Micrococci

J Mol Biol, 1984 Nov 15, 179(4), 607 - 28
Internal promoter elements of transfer RNA genes are preferentially exposed in chromatin; DeLotto R et al.; We have examined the chromatin organization of a cluster of transfer RNA genes at the cytogenic locus 90BC on the right arm of the third Drosophila melanogaster chromosome . We find that the internal promoter sequences are preferentially exposed to micrococcal nuclease and DNase I in chromatin . Moreover, these exposed sequences have an unusual single strand nuclease-sensitive conformation.

Biochemistry, 1984 Nov 6, 23(23), 5426 - 32
Mechanism of rat liver DNA methyltransferase interaction with anti-benzo{a}pyrenediol epoxide modified DNA templates; Ruchirawat M et al.; We investigated the methylation reaction catalyzed by 1500-fold purified rat liver DNA methyltransferase (DMase) on native Micrococcal luteus DNA (ML-DNA) and poly(dC-dG) templates containing covalently bound (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene (anti-BPDE), the strongly carcinogenic, principal metabolite of benzo{a}pyrene . Since eukaryotic DNA methyltransferases recognize the dinucleotide 5'd{CG} in DNA as a substrate for methylation, the model polynucleotide poly(dC-dG) was used to study in more detail the mode of interaction and effect on incorporation . With either of these BPDE-modified templates, a progressive inhibition of methylation was correlated with increasing amount of BPDE substitution . The effect of BPDE-dG adducts did not alter the apparent km with respect to the concentration of d{CG} in either unmodified or BPDE-modified poly(dC-dG) (km = 10 microM) but lowered the relative apparent Vmax . In assays in which perturbation by salt of preformed enzyme-DNA complex is measured, no change in the relative stability to either unsubstituted or the carcinogen-modified template was noted, thus, excluding any change in the ionic component of this interaction . However, in competition-type experiments, BPDE-DNA is an inhibitor of the methylation reaction on native DNA . When BPDE-DNA is allowed to interact with the enzyme before the addition of native competitor DNA, the methylation rate is not stimulated, suggesting very tight hydrophobic binding of the enzyme to BPDE-DNA and an inhibition in the dissociation of DMase from the template following a methylation event.(ABSTRACT TRUNCATED AT 250 WORDS)

Anal Biochem, 1984 Nov 1, 142(2), 497 - 503
Direct determination of uracil in {32P,uracil-3H}poly(dA.dT) and bisulfite-treated phage PM2 DNA; Green DA et al.; A simple but effective technique for determining the presence of uracil existing as either A:U base pairs or G:U base pairs in DNA was developed . DNA is degraded to deoxynucleoside 3'-monophosphates by a combination of micrococcal nuclease and spleen phosphodiesterase . The monophosphates are converted to 5'-end-labeled 32P-labeled diphosphates in a reaction catalyzed by T4 polynucleotide kinase . The resultant product is then converted to 5'-end-labeled deoxynucleoside monophosphates by P1 nuclease digestion, which specifically removes 3'-phosphates . Successful separation of labeled dUMP from conventional bases in DNA is achieved by two-dimensional polyethyleneimine chromatography, with its detection determined by autoradiography and liquid scintillation counting . The sensitivity of the technique described can detect a minimum 1 X 10(-16) mol of dUMP in DNA . Additionally, the detection of 5-methylcytosine in placental DNA demonstrates the flexibility of the technique for the analysis of modified bases in DNA.

J Biochem (Tokyo), 1984 Nov, 96(5), 1337 - 42
Two species of chromatin-RNA polymerase II complex are commonly present in nuclei of various tissues of rats; Inaba M et al.; When rat liver nuclei were digested with nuclease, we found that the chromatin-bound RNA polymerase II was liberated as two distinct complexes, peak 1 and peak 2, which seemed to reflect different functional states in cell nuclei . We further examined their occurrence in nuclear digests of various tissues of rats and the following results were obtained . Upon digestion with micrococcal nuclease of nuclei from brain, spleen, testis and kidney, chromatin-bound RNA polymerase II was liberated as two distinct forms which sedimented differently in a sucrose density gradient . The sedimentation rate of peak 1 varied depending on the tissue nuclei examined . After high salt or RNase treatment of the nuclear digests, peak 1 from liver, brain, spleen and testis nuclei showed the same sedimentation rate as did kidney peak 1, the rate for which remained unchanged by these treatments . The results suggested that peak 1 complexes from various tissue nuclei had basically the same structural organization, and we confirmed this by electrophoretic studies on RNase-treated liver and kidney nuclear digests . Peak 2 from various tissue nuclei exhibited identical sedimentation rates . Thus, the chromatin-bound RNA polymerase II seems to exist commonly in two distinct states in cell nuclei of rats.

Endocrinology, 1984 Nov, 115(5), 1705 - 9
Effects of cycloheximide, alpha-amanitin, and alpha-difluoromethylornithine on thyrotropin-induced increases in the micrococcal nuclease sensitivity of thyroid nuclear chromatin; Fucile NW et al.; Treatment of calf thyroid slices with TSH increases the nuclease sensitivity of nuclear chromatin, i.e . the amount of DNA released from nuclei by mild digestion with DNase I and micrococcal nuclease . Cycloheximide and alpha-amanitin were used to investigate the roles played by protein and RNA synthesis in mediating this effect of TSH; alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, was used to investigate the possible involvement of polyamines . Calf thyroid slices were incubated with or without TSH (50 mU/ml) for 5 h, in the presence or absence of inhibitors . Nuclei were then prepared, subjected to mild digestion with micrococcal nuclease, and centrifuged at 1200 X g . The amount of DNA in 1200 X g supernatants was increased by TSH; this was inhibited by cycloheximide (100 micrograms/ml) and alpha-amanitin (4 micrograms/ml) when these agents were present throughout incubations with TSH . In contrast, alpha-amanitin failed to inhibit the TSH effect when it was added to incubations 30 min or 2 h after the addition of TSH . These results indicate that RNA and protein synthesis play a part in mediating the effect of TSH on the micrococcal nuclease sensitivity of chromatin, and that the RNA synthesis involved takes place within the first 30 min of exposure of thyroid slices to TSH . alpha-Difluoromethylornithine (5 mM) inhibited the TSH-dependent development of micrococcal nuclease sensitivity; however, it also inhibited nuclease digestion when it was added directly to nuclei prepared from fresh thyroid tissue . This observation should serve as a warning against uncritical acceptance of the notion that all effects of alpha-difluoromethylornithine are the result of inhibition of ornithine decarboxylase.

J Virol, 1984 Nov, 52(2), 638 - 49
Structural organization and polypeptide composition of the avian adenovirus core; Li P et al.; CELO virus (fowl adenovirus 1) contained three core polypeptides of molecular weights 20,000, 12,000, and 9,500 . The core was similar to that of human adenoviruses, with some evidence of compact subcore domains . Micrococcal nuclease digestion of CELO virus cores produced a smear of DNA fragments of gradually decreasing size, with no nucleosome subunit or repeat pattern . Moreover, when digested cores were analyzed without protease treatment, there was again no evidence of a nucleosome substructure; neither DNA fragments nor core proteins entered a 4% polyacrylamide gel . The organization of the core is thus quite unlike that of chromatin . Restriction endonuclease analysis of the DNA from digested cores showed that the right end was on the outside of the core . We suggest that adenovirus DNA is condensed into the core by cross-linking and neutralization by the core proteins, beginning with the packaging sequence at the center of the core and ending with the right end of the DNA on the outside.

Nucleic Acids Res, 1984 Oct 11, 12(19), 7599 - 614
Synthesis and properties of poly 5-methylthiouridylic acid; Ho YK; In an effort to search for good methods for the enzymatic synthesis of polynucleotide analogs with antitemplate activity, 5-methylthiouridine-5'-diphosphate (ms5UDP) has been synthesized and investigated as a substrate for polynucleotide phosphorylase . While ms5UDP was polymerized at a very low rate to give a 6% yield of polynucleotides by the polynucleotide phosphorylase of Micrococcus luteus, it was utilized more efficiently by the corresponding enzyme of Escherichia coli resulting in a 15% yield of poly (5-methylthiouridylic) acid . Results of the co-polymerization of ms5UDP and UDP revealed that the ratio of 5-methylthiouridylate to uridylate residues in the polynucleotide product was lower than the ratio of ms5UDP to UDP in the substrate mixture . The 5-methylthio group conferred only minute changes on the conformation of the modified polyuridylic acid, and the complexes formed between poly-(5-methylthiouridylic) acid and poly(adenylic) acid possessed slightly higher Tm values than did the unmodified counterparts . Poly(5-methylthiouridylic) acid was a potent inhibitor of calf thymus DNA polymerase alpha.

J Biol Chem, 1984 Oct 10, 259(19), 12007 - 13
High-mobility group chromosomal proteins of wheat; Spiker S; Four proteins have been extracted from purified chromatin of wheat embryos with 0.35 M NaCl . These proteins are soluble in 2% (w/v) trichloroacetic acid and thus meet the original operational requirements to be classified as "high-mobility group" (HMG) chromosomal proteins . The proteins have been characterized by one- and two-dimensional electrophoresis, amino acid analysis, and peptide mapping . Three of the proteins (HMGb, c, and d) share the mammalian HMG characteristic of being rich in both acidic and basic amino acid residues . Unlike their putative mammalian counterparts, these plant HMG proteins contain less than 7 mol % proline . The fourth wheat protein (HMGa) is rich in both proline and in basic amino acid residues . This wheat protein, however, contains only about half the proportion of acidic residues found in mammalian HMG proteins--a characteristic also found in the trout testis HMG protein, H6 . Comparative peptide maps show that none of the wheat HMG proteins are degradation products of other HMG proteins or the H1 histones . The peptide maps have not, however, been useful in establishing homologies with mammalian HMG proteins . Wheat HMG proteins are released from DNase I-treated nuclei and co-isolate with micrococcal nuclease-sensitive chromatin fractions . Similar observations concerning the HMG proteins of vertebrate animals have been considered consistent with a role for these proteins as structural components of actively transcribed chromatin.

J Biol Chem, 1984 Oct 10, 259(19), 12084 - 91
Photoaffinity labeling of thyroid hormone nuclear receptors . Influence of n-butyrate and analysis of the half-lives of the 57,000 and 47,000 molecular weight receptor forms; Casanova J et al.; The thyroid hormone receptor is a nuclear-associated protein which appears to mediate the actions of 3,5,3'-triiodo-L-thyronine (L-T3) and 3,5,3',5'-tetraiodo-L-thyronine (L-T4) in mammalian cells . In a previous study we reported that N-2-diazo-3,3,3-trifluoropropionyl-3,5,3'-triiodo-L-thyronine (L-T3-PAL) serves as an effective photoaffinity label probe of the receptor in GH1 cells, a growth hormone producting rat pituitary cell line . Irradiation of cells at 254 nm covalently cross-links L-{125I}T3-PAL to two molecular weight (Mr) nuclear receptor forms, an abundant 47,000 Mr component and a less abundant 57,000 Mr species (Pascual, A., Casanova, J., and Samuels, H . H . (1982) J . Biol . Chem . 257, 9640-9647) . In this study we have explored a number of possible interrelationships of the different Mr receptor forms . Denaturing gel electrophoresis and autoradiography indicates that the 57,000 Mr form is a doublet species which differ in Mr by 1,000 to 2,000 . The various receptor forms are not an artifact of the L-{125I}T3-PAL probe, and identical forms can be labeled at 310 nm using underivatized L-{125I}T4 with a 15-fold lower coupling efficiency . The 57,000 and 47,000 Mr receptor forms are not generated by indiscriminate proteolysis, UV peptide cleavage, or zero length protein-protein cross-linking by irradiation at 254 nm . Micrococcal nuclease excises both the 57,000 and 47,000 Mr forms, and receptor is not identified in the residual nuclear matrix fraction . Receptor is also not detected in the cytoplasmic fraction . By coupling dense amino acid labeling and photoaffinity labeling of receptor we determined a half-life of 2.4 h for the 57,000 Mr species and 5.6 h for the 47,000 Mr form while both species have similar relative synthetic rates . n-Butyrate has been previously shown to decrease receptor levels in GH1 cells . We demonstrate that n-butyrate decreases receptor levels primarily by shortening the half-life of the 47,000 Mr form.

Biochemistry, 1984 Oct 9, 23(21), 5016 - 23
Association of poly(adenosine diphosphate ribosylated) nucleosomes with transcriptionally active and inactive regions of chromatin; Hough CJ et al.; We have investigated whether transcriptionally active or inactive gene sequences are associated in vivo with poly(adenosine diphosphate ribosylated) regions of chromatin . Soluble HeLa cell chromatin derived from nuclei treated either briefly or extensively with micrococcal nuclease was fractionated on an anti-poly(adenosine diphosphate ribose)-Sepharose column {Malik, N., Miwa, M., Sugimura, T., Thraves, P., & Smulson, M . E . (1983) Proc . Natl . Acad . Sci . U.S.A . 80, 2554-2558} to obtain fractions that were enriched or depleted in poly(ADP-ribosylated) chromatin . DNA obtained from these fractions was then probed for active and inactive gene sequences with a cDNA probe made from total cell mRNA and a probe for the beta-globin gene . Chromatin enriched in poly(ADP-ribosylated) nucleosomes contained both active and inactive gene sequences as detected by the probes and appeared to be more nuclease sensitive than that found in the fraction of chromatin depleted of poly(ADP-Rib) . Poly(ADP-ribosylated) chromatin from nuclei digested briefly with nuclease showed an enrichment in both active and inactive genes while that treated extensively with nuclease showed either no enrichment or a depletion of active and inactive genes . Actively transcribed chromatin was digested at a rate several times that of the bulk or inactive chromatin . Nevertheless, the enrichment of active genes in poly(ADP-ribosylated) nucleosomes derived from brief nuclease digestion was greater than that of inactive genes . These results are interpreted as showing that some, but not all, of actively transcribed chromatin contains associated poly(ADP-ribosylated) proteins . However, since poly(ADP-ribosylated) proteins are also associated with inactive genes, the function of this modification cannot be assigned solely to transcription.

Biochemistry, 1984 Oct 9, 23(21), 5024 - 9
Changes in histone H1 content and chromatin structure of cells blocked in early S phase by 5-fluorodeoxyuridine and aphidicolin; D'Anna JA et al.; We have measured changes in histone H1 content and changes in chromatin structure of Chinese hamster (line CHO) cells blocked in early S phase by sequential use of isoleucine deprivation and blockade with 5-fluorodeoxyuridine or aphidicolin . Both the H1:core histone ratio in isolated nuclei and the H1 content of the cell are reduced 20-60%, depending on the duration of the block . The new deoxyribonucleic acid (DNA) synthesized during S-phase block has a shorter nucleosome repeat length than that of bulk chromatin, but it is nearly equally resistant as bulk DNA to attack by micrococcal nuclease . During the time that H1 content is decreasing, bulk chromatin also undergoes structural changes so that its nucleosome cores appear to be more closely packed along the DNA chain . The losses in H1 content and changes in chromatin structure are similar to those reported for cells blocked in early S phase by hydroxyurea {D'Anna, J . A., & Prentice, D . A . (1983) Biochemistry 22, 5631-5640} . The results suggest that losses of H1 and changes in chromatin structure are general events which occur when the elongation of initiated replicons or the joining of intermediate-sized DNA fragments is retarded during replication . They are consistent with the notions that H1 is lost from initiated replicons and/or the loss of H1 is part of an alarm response in the cell which might facilitate events leading to gene amplification.

J Mol Biol, 1984 Oct 5, 178(4), 920 - 8
Psoralen-crosslinking of soluble and of H1-depleted soluble rat liver chromatin; Conconi A et al.; We purified soluble rat liver chromatin and H1-depleted chromatin and photocrosslinked its DNA with psoralen at pH 7 . Digestion of this chromatin with micrococcal nuclease produced a normal nucleosomal repeat . Chromatin was photoreacted in the presence of 0 to 700 mM-NaCl and was fractionated in sucrose gradients containing the same NaCl concentrations . The dissociation of H1 occurred as in the non-crosslinked controls and no preferential dissociation of core histones was observed . The samples between 100 and 500 mM-NaCl showed precipitation . In the electron microscope, the fibers appeared indistinguishable from the controls at low ionic strength . In the presence of 40 mM-NaCl, the fibers of the photoreacted chromatin were slightly more compact than the controls, and at 500 mM-NaCl, despite the complete dissociation of H1, there were still apparently intact fibers at this ionic strength . The disruption of the psoralen-treated chromatin fibers occurred only in 600 mM-NaCl, as opposed to 500 mM-NaCl in controls . The DNA of all the photoreacted samples was spread for electron microscopy under denaturing conditions . They revealed, for all the samples, single-stranded bubbles corresponding to 200 to 400 base-pairs in size . H1-depleted chromatin containing stoichiometric amounts of core histones was photoreacted at pH 10 and very low ionic strength . Under these conditions many of the nucleosomes appeared to be unraveled, although to a variable extent . In the electron microscope, the purified DNA from these samples showed extensive crosslinking when spread under denaturing conditions . These observations show that histone-DNA interactions different from those in intact nucleosomes may be created, which allow extensive access of psoralen to the DNA.

J Mol Biol, 1984 Oct 5, 178(4), 897 - 919
Psoralen-crosslinking of DNA as a probe for the structure of active nucleolar chromatin; Sogo JM et al.; Trimethylpsoralen was used to crosslink the extrachromosomal ribosomal DNA in nucleoli or nuclei of growing Dictyostelium discoideum cells . The DNA was extracted and was examined by spreading under denaturing conditions for electron microscopy . Intact 95,000 base ribosomal DNA molecules were seen, showing regularly spaced, single-stranded bubbles of about 200 to 400 bases in size, interrupted twice by 11,000 base heavily crosslinked stretches, which correspond to the known positions of the coding regions . The bubbles on the nontranscribed regions indicate the presence of nucleosomes during crosslinking . The DNA was digested with restriction enzymes and analysed by gel electrophoresis in parallel with DNA not treated with psoralen . Fragments from the non-coding region had the same mobility as untreated DNA, while those from the coding region had a markedly lower mobility, though not as low as that of crosslinked pure DNA . This shifting of the bands, specific to the coding region, was also seen when whole cells were treated with psoralen . Treatment of nucleoli with 2 m-NaCl (which is known to dissociate histones) before addition of psoralen led to strong crosslinking all along the ribosomal DNA, resulting in a decreased electrophoretic mobility of bands from the non-coding region, but no further retardation of those from the coding region . In differentiating Dictyostelium cells, slugs, where ribosomal RNA synthesis is very much reduced, the extent of psoralen-crosslinking in the coding region was reduced, but not completely to the level of that of the non-transcribed spacer . In order to test whether psoralen itself alters chromatin structure, crosslinked and non-crosslinked nucleoli from growing cells were lysed with heparin and spread for electron microscopy . There was no difference in the appearance or the frequency of the transcription units seen . Digestion of crosslinked nuclei with micrococcal nuclease indicated an undisturbed structure for bulk chromatin, as well as for the chromatin in the non-transcribed spacer of the ribosomal DNA . Thus psoralen-crosslinking does not lead to extensive disruption or distortion of the structure of either inactive or active chromatin . We conclude, taking the results presented in the Appendix into account, that the extent of psoralen-crosslinking in chromatin DNA is diagnostic for the structure of undistorted chromatin.(ABSTRACT TRUNCATED AT 400 WORDS)

Mikrobiyol Bul, 1984 Oct, 18(4), 208 - 12
{A cerebellar abscess caused by anaerobic and aerobic (mixed) microorganisms}; Tokatli A et al.; A 15 year old boy was admitted to hospital with five days history of fever, headache, vomiting and otorrhea . Findings on physical examination included high fever, purulent drainage from right ear, nuchal rigidity, Brudzinski's and Kernig's signs . Laboratory finding was polymorphonuclear leukocytosis . Computerized tomography (CT) of his brain was normal . A lumbar puncture disclosed purulent CSF . Chloramphenicol and Penicillin G were given intravenously as treatment for the meningitis . After five days of this therapy he continued to be febrile and nuchal rigidity, Brudzinski's and Kerning's signs increased . The second CT demonstrated the presence of an abscess in the cerebellum . The abscess was aspirated during mastoidectomy . In the cultures of the aspiration material Bacteroides species and gram positive micrococci grew out . Metronidazole, 500 mg qid per oral, was added to the therapy . During treatment, his condition was evaluated with serial computerized tomography scans of his brain and these studies showed progressive decrease in the size of the lesion . Metronidazole and antibiotics therapies were continued 45 days . The patient made an uneventful recovery.

Radiat Res, 1984 Oct, 100(1), 87 - 95
Inhibition by hyperthermia of repair synthesis and chromatin reassembly of ultraviolet-induced damage to DNA; Bodell WJ et al.; We have investigated the effects of hyperthermia treatment on sequential steps of the repair of UV-induced DNA damage in HeLa cells . DNA repair synthesis was inhibited by 40% after 15 min of hyperthermia treatment at 45 degrees C; greater inhibition of repair synthesis occurred with prolonged incubation at 45 degrees C . Enzymatic digestion of repair-labeled DNA with Exonuclease III indicated that once DNA repair was initiated, the DNA repair patch was synthesized to completion and that ligation of the DNA repair patch occurred . Thus the observed inhibition of UV-induced DNA repair synthesis by hyperthermia treatment may be the result of inhibition of enzymes involved in the initiating step(s) of DNA repair . DNA repair patches synthesized in UV-irradiated cells labeled at 37 degrees C with {3H}Thd were 2.2-fold more sensitive to micrococcal nuclease digestion than was parental DNA; if the length of the labeling period was prolonged, the nuclease sensitivity of the repair patch synthesized approached that of the parental DNA . DNA repair patches synthesized at 45 degrees C, however, remained sensitive to micrococcal nuclease digestion even after long labeling periods, indicating that heat treatment inhibits the reassembly of the DNA repair patch into nucleosomal structures.

Mol Immunol, 1984 Oct, 21(10), 929 - 37
Idiotypic manipulations: hierarchy in idiotype expression in rabbits immunized against Micrococcus luteus; Wikler M et al.; Idiotypic cross-reactions were analyzed among three series of anti-peptidoglycan antibodies of the Micrococcus luteus system . The reference idiotype Ab1 was an antibody fraction isolated from an isoelectric focusing preparative column . Cross-reactive idiotypes, Ab1', were induced through the immunization chain (Ab1-Ab2-Ab3) . Idiotypic antibodies of Ab1-F1 type were obtained from offspring of female rabbits, actively producing Ab3 during pregnancy . Finally, Ab1 CRI were cross-reactive idiotypes with Ab1 found in a random population of rabbits immunized with M . luteus . Three idiotopes could be characterized within Ab1 antibody . Ab1' usually expressed two of these idiotopes, but never the third specificity which is "private" to Ab1 . Ab1-F1 shared one or two idiotopes with Ab1 and Ab1' antibodies . Only one common idiotope appeared to be present on Ab1 CRI . Finally, this idiotope, IdX, could be detected by radioimmunoassay in 20% of rabbits immunized with micrococcal vaccine . It appears that a recurrent idiotype of anti-peptidoglycan antibodies can be preferentially amplified through idiotypic manipulations . On the other side, cascade immunizations lead to the expression on Ab1' and on some Ab1-F1 of a second idiotypic specificity, shared with Ab1 . This hierarchy of idiotype expression may well be important in the regulation of antibody synthesis through idiotypes.

J Mol Biol, 1984 Sep 15, 178(2), 249 - 71
Dispersive segregation of nucleosomes during replication of simian virus 40 chromosomes; Cusick ME et al.; The distribution of preformed ("old") histone octamers between the two arms of DNA replication forks was analyzed in simian virus 40(SV40)-infected cells following treatment with cycloheximide to prevent nucleosome assembly from nascent histones . Viral chromatin synthesized in the presence of cycloheximide was shown to be deficient in nucleosomes . Replicating SV40 DNA (wild-type 800 and capsid assembly mutant, tsB11) was radiolabeled in either intact cells or nuclear extracts supplemented with cytosol . Nascent nucleosomal monomers were then released by extensive digestion of isolated nuclei, nuclear extracts or isolated viral chromosomes with micrococcal nuclease . The labeled nucleosomal DNA was purified and found to hybridize to both strands of SV40 DNA restriction fragments taken from each side of the origin of DNA replication, whereas Okazaki fragments hybridized only to the strand representing the retrograde DNA template . In addition, isolated, replicating SV40 chromosomes were digested with two strand-specific exonucleases that excised nascent DNA from either the forward or the retrograde side of replication forks . Pretreatment of cells with cycloheximide did not result in an excess of prenucleosomal DNA on either side of replication forks, but did increase the amount of internucleosomal DNA . These data are consistent with a dispersive model for nucleosome segregation in which "old" histone octamers are distributed to both arms of DNA replication forks.

J Clin Endocrinol Metab, 1984 Sep, 59(3), 427 - 31
Increased micrococcal nuclease sensitivity and/or solubility in nuclei from Graves' disease thyroid tissue; Inaba M et al.; The sensitivity of nuclei to micrococcal nuclease was compared in thyroid tissue obtained from euthyroid patients with solitary cold nodules and from patients with Graves' disease . A significant increase in the solubility and/or the sensitivity of nuclei to the nuclease was found in thyroid tissue from patients with Graves' disease . Electrophoretic analysis of DNA in chromatin solubilized by the nuclease revealed that the amount of oligonucleosomal DNA was increased, and that of polynucleosomal DNA was even more increased, in nuclei from Graves' thyroids than in those from normal thyroids . Polyacrylamide gel electrophoretic analysis in Triton acid-urea showed that the extent of histone acetylation in nuclei from Graves' thyroids was almost the same as in those from normal thyroids . These findings suggest that the state of chromatin organization in Graves' thyroid nuclei is different from that in normal thyroid nuclei and is independent of the extent of histone acetylation.

Radiobiologiia, 1984 Sep-Oct, 24(5), 603 - 6
{Genetic control of the processes of postradiation repair of a compact chromosome in Micrococcus radiodurans cells}; Kudriashova NIu et al.; X-irradiation of Micrococcus radiodurans cells with sublethal doses caused disturbances in the structure of a membrane-bound compact chromosome . Recovery of the compact chromosome occurred during the postirradiation incubation of the wild type cells and cells of the UVS-17 mutant deficient in DNA-polymerase . This process was blocked in cells of rec-30 mutant with the impaired system of genetic recombination: this is indicative of an important role played by rec-30 gene product in the postirradiation recovery of the compact chromosome in M . radiodurans cells.

Mutat Res, 1984 Sep-Oct, 132(3-4), 129 - 38
Kinetics of ultraviolet induced DNA excision repair in rat and human fibroblasts; Vijg J et al.; To obtain more information on the well-documented low excision-repair capacity of rodent cells in comparison with human cells, we have studied this form of DNA repair in UV-irradiated human and rat skin fibroblasts . For this purpose, we have determined (i) unscheduled DNA synthesis (UDS), using autoradiography, (ii) the number and size of repaired sites with the bromodeoxyuridine (BrdU) photolysis assay and (iii) the removal of Micrococcus luteus UV-endonuclease susceptible sites (ESS) . We found rat cells to be quite capable of performing DNA-repair synthesis, as demonstrated by both UDS and BrdU photolysis, whereas they almost completely lacked the capacity to remove pyrimidine dimers, as indicated by the persistence of ESS . This discrepancy will be discussed in terms of the types of mechanisms by which mammalian cells may recognize and remove UV-induced photoproducts.

Cell, 1984 Sep, 38(2), 501 - 10
Chromatin structure of the molecular ends of Oxytricha macronuclear DNA: phased nucleosomes and a telomeric complex; Gottschling DE et al.; Oxytricha macronuclear DNA exists as approximately 24 X 10(6) gene-sized molecules terminating with a C4A4 repeat . DNA-protein interactions at the ends of bulk macronuclear molecules were probed with micrococcal nuclease and methidiumpropyl-EDTA X Fe(II) (MPE X Fe{II}) . The ends were indirectly labeled by hybridizing with (C4A4)2 . Alternatively, a novel method using MPE X FE(II) as a probe and directly labeling the 3' ends with terminal transferase was implemented . A terminal complex involving approximately 100 bp with nucleosomes phased inward from the complex was found to be characteristic of most or all of the ends . Analysis of two specific genes confirmed the pattern and showed that the special structure was on both ends of each molecule . We conclude that a DNA-protein complex involving 100 bp and terminating with the C4A4 repeat can be sufficient to provide the fundamental functions of telomeres, allowing linear DNA replication and conferring stability of linear DNA.

Mol Immunol, 1984 Sep, 21(9), 761 - 70
Anticentromere antibodies bind to trout testis histone 1 and a low molecular weight protein from rabbit thymus; Ayer LM et al.; Antibodies directed against centromeric chromatin characteristically occur in the sera of patients with the CREST variant of scleroderma . We have studied the in situ enzymatic sensitivity and solubility of the centromeric antigen and have isolated an antigenic moiety that reacts with anticentromere antibodies . The centromeric antigen in the human epithelial cell line, HEp-2, was sensitive to DNAase I and micrococcal nuclease but not affected by RNAase A, trypsin or amylase . It was insoluble in 0.15-4 M NaCl but was extracted from the HEp-2 cells by 4 M urea/2 M NaCl . Antigenic activity in a 4 M urea/2 M NaCl extract of rabbit thymus was demonstrated by immunoabsorption . Indirect immunofluorescence of the extract separated by polyacrylamide gel electrophoresis revealed a fluorescent band with a mol . wt of 33,000 . Calf thymus and trout testis histone preparations were fractionated by gel electrophoresis and transferred by blotting techniques to diazobenzyloxymethyl cellulose paper for autoradiography . Anticentromere antibodies bound to and were absorbed by trout testis histone 1 . We propose that the centromeric antigen may be a variant of histone 1 that is associated with condensed chromatin.

Mol Cell Biol, 1984 Sep, 4(9), 1890 - 9
Mg2+ induces a sharp and reversible transition in U1 and U2 small nuclear ribonucleoprotein configurations; Reveillaud I et al.; When U1 and U2 small nuclear ribonucleoproteins (snRNPs) purified by a procedure which preserves their immunoprecipitability by autoimmune antibodies (Hinterberger et al., J . Biol . Chem . 258:2604-2613, 1983), were submitted to extensive digestion with micrococcal nuclease, we found that their degradation pattern was sharply dependent upon magnesium concentration, indicating that they undergo a profound structural modification . At low Mg2+ (less than or equal to 5 mM), both particles only exhibit a core-resistant structure previously identified as being common to all but U6 snRNAs (Liautard et al., J . Mol . Biol . 162: 623-643, 1982) . At high Mg2+ (greater than or equal to 7 mM), U1 and U2 snRNPs behave differently from one another . In U1 snRNP, most U1 snRNA sequence is protected, except for the 10 5'-terminal nucleotides presumably involved in splicing and a short sequence between nucleotides 102 and 108 . Another region spanning nucleotides 60 to 79 is only weakly protected . This structural modification was demonstrated to be reversible . In U2 snRNP, the U2 snRNA sequence remains exposed in its 5' part up to nucleotide 92, and the 3'-terminal hairpin located outside the core structure becomes protected.

J Gen Virol, 1984 Sep, 65 ( Pt 9), 1611 - 5
Nuclease sensitivity of adenovirus type 2 chromatin in lytic infection; Toth M et al.; We have investigated the sensitivity of adenovirus type 2 naked DNA and chromatin at 5 h and 20 h after infection to digestion by DNase I, micrococcal nuclease and endogenous nuclease between map coordinates 11.3 and 18.0 (SmaI-F fragment) using a terminal labelling method . Infected cell nuclei were gently digested with nucleases, DNA was extracted and digested to completion with SmaI and the fragments shorter than the SmaI-F fragment mapped by hybridization with a 708 base pair probe co-terminal with the SmaI-F fragment . Early chromatin contained hypersensitive sites at 16.0 and 14.3 . These sites became minor cleavage sites in late chromatin and new hypersensitive sites appeared at 13.5 and 13.0 . The change in the location of the hypersensitive sites in the course of infection correlated with the early to late switch in the transcription pattern in this region and the early to late change in the overall structure of adenovirus chromatin.

Exp Cell Res, 1984 Sep, 154(1), 213 - 23
Diffusible factors are responsible for differences in nuclease sensitivity among chromatins originating from different cell types; Chambers SA et al.; We have examined the kinetics of nuclease digestion of chromatin from committed and uncommitted cells in experiments where the nuclei are mixed and co-digested . Cultures of the sea urchin, Arbacia punctulata, were grown to the 16-cell stage in either {3H}thymidine or {14C}thymidine and the macromere, mesomere, and micromere cell types separated . After isolation, sets of nuclei with two different blastomere types (each having different radionucleotide tagging) were mixed and co-digested with micrococcal nuclease or DNase . I . The extent of digestion was monitored by solubility in 5% perchloric acid (PCA) . We find no significant differences in initial digestion rates or limit digests among the different cell types when co-digested with either nuclease . Differences in nuclease sensitivity observed when nuclei are digested separately are abolished when nuclei are probed in a mixing experiment . The results support the hypothesis that phenotypic differences in digestibility among different cell types in vitro reflect differences in chromatin-condensing factors which can diffuse between nuclei.

Cell Biophys, 1984 Sep, 6(3), 183 - 96
Phase transitions in nuclei and chromatin . Is nuclear volume controlled by the chromatin or by the nuclear matrix?
Nicolini C, Carlo P, Finollo R, Vigo F, Cavazza B, Ledda A, Ricci E, Brambilla G.
Changes in the volume of rat liver nuclei have been monitored as a function of modifications in ionic environment (from 0 to 20 mM), temperature (from 4 to 37 degrees C), and pH (from 1 to 8) . An abrupt reduction of nuclear volume occurred with increasing ion concentration, this contraction being more pronounced with bivalent (either Ca2+ or Mg2+) than with monovalent (either Na+ or K+) cations . The lowering of pH produced a similar effect . Parallel changes in chromatin structure took place at the same time as phase-like transitions . Atomic absorption spectroscopy allowed determination of free and nuclei-bound ions, pointing to the presence of a sizeable number of free binding sites for chromatin-DNA even within intact nuclei . DNA-phosphate sites appear to be neutralized by ions strictly according to the size of the electric charge and polyelectrolyte theory . Partial digestion (by micrococcal nuclease) or simple breaks (by chemical carcinogens) of the chromatin-DNA fiber caused respectively elimination or reduction of the abrupt volume changes in the intact nuclei . The apparent role of chromatin structure versus nuclear matrix in determining the shape and volume of intact nuclei is briefly discussed.

Radiat Res, 1984 Sep, 99(3), 502 - 10
The contribution of hydroxyl radical to radiosensitization: a study of DNA damage; Skov KA; Using the radioprotector dimethylsulfoxide, DMSO, as a scavenger of hydroxyl radicals, the proportions of DNA damage caused by OH . were determined in mammalian cells irradiated in hypoxia with or without the radiosensitizers misonidazole and TAN or in air . Yields of both single-strand breaks (SSB) and base/sugar damage (MLS for Micrococcus luteus sensitive sites) were measured for each situation . Most of the damage enhanced by the sensitizers was found to be OH . dependent, for both MLS and SSB classes of damage: most breaks (greater than 80%) enhanced by oxygen and about two-thirds of the breaks enhanced by misonidazole (hypoxia) occur at OH.-damaged sites; most if not all base/sugar damage enhanced by the sensitizers misonidazole and TAN (in hypoxia) occurs only in the presence of OH., whereas in air, some (about one-quarter) of the enhanced MLS damage does not require OH. . The sensitizer enhancement ratios in the presence of scavenger and the degree of protection afforded by the scavenger determined for total (MLS + SSB) damage agree well with those derived from corresponding survival experiments.

Biochim Biophys Acta, 1984 Aug 28, 789(1), 63 - 8
Isolation of high-mobility-group proteins HMG1 and HMG2 in non denaturing conditions and comparison of their properties with those of acid-extracted proteins; Marekov LN et al.; We describe a method for isolation and purification of the chromosomal proteins HMG1 and HMG2 in non-denaturing conditions which overcomes the difficulties of the published methods concerning yield and purity . The method is based on salt extraction, selective precipitation with ammonium sulfate and DEAE-cellulose chromatography . All studied properties of these proteins (formation of protein tetramers, enhancement of micrococcal nuclease digestion of DNA and chromatin, and protection of 165-basepair DNA in chromatosome) differ significantly from the properties of HMG1 and 2 isolated under denaturing conditions.

