Mol Gen Genet, 1989 Sep, 218(3), 384 - 9 Micrococcus luteus, a bacterium with a high genomic G + C content, contains Escherichia coli-type promoters; Nakayama M et al.; The G + C content of Micrococcus luteus DNA is 74%, which is much higher than that of Escherichia coli (about 50%) . In order to understand the influence of GC-directed mutation pressure (GC pressure) on the promoter structure, the initiation sites for both in vitro and in vivo transcription of M . luteus streptomycin (str) and spectinomycin (spc) operons were identified by the reverse transcriptase mapping method . The promoter sequences of M . luteus are similar to those of E . coli, but have significantly higher G + C contents . In an in vitro run-off transcription assay using truncated DNA templates, RNA polymerases from both M . luteus and E . coli were able to transcribe correctly from both the str promoter and the spc major promoter of M . luteus. Mol Cell Biochem, 1989 Aug 15, 89(1), 37 - 46 Biochemical characterization of chromatin fractions isolated from induced and uninduced Friend erythroleukemia cells; Knosp O et al.; Chromatin fractions from Friend erythroleukemia cells after induction of differentiation by dimethylsulfoxide (DMSO) were compared in their biochemical characteristics to fractions from uninduced cells . Fractions were prepared by extracting chromatin from nuclei after mild micrococcal nuclease treatment with increasing concentrations of NaCl according to Sanders . This procedure has been found to release chromatin containing hyperacetylated histones preferentially . The fractions obtained by this procedure were analysed in respect to the amount of chromatin released, the amount of histone H1, the degree of acetylation of histone H4, the presence of non-histone proteins and the concentration of transcribed and non-transcribed sequences . It was found that the fractions differ in the amount of histone H1 present, in several non-histone proteins and in the acetylation of histone H4, regardless whether induced or uninduced cells were analysed . The distribution of transcribed sequences versus non-transcribed sequences among the fractions was the same, demonstrating that this fractionation procedure, although leading to fractions with biochemical differences, is not able to discriminate functional states of chromatin and that the biochemical characteristics of the fractions may be common to both, active as well as inactive states of chromatin. J Biol Chem, 1989 Aug 5, 264(22), 12772 - 9 Comparative spectral analysis of mammalian, fungal, and bacterial catalases . Resonance Raman evidence for iron-tyrosinate coordination; Sharma KD et al.; Resonance Raman spectra are reported for catalases from bovine liver, the ascomycete fungus Aspergillus niger, and the bacterium Micrococcus luteus . The vibrational frequencies of the oxidation-, spin-, and coordination number-sensitive spectral bands are indicative of high spin pentacoordinate hemes in the resting ferric enzymes of each of these organisms . This result is in accord with the crystal structure of bovine catalase (Fita, I., and Rossmann, M.G . (1985) J . Mol . Biol . 185, 21-37) . In contrast, the crystallographic study of catalase from the ascomycete Penicillium vitale (Vainshtein, B . K., Melik-Adamyan, W . R., Barynin, V . V., Vagin, A.A., Grebenko, A . I., Borisov, V . V., Bartels, K . S., Fita, I., and Rossmann, M . G . (1986) J . Mol . Biol . 188, 49-61) showed electron density on the distal side of the heme which could imply the presence of a sixth ligand, possibly a water molecule . However, both of these crystallographic studies showed the proximal ligand in catalase to be a tyrosine . The present study confirms tyrosinate coordination in each of the three catalases from the appearance of selected resonance-enhanced tyrosine vibrational modes . The most characteristic band is the tyrosinate ring mode at approximately 1612 cm-1 which is maximally enhanced with 488.0 nm excitation . The appearance of tyrosinate modes at 1607 and 1245 cm-1 in the resonance Raman spectra of M . luteus cyano catalase serves to identify tyrosine as an axial ligand in bacterial as well as eukaryotic catalases . Unlike non-heme iron tyrosinate proteins, whose resonance Raman spectra are dominated by several intense bands diagnostic of tyrosine ligation, the heme-linked tyrosine modes are not easily distinguished from the large number of porphyrin vibrations. J Biol Chem, 1989 Aug 5, 264(22), 13093 - 101 The actions of Neurospora endo-exonuclease on double strand DNAs; Fraser MJ et al.; Neurospora crassa endo-exonuclease, an enzyme implicated in recombinational DNA repair, was found previously to have a distributive endonuclease activity with a high specificity for single strand DNA and a highly processive exonuclease activity . The activities of endo-exonuclease on double strand DNA substrates have been further explored . Endo-exonuclease was shown to have a low bona fide endonuclease activity with completely relaxed covalently closed circular DNA and made site-specific breaks in linear double strand DNA at a low frequency while simultaneously generating a relatively high level of single strand breaks (nicks) in the DNA . Sequencing at nicks induced by endo-exonuclease in pBR322 restriction fragments showed that the highest frequency of nicking occurred at the mid-points of two sites with the common sequence, p-AGCACT-OH . In addition, sequencing revealed less frequent nicking at identical or homologous hexanucleotide sequences in all other 54 cases examined where these sequences either straddled the break site itself or were within a few nucleotides on either side of the break site . The exonucleolytic action of endo-exonuclease on linear DNA showed about 100-fold preference for acting in the 5' to 3' direction . Removal of the 5'-terminal phosphates substantially reduced this activity, internal nicking, and the ability of endo-exonuclease to generate site-specific double strand breaks . On the other hand, nicking of the dephosphorylated double strand DNA with pancreatic DNase I stimulated the exonuclease activity by almost 5-fold, but no stimulation was observed when the DNA was nicked by Micrococcal nuclease . Thus, 5'-p termini either at double strand ends or at nicks in double strand DNA are entry points to the duplex from which endo-exonuclease diffuses linearly or "tracks" in the 5' to 3' direction to initiate its major endo- and exonucleolytic actions . The results are interpreted to show how it is possible for endo-exonuclease to generate single strand DNA for switching into a homologous duplex either at a nick or while remaining bound at a double strand break in the DNA . Such mechanisms are consistent with current models for recombinational double strand break repair in eukaryotes. J Antimicrob Chemother, 1989 Aug, 24(2), 131 - 45 Inhibition of bacterial isoprenoid synthesis by fosmidomycin, a phosphonic acid-containing antibiotic; Shigi Y; The mechanism of action of fosmidomycin (FR-31564), a phosphonic acid containing antibiotic, was examined against Escherichia coli, Micrococcus luteus and other bacteria . It converted growing Esch . coli cells into spheroplasts or swollen cells, but did not inhibit the enzymatic reactions of cell wall synthesis in ether-treated cells . Unlike bicyclomycin, phenethyl alcohol and mitomycin C, it did not reduce the amount of envelope lipoprotein, phospholipids, or DNA, but did reduce the amount of menaquinones and ubiquinones in growing Esch . coli cells . It also inhibited the biosynthesis of both carotenoids and menaquinones in M . luteus cells, suggesting that inhibition of the biosynthesis of a common precursor of these isoprenoids (possibly farnesyl pyrophosphate) might be the main site of its antibacterial activity. J Cell Sci, 1989 Aug, 93 ( Pt 4), 593 - 603 Inhibitors of topoisomerases I and II arrest DNA replication, but do not prevent nucleosome assembly in vivo; Annunziato AT; Specific inhibitors of eukaryotic DNA topoisomerases I and II (camptothecin and VM-26, respectively) were used to examine the involvement of topoisomerases in DNA replication and chromatin assembly in vivo . When used singly, either camptothecin or VM-26 inhibited DNA synthesis in HeLa cells by more than 80%; when used simultaneously, the inhibitors effectively stopped replication, demonstrating that at least one class of topoisomerase must be active for fork propagation in vivo . To study nucleosome assembly during topoisomerase inhibition, three experimental strategies were employed: (1) pulse-chase experiments; (2) analyses of chromatin synthesized during residual replication in the presence of either camptothecin or VM-26; and (3) the assembly of previously replicated, unassembled DNA, generated in the presence of protein synthesis inhibitors . Using sensitivity to micrococcal nuclease and the maturation of non-nucleosomal replication intermediates as criteria, neither camptothecin nor VM-26, alone or in concert, inhibited nucleosome assembly under any experimental protocol tested . These data provide evidence that, although topoisomerase activity is essential for DNA replication, neither continuous fork propagation nor topoisomerase activity is required for chromatin assembly on new DNA. Shika Kiso Igakkai Zasshi, 1989 Aug, 31(4), 417 - 26 Intranuclear androgen-receptor complex binding sites of mouse submandibular gland; Sato N et al.; Nuclear androgen-receptor binding sites in chromatin regions of mouse submandibular gland were studied . Cytosol androgen receptors prelabeled with {3H}androgen interacted with the crude nuclei in mouse submandibular gland and activation of the receptor was a prerequisite for this interaction . After in vivo administration of {3H}androgen to female mice, radioactivities were found in the nuclei purified from submandibular gland tissues and the {3H}androgen-labeled purified nuclei were further digested with micrococcal nuclease . The androgen receptor was found in solubilized, active chromatin fractions which contained mono- and dinucleosomes . By an in vitro exchange assay, endogenous androgen-receptor complexes associated with chromatin binding sites in intact males were found in the solubilized fraction after micrococcal nuclear digestion, whereas such complexes were not found in females . These results suggest that the androgen receptors translocated to the nuclei and became associated with chromatin and that this association occurred in transcriptionally active chromatin regions that were preferentially sensitive to micrococcal nuclease. Int J Biol Macromol, 1989 Aug, 11(4), 241 - 8 Micrococcal nuclease digestion study of spermidine-condensed DNA; Marx KA et al.; Spermidine-condensed lambda DNA tertiary structures have been studied by micrococcal nuclease digestion . Broad but discrete DNA bands were observed in gel electrophoresis experiments of digests at sizes of: 1003 +/- 115 bp, 1972 +/- 190 bp and 3100 +/- 350 bp . These bands comprise an arithmetic series, similar to, but larger than, arithmetic DNA band series sizes we have observed previously in calf thymus and phi x-174 DNA condensates . The 1003bp monomer lambda DNA band size corresponds to wrapping B DNA once circumferentially about the toroidal-shaped tertiary structures, the predominant condensed structures present in these preparations, and is consistent with the measured electron microscopic dimensions for hydrated lambda DNA toruses previously presented . DNA fragment length stability was determined by release from the digested condensates . Fragments of 80-85bp and sizes below are thermodynamically unstable in the lambda DNA condensates . This fragment size agrees well with a recent determination of the cooperativity size in DNA condensates. Cell Biophys, 1989 Aug-Oct, 15(1-2), 149 - 57 A possible role of chromatin and tightly-bound chromatin proteins on enzyme-catalyzed methylation of DNA; Strom R et al.; Upon extensive digestion with DNAaseI of placenta chromatin matrix, previously "stripped" from its loosely-bound components by high-salt extraction, a fraction is obtained that contains almost no endogenous DNA methylase activity but whose DNA, if still included in this whole fraction--not if it has been purified to a protein-free condition--is a good substrate for externally added enzyme . This chromatin matrix can even cause a significant stimulation of methylation of single-stranded Micrococcus luteus DNA by placental methylase . In vivo, this phenomenon may have possible counterparts in the existence of highly-methylated regions of chromatin loops that appear to be protected by tightly-bound protein components from digestion of the "stripped loops" with DNAaseI. Biochemistry, 1989 Jul 25, 28(15), 6367 - 73 Primary structure and androgen regulation of a 20-kilodalton protein specific to rat ventral prostate; Ho KC et al.; Nuclear and cytosolic forms of a 20-kdalton rat ventral prostate protein were purified and partially sequenced from their N-termini . Isolated nuclei were treated with micrococcal nuclease and extracted in 0.6 M NaCl, and proteins were separated by affinity chromatography on Matrex gel green A, ammonium sulfate fractionation, and fast protein liquid chromatography on Superose 12 . The 43 amino acid N-terminal sequence of the nuclear 20-kdalton protein was identical with the cytosolic protein except it lacked 7 N-terminal amino acids present in the cytosolic form . The DNA sequence of a full-length complementary DNA clone isolated from a ventral prostate gt11 library extended the N-terminal sequence of the cytosolic form by an additional nine amino acids from the predicted initiation methionine . The cDNA included the nucleotide sequence for the 43 amino acid N-terminal sequence of the purified 20-kdalton protein and