter by the alkaline beta-elimination reaction . The saccharide and lipid moieties of the intermediates are linked through pyrophosphate . Thus, component A is P1-N-acetyl-alpha-D-glucosaminyl P2-undecaprenyl diphosphate, component B is P1-N-acetyl-D-mannosaminuronyl-(1, 3)-N-acetyl-alpha-D-glucosaminyl P2-undecaprenyl diphosphate, and component C is P1-N-acetyl-D-mannosaminuronyl-(1,4)-N-acetyl-D-mannosaminurony l-(1, 3)-N-acetyl-alpha-D-glucosaminyl P2-undecaprenyl diphosphate.

J Biochem Biophys Methods, 1984 Dec, 10(3-4), 211 - 9
Hydrophobic interaction chromatography of Micrococcus lysodeikticus F1-ATPase . A method to detect conformational flexibility of the molecule; Pivel JP et al.; Hydrophobic interaction chromatography of purified ATPase from Micrococcus lysodeikticus (E.C . 3.6.1.3.), a complex oligomeric protein, induces extensive conformational changes in it . In this report, we describe some physicochemical properties of the enzyme forms obtained . They can be summarized as follows . (1) The subunit stoichiometry of the enzyme is altered by the absorption and desorption process since most of the forms obtained are defective in gamma and delta subunits . An important reduction in the molar proportion of alpha subunit is also observed; (2) the fluorescence spectra of the different forms show progressive tyrosine residues which roughly correspond to the extent and strength of the interaction existing before elution of the enzyme; (3) circular dichroism measurements reveal changes of the secondary structure of the F1-ATPase undergoing an increase in alpha-helical content; (4) the ordered, active forms eluted from the hydrophobic chromatography columns are less stable than the native protein, as shown by dialysis experiments . These results while supporting the use of hydrophobic chromatography as a simplified model of membrane-membrane protein interaction, also indicate the need for caution in its application to the purification of complex membrane proteins.

J Hypertens Suppl, 1984 Dec, 2(3), S271 - 3
Angiotensin II receptors in chromatin; Re RN et al.; When isolated rat hepatic nuclei or bovine thymus nuclei were incubated with 125I-angiotensin II (ANG II) in the presence or absence of cold hormone, displaceable binding was consistently detected in micrococcal nuclease generated fragments Nanomolar concentrations of ANG II produced detectable displacement . Little or no specific binding was found when nuclei were first digested and then treated with hormone, suggesting that ANG II solubilizes its own receptor . The binding moiety was partially purified by DNP gel electrophoresis . These studies indicate the existence in chromatin of high affinity receptors for ANG II, and further suggest that hormone binding to these receptors produces conformational changes in chromatin similar to those seen during enhanced transcriptional activity . Thus, the present studies suggest the existence of functional intracellular renin-angiotensin systems.

J Gen Microbiol, 1984 Dec, 130 ( Pt 12), 3153 - 7
Two-dimensional gel electrophoretic separation of the proteins present in chromatin of Escherichia coli; Lossius I et al.; The polypeptides present in 35S-labelled chromatin prepared from Escherichia coli cells, and polypeptides present in the DNA and RNA complexes obtained by micrococcal nuclease digestion of the chromatin, were analysed by two-dimensional non-equilibrium polyacrylamide gel electrophoresis . Three hundred and thirty-five 35S-labelled polypeptides were detected in the chromatin whereas the DNA- and RNA-containing fractions of the micrococcal nuclease digest contained 126 and 183 polypeptides respectively . The major basic low-molecular-weight polypeptides were found in the DNA-containing fractions.

J Mol Biol, 1984 Nov 25, 180(1), 73 - 90
Site-specific phasing in the chromatin of the rDNA in Dictyostelium discoideum; Edwards CA et al.; The rDNA in Dictyostelium discoideum is organized in linear, extrachromosomal, palindromic dimers of approximately 88 X 10(3) bases in length . The dimers are repeated about 90 times per haploid genome . Using indirect end-labeling, we have mapped micrococcal nuclease and DNAase I-sensitive sites in the chromatin near the rDNA telomeres . This region is 3' to the 36 S rRNA coding region and contains a single 5 S rRNA cistron but is primarily non-coding . We have observed somewhat irregularly spaced but specific phasing of nuclease-sensitive sites relative to the underlying DNA sequence . Comparison of the sites in chromatin with those in naked DNA reveals an unusual and striking pattern: the sites in naked DNA that are attacked most readily by both nucleases, presumably because of the specificity of the nucleases for certain sequences or physical characteristics of the DNA, appear to be the same sites that are most protected in chromatin . This pattern extends over most of a 10(4) base region, from the sequence immediately distal to the 36 S rRNA coding region and extending to the terminus . Although much of the sequence-specific phasing is irregularly spaced, salt extraction data are consistent with the presence of nucleosomes . In addition, phasing in the terminal region may be directed partially by proteins that do not bind DNA as tightly as do core histones . We present a model for phasing in spacer regions in which the sequence preferences of nucleases such as micrococcal nuclease and DNAase I may be useful tools in predicting nucleosome placement.

Biochim Biophys Acta, 1984 Nov 22, 783(2), 152 - 7
Translation in micrococcal nuclease-treated cell-free extracts from Ehrlich ascites tumor cells . Stimulation by initiation factor eIF-2B; Person A et al.; Translation of exogenous mRNAs in micrococcal nuclease-treated extracts from Ehrlich ascites tumor cells is greatly stimulated by the addition of crude initiation factors or initiation factors eIF-2B and eIF-2 containing eIF-2B . The requirement for exogenous eIF-2B in micrococcal nuclease-treated extracts does not result from either loss or enhanced phosphorylation of eIF-2 during incubation.

J Mol Biol, 1984 Nov 15, 179(4), 651 - 66
Regions in the ribosomal minichromosome of Physarum polycephalum are protected from restriction nucleases; protection is insensitive to high salt in the G phase and sensitive in the M phase of the cell cycle; Kunzler P et al.; In the central spacer region of the extrachromosomal ribosomal DNA of the slime mold Physarum polycephalum, four small regions of related sequence are completely inaccessible to restriction endonucleases (HinfI and MboI) . In addition, some sequences neighboring the inaccessible ones, are partially inaccessible to restriction enzymes and micrococcal nuclease . Taking advantage of the natural synchrony of Physarum plasmodia, we found that this protection is present throughout the cell cycle . Treatment with high salt (2.5 M-NaCl) of nuclei from the G2 phase of the cell cycle left the protection essentially unchanged . When nuclei from the M phase were treated with salt, the protection was abolished . The inaccessible sites are located close to the origins of replication of the rDNA.

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