J Biol Chem, 1984 Aug 25, 259(16), 10582 - 9
Dissipative structures in proteoglycan solutions; Harper GS et al.; Diffusion in multicomponent solutions containing proteoglycan is shown to result in the formation of coherent, fluid structures (known as dissipative structures) and induction of rapid polymer transport . These phenomena occur over a wide range of conditions (i.e . varying solute distribution, concentration, size, and chemical composition) which are envisaged to occur in the extracellular matrix of connective tissues . A concentration gradient of chondroitin sulfate in a proteoglycan matrix of uniform concentration yields dissipative structures which transport the proteoglycan up to 300-fold faster than its transport in the absence of the gradient component . Similar behavior was observed with other polysaccharide and monosaccharide concentration gradient components . Amplification of structure formation and rapid transport was achieved by 1) increasing the concentration of proteoglycan matrix, 2) increasing the magnitude of the concentration gradient, 3) decreasing the molecular weight of the gradient-forming component, and 4) decreasing the concentration gradient of proteoglycan in the matrix . Dissipative structure morphology exhibits a marked dependence on the initial component distribution . Non-specific, excluded volume interactions between the proteoglycan and the gradient component are believed to induce coupled diffusive transport of the proteoglycan . This leads to microscopic density inversions which nucleate and develop into macroscopic convective flows . These results are similar to those previously observed in ternary solutions of uncharged polymers (i.e . dextran/polyvinylpyrrolidone) . We have demonstrated that dissipative structures may transport Micrococcus luteus cells as well as various solutes . Flows were also observed in proteoglycan solutions after localized addition of small amounts of either a proteolytic enzyme or hyaluronic acid . It is likely that the prerequisites for this spontaneous macroscopic self-organization, as manifested by the flow phenomenon, are present in the extracellular matrix of connective tissues.

J Mol Biol, 1984 Aug 25, 177(4), 715 - 33
Nuclease digestion of circular TRP1ARS1 chromatin reveals positioned nucleosomes separated by nuclease-sensitive regions; Thoma F et al.; TRP1ARS1 is a circular yeast DNA of 1453 base-pairs that contains the N-5'phosphoribosyl anthranilate isomerase (TRP1) gene and a sequence important for autonomous replication (ARS1) . It exists extrachromosomally in 100 to 200 copies/cell and is presumably packed in nucleosomes . TRP1ARS1 has been partially purified as chromatin from lysed spheroplasts of yeast using gel filtration . A structural analysis of mapping micrococcal nuclease and DNAase I cutting sites with an accuracy of +/- 20 base-pairs is presented . Comparison of nuclease cleavage sites in chromatin and in purified DNA reveals that regions which are protected against nuclease attack are not distributed randomly . These regions are big enough to accommodate nucleosome cores . Three nucleosomes are positioned in the so-called ARS sequences, and are stable at low and high levels of digestion . The TRP1 gene region is covered by four nucleosomes, but they are neither randomly arranged nor precisely positioned . They are not stable and rearrange or disintegrate during digestion . The nucleosomal regions are separated by two segments of DNA (A, B), each about 180 base-pairs long, which are very sensitive to DNAase I and micrococcal nuclease and therefore presumably not packed in nucleosomes . Region B is found 5' to the TRP1 gene and might be related to transcription, whereas region A is centered around the termination codon of the TRP1 gene and the putative origin of replication.

FEBS Lett, 1984 Aug 20, 174(1), 1 - 6
Evidence for a homogeneous lateral distribution of lipids in a bacterial membrane . A photo cross-linking approach using anthracene as a photoactivable group; de Bony J et al.; A new photo cross-linking method has been developed for the study of the lateral distribution of lipids in natural membranes, which uses anthracene as a photoactivable group . This method, which rests on the potentiality of anthracene to form covalently bound dimers upon irradiation around 340-380 nm has been applied to the membrane lipids (dimannosyl diacylglycerol, phosphatidylglycerol, phosphatidylinositol) of the bacterium Micrococcus luteus . These glyco- and phospholipids were anthracene labelled by metabolically incorporating the synthetic 9-(2-anthryl)nonanoic acid . The following sequential procedure was used: dimerization of the anthracene-labelled lipids in the membrane by irradiation of the intact cells at 360 nm; extraction of the lipids and thin-layer chromatography in the first dimension to separate the various lipid dimers from the monomers; partial dedimerization of the lipid dimers by illumination of the chromatogram at around 250-280 nm; chromatography in the second dimension to separate the native lipid monomers from the corresponding residual lipid dimers . On account of the occurrence of the 3 hetero dimers phosphatidylglycerol-dimannosyl diacylglycerol, phosphatidylinositol-dimannosyl diacylglycerol and phosphatidylglycerol-phosphatidylinositol after irradiating the cells, it is concluded that in this bacterial membrane, dimannosyl diacylglycerol, phosphatidylglycerol and phosphatidylinositol are homogeneously distributed.

Environ Res, 1984 Aug, 34(2), 335 - 42
Lysozyme levels in rabbit lung after inhalation of nickel, cadmium, cobalt, and copper chlorides; Lundborg M et al.; Groups of rabbits were exposed to chlorides of nickel, cadmium, copper, and cobalt at concentrations ranging from 0.2 to 0.6 mg/m3 (as metal) for 4-6 weeks (5 days/week, 6 hr/day) . Activity of lysozyme (muramidase) in lavage fluid, in alveolar macrophages, and in culture medium from macrophages incubated at 37 degrees C for 1 and 20 hr was estimated using the lyso-plate technique, agar plates with heat-killed Micrococcus lysodeikticus . In the nickel-exposed rabbits lysozyme activity in the mucous membrane from the left main bronchus was also estimated . Following nickel exposure the lysozyme level was significantly decreased in lavage fluid, macrophages, and in culture medium from incubated macrophages but remained unchanged in the mucous membrane . After exposure to cadmium, copper, and cobalt, lysozyme levels increased or were unchanged.

Endocrinol Jpn, 1984 Aug, 31(4), 509 - 22
An approach to searching for specific proteins associated with active genes in hen oviduct; Kato Y et al.; This study has examined an approach to searching for specific proteins associated with the altered nucleosome structure of transcriptionally active genes that are induced by steroid hormones in the hen oviduct . Hen oviduct nuclei were digested with micrococcal nuclease by the procedure which selectively excises nucleosomes from the ovalbumin gene . The oviduct nuclei, as well as chick erythrocyte nuclei, were also digested with DNAase I under conditions preferentially sensitive to the ovalbumin gene, as well as the globin gene . Released proteins were characterized by one- and two-dimensional polyacrylamide gel electrophoresis with detection by silver staining . Thus, high mobility group (HMG) proteins 14 and 17 were found in the three cases of nuclease digestion . Furthermore, about 10, 20 and 15 nonhistone protein spots, specific to each nuclease action, were observed in the cases of micrococcal nuclease to oviduct nuclei and DNAase I to oviduct and erythrocyte nuclei, respectively . Between these three series of protein spots, at least three spots were characterized to be common to those released by both nucleases from oviduct nuclei . These common proteins may be involved, as estrogen receptor proteins or others, in recognition of the ovalbumin DNA sequences, followed by a non-sequence-specific process in which the HMG proteins alter the structure of nucleosomes along the transcription unit.

J Appl Bacteriol, 1984 Aug, 57(1), 139 - 45
Scanning electron microscopy of dairy equipment surfaces contaminated by two milk-borne micro-organisms; Speers JG et al.; Ethanol dehydration followed by argon replacement induced drying (ARID) was found to be a suitable method for the preparation of glass, stainless steel and rubber surfaces which had been in contact with inoculated milk and which were to be examined using scanning electron microscopy (SEM) . This technique was used to examine samples of all three materials which had been subjected to both single and repeated inoculation with whole milk containing a Pseudomonas sp . or a Micrococcus sp . and incubated for various periods . Some samples were also prepared for SEM using a cryofixation technique . The Pseudomonas sp . was found to proliferate on glass and stainless steel surfaces but not on rubber . Due to the clumping tendency of the Micrococcus sp . proliferation of this organism was more difficult to assess accurately . In general there was no difference in results obtained between single and repeated inoculation . Various factors which may have aided attachment of micro-organisms to surfaces were identified viz., surface channels present in stainless steel, milk deposits and the production of extracellular material . The value of using both the cryofixation and chemical preparatory techniques for the identification of artifacts is discussed.

Biochem Int, 1984 Aug, 9(2), 251 - 8
Undermethylation of DNA in mononucleosomes solubilized by micrococcal nuclease digestion of HeLa cell nuclei; Hatayama T et al.; To examine the distribution of 5-methylcytosine in chromatin DNA, DNA of HeLa cells was labeled with {3H-methyl}methionine and {14C} thymidine and analyzed after extensive digestion of the nuclei with micrococcal nuclease . When the chromatin solubilized with the nuclease was fractionated on a sucrose density gradient, DNA in mononucleosomes was considerably depleted in 5-methylcytosine, as compared with polynucleosomes . Electrophoretic separation of DNA from the chromatin also revealed the depletion of 5-methylcytosine in the mononucleosomal size of DNA . This was confirmed by the chromatographic analysis of 5-methyldeoxycytidine after enzymatic digestion of the DNA to nucleosides . Thus the DNA in mononucleosomes solubilized by extensive micrococcal nuclease digestion is depleted in 5-methylcytosine, suggesting that 5-methylcytosine is preferentially missing from the DNA in the nucleosome core particles.

Radiat Res, 1984 Aug, 99(2), 363 - 71
Effect of irradiation and endogenous nucleases on rat liver chromatin; Gelderblom D et al.; These assessment of the consequences of irradiation on chromatin is complicated by endogenous nucleases . Isolation and prolonged storage of rat liver nuclei in buffers containing divalent metal ions activates these enzymes and promotes the degradation of chromatin . Irradiation of rat liver nuclei to dose levels of 20,000 rad under conditions in which endogenous nucleases are inhibited and analysis of the irradiated chromatin by sucrose density gradient centrifugation gave no evidence for monosomes or oligosomes . When chromatin from irradiated nuclei was digested with micrococcal nuclease, the levels of monosomes and oligosomes were identical to those of micrococcal nuclease, the levels of monosomes and oligosomes are identical to suggest that irradiation results in neither a direct fragmentation of linkers nor the sensitization of linkers for subsequent cleavage by micrococcal nuclease . Histones isolated from monosomes of irradiated and unirradiated nuclei were intact, showing no fragmentation or loss of residues, as judged by sodium dodecyl sulfate-polyacrylamide electrophoresis.

Virology, 1984 Jul 30, 136(2), 321 - 7
Protein composition of adenovirus nucleoprotein complexes extracted from infected cells; Weber J et al.; A viral nucleoprotein complex was extracted from the nuclei of human cells 20 hr after infection with adenovirus type 2 or several of its temperature-sensitive mutants . In its sedimentation property, density in CsCl, and digestion pattern with micrococcal nuclease, the complex resembled viral cores . The polypeptides V, PVII, 11K, and 36K were found associated with this complex which is formed prior to or in the absence of virus assembly . The results suggest that this nucleoprotein complex is a direct precursor to virus assembly.

Nucleic Acids Res, 1984 Jul 25, 12(14), 5693 - 706
Chicken reticulocyte polysomal messenger RNA-protein complex: absence of bound proteins in most of the coding region of beta globin mRNA; Chae CB et al.; The 15s globin mRNA-protein complex (mRNP) was isolated from chicken reticulocyte polyribosomes dissociated in EDTA . To determine protein binding sites, the mRNP was treated with micrococcal nuclease and the nuclease resistant RNA was mapped to the beta globin gene at the nucleotide level . As far as we can determine there is no bound protein from the Cap site to the poly A addition site of beta globin mRNA in the mRNP except for a short area in the coding region near the translation initiation site.

Virology, 1984 Jul 15, 136(1), 10 - 9
Histone H3 modification in BHK cells infected with foot-and-mouth disease virus; Grigera PR et al.; Infection of BHK cells with foot-and-mouth disease virus (FMDV) causes a thorough change in the electrophoretic profile of whole nuclear histones . It consists in the disappearance of histone H3 and the appearance of a new polypeptide (Pi) which migrates between histones H2A and H4 on SDS-polyacrylamide gels . Protein Pi is detected at 2 hr postinfection (pi), the time in which viral RNA synthesis begins to increase, and reaches equimolecular amounts with the remaining core histones 1 hr later, when the disappearance of histone H3 is almost complete . Labeling of cells prior to infection demonstrates that Pi is not a novo product but the result of a viral-induced processing of a host precursor synthetized beforehand . Protein Pi comigrates with histone H2A/B in acetic acid/urea polyacrylamide gels and it shares common major peptides with histone H3 under controlled proteolysis with protease V8 or trypsin . The mononucleosomal and nucleosomal DNA pattern analysis after micrococcal nuclease treatment of nuclei from infected and mock-infected cells did not show any significant differences even though after 3 hr (p.i.), protein Pi replaces histone H3 in the nucleosomal structure . It was concluded that FMDV infection is responsible for a specific modification in the nucleus of infected cells which leads, after 3 hr (p.i.), to a complete histone H3 protein Pi transition in the nucleosomes.

J Mol Biol, 1984 Jul 15, 176(4), 535 - 57
Structural specificities of five commonly used DNA nucleases; Drew HR; Five commonly used nucleases were surveyed for their ability to distinguish among several different DNA backbone configurations . The digestion data suggest that: (1) DNAase I binds across the minor groove; whereas (2) nuclease S1 and (3) micrococcal nuclease bind to an exposed single strand; (4) copper/phenanthroline seeks a base-pair step; and (5) DNAase II requires just a stacked single strand of limited exposure . Only micrococcal nuclease is demonstrably base-specific, with a strong preference for T, A over C, G in any structural context.

J Biol Chem, 1984 Jul 10, 259(13), 8558 - 63
Differential salt fractionation of active and inactive genomic domains in chicken erythrocyte; Rocha E et al.; We have utilized the Sanders salt fractionation technique (Sanders, M . M . (1978) J . Cell Biol . 79, 97-109) to analyze the products of micrococcal nuclease digestion of adult chicken erythrocyte nuclei . By dot-blot hybridization with specific gene probes, it is found that nucleosomes from the globin gene domain, including a region extending to about 10 kilobase pairs 5' to the beta p gene are selectively enriched in the fractions eluted at low salt . In contrast, a single copy sequence located at about 10 kilobase pairs 5' to the beta p gene was concentrated in the less salt-soluble fractions . The vitellogenin and ovalbumin genes, which are never expressed in erythroid tissues, are also concentrated in the less salt-soluble fractions . Some more generally expressed genes (histone H4, thymidine kinase) appear to be more uniformly distributed . The low salt fractions are depleted in H1/H5, enriched in high mobility group 14 and 17, and contain somewhat more highly acetylated histones.

J Biol Chem, 1984 Jul 10, 259(13), 8534 - 44
Differentiation-dependent chromatin alterations precede and accompany transcription of immunoglobulin light chain genes; Rose SM et al.; We have studied the nature of chromatin alterations along immunoglobulin light chain (IgL) genes during B cell development using cultured murine cell lines . Employing a chromatin fractionation procedure on micrococcal nuclease-treated nuclei, we demonstrate that transcriptionally active k IgL chromatin lacks a canonical nucleosomal repeat and exhibits a pronounced association with insoluble nuclear material but is processed by nuclease to a soluble nucleosomal component that apparently lacks histone H1 and is enriched in high mobility group proteins . Of particular significance, utilizing a variant plasmacytoma cell line that has transcriptionally inactivated one k allele via a promoter deletion, we demonstrate that transcription per se is not responsible for these novel alterations . Furthermore, we show that the chromatin encompassing germline (unrearranged) and transcriptionally silent lambda IgL alleles in k-producing plasmacytomas exhibit some of the same unusual properties that are displayed by k alleles . Finally, we demonstrate that these alterations only occur in cell lines of the lymphocyte lineage that have progressed past the early pre-B cell stage; when inactive, both k and lambda IgL genes possess typical nucleosomal packaging and co-fractionate with histone H1-containing chromatin . These findings lead us to propose a model that predicts B cell stage-specific alterations in IgL chromatin prior to gene rearrangement and transcription.

J Steroid Biochem, 1984 Jul, 21(1), 35 - 42
Characterization of nuclear estradiol receptors released by micrococcal nuclease and deoxyribonuclease I; Thomas T et al.; Interaction of estradiol receptor with chromatin was probed by nuclease digestion of receptor-chromatin complex . The complex was formed by incubating partially purified receptor with chromatin . Micrococcal nuclease digestion of the complex released a 7S form of receptor which could be converted to 2.8S form by DNAase I . Digestion of the complex with DNAase I yielded different forms of receptors ranging from 7S to 2.8S depending on the digestion time . Receptor distribution was also examined by isolating nuclei form tissue pre-incubated with radioactive estradiol . Micrococcal nuclease digestion of receptor-filled nuclei released 7S, 5.5S and 3.5S forms of receptors . Collectively, these results indicate that the 7S nuclear receptor may have an associated chromatin fragment which is sensitive to DNAase I activity . The desirable features of receptor-chromatin method over conventional methods for studies relating to receptor interaction at the gene site are discussed.

J Cell Biol, 1984 Jul, 99(1 Pt 1), 272 - 86
Differences of supranucleosomal organization in different kinds of chromatin: cell type-specific globular subunits containing different numbers of nucleosomes; Zentgraf H et al.; Fractions of homogeneously-sized supranucleosomal particles can be obtained in high yield and purity from various types of cells by brief micrococcal nuclease digestion (10 or 20 s) of condensed chromatin in 100 mM NaCl followed by sucrose gradient centrifugation and agarose gel electrophoresis . These chromatin particles, which contain only DNA and histones, differed according to cell type . Sea urchin spermatozoa (Paracentrotus lividus) gave rise to heavy particles (ca . 260 S) with a mean diameter (48 nm) . These resembled the unit chromatin fibrils fixed in situ, which contain an average of 48 nucleosomes, as determined both by electron microscopy after unraveling in low salt buffer and gel electrophoresis . In contrast, higher order particles from chicken erythrocyte chromatin were smaller (105 S; 36-nm diam) and contained approximately 20 nucleosomes . The smallest type of supranucleosomal particle was obtained from chicken and rat liver (39 S; 32-nm diam; eight nucleosomes) . Oligomeric chains of such granular particles could be recognized in regions of higher sucrose density, indicating that distinct supranucleosomal particles of globular shape are not an artifact of exposure to low salt concentrations but can be obtained at near-physiological ionic strength . The demonstration of different particle sizes in chromatin from different types of nuclei is contrary to the view that such granular particles are produced by artificial breakdown into "detached turns" from a uniform and general solenoid structure of approximately six nucleosomes per turn . Our observations indicate that the higher order packing of the nucleosomal chain can differ greatly in different types of nuclei and the supranucleosomal organization of chromatin differs between cell types and is related to the specific state of cell differentiation.

Biosci Rep, 1984 Jul, 4(7), 541 - 50
Binding of benzo{a}pyrene to different chromatin domains following activation at the nuclear membrane; Obi FO et al.; When isolated liver nuclei from methylcholanthrene-treated rats are incubated with benzopyrene, covalent adducts are formed between DNA and the ultimate carcinogen, benzopyrene diol epoxide . Brief digestion with DNaseI, or micrococcal nuclease has been used to demonstrate that benzopyrene metabolites bind more readily to DNA in chromatin regions with a more open, active conformation than to inactive chromatin.

Mol Biol Rep, 1984 Jul, 10(1), 31 - 9
Chromatin proteins associated with micrococcal nuclease-sensitive and nuclease-resistant chromatin fractions of Kirkman-Robbins hepatoma and hamster liver; Lipinska A et al.; Micrococcal nuclease-sensitive (SP) and nuclease-resistant (PP) chromatin fractions from Kirkman-Robbins hepatoma and hamster liver were obtained . The molecular distribution of three non-histone proteins (NHCP1, NHCP2 and NHCP3), histones, and chromatin-bound protease activity between SP and PP fractions of both tissues was compared . Differences, mainly of quantitative nature, among non-histone proteins of neoplastic and normal tissue were observed . Moreover, it was found that polypeptides with mol . wt 81 000 (NHCP1), 39 000 (NHCP2) and 21 000, 35 000, 37 000 (NHCP1), 70 000, 112 000, 141 000, 157 000 (NHCP2), 30 000-33 000 (NHCP3) were associated only with the nuclease-sensitive part of chromatin of hepatoma and normal tissue, respectively . A major difference in histone composition of hamster hepatoma and liver concerns histones H2A and H1 . Furthermore, an enrichment of high mobility group proteins as well as other soluble non-histone proteins in an acid extract of the SP fraction was observed . Apparently chromatin-bound protease activity can be found in both fractions of chromatin.

Mol Biol Rep, 1984 Jul, 10(1), 9 - 12
Removal of histone H1 exposes linker DNA in chromatin to DNAse I; Iovcheva C et al.; After removal of histone H1 about 40% of DNA in chromatin acquires the sensitivity of naked DNA to DNAse I . Digestion of H1-depleted chromatin with DNAse I leads to a qualitative change in the digestion pattern, generating DNA fragments of approx . 200 b.p . and multiples, similar to those obtained with micrococcal nuclease . Both effects are reversed upon reconstitution of purified H1 to H1-depleted chromatin.

Mol Biol (Mosk), 1984 Jul-Aug, 18(4), 1141 - 50
{Chromatin structure of ribosomal genes of Drosophila melanogaster . Random location of nucleosomes in DNA and characteristics of organization of non-transcribed spacer}; Kolchinskii AM et al.; Chromatin structure of ribosomal genes from nuclei of Drosophila melanogaster embryos was studied by using micrococcal nuclease cleavage . End-directed labelling with short cloned fragments of the ribosomal repeat was carried out . It shows, that the micrococcal nuclease prefers specific sites in naked DNA, and the pattern of DNA cleavage is essentially conserved when the nuclei are digested . Only minor differences are visible . Hence, there are no specific positions of nucleosomes on the ribosomal repeat . The DNA fragments from the nuclei treated with micrococcal nuclease were electrophoresed, transferred to DBM-paper and hybridized with different probes subcloned from the ribosomal repeat . The non-transcribed spacer and the region of the beginning of transcription are hydrolysed significantly faster than the coding region or inactive ribosomal insertion . The region of NTS and the beginning of transcription do not give normal nucleosomal fragment in the range of 145-185 bp; instead they produce a heterogeneous band 200-280 bp in length even after prolonged digestion . Dinucleosomal fragments are also slightly longer and more heterogeneous than in other parts of the ribosomal repeat . Higher oligomers are similar throughout the ribosomal repeat . We suggest that a hypothetical transcription factor interacts in a way with histones and protects unusual fragments of DNA from digestion.

Mol Biol (Mosk), 1984 Jul-Aug, 18(4), 1099 - 110
{Structural-functional organization of the SV40 virus chromosome . III . Nucleosome organization of free mini-chromosomes}; Nedospasov SA et al.; Micrococcal nuclease digestion and hybridization end labeling procedure have been used for analysis of nucleosomal organization of SV40 minichromosomes . Usual oligonucleosomal pattern containing long oligonucleosomes has been observed after digestion and DNA electrophoresis . All the regions of the minichromosome, including the "regulatory" one, are involved in nucleosome structure, as judged by hybridization analysis . This is valid at least for the major minichromosome fraction . On the other hand, the nucleosome ordering turned out to be higher in certain genome regions, for example around the site where the replication terminates . Data implying the existence of several discrete nucleosome "frames" in this region have been obtained . Possible artefacts due to micrococcal nuclease sequence-specificity are discussed.

EMBO J, 1984 Jul, 3(7), 1635 - 41
Chromatin structure of a hyperactive secretory protein gene (in Balbiani ring 2) of Chironomus; Widmer RM et al.; We examined the chromatin structure of a Balbiani ring (secretory protein gene) in the salivary glands of Chironomus larvae in its hyperactive state after stimulation with pilocarpine . For the inactive state of the gene an established tissue culture cell line, not expressing the gene, was used . Electron microscopy showed an RNA polymerase density of approximately 38/microns . Micrococcal nuclease digestion of purified nuclei followed by DNA transfer and hybridization revealed a smear with no recognizable discrete DNA fragments . Without pilocarpine stimulation a faint nucleosomal repeat was superimposed upon the smear, and in tissue culture cells a clear nucleosomal repeat was revealed . The restriction enzyme XbaI, which has a 6-bp recognition sequence, cut the gene in the hyperactive chromatin state, but not in its inactive conformation . The combined results are best explained by the absence of most of the nucleosomes in this hyperactive RNA polymerase II transcribed gene.

Nucleic Acids Res, 1984 Jun 25, 12(12), 5015 - 24
Reduced repeat length of nascent nucleosomal DNA is generated by replicating chromatin in vivo; Jakob KM et al.; Micrococcal nuclease digestion of nuclei from sea urchin embryos revealed transient changes in chromatin structure which resulted in a reduction in the repeat length of nascent chromatin DNA as compared with bulk DNA . This was considered to be entirely the consequence of in vivo events at the replication fork (Cell 14, 259, 1978) . However, a micrococcal nuclease-generated sliding of nucleosome cores relative to nascent DNA, which might account for the smaller DNA fragments, was not excluded . In vivo {3H}thymidine pulse-labeled nuclei were fixed with a formaldehyde prior to micrococcal nuclease digestion . This linked chromatin proteins to DNA and thus prevented any in vitro sliding of histone cores . All the nascent DNAs exhibiting shorter repeat lengths after micrococcal nuclease digestion, were resolved at identical mobilities in polyacrylamide gels of DNA from fixed and unfixed nuclei . We conclude that these differences in repeat lengths between nascent and bulk DNA was generated in vivo by changes in chromatin structure during replication, rather than by micrococcal nuclease-induced sliding of histone cores in vitro.

Biochim Biophys Acta, 1984 Jun 16, 782(2), 210 - 9
Non-histone proteins of soluble nucleoproteins released from mouse myeloma nuclei by mild micrococcal nuclease digestion; Chambers SA et al.; Mono- and dinucleosomes preferentially cleaved from mouse myeloma chromatin by very mild micrococcal nuclease digestion at 0 degree C are soluble and are released from nuclei under near-physiological conditions in which normal nucleosomes containing Hl are insoluble . These nucleosomes are highly enriched in RNA, high-mobility-group proteins and a unique subset of other non-histone proteins . They are nearly devoid of histone Hl and contain DNA significantly less methylated than whole myeloma DNA, indicating that they comprise a subset of genomic sequences . Previously we have shown that this fraction is enriched in transcribed DNA sequences . Non-histone proteins that co-sedimented with readily solubilized nucleosomes included many of the most basic, low-to-moderate molecular weight chromosomal proteins . Many of these proteins were also preferentially acetylated in vivo . The residual, pelleted chromatin was highly enriched in high molecular weight proteins (greater than 60 000), and very depleted in medium molecular weight proteins . Readily solubilized nucleoproteins sedimenting like mononucleosomes were partly resolved by electrophoresis, under non-denaturing conditions, into several subfractions differing significantly in non-histone protein contents . Methods described here should be useful for identifying and isolating non-histone proteins bound to nucleosomes and other chromatin regions that are structurally and functionally unique.

Biochim Biophys Acta, 1984 Jun 16, 782(2), 202 - 9
Enrichment of transcribed and newly replicated DNA in soluble chromatin released from nuclei by mild micrococcal nuclease digestion; Chambers SA et al.; A chromatin fraction solubilized from mouse myeloma nuclei under near-physiological ionic conditions by very mild micrococcal nuclease digestion at 0 degrees C is enriched at least 7-fold in DNA complementary to total myeloma polyadenylated mRNA, and 15-fold in DNA originating near the replication fork (labeled within 30 s) . Newly replicated DNA recovered in solubilized chromatin after brief labeling was incorporated mainly into particles sedimenting with, or faster than, mononucleosomes . A rapid decrease in enrichment of newly replicated DNA in readily released, soluble chromatin with increasing labeling times indicated that newly replicated chromatin matured within 90 s to a form that was partitioned similarly to bulk chromatin by this fractionation method . Previous studies showed that chromatin readily solubilized from myeloma nuclei is enriched in high-mobility-group (HMG) and other non-histone proteins, RNA and single-stranded DNA; and depleted in H1 and 5-methylcytosine, relative to bulk chromatin (Jackson, J.B., Pollock , J.M., Jr., and Rill , R.L . (1979) Biochemistry 18, 3739-3748) . Mild digestion of chicken erythrocyte nuclei with micrococcal nuclease yielded a soluble chromatin fraction (1-2% of the total DNA) with similar properties . This fraction was enriched at least 6-fold in DNA complementary to chicken globin mRNA, relative to total erythrocyte DNA.

J Mol Biol, 1984 Jun 15, 176(1), 131 - 54
Positioning of nucleosomes in satellite I-containing chromatin of rat liver; Bock H et al.; The location of nucleosomes on rat satellite I DNA has been investigated using a new approach . Nucleosome cores were prepared from rat liver nuclei with micrococcal nuclease, exonuclease III and nucleases S1 . From the total population of core DNA fragments the satellite-containing fragments were isolated by molecular cloning and the complete sequence of 50 clones was determined . The location of nucleosomes along the satellite sequence was found to be non-random . Our results show that nucleosomes occupy a number of positions on satellite I DNA . About 35 to 50% of all nucleosomes are positioned in two corresponding major sites, the remainder in about 16 less preferred sites . The major nucleosome positions are apparently strictly defined with the precision of a single base-pair . These results were confirmed by other approaches, including restriction nuclease digestion experiments . There are good indications of a defined long-range organization of the satellite chromatin fiber in two or more oligonucleosomal arrays with distinct nucleosome configurations.

J Mol Biol, 1984 Jun 15, 176(1), 105 - 29
Nucleosomes are positioned on mouse satellite DNA in multiple highly specific frames that are correlated with a diverged subrepeat of nine base-pairs; Zhang XY et al.; Nucleosome phasing on highly repetitive DNA was investigated using a novel strategy . Nucleosome cores were prepared from mouse liver nuclei with micrococcal nuclease, exonuclease III and nuclease S1 . The core DNA population that contains satellite sequences was then purified from total core DNA by denaturation of the DNA, reassociation to a low Cot value and hydroxyapatite chromatography to separate the renatured satellite fraction . After end-labeling, the termini of the satellite core DNA fragments were mapped with an accuracy of +/- 1 base-pair relative to known restriction sites on the satellite DNA . Sixteen dominant nucleosome positions were detected . There is a striking correlation between these nucleosome frames and an internal highly diverged 9 base-pair subrepeat of the satellite DNA . The results are consistent with a sequence-dependent association of histone octamers with the satellite DNA . Our finding that histone octamers can interact with a given DNA in a number of different defined frames has important implications for the possible biological significance of nucleosome phasing.

FEBS Lett, 1984 Jun 11, 171(2), 240 - 4
Thyroxine preferentially stimulates transcription by isolated neuronal nuclei in the developing rat brain cortex; Lindholm DB; The administration of thyroxine to neonatal rats stimulates RNA synthesis by neuronal nuclei isolated from the developing rat brain cortex . Glial nuclei are relatively resistant to thyroxine treatment . The activity of neuronal RNA polymerase II is particularly stimulated by the hormone . Thyroxine also affects neuronal chromatin structure as shown by changes in the relative proportion of different subnuclear fractions obtained by gentle micrococcal nuclease digestion of nuclei from hormone-treated rats.

Biochemistry, 1984 Jun 5, 23(12), 2627 - 33
Stopped-flow kinetic studies on the interaction between echinomycin and DNA; Fox KR et al.; The kinetics of association between the quinoxaline antitumor antibiotic echinomycin and DNA have been studied by stopped-flow methods . With natural DNAs, the reaction profile is completely described by a single exponential, the time constant for which varies linearly with the DNA concentration . This bimolecular rate constant is similar for both calf thymus and Micrococcus lysodeikticus DNA (k = 6 X 10(4) M-1 s-1 at 25 degrees C, I = 0.01) and is probably dominated by interaction with relatively weak but abundant binding sites from which the antibiotic dissociates fairly quickly . The observed single exponential suggests a molecular mechanism of binding in which both chromophores of the antibiotic become intercalated simultaneously rather than sequentially; no transition from a mono-intercalated state to a bis-intercalated state could be detected . The reaction is slowed by a factor of about 3 on raising the salt concentration from I = 0.01 to I = 0.5 . Binding to poly(dA-dT) is also described by a single exponential, the time constant for which is about 3 times faster than that seen with natural DNAs . By contrast, the interaction with poly-(dG-dC) requires two exponentials for a proper description, the faster of which is similar to that seen with natural DNAs . This may reflect the initial interaction of the antibiotic with two types of sequences, tentatively identified as GpC and CpG, from which it dissociates at very different rates . The differences in kinetic behavior may be explicable on the basis of an alternating B structure for poly(dA-dT) and a more classical B form for poly(dG-dC).

Biochem J, 1984 Jun 1, 220(2), 539 - 45
A chromosomal phosphoprotein is preferentially released by mild micrococcal-nuclease digestion; Liew CC et al.; {32P}Pi was administered to rats (5mCi/rat) 2h before the isolation of liver nuclei . The isolated nuclei were subjected to mild micrococcal-nuclease digestion for 2.5, 5 and 10 min at 37 degrees C, and the mononucleosomal fraction was subsequently isolated by sucrose-density-gradient centrifugation . The specific radioactivity of 32P-labelled mononucleosomal fractions decreased with increased digestion times . A phosphorylated chromosomal protein, B2 (Mr 68000, pI6.5-8.2), was demonstrated immunologically in the mononucleosomal fraction by using an antibody specific to this electrophoretically purified phosphoprotein . The incorporation of 32P into this phosphoprotein, previously shown to be mainly through covalent linkage, was revealed by antibody precipitation followed by gel electrophoresis . The rate of release of acid-soluble nucleotides by micrococcal-nuclease digestion of liver nuclei from partially hepatectomized rats 16 h after operation was strikingly higher than that for sham-operated controls . After partial hepatectomy, an increase in 32P incorporation into phosphoprotein in the monomer fractions specifically precipitated by this antibody was also found . This suggests that the phosphorylated non-histone chromatin protein B2 is preferentially associated with the transcriptionally active chromatin.

J Immunol, 1984 Jun, 132(6), 2904 - 8
Antibodies from patients with autoimmune disease react with a cytoplasmic antigen in the Golgi apparatus; Fritzler MJ et al.; In this study we report the identification of an antibody in the sera of some patients with autoimmune disease that reacted with a cytoplasmic antigen localized within the Golgi apparatus . The antibody reacted with all tissues investigated, which included pancreas, kidney, testis, liver, thymus, and spleen . In addition, it reacted with some human peripheral circulating lymphocytes, murine peritoneal macrophages, and a variety of tissue culture cell lines, which included HEp-2 cells (human epithelial carcinoma), baby hamster kidney cells, a canine thymus cell line, a primary kidney cell line, Ehrlich ascites cells, Wil-2 cells, and Raji cells . The antigen is located in the same region stained by the histochemical reaction for thiamine pyrophosphatase, thus indicating that the antigen is located within the Golgi apparatus . The antigen was not demonstrated by immunodiffusion of saline extracts of rabbit thymus, pancreas, or liver . The antigen in HEp-2 cells was resistant to RNase A, DNase I, micrococcal nuclease, and to extraction with 0.1 N HC1, but was sensitive to trypsin and Proteinase K . Eight patients with anti-Golgi antibodies have been identified . Six of the eight had systemic lupus erythematosus . Autoantibodies to a Golgi apparatus antigen might serve as a useful biologic marker to study the functional relationship of the Golgi apparatus to lymphocytes and macrophages.

Arch Biochem Biophys, 1984 Jun, 231(2), 303 - 8
Two human colon tumor cell lines with similar nuclease sensitivities have different ethidium bromide binding characteristics; Duhl DM et al.; Chromatin from two human colon adenocarcinoma cell lines (HT-29 and LoVo) showed similar digestion kinetics when sensitivities to DNase I and micrococcal nuclease were examined . Chromatin conformations were probed by examining the binding of ethidium bromide . A Scatchard plot revealed that both chromatins bound the same amount of ethidium bromide per mole of DNA, but the DNA from LoVo cells was more accessible to the intercalator . The results indicate that differences in chromatin conformation are not necessarily accompanied by different nuclease sensitivities.

EMBO J, 1984 Jun, 3(6), 1243 - 53
Assembly of transfected DNA into chromatin: structural changes in the origin-promoter-enhancer region upon replication; Cereghini S et al.; Chimeric SV40 DNA containing only the early region, or plasmid DNA harboring the origin-promoter-enhancer region of SV40, when introduced into CV-1 or Cos-1 monkey cells by DEAE-dextran mediated transfer are rapidly assembled in a typical chromatin structure revealed by the generation of a regular 190 bp repeat ladder after micrococcal nuclease digestion . DNA replication is not required for this assembly process . Chromatin-specific DNase I hypersensitive sites are observed in the enhancer region of these minichromosomes . The pattern of the sites differs between non-replicating and post-replicated chromatin . The latter is identical to that observed in the lytic cycle . The presence of large T antigen is not sufficient for the shift in the structure of the chromatin . These experiments suggest that replication can modulate protein-DNA interactions during viral infection or upon cell differentiation.