predicted molecular weights of 16,686, 17,521, and 18,650, respectively, for the nuclear, cytoplasmic, and nonprocessed proteins . Northern blot analyses of reproductive tract tissue RNAs using the 20-kdalton protein cDNA as probe revealed a single mRNA species of 0.92 kb detectable only in extracts of rat ventral prostate . Expression of the 0.92-kb mRNA was androgen dependent since the mRNA was undetectable in extracts obtained 4 days after castration and was restored 16 h after restimulation with androgen. Nucleic Acids Res, 1989 Jul 11, 17(13), 5017 - 28 Micrococcal nuclease as a probe for bound and distorted DNA in lac transcription and repression complexes; Zhang L et al.; Micrococcal nuclease (MNase) is used to probe the structure of transcription and repression complexes at the lac regulatory region in vitro . Both the lac operator, 01, and the pseudo-operator, 03, are found to be protected from MNase digestion by the lac repressor on supercoiled DNA, and hypersensitive sites appear on both strands around nucleotide (nt) -26 between 01 and 03 . This hyperreactive site is coincident with the site of the DNA kink shown previously to form within a loop caused by simultaneous repressor binding to 01 and 03 . MNase hypersites are also observed both upstream from cAMP receptor protein (CRP) and downstream from bound RNA polymerase in open promoter complexes . In both open and closed complexes the binding of polymerase partially protects the backbone from MNase attack . Catabolite activator protein is shown to be required for both closed and open complex formation . Taken together with previous footprinting data, the results suggest that lac transcription complexes involve DNA bent towards a protein core consisting of RNA polymerase and catabolite activator protein. FEBS Lett, 1989 Jul 3, 250(2), 419 - 24 Organization of the thyroid hormone receptor in the chromatin of C6 glial cells: evidence that changes in receptor levels are not associated with changes in receptor distribution; Ortiz-Caro J et al.; The association of {125I}T3-receptor complexes with C6 cell chromatin was analyzed after a limited digestion with micrococcal nuclease (MN) or DNase I . Both nucleases solubilized up to 60-70% of receptor and 0.4 M KCl extracted 70% of the non-digested receptor, thus showing that only a residual fraction of receptor is associated with the nuclear matrix . With DNase I the receptor was released 2-3-fold faster than the bulk of chromatin, whereas a preferential release of receptor over total chromatin was not observed with MN . The digestion of receptor with DNase I and MN occurred 14- and 6-fold faster, respectively, than the appearance of PCA-soluble chromatin . Preincubation for 48 h with 4 nM T3 of 2 mM butyrate significantly altered receptor levels but did not change sensitivity to the nucleases . These results suggest that the thyroid hormone receptor is associated with chromatin highly sensitive to nuclease digestion, and that changes in receptor number are not associated with changes in its distribution in chromatin. Anal Biochem, 1989 Jul, 180(1), 186 - 91 Quantitative determination of phospholipids using the dyes Victoria blue R and B; Eryomin VA et al.; Different phospholipids, except the choline-containing phospholipids phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, formed complexes with the dye Victoria blue R, which selectively partitioned into the chloroform phase of chloroform/ethylene glycol/glycerol biphasic solvent system, and were quantitatively estimated at 590 nm . Considerable amounts of water, alcohols, nonlipid phosphates, neutral lipids, free fatty acids, and some detergents did not interfere with the formation of phospholipid-dye complexes . This special advantage of the method described allowed combined phospholipid extraction and estimation procedures in one test tube . Because of its high sensitivity (about 24.00 OD units/mumol of phosphatidic acid and about 10.25 OD units/mumol of other phospholipids), specificity, and simplicity, the proposed phospholipid assay appears to be very useful for rapid analyses of lipid extracts as well as TLC spots or suspensions of biological materials, as demonstrated for membranes and cells of Micrococcus lysodeikticus . The applicability of the dye Victoria blue B to the quantitative determination of phospholipids, except phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, at 605 nm using chloroform/ethylene glycol/glycerol/water and pentane (hexane)/ethyl acetate/isopropanol/water biphasic solvent systems with similar sensitivities and of sodium dodecyl sulfate in the pentane-containing system with high sensitivity (22.96 OD units/mumol) is also shown . The adaptation of this phospholipid assay to the determination of phospholipases C and D and to the differential quantitation of choline-containing phospholipids using additional phospholipid estimation techniques is discussed. J Biochem (Tokyo), 1989 Jul, 106(1), 29 - 33 Assembly of transcriptionally inactive chromatin in vitro; Shanahan MM et al.; We have successfully uncoupled the previously interlocked activities of chromatin assembly and in vitro transcription promoted by the Xenopus oocyte S-150 cell-free extract . Our isolated fraction catalyzes extensive chromatin assembly measured both by changes in DNA topology and Micrococcal nuclease digestions . The assembly of chromatin is slowed by the exogenous addition of ATP . In the absence of exogenously added ATP, the fraction forms a chromatin template that is transcriptionally inert . Addition of small amounts of the HeLa cell extract (S-100) converts these templates into transcriptionally active ones without disrupting the chromatin structure . Our protocol defines a method for the isolation of a fraction from the Xenopus cell free extract that catalyzes the assembly of transcriptionally inactive chromatin . We characterize this reaction and establish conditions for the transcriptional activation of these inactive minichromosomes. Cell Differ Dev, 1989 Jul, 27(2), 137 - 46 Lens fiber differentiation correlated with activation of two different DNAases in lens embryonic cells; Counis MF et al.; In order to identify the different DNAases present in the lens differentiating tissue, we have used an assay which reveals their activity directly on DNA-containing gels after SDS polyacrylamide gel electrophoresis . DNAase renaturation from nuclear embryonic lens extracts does not occur after separation in 0.1% SDS polyacrylamide gel electrophoresis in contrast to that observed with purified micrococcal nuclease . When the SDS concentration in the running buffer and separating gel is decreased to 0.075%, renaturation of lens DNAase and enzyme activities are observed . Isoelectrofocusing was carried out in a polyacrylamide gel which was overlaid with an agarose gel containing DNA, permitting the visualization of the pI of DNAase activity . The presence of several DNAase isoenzymes was demonstrated in 11-day embryonic lenses . In epithelial lens nuclei, high molecular weight (MW) isoenzymes with basic pI were predominant . In post-mitotic fiber lens nuclei, two lower MW isoenzymes with acidic pI were detected as well as high MW activity with a basic pI. Radiat Res, 1989 Jul, 119(1), 57 - 72 Alterations of neuronal nuclear matrix and chromatin structure after irradiation under aerobic and anoxic conditions; Jaberaboansari A et al.; This study was undertaken to determine if structural alterations of the bulk chromatin and the amount of protein associated with the nuclear matrix in cerebellar neurons depend on radiation dose and a cell's state of oxygenation . After irradiation with 2.5 to 25.0 Gy under both aerobic and anoxic conditions, the sensitivity of the neuronal chromatin to m . nuclease digestion increase linearly with dose up to about 5 Gy, beyond which there was no further increase . The same increase in accessibility of chromatin to micrococcal nuclease digestion was observed when neuronal nuclei were irradiated at 4 degrees C . Neuronal nuclei were stained with propidium iodide (PI) for DNA and with fluorescein isothiocyanate (FITC) for protein, both before and after complete digestion with DNase I, and analyzed by flow cytometry . There was no change in either the PI (P greater than 0.4) or the FITC (P greater than 0.9) fluorescence of undigested nuclei after irradiation . For the DNase I digested nuclei, the PI fluorescence was unchanged after irradiation (P greater than 0.4), but the FITC fluorescence increased significantly (P less than 0.02) . This increase in the FITC fluorescence was linear with dose up to about 5 Gy, beyond which there was no further increase . The flow cytometry results from DNase I digested nuclei were identical for neurons irradiated under aerobic or anoxic conditions, indicating that this phenomenon is oxygen independent . This increase in FITC fluorescence after irradiation was inhibited at ice-cold temperatures and probably reflects an increase in protein content at the nuclear matrix that requires metabolism . This may explain our previously observed resistance of nuclear matrix-associated DNA to digestion by DNase I . This protein increase at the nuclear matrix appears to follow "saturation" kinetics identical to that previously reported for repair of DNA strand breaks in cerebellar neurons . However, the exact molecular nature of this process and its role in DNA repair or cell survival remains to be determined. Carcinogenesis, 1989 Jul, 10(7), 1231 - 9 A new sensitive 32P-postlabeling assay based on the specific enzymatic conversion of bulky DNA lesions to radiolabeled dinucleotides and nucleoside 5'-monophosphates; Randerath K et al.; A new sensitive 32P-postlabeling assay for DNA adducts has been developed in which DNA is hydrolyzed initially by nuclease P1 and prostatic acid phosphatase instead of micrococcal nuclease and spleen phosphodiesterase as employed in previous postlabeling procedures . When DNA containing bulky adducts, X1, X2, .....Xn, is digested with nuclease P1 at pH 5, normal nucleotides are released as 5'-monophosphates, pN, while adducts are excised as 5'-phosphorylated dinucleotides, pXipN, because internucleotide linkages on the 3' side of X resist attack by nuclease P1 . Addition of prostatic acid phosphatase to such a digest results in 5'-dephosphorylation of the nucleotides to normal nucleosides, N, and adducted dinucleotides, XipN, carrying a 5'-terminal free hydroxyl group . The dinucleotides but not nucleosides are converted to 5'-32P-labeled dinucleotides, {32P}pXipN, by T4 polynucleotide kinase-catalyzed {32P}phosphate transfer from {gamma-32P}ATP . Upon mapping on polyethyleneimine--cellulose anion-exchange TLC, the labeled dinucleotide adducts produce characteristic autoradiographic fingerprints . Alternatively, they are further digested with snake venom phosphodiesterase to yield 5'-monophosphates, {32P}pXi and pN . TLC profiles of the monophosphate adducts are distinct from those of the dinucleotides . These reactions provide the basis of the new 32P-postlabeling scheme, which is compared in this paper with a previously reported protocol yielding adducts in the form of 5'-32P-labeled 3',5'-bisphosphates, {32P}pXip . The results show that the availability of three different types of 32P-postlabeled derivatives for the same adduct aids in the analysis and chromatographic characterization of DNA adducts from diverse exogenous and endogenous sources. Mol Cell Biol, 1989 Jul, 9(7), 3136 - 42 Major histocompatibility complex class I genes in murine fibrosarcoma IC9 are down regulated at the level of the chromatin structure; Maschek U et al.; The fibrosarcoma IC9 is deficient in the expression of the major histocompatibility complex class I genes Kb, Kk, and Dk and expresses only the Db molecule . Because class I deficiency may enable tumor cells to escape the immune response by cytotoxic T lymphocytes, we investigated why the class I genes are not expressed . Expression of the silent class I genes could not be induced, but all known DNA-binding factors specific for class I genes could be detected in nuclear extracts of IC9 cells . After cloning of the silent Kb gene from the IC9 cells and subsequent transfection of this cloned Kb gene into LTK- and IC9 cells, normal Kb antigens were expressed on the cell surface of both cell lines . Digestion of the chromatin of IC9 cells with micrococcal nuclease and DNase I showed a decreased nuclease sensitivity of the silent class I genes in comparison with active genes and the absence of DNase I hypersensitive sites in the promoter region of the silent Dk gene . These findings demonstrate that class I expression is turned off by a cis-acting regulatory mechanism at the level of the chromatin structure. Eur J Biochem, 1989 Jul 1, 182(3), 687 - 98 Expression of muscle-specific proteins is necessary to regulate translation of the mRNA for a 40-kDa housekeeping polypeptide in rat L6 cells; Pramanik SK et al.; Translation of the mRNA for a housekeeping polypeptide of 40 kDa (P40) was found to be regulated under a variety of conditions in rat L6 cells . This mRNA was translated in the proliferating myoblasts but not in the non-proliferating myotubes . A number of chemicals, such as dimethyl sulfoxide, sodium butyrate and aphidicolin, were used to prevent expression of muscle-specific genes in mitogen-poor differentiation medium . In the absence of any detectable accumulation of muscle-specific alpha-actin mRNA, the P40 mRNA remained in the translated state . A fourth chemical, EGTA, a known inhibitor of fusion of muscle cells, blocked translation of muscle-specific actin and tropomyosin mRNAs . On the other hand, it showed no effect on the translation of P40 mRNA . Addition of Ca2+ to the EGTA-treated cultures, however, almost