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 . Ra