Biochemistry, 1984 May 22, 23(11), 2462 - 9
Isolation of a heme-controlled inhibitor of translation that blocks the interaction between messenger rna and eukaryotic initiation factor 2; Knoller S et al.; A heme-controlled inhibitor of translation was isolated from the S-100 of rabbit reticulocytes by a novel procedure including chromatography on double-stranded ribonucleic acid (dsRNA)-cellulose . The inhibitor thus purified is extremely active and functionally resembles previously studied heme-controlled inhibitor preparations in terms of kinetics and extent of inhibition of translation, relief of inhibition by eukaryotic initiation factor 2 (eIF-2), relief of inhibition by 2-aminopurine, and preferential inhibition of alpha-over beta-globin synthesis . The action of this inhibitor on translation is resistant to treatment with bacterial alkaline phosphatase, micrococcal nuclease, or trypsin and to incubation at 95 degrees C, pH 2 or pH 12 . The inhibitor not only is retained on DEAE-cellulose, phosphocellulose, and dsRNA-cellulose but also exhibits a high affinity for the dye Cibacron Blue, properties that suggest that it may be a protein . Unlike previously described heme-controlled inhibitor preparations, or preparations that did not pass over dsRNA-cellulose, the inhibitor recovered upon dsRNA-cellulose chromatography does not exhibit eIF-2 kinase activity . The inhibitor does not block ternary complex formation between eIF-2, methionyl-tRNAfMet, and GTP but inhibits the ability of eIF-2 to form a complex with labeled globin mRNA . In the presence of inhibitor, the formation of mRNA/eIF-2 complexes can be restored effectively by an excess of eIF-2 but not by an excess of mRNA . The inhibitor thus appears to block the interaction between eIF-2 and mRNA not by competing with eIF-2 for a binding site on mRNA but, instead, by acting on eIF-2 itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1984 May 15, 141(1), 105 - 8
Isolation of short interspersed repetitive DNA sequences present in the regions of initiation of mammalian DNA replication; Anachkova B et al.; Nascent DNA chains containing the putative replication origins were isolated from cells of human embryonic lung fibroblasts, Hela, Ehrlich ascites tumour and Guerin ascites tumour as described earlier { Russev , G., and Vassilev , L . (1982) J . Mol . Biol . 161, 77-87} . It was demonstrated that the synthesis of these nascent chains correlated with the ability of cells to initiate semiconservative DNA replication . Reassociation and electrophoretic analysis showed that the nascent chains from all four cell lines contained middle repetitive DNA in the form of short interspersed sequences . Mouse repetitive sequences were isolated and hybridized to Escherichia coli, chicken, calf and rat DNA and to homologous hnRNA . The kinetics of hybridization indicated that the repetitive sequences found in the vicinity of the replication origins were order-specific and were not heavily transcribed . Reassociation experiments, in which homologous DNA isolated from nuclei digested with micrococcal nuclease to different extents was used as a driver, showed that these repetitive sequences were organized into nucleosomes like the bulk of the chromatin.

Arch Biochem Biophys, 1984 May 15, 231(1), 169 - 74
HMG17 protein facilitates the DNA catenation reaction catalyzed by DNA topoisomerases; Tse YC et al.; HMG17 protein is shown to greatly facilitate the catention of double-stranded DNA rings catalyzed by DNA topoisomerases . Even at low DNA concentrations such that catenanes are not observable in the absence of HMG17, the addition of the protein promotes the catenation of greater than 95% of the input DNA into networks that do not enter the gel upon electrophoresis . Electron microscopy and restriction enzyme cleavage experiments indicate that these networks are large structures containing many catenated DNA rings . The HMG17-promoted DNA network formation has been observed with calf thymus type II DNA topoisomerase and the type I topoisomerases of Escherichia coli, Micrococcus luteus, and calf thymus.

J Mol Biol, 1984 May 15, 175(2), 113 - 30
Ribosomal RNA genes of Drosophila melanogaster have a novel chromatin structure; Udvardy A et al.; We have examined the chromatin organization of the Drosophila melanogaster ribosomal RNA genes using both micrococcal nuclease and DNase I . Several findings are of interest . First, the transcribed DNA segments of the rRNA repeat unit appear to be packaged into an unstable or "multiphasic" nucleosome structure . Second, the 5' end of the transcription unit is preferentially exposed to nuclease attack . Third, the non-transcribed spacer immediately upstream from the transcription start site has a novel chromatin organization with micrococcal nuclease and DNase I cleavage sites spaced at intervals of about 240 base-pairs . This unusual fragment distribution appears to reflect the underlying sequence organization of the spacer DNA segment, which consists of a series of tandemly repeated 239 base-pair sequence blocks . We have also examined the chromatin structure of the rRNA repeat unit after extraction of nuclei with different concentrations of salt . Our results suggest that the higher order structures may be of importance in determining the novel chromatin organization of the rRNA repeat unit.

J Biol Chem, 1984 May 10, 259(9), 5893 - 8
Ca/Mg-dependent endonuclease from porcine liver . Purification, properties, and sequence specificity; Stratling WH et al.; A rapid and reproducible method for the purification of the Ca/Mg-endonuclease from porcine and rat liver and for the stabilization of the enzyme activity is presented . The optimum conditions for enzyme activity were determined . The enzyme degrades double-stranded DNA endonucleolytically . In the course of digestion of form I closed circular SV 40 DNA, the form II nicked circular DNA is the prominent intermediate product . Digestion of hen erythrocyte nuclei with added endonuclease produces a ladder of mono- and oligonucleosomal fragments similar to that generated by micrococcal nuclease digestion . Determination of the 5'-terminal nucleotides indicates the absence of a significant base specificity . Analyzing the cleavage pattern of end-labeled pBR322 restriction fragments on sequencing gels shows that the enzyme exhibits a weak preference for dinucleotides with A in the 5'-position; dinucleotides with 5%-C are less readily cleaved . Digestion of end-labeled pBR322 DNA, followed by electrophoresis in agarose gels produces a "smear"-like fragmentation pattern with weak superimposed bands, while micrococcal nuclease generates a different and much more distinct pattern . These data show that the sequence specificity of the enzyme is less pronounced than that of micrococcal nuclease and that the sites preferentially cleaved are not the same.

Appl Environ Microbiol, 1984 May, 47(5), 915 - 8
Effect of physiological age on radiation resistance of some bacteria that are highly radiation resistant; Keller LC et al.; Physiological age-dependent variation in radiation resistance was studied for three bacteria that are highly radiation resistant: Micrococcus radiodurans, Micrococcus sp . isolate C-3, and Moraxella sp . isolate 4 . Stationary-phase cultures of M . radiodurans and isolate C-3 were much more resistant to gamma radiation than were log-phase cultures . This pattern of relative resistance was reversed for isolate 4 . Resistance of isolate 4 to UV light was also greater during log phase, although heat resistance and NaCl tolerance after heat stress were greater during stationary phase . Radiation-induced injury of isolate 4 compared with injury of Escherichia coli B suggested that the injury process, as well as the lethal process, was affected by growth phase . The hypothesis that growth rate affects radiation resistance was tested, and results were interpreted in light of the probable confounding effect of methods used to alter growth rates of bacteria . These results indicate that dose-response experiments should be designed to measure survival during the most resistant growth phase of the organism under study . This timing is particularly important when extrapolations of survival results might be made to potential irradiation processes for foods.

Mol Cell Endocrinol, 1984 May, 35(2-3), 205 - 14
Bovine estrogen receptor binds chromatin at pre-existing nuclease hypersensitive sites; Pratt K et al.; Partially purified estrogen receptor prepared from heifer uterine cytosol, and labeled in vitro with tritiated estradiol, was used to locate receptor binding sites in target and non-target nuclei from various bovine tissues . Nuclei were digested to various extents with bovine pancreatic deoxyribonuclease I, micrococcal nuclease or endogenous nuclease and then assessed for their ability to bind charged estrogen receptor . After very brief digestion with DNAase I, such that only hypersensitive sites were cleaved, calf uterus nuclei were no longer able to bind estrogen receptor . Brief digests with micrococcal nuclease or endogenous nuclease, such that most DNA was still of polynucleosomal length, eliminated the binding ability of both calf and heifer uterus nuclei . These results suggest that estrogen receptor binds to pre-existing nuclease hypersensitive sites . Interestingly, nuclei digested by HaeIII restriction endonuclease, which cleaves at specific sequences, demonstrated no loss of labeled estrogen receptor binding, even though digestion products were of similar size to those obtained from nuclei after treatment with the other nucleases . Since nuclease hypersensitive sites occur in regulatory regions of actively transcribed genes, including estrogen-inducible genes, binding of estrogen receptor at these sites, in vivo, may be part of the mechanism by which transcription is induced.

EMBO J, 1984 May, 3(5), 1193 - 9
Rat liver HMG1: a physiological nucleosome assembly factor; Bonne-Andrea C et al.; Incubation of rat liver single-stranded DNA-binding protein HMG1 with the four core histones at 0.15 M NaCl favors histone association primarily into tetramers and, to a lesser extent, into octamers . The assembly of pre-formed histone-HMG1 complexes with DNA yields nucleosome-like subunits which satisfy most of the criteria defining native core particles: (i) the circular DNA extracted from the complexes is supercoiled indicating that the initially relaxed DNA acquired superhelical turns during complex formation in the presence of topoisomerase I; (ii) the digestion of the complexes with micrococcal nuclease yields a DNA fragment of approximately 140 bp in length; (iii) electron microscopy of the reconstituted complexes shows a beaded structure with the DNA wrapped around the histone cores, leading to a reduction in the contour length of the genome compared with free DNA . Moreover, in the presence of HMG1, nucleosome assembly occurs rapidly at 0.15 M NaCl . Therefore, in addition to its DNA-binding properties, HMG1 mediates the assembly of nucleosomes in vitro under conditions of physiological ionic strength . The possible involvement of these properties in the DNA replication process is discussed.

EMBO J, 1984 May, 3(5), 1137 - 44
A close association between sites of DNase I hypersensitivity and sites of enhanced cleavage by micrococcal nuclease in the 5'-flanking region of the actively transcribed ovalbumin gene; Kaye JS et al.; The organization of chromatin was analysed in a segment of the chicken ovalbumin gene extending 6.5 kb upstream from the start site of transcription . Nuclei of chicken oviduct cells and of erythrocytes, and preparations of 'naked' DNA were digested with DNase I and with micrococcal nuclease . The locations of specific nuclease cleavage sites were determined by analyzing the fragments obtained with an indirect end-labeling technique . In oviduct nuclei there are four regions of DNase I hypersensitivity centered at approximately 0.15, 0.80, 3.2 and 6.0 kb upstream from the mRNA cap site . DNase I hypersensitive regions are absent from the 5'-flanking regions of erythrocyte nuclei . Micrococcal nuclease cleavage sites were found that are unique to oviduct nuclei and others that are enhanced in oviduct nuclei, relative to erythrocyte nuclei and to naked DNA . The locations of these micrococcal nuclease cleavage sites are closely associated with the DNase I hypersensitive regions . Nuclease hypersensitivity in the 5'-flanking region of oviduct nuclei reflects alterations in chromatin structure that are specifically correlated with gene expression . Our results suggest the presence at hypersensitive regions of specific proteins which alter the chromatin structure, making the DNA more accessible to nuclease attack.

J Biol Chem, 1984 Apr 25, 259(8), 4833 - 9
Characterization of the glucocorticoid receptor . Comparison of wild type and variant receptors; Gruol DJ et al.; We have measured the size of the glucocorticoid receptors from two murine lymphoid cell lines, one displaying a wild type cytolytic response to hormone, the other a resistant variant . Using radiation inactivation and target analysis, we first compared the nuclear and cytoplasmic forms of the steroid receptors in a wild type line, WEHI 7.1 (W7) . Within the variation expected for this type of measurement (+/- 14%), the nuclear and cytoplasmic forms have the same size, 75,000 and 79,000 daltons, respectively . We have also measured the size of the receptor in a hormone-insensitive "nuclear transfer-increased" (nti) variant (S49 143R) . In contrast to reports indicating that the nti phenotype is associated with a much smaller cytoplasmic receptor, we found little or no difference in sizes of translocated receptor in wild type and nti cells . We have found significant differences, however, in the release of wild type and nti receptors from nuclei by nuclease digestion, salt, and spermidine . Approximately 80% of the nti receptor was readily released from nuclei incubated with micrococcal nuclease, while only 40-50% of the wild type receptor was released under similar conditions . The wild type nuclei also contained a fraction of receptor (approximately 30%) which was resistant to extraction by NaCl and spermidine . This fraction was greatly diminished in the nti nuclei . Thus, a portion of the wild type receptors appears to be stabilized within the nuclei, possibly through a type of interaction which cannot be sustained by the nti receptor.

Nucleic Acids Res, 1984 Apr 25, 12(8), 3473 - 90
Human placental DNA methyltransferase: DNA substrate and DNA binding specificity; Wang RY et al.; We have partially purified a DNA methyltransferase from human placenta using a novel substrate for a highly sensitive assay of methylation of hemimethylated DNA . This substrate was prepared by extensive nick translation of bacteriophage XP12 DNA, which normally has virtually all of its cytosine residues replaced by 5-methylcytosine (m5C) . Micrococcus luteus DNA was just as good a substrate if it was first similarly nick translated with m5dCTP instead of dCTP in the polymerization mixture . At different stages in purification and under various conditions (including in the presence or absence of high mobility group proteins), the methylation of m5C-deficient DNA and that of hemimethylated DNA were compared . Although hemimethylated , m5C-rich DNAs were much better substrates than were m5C-deficient DNAs and normal XP12 DNA could not be methylated, all of these DNAs were bound equally well by the enzyme . In contrast, from the same placental extract, a DNA-binding protein of unknown function was isolated which binds to m5C-rich DNA in preference to the analogous m5C-poor DNA.

FEBS Lett, 1984 Apr 24, 169(2), 309 - 12
On the structure of active chromatin . A flow linear dichroism study of chromatin fractionated by nuclease digestion; Matsuoka Y et al.; Nuclei from Ehrlich ascites cells were treated with micrococcal nuclease or DNase I and extracted with 1 mM EDTA . The chromatin fraction released by this procedure showed positive flow linear dichroism (LD) at low salt (2 mM NaCl) while the non-released fraction had negative LD . Furthermore, the chromatin structure responsible for the positive LD was found to be labile: The LD was reduced by heat (37 degrees C) or RNase treatment and inverted to a negative LD by electric fields (10 kV/cm) and by the presence of DNA binding dyes.

Nucleic Acids Res, 1984 Apr 11, 12(7), 3201 - 17
Physical structure of gene-sized chromatin from the protozoan Oxytricha; Butler AP et al.; Oxytricha nova is a hypotrichous ciliate containing a transcriptionally active macronucleus and a transcriptionally inactive micronucleus . Two-dimensional gel electrophoresis shows that macronuclei contain a normal complement of inner histones . However, despite extensive efforts, no classical H1-like protein has been detected . Micrococcal nuclease digestion indicates a nucleosome repeat length of approximately 220 bp for macronuclear chromatin . Thermal denaturation profiles of macronuclear chromatin in 0.2 mM EDTA display four transitions at about 46, 57, 64, and 79 degrees C . The lowest of these shifts to higher temperature as the ionic strength is raised to 3-5 mM Na phosphate . These results are consistent with the absence of H1 and a nucleosome repeat of 220-230 bp . Circular dichroism (CD) results agree with these findings . By contrast, micronuclear chromatin displays a much smaller premelt and a more suppressed DNA CD signal at 285 nm, consistent with a micronuclear chromatin repeat of 165-185 bp as determined by micrococcal nuclease digestion.

J Biol Chem, 1984 Apr 10, 259(7), 4609 - 15
Alterations in globin gene chromatin conformation during murine erythroleukemia cell differentiation; Smith RD et al.; Adult beta-globin gene chromatin in murine erythroleukemia (MEL) cells acquired increased sensitivity to both micrococcal nuclease and DNase I during hexamethylenebisacetamide-induced erythoid differentiation . The DNase I hypersensitivity of the globin genes accompanied their actual transcription and was strongly correlated with commitment events . On the other hand, the rate of micrococcal nuclease digestion was closely related to the rate of globin gene transcription . Two distinct DNase I hypersensitive sites were found on the 5' side of the beta-major globin gene in HMBA-induced cells . One site was located near the 5' side of the beta-major globin gene and the second site was located approximately 3 kilobases upstream of the beta-major cap site . Following the commitment of MEL cells to differentiate, DNase I sensitivity was stably inherited in the absence of inducer . In contrast to HMBA, another inducer, hemin, known to cause the accumulation of globin-specific mRNA in MEL cells by a post-transcriptional mechanism, did not elicit alterations of beta-globin gene chromatin . The addition of dexamethasone, a hormone known to inhibit MEL cell commitment, blocked the formation of general and site-specific nuclease sensitivity of beta-globin gene chromatin prior to but not after cell commitment.

Ann Neurol, 1984 Apr, 15(4), 329 - 34
Chromatin structure in dementia; McLachlan DR et al.; Nuclei extracted from neocortex of patients with Alzheimer's disease and treated with micrococcal nuclease release a population of dinucleosomes that contain an increase in the linker histones H1o and H1oo . Five other degenerative brain diseases that clinically resemble Alzheimer's disease do not result in these changes, although Pick's disease is associated with an increase in H1 on dinucleosomes . Histones from nuclei of patients with Alzheimer's disease are also more resistant to salt-induced release from chromatin than are those from age-matched control subjects . These results support the hypothesis that an alteration in chromatin structure is a marker for Alzheimer's disease.

J Appl Bacteriol, 1984 Apr, 56(2), 349 - 50
A note on the temperature tolerance of Legionella; Dennis PJ et al.; A strain of Legionella pneumophila serogroup 1 isolated from the environment had a decimal reduction time in water at 50 degrees C (D50) of 111 min, a D54 of 27 min and a D58 of 6 min . There was little loss of viability at 46 degrees C . Other environmental organisms, a Pseudomonas sp., a Micrococcus sp . and a coliform survived less well at these temperatures . A species of Sarcina had a survival time greater than the L . pneumophila at all the temperatures tested . Other strains of legionellas were tested at 50 degrees C and decimal reduction times calculated . These ranged from 80 min for another strain of L . pneumophia serogroup 1 to 216 min for L . bozemannii . Legionella micdadei did not survive well at 50 degrees C.

Biull Eksp Biol Med, 1984 Apr, 97(4), 414 - 5
{Proteolytic resistance and thermostability of catalase and histidine decarboxylase from Micrococcus sp . n.}; Romantsev FE et al.; Catalase from Micrococcus sp . n . is not hydrolyzed by trypsin at the protein/protease (pn/pe) exceeding 10/1 . Histidine decarboxylase (HDC) loses 50% of activity after the first nine minutes of hydrolysis at the pn/pe ratio equal to 6/1, followed by a slow linear inactivation . Investigation of thermal stability at varying pH and temperatures demonstrated that catalase and HDC preserve 70-80% of activity after 5 minutes of incubation at 72 degrees C only at pH approaching the optimal pH of enzymatic activity (pH 7.45 for catalase and pH 5.55 for HDC).

Proc Natl Acad Sci U S A, 1984 Apr, 81(7), 2092 - 6
Normal liver chromatin contains a firmly bound and larger protein related to the principal cytosolic target polypeptide of a hepatic carcinogen; Vinores SA et al.; A 14,000-dalton polypeptide was previously reported to be the principal protein target of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene) in liver cytosol at the start of hepatocarcinogenesis in rats . The 14,000-dalton polypeptide was purified to homogeneity according to gel electrophoreses in both NaDodSO4-containing medium and acetic acid/urea and also by immunogenicity . An immunologically related form of the cytosolic target polypeptide has now been found to be present in the nuclei of normal rat liver as a 17,500-dalton polypeptide that is firmly and ionically bound to chromatin . Serial salt extractions of isolated liver nuclei or chromatin at 0.15 and 0.35 ionic strengths fail to dissolve the bound polypeptide, according to electrophoretic transfer immunoblot analyses . Most of the 17,500-dalton polypeptide is extracted at 0.65 ionic strength, the remainder at 1.2, and none at 2.0, nor thereafter in 8 M urea . In addition, short-term digestion of purified liver nuclei with micrococcal nuclease solubilizes the 17,500-dalton polypeptide . All three protocols also solubilize low levels of intermediate 17,500- to 14,000-dalton species, the latter size being the same as that of the cytosolic protein target of the carcinogen . The presence of protease inhibitors during the isolations and extractions of the nuclei and chromatin reduces the amounts of these smaller polypeptides . In normal rat liver only nuclei and cytoplasm of hepatocytes contain reactive antigen according to peroxidase-antiperoxidase immunohistochemistry, staining most intensely perilobularly, less in the lobular midzone, and least centrilobularly . The nuclei of the perilobular hepatocytes constitute the strongest staining compartment within all of normal liver . Of 22 nonhepatic tissues of normal rats, 16 contain relatively few cells with immunoreactive cytoplasm . Nonhepatic nuclear antigen is present only in villar crest cells of duodenum (which are normally exposed to liver bile), also having cytoplasmic antigen as well . Five kinds of evidence appear to connect the chromatin-bound 17,500-dalton polypeptide of normal liver nuclei to the cytosolic 14,000-dalton polypeptide that is the principal target of the carcinogen early during hepatocarcinogenesis in rats . The present findings indicate a direct connection between a chromosomal protein and the immediate principal cytosolic protein target of a carcinogen.

Cell Biochem Funct, 1984 Apr, 2(2), 78 - 84
Distribution of non-histone proteins between micrococcal nuclease sensitive and nuclease resistant chromatin from chicken cells with active and inactive genomes; Kilianska Z et al.; Chicken liver and erythrocyte nuclei were separated by mild treatment with micrococcal nuclease into nuclease sensitive (NS) and nuclease resistant (NR) fractions, differing in chemical composition and transcriptional activity in vitro . Nuclei, NS and NR fractions of both tissues were fractionated by hydroxyapatite chromatography into three groups of non-histone chromatin proteins (NHCP) and characterized by SDS-polyacrylamide gel electrophoresis . Some differences in the molecular distribution of non-histone proteins of chicken liver and erythrocytes between nuclease sensitive and resistant parts of chromatin have been described.

Biochem J, 1984 Apr 1, 219(1), 165 - 71
Mechanism of enhanced RNA synthesis in acute-phase rat liver and its relationship to chromatin structure; Schiaffonati L et al.; Nuclei isolated from the liver of rats undergoing an acute inflammatory reaction induced by turpentine treatment show increased RNA synthesis . This increase is essentially determined by a faster polyribonucleotide-elongation rate while the number of transcribing polymerase molecules is unchanged . The sensitivity of chromatin to micrococcal-nuclease digestion and the composition of chromosomal proteins are not affected by the acute-phase process . Therefore the increased RNA synthesis by liver nuclei from acutely inflamed rats does not seem to correlate with major changes in chromatin structure.

Science, 1984 Mar 30, 223(4643), 1420 - 3
Appearance of a new nucleosomal protein during differentiation of human leukemia (HL-60) cells; Chou RH et al.; A 60-kilodalton protein was identified in chromatin digested by micrococcal nuclease during retinoic acid-induced differentiation of human leukemia (HL-60) cells to mature-like granulocytes . The protein was not detected in a retinoic acid-resistant variant of the HL-60 cell line treated with retinoic acid, in HL-60 cells induced with dimethyl sulfoxide, or in normal human granulocytes . This protein may have an important role in the regulation of retinoic acid-induced leukemic cell differentiation.

Nucleic Acids Res, 1984 Mar 26, 12(6), 2691 - 704
Compact structure of ribosomal chromatin in Xenopus laevis; Spadafora C et al.; Micrococcal nuclease digestion was used as a tool to study the organization of the ribosomal chromatin in liver, blood and embryo cells of X . laevis . It was found that in liver and blood cells, ribosomal DNA is efficiently protected from nuclease attack in comparison to bulk chromatin . Although ribosomal chromatin is fragmented in a typical nucleosomal pattern, a considerable portion of ribosomal DNA retains a high molecular weight even after extensive digestion . A greater accessibility of the coding region in comparison to the non-coding spacer was found . In embryos, when ribosomal DNA is fully transcribed, these genes are even more highly protected than in adult tissues: in fact, the nucleosomal ladder can hardly be detected and rDNA is preserved in high molecular weight . Treatment of chromatin with 0.8 M NaCl abolishes the specific resistance of the ribosomal chromatin to digestion . The ribosomal chromatin, particularly in its active state, seems to be therefore tightly complexed with chromosomal proteins which protect its DNA from nuclease degradation.

J Biol Chem, 1984 Mar 10, 259(5), 3343 - 9
Nick translation of HeLa cell nuclei as a probe for locating DNase I-sensitive nucleosomes; Javaherian K et al.; The technique of nick translation of nuclei (Levitt, A., Axel, R., and Cedar, H . (1979) Dev . Biol . 69, 496-505) has been used in HeLa cells to label DNase I-sensitive regions . Micrococcal nuclease digestion of the nick translated nuclei was followed by a low ionic strength gel electrophoresis system which separates different types of mononucleosomes . The major label was observed in the vicinity of high mobility group protein containing mononucleosomes . However, further analysis revealed that the particle does not sediment in the position of mononucleosomes on a sucrose gradient . Two alternative explanations are discussed as the possible source of this particle . It is either a high mobility group protein containing nucleosome in some unfolded conformation or the labeled particle originates from discrete DNA fragments, wrapped around some nonhistone proteins, located in a highly DNase I-sensitive region, which is resistant to micrococcal nuclease digestion.

J Antibiot (Tokyo), 1984 Mar, 37(3), 207 - 10
{R-(Z)}-4-amino-3-chloro-2-pentenedioic acid, a new antibiotic . Fermentation, isolation and characterization; Chaiet L et al.; A new unsaturated glutamic acid analog, 4-amino-3-chloro-2-pentenedioic acid ( ACPA ) was isolated from a fermentation broth produced by a strain of Streptomyces . ACPA has a very narrow antibacterial spectrum, which is virtually limited to Micrococcus luteus.

Tsitologiia, 1984 Mar, 26(3), 343 - 8
{Daughter DNA synthesis in UV-resistant and UV-sensitive Chinese hamster clones cells under UV irradiation}; Barenfel'd LS; By means of ultracentrifugation in alkaline sucrose gradients it has been shown that the size of DNA fragments synthesized in Chinese hamster cells of UV-sensitive clone (CHS-1) after exposure to UV light was equal to the distance between pyrimidine dimers in the parental DNA determined using endonuclease of Micrococcus luteus . With the UV-resistant clone (V-79), the length of fragments of the newly synthesized DNA was much longer than that between pyrimidine dimers in the parental DNA . The data obtained support the model according which DNA synthesis on the UV-irradiated template gives rise to gaps opposite to pyrimidine dimers.

Eur J Biochem, 1984 Mar 1, 139(2), 381 - 7
Dietary regulation of levels of active mRNA coding for amylase and serine protease zymogens in the rat pancreas; Wicker C et al.; The mechanism by which changes in diet mediate levels of exportable enzymes and proenzymes in pancreatic tissue were studied in rats . The relative levels of mRNA coding for pancreatic amylase, lipase, procarboxypeptidases A and B, and the family of serine protease zymogens have been determined by the ability of isolated RNA to direct the synthesis of these products in a high-fidelity micrococcal nuclease-treated reticulocyte-lysate translation system . Translation products synthesized in vitro correlated directly with products synthesized in vivo in pancreatic lobules . Dietary adaptation was observed when dietary carbohydrate was increased from 0 to 58% at the expense of protein (81-23%) . The increase in dietary carbohydrate over this range resulted in a 2-fold increase in amylase synthesis in pancreatic lobules and a 1.8-fold increase in mRNA-directed synthesis of amylase in the translation system in vitro . Concomitant with the decrease in dietary protein, synthesis of serine protease zymogens in pancreatic lobules and in the system in vitro decreased by approximately 50% . Over this range of dietary manipulation, ratios of amylase to serine proteases showed a 3.6-fold change . When dietary carbohydrate was further increased to 81% and protein reduced to 0, non-adaptive changes were observed since there was a decrease in amylase synthesis under conditions both in vivo and in vitro . mRNAs coding for pancreatic lipase and procarboxypeptidases A and B were unaffected by the dietary changes . These findings indicate that nutritional regulation in the tissue levels of pancreatic enzymes and proenzymes is mediated by changes in the content of active cytoplasmic mRNAs.

J Immunol, 1984 Mar, 132(3), 1216 - 22
Antibodies from patients with mixed connective tissue disease react with heterogeneous nuclear ribonucleoprotein or ribonucleic acid (hnRNP/RNA) of the nuclear matrix; Fritzler MJ et al.; The sera of patients with mixed connective tissue disease (MCTD) have high titers of antibodies directed against nuclear U1-ribonucleoprotein (U1-RNP) . This antigen is easily extracted from nuclear preparations with physiologic saline and from tissue sections with 0.1 HCl, leaving the nucleic acids and nuclear matrix behind . When U1-RNP is extracted from HEp-2 cells with 0.1 N HCl, the sera of 32/32 patients with MCTD react with another antigen that is exposed by the extraction procedure . This antigen is not destroyed by trypsin and deoxyribonuclease 1 treatment but is sensitive to both purified ribonuclease A and purified micrococcal nuclease . Absorption studies showed that the antibody reacting with this antigen cannot be absorbed by sheep red blood cells coated with extracts of rabbit thymus that contain U1-RNP . Radioimmunoassay showed that the reaction of the unadsorbed antibody was with heterogeneous nuclear ribonucleoprotein or ribonucleic acid (hnRNP/RNA) and not with transfer RNA or ribosomal RNA . The hnRNP/RNA antigen is demonstrated as discrete particles in the internucleolar chromatin of interphase cells, but in metaphase cells the antigen is diffusely dispersed . The distribution, solubility, and biochemical characteristics suggest that the antigenic moiety is part of the nuclear matrix . Therefore, MCTD sera contain antibodies that react with at least two species of nuclear RNP: small nuclear RNP (snRNP), as described by others, and a high m.w . hnRNP/RNA bound to the nuclear matrix.

Biochem Biophys Res Commun, 1984 Feb 29, 119(1), 220 - 7
Angiotensin II receptors in chromatin fragments generated by micrococcal nuclease; Re RN et al.; Rat liver nuclei were digested with micrococcal nuclease following incubation with 125I-angiotensin II (AII) or with 125I-AII and excess unlabeled hormone . Chromatin enriched in 125I was solubilized after 3 min and was applied to a BIO-GEL A-5 M column . Labeled hormone was 40-60% displaceable by unlabeled hormone, in nucleoprotein eluting with a V/Vo near 1.9, indicating that these solubilized chromatin fragments contained specific receptors for AII . Furthermore, a discrete AII binding nucleoprotein particle was resolved on DNP gel electrophoresis . Additionally, binding to specific AII nuclear receptors appeared to bring about changes in chromatin structure consistent with the induction of transcriptional activity.

Nucleic Acids Res, 1984 Feb 24, 12(4), 2111 - 26
Adaptive response of Micrococcus luteus to alkylating chemicals; Ather A et al.; Wild type M . luteus cells have been adapted by a step-wise treatment with sub-lethal concentrations of MNNG . The adapted cells exhibit 5.7 fold increased resistance to the killing effects of the mutagen and a simultaneous efficient removal of various base modifications present in cellular DNA . A protein extract prepared from adapted cells contains inducible repair functions which can reduce 80-90% of the alkylated DNA content of 06-MeG is effected by a transmethylase and there is no concomitant release of the modified base . However, N-3 MeG is released as a free modified base through the action of a DNA glycosylase . The release of N-3 MeA is unaffected by the induction treatment whereas that of N-7 methylpurine is slightly improved in the adapted cells.

Biochemistry, 1984 Feb 14, 23(4), 785 - 90
Chromatin structure of the beta-globin gene family in murine erythroleukemia cells; Smith RD et al.; We have analyzed the chromatin structure of the beta-major globin gene and other related beta-globin genes in induced and uninduced murine erythroleukemia (MEL) cell nuclei . Nuclei were digested with either DNase I or micrococcal nuclease, and the purified DNA was hybridized to a set of cloned genomic DNA fragments covering the beta-globin gene region . This region consisted of two distinct domains as characterized by sensitivity to DNase I digestion . One domain was relatively sensitive and contained the potentially active or actively transcribed beta-major and beta-minor globin genes . The other, relatively insensitive domain contained the nontranscribed embryonic and beta-globin homologous genes . The sensitivity of these domains was not altered during erythroid differentiation . In nonerythroid cells, the entire globin gene family, including the adult and embryonic globin genes, was contained in a single relatively resistant domain . Micrococcal nuclease (MNase) also defined two general domains of nuclease sensitivity that coincided with those of DNase I . However, the relatively sensitive MNase domain containing the beta-major and beta-minor genes became more sensitive upon chemically stimulated erythroid differentiation . A detailed examination of the beta-major globin gene revealed that the actual coding region became increasingly sensitive to micrococcal nuclease after differentiation while the 5'-flanking DNA did not . Thus, micrococcal nuclease was able to accurately define the primary transcription unit of the beta-major gene.

Nucleic Acids Res, 1984 Feb 10, 12(3), 1391 - 400
Chromatin prepared using micrococcal nuclease retains the enzyme activity which can be removed with cation-exchange resin AG 50 WX2; Goel SB et al.; Micrococcal nuclease used to digest nuclei remains bound to the chromatin in an inactive form . The enzyme activity can be restored in situ by addition of divalent cations such as Ca++ or Mg++ resulting in continued digestion of high molecular weight chromatin into smaller fragments as demonstrated by two dimensional gel electrophoresis of samples as chromatin and DNA in the first and second dimensions, respectively . The bound enzyme can be selectively removed from the chromatin by treatment with cation exchange resin AG 50 WX2 at low salt concentration without altering the electrophoretic mobility of the chromatin.

Biokhimiia, 1984 Feb, 49(2), 209 - 15
{Physico-chemical characteristics of catalases from Micrococcus sp . n.}; Prozorovskii VN et al.; The physico-chemical properties of heme-containing and non-heme catalases isolated from the cell culture of Micrococcus sp . n . grown under intensive aeration were studied . The enzyme preparations were homogenous during polyacrylamide disc electrophoresis . The spectral and functional properties of the enzymes (e . g . specific activity, subunit molecular weight, quaternary structure, amino acid composition, immunoprecipitability, N-terminal amino acid sequences) were investigated . Monocrystals of non-heme catalase applicable for an X-ray analysis were grown and examined by X-ray spectroscopy . Both enzymes were stable upon storage in 40% ammonium sulfate for 2 months and resistant to lyophylization without any significant loss of their activity . Non-heme catalase is apparently an independent enzyme which is not derived from heme-containing catalase via dissociation, limited proteolysis or heme cleavage.

Cell, 1984 Feb, 36(2), 423 - 31
Chromatin structure of hsp 70 genes, activated by heat shock: selective removal of histones from the coding region and their absence from the 5' region; Karpov VL et al.; The presence of histones in hsp 70 genes was studied by "protein-image" hybridization technique after crosslinking histones to DNA . With increasing transcription of the genes, the coding region was demonstrated to be depleted first of H1 and then of all histones . This probably accounts for unraveling the 25 nm silent chromatin fiber to moderately and actively transcribed 10 nm fiber and linearized DNA . No histones were found in the 5'-terminal DNAase I-hypersensitive region, which may be a prerequisite to gene activation . The absence of histones on DNA correlates well with the high nuclease sensitivity and disappearance of the regular pattern in micrococcal nuclease digests of chromatin.

Zh Mikrobiol Epidemiol Immunobiol, 1984 Feb, (2), 27 - 8
{Method of determining the antilysozyme activity of microorganisms}; Bukharin OV et al.; The method for the determination of the antilysozyme activity of microorganisms is described . The method consists in the cultivation of the strains under study in a lysozyme-containing medium, and the effect of lysozyme inactivation is determined from the growth of Micrococcus luteu S indicator strain adjacent to active strains . The quantitative evaluation of this property is presented . The study of 1 296 strains belonging to 9 genera has disclosed that antilysozyme activity occurs most frequently among Gram-negative bacteria.

J Biochem (Tokyo), 1984 Feb, 95(2), 485 - 93
Purification and characterization of a factor stimulating DNA polymerase alpha activity from mouse FM3A cells; Kawasaki K et al.; A protein factor which stimulates DNA polymerase alpha activity on heat-denatured DNA has been purified from mouse FM3A cells . The final preparation had a specific activity of 43,000 units/mg protein and lacked detectable DNA polymerase, RNA polymerase, DNA-dependent- and independent ATPase, exo- and endodeoxyribonuclease and phosphatase activities . The stimulating factor sedimented at 2.9S in a glycerol gradient . Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the glycerol gradient fraction revealed the presence of a major band of 36,000 daltons, the amount of which corresponded well with the level of stimulating activity . The stimulation by the factor was specific for heat-denatured DNA, and a little or no stimulation was observed with native DNA, ribo- and deoxyribohomopolymers and single stranded circular DNA . Alkaline sucrose gradient sedimentation analysis of the reaction products revealed that newly synthesized DNA was covalently linked to the termini of heat-denatured DNA . The average chain length of the elongated span determined by the digestion with micrococcal nuclease and phosphodiesterase II, did not differ between in the presence and absence of the stimulating factor, suggesting that the stimulation by the factor was due to the increase in the initiation frequency of DNA synthesis from the 3'-hydroxyl terminus of heat-denatured DNA.