completely reversed the block of translation of actin tropomyosin mRNAs within four days . Concomitant to Ca2+ reversal of the translational block of muscle mRNAs, P40 mRNA entered the non-translated state . An inverse relationship, therefore, was observed between the translation of housekeeping P40 mRNA and muscle-specific mRNAs . The ability to mimic in vivo regulation of P40 mRNA translation was examined in mRNA-dependent micrococcal-nuclease-treated homologous cell-free extracts . The extracts from myoblasts and myotubes were able to translate P40 mRNA . Furthermore, ribosomes of both myoblast and myotube extracts containing endogenous mRNAs were also able to bind to P40 and actin mRNAs . Myotube extract, however, showed a lower binding ability to P40 mRNA than to the actin mRNA . The ability of ribosomes of myotube extract to bind P40 mRNA was somewhat enhanced by addition of proteins derived from washing these ribosomes with a high-ionic-strength buffer . In order to elucidate the role of interaction between mRNA and proteins in translational control of P40 mRNA, the polypeptide complements of polysomal and free P40 mRNA-protein (mRNP) complexes were also examined . Hybrid selection of polysomal and free P40 mRNP complexes followed the covalent joining of the RNA and protein moieties of mRNP complexes by ultraviolet irradiation of rat L6 cells . Analysis of buoyant densities of these complexes showed that free P40 mRNP had slightly less protein than polysomal P40 mRNP . Furthermore, analysis of the polypeptide complements of both free and polysomal P40 mRNP complexes showed that they were composed of identical polypeptides . The only detectable difference between the polypeptide complements of these complexes was that two polypeptides of 72 kDa and 55 kDa were more abundant in the polysomal P40 mRNP than free P40 mRNP. Biochemistry, 1989 Jun 27, 28(13), 5670 - 80 Binding of adenosine diphosphoribosyltransferase to the termini and internal regions of linear DNAs; Sastry SS et al.; Binding mechanisms of ADPR-transferase to restricted double-stranded DNA fragments of SV40 and pBR322 DNA were determined by nuclease protection techniques . Top and bottom strands of double-stranded DNA were identified by specific labeling with 32P . Protection against specific exonucleases identified binding of ADPR-transferase to DNA termini, whereas binding to internal regions of linear DNAs was probed by protection against endonucleases . ADPR-transferase protein protected against exonucleolytic attack from lambda exo and exoIII in all DNA fragments tested, demonstrating that the enzyme protein binds indiscriminately to all DNA termini . Extending earlier results {Sastry, S.S., & Kun, E . (1988) J . Biol . Chem . 263, 1505-1512}, the modifying effect of the binding of ADPR-transferase to DNA induced changes in DNA conformation, as evident from altered pause sites that appeared following digestion of DNA fragments by lambda exonuclease in the presence of ADPR-transferase . In contrast to the nonselective binding of ADPR-transferase to DNA termini, ADPR-transferase conferred protection endonuclease attack (DNase I and micrococcal nuclease) only to the 209-bp EcoRI-PstI SV40 DNA fragment . These results indicate that binding of ADPR-transferase to relatively rare internal regions of restricted DNA fragments exhibits some degree of specificity . Specificity of binding appears to be related to the coincidental relative A+T-rich regions in DNA, and to DNA bending, both identified in the 209-bp SV40 DNA fragment . Synthetic polydeoxyribonucleotides containing dA-dT bind ADPR-transferase stronger than polydeoxyribonucleotides containing dG-dC . It was deduced from endonuclease protection patterns that binding of the enzyme protein leaves no defined footprints on the 209-bp SV40 DNA fragment, but there is significant modification of DNA structure following binding of the enzyme protein . Methylation protection assays and the prevention of the binding of ADPR-transferase to T4 DNA by its glucosylation indicate that the enzyme binds in the major groove of DNA . The 36-kDa A peptide fragment of ADPR-transferase {Buki, K . G., & Kun, E . (1988) Biochemistry 27, 5990-5995} exhibits the same protection against endonucleolytic enzymes as the intact ADPR-transferase molecule. Nucleic Acids Res, 1989 Jun 26, 17(12), 4817 - 28 U2 small nuclear RNP assembly in vitro; Kleinschmidt AM et al.; Incubation of a SP6-transcribed human U2 RNA precursor molecule in a HeLa cell S100 fraction resulted in the formation of ribonucleoprotein complexes . In the presence of ATP, the particles that assembled had several properties of native U2 snRNP, including resistance to dissociation in Cs2SO4 gradients, their buoyant density, and pattern of digestion by micrococcal nuclease . These particles also reacted with Sm monoclonal antibody and a human autoantibody with specificity for the U2 snRNP-specific proteins A' and B", but not with antibodies for U1 snRNP-specific proteins . In contrast, the particles that formed in the absence of ATP did not have these properties . ATP analogs with non-hydrolyzable beta-gamma bonds did not substitute for ATP in U2 snRNP assembly . Additional experiments with a mutant U2 RNA confirmed that nucleotides 154-167 of U2 RNA are required for binding of the U2 snRNP-specific proteins but not of the "Sm" core proteins . Pseudouridine formation, a major post-transcriptional modification of U2 RNA, was enhanced under assembly permissive conditions. Nucleic Acids Res, 1989 Jun 26, 17(12), 4647 - 60 In vitro 3' end processing and poly(A) tailing of RNA in Trypanosoma cruzi; Zwierzynski TA et al.; Pre-mRNA in kinetoplastids is processed to maturity following unique pathways requiring a transplicing event that links a common 39 nucleotide leader to the 5' termini of the mature mRNAs . The mechanisms of this reaction and other steps of mRNA processing; i.e., 5' capping and 3' cleavage and polyadenylation, have not been resolved . Herein, we describe a 3' polyadenylation activity in cell-free extracts prepared from nuclei isolated from Trypanosoma cruzi, the kinetoplastid agent of Chagas' Disease . Synthetic RNA transcripts incubated in these extracts in the presence of ATP are 3' polyadenylated . This polyadenylation activity is sensitive to heat or pre-treatment of the extract with Micrococcal nuclease, suggesting that an RNA-protein complex is required . As these are characteristics of polyadenylation activities in other eukaryotes, we believe that this activity may participate in the in vivo trypanosome mRNA polyadenylation system . Several other modification activities specific for RNA 3' termini, including terminal nucleotide transferases, a tRNA CCA maturation activity, and a 3' exonuclease were also identified in these T . cruzi nuclear extracts. Biochem J, 1989 Jun 15, 260(3), 697 - 704 Polyamine depletion is associated with altered chromatin structure in HeLa cells; Snyder RD; HeLa cells depleted of polyamines by treatment with alpha-difluoromethylornithine (DFMO), methylglyoxal bis(guanylhydrazone) (MGBG) or a combination of the two, were examined for sensitivity to micrococcal nuclease, DNAase I and DNAase II . The degrees of chromatin accessibility to DNAase I and II appeared enhanced somewhat in all three treatment groups, and the released digestion products differed from those in non-depleted cells . DNA released from MGBG- and DFMO/MGBG-treated cells by DNAase II digestion was enriched 4-7-fold for Mg2+-soluble species relative to controls . DNA released by micrococcal nuclease digestion from all three treatment groups was characterized as consisting of higher-order nucleosomal structure than was DNA released from untreated cells . At least some of the altered chromatin properties were abolished by a brief treatment of cells with polyamines, notably spermine . These studies provide the first demonstration in vivo of altered chromatin structure in cells treated with inhibitors of polyamine biosynthesis. J Biol Chem, 1989 Jun 15, 264(17), 9891 - 6 Tissue-specific undermethylation of DNA sequences at the 5' end of the human apolipoprotein B gene; Levy-Wilson B et al.; The methylation-sensitive restriction endonucleases HpaII and HhaI and the methylation-insensitive enzyme MspI were used to examine the methylation status of the 5' end of the human apolipoprotein B gene in cells in which the gene is transcriptionally active, such as HepG2 and CaCo-2 cells, and in HeLa cells, which do not express the gene . Several CCGG and GCGC sequences present in the region from -899 to +121 relative to the transcriptional start of the gene were undermethylated in HepG2 and CaCo-2 cells but methylated in HeLa cells . Because the region between -899 and +121 had previously been shown to exhibit tissue-specific promoter activity and to contain many DNaseI- and micrococcal nuclease-hypersensitive sites (Levy-Wilson, B., Fortier, C., Blackhart, B.D., and McCarthy, B.J . (1988) Mol . Cell . Biol . 8, 71-80), the current results suggest a correlation between undermethylation and nuclease sensitivity at the 5' end of the apolipoprotein B gene and expression of the gene. J Mol Biol, 1989 Jun 5, 207(3), 615 - 9 Linkage reduction allows reconstitution of nucleosomes on DNA microdomains; Negri R et al.; We have established an experimental system for reconstitution of an individual nucleosome on a closed DNA microdomain (operationally defined as a DNA domain of a size so small as to be unable to establish titratable superhelical turns) . The microdomain (185 base-pairs (bp), composed of 128 bp encompassing the central part of the Saccharomyces cerevisiae ADH II promoter plus 57 bp of a polylinker) was obtained by ligation under conditions that produced three circularized forms characterized by different linkage numbers . These linkomers were tested for nucleosome reconstitution with S . cerevisiae histones . It was observed that only microcircles with linkage reduction (delta Lk = 1 or 2) could form a nucleosome, as defined by protection of a 145(+/- 2) bp DNA fragment from micrococcal nuclease, relaxed forms (open or closed circles) could not. Microbiologia, 1989 Jun, 5(1), 53 - 5 Actual identity of six micrococcal strains selected as potential starter for dry fermented sausages production; Selgas MD et al.; The DNA guanine + cytosine contents of six strains previously selected as potential starters for their use for the dry fermented sausages production have been determined . Five strains were characterized as Micrococcus spp . and the remainder as incertae sedis. Exp Cell Res, 1989 Jun, 182(2), 436 - 44 Analysis of supra-nucleosome particles from unfertilized eggs of sea urchins; Imschenetzky M et al.; Two discrete supranucleosomal particles that differ in their electrophoretic migration on 1% agarose gels were isolated from unfertilized sea urchin eggs . Both particles contain the same complement of cleavage stage (CS) chromosomal proteins, which is identical to the complete set of basic proteins isolated directly from chromatin by extraction with 0.25 N HCl . DNA fragments between 210 and 1500 bp were found in both particles, and the basic unit of DNA repeat length determined by micrococcal nuclease digestion was 126 +/- 3 bp . The isolated nucleoparticles are electrophoretically stable over a wide range of DNA sizes (126-1500 bp) indicating that their structure is maintained by internal interactions among the CS chromosomal proteins, previously designated CS A through CS G . Based on these results we conclude that the CS chromosomal proteins are functionally equivalent to classical histones in their ability to direct higher ordered structures of chromatin. Oncogene, 1989 Jun, 4(6), 691 - 7 The c-ets-1 protein is chromatin associated and binds to DNA in vitro; Pognonec P et al.; The c-ets-1 proto-oncogene is expressed at high levels in proliferating lymphoid cells . We show here that the chicken and murine c-ets-1 proteins are predominantly localized in the cell nucleus . Over 90% of the c-ets-1 protein can be released from isolated thymocyte nuclei by treatment with low salt buffer . Release from nuclei is also observed after treatment with micrococcal nuclease, but not with RNaseA, in conditions where digestion of only a minor fraction of chromatin occurs . c-ets-1 proteins exhibit DNA binding activity, suggesting that the association to chromatin is mediated at least in part by their association to DNA . We previously showed that mitogenic stimulation of thymocytes is accompanied by the rapid calcium-dependent phosphorylation of c-ets-1 proteins . We demonstrate here that these phosphorylation events abolish the ability of c-ets-1 proteins to bind to DNA in vitro and reduce their affinity for chromatin, lending further support to the importance of these modifications in the regulation of c-ets-1 protein function. Mol Gen Genet, 1989 Jun, 217(2-3), 332 - 40 Micrococcus luteus homolog of the Escherichia coli uvrA gene: identification of a mutation in the UV-sensitive mutant DB7; Shiota S et al.; Restriction fragments of Micrococcus luteus DNA containing the gene affected by a mutation in the UV-sensitive mutant DB7 were cloned both from the wild type and from the mutant in an Escherichia coli host-vector system . The wild-type fragment was able to reverse the multiple sensitivity of the mutant to UV, mitomycin C, and 4-nitroquinoline 1-oxide by a one-step transformation . Determination of the nucleotide sequences revealed a potential open reading frame coding for a protein of 992 (tentative) amino acid residues, within which the DB7 mutation was identified as a CG-to-TA transition causing a translation termination . The putative product of the open reading frame shares an extensive amino acid sequence homology with the E . coli UvrA protein comprising 940 residues . The homology extends over the greater part of both polypeptides except for two extra sequences of 31 and 24 amino acid residues located at the amino-terminal and in the interior, respectively, of the M . luteus protein . In the homologous region, 56.7% and 16.7% of the 933 pairs of the aligned amino acids were accounted for by conserved residues and conservative substitutions, respectively . These results indicate that the gene defined by the mutation in DB7 represents a homolog of the E . coli uvrA gene . Hence, it has to be concluded that DB7, known for its deficiency in UV endonuclease (pyrimidine dimer DNA glycosylase/apurinicapyrimidinic endonuclease) activity, is a double mutant which is also defective in an enzyme complex similar to the E . coli UvrABC excinuclease. Exp Hematol, 1989 Jun, 17(5), 405 - 10 Hemin and cyclic AMP stimulate message-dependent translation in lysates from Friend erythroleukemia cells; Chinchar VG et al.; A message-dependent, cell-free translation system was prepared from both uninduced and N,N'-hexamethylene-bis-acetamide-induced Friend erythroleukemia cells following modification of standard protocols . Active extracts were prepared by lysing Friend erythroleukemia cells in hypotonic buffer containing 50 microM hemin and 10 mM cyclic adenosine monophosphate (cAMP), and assaying translation in vitro in the absence of exogenous nucleoside triphosphates . Both hemin and cAMP were required for full activity and appeared to act by prolonging initiation in vitro . Following treatment of cell extracts with micrococcal nuclease, brome mosaic virus, globin, equine herpesvirus type 1, and frog virus 3 mRNA were efficiently translated to yield full-sized products . Using brome mosaic virus as a test message, levels of translation equivalent to 30%-50% of that seen in mock-treated lysates were obtained . These results attest to the translational efficiency of extracts prepared and assayed in the manner described above, and suggest that such extracts may be useful both for routine translational studies, and as a tool to dissect translational changes accompanying erythroid differentiation in vitro. J Bacteriol, 1989 Jun, 171(6), 3530 - 8 Rolling-circle replication of UV-irradiated duplex DNA in the phi X174 replicative-form----single-strand replication system in vitro; Shavitt O et al.; Cloning of the phi X174 viral origin of replication into phage M13mp8 produced an M13-phi X174 chimera, the DNA of which directed efficient replicative-form----single-strand rolling-circle replication in vitro . This replication assay was performed with purified phi X174-encoded gene A protein, Escherichia coli rep helicase, single-stranded DNA-binding protein, and DNA polymerase III holoenzyme . The nicking of replicative-form I (RFI) DNA by gene A protein was essentially unaffected by the presence of UV lesions in the DNA . However, unwinding of UV-irradiated DNA by the rep helicase was inhibited twofold as compared with unwinding of the unirradiated substrate . UV irradiation of the substrate DNA caused a strong inhibition in its ability to direct DNA synthesis . However, even DNA preparations that contained as many as 10 photodimers per molecule still supported the synthesis of progeny full-length single-stranded DNA . The appearance of full-length radiolabeled products implied at least two full rounds of replication, since the first round released the unlabeled plus viral strand of the duplex DNA . Pretreatment of the UV-irradiated DNA substrate with purified pyrimidine dimer endonuclease from Micrococcus luteus, which converted photodimer-containing supercoiled RFI DNA into relaxed, nicked RFII DNA and thus prevented its replication, reduced DNA synthesis by 70% . Analysis of radiolabeled replication products by agarose gel electrophoresis followed by autoradiography revealed that this decrease was due to a reduction in the synthesis of progeny full-length single-stranded DNA . This implies that 70 to 80% of the full-length DNA products produced in this system were synthesized on molecules that carried photodimers . Thus, similarly to its activity on UV-irradiated single-stranded DNA, DNA polymerase III holenzyme can bypass pyrimidine photodimers in the more complex replicative form --->single-strand replication, which involves, in addition to the polymerizing activity, the unwinding of the duplex by the rep helicase and the participation of a more complex multiprotein replisome. Antibiot Khimioter, 1989 Jun, 34(6), 425 - 9 {Study of the adaptation resistance in bacteria to membrane active polypeptide antibiotics}; Bulgakova VG et al.; Variants of Micrococcus lysodeikticus resistant to 100 micrograms/ml of gramicidin S with preserved resistance in subcultures on media without the antibiotic were isolated as a result of prolonged adaptation on a solid medium with increasing concentrations of gramicidin . The sensitive and resistant cells did not differ by their ability to bind gramicidin . Under the antibiotic effect permeability of the cytoplasmic membranes of the intact cells in the sensitive bacteria appeared to be impaired to a greater extent than that of the membranes of the cells in the resistant variant . Comparison of the lytic activity of gramicidin and its derivatives with respect to the protoplasts prepared with the cells of the initial and resistant variants of M . lysodeikticus revealed much higher resistance of the resistant variant protoplasts to the membrane-disorganizing effect of the preparations . Malate dehydrogenase and NADH-oxidase in the membrane preparations of the resistant variant cells differed from analogous enzymes from the membranes of the initial strain by the levels of their activity and sensitivity to gramicidin . It is likely that during adaptation of M . lysodeikticus to gramicidin significant changes in the cell cytoplasmic membranes occurred. J Mol Biol, 1989 May 5, 207(1), 183 - 92 Formation, stability and core histone positioning of nucleosomes reassembled on bent and other nucleosome-derived DNA; Pennings S et al.; DNA originating from chicken erythrocyte mononucleosomes was cloned and sequenced . The properties of nucleosome reconstruction were compared for two cloned inserts, selected on account of their interesting sequence organization, length and difference in DNA bending . Cloned fragment 223 (182 base-pairs) carries alternatively (A)3-4 and (T)4-5 runs approximately every ten base-pairs and is bent; cloned fragment 213 (182 base-pairs) contains a repeated C4-5ATAAGG consensus sequence and is apparently not bent . Our experiments indicate the preference of the bent DNA fragment 223 over fragment 213 to associate in vitro with an octamer of histones under stringent conditions . We provide evidence that the in vitro nucleosome formation is hampered in the case of fragment 213, whereas the reconstituted nucleosomes were equally stable once formed . For the correct determination of the positioning of the histone octamer with regard to the two nucleosome-derived cloned DNA sequences, the complementary use of micrococcal nuclease, exonuclease III and DNase I is a prerequisite . No unique, but rotationally related, positions of the histone octamer were found on these nucleosome-derived DNA fragments . The sequence-dependent anisotropic flexibility, as well as intrinsic bending of the DNA, resulting in a rotational setting of the DNA fragments on the histone core, seems to be a strong determinant for the allowed octamer positions, Exonuclease III digestion indicates a different histone-DNA association when oligo(d(C.G)n) stretches are involved . The apparent stagger near oligo(d(A.T)n) stretches generated by DNase I digestion on reconstituted nucleosome 223 was found to be inverted from the normal two-base 3' overhang to a two-base 5' overhang . Two possibilities of the oligo(d(A.T)n) minor groove location relative to the histone core are envisaged to explain this anomaly in stagger. Biochemistry, 1989 May 2, 28(9), 3728 - 37 Transverse and lateral distribution of phospholipids and glycolipids in the membrane of the bacterium Micrococcus luteus; de Bony J et al.; The photodimerization of anthracene was used to investigate the transverse and lateral distribution of lipids in the membrane of the Gram-positive bacterium Micrococcus luteus . 9-(2-Anthryl)nonanoic acid (9-AN) is incorporated at a high rate into various membrane lipids of M . luteus . On irradiation of intact bacteria at 360 nm, anthracene-labeled lipids form stable photodimers which can be extracted and separated by thin-layer chromatography . We present here the results of a study on the distribution of two major lipids, phosphatidylglycerol (PG) and dimannosyldiacylglycerol (DMDG), within each leaflet of the membrane lipid bilayer . After metabolic incorporation of a tritiated derivative of 9-AN in M . luteus, the radioactivity associated with the photodimers issued from PG and DMDG was counted . In the bacterial membrane, the ratio of PG-DMDG heterodimer with respect to PG-PG and DMDG-DMDG homodimers is around half of what should be obtained for a homogeneous mixture of the two lipids . In order to find out whether this was due to an asymmetric distribution of the two lipids between the two membrane leaflets or a heterogeneous distribution of the two lipids within the same membrane leaflet, the transverse distribution of PG and DMDG was also investigated . This was carried out by following the kinetics of oxidation of the two lipids by periodic acid in the membrane of M . luteus protoplasts . PG predominated slightly in the outer layer (60%), while DMDG was found to be symmetrically distributed between the two leaflets . By itself, this lipid asymmetry cannot account for the lipid distribution determined from the photodimerization experiments . This indicates that PG and DMDG are not homogeneously distributed in the plane of the bacterial membrane. J Cell Physiol, 1989 May, 139(2), 245 - 52 Detection of colony-stimulating factor messenger RNA in single T cells by in situ hybridization; Williamson DJ et al.; In situ hybridization has been used to study the accumulation of colony-stimulating factor (CSF) mRNA in single cells of a T lymphocyte clone (E9.D4) following antibody-mediated (F23.1) activation via the Ti-T3 complex in filler-independent bulk cultures . The specificity of hybridization for cellular RNA was demonstrated by pretreating the cells with the Ca2+-dependent enzyme micrococcal nuclease by using a novel protocol developed for use with riboprobes . Maximal levels of granulocyte-macrophage (GM) and multipotential-CSF (interleukin 3) mRNA were detected after 8-10 h, with GM-CSF mRNA being detected earlier and at a lower concentration of stimulus . The rise in intracellular mRNA was accompanied by an increase in the corresponding CSF bioactivity in the supernatant . In situ hybridization was of comparable sensitivity to Northern blot analysis and revealed significant heterogeneity in the accumulation of CSF mRNA within individual cells of the clone following stimulation with F23.1 . This could account for the corresponding heterogeneity in CSF production by single cells . Under optimal conditions at least 25% of cells contained both transcripts . The method has been used to examine CSF production by normal spleen cells and should be useful in the further analysis of lymphokine gene regulation in single T cells. Vopr Med Khim, 1989 May-Jun, 35(3), 112 - 6 {Modification of micrococcal histidine decarboxylase with tetranitromethane}; Gonchar NA et al.; Tetranitromethane inhibited distinctly the histidine decarboxylase activity at pH above 7.0 . Spectral and fluorescence properties as well as amino acid composition of the transformed enzyme were studied . Nitration of tyrosine residues occurred simultaneously with oxidation of cysteine in a molecule of histidine decarboxylase treated with tetranitromethane, while the other amino acids such as tryptophane, histidine and methionine were not altered . The histidine decarboxylase pretreated with dithiothreitol, became insensitive to the tetranitromethane effect but complete inactivation of the enzyme was observed under these conditions . Possible mechanisms of the histidine decarboxylase inactivation are discussed. Mol Cell Biol, 1989 May, 9(5), 1996 - 2006 Regulation of c-myc mRNA stability in vitro by a labile destabilizer with an essential nucleic acid component; Brewer G et al.; The turnover rates of some mRNAs vary by an order of magnitude or more when cells change their growth pattern or differentiate . To identify regulatory factors that might be responsible for this variability, we investigated how cytosolic fractions affect mRNA decay in an in vitro system . A 130,000 X g supernatant (S130) from the cytosol of exponentially growing erythroleukemia cells contains a destabilizer that accelerates the decay of polysome-bound c-myc mRNA by eightfold or more compared with reactions lacking S130 . The destabilizer is deficient in or absent from the S130 of cycloheximide-treated cells, indicating that it is labile or is repressed when translation is blocked . It is not a generic RNase, because it does not affect the turnover of delta-globin, gamma-globin, or histone mRNA and does not destabilize a major portion of polysomal polyadenylated mRNA . The destabilizer accelerates the turnover of the c-myc mRNA 3' region, as well as subsequent 3'-to-5' degradation of the mRNA body . It is inactivated in vitro by mild heating and by micrococcal nuclease, suggesting that it contains a nucleic acid component . c-myb mRNA is also destabilized in S130-supplemented in vitro reactions . These results imply that the stability of some mRNAs is regulated by cytosolic factors that are not associated with polysomes. Proc Natl Acad Sci