J Bioenerg Biomembr, 1984 Feb, 16(1), 61 - 8
Respiratory control in Micrococcus lysodeikticus; Rosenberg M et al.; The respiration rate of Pi-deprived cells of Micrococcus lysodeikticus is markedly increased by Pi, and returns to the original level following Pi consumption . The stimulation of the respiration was found to be specific for Pi and arsenate . Although succinate and valinomycin enhanced the respiration of both Pi-grown and Pi-deprived cells, only the latter could be further stimulated by Pi . The effect of Pi on the respiration rate was found to be concentration dependent . The control of respiration by Pi is due to its rapid uptake and its subsequent polymerization to polyphosphate via ATP . Both of these processes are coupled to proton influx into the cell, and thus stimulate the proton efflux and the respiration rate.

J Biol Chem, 1984 Jan 10, 259(1), 497 - 503
Role of histone tyrosines in nucleosome formation and histone-histone interaction; Kleinschmidt AM et al.; We have studied the functional properties of iodinated histones . Isolated, denatured histones were iodinated at trace levels and then renatured together with carrier histones and high molecular weight DNA to form nucleohistone . Nucleosomes were prepared from the reconstitute using micrococcal nuclease, and the relative representations of the individual iodinated tyrosines of the histones in the reconstituted nucleosomes were determined . Our principal findings are 1) that denatured histones can be iodinated at any tyrosine without interfering in subsequent nucleosome reconstitution and 2) that the resulting reconstituted nucleosomes nevertheless possess histone cores of altered stability, being either more or less stable depending on the particular tyrosine which is iodinated . We show that tyrosines 37, 40, and 42 of H2B are protected from iodination in intact core particles, as expected since these tyrosines lie within the H2B-H2A binding site . Yet iodination of these tyrosines in denatured H2B does not interfere with nucleosome assembly . However, the histone cores isolated from these reconstituted nucleosomes are of diminished stability as assayed by Sephadex column chromatography in 2 M salt . In contrast, iodination of tyrosines 83 and 121 of H2B, as well as iodination of the tyrosines of H2A, increases the stability of the histone octamer core . Iodination of H4 tyrosine 72 is without effect on histone octamer stability . Tyrosine iodination constitutes a profound amino acid alteration in the context of the absolute evolutionary conservation of most histone tyrosines . For example, all H2Bs sequenced to date, from fungi to mammals, possess tyrosines at positions 37, 40, and 42 . Our results suggest that the immutability of these tyrosines reflects some sophisticated function of the nucleosome histone core beyond the assembly and mere maintenance of a compact structure.

J Biol Chem, 1984 Jan 10, 259(1), 31 - 40
Phosphorylation of the C-proteins of HeLa cell hnRNP particles . Involvement of a casein kinase II-type enzyme; Holcomb ER et al.; The phosphorylation of the proteins of heterogeneous nuclear ribonucleoprotein particles has been investigated in HeLa cells . 32Pi labeling of intact cells indicated that, of the six major particle proteins, the most heavily phosphorylated was the C1-protein (Mr = 42,000) . This protein, together with C2 (Mr = 44,000), is also phosphorylated by {gamma-32P}ATP in particle extracts and in particles that are purified by sedimentation or exclusion chromatography . The C-proteins, together with their particle-associated kinase, were partially purified from isolated particles following dissociation with micrococcal nuclease . Proteins C1 and C2 co-purify on phosphocellulose chromatography, and their peak overlaps with that of a casein kinase activity . Evidence suggesting that this kinase activity is responsible for C-protein phosphorylation includes 1) the phosphorylation of C-proteins in the fractions where they overlap with the kinase, 2) the phosphorylation of added C-protein by fractions of the casein kinase which lack detectable C-protein, and 3) the similarities in catalytic properties of the casein kinase- and C-protein-phosphorylating activities . The purified kinase activity is cyclic nucleotide and Ca2+ independent . It is stimulated by polyamines, inhibited by heparin, and utilizes either GTP or ATP with high affinity . Serine residues are the major phosphate acceptors . These properties indicate that the kinase is casein kinase II or a closely related enzyme . Moreover, purified casein kinase II from rabbit liver effectively phosphorylates C-protein . These results suggest that C-proteins may be natural substrates for nuclear casein kinase II.

Anticancer Res, 1984 Jan-Apr, 4(1-2), 53 - 8
Interaction of cyclophosphamide with DNA in isolated rat liver cell nuclei; Lindemann H; Isolated rat liver cell nuclei were incubated with the alkylating cytostatic drug cyclophosphamide (CPA) in the presence of a microsomal activation system . Digestion of the 3H-CPA-treated nuclei with DNase I and micrococcal nuclease, respectively, showed that the CPA-modified DNA apparently has become resistant against such enzymatic attack . For analysis of the DNA-CPA reaction products, the DNA was isolated under mild conditions and degraded enzymatically . In cell nuclei whose DNA had been prelabeled with 32P in vivo, a predominant binding of 3H-CPA (about 50%) to the DNA phosphate groups was observed . Terminal phosphate groups apparently play an important role in this reaction . Neutral heating of DNA from 3H-CPA-treated nuclei liberated up to 60% of the initially bound 3H-radioactivity . The results are discussed in relation to the recent data concerning the route of decomposition of activated CPA to phosphoramide mustard and acrolein.

Mol Biol Rep, 1984 Jan, 9(4), 253 - 7
Conformational changes in the chromatin of the brain of developing rats and its modulation by zinc chloride; Supakar PC et al.; Conformational changes in the chromatin of the brain were studied during the development of the rat (3-, 14- and 30-day old) using micrococcal nuclease (MCN) and DNase I . The rate and extent of digestion of chromatin by MCN is not altered during development . However, pre-incubation of slices of the cerebral cortex with ZnCl2 increases the initial rate of digestion by MCN by 2-3-fold, and also enhances the production of monomer DNA . The rate and extent of digestion of chromatin by DNase I is greater in an early stage of development . The initial rate of digestion by DNase I is stimulated by 3-4-fold after ZnCl2 treatment . These data show that changes occur in the conformation of chromatin, particularly in the internucleosomal region of brain cells as they pass from dividing to the non-dividing state.

Mol Biol Rep, 1984 Jan, 9(4), 245 - 51
Analysis of chromatin of skeletal muscle of developing rats using micrococcal nuclease and DNase I; Pandey RS et al.; Conformational changes in the chromatin of skeletal muscle of 3-, 14- and 30 day-old developing rats have been studied using DNase I and micrococcal nuclease (MCN) . Purified nuclei were digested separately by MCN and DNase I . The rate and extent of digestion by MCN decreases gradually as development proceeds . The electrophoretic pattern of MCN digested DNA, however, shows no change . The kinetics of digestion of nuclei by DNase I show no change with development . However, the electrophoretic pattern of DNase I digested DNA shows a gradual decrease in the amount of 10-30 bp fragments with progressive development . These studies show that the chromatin of the skeletal muscle undergoes certain conformational changes during postnatal development, and such changes in chromatin may be necessary for terminal differentiation of this tissue.

Mol Biol Rep, 1984 Jan, 9(4), 231 - 4
O-phenantroline-CuSO4-induced redistribution of nuclear proteins; Poznanovic G et al.; Incubation of rat liver nuclei with the o-phenantroline-CuSO4 (OP-Cu) complex under conditions not causing any DNA cleavage, enhanced the susceptibility of chromatin to the action of micrococcal nuclease . The released nucleosomal fraction had less coextracted nonhistone proteins, while the nuclear matrix was enriched in nonhistone proteins when compared with the controls . These changes were interpreted as the consequence of a displacement of nonhistone proteins from their closer association with the chromatin complex and a concomitant exposure of chromatin regions in a state less protected by nonhistone proteins.

Acta Biochim Pol, 1984, 31(3), 307 - 16
In vitro rabbit mammary gland chromatin replication studied by micrococcal nuclease digestion; Kleczkowska D et al.; Isolated nuclei from pregnant rabbit mammary glands were labelled with {3H}dTTP under conditions for DNA synthesis and subsequently digested with micrococcal nuclease . Replicating chromatin was found to exhibit increased susceptibility towards the nuclease . Analysis of chromatin digestion products by sucrose density gradient centrifugation demonstrated the association of in vitro replicated DNA with nucleosomes . Furthermore, the distribution of DNA polymerizing activity was studied in isolated nuclease-digested mammary gland chromatin . About 90% of all recovered nuclear DNA polymerizing activity cosedimented with nucleosomal particles, mainly with mononucleosomes . The distribution of DNA polymerases alpha and beta in chromatin isolated from the mammary glands of pregnant and lactating rabbits was compared . In these physiological states the mononucleosome-associated DNA polymerase alpha activity varied in accordance with the rate of DNA synthesis.

J Steroid Biochem, 1984 Jan, 20(1), 57 - 65
Interaction of androgen receptors with chromatin and DNA; Davies P et al.; Controlled digestion of rat ventral prostate nuclei by careful adjustment of conditions of temperature, divalent cation concentration, ionic strength and micrococcal nuclease:DNA ratios yielded oligonucleosome fractions corresponding to less than 10% of the total genome which contain the majority of RNA polymerase B activity and androgen-receptor complexes of the nucleus . These parameters were affected acutely by androgen withdrawal and administration: furthermore, such manipulations affected the susceptibility to micrococcal nuclease release of prostate binding protein gene sequences . This transcriptionally-active androgen-influenced fraction was considered ideal for studies of interaction of chromatin components with androgen receptor protein . Androgen receptor was purified approximately 20 000-fold from rat prostate cytosol . The purified protein retained its ability to stimulate RNA polymerase B activity in prostate nuclei and chromatin fractions, and its properties of binding to chromatin and to DNA . However, although purified receptor protein showed tissue-specific binding to prostate chromatin and enhanced binding to fractions released by low nuclease digestion, no such specificity was indicated by binding to total DNA, DNA from specific fractions or cloned prostatic binding protein cDNA.

Microbios, 1984, 41(164), 91 - 6
Preferential nuclease digestion of the non-messenger sequences of HnRNA upon micrococcal nuclease digestion of HnRNP particles; Pironcheva GL et al.; HnRNP particles from rat liver were digested with micrococcal nuclease to render about 50% of HnRNA acid soluble . Hybridization experiments, in which cDNA complementary to rat liver poly(A)+ mRNA was annealed to an excess of HnRNA, showed that the micrococcal nuclease resistant HnRNA fraction was enriched in mRNA-homologous sequences.

Vopr Med Khim, 1984, 30(6), 94 - 7
{Specific binding of fluorescein bimercuric acetate by histidine decarboxylase from Micrococcus}; Gonchar NA et al.; The SH-groups of histidine decarboxylase were modified by means of fluorescent probe--fluorescein bimercuric acetate (FMA) . Native histidine decarboxylase bound 3 molecules of FMA with complete inhibition of the enzymatic activity . Binding of FMA with the enzyme was accompanied by distinct alteration in absorption and fluorescence spectra . The FMA was also used in studies of SH-containing peptides of histidine decarboxylase; acid SH-peptides obtained after tryptic hydrolysis appears to contain the cysteine of the enzyme active site.

Nauchnye Doki Vyss Shkoly Biol Nauki, 1984, (9), 30 - 3
{Catalase and superoxide dismutase activity in isogenous bacterial strains with different degrees of radioresistance}; Vasil'eva EI et al.; Superoxide dismutase and catalase activity has been studied in isogenous strains of various radioresistance bacteria . In mutants Micrococcus radiodurans having defects in the systems of DNA repair the superoxide dismutase activity is lower than in cells of wild type . The changes of catalase and superoxide dismutase activity have not been revealed in investigated strains Escherichia coli differing in radioresistance . It has been concluded that the survival of bacteria exposed to ionizing radiation is determined by the effectiveness of DNA repair systems realiability of which depends on the catalase and superoxide dismutase activity.

Cell, 1984 Jan, 36(1), 121 - 9
Patterns of DNA structural polymorphism and their evolutionary implications; Keene MA et al.; The pattern of sites within purified DNA that are highly susceptible to double-stranded cleavage by micrococcal nuclease has been analyzed in the vicinity of over 20 genes from widely separated loci in Drosophila . These genes have uniformly exhibited a distinctive organization of cleavage sites such that at early times of digestion major sites are observed in the spacer regions surrounding the genes, but not within the protein coding regions themselves . Examples examined include Drosophila genes for heat-shock proteins, cytoplasmic actin, ribosomal protein 49, alcohol dehydrogenase, Sgs 4 glue protein, and other developmentally regulated transcripts, a human beta-globin gene, and mouse alpha 3-globin pseudogene . It seems probable that this gene/spacer pattern will be a general one in the genomes of eucaryotes, but not in the genomes of procaryotes, since neither pBR322 nor phage lambda DNA display such a pattern . One observes a nonrandom spacing of strong cleavage sites in Drosophila DNA, with the most frequent intervals being 195 bp and 411 bp . Such a pattern of variation in DNA structure may have evolved to facilitate the packaging of eucaryotic DNA into chromatin.

Biol Cell, 1984, 52(2), 91 - 101
Anchorage of the nucleolus in the pore complex-lamina by a DNA-bearing structure masked in situ in rat liver nuclei; Hubert J et al.; We have developed a method by which nuclear shells containing nucleoli can be isolated from membrane-depleted rat liver nuclei . This method involves the removal of the internal chromatin . This chromatin is expelled from the nuclear shell using combinations of low and high ionic strength buffers . The expelled internal part is subsequently digested with DNase I or micrococcal nuclease . Examination by electron microscopy of the nuclear and the nucleolar structures at various steps of the isolation procedure shows that the nucleoli are anchored in the peripheral lamina by a pedicle that is continuous with an intranucleolar network . This network is masked in situ by nucleolar granules . The pedicle and the network which support the nucleolar DNA are composed mainly of non-histone proteins insoluble in 2M NaCl.

J Steroid Biochem, 1984 Jan, 20(1), 387 - 90
Interaction of calf uterine estrogen receptors with chicken target cell nuclei; de Boer W et al.; The calf uterine estrogen receptor (ER) was used to study the capacity and the characteristics of the acceptor sites in chicken target cell nuclei . The temperature-activated ER is bound at 0 degrees C with a high affinity to all chicken cell nuclei tested (Kd = 0.4-1.0 nM) . The nuclear binding displayed tissue specificity: oviduct greater than liver, heart greater than spleen greater than erythrocytes and was salt-dependent . ER binding to liver nuclei measured in 0.15 M KCl varied between 3000 and 6000 acceptor sites per nucleus . Liver nuclei isolated from estrogen-treated cockerels showed a 2-fold lower binding capacity than nuclei from non-treated chickens . When nuclei were incubated with {3H}ER from embryo liver and increasing concentrations of uterine non-radioactive-ER a progressive inhibition of the binding of the liver ER was found . These experiments suggest that liver and uterine ER compete for a common acceptor site . Liver nuclei charged in vitro with calf uterine ER were digested at 0 degree C with DNAase I and micrococcal nuclease . Both enzymes excised the ER in the form of a chromatin-ER complex . A considerable portion was associated with nucleosomal subunits and a minor fraction was associated with a nuclease-sensitive, protein-poor fraction of the chromatin.

Cell Biol Int Rep, 1984 Jan, 8(1), 55 - 63
Response of isolated nuclei to phospholipid vesicles: analysis of chromatin sensitivity to DNase I and micrococcal nuclease; Cocco L et al.; Phosphatidylserine (PS) and phosphatidylcholine (PC) multilamellar vesicles (MLV) affect chromatin structure as analysed by DNase I sensitivity . The kinetics of DNA solubilisation during the digestion of nuclei indicates that phosphatidylserine causes an increase in DNase accessibility while phosphatidylcholine slightly reduces this accessibility . The effect of phosphatidylserine has also been analysed by means of isokinetic sucrose gradients and agarose gel electrophoresis of nuclear DNA solubilised by micrococcal nuclease . This analysis indicates that phosphatidylserine induces a very rapid production of mononucleosome subunits as compared with untreated nuclei.

Adv Exp Med Biol, 1984, 179, 127 - 41
Chromatin structure and DNA replication; Pulm W et al.; The structure of (3H) thymidine pulse labeled, replicating chromatin differs from that of non replicating chromatin by several operational criteria which are related to the higher nuclease sensitivity of replicating chromatin . We summarize the structural changes that we observe using replicating chromatin as substrate for micrococcal nuclease . The data suggest a more extended configuration of replicating compared to non replicating chromatin . We use these data to discuss a model of the chromatin structure in the vicinity of replication forks . Finally, we present data to show that the reversion of structural changes in replicative chromatin depends on continued DNA replication.

Biochim Biophys Acta, 1983 Dec 22, 741(3), 279 - 87
Ion competition and micrococcal nuclease digestion studies of spermidine-condensed calf thymus DNA . Evidence for torus organization by circumferential DNA wrapping; Marx KA et al.; Spermidine-condensed calf thymus DNA structures have been studied by ion competition using a sedimentation assay and by micrococcal nuclease digestion . Competitor ions Mg2+, Ca2+ and putrescine2+ show specific ion effects; but all three appear to affect the DNA condensation-decondensation equilibrium caused by spermidine3+ in a qualitatively similar manner, suggesting the spermidine3+-DNA interaction is largely electrostatic . Our data show a hysteresis in condensation and decondensation transition directions . We interpret this in terms of a kinetic block in the condensation direction with decondensation representing the equilibrium state of the system . These results agree with results obtained from related systems using different measurement techniques . Micrococcal nuclease digestion of spermidine-condensed calf thymus DNA produces broad but discrete bands in gel electrophoresis experiments . At least two bands determined to be 760 +/- 87 bp and 1355 +/- 135 bp, possess the size ratio 1:1.8 +/- 0.4 consistent with their forming the monomer and dimer fragments of an arithmetic band series . We rationalize this result in terms of a localized micrococcal nuclease cleavage model of circumferentially-wrapped DNA toruses proposed previously by Marx, K.A . and Reynolds, T.C . (Proc . Natl . Acad . Sci . (1982) 79, 6484-6488) . The arithmetic series monomer band (760 +/- 87 bp), corresponding to wrapping B DNA once circumferentially about the torus, is in agreement with the electron microscopic measurements of hydrated calf thymus DNA torus circumferences presented by Marx, K.A . and Ruben, G.C . (Nucleic Acids Res . (1983) 11, 1839-1853).

Biochim Biophys Acta, 1983 Dec 22, 741(3), 288 - 96
DNAase I and cellular factors that affect chromatin structure; Prentice DA et al.; The use of DNAase I as a probe of chromatin structure is frequently fraught with problems of irreproducibility . We have recently evaluated this procedure, documented the sources of the problems, and standardized the method for reproducible results (Prentice and Gurley (1983) Biochim . Biophys . Acta 740, 134-144) . We have now used this probe to detect differences in chromatin structure between cells blocked (1) in G1 phase by isoleucine deprivation, or (2) in early S phase by sequential use of isoleucine deprivation followed by release into the presence of hydroxyurea . The cells blocked in G1 phase have easily-digestible chromatin, while cells blocked in early S phase have chromatin which is much more resistant to DNAase I . These differences were found to be the result of diffusible factors found in the cytoplasm and nuclei of G1- and S-phase cells, respectively . The G1 cells contained a cytoplasmic factor which modulates the chromatin structure of S-phase nuclei to a more easily digestible state, while cells blocked in S phase contain a nuclear factor which modulates the chromatin structure of G1 nuclei to a state more resistant to digestion . DNAase I is much more sensitive to these cell cycle-specific chromatin changes than is micrococcal nuclease . The results indicate that, under controlled conditions, DNAase I should be a valuable probe for detecting chromatin structural changes associated with cell cycle traverse, differentiation, development, hormone action and chemical toxicity.

Biochemistry, 1983 Dec 20, 22(26), 6175 - 80
Histone deacetylase from HeLa cells: properties of the high molecular weight complex; Hay CW et al.; In previous work {Hay, C . W., & Candido, E . P . M . (1983) J . Biol . Chem . 258, 3726-3734}, we have shown that the histone deacetylase from HeLa cell nuclei is associated with a high molecular weight, nuclease-resistant complex . This complex was found to contain a variety of non-histone proteins, and indirect evidence for the importance of protein-protein interactions in the maintenance of its structure was obtained . In the present report, we examine the effects of beta-mercaptoethanol and neocuproine on the deacetylase complex and present data on the level of histone acetylation and the presence of satellite DNA sequences in this material . HeLa cell histone deacetylase complex partially dissociates in 10 mM beta-mercaptoethanol, resulting in a loss of non-histone proteins . The presence of 10 mM beta-mercaptoethanol during the micrococcal nuclease digestion of HeLa cell nuclei results in a greatly reduced yield of histone deacetylase complex, with a correspondingly large increase in the production of small oligonucleosomes and mononucleosomes . Histone deacetylase activity on endogenous labeled histone within the complex is strongly inhibited by either 1 or 10 mM beta-mercaptoethanol or 3 mM neocuproine . This loss of histone deacetylase activity does not seem to be due to an inactivation of the enzyme but appears to be a consequence of the disruption of the structure of the deacetylase complex itself . Histone H4 in the deacetylase complex prepared from HeLa cell nuclei by micrococcal nuclease digestion was more highly acetylated than H4 in bulk nucleosomes . Restriction enzyme analysis of the DNA associated with the histone deacetylase complex revealed neither an enrichment nor a depletion of major satellite sequences in this material.

J Biol Chem, 1983 Dec 10, 258(23), 14309 - 14
A simple method for the preparation of extracts from animal cells which catalyze efficient in vitro protein synthesis; Brown GD et al.; A rapid, simple procedure is described for the preparation of cell-free extracts of both uninfected and virus-infected pig kidney and baby hamster kidney cells which are very active in the in vitro translation of not only endogenous mRNA, but also of exogenous mRNA added to extracts that had been depleted of endogenous mRNA by treatment with micrococcal nuclease . This procedure appears to be adaptable with only minor variations to many eukaryotic cell lines and should greatly facilitate in vitro molecular studies of protein synthesis and gene regulation at the level of translation.

J Biochem (Tokyo), 1983 Dec, 94(6), 1781 - 7
Studies on histone oligomers . V . Reconstitution of chromatin from purified DNA and acid-extracted histones; Kawashima S et al.; DNA-histone complexes were reconstituted from DNA and acid-extracted core histones and the products were characterized by micrococcal nuclease digestion to examine whether proper nucleosome structure had been reconstituted . No nucleosome structure was produced starting from the mixture of acid-extracted histones and purified DNA in 2 M NaCl-5 M urea, while the reassociation of chromatin by the same procedures was successful . This was due to the inappropriate conformation of acid-extracted histones, which was preserved in 2 M NaCl even in the presence of 5 M urea . If acid-extracted histones were reannealed from the completely denatured state, such as in 5 M urea, 6 M guanidine hydrochloride or 0.6 M NaCl-5 M urea, reconstitution of nucleosome structure was always successful.

Biokhimiia, 1983 Dec, 48(12), 2049 - 55
{Interrelation between the available boundary lipids in the bacterial membrane and the respiratory chain function}; Kaprel'iants AS et al.; In order to establish a possible correlation between the expression of the boundary lipid and the NADH-oxidase activity, the temperature dependences of the membranes of bacteria grown at 14 and 38 degrees C were investigated . The Tmelt . for the boundary lipid determined by comparing the excimerization parameters of the fluorescent probe pyrene in the vicinity of the proteins and in the total lipid phase was directly correlated with Tgrowth . A similar temperature dependence was observed with the NADH-oxidase activity, i . e . inhibition of activity at T greater than Tmelt . coincided with the disappearance of the boundary lipid . Incorporation of a cis-unsaturated fatty acid (linoleic acid) into the membranes markedly decreased the structural heterogeneity of membrane lipids and caused a simultaneous inhibition of the NADH-oxidase activity . No structural-functional changes were observed in the case of saturated fatty acids (stearic acid) . It was assumed that the presence of boundary lipids in the membrane is essential for the normal functioning of the multienzyme system of the respiratory chain . Presumably, the state of the immediate lipid environment controls the function of the micrococcal respiratory chain at the level of interaction between the carriers in the membrane.

Biokhimiia, 1983 Dec, 48(12), 2023 - 7
{Isolation from Micrococcus sp . n . of a homogeneous heme-containing catalase and a crystalline protein with catalase activity}; Romantsev FE et al.; A method for isolation and purification of catalases from the culture of Micrococcus sp . n . grown under aeration conditions is described . Heme-containing catalase (I) and the protein possessing a catalase activity (II) were separated by fractionation with ammonium sulfate . The specific activity of the highly purified protein causing degradation of H2O2 is 200 times less than that of the heme-containing enzyme . The molecular weights of catalases I and II as determined by electrophoresis in polyacrylamide gel gradient 4/30% are 240000 and 130000, respectively . The method described is designed at rapid isolation of preparative amounts of catalases from Micrococcus sp . n.

Anal Biochem, 1983 Dec, 135(2), 401 - 8
Characterization of messenger RNA by direct translation from agarose gels; Brandt TL et al.; A method for characterizing nanogram quantities of poly(A)-containing messenger RNAs that have been fractionated according to size by electrophoresis through agarose gels has been developed . The mRNAs from Friend leukemia cells were identified by the protein products they encode, as determined by slicing the agarose gel and directly translating the enclosed mRNA with an extract from rabbit reticulocytes that had been treated with micrococcal nuclease . A number of parameters which affect the efficiency of translation in this system have been examined . These include the sensitivity of the in vitro translational system to RNA, the agarose concentration, the incubation temperature, and the addition of either exogeneous tRNA or RNasin . The procedure is rapid, simple, reproducible, and applicable for the fractionation and characterization of mRNAs from any source.

Cancer Res, 1983 Dec, 43(12 Pt 1), 5747 - 53
Release of 3-methyladenine from linker and core DNA of chromatin by a purified DNA glycosylase; Heller EP et al.; Oligonucleosomes were isolated from {14C}thymidine-labeled HeLa cells by digestion of the nuclei with micrococcal nuclease and were then alkylated with {3H}methylnitrosourea . Nucleosome core particles were also prepared by further digestion of the oligonucleosomes . The distribution of 3H-labeled methyl groups in the linker versus the core DNA was established by a determination of 3H:14C ratios in oligonucleosome and core DNA . The ratios in the core DNA of 145 and 165 base pair DNA fragments were 5.2 and 5.4, respectively, while the ratio in the oligonucleosomal DNA was 8.2 . Assuming an equal mixture (as determined) of 145 and 165 base pair fragments of DNA in the 185 base pair repeat, the relative concentration of 3H methyl groups in the linker versus the core DNA was 4.2 . Thus, 45% of the 3H methyl groups were in the linker DNA, and 55% were in the core DNA . Some shielding of the DNA was evident during alkylation . The concentrations of alkyl groups on the linker and core DNA were 67 and 12% of that found on free DNA alkylated under comparable conditions . No evidence for preferential shielding of the major or minor groove was observed . The purified 3-methyladenine DNA glycosylase I of Escherichia coli released approximately 37% of the 3-methyladenine from the linker DNA and 13% from the core DNA . The limited enzymatic removal of 3-methyladenine in vitro compared to the efficient removal in vivo suggests that conformational changes of the oligonucleosome and core structure must occur for total repair.

Cancer Lett, 1983 Dec, 21(2), 125 - 31
Resistance of H1 histone to proteolytic attack in chromatin from rat-ascites hepatoma; Kishida K et al.; From rat-liver and ascites-hepatoma chromatins, NaCl-soluble fractions were prepared . The 0.35 M NaCl-soluble fraction from the hepatoma (AH) chromatin contained much non-histone protein of high-molecular weight, compared with the fraction from the rat-liver (RL) chromatin . The 0.35 M NaCl-insoluble, but 2 M NaCl/5 M urea-soluble fraction was composed mainly of 5 classes of histones . These histones were quantitatively not different between AH and RL chromatins . However, H1 histone was rather protease-resistant in AH chromatin, but not in RL chromatin . The proteolytic capacity was also lower in AH chromatin . In addition, in the micrococcal-nuclease digest of AH nuclei, the oligonucleosomes were considerably retained even by long-time digestion, but not in that of RL nuclei.

Eur J Biochem, 1983 Dec 1, 137(1-2), 191 - 6
Characterization of succinate dehydrogenase from Micrococcus luteus (lysodeikticus) by electron-spin-resonance spectroscopy; Crowe BA et al.; Low-temperature electron spin resonance spectroscopy has been used to study the biophysical properties of succinate dehydrogenase from the gram-positive bacterium Micrococcus luteus . The paramagnetic redox centres of the enzyme were identified in a succinate-dehydrogenase--antigen complex, which had been purified with the aid of monospecific serum from membranes solubilized with Triton X-100 . The centres were characterized in further detail using the membrane-bound and Triton-solubilized forms of the enzyme . These studies distinguished two types of iron-sulphur centres, viz . a {4Fe-4S}3+ cluster displaying a narrow signal at g = 2.01 in the oxidized state (conventionally termed centre S-3) and a {2Fe-2S )0 cluster with an axial signal at g = 2.03 and 1.93 in the reduced state (conventionally termed centre S-1) . Centre S-3 had a mid-point redox potential of +10 mV, a comparatively low value for this type of cluster . The behaviour of the g = 1.93 signal of centre S-1 was a complex function of the redox potential, microwave power and temperature of measurement . When measured at low power (i.e . non-saturating conditions), the intensities observed for the g = 1.93 signal poised at various critical potentials in the redox titration were similar . However, the corresponding intensities differed markedly at high power, where conditions were saturating . It is proposed that under saturating conditions the spin-lattice relaxation of the {2Fe-2S} cluster S-1 (mid-point potential +70 mV) is enhanced by centre S-3 between the potential range +10-+70 mV and by an ESR-silent centre, termed centre S-2, with a mid-point potential of -295 mV.

Eur J Biochem, 1983 Dec 1, 137(1-2), 185 - 90
Study of the respiratory chain in Micrococcus luteus (lysodeikticus) by electron-spin-resonance spectroscopy; Crowe BA et al.; Low-temperature electron spin resonance spectroscopy was used to investigate the redox centres of Micrococcus luteus membranes . Three different types of iron-sulphur centres were distinguished . Two of these, a {4Fe-4S}3+-type cluster giving rise to a signal at g = 2.01 in the oxidized state and a {2Fe-2S} cluster with a spectrum at g = 2.03 and 1.93 in the reduced state, were attributable to succinate dehydrogenase . Another, generating signals in the reduced state at g = 2.027, 1.90 and 1.78 was identified as a 'Rieske' iron-sulphur centre . This latter cluster had a mid-point potential (pH 7.0) of +130 mV . In addition, signals characteristic of high-spin ferric haem (g = 6.20), low-spin ferric haem (g = 3.67, 3.36 and 3.01) and Cu2+ (g = 2.18 and 2.02) were also detected . The ferric-haem features, together with the Cu2+ and 'Rieske' centres, were enriched in membrane residues insoluble in Triton X-100, which are known from difference spectroscopy to contain cytochromes b-560, c-550 and a-601 (aa3 oxidase) . The signals demonstrated by electron spin resonance for M . luteus membranes showed marked similarities to those documented for the complexes II, III, and IV of mitochondria . However, signals analogous to complex I (NADH-ubiquinone reductase) could not be demonstrated for M . luteus membranes.

J Biomol Struct Dyn, 1983 Dec, 1(3), 689 - 704
Simian virus 40 chromatin interaction with the capsid proteins; Bina M et al.; It has been established that both in virions and in infected cells, the cellular core histones fold the SV40 DNA into nucleosomes to form the SV40 chromosome or chromatin . We and others have begun to examine how the capsid proteins assemble the SV40 chromatin into virions and to investigate whether these proteins interact with the encapsidated chromatin . To follow the pathway of virus assembly, we have analyzed the nucleoproteins which accumulate in cells infected with the SV40 mutants temperature-sensitive in assembly: tsC, tsBC, and tsB . (The temperature-sensitivity of these mutants result from alterations in the amino acid sequence of the major capsid protein VP1) . We have found that mutants belonging to the same class accumulate similar types of nucleoproteins at the nonpermissive temperature (40 degrees C) and thus, share characteristics in common . For example, the tsC mutants accumulate only the 75 S chromatin . Both tsBC and tsB mutants produce in addition to chromatin, nucleoprotein complexes which sediment broadly from 100-160 S and contain all the three capsid proteins VP1, VP2, and VP3 . These nucleoproteins can be distinguished morphologically, however . Under the electron microscope, the tsBC 100-160 S nucleoproteins appear as chromatin to which a small cluster of the capsid proteins is attached; the tsB nucleoproteins appear as partially assembled virions . In addition, we find that the 220 S virions are assembled in cells coinfected with tsB and tsC mutants at 40 degrees C, in agreement with genetic analysis . Our observations favor the hypothesis that the VP1 protein contains three discrete domains . We speculate that each domain may play a specific function in SV40 assembly . To gain more insight into VP1-VP1 interactions, we have examined the nucleoproteins which result from treatment of the mature wild-type virions with increasing concentrations of the reducing agent DTT . In the presence of as low a concentration of DTT as 0.1 mM, the virion shell can be penetrated by micrococcal nuclease, which then cleaves the viral DNA . This result indicates that some of the disulfide bonds bridging the VP1 proteins are on the virion surface.

J Biol Chem, 1983 Nov 25, 258(22), 13986 - 91
Chromatin structure and transcriptional activity of an X-linked heat shock gene in drosophila pseudoobscura; Eissenberg JC et al.; Nuclease digestion of isolated nuclei was used to test whether differential chromatin structure exists for a dosage-compensated heat shock gene in Drosophila pseudoobscura . No differences were observed in nuclease sensitivity at this locus in males and females, either under heat shock or non-heat shock conditions, using micrococcal nuclease or DNase I . Although the higher level of nuclease sensitivity characterized by the induced state was removed when nuclei were prepared in high salt (0.45 M sodium chloride), this procedure did not reveal covert differences in X-linked chromatin structure between males and females . However, a clear difference was observed in the nuclease sensitivity at low level (uninduced) and high level (heat-induced) expression of the X-linked heat shock gene, suggesting that the same gene transcribed at two steady state rates can have different chromatin structures.

J Biol Chem, 1983 Nov 25, 258(22), 13478 - 85
Structural organization of the meiotic prophase chromatin in the rat testis; Rao BJ et al.; Pachytene nuclei were isolated from rat testes by the unit gravity sedimentation technique and contained histone variants H1a, H1t, TH2A, TH2B, and X2 in addition to the somatic histones H1bde, H1c, H2A, H2B, H3, and H4 . The basic organization of the pachytene chromatin namely the nucleosome repeat length and the accessibility to micrococcal nuclease, was similar to that of rat liver interphase chromatin . However, when digested by DNase I, the susceptibility of pachytene chromatin was 25% more than liver chromatin under identical conditions . Nucleosome core particles were isolated from both liver and pachytene nuclei and were characterized for their DNA length and integrity of the nucleoprotein on low ionic strength nucleoprotein gels . While liver core particles contained all the somatic histones H2A, H2B, H3, and H4, in the pachytene core particles, histone variants TH2A, X2, and TH2B had replaced nearly 60% of the respective somatic histones . A comparison of the circular dichroism spectra obtained for pachytene and liver core particles indicated that the pachytene core particles were less compact than the liver core particles . Studies on the thermal denaturation properties of the two types of core particles revealed that the fraction of the pachytene core DNA melting at the premelting temperature region of 55-60 degrees C was significantly higher than that of the liver core DNA.

Biochemistry, 1983 Nov 22, 22(24), 5631 - 40
Chromatin structural changes in synchronized cells blocked in early S phase by sequential use of isoleucine deprivation and hydroxyurea blockade; D'Anna JA et al.; We have investigated the loss of histone H1 from chromatin {D'Anna, J . A., Gurley, L . R., & Tobey, R . A . (1982) Biochemistry 21, 3991-4001} and the structure of chromatin from Chinese hamster (line CHO) cells blocked in early S phase by sequential use of isoleucine deprivation G1 block and 1 mM hydroxyurea (HU) blockade . Measurements of H1 content in the cell and histone turnover indicate that H1 is lost from the cell and that there is negligible replacement synthesis of H1 during the period of the S-phase block . As H1 is lost, chromatin appears to undergo structural change . After 10 h of HU block, the new deoxyribonucleic acid (DNA) and a portion of the old DNA have measured nucleosome repeat lengths (37 degrees C digestion) which are less than those of controls and similar to those observed by Annunziato and Seale {Annunziato, A . T., & Seale, R . L . (1982) Biochemistry 21, 5431-5438} for new immature chromatin in the absence of HU . By 24 h of HU block, nearly all of the chromatin has assumed a pseudoimmature conformation in which the nucleosome cores appear to be more closely packed along the DNA chain, but the new DNA is slightly more resistant than old DNA to attack by micrococcal nuclease . Electrophoretic analysis of nucleoprotein particles produced by micrococcal nuclease digestion of nuclei indicates that (1) the distribution of mononucleosome species changes during HU block and (2) some mononucleosome species appear to be enriched in normally minor proteins which may determine the electrophoretic mobility of the nucleoprotein particles in agarose-acrylamide gels . The results raise the possibility that (1) during the early stages of replication (or prior to the passage of the replication fork), H1 is dissociated from initiated replicons and (2) H1 does not reassociate in a concerted fashion with the H1-depleted chromatin until the replication fork has passed and, perhaps, a substantial portion of the replicon has been replicated.