U S A, 1989 May, 86(10), 3499 - 503 DNA strand breaks alter histone ADP-ribosylation; Boulikas T; Histone ADP-ribosylation was studied using two-dimensional gel electrophoresis after cleavage of the nuclear DNA with nucleases . Modified histones carrying different numbers of ADP-ribose groups form a ladder of bands above each variant histone . Cellular lysates containing unfragmented DNA mainly synthesize mono(ADP-ribosylated) histones . Cleavage of the DNA with either DNase I or micrococcal nuclease to fragments of an average size of 10-20 kilobases (kb) dramatically induces the formation of poly(ADP-ribosylated) species of histones in nuclei . As the number of DNA strand breaks produced by either DNase I or micrococcal nuclease increases and a great number of DNA cuts is introduced (fragments of 0.4-0.2 kb), the size of the poly(ADP-ribose) chains on the histones decreases . Finally, in the presence of 10 mM cAMP as an inhibitor of poly(ADP-ribose) glycohydrolase, human lymphoid nuclei synthesize hyper(ADP-ribosylated) histone H2B with at least 40 ADP-ribose groups attached to it . Lateral ladders emanating at precise points of the linear ladder on hypermodified H2B can arise from branching of poly(ADP-ribose) or from multiple monomodifications of glutamic (or aspartic) acid residues . Branching or de novo monomodifications occur after a precise number of ADP-ribose groups have been added to a histone molecule . Poly(ADP-ribosylated) histones thus appear to be intermediates in nuclear processes involving DNA strand breaks. Biochem Int, 1989 May, 18(5), 971 - 9 Release of nucleosomes from nuclei by bleomycin-induced DNA strand scission; Hamana K et al.; Mononucleosomes were released from both isolated mammalian (hog thyroid) and protozoan (Tetrahymena) nuclei by the bleomycin-induced DNA-strand breaking reaction . Trout sperm nuclei, on the other hand, were protected from the bleomycin-mediated DNA degradation . The mononucleosomes released from the bleomycin-treated nuclei contained the core histones H2A, H2B, H3, and H4; while HMG1 and HMG2 proteins, in addition to the core histones, were detected in the mononucleosomes obtained by micrococcal nuclease digestion of nuclei . HMGs, but not H1 histone, were dissociated into the supernatant by cleavage of chromatin DNA with bleomycin, whereas both HMGs and H1 were found in that fraction by digestion of nuclei with micrococcal nuclease . HMG1 and HMG2 were exclusively dissociated from chromatin with 1 mM bleomycin under the solvent condition where the DNA strand-breaking activity of the drug is repressed . These observations suggest the possibility that bleomycin preferentially binds to linker DNA regions not occupied by H1 histone in chromatin and exclusively dissociates HMG proteins and breaks the DNA strand . The results of the effects on bleomycin-induced DNA cleavage of nuclei of various drugs including polyamines, chelating agents, intercalating antibiotics such as mitomycin C or adriamycin, and radical scavengers are also presented. Biochem J, 1989 May 1, 259(3), 751 - 9 Bacteriophage-T4 and Micrococcus luteus UV endonucleases are not endonucleases but beta-elimination and sometimes beta delta-elimination catalysts; Bailly V et al.; Bacteriophage-T4 UV endonuclease nicks the C(3')-O-P bond 3' to AP (apurinic or apyrimidinic) sites by a beta-elimination reaction . The breakage of this bond is sometimes followed by the nicking of the C(5')-O-P bond 5' to the AP site, leaving a 3'-phosphate end; delta-elimination is proposed as a mechanism to explain this second reaction . The AP site formed when this enzyme acts on a pyrimidine dimer in a polynucleotide chain undergoes the same nicking reactions . Micrococcus luteus UV endonuclease also nicks the C(3')-O-P bond 3' to AP sites by a beta-elimination reaction . No subsequent delta-elimination was observed, but this might be due to the presence of 2-mercaptoethanol in the enzyme preparation. Biochem Biophys Res Commun, 1989 Apr 28, 160(2), 448 - 52 Formation of a stable and catalytically active complex of the two essential components of hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26; Yoshida I et al.; Formation of a stable complex of the two essential components of hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26, which represents the catalytically active state of this enzyme, is observed in the presence of a relatively high concentrations of inorganic pyrophosphate or one of the substrates, isopentenyl diphosphate or farnesyl diphosphate . The apparent molecular mass of the complex is estimated to be about 50 kDa by gel filtration with Superose 12. Biochim Biophys Acta, 1989 Apr 12, 1007(3), 318 - 24 Reconstitution of nucleosomes in vitro with a plasmid carrying the long terminal repeat of Moloney murine leukemia virus; Kimura T et al.; The potential of nucleosome assembly along the sequence of a plasmid carrying the long terminal repeat (LTR) and its flanking region of Moloney murine leukemia virus was analyzed by in vitro reconstitution experiments with histones from chicken erythrocytes . The results of electrophoretic mobility-shift and micrococcal nuclease-digestion assays indicated that the plasmid DNA contained four preferred sites for nucleosome formation . However, all of these sites were mapped on the vector moiety but not on the LTR moiety . Computer analysis of the sequences in the four preferred sites, each spanning about 150 bp, indicated that short runs of (dA,dT) containing two kinds of triplets, AAA/TTT and AAT/ATT, occurred frequently . Furthermore, many of these triplets tended to occur in the same side of the DNA helix, suggesting that DNA curvature was involved in the preferred sites for nucleosome assembly . Consistent was the observation that DNA fragments carrying these preferred sites showed anomalous electrophoretic mobilities at a low temperature. Nucleic Acids Res, 1989 Apr 11, 17(7), 2819 - 33 DNA topology of the ordered chromatin domain 5' to the human c-myc gene; Kumar S et al.; DNA restriction fragments located 5' to the human c-myc gene display anomalous electrophoretic mobility on polyacrylamide gels . Computer modeling of the c-myc flanking DNA suggests that the slow-moving DNA fragments spanning nucleotides -1690 to -1054 (relative to c-myc promoter P1) and -718 to -452 form large left handed superhelices or curved structures while the fast-moving DNA fragment spanning nucleotides -407 to +78 has an unusually straight structure . These analyses also predict a periodic array of localized regions of bending through the superhelical domains . Micrococcal nuclease digestion of isolated nuclei reveals that the slow-moving DNA fragments exist in an ordered chromatin structure stable to nuclease, whereas the digestion pattern of the fast-moving DNA fragment suggests a less ordered array of nucleosomes or a non-nucleosomal chromatin structure. Biochim Biophys Acta, 1989 Apr 6, 995(2), 138 - 43 Protection of hexaprenyl-diphosphate synthase of Micrococcus luteus B-P 26 against inactivation by sulphydryl reagents and arginine-specific reagents; Yoshida I et al.; Hexaprenyl-diphosphate synthase from Micrococcus luteus B-P 26 has been shown to comprise two essential components, designated as components A and B . Treatment of the synthase with sulphydryl reagents (N-ethylmaleimide, iodoacetamide or p-chloromercuribenzoate) or arginine-specific reagents (2,3-butanedione, 1,2-cyclohexanedione or phenylglyoxal) resulted in a rapid loss of the component B activity . In contrast, component A was resistant to treatment with such reagents, retaining the initial activity almost completely . Farnesyl diphosphate, isopentenyl diphosphate, farnesyl monophosphate and inorganic pyrophosphate protected the synthase against the inactivation by N-ethylmaleimide, farnesyl diphosphate being the most effective . The presence of Mg2+ was essential for the protection by isopentenyl diphosphate and inorganic pyrophosphate . For protection of the synthase activity against the inactivation by 2,3-butanedione, the presence of farnesyl diphosphate, isopentenyl diphosphate and Mg2+ was more effective than that of the individual substrates and Mg2+ . Inorganic pyrophosphate provided substantial protection . In the absence of component A, the component B activity was not protected by any substrates or its analogue . These results suggest that the catalytic site of the synthase is formed by cooperative interaction between components A and B, and that cysteine and arginine residues on component B play important roles in the synthase activity. Photochem Photobiol, 1989 Apr, 49(4), 419 - 22 More efficient excision repair of pyrimidine dimers in the specific DNA sequence than in the genome overall in goldfish cells; Komura J et al.; Excision repair of pyrimidine dimers induced by 254 nm UV was examined in the genome overall and in a specific sequence containing a transfected gene for hygromycin B resistance, in RBCF-1 cells derived from a goldfish, by the use of UV endonuclease of Micrococcus luteus and alkaline agarose gel electrophoresis . More than 40% of dimers were removed from the specific sequence, while about 20% were removed from the genome overall, within 24 h after exposure to UV (2.5-7.5 J/m2). Yeast, 1989 Apr, 5 Spec No, S525 - 35 Yeast as source of oncoproteins; Shalitin C; The properties of a Saccharomyces cerevisiae 20 kDa polypeptide (Yp20) and its relationship to human ras antigen were tested . Yp20 was isolated from commercial yeast cells by the procedure of Sommer (1978) . Proteins associated with yeast chromatin were released by micrococcal nuclease digestion and purified by sucrose gradient centrifugation . Rabbit polyclonal and mouse monoclonal antibodies specifically detecting the Yp20 antigen have been generated . We observed that Yp20 was recognized by anti-ras polyclonal and monoclonal antibodies . Mammalian Ha-ras and Ki-ras proteins were specifically detected by anti-Yp20 antibodies . Based on immunological cross-reactivities, we believe that Yp20 may share some homology with the yeast YP2 gene previously described . Anti-Yp20 antibodies will be used to isolate the gene that encodes the protein . Practical applications of our antibodies for the detection of tumor specific antigens will be discussed. Mol Cell Biol, 1989 Apr, 9(4), 1721 - 32 Upstream activation sequence-dependent alteration of chromatin structure and transcription activation of the yeast GAL1-GAL10 genes; Fedor MJ et al.; Conversion of the positioned nucleosome array characteristic of the repressed GAL1-GAL10 promoter region to the more accessible conformation of the induced state was found to depend on the upstream activation sequence, GAL4 protein, a positive regulator of transcription, and galactose, the inducing agent . The effect of the GAL4 protein-upstream activation sequence complex on the structure of adjacent chromatin required no other promoter sequences . Although sequences protected by histones in the repressed state became more accessible to micrococcal nuclease and (methidiumpropyl-EDTA)iron(II) cleavage following induction of transcription, DNA-protein particles containing these sequences retained the electrophoretic mobility of nucleosomes, indicating that the promoter region can be associated with nucleosomes under conditions of transcription activation. Zentralbl Bakteriol Mikrobiol Hyg {B}, 1989 Apr, 187(4-6), 404 - 13 {Efficient hygiene precautions in the household today}; Borneff J; The results of epidemiological investigations justifies the assumption that increasing health defects, especially enteritis infectiosa, are caused inter alia by inadequate hygienic conditions in households . The number of such diseases ranges between 100,000 and more than 1,000,000 cases per year in the FRG . Responsible for this development is a lack of information about the behaviour of microorganisms in the environment and its pathways of distribution . In addition risks are growing with the recommendation of cleansing methods, which had been adequate for the kitchen techniques in former centuries, but must fall under the conditions of the modern supply, processing and conservation . The described investigations are directed at the determination of the distribution of germs by working in normal household kitchens and at the effectiveness of surface-decontamination-cleansers (so-called FD-preparations) . Test principle was the production of a complete dinner by each of 79 housewives with use of minced meat, which was contaminated with micrococcus luteus . After final cleaning of the kitchen we determined the degree of contamination of surfaces, machines and of the components of the meal with use of rodacplates, swabs and quantitative cultures respectively . The experiments are completed by interviews with the housewives . The results let conclude that the use of household cleansers with germicidal properties even in the hand of housewives will reduce the distribution of unwanted microorganisms in the kitchens . In this respect surfaces, on which components of the meal are prepared, and the machines, like cutting machines, waring blenders a.o., are of utmost importance . Disinfections of other parts of the flats including toilets are unnecessary (exception: severe infectious diseases) . Therefore the use of FD-preparations outside of the kitchens is not required, but acceptable (it is not necessary to use a cleanser in the kitchen, another one in the toilet and a third one for the bath tube etc.) . We do not recommend adapting cleaning and disinfective methods of the hospitals to the normal households . Instead of medical disinfections one should use detergents with nontoxic germicidal additives (e.g . H2O2) in this area, which do not require changing the traditional cleaning techniques . They also