Biochemistry, 1983 Nov 22, 22(24), 5626 - 31
Shortest nucleosomal repeat lengths during sea urchin development are found in two-cell embryos; Chambers SA et al.; Prior to fertilization, sperm possess one of the longest nucleosome repeat lengths yet determined {approximately 250 base pairs (bp) for the sea urchin Strongylocentrotus purpuratus} . We show here that the two-cell embryo has an average repeat size of 189 +/- 2 bp as probed by micrococcal nuclease; this is the shortest average nucleosomal subunit reported for S . purpuratus . By the eight-cell stage, the average nucleosome repeat increases to 201 +/- 2 bp, and it subsequently increases further during development . These results indicate that a dramatic rearrangement of chromatin occurs upon fertilization and that this chromatin remodeling continues through early development . When two-cell embryos are labeled for 30 min with {3H}thymidine and digested briefly, they exhibit nuclease-hypersensitive fragments averaging 308 bp in size, which are consistent with the size of protected DNA units in replication intermediate complexes at blastula stage (as described by Levy and Jacob {Levy, A., & Jacob, K . M . (1978) Cell (Cambridge, Mass.) 14, 259}) . Our results are consistent with two general propositions: (1) long repeat lengths are found in highly differentiated cells, and (2) short repeat lengths are characteristic of cells more active in cell division . Our data would also imply that a rapid increase in the DNA complement, e.g., in the transition from haploid to diploid state following fertilization, is accompanied by a shortening of the average size of DNA in a nucleosome after replication.

Biochem Biophys Res Commun, 1983 Nov 15, 116(3), 952 - 8
Endodeoxyribonuclease from nuclei of bovine small intestinal mucosa: further studies on intranuclear localization and cleavage mechanism of the enzyme; Nagae S et al.; Most of the activity of endodeoxyribonuclease was extracted from isolated chromatin with buffer containing 0.6 M NaCl, indicating that the endonuclease is present as a chromatin-bound form . When nuclei or chromatin of calf thymus or rat liver was digested with the bovine nuclear enzyme in the presence of 2 mM EGTA, to suppress endogenous Ca, Mg-dependent DNase activity, discrete DNA bands with integral multiples of 200 base pairs in length were produced, but no acid-soluble nucleotide was detected . The enzyme made single-strand breaks in pBR322 DNA and degraded it to fragments of limited size . The size of the final products of DNA's of Micrococcus luteus, rat liver nuclei, and calf thymus nuclei was about 3,000, 200, and 160 base pairs, respectively, but the enzyme showed no base specificity . Thus the endonuclease seems preferentially to recognize AT-rich regions of double-stranded DNA and to make single-strand breaks.

Nucleic Acids Res, 1983 Nov 11, 11(21), 7363 - 74
The organization of the 7SL RNA in the signal recognition particle; Gundelfinger ED et al.; Digestion of the signal recognition particle (SRP) of dog pancreas with micrococcal nuclease results in the stepwise cleavage of the 300 nucleotide 7SL RNA moiety producing five major fragments approximately 220 (1), 150 (2), 72 (3), 62 (4) and 45 (5) nucleotides long . The RNA molecule is initially cut once yielding fragments 1 and 3 . Further degradation releases fragments 2, 4 and 5 . The introduction of the first nick into the 7SL RNA does not alter the structure nor the function of the SRP . Further degradation of the RNA results in disruption and loss of activity of the particle . The sequence of the RNA fragments shows that the nuclease causes discrete cuts in the RNA with minimal nibbling indicating that only few sites are accessible to the action of the enzyme . The five major products of nuclease digestion together span almost the entire length of the 7SL RNA . Nicking occurs mainly around the boundary region between the central S sequence and the flanking Alu sequences constituting the 7SL RNA (1) . The S fragment is bound to the four largest polypeptides while the 5' and 3' Alu fragments are associated with the two smallest protein constituents of the SRP.

J Biol Chem, 1983 Nov 10, 258(21), 13321 - 7
Pathways of assembly of nucleohistone complexes formed in vitro under physiological conditions . Implications for the structure of the nucleosome; Ellison MJ et al.; The composition and structure of three discrete nucleohistone particles produced by micrococcal nuclease digestion of the complex formed when the four core histones are mixed directly with DNA under conditions that are close to physiological have been reported in accompanying articles (Ellison, M . J., and Pulleyblank, D . E . (1983) J . Biol . Chem . 258, 13307-13313; 13314-13320) . These include a peak containing a compact hexameric complex composed of one pair of (H3,H4) and two pairs of (H2A,H2B) (P2) and a peak containing complexes with the composition and sedimentation properties of nucleosome cores (P3) . We have examined the pathways of assembly of P2 and P3 by determining the effect of order of histone addition on the yields of these species . The assembly of P2 is initiated by the binding of an (H2A,H2B) pair to the DNA which then directs the placement of an (H3,H4) pair . The incorporation of the final (H2A,H2B) pair to complete the particle is probably cooperative . The assembly of P3 is less dependent than the assembly of P2 upon the order in which the histone pairs are added to the DNA . More than one pathway has been implicated in the formation of these particles . P3 contains a component which has many but not all of the properties of nucleosome cores assembled at elevated ionic strength . Alternative pathways of assembly of histone pairs onto DNA are discussed which could lead to the formation of the discrete histone DNA structures that have been isolated here . A model is proposed in which the nucleosome unfolds and refolds into an alternative configuration.

J Biol Chem, 1983 Nov 10, 258(21), 13307 - 13
The assembly of an H2A2,H2B2,H3,H4 hexamer onto DNA under conditions of physiological ionic strength; Ellison MJ et al.; A novel nucleohistone particle is generated in high yield when a complex of DNA with the four core histones formed under conditions that are close to physiological (0.15 M NaCl, pH 8) is treated with micrococcal nuclease . The particle was found to contain 102 base pairs of DNA in association with six molecules of histones in the ratio 2H2A:2H2B:1H3:1H4 after relatively brief nuclease treatment . Prolonged nuclease digestion resulted in a reduction in the DNA length to a sharply defined 92-base pair fragment that was resistant to further degradation . Apparently normal nucleosome core particles containing two molecules each of the four core histones in association with 145 base pairs of DNA and a particle containing one molecule each of histones H2A and H2B in association with approximately 40 base pairs of DNA were also generated during nuclease treatment of the histone-DNA complexes formed under physiological ionic strength conditions . Kinetic studies have shown that the hexamer particle is not a subnucleosomal fragment produced by the degradation of nucleosome core particles . Furthermore, the hexamer particle was not found among the products of nuclease digestion when histones and DNA were previously assembled in 0.6 M NaCl . The high sedimentation coefficient of the hexameric complex (8 S) suggests that the DNA component of the particle has a folded conformation.

Prikl Biokhim Mikrobiol, 1983 Nov-Dec, 19(6), 788 - 94
{Action of the steroid glycoside deltonin on the bacterial membranes of Micrococcus lysodeikticus}; Turgenbaeva DA et al.; A slight detergent-like effect of steroid glycoside deltonine from Dioscorea deltoidea on the bacterial membranes of Micrococcus lysodeikticus was detected which resulted in the breaking of the osmotic barrier of protoplasts and in the loss from the membranes of small fragments containing the dehydrogenases of the respiratory chain but without cytochromes . These small fragments still retained the membrane structure.

Biokhimiia, 1983 Nov, 48(11), 1927 - 32
{Study of a free radical compound from the cells of the bacteria Micrococcus lysodeikticus}; Biniukov VI et al.; Some physico-chemical properties of a compound previously detected in M . lysodeikticus cells were studied . This compound is hydrophilic, has hydrophilic, has a low molecular weight and does not contain The redox potentials of the free radical formation in the cytoplasm and in purified preparations were determined . A hypothetical reaction scheme is proposed.

Arch Toxicol, 1983 Nov, 54(3), 215 - 25
Non-random effect on RNA synthesis in liver chromatin by administration of dimethylnitrosamine to mice; Klaude M et al.; The effect of dimethylnitrosamine on functional activities of liver chromatin was studied in mice . After a single dose of dimethylnitrosamine injected i.v . (25 mg/kg body wt, 45 min before sacrifice) liver nuclei were isolated and incubated with micrococcal nuclease (EC 3.1.4.7) to an acid-solubility of 2.5% of total DNA . Chromatin was fractionated into a 1,200 g pellet P1, 102,000 g pellet P2 and supernatant fraction S2 . Chromatin-bound RNA polymerase I plus III activity decreased 15% in the P1 and 25% in the P2 fraction . No changes in activity were observed in the S2 fraction . Chromatin-bound RNA polymerase II activity decreased 19% in the P1, 49% in the P2 and 32% in the S2 fraction . Heparin stimulated RNA polymerase II activity decreased 10% in the P1 and 44% in the P2 fraction . Formation of initiation in nuclear lysates with RNA polymerase from Escherichia coli increased after administration of dimethylnitrosamine suggesting an increase in the number of sites available for the start of new RNA chains . The results show that limited digestion of nuclei with endonuclease cleaves chromatin regions which are more affected by dimethylnitrosamine than the total chromatin suggesting a non-random effect of the hepatotoxin on chromatin . Modifications of the DNA template by dimethylnitrosamine is indicated by the change in number of initiation complexes.

J Mol Biol, 1983 Oct 25, 170(2), 423 - 46
Location of the primary sites of micrococcal nuclease cleavage on the nucleosome core; Cockell M et al.; The positions and relative frequencies of the primary cleavages made by micrococcal nuclease on the DNA of nucleosome core particles have been found by fractionating the double-stranded products of digestion and examining their single-stranded compositions . This approach overcomes the problems caused by secondary events such as the exonucleolytic and pseudo-double-stranded actions of the nuclease and, combined with the use of high resolution gel electrophoresis, enables the cutting site positions to be determined with a higher precision than has been achieved hitherto . The micrococcal nuclease primary cleavage sites lie close (on average, within 0.5 nucleotide) to those previously determined by Lutter (1981) for the nucleases DNase I and DNase II . These similarities show that the accessible regions are the same for all three nucleases, the cleavage sites being dictated by the structure of the nucleosome core . The differences in the final products of the digestion are explained in terms of secondary cleavage events of micrococcal nuclease . While the strongly protected regions of the nucleosome core DNA are common to all three nucleases, there are differences in the relative degrees of cutting at the more exposed sites characteristic of the particular enzyme . In particular, micrococcal nuclease shows a marked polarity in the 3'-5' direction in the cutting rates as plotted along a single strand of the nucleosomal DNA . This is explained in terms of the three-dimensional structure of the nucleosome where, in any accessible region of the double helix, the innermost strand is shielded by the outermost strand on the one side and the histone core on the other . The final part of the paper is concerned with the preference of micrococcal nuclease to cleave at (A,T) sequences in chromatin.

Biochemistry, 1983 Oct 25, 22(22), 5150 - 6
Composition and structure of zinc-deficient Euglena gracilis chromatin; Stankiewicz AJ et al.; The histone content of zinc-deficient (-Zn) Euglena gracilis decreases while, concomitantly, DNA content increases and the transcription rate is reduced markedly {Mazus, B., Falchuk, K . H., & Vallee, B . L . (1983) Biochemistry (in press); Falchuk, K . H., Fawcett, D . W., & Vallee, B . L . (1975) J . Cell Sci . 17, 57-78} . The effects on major constituents of the genome have been examined by studying the rate and extent of hydrolysis of +Zn and -Zn chromatin by micrococcal nuclease, DNase I, or DNase II . The size of hydrolyzed DNA fragments suggests similarity of the +Zn E . gracilis chromatin organization to that of other eukaryotes . The major protein constituent of -Zn chromatin is a polypeptide of less than 3000 daltons whose electrophoretic mobility differs from that of any known histone components of chromatin, the latter described elsewhere (K . H . Falchuk et al., unpublished results) . This protein profoundly affects the structure of -Zn chromatin, which is about 10-30-fold more resistant to micrococcal nuclease hydrolysis than +Zn chromatin . Moreover, the resultant DNA fragments {2000 base pairs (bp)}, are much larger than those of +Zn cells . Under conditions which hydrolyze +Zn chromatin into DNA fragments smaller than 50 bp, only 50% of -Zn chromatin is digested into fragments less than 2000 bp, i.e., in the range of those expected for oligonucleosomes . Removal of the low molecular weight protein from -Zn chromatin reverses its enhanced resistance to nucleolysis and results in extensive hydrolysis . Conversely, addition of the low molecular weight protein to +Zn chromatin increases the resistance of this complex to digestion.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1983 Oct 25, 258(20), 12675 - 84
Histone deacetylation is required for the maturation of newly replicated chromatin; Annunziato AT et al.; The effects of inhibiting histone deacetylation on the maturation of newly replicated chromatin have been examined . HeLa cells were labeled with {3H}thymidine in the presence or absence of sodium butyrate; control experiments demonstrated that butyrate did not significantly inhibit DNA replication for at least 70 min . Like normal nascent chromatin, chromatin labeled for brief periods (0.5-1 min) in the presence of butyrate was more sensitive to digestion with DNase I and micrococcal nuclease than control bulk chromatin . However, chromatin replicated in butyrate did not mature as in normal replication, but instead retained approximately 50% of its heightened sensitivity to DNase I . Incubation of mature chromatin in butyrate for 1 h did not induce DNase I sensitivity: therefore, the presence of sodium butyrate was required during replication to preserve the increased digestibility of nascent chromatin DNA . In contrast, sodium butyrate did not inhibit or retard the maturation of newly replicated chromatin when assayed by micrococcal nuclease digestion, as determined by the following criteria: 1) digestion to acid solubility, 2) rate of conversion to mononucleosomes, 3) repeat length, and 4) presence of non-nucleosomal DNA . Consistent with the properties of chromatin replicated in butyrate, micrococcal nuclease also did not preferentially attack the internucleosomal linkers of chromatin regions acetylated in vivo . The observation of a novel chromatin replication intermediate, which is highly sensitive to DNase I but possesses normal resistance to micrococcal nuclease, suggests that nucleosome assembly and histone deacetylation are not obligatorily coordinated . Thus, while deacetylation is required for chromatin maturation, histone acetylation apparently affects chromatin organization at a level distinct from that of core particle or linker, possibly by altering higher order structure.

Biochim Biophys Acta, 1983 Oct 18, 760(2), 309 - 17
Double-stranded RNA-stimulated enzyme activities isolated from human placental extracts; Borelli TJ et al.; A (2'-5')An synthetase activity was isolated from human placental extracts by affinity chromatography on poly(rI) . poly(rC)-agarose . The oligonucleotide (2'-5')An was identified by (1) chromatography on PEI-cellulose and DEAE-cellulose, (2) inhibition of polypeptide synthesis in lysed rabbit reticulocytes (3) competition of the binding of pppA(pA)3,3'-{32P}pCp to rabbit reticulocyte lysates, and (4) alkaline phosphatase digestion . The synthetase activity in most placental preparations is activated by natural or synthetic dsRNA . However, in a few placental synthetase preparations, dsRNA is only marginally stimulatory and only becomes effective by prior treatment of the enzyme preparations with the calcium-dependent micrococcal nuclease . This suggests that there is an endogenous placental dsRNA contaminant in the enzyme preparations . In some synthetase preparations, a second dsRNA-stimulated product, tentatively identified as the nucleotide 5'-IMP, is also observed . Because the specific AMP deaminase inhibitor coformycin (10 microM) blocks the formation of IMP from ATP and causes a quantitative accumulation of AMP, and because the formation of IMp becomes independent of dsRNA when ADP or AMP is used in place of ATP, the presence of a dsRNA-stimulated ATP phosphohydrolase (ATPase) activity in human placenta is suggested.

Exp Cell Res, 1983 Oct 15, 148(2), 363 - 76
Nucleosome DNA repeat length and histone complement in a fungus exhibiting condensed chromatin; Ralph-Edwards A et al.; Fungal chromatins are reported to exhibit unusually short nucleosomal DNA repeat lengths . To test whether this is a phylogenetic feature of fungi or rather is correlated with an apparent absence of condensed chromatin in the organisms studied, we have examined the chromatin organization and the complement of basic nuclear proteins in the fungus Entomophthora, an organism which exhibits marked chromatin condensation . Micrococcal nuclease digestion of Entomophthora chromatin revealed a nucleosomal DNA repeat length of 197 +/- 1.2 base pairs (bp) . This repeat length is 20-40 bp longer than that reported for any fungus . Entomophthora nucleosomes exhibited an HI-like protein which was much less basic than the HI histones reported for higher eukaryotes but which was similar in basicity to the HI histone reported for the fungus Neurospora . However, the nucleosomal DNA repeat length of Neurospora chromatin is reported to be unusually short, whereas that of Entomophthora was found to be typical of the repeat lengths observed for chromatins of higher eukaryotes . Thus, repeat length, at least in fungi, would not appear to be directly determined by the basicity of the fungal cognate of histone HI.

Biochem Biophys Res Commun, 1983 Oct 14, 116(1), 312 - 20
Transcription renders chromatin resistant to micrococcal nuclease digestion; Kohno K et al.; When isolated HeLa cell nuclei were preincubated under transcription conditions with excess E . coli RNA polymerase, chromatin DNA became relatively resistant to digestion by micrococcal nuclease . Quantitation of the DNA content in nuclei after enzyme digestion revealed that approximately twice as much nuclease was required to give the same levels of release of DNA fragments from transcribed as from untranscribed nuclei . Resistance increased with the amount of polymerase and with the time of preincubation . Since the resistance to nuclease was not observed in the presence of rifampicin or by preincubation without UTP, both RNA chain initiation and elongation were considered to be essential for the manifestation of resistance . However, when DNase 1 was used as a probe, such a change in chromatin DNA was not detected.

Biochemistry, 1983 Oct 11, 22(21), 5008 - 15
High mobility group proteins: abundance, turnover, and relationship to transcriptionally active chromatin; Seale RL et al.; We have measured the abundance of high mobility group (HMG) proteins 14 and 17 in HeLa cell chromatin and their fractionation with respect to transcriptionally active sequences . HMG protein 17 constitutes 10-20% of the mass of an individual core histone; HMG 14 is approximately one-tenth the mass of HMG 17 . The enrichment of HMG proteins, relative to bulk chromatin, is less than 2-fold in the chromatin fraction enriched 6-fold in active sequences . The digestion characteristics of HMG nucleosomes indicate that they are interspersed with H1 nucleosomes and other monomer species . The HMG monomers are quite resistant to degradation by micrococcal nuclease and can be resolved as distinct nucleoprotein entities after trimming of the DNA to core length . Turnover measurements showed that HMG proteins 14 and 17 are stable for at least 24 h . When nucleosome monomers are reconstituted with a 0.35 M NaCl nuclear protein extract, each nucleosome subtype can be reconstituted; however, this is a function of both the amount of extract added and the DNA length of the nucleosomes . When the kinetics of reconstitution of bulk vs . coding sequences were measured with cDNA, there was no significant enrichment of active sequences in the HMG-containing mononucleosomes of HeLa cells at any ratio of extract to monomer employed . In Friend cells, the abundance of sequences among mononucleosome species was the same for the transcribed beta-major globin gene, a transcriptionally inactive embryonic globin, and an inactive immunoglobulin gene . There was little correlation of HMG content with transcriptionally active chromatin, either native or reconstituted.

Nucleic Acids Res, 1983 Oct 11, 11(19), 6631 - 46
Interaction of snRNAs with rapidly sedimenting nuclear sub-structures (hnRNPs) from HeLa cells; Sri-Widada J et al.; We have shown previously (Liautard et al., 1982, J . Mol . Biol., 162, 623-643) that digestion with micrococcal nuclease under drastic conditions of a pure U1 snRNP, as well as a mixture containing U2, U1, U4, U5 and U6 snRNPs, gives rise to resistant RNA fragments derived from all but U6 snRNAs . As an attempt to elucidate the way in which snRNPs are attached to their native structure, the same approach was applied to hnRNP which are known to contain snRNP (Guimont-Ducamp et al., 1977, Biochimie, 59, 755-758) . Micrococcal nuclease digestion of hnRNPs yielded a population of 15-50 nucleotides long resistant fragments of snRNAs . Sequence analyses showed that all fragments previously identified in core snRNPs were also present . Only U2 and U5 snRNAs were further protected as a result of their association with the hnRNP complex (from the cap to nucleotide 32 for U2 and from nucleotide 22 to nucleotide 70 for U5) . No additional protected fragment derived from U1, U4 and U6 snRNAs was found . This finding confirms that the 5' terminal region of U1 snRNP remains available for base-pairing interaction with the premessenger RNA, as predicted by the model of Lerner et al . (Nature, 1980, 283, 220-224).

FEBS Lett, 1983 Oct 3, 162(1), 161 - 6
Fate of newly synthesized histones in G1 and G0 cells; Wu RS et al.; We have shown that quiescent cells as well as those in the G1 phase of the cell cycle synthesize histones at a reduced but significant rate . Now, we show that the histones synthesized during G0 and G1 are stably incorporated into nuclei soon after synthesis . Micrococcal nuclease digestion of nuclei isolated from cells in G0 and G1 revealed that the specific histone variants synthesized in these different physiological states are found associated with DNA as nucleosomes . Nucleosomes were separated by polyacrylamide gel electrophoresis in a reducing buffer so that histone spot morphology, particularly that of the H3s was improved.

Cancer Res, 1983 Oct, 43(10), 4913 - 9
Effects of divalent metal cations on composition and neoplasia-specific antigenicity of chromatins; Dupere SL et al.; The antigenicity and composition of chromatins differ markedly in chromatin preparations obtained by different procedures . Rat Novikoff hepatoma chromatin (NC) obtained by the "salt precipitation" and the micrococcal nuclease digestion procedures using significant levels of EDTA and NaCl each shows a common complement fixation (CF) capacity, exceeding chromatin preparations obtained from normal rat liver when tested with rabbit antisera raised to dehistonized NC . In contrast, "structured" NC preparations, which have been postulated to retain a native physical conformation, show minimal CF capacity when tested with the same antiserum but show high CF following elution of histones . While further progressive elution of non-histone proteins (NHPs) did not alter the CF capacity per unit DNA, the completely separated DNA and NHP fractions each showed minimal CF . The data suggest that the antigens detected in the CF assay predominantly represent an artifactual but specific complex of DNA and NHP arising from a denaturation of the native chromatin following elution of metal ions or histones . A qualitatively similar profile of NHPs in salt-precipitated NCs shows a range of total protein/DNA ratios, suggesting that the NHPs found in chromatin preparations may not be intrinsic to the native chromatin structure.

Tsitologiia, 1983 Oct, 25(10), 1173 - 8
{Excision of UV-induced pyrimidine dimers from the DNA of Chinese hamster cells of the CHO line}; Barenfel'd LS et al.; Using DNA sedimentation in alkaline sucrose gradients, the excision of pyrimidine dimers from DNA of CHO cells with the help of UV-endonuclease from Micrococcus luteus has been studied . A considerable proportion (approximately 80%) of pyrimidine dimers was shown to be excised within 26 hours, whereas during the initial 17 hours after the UV-fluence (4 J/M2) the excision was slight (approximately 15%).

J Endocrinol, 1983 Oct, 99(1), 51 - 61
Extraction of androgen-receptor complexes from regions of rat ventral prostate nuclei sensitive or resistant to nucleases; Davies P; Rat ventral prostate nuclei contain androgen-binding sites which are susceptible or resistant to excision by endonucleolytic action . Those which were susceptible were associated both with oligonucleosomal and subnucleosomal particles . The sedimentation profile characteristic of a nuclear androgen-receptor complex could be obtained by exhaustive nucleolytic digestion or by treatment of fractions with KCl (0.6 mol/l) . Androgen-binding sites resistant to DNAase I were also resistant to KCl, whereas those sites resistant to micrococcal nuclease were partially extractable with KCl . Nuclease-resistant sites could be extracted with heparin (10 mg/ml) . Androgen-receptor complexes obtained from nuclease-sensitive or nuclease-resistant regions by extraction with KCl or heparin were indistinguishable by routine sedimentation analysis.

J Virol, 1983 Oct, 48(1), 79 - 87
Chromatin-like structure of adeno-associated virus DNA in infected cells; Marcus-Sekura CJ et al.; Adeno-associated virus (AAV) is a single-stranded DNA virus which requires adenovirus as a helper for productive infection . We studied whether intracellular AAV DNA in KB cells was present in a chromatin-like structure by digesting infected cell nuclei with micrococcal nuclease . Virus DNA was detected by agarose gel electrophoresis followed by blotting and hybridization to nick-translated {32P}DNA probes . After coinfection with adenovirus, AAV DNA was present in nucleosome-like structures which were similar to cell nucleosomes and were double stranded as judged by insensitivity to S1 nuclease digestion . In the absence of adenovirus, intracellular AAV DNA also formed similar nucleosome-like structures which were also insensitive to S1 digestion and were formed in both the presence and absence of hydroxyurea . These latter structures probably formed on AAV duplexes created either by reassociation of infecting parental single-stranded DNA or by covalent integration into the cell genome rather than by de novo AAV DNA synthesis . These results have implications for the mechanism of AAV genome replication, transcription, and integration into the cell genome.

Mol Cell Biol, 1983 Oct, 3(10), 1724 - 9
Analysis of DNA structural patterns and sequence organization at the larval cuticle locus in Drosophila melanogaster; Eissenberg JC et al.; We examined the pattern of DNA organization at the larval cuticle gene complex 44D of Drosophila melanogaster, using micrococcal nuclease and the 1,10-phenanthroline-cuprous complex . The initial cleavage patterns obtained with both reagents exhibited "gaps" at the positions of each of the genes examined, as well as at a pseudogene sequence contained within the complex . An additional gap for which no gene exists was observed for both patterns . The cleavage pattern obtained with micrococcal nuclease was unaltered, at a level of resolution of +/- 50 base pairs, in a mutant containing a transposable element . Analysis of the sequence data from this 5.5-kilobase gene cluster indicated that the sequence per se, and not the general base composition, is a dominant factor in determining the patterns observed.

J Biol Chem, 1983 Sep 25, 258(18), 11274 - 9
Changes in chromatin structure at the replication fork . DNase I and trypsin-micrococcal nuclease effects on approximately 300- and 150-base pair nascent DNAs; Galili G et al.; DNase I, trypsin, and micrococcal nuclease are used to further probe the structure of nascent deoxyribonucleoprotein (DNP) fractions which appear after in vivo 20-s pulse labeling of sea urchin embryos with {3H}thymidine . We present evidence that the large nascent DNP which protects the approximately 300-base pair large nascent DNA consists of at least one nucleosome core . This is based on fractionation in denaturing polyacrylamide gels of DNA extracted from large nascent DNP fractions of a micrococcal nuclease + DNase I digest of nuclei . The data also suggest the existence of a DNase I-hypersensitive site(s) within the large nascent DNP; this is consistent with the hypothesis that the latter consists of closely packed dinucleosome cores . Histone H1 and non-histone proteins do not account for the previously reported unusual hyperresistance of the large nascent DNA against micrococcal nuclease . The protection offered this approximately 300-base pair nascent DNA was not eliminated by an 0.2-microgram/ml trypsin pretreatment which removes the above proteins from the chromatin . However, 5-10 micrograms/ml of trypsin, which remove a portion of the NH2 termini of the four core histones of nucleosomes, eliminate the hyperresistance of the large nascent DNA to subsequent micrococcal nuclease digestion, while nascent and bulk monomer DNAs remain unaffected . This indicates histone-histone and/or histone-DNA interactions within the large nascent DNP which differ from those of nascent and bulk mononucleosome cores.

Biochemistry, 1983 Sep 13, 22(19), 4592 - 600
In vitro reconstitution of 35S ribonucleoprotein complexes; Wilk HE et al.; Ribonucleoprotein complexes (hnRNP) sedimenting at 30-40 S and containing fragments of heterogeneous nuclear RNA (hnRNA) have been extracted from HeLa cell nuclei . Besides hnRNA fragments (8-12 S), the complexes contain eight mostly basic core proteins of Mr 31 000-41 000 as shown by two-dimensional gel electrophoresis . Other proteins (mostly of higher molecular weight) seem to be peripherally associated since they are lost after pelleting and recentrifugation of the hnRNP complexes . The particle dissociates into its protein components after digestion of the endogenous hnRNA fragments by micrococcal nuclease . After inactivation of the nuclease and addition of a wide variety of exogenous RNAs {MS2 phage RNA, poly(U), poly(C), poly(A), and poly(A,U)}, a RNP particle is re-formed which resembles the native hnRNP complex according to its sedimentation value (35 S), its appearance in the electron microscope, its density in metrizamide, and its protein composition . No particles are formed on double-stranded RNA {poly(A) . poly(U)} or native DNA whereas denatured DNA allows complex formation . On MS2 RNA (3569 nucleotides), the formation of tri- and tetrameric complexes is observed . This indicates the presence of 900-1200 nucleotides per particle . In vivo, 40S hnRNP particles are a unit component of larger RNP structures . Hence, we conclude from our results that the hnRNP core proteins have the intrinsic capability to associate with nascent single-stranded hnRNA regions to form these RNP complexes . Because of the lack of any sequence specificity, the complexes may function in packaging of the hnRNA and in connection with other nuclear components may provide a scaffold for subsequent processing reactions.

Nucleic Acids Res, 1983 Sep 10, 11(17), 6107 - 20
Poly(2-methylthio-7-deazainosinic acid)--hydrophobic stabilization of polynucleotide secondary structure by the 2-methylthio group; Seela F et al.; Poly(2-methylthio-7-deazainosinic acid) {poly(ms2c7I)} was enzymatically synthesized by polymerization of 2-methylthio-7-deazainosine 5'-diphosphate with polynucleotide phosphorylase from Micrococcus luteus in high yield . The homopolymer shows much higher thermal stability than its parent polynucleotides poly(7-deazainosinic acid) {poly(c7I)} and poly(I) . Its sigmoidal melting curve and pronounced hypochromicity imply a rigid, ordered structure . Poly(ms2c7I), like poly(2-methylthio-inosinic acid) {poly(ms2I)}, does not form a complex with poly(C) because of the bulky 2-methylthio substituent . On the other hand, two poly(ms2c7I) strands form very rigid triple strands with poly(A) . Different from poly(I) and poly(c7I) the homopolymer poly(ms2c7I) is very stable against cleavage by nuclease S1 and ribonuclease T2 as expected from its rigid secondary structure.

Cancer Res, 1983 Sep, 43(9), 4091 - 7
Characterization of a unique nuclear estrogen-binding component in an estrogen-responsive mouse Leydig cell tumor; Maeda Y et al.; An estrogen-responsive mouse Leydig cell tumor line (Tumor 124958) has been shown to contain only a low-affinity binder for estradiol in the cytosol fraction . This differed from the putative estrogen receptor in terms of its hormone-binding specificity as well as affinity . In addition, the possibility that an estrogen receptor-like molecule exists in the nuclei even without hormonal stimuli was examined using purified nuclei . Scatchard plot analyses showed that these nuclei possessed a large amount of estrogen binder having a high affinity for estradiol and diethylstilbestrol . The content of this nuclear binding component was not diminished by using molybdate, a potent inhibitor for receptor activation, and in vitro incubation of collagenase-dispersed cells with estradiol did not cause significant increase in the number of nuclear binding sites when compared with the values obtained by direct incubation of isolated nuclei with estradiol . These results support the view that this nuclear estrogen binder is not due to artificial migration of the cytosol receptor into nuclei during homogenization . The characterization of this nuclear binding component under cell-free conditions revealed that its affinity for estradiol in Mg2+-containing buffer was temperature dependent (Kd 3 nM at 30 degrees and 12 nM at 0 degrees) without significant alteration in the number of maximum binding sites . Introduction of a chelating agent (ethylenediaminetetraacetate) into the buffer system abolished the temperature effect on the affinity, resulting in high affinity for estradiol at both low and high temperatures . These Mg2+ and temperature effects were reversible . In addition, when compared with putative nuclear estrogen receptors, this nuclear binding was observed to be relatively resistant to high salt or micrococcal nuclease treatments in relation to solubilization from nuclei . However, trypsin digestion was found to result in a marked decrease in the nuclear binding sites, indicating that this unique nuclear binding component contains a protein unit(s) . These results suggest the possibility that this tumor line contains a unique unoccupied nuclear estrogen binder which might be able to transmit estrogen signals to tumor cell nuclei with regard to tumor growth.

J Biochem (Tokyo), 1983 Sep, 94(3), 735 - 44
A protein which facilitates assembly of nucleosome-like structures in vitro in mammalian cells; Ishimi Y et al.; An activity which facilitates assembly of nucleosome-like structures in vitro at physiological ionic strength was detected both in human HeLa S3 cells and mouse FM3A cells . The assembly protein was purified from FM3A cells by fractionation with ammonium sulfate, DEAE-cellulose, phosphocellulose, Sephadex G-150 column chromatography, and sucrose gradient centrifugation . In the sucrose gradient, the activity was detected at 5S and the active fraction contained three peptides of 59,000, 65,000, and 102,000 daltons . When core histones were mixed with these peptides, the 59,000 peptide sedimented at the 6S and 10S positions, where the histones co-sedimented . The 6S fraction contained H2A, H2B, and A24 proteins, and the 10S fraction contained four kinds of core histones in equal amounts . Nucleosomes were formed by mixing DNA with the 10S fraction, but were not formed with the 6S fraction . The nucleosome structure assembled was assessed using the sensitivity to micrococcal nuclease.

Biosci Rep, 1983 Sep, 3(9), 847 - 56
Increased thermal stability of solubilized chromatin after poly(ADP-ribose) synthesis; States JC et al.; A hyperthermic shift in the hyperchromicity curve of thermally denatured swine aortic-smooth-muscle-cell chromatin solubilized by digestion of nuclei with micrococcal nuclease was observed after the chromatin was incubated under conditions to allow poly-(ADP-ribose) synthesis by the endogenous poly(ADP-ribose) polymerase . When the order of solubilization and poly(ADP-ribosyl)ation was reversed, a smaller proportion of the solubilized chromatin exhibited greater thermal stability . Nuclease digestion of nuclei preincubated for poly(ADP-ribose) synthesis revealed no difference in kinetics of digestion or fragment size distribution compared to that of control nuclei . Poly(ADP-ribose) synthesis in these nuclei was proportionately greater in the chromatin fraction most resistant to solubilization by micrococcal nuclease treatment.

Biochem Pharmacol, 1983 Sep 1, 32(17), 2571 - 5
Physical association of oestrogens and other steroids with DNA; Blackburn GM et al.; 4C-Labelled mestranol, 3-O-methyl oestrone and cholesterol-5 alpha,6 alpha-oxide have been prepared . Along with the natural oestrogens, E1 and E2, and other steroids, these compounds have been used to determine the extent of their physical association with DNA . Analysis of binding both by equilibrium solubilization and by caesium chloride density gradient centrifugation showed the same relative order of binding: mestranol greater than 3-O-methyl oestrone greater than oestrone greater than cholic acid greater than cholesterol-5 alpha,6 alpha-oxide greater than progesterone, testosterone greater than oestradiol . DNA from Micrococcus lysodeikticus showed a higher affinity for cholic acid than did calf thymus DNA, while pretreatment of the latter with proteinase K somewhat reduced the level of physical binding of oestrone.

Eur J Immunol, 1983 Sep, 13(9), 772 - 8
The immunomodulatory effect of anti-Micrococcus luteus antibodies . I . Effect on in vitro rabbit T cell functions; Vaeck M et al.; A range of purified rabbit anti-Micrococcus luteus antibodies (anti-MCAb) were tested for their ability to interfere with a variety of in vitro immune responses . Such antibodies strongly inhibited the secondary IgG antibody response to sheep red blood cells without affecting the IgM response or the proliferative responses to mitogens and antigens . By exposing lymphocyte populations to anti-MCAb, it was found that such reagents exerted a strong mitogenic effect on rabbit T lymphocytes, provided these cells were derived from antigen-activated lymph nodes . This mitogenic effect was also obtained with F(ab')2 fragments of anti-MCAb and with hybridoma-derived anti-MCAb . Collectively, these data indicate that anti-MCAb inhibit the initiation of IgG synthesis possibly through the expansion of immunoregulatory T cell subsets.

Biokhimiia, 1983 Sep, 48(9), 1568 - 79
{Generation of membrane potential by aerobic bacteria Micrococcus lysodeikticus . Correlation between coupled and uncoupled respiration}; Artsatbanov VIu et al.; The effects of cyanide and nonylhydroxyquinoline-N-oxide on membrane potential generation and oxidase activities in intact cells and membrane particles of M . lysodeikticus were studied . Low concentrations of the inhibitors interacting with the components of one branch of a branched respiratory chain of M . lysodeikticus strongly inhibit the membrane potential generation and only slightly reduce the total respiration rate . It was assumed that the generation of delta psi takes place in the b-c locus of the respiratory chain branch terminated with cytochrome oxidase; over 90% of total O2 uptake provides for the uncoupled respiration via cytochrome omicron . Inside the b-c locus two sites responsible for delta psi generation were singled out; these sites differ in their sensitivities towards nonylhydroxyquinoline-N-oxide . Based on the data obtained and the effect of "extra-reduction" of cytochrome b566 a hypothetical model of the respiratory chain of M . lysodeikticus was developed, which includes the branching of the electron flux to two terminal pathways and redox equivalent transport cyclization via the menaquinone cycle . The physiological role of uncoupled respiration through cytochrome omicron is discussed.

Ukr Biokhim Zh, 1983 Sep-Oct, 55(5), 483 - 8
{Accumulation of one of the histone H1 subfractions and increase in chromatin resistance to the nuclease effect in the course of carp and grass carp spermatogenesis}; Kadura SN et al.; Chromatin of immature testes and sperma of grass carp and carp was hydrolyzed by micrococcal nuclease and DNA I . The hydrolysis kinetics was studied . Chromatin of sperm maintains the nucleosomal repeat typical to somatic cells and is more resistant to the nuclease effect than chromatin of testes . Histones H1 and H2B of grass carp differ from the respective histones of the calf thymus . The core histones of sperm testes and liver chromatin in grass carp and carp have similar electrophoretic mobility . However the amount of slowly moving subfraction of histone H1 increases in the course of spermatogenesis . The amino acid composition of histone H1 from the carp sperm is characterized by a high lysine (34.6%) and low glycine (4.5%) content . A histone H1 molecule contains one tyrosine . The lysine/arginine ratio is 21.6 which is considerably higher than in H1-like histones of sperma in other species (for instance of Echinodermata).

Eur J Biochem, 1983 Sep 1, 135(1), 61 - 8
A set of non-histone proteins isolated from the nuclei of various rat tissues; Inoue A et al.; A set of non-histone proteins has been identified in the nuclei from liver, brain, spleen and testis tissues of the rat . Following moderate digestion of thoroughly washed nuclei with DNase I or micrococcal nuclease, EDTA was added to 5 mM to the reaction mixture and the preparation centrifuged . We found that the supernatant contained a limited amount of non-histone proteins (fraction S1) . Sodium dodecyl sulfate (SDS) gel electrophoresis revealed S1 to be composed of a remarkably simple set of proteins resolved into four groups (A-D) each possessing closely spaced doublets or a triplet . Their molecular weights were A, 76 100-80 000; B, 48 200-49 500; C, 44 500-45 200 and D, 39 500-41 500 . The yield suggested that these proteins were structural constituents; however, they did not coincide with the known structural proteins of the cell nucleus . Two-dimensional gel electrophoresis further resolved each of the SDS bands into as many as nine spots, according to various charges . Some were labelled with {32P}orthophosphate in vivo, or with {gamma-32P}ATP and purified nuclear protein kinase NII in vitro . The released proteins B-D had fairly constant relative molar ratios at various times of digestion, thereby indicating possible localizations at similar sites in the nucleus . The kinetic data together with the aggregation property at neutral pH values and the solubility in 5 mM EDTA suggest that proteins B-D constitute a group of proteins that have several common characteristics.

Biochim Biophys Acta, 1983 Aug 31, 724(2), 230 - 40
Immunochemical analysis of Micrococcus lysodeikticus (luteus) F1-ATPase and its subunits; Urban C et al.; The F1-ATPase from Micrococcus lysodeikticus has been purified to 95% protein homogeneity in this laboratory and as all other bacterial F1S, possesses five distinct subunits with molecular weights ranging from 60 000 to 10 000 (Huberman, M . and Salton, M.R.J . (1979) Biochim . Biophys . Acta 547, 230-240) . In this communication, we demonstrate the immunochemical reactivities of antibodies to native and SDS-dissociated subunits with the native and dissociated F1-ATPase and show that: (1) the antibodies generated to the native or SDS-dissociated subunits react with the native molecule; (2) all of the subunits comprising the F1 are antigenically unique as determined by crossed immunoelectrophoresis and the Ouchterlony double-diffusion techniques; (3) antibodies to the SDS-denatured individual delta- and epsilon-subunits can be used to destabilize the interaction of these specific subunits with the rest of the native F1; and (4) all subunit antibodies as well as anti-native F1 were found to inhibit ATPase activity to varying degrees, the strongest inhibition being seen with antibodies to the total F1 and anti-alpha- and anti-beta-subunit antibodies . The interaction of specific subunit antibodies may provide a new and novel way to study further and characterize the catalytic portions of F1-ATPases and in general may offer an additional method for the examination of multimeric proteins.

Carbohydr Res, 1983 Aug 16, 120, 159 - 70
Biosynthesis of a D-glucosyl polyisoprenyl diphosphate in particulate preparations of Micrococcus lysodeikticus; Yamazaki T et al.; Particulate fractions of Micrococcus lysodeikticus incubated with UDP-D-{14C}glucose incorporated radioactivity into a chloroform - methanol-soluble, low-mol . wt . compound, and into a polymer . The low-mol . wt . compound consisted of a glucolipid that was extremely labile to mild acid hydrolysis with the formation of D-{14C}glucose, and to mild alkali, yielding 14C-labeled alpha-D-glucopyranose 1,2-phosphate and D-glucose 2-phosphate . The labeled glucolipid was eluted from a DEAE-cellulose column at a salt concentration higher than that required by synthetic ficaprenyl (D-glucopyranosyl phosphate), and it migrated more slowly than the latter compound in t.l.c . Formation of the glucolipid was stimulated by exogenous ficaprenyl phosphate, but not by C55-dolichyl phosphate . These results suggest that the {14C}glucolipid has the characteristic properties of a polyisoprenyl glucosyl diphosphate.

Nature, 1983 Aug 11-17, 304(5926), 552 - 4
Recombination of parent and daughter strand DNA after UV-irradiation in mammalian cells; Fornace AJ Jr; The mechanism by which mammalian cells replicate DNA containing pyrimidine dimers is poorly understood . When DNA synthesis is initiated after UV-irradiation in bacteria, parental DNA containing pyrimidine dimers has been shown to 'exchange' into the daughter strand DNA by a recA-dependent mechanism . In earlier experiments, when growing mammalian cells were UV-irradiated and then incubated with labelled thymidine, pyrimidine dimers were initially interpreted to be in the newly synthesized DNA, but later were found to be only adjacent to newly synthesized DNA in daughter strand present before UV treatment . A way to avoid these problems of interpretation would be to use cells in G0 or G1 which are not synthesizing DNA at the time of irradiation . UV damage could then be detected in a very sensitive quantitative assay such as that recently described using alkaline elution and an endonuclease preparation from Micrococcus luteus . I have now used this approach and report that up to 3 dimers per 10(9) daltons of daughter strand DNA could be detected 34-45 h after UV-irradiation in human peripheral blood lymphocytes (PBL), normal human fibroblasts, group A xeroderma pigmentosum (XP) fibroblasts and mouse 3T3 cells . This represents approximately 1-3% of the dimers present in the parent strand at this time after UV.

FEBS Lett, 1983 Aug 8, 159(1-2), 251 - 5
Distribution of nucleosomes on reconstituted chromatin from cloned mouse beta-globin DNA; Nose K et al.; Relative abundance of nucleosomes on reconstituted chromatin was estimated with cloned mouse beta-globin gene DNA . Mononucleosomal DNA was isolated from reconstituted chromatin after digestion with micrococcal nuclease, nick-translated and used as a probe for blot hybridization . DNA fragments of restriction nuclease-digested globin DNA were transferred to DBM-paper and hybridized with mononucleosomal {32P} DNA probe . The results showed non-random distribution of nucleosomes.

Biochemistry, 1983 Aug 2, 22(16), 3873 - 84
Structure of chromatin at deoxyribonucleic acid replication forks: nuclease hypersensitivity results from both prenucleosomal deoxyribonucleic acid and an immature chromatin structure; Cusick ME et al.; Relative to nonreplicating DNA in mature simian virus 40 (SV40) chromosomes, newly synthesized DNA in replicating SV40 chromosomes was found to be hypersensitive to the nonspecific endonucleases, micrococcal nuclease (MNase), DNase I, and DNase II . Nascent DNA, pulse labeled in either intact cells or nuclear extracts supplemented with cytosol, was digested about 5-fold faster and about 25% more extensively than uniformly labeled DNA in mature viral chromosomes . Pulse-chase experiments in vitro revealed a time-dependent chromatin maturation process that involved two distinct steps: (i) conversion of prenucleosomal DNA (PN-DNA) into immature nucleosomal oligomers and (ii) maturation of newly assembled chromatin into a structure with increased nuclease resistance . PN-DNA was hypersensitive to MNase, releasing short DNA fragments which were subsequently solubilized by the nuclease . However, when the nascent PN-DNA was specifically removed by digestion of replicating viral chromosomes with Escherichia coli exonuclease III (3'-5') and phage T7 exonuclease (5'-3'), subsequent digestion of the remaining chromatin with MNase revealed the same degree of hypersensitivity observed prior to exonuclease treatment . Furthermore, newly assembled nucleosomal oligomers, isolated after a brief MNase digestion of replicating viral chromosomes, were also hypersensitive to MNase relative to oligomers isolated from mature chromosomes . Hybridization analysis of the DNA in these immature oligomers revealed that it originated from both sides of replication forks . Inhibition of DNA polymerase alpha by aphidicolin inhibited conversion of PN-DNA into nucleosomes but did not inhibit loss of nucleosomal hypersensitivity to MNase . In contrast, components in the soluble fraction of the subcellular system ("cytosol") were required for both DNA replication and chromatin maturation . Analysis of the nucleoprotein products from a MNase digestion of replicating and mature SV40 chromosomes failed to detect a change in nucleosome structure that corresponded to the loss of nuclease hypersensitivity . However, the results presented demonstrate that both PN-DNA and newly assembled immature chromatin, present on both arms of SV40 replication forks, contribute to the commonly observed hypersensitivity of newly replicated chromatin to endonucleases.

J Neurochem, 1983 Aug, 41(2), 516 - 23
Chromatin structure in neuronal and neuroglial cell nuclei as a function of age; Berkowitz EM et al.; Nuclei from the cerebral cortices of animals of different ages were separated into neuronal and neuroglial populations . Nuclei from cerebellar neurons were also studied . Using the enzyme micrococcal nuclease as a probe for chromatin structure, we found that the DNA from both neuronal preparations showed a decreased susceptibility to digestion during aging, although the onset of this alteration varies . In addition, both neuronal populations showed dramatic increases in the nucleosome spacing of the chromatin . Cerebral neuronal chromatin has a repeat length (nucleosome core and linker region) of 164 base pairs at 22 days and 11 months, 186 base pairs at 24 months, and 199 base pairs at 30 months . Cerebellar neuronal chromatin has a repeat of 188 base pairs at both 22 days and 11 months, 208 base pairs at 24 months, and 243 base pairs at 30 months . Neuroglial chromatin, on the other hand, showed no change in either accessibility to nuclease or repeat length.

Eur J Biochem, 1983 Aug 1, 134(2), 365 - 70
Some properties of a partially purified inhibitor(s) of protein synthesis from rat-liver mitochondria; Wu JM et al.; We have previously described an inactive inhibitor of protein synthesis from rat liver mitochondria and its activation by brief incubation with N-ethylmaleimide {Wu, J . M . and Ibrahim, N.G . (1980) FEBS Lett . 119, 25-28} . To study the mode of action of the mitochondrial translational inhibitor (MTI), the relative distribution of monosomes and polysomes in rabbit reticulocyte lysates has been analysed by sucrose gradient centrifugation . These studies show that MTI causes a significant decrease in the amount of polysomes with marginal effect on the polysome profile . Under identical experimental conditions, addition of partially purified heme-regulated inhibitor results in a complete disaggregation of polysomes . Studies with micrococcal-nuclease-treated rabbit reticulocyte lysates suggest that the primary target of MTI is the inactivation of globin mRNA with relatively little effect on other components of the translational machinery . The inhibitor also degrades poly(U), vesicular stomatis virus mRNA, reovirus mRNA, but appears to be inactive against poly(A), Escherichia coli 16S rRNA, HeLa cell rRNA or chick embryo DNA . Chromatography of MTI on heparin-agarose results in the resolution of at least two inhibitory activities . The first inhibitory activity (eluted with 250 mM KCl) can be reversed (50-70%) with high concentrations of glucose 6-phosphate or MgGTP (0.7 mM or 3.3 mM) whereas the second inhibitory activity can only be partially reversed with poly(A)-rich RNA.

Biokhimiia, 1983 Aug, 48(8), 1319 - 23
{Dynamic organization of the electron transport chain in bacterial membrane by radiation inactivation}; Zinov'eva ME et al.; A complete cross-linking of proteins in isolated Micrococcus lysodeikticus membranes under effect of glutaric aldehyde causes 50% inhibition of the NADH-oxidase activity . Using the irradiation inactivation procedure, it was demonstrated that the size of the irradiation target for NADH-oxidase coincides with that for NADH-dehydrogenase and makes up to about 50 KD . In glutaric aldehyde-treated membranes the target size for NADH-oxidase is 3 times more than that, i.e . 150 KD . It is assumed that the effective electron transfer is mediated by a carrier assembly united into a supramolecular complex with a terminal life-time . Different assemblies exchange their components due to lateral diffusion of proteins in the membrane, which can account for the small size of the irradiation target for the oxidase activity.

J Gen Microbiol, 1983 Aug, 129 (Pt 8), 2437 - 45
Isolation and properties of strains of Micrococcus (Deinococcus) radiodurans unable to excise ultraviolet light-induced pyrimidine dimers from DNA: evidence for two excision pathways; Moseley BE et al.; A mutant of Deinococcus (formerly Micrococcus) radiodurans (strain 302, mutant in mtcA) sensitive to both the lethal effect of mitomycin C and the mutagenic effect of simple alkylating agents, but having wild-type resistance to UV light, was treated with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine in an attempt to isolate strains deficient in the ability to excise UV-induced pyrimidine dimers . Three strains were isolated that were UV-sensitive, but had wild-type resistance to the lethal effect of methyl methanesulphonate and all were shown to be unable to excise pyrimidine dimers . The three strains UVS9, UVS25 and UVS78 had, in addition to the mutation in mtcA, mutations in loci designated uvsC, uvsD and uvsE, respectively . When the mutant mtcA gene was replaced by its wild-type allele in all three strains they became UV- and mitomycin C-resistant . On incubating the double mutants UVS9, UVS25 and UVS78 with wild-type DNA about 50% of the transformants selected for UV resistance were mitomycin C-sensitive and about 50% resistant depending on whether the mutant mtcA or the uvsC, D or E genes had been replaced by their wild-type alleles . Although strains mutant singly in uvsC, D or E were UV-resistant the rates of excision of pyrimidine dimers differed between them and was slower in all of them than in the wild-type and strain 302 . The results indicate that wild-type D . radiodurans possesses two pathways for the excision of pyrimidine dimers and that mutational blocks in both must exist for the excisionless phenotype to be expressed.

Gene, 1983 Aug, 23(2), 175 - 83
A simple procedure for parallel sequence analysis of both strands of 5'-labeled DNA; Razvi F et al.; Ligation of a 5'-labeled DNA restriction fragment results in a circular DNA molecule carrying the two 32Ps at the reformed restriction site . Double digestions of the circular DNA with the original enzyme and a second restriction enzyme cleavage near the labeled site allows direct chemical sequencing of one 5'-labeled DNA strand . Similar double digestions, using an isoschizomer that cleaves differently at the 32P-labeled site, allows direct sequencing of the now 3'-labeled complementary DNA strand . It is possible to directly sequence both strands of cloned DNA inserts by using the above protocol and a multiple cloning site vector that provides the necessary restriction sites . The simultaneous and parallel visualization of both DNA strands eliminates sequence ambiguities . In addition, the labeled circular molecules are particularly useful for single-hit DNA cleavage studies and DNA footprint analysis . As an example, we show here an analysis of the micrococcal nuclease-induced breaks on the two strands of the somatic 5S RNA gene of Xenopus borealis, which suggests that the enzyme may recognize and cleave small AT-containing palindromes along the DNA helix.

Proc Natl Acad Sci U S A, 1983 Aug, 80(15), 4609 - 13
(2'-5')Oligoadenylate in rat liver: modulation after partial hepatectomy; Etienne-Smekens M et al.; (2'-5')Oligoadenylate synthetase {(2'-5')A synthetase}, which synthesizes a series of oligoadenylates ppp-(A2'p)n5'A {collectively referred to as (2'-5')A}, has been described previously in rat liver cells, where its concentration varied with the growth status of this organ--i.e., it decreased during the early phase of rat liver regeneration after partial hepatectomy . Because double-stranded RNA, the only known activator of this enzyme, has been detected in rat liver nuclei, (2'-5')A synthesis could occur in this tissue in vivo . Analysis of rat liver cell extract after HPLC by the endonuclease-based radiobinding assay revealed several components with retention times similar to (2'-5')A trimer- and tetramer-like material . A further characterization of these compounds by their susceptibility to alkaline phosphatase and snake venom phosphodiesterase, their resistance to micrococcal nuclease, and their ability to activate an endonuclease indicated the natural occurrence of oligonucleotides indistinguishable from authentic (2'-5')A in rat liver cells . Using the combination of the radiobinding assay and a simplified (2'-5')A extraction procedure that does not involve HPLC, we further show that the early drop of (2'-5')A synthetase activity during rat liver regeneration was accompanied by a similar decrease in intracellular (2'-5')A concentration . The three characteristic phases of the (2'-5')A synthetase kinetics during the first 40 hr of liver regeneration were mimicked by the kinetics of the synthesis of the (2'-5')A oligonucleotides themselves: between 6 and 20 hr after hepatectomy, there was a sharp decrease in (2'-5')A concentration; between 20 and 24 hr, the concentration of (2'-5')A reached a minimum; at 36 hr or after the first wave of DNA synthesis (the major event of liver regeneration), the (2'-5')A concentration returned to normal . In this characterization of the (2'-5')A oligonucleotide family in a functional tissue of an animal that had not been previously treated with interferon or infected with virus, the data are compatible with a physiological role of the (2'-5')A system acting as an intracellular component of the regulatory mechanisms leading to cell proliferation or differentiation.

FEBS Lett, 1983 Jul 25, 158(2), 281 - 4
Transcribing chromatin is not preferentially enriched with acetylated histones; Yukioka M et al.; Chromatin fragments of the RNA polymerase II-transcriptional complex were purified from the micrococcal nuclease digest of rat liver nuclei in the presence of n-butyrate, a potent histone deacetylase inhibitor . Polyacrylamide gel electrophoretic analysis in Triton acid-urea revealed that the extent of histone acetylation of the complex did not differ markedly from that of the total chromatin.

J Biol Chem, 1983 Jul 25, 258(14), 8554 - 60
Association of 1,25-dihydroxyvitamin D3 with cultured 3T6 mouse fibroblasts . Cellular uptake and receptor-mediated migration to the nucleus; Pike JW et al.; The uptake of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) by intact cells was investigated using the cultured embryonic 3T6 mouse fibroblast as a model . Suspended cells, incubated for 60-90 min in serum-containing culture medium supplemented with 1,25-(OH)2D3 (2 nM), maximally accumulate hormone which becomes bound to a typical vitamin D 3.3 S receptor protein . Incubation of cells with varying concentrations of 1,25-(OH)2D3 reveals the presence of 21,000 receptor molecules/3T6 cell, with an apparent uptake constant of 6-8 X 10(-10) M at 37 degrees C . This value contrasts with the equilibrium dissociation constant (Kd) for 1,25-(OH)2D3 binding of 6 X 10(-11) M as determined at 2 degrees C in disrupted cell cytosol . The distribution of unoccupied (R0) receptors is predominantly (greater than 85%) cytosolic in the hormone-deprived state (1,25-(OH)2D3 less than 0.05 nM), whereas exposure to 1,25-(OH)2D3 (2 nM) leads to almost complete nuclear localization of the occupied receptor at both 2 and 37 degrees C . This phenomenon was similarly supported through reconstitution of receptor and purified 3T6 nuclei in vitro in which binding also occurs at 2 degrees C . The majority (65%) of intact cell-formed receptor-nuclear complexes can be solubilized by micrococcal nuclease treatment, suggesting the participation of DNA in the acceptor binding site for the 1,25-(OH)2D3 receptor . Consistent with these data, DNA-binding of receptor also occurred in vitro at 2 degrees C and was a characteristic of both occupied (Rs) and unoccupied receptors . However, elution of the latter occurred at reduced ionic strength, implying that the hormone does physically alter the receptor protein . This binding was also sensitive to prior ethidium bromide saturation of DNA-cellulose, but not phosphocellulose . Although the biologic effects of the 1,25-(OH)2D3 hormone in 3T6 fibroblasts are as yet unknown, the present findings support previous work with 1,25-(OH)2D3 receptors and suggest that this cell represents a good model for the study of nuclear events associated with the molecular action of 1,25-(OH)2D3.

Arch Biochem Biophys, 1983 Jul 15, 224(2), 718 - 27
The effect of growth temperature on the membrane lipid environment of the psychrophilic bacterium Micrococcus cryophilus; Foot M et al.; The relationship between the delta 9-desaturase activity of the psychrophilic bacterium Micrococcus cryophilus grown at different temperatures and the physical state of its membrane lipids as measured by ESR spectroscopy has been studied . Arrhenius plots of desaturase activity were biphasic with a discontinuity at a temperature which depended upon the bacterial growth temperature . Changes in the desaturase activation energy, which increased as the growth temperature was lowered, are discussed in the context of membrane lipid fluidity adaptation to changing environmental temperature . The fluidity of membranes and isolated lipids was measured using nitroxide-labeled fatty acids . The spectra of 2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinoxyl in membranes indicated that there were two lipid environments within the membrane whose relative proportions were dependent both on temperature of measurement and on bacterial growth temperature . In contrast, 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinoxyl spectra showed a single lipid environment and plots of log order parameter (S3) vs 1/T were biphasic with inflexion temperatures which were closely related to the bacterial growth temperature . As with membranes, plots of log S3 vs 1/T for total lipids, phosphatidylglycerol and cardiolipin, but not phosphatidylethanolamine, were biphasic and showed inflexions which correlated well with bacterial growth temperature . These results are interpreted as being consistent with a location for the desaturase within the bulk lipid of the membrane rather than in association with specific lipid types.

Nucleic Acids Res, 1983 Jul 11, 11(13), 4287 - 306
Eight different highly specific nucleosome phases on alpha-satellite DNA in the African green monkey; Zhang XY et al.; The question of nucleosome phasing on African Green Monkey (AGM) alpha-satellite DNA has been addressed by employing a new approach . Nucleosome cores were prepared from AGM nuclei with micrococcal nuclease, exonuclease III and nuclease S1 . The core DNA population derived from alpha-satellite DNA containing chromatin was purified from total core DNA by denaturation of the DNA, reassociation to a low Cot value, and hydroxyapatite chromatography to separate the renatured satellite fraction . After end-labeling the termini of the alpha-satellite containing core DNA fragments were mapped by high resolution gel electrophoresis relative to known restriction sites along the 172 bp repeat unit of the satellite DNA . The results show that nucleosomes occupy eight strictly defined positions on the alpha-satellite DNA which could be determined with an accuracy of +/- 1 base pair . Approximately 35% of all nucleosomes are organized in one of these frames while the other seven registers contribute about 10% each.

Nucleic Acids Res, 1983 Jul 11, 11(13), 4275 - 85
Sequence specific cleavage of African green monkey alpha-satellite DNA by micrococcal nuclease; Horz W et al.; The sequence specificity of micrococcal nuclease complicates its use in experiments addressed to the still controversial issue of nucleosome phasing . In the case of alpha-satellite DNA containing chromatin from African green monkey (AGM) cells cleavage by micrococcal nuclease in the nucleus was reported to occur predominantly at only one location around position 126 of the satellite repeat unit (Musich et al . (1982) Proc . Natl . Acad . Sci . USA 79, 118-122) . DNA control experiments conducted in the same study indicated the presence of many preferential cleavage sites for micrococcal nuclease on the 172 bp long alpha-satellite repeat unit . This difference was taken as evidence for a direct and simple phase relationship between the alpha-satellite DNA sequence and the position of the nucleosomes on the DNA . We have quantitatively analyzed the digestion products of the protein-free satellite monomer with micrococcal nuclease and found that 50% of all cuts occur at positions 123 and 132, 5% at position 79, and to a level of 1-3% at about 20 other positions . We also digested high molecular weight alpha-satellite DNA from AGM nuclei with micrococcal nuclease . Again cleavage occurred mostly at positions 123 and 132 of the satellite repeat unit . Thus digestion of free DNA yields results very similar to those reported by Musich et al . for the digestion of chromatin . Therefore no conclusions on a possible phase relationship can be drawn from the chromatin digestion experiments.

Cell, 1983 Jul, 33(3), 843 - 8
Major changes in the 5' and 3' chromatin structure of sea urchin histone genes accompany their activation and inactivation in development; Bryan PN et al.; The major histone gene repeat (h22) of the sea urchin Psammechinus miliaris is transiently expressed for several hours during early embryonic development . Several major alterations in chromatin structure coincide with changes in the pattern of early histone gene expression and are reversed later . During the early (128-cell) blastula stages when h22 DNA is maximally expressed, promoter regions of all five histone genes are sensitive to both micrococcal nuclease and DNAase I . Hypersensitivity to micrococcal nuclease remains the same throughout the early hours of development, but disappears abruptly at hatching blastula stage when transcription has completely ceased . Some sequences near the 3' end of active histone genes are very resistant to micrococcal nuclease cutting and map just downstream of the inverted DNA repeats essential for generating faithful 3' ends of histone mRNAs . These same histone DNA sequences are readily cut in free DNA or when histone genes are transcriptionally inactive.

Biochem Pharmacol, 1983 Jul 1, 32(13), 2089 - 94
Interaction between a 3-nitrobenzothiazolo (3,2-a) quinolinium antitumour drug and deoxyribonucleic acid; Baez A et al.; The interaction of 3-nitrobenzothiazolo (3,2-a) quinolinium (NBQ) perchlorate with DNA was studied by u.v.-visible and fluorescence spectrophotometry as well as by hydrodynamic methods . On binding to DNA, the absorption spectrum underwent bathochromic and hypochromic shifts, and the fluorescence was quenched . Binding parameters, determined from spectrophotometric measurements by Scatchard analysis according to an excluded-site model, indicated a binding constant of 2.4 X 10(5)M-1 for calf thymus DNA at ionic strength 0.01 . The interaction was markedly suppressed by increasing the salt concentration . Binding to the GC-rich DNA of Micrococcus lysodeikticus was weaker than the binding to calf thymus DNA at ionic strength 0.01 NBQ increased the viscosity of sonicated rod-like DNA fragments, producing a calculated increment in length of 2.4 A/bound drug molecule . It removed and reversed the supercoiling of closed circular duplex plasmid pBR322 DNA by virtue of a helix-unwinding angle estimated as approximately 13 degrees/bound ligand molecule . We conclude that the binding of NBQ to DNA occurs by a mechanism of intercalation, which probably accounts for its reported antitumor activity.

Radiat Res, 1983 Jul, 95(1), 205 - 10
Effect of cordycepin(3'-deoxyadenosine) on excision repair of 5,6-dihydroxy-dihydrothymine-type products from the DNA of Micrococcus radiodurans; Patil MS et al.; Cordycepin(3'-deoxyadenosine), a nucleoside analog, has been shown to enhance radiation-induced cell killing . In an effort to elucidate the possible mechanism for enhancement of cell killing, the effect of cordycepin on the excision repair of radiation-induced 5,6-dihydroxy-dihydrothymine-type (t') products from the DNA of wild type Micrococcus radiodurans was investigated . The capacity of M . radiodurans to excise nondimeric (t') products from its DNA was significantly impaired after cordycepin treatment . The results suggest that the increased radiation sensitivity of cordycepin-treated cells could be due to alterations in cellular processes that repair DNA damage.

Mol Immunol, 1983 Jul, 20(7), 753 - 61
Structural correlates to the rabbit immunoglobulin heavy chain a100 allotype; Tonnelle C et al.; Three a100/a100 homozygous rabbits immunized with Micrococcus lysodeikticus produced large amounts of anti-polysaccharide antibodies of restricted heterogeneity . These antibodies were purified by either immunoabsorption or ion exchange chromatography . The almost complete sequence of one heavy chain spanning residues 1-123, with the exception of 10 residues (66-67 and 79-86), was determined . Partial sequence data were also obtained for the two other heavy chains . The identity of these three sequences in the first framework region unraveled a prototype sequence of the a100 allotype that differs from homologous sequence of VH regions that determine other allotypic specificities . The gradient of sequence conservation was found to be a100 greater than a3 greater than a1 greater than a- greater than a2 . Homologies in sequence paralleled previously described serological cross-reactions observed between a100, a3 and a1 specificities . This remarkable conservation of framework residues suggests that the VH regions of the rabbit immunoglobulins represent a paucigene system, in which each basic allotypic specificity might be encoded a discrete subgroup of genes.

Mol Pharmacol, 1983 Jul, 24(1), 97 - 102
The effects of the antitumor protein auromomycin on HeLa S3 nuclei . Release of soluble chromatin; Rauscher F 3rd et al.; The protein antitumor agent auromomycin releases chromatin from intact HeLa S3 nuclei by introducing strand scissions in DNA linker regions . The ability of auromomycin to solubilize nuclear chromatin was compared with the well-characterized action of micrococcal nuclease . Production of acid-soluble material (DNA 10-20 base pairs or less) did not increase significantly over a range of drug levels or incubation times . Release of chromatin from auromomycin-treated nuclei was not affected by EDTA but required dithiothreitol for optimal activity . The soluble chromatin, when resolved by agarose gel electrophoresis, consisted of discrete nucleosome bands ranging from monomer to hexamer size and unresolved higher molecular weight oligomers . Sufficiently high concentrations of drug convert most of the higher molecular weight oligonucleosomes to dimer and monomer material . These nucleosome bands were quite broad and could indicate strand scission occurring at random throughout the linker region . The average size of the drug-generated mononucleosomes from multiple experiments was 175 +/- 4 base pairs . In contrast to micrococcal nuclease, higher concentrations of drug did not degrade mononucleosomes to core particles . Solubilized chromatin is detectable following drug treatments as short as 5 min . Chromatin produced at early time points was mostly small and very heterogeneous . Chromatin from drug-digested nuclei analyzed under denaturing conditions appeared as discrete bands similar to those observed on native gels . The ratio of single- to double-stranded breaks may be close to 2:1 . When chromophore isolated from the holantibiotic was incubated with nuclei, a similar pattern of chromatin degradation was observed.

Nucleic Acids Res, 1983 Jun 25, 11(12), 4065 - 75
Histone hyperacetylation has little effect on the higher order folding of chromatin; McGhee JD et al.; HeLa cells were grown in the presence of 10 mM sodium butyrate and soluble chromatin containing hyperacetylated histones was prepared by mild micrococcal nuclease digestion and sucrose gradient fractionation . Sedimentation and electric dichroism were used to study the cation-induced folding of this acetylated chromatin from the 10 nm filament to the 30 nm solenoid conformation . Although under some conditions acetylated chromatin appears slightly less condensed than control chromatin, the major conclusion is that hyperacetylation of histones does not in itself prevent the formation of the higher order chromatin solenoid.

J Biol Chem, 1983 Jun 25, 258(12), 7623 - 30
Isolation of 3 S androgen receptors from salt-resistant fractions and nuclear matrices of prostatic nuclei after mild trypsin digestion; Rennie PS et al.; The physical properties of two types of androgen-binding sites in prostatic nuclei were compared and found to be identical . The first type was released from chromatin by micrococcal nuclease digestion and solution in 0.6 M NaCl; the second resisted such treatment and remained associated with nuclear structures . After in vivo administration of {1,2-3H}testosterone to 24-h castrated rats and sonication of purified nuclei, 90% of the nuclear radioactivity was extracted with nuclease/salt treatment and was found by sucrose density gradient analysis to be associated with a 3 S androgen receptor . If sonication was omitted, 50 to 60% of the nuclear radioactivity was recovered in the nuclease/salt-resistant pellets or bound to nuclear matrices . Mild digestion of either of these particulate fractions with trypsin resulted in the release of a 3 S androgen receptor . After in vitro isotope-exchange labeling with {1,2-3H}dihydrotestosterone, the sedimentation coefficient, steroid specificity, and dissociation constant of the androgen receptors released by trypsin digestion of nuclease/salt-resistant pellets or nuclear matrices were similar to those of the receptors extracted by nuclease/salt treatment . These results indicate first, that all androgen-binding sites in prostatic nuclei can be released, either with nuclease/salt or trypsin digestion procedures to yield a 3 S androgen receptor with uniform binding characteristics, and second, that the androgen receptors are distributed between two intra-nuclear pools--one containing about 10,000 molecules/nucleus sensitive to micrococcal nuclease digestion and salt and the other containing about 8,000 to 13,000 androgen receptors tightly bound to the nuclear matrix.

Nucleic Acids Res, 1983 Jun 25, 11(12), 4035 - 47
A destabilized DNA conformation associated with tightly bound nuclear proteins in active genes of rat myoblast; Leibovitch SA et al.; Purified nuclei from tissue cultured myoblasts were disrupted and centrifuged to equilibrium in a sarcosyl-caesium chloride gradient . A small portion (1.3% - 1.9%) of the non histone proteins (NHP) were banded with DNA in a high density region of the gradient . The DNA tightly bound to proteins representing about 0.6% of the total nuclear DNA was degraded after treating cell nuclei with S1 nuclease or DNAse I but resisted to mild micrococcal nuclease digestion . A large portion of the DNA sequences complementary to homologous RNA was concentrated in this DNA-proteins fraction . These finding suggest that a subset of NHP strongly associated to the active DNA regions play a role in the destabilisation of the double helical DNA during transcriptional processes.

Biochim Biophys Acta, 1983 Jun 24, 740(2), 179 - 84
Effects of thyrotropin on thyroid chromatin . Enhanced sensitivity to micrococcal nuclease and increased nuclear protein phosphorylation; Cooper E et al.; Thyroid slices were incubated with or without TSH for 2 or 5 h . Nuclei were then prepared, subjected to mild digestion with micrococcal nuclease, and centrifuged at 1200 X g . The amount of DNA in 1200 X g supernatants was increased by TSH at 5 h, but not at 2 h . In parallel studies, thyroid slices were incubated with 32Pi and labeling of acid-soluble nuclear proteins was examined . TSH-dependent increases in labeling of histones H1 and H3, and of the high mobility group protein HMG 14, were observed at 2 h; however, there were no apparent changes in TSH-dependent labeling between 2 and 5 h, in nuclease-sensitive or in bulk chromatin . These results suggest that the observed TSH-dependent changes in the micrococcal nuclease-sensitivity of thyroid nuclear chromatin were not induced directly by changes in the phosphorylation of the histones or HMG 14.

Biochim Biophys Acta, 1983 Jun 24, 740(2), 134 - 44
Nuclease digestibility of chromatin is affected by nuclei isolation procedures; Prentice DA et al.; Experiments using nucleases as probes of chromatin structure take place in two stages: (1) nuclei isolation, and (2) nuclease digestion . The parameters of the nuclease digestion stage are usually strictly controlled because of nuclease sensitivity to them . However, there have been no reports on whether parameters in the nuclei isolation stage affect the subsequent nuclease digestions . We have evaluated a typical nuclei isolation technique with respect to how changes in the isolation parameters affect nuclease digestion kinetics . Our observations point out that various parameters encountered in the nuclei isolation stage have a significant effect on the subsequent nuclease digestion kinetics of DNAase I . These parameters include the concentration of cells, divalent cations and phosphatase inhibitors . The pH, concentration of NaCl and concentration of detergent had little effect . Micrococcal nuclease was relatively unaffected by changes in the nuclei isolation parameters . The importance of this report lies in the demonstration that lack of control of seemingly insignificant parameters, such as cell concentration during the nuclei isolation stage, leads to subsequent irreproducible results in the DNAase I digestion . These findings indicate that great care must be exercised in the nuclei isolation stage if reproducible work is to be performed with DNAase I.