should guarantee that cleanliness, absence of odor and minimization of germs are achieved . In addition normal kitchen soaps should be replaced by HD-preparations. J Biol Chem, 1989 Mar 25, 264(9), 5245 - 52 Factor C from rabbit liver . A new poly(dC) and poly{d(G-C)} template-selective stimulatory protein of DNA polymerases; Asna N et al.; We have undertaken a search for mammalian DNA-binding proteins that enhance the activity of DNA polymerases in a template sequence-specific fashion . In this paper, we report the extensive purification and characterization of a new DNA-binding protein from rabbit liver that selectively stimulates DNA polymerases to copy synthetic poly{d(G-C)} and the poly(dC) strand of poly(dC).poly(dG) as well as single-stranded natural DNA that contains stretches of oligo(dC) . The enhancing protein, a polypeptide of 65 kDa designated factor C, stimulates the copying of the two synthetic templates by Escherichia coli DNA polymerase I, Micrococcus luteus polymerase, and eukaryotic DNA polymerases alpha and beta, but not by avian myeloblastosis virus polymerase . Factor C, however, does not affect utilization by these polymerases of the poly(dG) strand of poly(dC).poly(dG), of poly(dC) primed by oligo(dG), or of poly(dA).poly(dT) and poly{d(A-T)} . With polymerase I, Michaelis constants (Km) of poly{d(G-C)} and of the poly(dC) strand of poly(dC).poly(dG) are decreased by factor C 37- and 4.7-fold, respectively, whereas maximum velocity (Vmax) remains unchanged . By contrast, neither the Km value of the poly(dG) strand of poly(dC).poly(dG) nor the Vmax value with this template is altered by factor C . Rates of copying of activated DNA, denatured DNA, or singly primed M13 DNA are not affected significantly by factor C . However, primer extension analysis of the copying of recombinant M13N4 DNA that contains runs of oligo(dC) within an inserted thymidine kinase gene shows that factor C increases processivity by specifically augmenting the efficiency at which polymerase I traverses the oligo(dC) stretches . Direct binding of factor C to denatured DNA is indicated by retention of the protein-DNA complex on columns of DEAE-cellulose . Binding of factor C to poly{d(G-C)} is demonstrated by the specific adsorption of the enhancing protein to columns of poly{d(G-C)}-Sepharose . We propose that by binding to poly{d(G-C)} and to poly(dC).poly(dG), factor C enables tighter binding of some DNA polymerases to these templates and facilitates enzymatic activity. Nucleic Acids Res, 1989 Mar 25, 17(6), 2315 - 32 The effects of transcription on the nucleosome structure of four Dictyostelium genes; Pavlovic J et al.; Micrococcal nuclease digestion of Dictyostelium discoideum nuclei from various developmental stages was used to investigate transcription-related changes in the chromatin structure of the coding region of four genes . Gene activity was determined by Northern blotting and nuclear run on experiments . During strong transcription of the developmentally regulated cysteine proteinase I gene, a smear superimposed on a nucleosomal ladder was observed, indicating perturbation of nucleosomal structure was occurring . However, two other developmentally regulated genes, discoidin I and pSC253, showed only slight nucleosome disruption during high levels of transcription . The chromatin structure of a fourth gene (pCZ22) was disrupted throughout development, even at those stages where transcription was greatly reduced . We suggest that although nucleosome structure can be transiently perturbed by the passage of the transcription complex in vivo, the degree of perturbation and the speed with which nucleosomes reassemble is also influenced by the DNA sequence. J Biol Chem, 1989 Mar 5, 264(7), 4045 - 51 In vitro RNA processing generates mature 3' termini of yeast 35 and 25 S ribosomal RNAs; Yip MT et al.; 35 S rRNA is the major intracellular precursor to 18, 5.8, and 25 S rRNAs in Saccharomyces cerevisiae . In this report, we show that the 3' termini of 35 S rRNA as well as 25 S rRNA are generated by post-transcriptional RNA processing rather than transcription termination . Using a partially purified yeast whole cell extract, efficient site-specific cleavage of a synthetic rRNA precursor was demonstrated in vitro . The 3' termini of the processed precursor were established by S1 nuclease protection analysis . RNA molecules containing the mature 3' termini of 35 and 25 S rRNA as well as molecules with a 3' terminus located 12 nucleotides beyond the 3' terminus of 25 S rRNA were the major products of the in vitro processing reaction . Processing activity required Mg2+ but was independent of ribonucleotides . Pretreatment of the yeast whole cell extract with proteinase K abolished processing activity, whereas micrococcal nuclease pretreatment of the extract had no effect on processing activity . These results show that RNA polymerase I-dependent transcription of yeast ribosomal cistrons continues beyond sequences that encode the 3' terminus of 35 S rRNA into the spacer region that separates 35 S rRNA transcription units. Mutat Res, 1989 Mar, 217(2), 101 - 7 Excision repair characteristics of denV-transformed xeroderma pigmentosum cells; Ley RD et al.; Introduction of the denV gene of phage T4, encoding the pyrimidine dimer-specific endonuclease V, into xeroderma pigmentosum cells XP12RO(M1) was reported to result in partial restoration of colony-forming ability and excision repair synthesis . We have further characterized 3 denV-transformed XP clones in terms of rates of excision of pyrimidine dimers and size of the resulting resynthesized regions following exposure to 100 J/m2 from an FS-40 sunlamp . In the denV-transformed XP cells we observed 50% dimer removal within 3-6 h after UV exposure as compared to no measurable removal in the XP12RO(M1) line and 50% dimer excision after 18 h in the GM637A human, control cells . Dimer removal was assayed with Micrococcus luteus UV-endonuclease in conjunction with sedimentation of treated DNA in alkaline sucrose gradients . The size of the resulting repaired regions was determined by the bromouracil photolysis technique . Based on the photolytic sensitivity of DNA repaired in the presence of bromodeoxyuridine, we calculated that the excision of a dimer in the GM637A cells appears to be accompanied by the resynthesis of a region approximately 95 nucleotides in length . Conversely, the resynthesized regions in the denV-transformed clones were considerably smaller and were estimated to be between 13 and 18 nucleotides in length . These results may indicate that either the endonuclease that initiated dimer repair dictated the size of the resynthesized region or that the long-patch repair observed in the normal cells resulted from the repair of non-dimer DNA lesions. J Biochem (Tokyo), 1989 Mar, 105(3), 484 - 9 Purification and characterization of chitinase produced by Streptomyces erythraeus; Hara S et al.; A chitinase was purified from the culture filtrate of Streptomyces erythraeus (SE) . The enzyme (SE chitinase) has a molecular weight of 30,000 and pI 3.7, and shows optimal activity at pH 5.0 with an optimal ionic strength of less than 0.2 M NaCl . SE chitinase could hydrolyze chitin and its derivatives, but could not hydrolyze cell walls of Micrococcus lysodeikticus . The substrate specificity of SE chitinase was compared with those of hen egg white (HEW) and SE lysozymes . The binding mode of the chitinase to substrates was investigated using chitooligosaccharides and their derivatives . The results showed that the binding mode of SE chitinase to the substrate is similar to that of HEW and SE lysozymes. Eur J Biochem, 1989 Feb 15, 179(3), 573 - 9 A structural requirement in the subsite F of lysozyme . The role of arginine 115 in human lysozyme revealed by site-directed mutagenesis; Muraki M et al.; Arginine 115 in the subsite F of human lysozyme (peptidoglycan N-acetylmuramoylhydrolase, EC 3.2.1.17) was replaced with lysine, histidine, glutamine or glutamine acid by site-directed mutagenesis . The conversions which conserve positive charge, Arg115 to Lys or His (at acidic pH), have little affected on either the kinetic parameters for Micrococcus lysodeikticus cells or the activity against glycol chitin, nor on the cleavage patterns of hexa(N-acetylglucosamine) {(GlcNAc)6} and penta(N-acetylglucosamine) {(GlcNAc)5} . On the other hand, the conversions which cause loss of the positive charge, Arg115 to His (neutral and alkaline pH), Gln or Glu, not only reduced the activity against glycol chitin but also changed the cleavage patterns for (GlcNAc)6 and (GlcNAc)5 . These results suggest that Arg115 is structurally required not for the specific hydrogen bonding interaction with a sugar residue but for the positively charged character in the construction of subsite F in human lysozyme. J Virol, 1989 Feb, 63(2), 943 - 7 During latency, herpes simplex virus type 1 DNA is associated with nucleosomes in a chromatin structure; Deshmane SL et al.; Latent herpes simplex virus type 1 DNA has a nucleosomal structure similar to that of cellular chromatin, as determined by micrococcal nuclease digestion . All of the major regions, including the transcriptionally active region of the genome, were found to be associated with nucleosomes . Such a chromatin structure is likely to be an important element in the control of herpes simplex virus type 1 gene expression during latency. EMBO J, 1989 Feb, 8(2), 607 - 11 Antibiotic interactions at the GTPase-associated centre within Escherichia coli 23S rRNA; Egebjerg J et al.; A comprehensive range of chemical reagents and ribonucleases was employed to investigate the interaction of the antibiotics thiostrepton and micrococcin with the ribosomal protein L11-23S RNA complex and with the 50S subunit . Both antibiotics block processes associated with the ribosomal A-site but differ in their effects on GTP hydrolysis, which is inhibited by thiostrepton and stimulated by micrococcin . The interaction sites of both drugs were shown to occur within the nucleotide sequences A1067-A1098 within the protein L11 binding site on 23S RNA . This region of the ribosome structure is involved in elongation factor-G-dependent GTP hydrolysis and in the stringent response . No effects of drug binding were detected elsewhere in the 23S RNA . In general, the two drugs afforded 23S RNA similar protection from the chemical and nuclease probes in accord with their similar modes of action . One important exception, however, occurred at nucleotide A1067 within a terminal loop where thiostrepton protected the N-1 position while micrococcin rendered it more reactive . This difference correlates with the opposite effects of the two antibiotics on GTPase activity. Biochim Biophys Acta, 1989 Jan 23, 1007(1), 23 - 9 The protamine gene chromatin in developing trout testis exists in an altered state; Nickel BE et al.; Micrococcal nuclease was used to probe the nucleosomal organization of the rainbow trout germ-line-specific protamine multi-gene family in testis and erythrocytes . In erythrocyte chromatin, the repressed protamine genes show a distinct nucleosomal repeat pattern . However, in early-stage testis chromatin, where the protamine genes are expressed, they lack a distinct nucleosomal repeat pattern, indicating that the disrupted chromatin structure is related to their transcriptional activity . Micrococcal nuclease-digested testis and erythrocyte chromatin was separated into soluble and insoluble fractions . Transcriptionally active/competent genes of testis that had been labeled by nuclear nick-translation were enriched in the low-salt eluted, micrococcal nuclease-sensitive chromatin fraction . This fraction was not enriched in protamine DNA sequences . In testis, but not erythrocytes, protamine DNA sequences were slightly enriched in chromatin that fractionated with insoluble nuclear material, suggesting that transcriptionally active protamine gene chromatin has an insoluble character . Since the different protamine genes may not be simultaneously expressed, our results show the distribution of both transcriptionally active and inactive protamine genes . However, our observations indicate that the active germ-line-specific protamine gene chromatin shares several, but not all, of the features associated with other active tissue-specific genes. J Med Chem, 1989 Jan, 32(1), 84 - 93 Inhibition of 125I-labeled ristocetin binding to Micrococcus luteus cells by the peptides related to bacterial cell wall mucopeptide precursors: quantitative structure-activity relationships; Kim KH et al.; Quantitative structure-activity relationships (QSAR) of N-Ac amino acids, N-Ac dipeptides, and N-Ac tripeptides in inhibition of 125I-labeled ristocetin binding to Micrococcus luteus cell wall have been developed to probe the details of the binding between ristocetin and N-acetylated peptides . The correlation equations indicate that (1) the binding is stronger for peptides in which the side chain of the C-terminal amino acid has a large molar refractivity (MR) value, (2) the binding is weaker for peptides with polar than for those with nonpolar C-terminal side chains, (3) the N-terminal amino acid in N-Ac dipeptides contributes 12 times that of the C-terminal amino acid to binding affinity, and (4) the interactions between ristocetin and the N-terminal amino acid of N-acetyl tripeptides appear to be much weaker than those with the first two amino acids. Comp Biochem Physiol B, 1989, 93(4), 803 - 6 Cell-free translation in lysates from Spodoptera frugiperda (Lepidoptera: Noctuidae) cells; Swerdel MR et al.; 1 . Conditions for in vitro translation of mRNA in cell-free extracts from cultured Spodoptera frugiperda cells were defined . 