J Mol Biol, 1983 Jun 15, 167(1), 133 - 55
Varigated chromatin structures of mouse ribosomal RNA genes; Davis AH et al.; We have employed a chromatin fractionation procedure on micrococcal nuclease-digested nuclei to examine the chromatin structure of mouse ribosomal RNA genes in two systems that differ by at least 14-fold in the level of ribosomal RNA transcription . In a cultured cell line enriched in transcriptionally active ribosomal chromatin, most ribosomal sequences are preferentially sensitive to digestion by micrococcal nuclease, reside in an insoluble chromatin fraction, and lack typical nucleosomal packaging; only minor amounts of ribosomal sequences are packaged into soluble, nucleosomal chromatin . By contrast, in adult liver, which is enriched in transcriptionally inactive ribosomal chromatin, the majority of ribosomal genes are packaged into soluble, nucleosomal chromatin . However, a significant fraction of liver ribosomal chromatin is insoluble and possesses a non-nucleosomal structure . Therefore, within a single cell population or tissue, mouse ribosomal RNA genes are organized into both nucleosomal and non-nucleosomal chromatin structures . We suggest that these structures have functional significance.

J Mol Biol, 1983 Jun 15, 167(1), 1 - 21
Characterization of satellite DNA in Trypanosoma brucei and Trypanosoma cruzi; Sloof P et al.; We have determined the properties of the simple-sequence satellite DNAs from two protozoa, Trypanosoma brucei and Trypanosoma cruzi . The T . brucei satellite DNA contains 29 mol% guanine plus cytosine and is made up of long tandem arrays of a 177 base-pair repeat . Sequence heterogeneity in these repeats is limited and restricted to certain positions as shown by sequence analysis, restriction enzyme digestion and two-dimensional analysis of nucleotides bordering the AluI and HhaI recognition sites in the repeat . The repeat contains two copies of a 19 base-pair sequence differing by a single base-pair substitution and several additional copies of part of this sequence . Sequence variants of the repeat are clustered in the DNA . Satellite DNA is not detectably linked to other DNA and no transcripts of this DNA are found in T . brucei . The T . cruzi satellite DNA repeat is 196 base-pairs long and contains 53 mol% guanine plus cytosine . Direct repetitions longer than eight base-pairs were not observed in the nucleotide sequence of this repeat . The nucleotide sequences of the satellites of T . brucei and T . cruzi are not related . In cell fractionation experiments, the T . brucei and T . cruzi satellite DNAs were recovered from the nuclear fraction . Micrococcal nuclease digestion of nuclear fractions yielded 193 and 197 base-pair nucleosomal oligomers in T . brucei and T . cruzi, respectively; these oligomers contained satellite DNA but not the extranuclear kinetoplast DNA . The 193 base-pair nucleosomal repeat of T . brucei is significantly different from the 177 base-pair satellite repeat . Satellite and nucleosomal repeats are, therefore, not in phase in T . brucei . These satellite DNAs are the first to be observed in protozoa, and we conclude that their properties are similar to those of satellites from animals or plants.

Nucleic Acids Res, 1983 Jun 11, 11(11), 3717 - 36
The organization of oligonucleosomes in yeast; Szent-Gyorgyi C et al.; We have developed a method of preparing yeast chromatin that facilitates the analysis of nucleoprotein organization . Yeast chromatin, isolated as an insoluble complex, is digested with micrococcal nuclease and fractionated into major insoluble and soluble fractions . No nucleosomal repeat is seen early in digestion for the insoluble fraction . Nucleosomal complexes of the soluble fraction are excised by nuclease in a distinctive non-random pattern; they are markedly depleted in mononucleosomes . When we analyze the soluble material by high resolution native electrophoresis, we find that the nucleoproteins resolve into two bands for each DNA multimer of the nucleosomal repeat . Our results suggest that there are structural similarities between bulk yeast chromatin and chromatin configurations found in transcribing genes of complex eukaryotes.

Nucleic Acids Res, 1983 Jun 11, 11(11), 3593 - 612
Protection of expressed immunoglobulin genes against nuclease cleavage; Weischet WO et al.; Fragmentation of the actively transcribed kappa immunoglobulin gene in mouse myeloma nuclei with micrococcal nuclease and the restriction nuclease Bsp RI reveals a chromatin structure without the regularity of repeating nucleosomes found in bulk chromatin . Such regularity is restored about 2.2 kb 3' of the coding region . An only moderately increased micrococcal nuclease sensitivity and a 65% average protection of the Bsp RI sites indicates a DNA-protein interaction in the transcribed region which is not very different from that of an inactive gene . As determined by indirect endlabeling the frequency of Bsp RI cleavage both, after very mild and exhaustive digestion, varied moderately from site to site along the gene . In addition, it was not in each case the same at analogous sites on both alleles which are both transcribed . Thus, the experiments demonstrate differences between the chromatin structures of the genes which may be related to regulatory phenomena and thereby corroborate earlier findings made with DNAase I.

J Biol Chem, 1983 Jun 10, 258(11), 7223 - 7
Modulation of chromatin structure associated with derepression of the acid phosphatase gene of Saccharomyces cerevisiae; Bergman LW et al.; We have analyzed the chromatin structure of a phosphate-repressible acid phosphatase gene (PHO5) within yeast nuclei . Under derepressed conditions (low Pi media), the gene is much more sensitive to either DNAse I or micrococcal nuclease digestion than is the repressed gene . We have mapped DNase I hypersensitive sites unique to the active gene near the 5'-end of the acid phosphatase mRNA and within a region presumed to function in the regulation of the gene by Pi . Although the gene is packaged into regularly spaced nucleosomes, no detectable phase relationship exists between nucleosomes and DNA sequence under derepressed conditions, whereas in the repressed state the nucleosomes occur in one predominant phase . These results demonstrate reversible changes in the chromatin structure of a eukaryotic gene system that directly correlate with the functional state of the gene.

Mol Cell Endocrinol, 1983 Jun, 30(3), 267 - 78
Progesterone, glucocorticoid and estradiol receptors in MCF-7 cells bind to chromatin; Sun LH et al.; MCF-7 cells contain progesterone, estradiol and glucocorticoid receptors . Following addition of these hormones to the growth medium of the cells, hormone-receptor complexes were found to sediment with chromatin fragments produced by trace digestion with micrococcal nuclease . The binding in all cases could be competed by excess unlabeled hormone . In each case the fragments with which the hormone-receptor complexes were associated tended to be smaller than the bulk chromatin fragments, indicating a greater sensitivity of those chromatin regions to the nuclease . The mononucleosomes released by more extensive digestion with micrococcal nuclease contained different amounts of each of the three hormone-receptor complexes . Progesterone could usually be detected on mononucleosomes only after very brief sedimentation analyses, whereas glucocorticoid- and estradiol-labeled mononucleosomes were stable during long centrifugations . Comparison of glucocorticoid- and estradiol-labeled mononucleosomes indicated that their sedimentation rates differed from one another and from bulk nucleosomes . Estradiol nucleosomes from MCF-7 cells and rat uterus (Senior and Frankel, 1978) sediment significantly faster than bulk nucleosomes, while glucocorticoid nucleosomes from MCF-7 cells and rat hepatoma cells sediment with, or even fractionally slower than, bulk nucleosomes.

Proc Natl Acad Sci U S A, 1983 Jun, 80(11), 3213 - 7
Cleavage of chromatin with methidiumpropyl-EDTA . iron(II); Cartwright IL et al.; Methidiumpropyl-EDTA . iron(II) {MPE . Fe (II)} cleaves double-helical DNA with considerably lower sequence specificity than micrococcal nuclease . Moreover, digestions with MPE . Fe(II) can be performed in the presence of certain metal chelators, which will minimize the action of many endogenous nucleases . Because of these properties MPE . Fe(II) would appear to be a superior tool for probing chromatin structure . We have compared the patterns generated from the 1.688 g/cm3 complex satellite, 5S ribosomal RNA, and histone gene sequences of Drosophila melanogaster chromatin and protein-free DNA by MPE . Fe(II) and micrococcal nuclease cleavage . MPE . Fe(II) at low concentrations recognizes the nucleosome array, efficiently introducing a regular series of single-stranded (and some double-stranded) cleavages in chromatin DNA . Subsequent S1 nuclease digestion of the purified DNA produces a typical extended oligonucleosome pattern, with a repeating unit of ca . 190 base pairs . Under suitable conditions, relatively little other nicking is observed . Unlike micrococcal nuclease, which has a noticeable sequence preference in introducing cleavages, MPE . Fe(II) cleaves protein-free tandemly repetitive satellite and 5S DNA sequences in a near-random fashion . The spacing of cleavage sites in chromatin, however, bears a direct relationship to the length of the respective sequence repeats . In the case of the histone gene sequences a faint, but detectable, MPE . Fe(II) cleavage pattern is observed on DNA, in some regions similar to and in some regions different from the strong chromatin-specified pattern . The results indicate that MPE . Fe(II) will be very useful in the analysis of chromatin structure.

Mutat Res, 1983 Jun, 115(2), 149 - 73
Genetic toxicology of ethylenediaminetetraacetic acid (EDTA); Heindorff K et al.; EDTA and its salts have a number of applications in medicine and pharmacy . EDTA is used to remove calcium from the human body, and serves as an anticoagulant and as a detoxicant after poisoning by heavy metals . It is often used in analytical chemistry for complexometric titrations and many other purposes . Because the compound is of rather low toxicity, it is used as a food additive to bind metal ions . EDTA affects the inhibition of DNA synthesis in primary cultures of mammalian cells . This may be due to impairment of enzymes involved in DNA replication . Some early studies have shown that EDTA leads to morphological changes of chromatin and chromosome structure in plant and animal cells . These alterations consist of dispersion or swelling of chromosomes or a loss of interphase chromatin structure . For several test systems, a low chromosome-breaking activity of EDTA has been reported . A weak activity in the induction of gene mutations has also been observed . It is well established that EDTA influences chromosome breakage by mutagenic agents . In particular, when applied in combination with chemical mutagens, EDTA enhances mutagen-induced aberration frequencies . Furthermore, the chelating agent is able to increase the incidence of meiotic crossing-over . This has been demonstrated for many gene loci in Drosophila melanogaster, Chlamydomonas reinhardi, Neurospora crassa and Zea mays . EDTA interferes with DNA repair processes that take place after exposure to mutagens . In E . coli or Micrococcus radiodurans as well as in Chinese hamster cells, the fast repair component detectable after treatment with ionizing radiation or bleomycin is inhibited by EDTA . In plant cells exposed to gamma-rays, EDTA inhibits unscheduled DNA synthesis . The mechanism by which EDTA causes these effects remains poorly understood . The sequestering of metal ions by the chelating agent is obviously responsible for functional and structural alterations of the genetic material . Although EDTA produces a whole set of genetic effects it seems to be a harmless compound to man as far as genotoxicity is concerned . The data presently at hand, however, are not sufficient for a reliable risk assessment.

Immunology, 1983 Jun, 49(2), 273 - 9
Response of the rabbit VH subgroups a and y following antigenic stimulation; Laster SM et al.; In order to test the relative immune competence of a minor VH subgroup (y), the concentrations of rabbit Ig bearing the VH subgroups a and y were determined in experimentally immunized rabbits . Three groups of three rabbits each were immunized with either Micrococcus luteus, bovine serum albumin or an alloantigenic determinant (b4) of the rabbit kappa light chain . The ratios of VHy to VHa Ig in the pre-immune sera ranged from 1:8 to 1:283 . In five of the nine rabbits, the VHy to VHa ratios in affinity purified antibody (Ab) closely reflected the ratios of VHy to VHa in pre-immune serum . Of the remaining four rabbits, three produced disproportionately low amounts of VHy Ab, when compared to pre-immune values, while one rabbit produced virtually no VHy Ab . Regardless of the levels of VHy and VHa Ab detected, the kinetics of Ab production by each subgroup during the course of the immune response were similar to each other . When total serum levels of the subgroups were analysed, all rabbits displayed increases (6-450%) in total serum levels of both VHa Ig and total IgG following immunization, while in eight out of nine rabbits the level of VHy Ig remained unchanged . Our observations suggest that the immune repertoire of the minor VHy subgroup is sufficiently diverse to generate VH regions which react with a variety of antigens, and in five out of nine rabbits, VHy Ab-producing clones compete well with VHa Ab-producing clones . In addition, it appears that clones of the major and minor VH subgroups, once triggered, are under similar regulatory controls with respect to specific Ab, but differ with respect to non-specific Ig production.

Cell Differ, 1983 Jun, 12(6), 307 - 16
Fractionation by micrococcal nuclease digestion of Drosophila embryo chromatin: isolation of a fraction enriched in two major nonhistone proteins; Guerrero I et al.; Limited digestion of Drosophila melanogaster embryo nuclei with mitochondrial nuclease, followed by selective solubilization in 0.1 M NaCl, yields a soluble nucleoprotein fraction (S3) enriched in two dominant protein bands of apparent molecular weight of 44000 and 48000 . The analysis of the nucleosome monomer and multimer peaks, separated on sucrose gradients after slight digestion with micrococcal nuclease, shows that these proteins are associated with chromatin subunits, and that they are principally found in the subnucleosome region of fraction S3 . This doublet is tentatively identified as a member of the noncanonical HMG of Drosophila . The thermal denaturation and the circular dichroism spectra of the fraction soluble in 0.1 M NaCl (S3) and insoluble fraction (P3) show that both fractions are also structurally different.

Biochem Biophys Res Commun, 1983 May 31, 113(1), 24 - 9
Decreased micrococcal nuclease sensitivity of nuclei from nerve growth factor-treated PC12 cells; Vinores SA et al.; Nuclei from nerve growth factor-treated PC12 cells are more resistant to digestion with micrococcal nuclease than are nuclei from control cells . The production of oligosomal fragments is decreased, as is the generation of Mg2+-soluble products . One interpretation of the data is that differentiation of these cells due to treatment with nerve growth factor involves a decrease in the total number of DNA sequences transcribed.

J Mol Biol, 1983 May 25, 166(3), 361 - 81
Chromatin structure along the ribosomal DNA of Dictyostelium . Regional differences and changes accompanying cell differentiation; Ness PJ et al.; The ribosomal genes of Dictyostelium discoideum are extrachromosomal palindromic DNA molecules situated in the nucleolus . Each molecule comprises ribosomal RNA coding regions and non-transcribed spacer regions . We used both biochemical and electron microscopic approaches to investigate the structure of transcribing and non-transcribing chromatin . Nucleoli from exponentially growing cells were digested with micrococcal nuclease, and the resulting DNA fragments were separated by gel electrophoresis and transferred to DBM paper . They were hybridized with cloned EcoRI fragments derived from different parts of the ribosomal gene . Probes of the coding region showed a smear, while probes of the non-transcribed regions gave pronounced banding patterns more complex than typical nucleosome repeats, but not due solely to sequence-specific cutting by micrococcal nuclease . The DNA of the coding region was digested more quickly than that of the non-transcribed ones . When nucleoli were digested with restriction enzymes, sites within the coding region were accessible and sites in the non-transcribed region were protected . The structure of ribosomal chromatin in differentiating cells, in which the rate of ribosomal RNA synthesis is reduced, was examined using essentially the same methods . The coding region, probed by hybridization to micrococcal digests, then showed a typical DNA repeat pattern indicating that this region had become condensed into nucleosomes, and its accessibility to restriction enzymes was very much reduced . On electron micrographs of lysed nucleoli from exponentially growing cells, two types of chromatin were observed, one with a beaded nucleosomal appearance, the other with putative RNA polymerase molecules attached to fibres indistinguishable from free DNA adsorbed to the same grid . The combined results suggest that whereas regions that are not transcribed are packaged with proteins that protect them from nuclease digestion, actively transcribing ribosomal genes are associated with few macromolecular constituents apart from those required for transcription and its regulation.

Nucleic Acids Res, 1983 May 25, 11(10), 3255 - 67
Mouse hepatic metallothionein-I gene cleavage by micrococcal nuclease is enhanced after induction by cadmium; Koropatnick J et al.; Micrococcal nuclease has been shown to preferentially cleave chromatin in the region of genes actively engaged in transcription . We have used this preferential cleavage to show that the metallothionein (MT) gene in adult mouse liver, when induced to produce mRNA by injection of cadmium, becomes more susceptible to nuclease cleavage . However, the MT gene in uninduced liver, and the alphafoetal protein (AFP) gene in both induced and uninduced liver, remain relatively resistant to nuclease cleavage . The AFP gene is not normally expressed in cadmium induced or uninduced liver . Thus, susceptibility of genes to nuclease cleavage appears to rise with increasing transcription of the gene.

J Mol Biol, 1983 May 15, 166(2), 159 - 81
Structural polymorphism in DNA; Udvardy A et al.; We have used the enzyme micrococcal nuclease and the methylating reagent dimethyl sulfate to examine the structural properties of eukaryotic DNAs . Our studies demonstrate extensive structural polymorphism in the DNA double helix . Moreover, we find that the distribution of helical variants is in some instances correlated with the functional organization of the DNA . These observations raise the possibility that eukaryotic DNAs may be organized into discrete functional units having characteristic structural properties . In addition, we find that boundaries between different functional units are typically marked by DNA segments having unusual conformational properties . Such structural perturbations could serve as signals in the utilization of genetic information in eukaryotes, and may be important in a variety of different protein-DNA interactions.

Nucleic Acids Res, 1983 May 11, 11(9), 2779 - 800
Biochemical characterization of topoisomerase I purified from avian erythrocytes; Trask DK et al.; A type I topoisomerase has been purified from avian erythrocyte nuclei . The most pure fraction contains one major polypeptide of Mr = 105,000 (80% of total) and several minor ones of lower molecular weight . Active forms of the topoisomerase were identified by covalently binding the enzyme to 32P-DNA, digesting with nuclease and detecting 32P labeled peptides by sodium dodecyl sulfate polyacrylamide gel electrophoresis . Topoisomerase activity, as measured by the ability to covalently bind DNA, is associated with the following peptides: Mr = 105, 83, 54 and 30,000 . The similar chromatographic properties of the various forms of topoisomerase suggests a common structural identity as previously proposed for the HeLa topoisomerase I (Liu, L.F . and Miller, K.G . (1981) Proc . Natl . Acad . Sci . USA 78, 3487-3491) . The avian enzyme is similar to other eucaryotic type I DNA topoisomerases in that it covalently binds double and single stranded DNA forming an enzyme linked to the 3'-phosphoryl end and after binding to single stranded DNA it can transfer the single stranded donor DNA to an acceptor DNA possessing 5'-OH end groups . The binding site size of topoisomerase on DNA has also been determined using micrococcal nuclease to digest unprotected DNA in the native enzyme/DNA complex . The enzyme blocks access to the helix over a span of 25 bp . These findings are discussed in light of the distribution and function of topoisomerase I in chromatin.

Can J Microbiol, 1983 May, 29(5), 488 - 96
Studies on the ring-cyclization and ring-expansion enzymes of beta-lactam biosynthesis in Cephalosporium acremonium; Kupka J et al.; Micrococcus luteus was found to be very sensitive to isopenicillin N and was used as assay organism for purification of the enzyme isopenicillin N synthetase, which cyclizes delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N . Purification of the enzyme from the crude extract obtained by sonication of mycelia of Cephalosporium acremonium CW-19 was carried out by ammonium sulfate precipitation, desalting with Sephadex G-25, gel filtration on LKB ultrogel AcA44 or ion-exchange chromatography on DEAE-Sepharose . The cyclization enzyme was separated from the ring-expansion enzyme and was purified considerably more than 50-fold by this procedure . Using the purified enzyme, we found that the disulfide bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine required reduction to delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine in order to behave as a substrate . The enzyme activity was stimulated by FeSO4 and ascorbate, but other cofactors, including alpha-ketoglutarate, were inactive . In addition to delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, the enzyme converted adipyl-L-cysteinyl-D-valine, N-acetyl-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, and glycyl-delta-(L-alpha-aminoadipyl)L-cysteinyl-D-valine to penicillins . All of these latter peptides were competitive inhibitors of the cyclization reaction . The Km of the cyclization enzyme is 10 times higher than that of the ring-expansion enzyme, deacetoxycephalosporin C synthetase . The pH and temperature optima of the two enzymes were rather similar . Phosphate inhibited ring expansion, but not cyclization . Both enzymes appear to be soluble enzymes of about 31 000 molecular weight.

Proc Natl Acad Sci U S A, 1983 May, 80(10), 2874 - 8
Lack of Z-DNA conformation in mitomycin-modified polynucleotides having inverted circular dichroism; Tomasz M et al.; Poly(dG-dC) . poly(dG-dC) and Micrococcus lysodeikticus DNA were modified by exposure to reductively activated mitomycin C, an antitumor antibiotic . The resulting covalent drug-polynucleotide complexes displayed varying degrees of CD inversions, which are strikingly similar to the inverted spectrum observed with Z-DNA . The following criteria have been used to establish, however, that the inverted CD pattern seen in mitomycin C-polynucleotide complexes does not reflect a Z-DNA conformation . (i) The ethanol-induced transition of poly(dG-dC) . poly(dG-dC) from B to Z conformation is not facilitated but rather is inhibited by mitomycin C modification . This may be due to the presence of crosslinks, (ii) Radioimmunoassay indicated no competition for Z-DNA-specific antibody by any of the mitomycin C-modified polynucleotides, (iii) 31P NMR of the complexes yielded a single relatively narrow resonance, which is inconsistent with the dinucleotide repeat characteristic of Z-DNA . Alternative explanations for the inverted CD pattern include a drug-induced left-handed but non-Z conformational change or the superposition of an induced CD onto the CD of B-DNA due to drug-base electronic interactions . These results illustrate the need for caution in interpreting CD changes alone as an indication of Z-DNA conformation.

J Virol, 1983 May, 46(2), 413 - 23
Biochemical mapping of the simian rotavirus SA11 genome; Mason BB et al.; Biochemical mapping experiments of the simian rotavirus SA11 genome were performed to determine which double-stranded RNA segment coded for each of the viral polypeptides . Viral RNA transcripts were synthesized in vitro by using the endogenous viral RNA polymerase and fractionated by electrophoresis in acid-urea agarose gels . The fractionated transcripts were translated in two cell-free systems: micrococcal nuclease-treated reticulocyte lysates and wheat germ extracts . The polypeptide products were identified by polyacrylamide gel electrophoresis and partial peptide analysis and compared with polypeptides synthesized in infected cells or found in purified virus . The RNA segment that coded for each transcript was determined by hybridization of the fractionated transcripts to the double-stranded RNA genome and analysis of the hybrids by electrophoresis in polyacrylamide gels . Primary gene products were assigned for 10 of the rotavirus transcripts and 10 of the double-stranded RNA segments . The coding assignments are as follows: the inner-capsid polypeptides, VP1, VP2, and VP6, were assigned to segments 1, 2, and 6, respectively; the major outer-capsid polypeptides, VP3 and VP7, were assigned to segments 4 and 9, respectively; segments 5, 7, and 8 coded for nonstructural polypeptides with molecular weights of 53,000, 34,000, and 35,000, respectively; segment 10 coded for the 20,000-molecular-weight precursor to the 29,000-molecular-weight glycosylated nonstructural polypeptide; and segment 11 coded for a 26,000-molecular-weight polypeptide that may be the precursor to the minor outer-capsid polypeptide VP9 . Several methods were used to determine the product of gene segment 3, and the problems associated with the identification of this gene product are discussed.

Endocrinology, 1983 May, 112(5), 1816 - 22
Effects of thyrotropin on the phosphorylation of histones and nonhistone phosphoproteins in micrococcal nuclease-sensitive and resistant thyroid chromatin; Cooper E et al.; Actively transcribed regions of chromatin are more susceptible than bulk chromatin to digestion by nucleases, and useful information about the composition and structure of active chromatin may be obtained by studying the chromatin fragments released from nuclei by limited nuclease digestion . In the present study, we have used micrococcal nuclease to investigate the effects of TSH on protein phosphorylation in nuclease-sensitive fractions of calf thyroid chromatin . Batches of calf thyroid slices were incubated for 2 h with 32Pi, with or without 50 mU/ml TSH . Nuclei were then prepared and the distribution of 32P-labeled histones, high mobility group (HMG) proteins, and other acid-soluble phosphoproteins between micrococcal nuclease-sensitive and resistant fractions of chromatin was examined . TSH increased the amount of 32P incorporated into HMG 14 and the histones H1 and H3 . Hormone-dependent increases in the 32P-labeling of H1 and H3 were not selectively associated with micrococcal nuclease-sensitive chromatin . In contrast, {32P} HMG-14 was preferentially solubilized from nuclei by micrococcal nuclease . This lends support to the view that TSH-induced effects on the structure and function of transcriptionally active chromatin may be mediated in part by phosphorylation of HMG 14.

Biochim Biophys Acta, 1983 Apr 15, 739(3), 286 - 90
Estimation of domain length of chicken erythrocyte chromatin; Ganguly A et al.; Chromatin of chicken erythrocyte nuclei was extracted by digestion with micrococcal nuclease . The length distribution of the soluble chromatin was determined by gel electrophoresis and electron microscopy . These results were fitted with a theoretical distribution which was an outcome of the domain model proposed by Igo-Kemenes and Zachau (Igo-Kemenes, T . and H.G . Zachau (1977) Cold Spring Harbour Symp . Quant . Biol . 42, 109-118) . A domain length of 45 kbp was obtained.

Biochemistry, 1983 Apr 12, 22(8), 1783 - 9
Histone H5 can increase the internucleosome spacing in dinucleosomes to nativelike values; Kunzler P et al.; Chicken erythrocyte chromatin was assembled with inner histones at about 60% of the ratio found in vivo and subsequently incubated with histone H5 (or H1 + H5) in a solution containing 0.1 M NaCl and poly(glutamic acid) . Micrococcal nuclease digestion produced dinucleosomes of 360-390 base pair (bp) DNA content, similar to those from native chromatin and contrasting with the 270-280 bp species found in material incubated without H5 . On sucrose gradients a dinucleosome sedimenting at 16 S containing 360 bp DNA was isolated . Removal of H1 + H5 after reconstitution did not change these results; H5 thus can induce rearrangements of nucleosome cores with respect to their neighbors . The results are interpreted as an H5-induced "sliding apart" of histone octamers, complementary to the "sliding together" found in native chromatin after removal of H1 + H5.

Nucleic Acids Res, 1983 Apr 11, 11(7), 2077 - 91
Transcribed and non-transcribed regions of Tetrahymena ribosomal gene chromatin have different accessibilities to micrococcal nuclease; Palen TE et al.; DNA renaturation kinetics was used to examine the relative accessibility of various regions of the Tetrahymena ribosomal RNA gene (rDNA) chromatin to micrococcal nuclease . In nuclei from cells active in rRNA transcription, the transcribed region of the rDNA chromatin was as much as 5-fold more accessible than the average of the total chromatin . As few as 20% inactive genes in the population could have accounted for all of the hybridization, so the transcribed region of the active units may be totally unprotected from nuclease degradation . The terminal non-transcribed spacer downstream from the transcription unit was also preferentially digested, but to a smaller degree . The central non-transcribed spacer was degraded to the same extent as total chromatin after a high degree of nuclease digestion . In nuclei from starved cells, which have 96% reduced rRNA transcription, the transcribed and terminal spacer regions of the rDNA were again more accessible than the total chromatin from the same nuclei, but the difference did not exceed 2-fold . We conclude that transcriptional activation is accompanied by major changes in the structure of the ribosomal gene chromatin, and that the extent and/or type of structural alteration differs in each functionally defined region of the rDNA.

Eur J Biochem, 1983 Apr 5, 131(3), 545 - 50
Translational competition by mRNA species encoding albumin, ferritin, haemopexin and globin; Kaempfer R et al.; Messenger RNA from rat liver was translated in a micrococcal-nuclease-treated reticulocyte lysate supplemented with liver tRNA . Synthesis of the liver proteins haemopexin, ferritin and albumin was analyzed by quantitative immunoprecipitation . The relative translation yield of these proteins changed as a function of the amount of mRNA present during protein synthesis, revealing the existence of translational competition between individual species of mRNA from the liver . The results show that the mRNA species encoding haemopexin, ferritin and albumin possess distinctly different abilities to compete for one or more critical components in translation, with competitive strength increasing in this order . Although on a weight basis total liver mRNA is apparently as effective a template for protein synthesis as is globin mRNA, the latter displays a greater resistance to inhibition of its translation by KCl . In analogy with the translation properties of alpha-globin and beta-globin mRNA {Di Segni, G., Rosen, H . and Kaempfer, R . (1979) Biochemistry, 18, 2847-2854}, this finding suggests that globin mRNA possesses greater competitive strength than does total liver mRNA . Increasing amounts of globin mRNA competitively inhibit the translation of albumin and ferritin mRNA present in total liver mRNA . The competition is relieved by the addition of eukaryotic initiation factor eIF-2 . Translation of ferritin mRNA responds more vigorously to relief by eIF-2 than does translation of albumin mRNA, a finding consistent with the observation that albumin mRNA competes more effectively than ferritin mRNA in translation . The results support the assumption that albumin mRNA possesses a greater affinity for eIF-2 than does ferritin mRNA.

FEBS Lett, 1983 Apr 5, 154(1), 151 - 5
Isolation of a 167 basepair chromatosome containing a partially digested histone H5; Puigdomenech P et al.; A test has been made of the proposal that protection of the 167 basepair DNA length in the 'chromatosome' is due only to the central globular domain of the lysine-rich histones . Chicken erythrocyte chromatin was treated with trypsin to leave only the limit peptide from histones H1 and H5 . Nucleosome monomers were then isolated on sucrose gradients following micrococcal nuclease digestion and were found to contain the 167 basepair DNA band as in intact chromatin . The presence of the limit peptide from H5 on the monomers was confirmed using an antibody to H5 . It is concluded that the trypsin-susceptible domains of the lysine-rich histones are not involved in the protection of the 2-turn 167 basepair length of DNA in the nucleosome.

Mol Immunol, 1983 Apr, 20(4), 475 - 82
Destruction of antibody idiotopes with ultra-low concentrations of reducing agents; Binion SB et al.; The nature of the idiotopes present on F(ab')2 fragments prepared from rabbit anti-micrococcal carbohydrate antibodies and the loss of idiotypic reactivity of these F(ab')2 fragments upon iodination were examined . Rabbit anti-micrococcal idiotopes were shown to be exquisitely sensitive to treatment with very low concentrations of sodium metabisulfite or 2-mercaptoethanol . The treatment destroyed anti-micrococcal idiotopes, as shown by the loss of idiotopes on F(ab')2 fragments after reduction; the allotype epitopes and the antigen binding capacity of the F(ab')2 fragments were unaffected . The destruction of the idiotopes by very low concentrations of reducing agents indicated that an extremely labile disulfide bond is involved in the structure of the idiotope or in the maintenance of the conformation of the anti-micrococcal idiotopes . Identical reduction-sensitive anti-micrococcal idiotopes have been demonstrated in a number of related outbred rabbits, and in each case they induced a natural auto-anti-idiotype (AAI) antibody response . Recognition of the existence of these reduction-sensitive idiotopes and their properties could provide a basis for further study of these idiotopes and may lead to a better understanding of the idiotope network.

Mol Cell Biol, 1983 Apr, 3(4), 720 - 30
Regular arrangement of nucleosomes on 5S rRNA genes in Xenopus laevis; Young D et al.; The chromatin structure of the oocyte-type 5S RNA genes in Xenopus laevis was investigated . Blot hybridization analysis of DNA from micrococcal nuclease digests of erythrocyte nuclei showed that 5S DNA has the same average nucleosome repeat length, 192 +/- 4 base pairs, as two Xenopus satellite DNAs and bulk erythrocyte chromatin . The positions of nuclease-sensitive regions in the 5S DNA repeats of purified DNA and chromatin from erythrocytes were mapped by using an indirect end-labeling technique . Although most of the sites cleaved in purified DNA were also cleaved in chromatin, the patterns of intensities were strikingly different in the two cases . In 5S chromatin, three nuclease-sensitive regions were spaced approximately a nucleosome length apart, suggesting a single, regular arrangement of nucleosomes on most of the 5S DNA repeats . The observed nucleosome locations are discussed with respect to nucleotide sequences known to be important for expression of 5S RNA . Because the preferred locations appear to be reestablished in each repeating unit, despite spacer length heterogeneity, we suggest that the regular chromatin structure reflects the presence of a sequence-specific DNA-binding component on inactive 5S RNA genes.

Zentralbl Bakteriol Mikrobiol Hyg {B}, 1983 Apr, 177(3-4), 251 - 6
{Production and characteristics of bacteria-labeled talc dust for experimental air hygiene studies}; Ohgke H et al.; Freeze-drying of suspensions of Micrococcus luteus together with talc yields bacteria-labelled dust . This material can be used in experimental air hygiene . Loss of viability due to drying in air during experiments can be expected to be negligible . A wide range of particle diameters (1 to greater than 23 micron) is available . Scanning electron microscopy shows the bacteria sticking on talc particles after freeze-drying (Fig . 3a + b) . Viable counts of the material decreased very slowly on storage.

Acta Physiol Scand, 1983 Apr, 117(4), 519 - 25
Lysine deficiency reduces transcription activity and concentration of chromatin proteins reversibly in rat liver; Astrom S et al.; The reversible effect of dietary lysine deficiency was studied in young adult rats . During 6 days on a lysine diet the rats maintained the same body weight . During 2 days of recovery body weight gain was that of the controls . Liver nuclei were isolated, incubated with micrococcus nuclease and chromatin fractionated in to a 2 000 X g pellet, 102 000 X g pellet and supernatant fraction . Chromatin-bound RNA polymerase 1 plus III activity decreased by 15% per mg of fractional and nuclear DNA and by 30% per total liver . The corresponding decrease of RNA polymerase II activity was 30% and 40% . Recovery from lysine deficiency was complete after 2 days of refeeding the amino acid . Chromatin proteins of the 102 000 X g pellet were characterized by polyacrylamide gel electrophoresis in sodium dodecylsulfate and by 2-dimensional gel electrophoresis . Quantitative but no qualitative differences between the proteins of the dietary groups were observed . Relative to DNA the non-histone proteins decreased in the lysine deficient group by 43% and histones by 10% . It is concluded that RNA synthesis is restored to its original level within 2 days of refeeding lysine after 6 days of lysine deficiency.

J Bacteriol, 1983 Apr, 154(1), 452 - 9
Tactic behavior of Myxococcus xanthus; Dworkin M; With time-lapse videomicroscopy it was demonstrated that cells of Myxococcus xanthus are capable of directed (tactic) movement toward appropriate targets . Mutants that had lost A motility (J . Hodgkin and D . Kaiser, Mol . Gen . Genet . 171:177-191, 1979) were unable to show directed movement . Cells showed directed movement to polystyrene latex beads and to glass beads, as well as to clumps of Micrococcus luteus . This is consistent with other observations in an accompanying paper (M . Dworkin and D . Eide, J . Bacteriol . 154:437-442, 1983) that indicate that M . xanthus does not perceive chemical gradients.

Cell, 1983 Apr, 32(4), 1205 - 15
Another potential artifact in the study of nucleosome phasing by chromatin digestion with micrococcal nuclease; McGhee JD et al.; We show that, contrary to expectations, restriction enzyme cleavage of chicken erythrocyte nucleosome core particle DNA generates a series of distinct subnucleosome fragments . These fragments do not result from bulk nucleosome phasing in vivo, but arise from micrococcal nuclease cleavages internal to the core particle, at roughly 10-base pair intervals and at AT-rich sequences . Those 145-base pair DNA fragments remaining intact are a biased population in which the guanine content can fluctuate by as much as 10%, with a 10-base pair period . We suggest that these same considerations, when applied to a unique DNA sequence, are the true explanation for several previous claims for nucleosome phasing.

Proc Natl Acad Sci U S A, 1983 Apr, 80(7), 1787 - 91
Inhibition of Micrococcus luteus DNA topoisomerase I by UV photoproducts; Pedrini AM et al.; The activity of Micrococcus luteus DNA topoisomerase I on UV-irradiated supercoiled DNA was studied under either processive or distributive reaction conditions . Changes in DNA structure caused by UV irradiation reduce the rate of DNA relaxation at very low concentration of photoproducts . Under processive conditions the inhibition of the topoisomerase I by photoproducts can be quantitated by measuring the amount of substrate left in the replicative form I band . The mode of action of DNA topoisomerase I was affected by the presence of photoproducts in the DNA substrate, although the ability of the enzyme to form a covalent complex with UV-irradiated supercoiled DNA was not changed . The inhibition of topoisomerase I by UV photoproducts has been compared to the effects of single-stranded DNA and UV-irradiated duplex linear DNA on the enzyme, and the results suggest that the inhibition by photoproducts is caused by changes in the conformation of the supercoil . Our findings indicate the possibility that DNA topoisomerase I plays a role in repair.