2 . Incorporation of {35S}methionine into acid-precipitable material increased for approximately 1 hr, and was sensitive to the protein synthesis inhibitors pactamycin and cycloheximide . 3 . Micrococcal nuclease-treated lysate, primed with purified rabbit globin mRNA, synthesized a major protein with the size of full length globin, indicating that the lysate supported correct initiation and elongation of polypeptides. Microbiol Immunol, 1989, 33(3), 165 - 74 Cell surface of Micrococcus luteus: chemical treatment of the cells and teichuronic acids on the surface; Monodane T et al.; Micrococcus luteus IFO 3333 cells, both treated with chemical reagents and non-treated, were observed with a scanning electron microscope (SEM) . The agglutinability of the cells with antiserum containing anti-teichuronic acid antibody was examined . The binding of protein A-gold particles to the cells, mediated with the antiserum, was also observed with SEM . The surface of a M . luteus cell consisted of two or three areas with borders--the rough and the smooth areas, or the rough, the slightly rough, and the smooth areas; fluffy materials were clearly seen in the rough area . Gold particles were observed uniformly and densely on the whole cell surface . However, either mild acid treatment or mild Smith degradation of the cells altered the fluffy rough area to a rough one, and extremely decreased the agglutinability and the binding of protein A-gold particles . Teichuronic acids appeared to be distributed uniformly on the whole cell surface of M . luteus IFO 3333. Comp Biochem Physiol B, 1989, 92(3), 523 - 7 Lysozyme activity in animal extracts after sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Audy P et al.; 1 . Lysozyme activity was detected after electrophoresis in sodium dodecyl sulfate-polyacrylamide gels containing 0.2% (W/V) autoclaved Micrococcus lysodeikticus cells as substrate . 2 . Lysozyme activity appeared as clear lysis zones after incubation of opaque gels at 37 degrees C in buffered Triton X-100 . 3 . As low as 0.1 pg of purified hen egg white lysozyme could be detected after 16 hr incubation at pH 6.5 . 4 . Bands with lytic activity from kidney and pancreas acetone powders, bird's egg whites and vitelline membranes, animal sera and human saliva corresponded to c-type (Mr 14,500), g-type (Mr 20,500) or both lysozymes as far as molecular weight is concerned . 5 . Some extracts, like porcine kidney, exhibited more than two bands . 6 . Bands with lytic activity migrating at the level of g-type lysozymes were detected in some kidney and pancreas extracts. Eisei Shikenjo Hokoku, 1989, (107), 145 - 7 {Lysozyme Reference Standard (Control 871) of the National Institute of Hygienic Sciences}; Komuro T et al.; A candidate for the Lysozyme Reference Standard (Control 871) of the National Institute of Hygienic Sciences was prepared . Purity of the standard material examined electrophoretically was more than 99.5% . Lysozyme potency of the standard material was assayed turbidimetrically using dry-cells of Micrococcus luteus as a substrate and compared with that of the Lysozyme Reference Standard (Control 865) . Potency of the standard material was in satisfactory agreement with that of the standard and was defined as 1 mg {potency} per mg. Gerontology, 1989, 35(5-6), 268 - 74 Characteristics of lymphocyte chromatin from Alzheimer's disease patients and from young and old normal individuals; Smith TA et al.; We have examined chromatin in lymphocytes from patients with Alzheimer's disease (AD) and from normal individuals of a range of ages . We have found that the neucleosome repeat length does not vary with age for normal individuals over the range 24-78 years and that there is no difference between the value for AD cells (mean and standard deviation, 202 +/- 7 base pairs, bp) and for normals (207 +/- 5 bp) . The rate of digestion of chromatin in lymphocyte nuclei by micrococcal nuclease does not appear to differ significantly between old and young normals and AD patients. Proc Natl Acad Sci U S A, 1989 Jan, 86(1), 133 - 7 Site-directed mutagenesis of the catalytic residues Asp-52 and Glu-35 of chicken egg white lysozyme; Malcolm BA et al.; The roles of the catalytic active-site residues aspartic acid-52 and glutamic acid-35 of chicken lysozyme (EC 3.2.1.17) have been investigated by separate in vitro mutagenesis of each residue to its corresponding amide (denoted as D52N and E35Q, respectively) . The mutant enzyme D52N exhibits approximately 5% of the wild-type lytic activity against Micrococcus luteus cell walls, while there is no measurable activity associated with E35Q (0.1% +/- 0.1%) . The measured dissociation constants for the chitotriose-enzyme complexes were 4.1 microM (D52N) and 13.4 microM (E35Q) vs . 8.6 microM for wild type, indicating that the alterations in catalytic properties may be due in part to binding effects as well as to direct catalytic participation of these residues . The mutant lysozymes have been expressed in and secreted from yeast and obtained at a level of approximately 5 mg per liter of culture by high-salt elution from the cell walls. Allerg Immunol (Leipz), 1989, 35(1), 51 - 8 {Investigations on the standardization of the lysoplate technique for assay of lysozyme}; Stelzner A et al.; In this paper studies for the standardization of the estimation of lysozyme in low concentrations concerning the lysoplate technique are reported . The influence of the conditions of diffusion, the concentration and the age of the bacteria as well as the judgement of different pictures of lysis and contrast resp . have been measured and compared . Sufficient results have been obtained with "Purified agar" (Difco) in a concentration of 0.9% and with living, 48 h old strains from Micrococcus luteus . After this a mixture of the bacteria in a concentration of 2.75 x 10(8)/ml with 5 ml "Purified agar" at 60 degrees C can be recommended . Using conditions of incubation with 50 degrees C and a final time of 4 h lysozyme can be estimated certainly with a sensitivity of 1 microgram/ml. J Pharm Biomed Anal, 1989, 7(7), 865 - 9 Flow microcalorimetric assay of antibiotics--III . Zinc bacitracin and its combinations with polymyxin B sulphate and neomycin sulphate on interaction with Micrococcus luteus; Joslin Kjeldsen N et al.; A flow microcalorimetric assay for zinc bacitracin has been developed which has better reproducibility (relative standard deviation less than 2%) and sensitivity (0.02 micrograms ml-1) than conventional microbiological assays, and requires an assay time of between 7.5-9 h . The assay is not suitable for zinc bacitracin determinations in the presence of equimolar concentrations of polymyxin B sulphate or neomycin sulphate, or of these antibiotics in the proportions in which they occur in the commercial preparation Trisep (ICI, Macclesfield, UK). Int J Biochem, 1989, 21(11), 1235 - 40 Characteristics of chromatin release during digestion of nuclei with micrococcal nuclease: preferential solubilization of nascent RNA at low enzyme concentration; Telford DJ et al.; 1 . Extensive digestion of nuclei with micrococcal nuclease (MNase), commonly used in the analysis of chromatin structure, results in the production of mono- and dinucleosomal chromatin fragments . 2 . Digestion of nuclei from a range of cell types with low enzyme concentrations solubilized high molecular weight polynucleosomal fragments, some greater than 22 kb long . 3 . Such digestion conditions also resulted in extensive solubilization of nascent RNA which contributed considerably to the nucleic acid content of the soluble fraction . 4 . We conclude that the contribution of RNA to total nucleic acid content of the soluble fraction should be taken into consideration when nuclei are digested with low concentrations of MNase. J Biochem (Tokyo), 1989 Jan, 105(1), 84 - 7 o-Phosphotyrosyl glutamine synthetase: modification of the nucleotide ligation site of adenylylated glutamine synthetase; Kimura K et al.; The nucleotide ligation site of adenylylated glutamine synthetase, which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP, was studied by digestion with three hydrolytic enzymes . The products on micrococcal nuclease digestion were adenosine and o-phosphotyrosyl glutamine synthetase . The Km for this macromolecular substrate with the nuclease was 40 microM, at pH 8.9 . The glutamine synthetase activity was not affected by deadenosylation with the nuclease, in contrast to SVPDE digestion, with which the glutamine synthetase activity was markedly increased . The Km for the native adenylylated glutamine synthetase with the SVPDE was 36 microM, i.e., similar to that for the nuclease . When the isolated o-phosphotyrosyl enzyme was incubated with alkaline phosphatase at pH 7.2, the glutamine synthetase activity rapidly increased to the same level as that of the SVPDE treated enzyme . Furthermore, kinetic properties of the o-phosphotyrosyl glutamine synthetase were compared with those of the adenylylated enzyme . The optimum pH, apparent Km for each of three substrates, glutamate, ATP, and NH3, and Vmax were in good agreement, as to either Mg2+- or Mn2+-dependent biosynthetic activity . From these results we can conclude that the regulation of glutamine synthetase activity simply requires the phosphorylation of the tyrosyl residue in each subunit, without recourse to adenylylation. Cell, 1988 Dec 23, 55(6), 945 - 53 Chromatin folding modulates nucleosome positioning in yeast minichromosomes; Thoma F et al.; Based on the chromatin structures of the yeast URA3 gene and the TRP1ARS1 circle, we have designed circular minichromosomes of different sizes that should each form a tight tetranucleosome . This structure was assumed to be stiff and bulky and therefore likely to be sensitive to packaging into a three-dimensional structure . The structures of the minichromosomes were determined using micrococcal nuclease . Only one of the minichromosomes showed a protected region of about 570 bp, compatible with the predicted tight tetranucleosome, while all other constructs showed alternative structures . A comparison of the structures revealed that neither histone-DNA interactions nor influences from flanking boundaries are sufficient determinants of nucleosome positions . The data strongly suggest that chromatin folding modulates the nucleosome arrangement along the DNA. EMBO J, 1988 Dec 20, 7(13), 4355 - 65 Assembly of spaced chromatin involvement of ATP and DNA topoisomerase activity; Almouzni G et al.; Undiluted extracts from eggs or oocytes of Xenopus laevis support the assembly of chromatin with physiologically spaced nucleosomes . Micrococcal nuclease and DNase I digestion experiments show that nucleosome formation as well as supercoiling of circular DNA concomitant to assembly do not require ATP or Mg2+ . However these factors are essential for the stability and the physiological spacing of the assembled chromatin . gamma-S-ATP can substitute for ATP in this process . With topoisomers of defined linking number topological interconversions proceed by steps of unity, both in vitro as well as in vivo, indicating that topoisomerase I is dominantly acting in this process . Novobiocin sensitivity occurred only with diluted extracts and was unrelated to an inhibition of topoisomerase II . Finally, nucleosome assembly occurs efficiently on linear DNA although the assembled DNA is less stable than with circular DNA . From these results we propose that mature chromatin is formed in a two-step reaction . In the first step, nucleosome deposition occurs independently of ATP and Mg2+ . Thus, nucleosome formation can be uncoupled from their spacing . In this step, topoisomerase activity is involved in the relaxation of the topological constraints generated by chromatin assembly rather than in the process of assembly itself . The second step, requiring ATP and Mg2+, generates properly spaced chromatin. Cancer Res, 1988 Dec 15, 48(24 Pt 1), 7146 - 9 Gene-specific differences in the aflatoxin B1 adduction of chicken erythrocyte chromatin; Delcuve GP et al.; Mature and immature chicken erythrocyte nuclei were treated with activated aflatoxin B1 (2,3-dichloroaflatoxin B1), producing covalently bound DNA adducts . This reaction produces alkali-labile sites in the DNA which can be identified by using a variation of the Maxam-Gilbert sequencing procedure . We determined the aflatoxin B1 accessibility of defined regions of the erythroid genome by using different specific probes and monitoring the disappearance of similar-sized fragments generated by restriction enzyme digestion . The genes studied were the erythroid-specific beta-globin and histone H5 genes, which are potentially active in mature erythroid nuclei and transcriptionally active in immature erythrocytes, and the vitellogenin and ovalbumin genes, which are both transcriptionally inactive in these cells . The beta-globin and histone H5 genes were more accessible than the repressed vitellogenin and ovalbumin genes to aflatoxin B1 modification in mature and immature erythroid chromatin . Micrococcal nuclease was used to probe the nucleosomal organization of active (beta-globin and histone H5) and repressed (vitellogenin and ovalbumin) genes in chicken erythrocytes . The vitellogenin and ovalbumin genes show a canonical nucleosome repeat pattern in mature and immature chicken erythrocyte nuclei . In contrast, the beta-globin and histone H5 genes lack a distinct nucleosomal repeat pattern in these cells . These results support the hypothesis that transcriptionally active genes are preferentially accessible to carcinogen modification because of their disrupted chromatin structure. Nucleic Acids Res, 1988 Dec 9, 16(23), 11187 - 205 The structure of nucleosomal core particles within transcribed and repressed gene regions; Studitsky VM et al.; The arrangement of