J Biol Chem, 1983 Mar 25, 258(6), 3726 - 34
Histone deacetylase . Association with a nuclease resistant, high molecular weight fraction of HeLa cell chromatin; Hay CW et al.; The chromatin-bound histone deacetylase of HeLa cells has been studied using endogenous {3H}acetyl-labeled polynucleosomes containing the enzyme, prepared in the presence of 40 mM butyrate . Histone deacetylase was assayed upon removal of the butyrate, and it was found that active enzyme is found only in association with a high molecular weight complex . This deacetylase-containing complex is relatively resistant to digestion with micrococcal nuclease . No activity is found on mononucleosomes or oligonucleosomes . Up to 90% of labeled acetyl groups are removed from histone deacetylase complexes incubated in the absence of butyrate . Free histones are a poor substrate under these conditions, but histones in mononucleosomes are deacetylated when they are incubated with histone deacetylase complex . Histone deacetylase remains bound to this complex in 1-2 M NaCl and does not dissociate from it during its reaction with acetylated core histones . Under typical nuclease digestion conditions, the histone deacetylase complex contains DNA with a size distribution of 5-11 kilobase pairs and a variety of nonhistone proteins . Comparison of the protein composition of histone deacetylase complexes with that of nuclear matrix preparations shows some similarities . Taken together, the results on the chromatographic behavior, the DNA fragment sizes, and the protein composition of the deacetylase complex suggest that protein-protein interactions may be important in maintaining its structure and also in the binding of the deacetylase itself to the complex.

Eur J Biochem, 1983 Mar 15, 131(2), 283 - 8
Structural characterization of nuclear poly(A)-protein particles in rat liver; Tomcsanyi T et al.; Poly(A)-protein particles were prepared from rat liver nuclear extract after digestion with pancreatic ribonuclease and ribonuclease T1 by sucrose gradient centrifugation . The particles were sedimented in a range of 9-23S with a peak at 16S . The particles isolated in this manner were 99-100% resistant to further pancreatic ribonuclease treatment and contained more than 90% adenylic acid . In CsCl density gradient the nuclear poly(A)-protein particles banded in a narrow density range of 1.28-1.32 g/cm3 with a peak at 1.30 g/cm3, which corresponds to about 90% of protein in the particles . The average length of the poly(A) molecules prepared from the 16-S particles was about 140 nucleotides . Urea/sodium dodecyl sulphate/polyacrylamide gel electrophoresis demonstrated two major polypeptide components with Mr of 63 000 and 90 000 and at least ten minor polypeptides in the 45 000-130 000-Mr range . In sodium dodecyl sulphate/polyacrylamide gels the 63 000-Mr polypeptide was the only one major component . Amino acid analysis of the polypeptides bound to nuclear poly(A) revealed that the polypeptides contained a relatively large amount of aspartic acid + asparagine and glutamic acid + glutamine (24%) . Treatment of glutaraldehyde-fixed particles with micrococcal nuclease showed that more than 90% of the poly(A) was accessible to the enzyme, thus almost the entire poly(A) should be located on the surface of the particles . On the basis of the results a model for the 'average' 16-S particle was constructed.

J Biol Chem, 1983 Mar 10, 258(5), 3309 - 18
Phosphorylation of nonhistone proteins during the HeLa cell cycle . Relationship to DNA synthesis and mitotic chromosome condensation; Song MK et al.; Cell cycle variations in the phosphorylation of chromatin-associated nonhistones were determined . Cells were radiolabeled with {32P}orthophosphate and chromatin was obtained by mild digestion of nuclei with micrococcal nuclease . The experiments were performed in the presence of a substrate inhibitor of alkaline phosphatase, beta-glycerophosphate . The results show that, while similar molecular weight species of phosphorylated nonhistones are associated with interphase chromatin through the HeLa cell cycle, the incorporation (32P cpm/micrograms of protein) profiles of selected major phosphononhistones show substantial changes . The most prominent peaks of specific radioactivity occur in the DNA synthesis phase (S phase) . The phosphorylation states of the proteins of isolated metaphase chromosomes were also determined . Nonhistone proteins of isolated metaphase chromosomes are strikingly dephosphorylated, especially in comparison to histone H1 . The phosphorylation of the major phosphononhistone of chromatin, which has a molecular weight of 55,000, was further characterized by techniques that included one-dimensional peptide mapping in sodium dodecyl sulfate-polyacrylamide gels and nonequilibrium pH gradient slab gel electrophoresis . Phosphoproteins are also components of the nuclear scaffold, and cell cycle variations in these proteins were investigated . The primary phosphorylated species has a molecular weight of 119,000 . As with chromatin-associated nonhistones, this nuclear scaffold protein shows substantial incorporation of 32P in S phase, and a high level of incorporation also occurs close to mitosis.

Exp Cell Res, 1983 Mar, 144(1), 63 - 72
Isolation and preliminary characterization of the synaptonemal complex from rat pachytene spermatocytes; Li S et al.; A method for preparation of the morphologically intact synaptonemal complex from rat pachytene spermatocytes is described . Pachytene spermatocytes were fractionated from rat testicular cells by centrifugal elutriation . Nuclei from fractionated pachytene cells were prepared and extensively digested with micrococcal nuclease . The digested nuclei were sedimented through 20% (w/v) sucrose containing 2 M NaCl by centrifugation . About 10% of total nuclear proteins and 1-2% of total genomic DNA was found to be associated with the residual structure . The residual structure, which contains mainly the synaptonemal complex, but may be still contaminated with other nuclear components including membrane and matrix, was stained with silver and examined under light microscopy . It was found that a silver-staining component of the synaptonemal complex is not grossly different from that in pachytene nuclei not subjected to digestion and extraction . Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed that virtually all the proteins in the residual structure are non-histones . The DNA isolated from the residual structure was about 135 base pairs (bp), long . The DNA was end-labeled and hybridized with a large excess of sonicated rat genomic DNA . The hybridization displayed a kinetics virtually identical to that of total nuclear DNA . We also prepared restricted DNA fragments associated with the residual structure . Southern blot analyses using a probe made from a recombinant DNA clone containing the albumin gene revealed that the DNA associated with the residual structure was not enriched (or depleted) in this gene sequence . Our results strongly suggest that (1) the synaptomenal complex may play a structural role to support the chromatin domains inside pachytene nucleus; and (2) a simple common DNA sequence in the chromatin domain is not required for association with the residual structure which contains morphologically intact synaptonemal complex in rat spermatocytes.

Mutat Res, 1983 Mar, 108(1-3), 57 - 66
Induction of mutation in Micrococcus radiodurans by N-methyl-N'-nitro-N-nitrosoguanidine; Rebeyrotte N; Micrococcus radiodurans was highly resistant to the lethal effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) but it was sensitive to the mutagenic action of this chemical . The induction of mutation was not significantly modified by the culture growth phase . This last finding leads to the assumption that the mutation takes place at some distance from the replication fork . Moreover, a low concentration of MNNG induced mutations that were added to those subsequently obtained from a second exposure to a higher concentration of the alkylating agent . Thus, M . radiodurans does not seem to have an inducible error-free repair system for alkylation damage . Furthermore, incubation in the presence of chloramphenicol did not modify the mutation rate, indicating that protein synthesis is not involved in the mutagenic process.

J Nutr, 1983 Mar, 113(3), 557 - 65
Nucleosomal repeat length in rat liver nuclei is decreased by a high carbohydrate, fat-free diet; Castro CE; Liver nuclei of Sprague-Dawley rats fed a high carbohydrate, fat-free diet (diet 1), a low carbohydrate, protein-free diet (diet 2), or a commercial stock diet were purified and mildly incubated with micrococcal nuclease (MN) (EC 3.1.4.7) until 5-6% of the chromatin was acid-soluble . Chromatin fragments generated by MN incubation were deproteinized and sized by electrophoresis . The mobilities of the DNA fragments were used to calculate by two independent methods nucleosomal repeat length . The nucleosome is the fundamental packaging unit of eucaryotic chromatin . When nuclei were incubated with MN for various lengths of time, the nucleosome repeat length for rats fed diet 1 was invariably shorter than that for stock diet-fed rats regardless of method of calculation . After incubation with MN for 10 minutes, the nucleosome repeat length of nuclei from rats fed diet 2 was virtually identical to that for stock diet-fed rats . In addition, after a 30-minute incubation, 32.4% of chromatin from nuclei of rats fed a lipogenic diet was of mononucleosome size, whereas only 9.9% and 20.6% was of the same size in nuclei of rats fed diet 2 or stock diet, respectively . These observations suggest that liver chromatin of rats fed a lipogenic diet may be in a different configuration than that from rats fed diet 2 or stock diet.

J Invest Dermatol, 1983 Mar, 80(3), 188 - 91
Induction of pyrimidine dimers in epidermal DNA of hairless mice by UVB: an action spectrum; Ley RD et al.; An action spectrum for the induction of pyrimidine dimers in the epidermis of hairless mice was determined between 288 and 307 nm . The presence of pyrimidine dimers in tritium-labeled DNA extracted from exposed SKH:hairless-1 mouse skin was determined using dimer-specific nucleases from Micrococcus luteus in conjunction with sedimentation of the irradiated DNA in alkaline sucrose gradients . The rate of induction of pyrimidine dimers was maximal at 293 nm . These values were used to propose a UVB transmission curve for mouse epidermis.

Bull Eur Physiopathol Respir, 1983 Mar-Apr, 19(2), 179 - 87
Modulation of the immune response by antibacterial antibodies; Hamers R et al.; Antibodies induced by the gram+ bacteria Micrococcus lysodeikticus exhibit different carbohydrate specificities and hence might cross-react with membrane glycoproteins and/or glycolipids of mammalian cells . Using a T cell derived lymphoid line, these antibodies were found to detect a membrane marker which is only exposed in confluent culture conditions on non-dividing cells . Such confluence related antigen (Cag) is a cryptic membrane antigen, which can be unmasked through membrane perturbating agents such as p-formaldehyde or through interactions with macrophages and macrophage derived factors . Anti-micrococcus antibodies appear also to affect functionally normal T lymphocytes . Thus such reagents drastically inhibit the murine T cell reactivity towards mitogens such as Con A and PHA provided, however, the mitogenic signal is delivered through peritoneal macrophages . Furthermore, anti-micrococcus antibodies induce activated T lymphocytes into mitogenesis, but not unprimed resting T cells . Hence the physiological activity of anti-micrococcus antibodies depends on the state of activation of the T lymphocyte, indicating the involvement of cryptic membrane molecules . The relevance of such phenomena to host-bacteria and host-parasite interaction will be discussed.

J Bacteriol, 1983 Mar, 153(3), 1493 - 501
Molecular properties of succinate dehydrogenase isolated from Micrococcus luteus (lysodeikticus); Crowe BA et al.; Succinate dehydrogenase (EC 1.3.99.1) of Micrococcus luteus was selectively precipitated from Triton X-100-solubilized membranes by using specific antiserum . The precipitated enzyme contained equimolar amounts of four polypeptides with apparent molecular weights of 72,000, 30,000, 17,000, and 15,000 . The 72,000 polypeptide possessed a covalently bound flavin prosthetic group and appeared to be strongly antigenic as judged by immunoprinting experiments . Low-temperature absorption spectroscopy revealed the presence of cytochrome b556 in the antigen complex . By analogy with succinate dehydrogenase purified from other sources, the 72,000 and 30,000 polypeptides were considered to represent subunits of the succinate dehydrogenase enzyme, whereas one (or both) of the low-molecular-weight polypeptides was attributed to the apoprotein of the b-type cytochrome . A succinate dehydrogenase antigen cross-reacting with the M . luteus enzyme complex could be demonstrated in membranes of Micrococcus roseus, Micrococcus flavus, and Sarcina lutea, but not in the membranes isolated from a wide variety of other gram-positive and gram-negative bacteria.

Biochemistry, 1983 Mar 1, 22(5), 1245 - 50
Distribution of thyroid hormone receptors, glucocorticoid receptors, and growth hormone gene sequences in chromatin from cultured rat pituitary cells; Levy-Wilson B; Chromatin from rat pituitary tumor (GH3) cells was fractionated into transcriptionally active and inactive domains . When the various chromatin fractions were assayed for their content of growth hormone gene sequences, it was found that these sequences were highly enriched in those chromatin fractions most sensitive to micrococcal nuclease . Fraction S1 showed the highest enrichment in growth hormone genes and was impoverished in histones H3 and H4 . The distribution of specifically bound thyroid and glucocorticoid receptors in chromatin fractions enriched and depleted in growth hormone gene sequences was also examined . Both thyroid and glucocorticoid receptors are enriched to different extents in transcriptionally active chromatin . Within active chromatin, both types of receptors exist in more than one molecular form.

Biochem Int, 1983 Mar, 6(3), 357 - 63
Analysis of chromatin of the brain of young and old rats by micrococcal nuclease and DNase I; Chaturvedi MM et al.; Micrococcal nuclease (MCN) and DNase I were used to study the conformational changes in chromatin of the brain of rats of different ages . Purified nuclei and chromatin were digested separately by MCN and DNase I . Kinetics of digestion of chromatin by MCN are similar for young, adult and old rats . Also agarose gel electrophoresis of DNA fragments do not show any differences . The kinetics of digestion with DNase I, on the other hand, are greater and faster for 20-week old rats than for 90-week old rats . High performance denaturing polyacrylamide gel electrophoresis reveals that a greater amount of smaller fragments of DNA are produced in the 20-week old rats than in the 90-week . These conformational changes occur in the chromatin during aging.

Mech Ageing Dev, 1983 Mar-Apr, 21(3-4), 273 - 82
The effects of histones, in vitro age, and culture state on the digestion of DNA by micrococcal nuclease and deoxyribonuclease I; Dell'Orco RT et al.; Nuclei from confluent and mitotically arrested populations of human diploid fibroblast-like cells were subjected to digestion by micrococcal nuclease and deoxyribonuclease (DNase I) following the removal of various histone components by salt extraction . There was no age or culture state variation in the susceptibility of DNA to micrococcal nuclease digestion . There was an age related inhibition of DNA digestion by DNase I in nuclei from older confluent cells before and after the removal of H1 histone but not after the removal of core particle histones . This inhibition was not detected in older arrested populations . These results indicate that an age-related masking by nucleosome core histones may limit the accessibility of DNA to enzymatic activities in older confluent cells . Since this inhibition was absent in older arrested populations, the importance of limited DNA accessibility as a primary cause of cellular senescence is questionable.

Mol Pharmacol, 1983 Mar, 23(2), 493 - 9
Effects of neocarzinostatin on chromatin in HeLa S3 nuclei; Beerman TA et al.; Neocarzinostatin solubilizes chromatin from HeLa S3 nuclei by introducing strand scissions in linker regions . Multimeric nucleosome patterns are seen on both native and denaturing gel analysis . The mechanism of drug action differs from the type of chromatin digestion seen with micrococcal enzyme in that DNA damage occurs through single-strand breaks and less acid-soluble material is produced . In addition, drug-induced release of soluble chromatin from the nuclei is not very dependent upon the addition of EDTA . The monomer repeat size is larger than that found for micrococcal enzyme and contains linker regions that are partially single-stranded . Core histone proteins as well as histone H1 do not appear to be altered by drug action, although there is clear evidence that DNA damage can occur in nucleosome cores . The chromophore portion of the drug degrades chromatin as effectively as the holoantibiotic.

Biochemistry, 1983 Feb 15, 22(4), 745 - 9
Quantitation of pyrimidine dimer contents of nonradioactive deoxyribonucleic acid by electrophoresis in alkaline agarose gels; Sutherland BM et al.; We have developed a method of quantitating the pyrimidine dimer content of nonradioactive DNAs . DNA samples are treated with the UV-endonuclease from Micrococcus luteus and then separated according to molecular weight by electrophoresis on alkaline agarose gels . From their migration relative to known molecular weight standards, their median molecular weights and thus the number of dimers per DNA molecule in each sample can be calculated . Results of action spectra for dimer formation in T7 bacteriophage measured by this method agree well with action spectra for T7 killing . In addition, the method gives dimer yields in good agreement with those obtained by others using alkaline sucrose gradient sedimentation.

Nucleic Acids Res, 1983 Feb 11, 11(3), 823 - 35
Modulations of prolactin and growth hormone gene expression and chromatin structure in cultured rat pituitary cells; Levy-Wilson B; I have measured the effect of hormones and other regulatory factors present in the serum component of the culture medium on the levels of growth hormone and prolactin mRNAs in rat pituitary (GH4) cells . Hybridization of cytoplasmic RNA with growth hormone or prolactin cDNA clones indicate that serum depletion reduces significantly the amount of these two mRNAs . The localization of these two genes in chromatin was also analysed using micrococcal nuclease as a probe . At intermediate levels of digestion (about 10% of the input A260 released into a soluble supernatant S1), the bulk of both growth hormone and prolactin genes are rapidly solubilized by the nuclease and appear in the soluble supernatant S1 . Nevertheless, at low levels of digestion (less than 4% of the input A260 released into S1) the growth hormone gene remains exquisitively sensitive to micrococcal nuclease while the sensitivity of the prolactin gene is reduced considerably . When one compares the distribution of growth hormone and prolactin genes in chromatin fractions differing in nuclease sensitivity and derived from cells grown in control medium or in depleted medium, it appears that markedly reduced transcriptional activity of the prolactin gene shows no correlation with altered chromatin structure . On the other hand, the chromatin structure of the growth hormone gene is significantly altered when transcription is markedly reduced.

Nucleic Acids Res, 1983 Feb 11, 11(3), 753 - 72
The nuclease sensitivity of active genes; Nicolas RH et al.; Brief micrococcal nuclease digestion of chick embryonic red blood cells results in preferential excision and solubilization of monomer nucleosomes associated with beta-globin sequences and also 5'-sequences flanking the beta-globin gene . Both regions are DNAse-I sensitive in nuclei . Such salt-soluble nucleosomes are enriched in all four major HMG proteins but HMG1 and 2 are only weakly associated . These nucleosomes appear to have lost much of the DNAse-I sensitivity of active genes . The HMG14 and 17-containing salt-soluble nucleosomes separated by electrophoresis are not DNAse-I sensitive and contain inactive gene sequences as well as active sequences . Reconstitution of HMG proteins onto bulk nucleosomes or chromatin failed to reveal an HMG-dependent sensitivity of active genes as assayed by dot-blot hybridization and it was found that the DNAse-I sensitivity of ASV proviral sequences as assayed by dot-blot hybridization was not HMG-dependent . These results indicate that higher order chromatin structures might be responsible for nuclease sensitivity of active genes.

Biochem Biophys Res Commun, 1983 Feb 10, 110(3), 811 - 8
Assembly kinetics of replicating chromatin: isolation and characterization of prenucleosomal and nucleosomal DNA; Vaury C et al.; Replicating chromatin is known to be more sensitive to micrococcal nuclease than bulk chromatin . We have used this property and a fractionation procedure based on the specific release of replicating material under mild micrococcal nuclease digestion, in order to analyse both the kinetics of maturation of newly replicated DNA into nucleosomes and the structure of the replicating material . As other authors, we initially observed that repetitive unit of newly replicated chromatin was shorter than that of bulk chromatin, however this result appears to be due to sliding of nucleosomes along the chromatin fibers close to the replicating fork . Replicative chromatin was fractionated and analysed . A prenucleosomal peak was observed and preliminary characterized.

J Biol Chem, 1983 Feb 10, 258(3), 1536 - 43
Purification and characterization of DNase VIII . A 5'-3' directed exonuclease from human placental nuclei; Pedrini AM et al.; DNase VIII is an exonuclease purified from human placenta trophoblast nuclei . The enzyme has a pH optimum of 9.5 and requires a divalent cation . It is inhibited by salt and stimulated by Triton X-100 . Glycerol gradient analysis of the activity indicates a sedimentation coefficient of 2.8 S (31,000 daltons if globular) . This enzyme initiates hydrolysis from 5'-phosphorylated termini of single-stranded DNA and acts at internal phosphodiester bonds liberating 5'-phosphorylated oligonucleotides . It degrades polynucleotides of repeating base sequence as well as single-stranded DNA, yielding oligonucleotides of even number, in which the main reaction products are dinucleotides . The activity on denatured DNA is not inhibited by the presence of ultraviolet-induced photoproducts . DNase VIII can also initiate hydrolysis at those distorted termini produced by the action of Micrococcus luteus dimer specific endonuclease on duplex DNA, which contains cyclobutane dimers.

J Biol Chem, 1983 Feb 10, 258(3), 2005 - 9
Proteolytic processing of presecretory proteins is required for development of biological activities in pancreatic exocrine proteins; Scheele G et al.; The biological activities of pancreatic presecretory and secretory proteins synthesized in vitro were compared in studies of (a) the binding of nascent amylase to its substrate, glycogen, (b) the binding of nascent trypsinogen 1, trypsinogen 2+3, and chymotrypsinogen 1 to Sepharose-bound soybean trypsin inhibitor, and (c) the activation of nascent trypsinogen by porcine enterokinase . Nascent secretory proteins synthesized in vitro using a mRNA-dependent gel-filtered reticulocyte lysate translation system supplemented with canine pancreas rough microsomes or canine pancreas mRNA and micrococcal nuclease-treated microsomal membranes showed biological activities similar to authentic secretory proteins if oxidized glutathione was added during their synthesis . Proteins synthesized in the presence of membranes and the absence of glutathione showed significantly less biological activity due to incorrect development of conformation . Presecretory proteins synthesized in vitro with canine pancreas mRNA in the absence of microsomal membranes had little or no activity after translation in either the absence or presence of glutathione . These and previous findings (Scheele, G . A., and Jacoby, R . (1982) J . Biol . Chem . 257, 12277-12282) indicate that proteolytic removal of the NH2-terminal transport peptide is necessary to allow correct conformational development, including the formation of native disulfide bonds, which not only stabilizes the molecule but allows expression of authentic biological and probiological activity.

Biochem J, 1983 Feb 1, 209(2), 345 - 53
Some properties, including the substrate in vivo, of the delta 9-desaturase in Micrococcus cryophilus; Foot M et al.; The delta 9-desaturase of the psychrophilic bacterium Micrococcus cryophilus is shown to be a membrane-bound enzyme that is probably linked to a cyanide- (and azide-) sensitive respiratory chain with oxygen as the final acceptor . It has a pH optimum of 8.7 and contains an essential thiol group, but has no special ion requirements . The desaturase activity of washed membranes could not be increased by adding supernatant or NADH and NADPH, possibly owing to the endogenous generation of reduced cofactors by the membranes . The substrate for the desaturase is not acyl-CoA and is probably not acyl-acyl-carrier protein . Evidence is presented that the substrate in vivo is saturated phospholipid and a scheme for the possible routes of incorporation of exogenous stearic acid into oleoyl-phospholipid is presented.

J Biochem (Tokyo), 1983 Feb, 93(2), 513 - 23
Artificial structure of chromatin derived in the preparation process; Ohba Y et al.; Nuclei were isolated from mouse lymphoma L5178Y cells in the exponential growth phase, and chromatin was prepared by mechanical treatment of the nuclei . The nuclei and the chromatin were then digested to various extents with micrococcal nuclease and the resulting mono- and dinucleosome fractions of the two preparations were compared . During progressive digestion mononucleosomes from chromatin retained H1 histone and a DNA length of 165 base pairs, whereas those from nuclei released H1 histone and the length of their DNA decreased to 140 base pairs at an early stage of digestion . These nucleosomal preparations were always associated with nonhistone proteins . The dinucleosomes from nuclei contained larger amounts of nonhistone proteins than those from chromatin, but half of these proteins was released during the process of cleavage into mononucleosomes . The final mononucleosome preparation from nuclei retained 20% less nonhistone proteins than that from chromatin . The contents of nonhistone proteins in mono- and dinucleosomes from chromatin were the same . The electrophoretical distributions of molecular species of nonhistone proteins in mononucleosomes from nuclei and chromatin were different from each other: during digestion the profile of the former changed, whereas that of the latter remained constant . It is tentatively concluded that both H1 histone and nonhistone proteins were bound to nucleosomes more or less loosely in intact nuclei in situ, but that when the nuclear structure was disrupted these proteins became more tightly bound.

J Gen Microbiol, 1983 Feb, 129 (Pt 2), 413 - 22
Release of high molecular weight DNA from Neurospora crassa using enzymic digestions; Calza RE et al.; Methods are described that allow extraction of high molecular weight DNA from germinated conidia of Neurospora crassa . By labelling DNA with ribonucleosides, early conidia were shown to be active in DNA synthesis . These cells when treated with the enzyme Zymolyase became fragile and could be readily lysed with ionic detergents to release high molecular weight DNA . The DNA extracted from Zymolyase treated cells on to alkaline sucrose gradients sedimented as a heterogeneous species of up to 150 x 10(6) molecular weight . A minor DNA species (presumably mitochondrial) of 20 x 10(6) molecular weight comprised 2-7% of the total . The identity of the DNA was confirmed by sensitivity to DNAase, the diphenylamine assay and TLC . Sedimentation patterns were unaffected by protease digestions and no anomalous high speed rotor effects were evident . Isopycnic gradients suggested that the DNA released was uncomplexed with either protein or carbohydrates . Sepharose chromatography of extracted, RNAase-treated Zymolyase lysate resulted in clearly separate high molecular weight DNA and RNA-protein elution profiles . UV light preferentially inhibited nuclear DNA synthesis and drastically reduced the size and amount of nascent DNA being synthesized in the excision defective uvs-2 mutant . Sites in parental DNA sensitive to Micrococcus luteus UV endonuclease were measured in cells made permeable with Triton X-100.

Arch Biochem Biophys, 1983 Feb 1, 220(2), 584 - 93
Tissue and cell-specific antigens in chromatin from cultured rat Sertoli cells; Schmidt WN et al.; Cell-specific chromatin antigens have been detected in rat Sertoli cells . Antisera were raised in rabbits to dehistonized chromatin prepared from 5- to 6-day cultures of rat Sertoli cells and immunoreactivity was assessed with microcomplement fixation tests or immunoidentification of antigens separated electrophoretically and transferred to nitrocellulose sheets . Tissue specificity was confirmed further by immunoabsorption . These antisera recognized only components of Sertoli cell chromatin; chromatins prepared from rat liver, kidney, thymus, Novikoff hepatoma, a testes germinal cell fraction, or purified rat DNA showed little or no immunoreactivity . Nitrocellulose transfers of total chromosomal proteins revealed the presence of two high-molecular-weight antigens (greater than 200,000) and a broad range of weaker immunologic species (M tau about 50,000-200,000) in Sertoli cell chromatin but not liver or thymus chromatin . Deproteinization of Sertoli cell chromatin with concentrated salt and urea at pH 6 or 8 produced an increase in complement-fixing activity of the fraction sedimenting with DNA and micrococcal nuclease digestion studies showed these fractions to depend in part on DNA for antigenicity . Individual antigens in the protein-DNA pellets of pH 8 salt and urea fractionated chromatin could not be identified with antigens on nitrocellulose sheets . Collectively, these observations suggest that at least two groups of cell-specific antigens exist in Sertoli cell chromatin; one group is detected after electrophoretic separation and transfer to nitrocellulose and the other group, reactive in complement fixation, cannot be detected through this method, apparently becoming antigenically inactive once the complex of protein and DNA is dissociated.

Biochem Biophys Res Commun, 1983 Jan 27, 110(2), 470 - 6
Ribonuclease H from chick embryos cleaves precisely at the junction between the RNA and DNA portion of the hybrid helix; Sawai Y et al.; DNA polymerase from Micrococcus luteus and RNA polymerase from E . coli catalyze the synthesis of poly(dA) with poly(dT) template, in the presence of ATP and {alpha-32P}dATP . The reaction is completely dependent on poly(A) primer synthesis . Poly(A) chains are covalently extended by DNA polymerase . Primer poly(A) is linked to the product poly(dA) via a 3':5'-phosphodiester bond, and can be specifically removed by ribonuclease H from chick embryos, leaving a 5'-phosphate end of poly(dA) . The length of RNA and DNA products appears to be relatively variable . The size of the DNA is less than 3 000 nucleotides.

Nucleic Acids Res, 1983 Jan 25, 11(2), 441 - 60
The structure of nucleoprotein cores released from adenovirions; Vayda ME et al.; The morphology, protein composition and DNA organization of nucleoprotein core complexes isolated from type 5 adenovirions have been examined by electron microscopy and biochemical techniques . The morphology of such core structures is in some ways strikingly similar to that exhibited by cellular chromatin . 'Native' core preparations contain compact and less highly-folded forms: the latter appear as thick fibres, 150-300A in diameter . Upon exposure to 0.4M NaCl, adenovirus cores undergo a transition to a beaded string form, reminiscent of nucleosomes . Of the three arginine-rich proteins, polypeptides V, VII and mu present in 'native' cores, only polypeptide VII remains associated with viral DNA in the presence of 0.4M NaCl . We therefore conclude that the nucleosome-like beads are constructed solely of polypeptide VII . The results of micrococcal nuclease digestion experiments suggest that polypeptide VII is sufficient to protect some 100-300bp of adenoviral DNA.

Biochim Biophys Acta, 1983 Jan 20, 739(1), 17 - 26
Effect of aphidicolin on de novo DNA synthesis, DNA repair and cytotoxicity in gamma-irradiated human fibroblasts . Implications for the enhanced radiosensitivity in ataxia telangiectasia; Smith PJ et al.; The antibiotic, aphidicolin, is a potent inhibitor of DNA polymerase alpha and consequently of de novo DNA synthesis in human cells . We report here that in gamma-irradiated normal human cells, aphidicolin (at 5 micrograms/ml and less) had no significant effect on the rate of the rejoining of DNA single strand breaks or rate of removal of DNA lesions assayed as sites sensitive to incising activities present in crude protein extracts of Micrococcus luteus cells . gamma-irradiated human ataxia telangiectasia cells are known to demonstrate enhanced cell killing and exhibit resistance to the inhibiting effects of radiation on DNA synthesis . Under conditions of minimal aphidicolin cytotoxicity but extensive inhibition of de novo DNA synthesis, the radiation responses of neither normal nor ataxia telangiectasia cells were significantly modified by aphidicolin . Firstly, we conclude that human DNA polymerase alpha is not primarily involved in the repair of the two classes of radiogenic DNA lesions examined . Secondly, the radiation hypersensitivity of ataxia telangiectasia cells cannot be explained on the basis of premature replication of damaged cellular DNA resulting from the resistance of de novo DNA synthesis to inhibition by ionizing radiation.

Biochem Biophys Res Commun, 1983 Jan 14, 110(1), 61 - 8
Nuclear-hormone mediated changes in chromatin solubility; Re RN et al.; Rat liver nuclei were incubated with either thyroid hormone or angiotensin (AII) at varying concentrations or with buffer (control) prior to digestion with micrococcal nuclease . Concentrations of hormones greater than 10(-10)M were effective in increasing the solubilization of chromatin with physiological levels (10(-9)M) of AII showing an approximate 2.4 fold increase over control . Nuclei were also isolated from animals treated in-vivo with either AII or buffer (control) and chromatin solubility was increased in the AII treated nuclei even prior to the addition of exogenous nuclease, presumably from the action of endogenous nucleases . The data suggest that hormone-induced increases in solubility are a reflection of structural changes in chromatin which enhance the accessibility of DNA to endonuclease attack.

Nucleic Acids Res, 1983 Jan 11, 11(1), 57 - 75
Control of 5S RNA transcription in Xenopus somatic cell chromatin: activation with an oocyte extract; Reynolds WF et al.; A chromatin fraction enriched for Xenopus 5S RNA genes has been isolated by restriction endonuclease digestion and sucrose gradient velocity sedimentation . Soluble chromatin sedimenting at 70-80S contains approximately 50% of the oocyte-expressed 5S RNA genes and only 1.5-3% of total chromatin DNA; this represents a 15- to 30-fold purification of the 5S genes . Such chromatin isolated from somatic cells (blood and cultured kidney cells) retains the transcriptionally-inactive state of the oocyte-expressed 5S genes . Soluble chromatin from somatic cells prepared by micrococcal nuclease digestion also retains the inactive state of the oocyte-type 5S genes . It is likely that the level of chromatin structure responsible for inactivity of the oocyte genes in somatic cells is the nucleosome or short chains of nucleosomes and not supranucleosomal structures . The oocyte-type genes can be rendered transcriptionally active in somatic cell chromatin either by salt extraction of some chromosomal proteins or by treatment with the ion exchange resin Dowex A50W-X2 . Alternatively, activation of these genes can be achieved by incubating somatic cell chromatin or nuclei with an extract prepared from Xenopus oocytes . This effect is not specific for 5S RNA genes as the transcription of other small RNAs (including pre-tRNA) is stimulated by the oocyte extract . The activating factor(s) is resistant to micrococcal nuclease, nondialyzable, heat labile and sensitive to trypsin; thus it is highly likely to be a protein or a group of proteins . Partial purification of the activating factor(s) has been achieved by ion exchange chromatography.

Int J Biochem, 1983, 15(6), 855 - 9
Intranuclear localization of DNA polymerases alpha and beta in regenerating rat liver; Mikhailov VS et al.; 1 . Nuclei isolated from regenerating rat liver were digested with micrococcal nuclease and fractionated on glycerol gradients into soluble chromatin fragments and chromatin associated with the nuclear skeleton . 2 . Distributions of DNA polymerases alpha and beta in these fractions were different . While beta polymerase followed closely the distribution of the chromatin fragments, alpha polymerase associated preferentially with the skeleton-chromatin complex . 3 . At least 20% of total alpha polymerase in the nuclei was shown to be bound to the skeleton . In nuclei extracted with isotonic sucrose buffer containing 50 or 100 mM Tris-Cl the portion of the skeleton associated enzyme was increased to 40-50% . 4 . These data show that the skeleton bound alpha polymerase was preferentially retained in the nuclei during salt extraction . 5 . Contrary to the replicational DNA polymerase alpha, DNA polymerase beta did not show any affinity to the skeleton.

J Bacteriol, 1983 Jan, 153(1), 498 - 505
Immunochemical analysis of respiratory-chain components of micrococcus luteus (lysodeikticus); Crowe BA et al.; Membrane-bound antigens of the respiratory chain of Micrococcus luteus were analyzed by crossed immunoelectrophoresis after growth of the organism in the presence of 59Fe, the flavin adenine dinucleotide-flavin mononucleotide precursor D-{2-14C}riboflavin, or the heme precursor 5-amino-{4-(14)C}levulinic acid . Using zymograms and procedures of selective extraction in conjunction with autoradiography, it was possible to resolve and partially characterize a number of antigens . Succinate dehydrogenase (EC 1.3.99.1) was shown to possess covalently bound flavin and nonheme iron and was possibly present as a complex with cytochrome . Three other dehydrogenases, namely, NADH dehydrogenase, NAD(P)H dehydrogenase (EC 1.6.99.3), and malate dehydrogenase (EC 1.1.1.37), contained flavin in noncovalent linkage, the NAD(P)H dehydrogenase also possessing nonheme iron . Four other discrete antigens (or antigen complexes) containing both iron and heme centers also resolved, as were two minor immunogens possessing iron as the sole detectable prosthetic group.

Carcinogenesis, 1983, 4(2), 179 - 84
DNA excision repair in permeable human fibroblasts; Kaufmann WK et al.; U.v . irradiation of confluent human fibroblasts activated DNA repair, aspects of which were characterized in the cells after they were permeabilized . Incubation of intact cells for 20 min between irradiation and harvesting was necessary to obtain a maximum rate of reparative DNA synthesis . Cells harvested immediately after irradiation before repair was initiated displayed only a small stimulation of DNA synthesis, indicating that permeable cells have a reduced capacity to recognize pyrimidine dimers and activate repair . The distribution of sizes of DNA strands labeled during 10 min of reparative DNA synthesis resembled that of parental DNA . However, during a 60-min incubation of permeable cells at 37 degrees C, parental DNA and DNA labeled by reparative DNA synthesis were both cleaved to smaller sizes . Cleavage also occurred in unirradiated cells, indicating that endogenous nuclease was active during incubation . Repair patches synthesized in permeable cells displayed increased sensitivity to digestion by micrococcal nuclease . However, the change in sensitivity during a chase with unlabeled DNA precursors was small, suggesting that reassembly of nucleosome structure at sites of repair was impaired . To examine whether this deficiency was due to a preponderance of incomplete or unligated repair patches, 3H-labeled (repaired) DNA was purified, then digested with exonuclease III and nuclease S1 to probe for free 3' ends and single-stranded regions . About 85% of the {3H}DNA synthesized during a 10-min pulse resisted digestion, suggesting that a major fraction of the repair patches that were filled were also ligated . U.v . light-activated DNA synthesis in permeable cells, therefore, appears to represent the continuation of reparative gap-filling at sites of excision repair activated within intact cells . Gap-filling and ligation were comparatively efficient processes in permeable cells, whereas activation of reparative DNA synthesis and reassembly of native chromatin structure upon completion of repair were not.

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