histones along DNA in nucleosomal core particles within transcribed heat shock gene (hsp 70) region and repressed insertion within ribosomal genes of Drosophila was analysed by using protein-DNA crosslinking methods combined with hybridization tests . In addition, two-dimensional gel electrophoresis was employed to compare the overall nucleosomal shape and the nucleosomal DNA size . The arrangement of histones along DNA and general compactness of nucleosomes were shown to be rather similar in transcriptionally active and inactive genomic regions . On the other hand, nucleosomes within transcriptionally active chromatin are characterized by a larger size of nucleosomal DNA produced by micrococcal nuclease digestion and some peculiarity in electrophoretic mobility. J Mol Biol, 1988 Dec 5, 204(3), 549 - 59 The upstream limit of nuclease-sensitive chromatin in Dictyostelium rRNA genes neighbors a topoisomerase I-like cluster; Bettler B et al.; In the chromatin of Dictyostelium ribosomal RNA (rRNA) genes, the coding and upstream flanking regions are sensitive to endonucleases . This sensitivity stops about 2.3 x 10(3) bases upstream from the transcription start, at a point we call the structural boundary . Upstream from the boundary an 850 base-pair region is strongly protected against micrococcal nuclease cleavage, particularly in rapidly transcribing vegetative cells, and upstream from this the pattern of nuclease protection suggests that positioned nucleosomes are present . On the gene side of the structural boundary nucleosomes are known to be absent in vegetative cells but present in differentiating slug cells where the rRNA synthesis rate is lower . We show that in slugs these nucleosomes are randomly distributed, in contrast to those upstream from the boundary . Close to the gene side of the boundary is a duplication of the putative promoter located 29 base-pairs distant from four clustered topoisomerase I recognition sequences, which are cleaved by endogenous topoisomerase I-like activity . An additional topoisomerase I recognition sequence found upstream from the structural boundary is not cleaved in chromatin . The possible significance of these sequences and structures in transcription is discussed. Eur J Cancer Clin Oncol, 1988 Dec, 24(12), 1861 - 8 Differential sensitivity of histone acetylation in nitrogen-mustard sensitive and resistant cells . Relation to drug uptake, formation and repair of DNA-interstrand cross-links; Helliger W et al.; Cultivation of Ehrlich-ascites tumor cells in the presence of N-mustard leads to a selection of cells with a defective choline carrier . As N-mustard employs the choline carrier for transport, this results in reduced drug uptake and in a decrease in drug sensitivity which is specific for N-mustard . Walker carcinoma cells with a stable pleiotropic resistance to a variety of alkylating agents and adriamycin exhibit no evidence for an impaired drug transport and show the same frequency of DNA-interstrand cross-links as the sensitive parental line . Both sensitive and resistant Walker cells exhibit equal capacities for repair of N-mustard induced DNA-interstrand cross-links . The inhibition of histone acetylation by N-mustard, however, was found to be significantly lower in the resistant Walker or Ehrlich cells compared to sensitive counterparts . Although the difference between N-mustard concentrations leading to half maximal inhibition of histone acetylation in sensitive and resistant cells is considerably smaller than the difference between N-mustard doses required for half maximal inhibition of cell proliferation the data suggest that--besides DNA-DNA cross-linking--the inhibition of histone acetylation has to be considered as an important alternative mechanism responsible for the cytotoxic activity of alkylating agents . Inhibition of histone acetylation is not due an accelerated deacetylation and is predominantly expressed in chromatin fractions soluble in 0.1 M NaCl after digestion with micrococcal nuclease. Experientia . 1988 Dec 1;44(11-12):1021. A marine Micrococcus produces metabolites ascribed to the sponge Tedania ignis; Stierle AC et al.; Extracts of the sponge Tedania ignis have been reported to contain several diketopiperazines . As part of an investigation of the commensal and symbiotic microflora of sponges, we have consistently isolated, from specimens of T . ignis, a Micrococcus sp . which produces diketopiperazines in laboratory cultural media . This is the first demonstration that a bacterium associated with a sponge produces secondary metabolites ascribed to the sponge host. J Biochem Biophys Methods, 1988 Dec, 17(4), 249 - 52 A rapid fluorimetric test for lysozyme with purified fluorescamine-labelled peptidoglycan; Moser I et al.; A sensitive and rapid fluorometric lysozyme assay is described . It is based on the hydrolysis of fluorescamine-labelled peptidoglycan from Micrococcus luteus cell walls . Lysozyme levels as low as 0.1 microgram can be detected. Proc Natl Acad Sci U S A, 1988 Dec, 85(24), 9411 - 5 Secretion in yeast of human lysozymes with different specific activities created by replacing valine-110 with proline by site-directed mutagenesis; Kikuchi M et al.; Computer graphics indicate that a steric hindrance exists between valine-110 side chain of human lysozyme (EC 3.2.1.17) and an acetyl group of a modified substrate that contains N6,O-diacetylmuramic acid . To alter the substrate specificity of human lysozyme to be effective on the modified substrate, we replaced the valine-110 residue with various amino acids by site-directed mutagenesis . One of the mutant proteins (valine residue replaced with proline:P110) was secreted in Saccharomyces cerevisiae as at least four components (P110-A, P110-B, P110-C, and P110-D) with different specific activities . Two components, P110-B and P110-D, were isolated in a pure form and structurally characterized . The results suggest that this mutation lowered the lytic activity against Micrococcus lysodeikticus by changing a local conformation of the catalytic site while keeping almost the same substrate binding sites . Our results also indicate that cis/trans isomerization of prolyl peptide bonds probably occurs in vivo and that the conformational change of protein as well as point mutations in genes might influence the molecular evolution of the protein. Photodermatol, 1988 Dec, 5(6), 243 - 7 Sunscreen protection against UV-induced pyrimidine dimers in DNA of human skin in situ; Freeman SE et al.; We have determined the ability of a chemical sunscreen with a sun protection factor (SPF) of 15 to protect human skin from ultraviolet (UV) radiation-induced DNA damage . The DNA damage was susceptible to cleavage by Micrococcus luteus UV endonuclease, which recognizes pyrimidine dimers in DNA . An alkaline agarose gel electrophoresis method was used to quantify the number of pyrimidine dimers in nonradioactive DNA from skin biopsies of 5 individuals irradiated with UV from a solar simulator . After exposure to an equivalent dose of UV, the number of pyrimidine dimers was 0.8 per 10(7) bases in sunscreen-treated skin as compared with 32 dimers per 10(7) bases in untreated skin . This assay provides a means of determining the efficacy of sunscreens in protecting skin from UV-induced DNA damage. Nucleic Acids Res, 1988 Nov 25, 16(22), 10493 - 509 An in vitro interaction between the human U3 snRNP and 28S rRNA sequences near the alpha-sarcin site; Parker KA et al.; Model transcripts containing mammalian pre-rRNA sequences were incubated with a HeLa cell extract, digested with T1 RNase, and immunoprecipitated with anti-(U3)RNP or control antibodies . Two overlapping fragments derived from the 3' domain of human 28S rRNA were specifically immunoprecipitated although transcripts which spanned the transcription initiation site, the ETS processing site, the 5' end of 18S, and both termini of 5.8S yielded no protected fragments . The sequence of these fragments was determined using a novel technique in which the {32P}-labeled fragment was co-finger-printed with {3H}-labeled total transcript serving as an internal marker . The fragments immunoprecipitated derive from nucleotides 4570-4590 and 4575-4590 of human 28S and are adjacent to the alpha-sarcin site . Protection most likely involves the U3 RNA since it is sensitive to pretreatment of the extract with micrococcal nuclease . Complementarity between U3 and this rRNA region is phylogenetically conserved in species ranging from human to S . cerevisiae . The possible significance of this finding is discussed. Sci Sin {B}, 1988 Nov, 31(11), 1319 - 24 Alteration in chromatin conformation of proliferating and differentiating enzyme genes in normal mouse liver and ascites hepatoma cells and its relation to the gene expression of enzymes; Li S et al.; Nuclei from the normal mouse liver were partially digested with micrococcal nuclease, followed by DNA extraction, agarose gel electrophoresis and dot blot hybridization with 32P-labeled cDNA probes of CPS1 and ACT complex . It was clearly shown that the CPS1 genes were distributed on the monomer, dimer . and trimer of nucleosomes, while the genes coding for ACT complex were distributed on the condensed oligonucleosomes . An opposite manner of distribution of CPS1 and ACT complex genes was, however, noted in the case of ascites hepatoma cells, in which the specific activity of ACT was 13 times higher than that in the normal liver, while that of CPS1 was remarkably reduced . Similar patterns of change in mRNA level of CPS1 and ACT complex were observed in the normal mouse liver and ascites hepatoma cells, indicating a close relationship between chromatin structure and gene expression of these enzymes. Biochem Int, 1988 Nov, 17(5), 863 - 75 Do tightly-bound chromatin proteins play a role in DNA methylation? Caiafa P, Mastrantonio S, Attina M, Rispoli M, Reale A, Strom R. When chromatin matrix, "stripped" from its loosely-bound components by extraction with 3 M NaCl, is extensively digested with DNAase I, a fraction is obtained, which carries no endogenous DNA methyltransferase activity but which is a good substrate for externally added enzyme . Under the same conditions, protein-free DNA isolated from this fraction can instead hardly be methylated, this different behaviour pointing to a role of DNA-tightly-bound proteins in favoring or promoting the catalytic action of the enzyme . A similar stimulation of enzymatic methylation could also be shown when, in the presence of this same fraction, single stranded Micrococcus luteus DNA was incubated with placental methyltransferase, using S-adenosylmethionine as a methyl donor . This finding can be correlated to the existence, in chromatin loops, of small regions which resist digestion by DNAase I also after high-salt removal of their loosely-bound components (presumably because of the presence of tightly-bound proteins) and whose DNA is characterized by high methylation levels and, at the same time, by high relative content of thymine. Biokhimiia, 1988 Nov, 53(11), 1876 - 82 {Study of the age-related features of the structural state of rat liver cell chromatin using micrococcal nuclease and circular dichroism}; Konoplia EF et al.; The structural state of liver cell chromatin of young (6 months-old) and aged rats was evaluated, using circular dichroism and DNA hydrolysis by micrococcal nuclease . It was found that the chromatin condensation ratio in active and non-active fractions increased upon ageing . The length of the DNA nucleosomal repeat did not change upon ageing in either chromatin fraction . An elevated chromatin condensation ratio was observed in aged rats both for the unfolded nucleosome chain and the supernucleosomal structures . The mechanisms of age-related increase in the chromatin compactization ratio at different levels of organization and in dissimilarly active fractions are different . It was suggested that the age-related differences in the degree of compactization may stimulate a decrease of the transcriptional activity of liver cell nuclei upon ageing. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1988 Nov, 270(1-2), 326 - 8 Stomatococcus mucilaginosus as an agent of CAPD peritonitis; Lanzendorfer H et al.; We report a case of bacterial CAPD peritonitis in a 57 year old immunosuppressed woman with renal insufficiency caused by a rare species of the Micrococcaceae, Stomatococcus mucilaginosus . This uncommon case shows that the presence of gram-positive cocci in pairs, tetrads and clusters forming whitish-gummy colonies should remind us of the possibility of Stomatococcus mucilaginosus. Eur J Biochem, 1988 Nov 1, 177(2), 383 - 94 Investigation of protein structure by means of 19F-NMR . A study of hen egg-white lysozyme; Adriaensens P et al.; A 19F-labeled derivative of hen egg-white lysozyme, in which the six epsilon-amino groups are trifluoroacetylated (LF6), was prepared by reaction of lysozyme with S-ethyltrifluorothioacetate . The reaction mixture was fractionated by cation-exchange chromatography at pH 7.3 . A comparison of the circular dichroic spectra and the activity towards Micrococcus lysodeikticus of both LF6 and native lysozyme reveals that the labeling causes no major conformational changes of the polypeptide backbone . Assignment of the six resonances present in the 19F-NMR spectrum of LF6 was accomplished by using a variety of techniques: specific chemical modifications, the effect of the inhibitor (GlcNAc)3, 19F-shift/pH information and relaxation parameters. Radiat Res, 1988 Nov, 116(2), 245 - 53 Proximity of repair patches to persistent pyrimidine dimers in DNA of normal human and xeroderma pigmentosum cells; Cleaver JE; The proximity of repair patches to persistent pyrimidine dimers in normal human cells and xeroderma pigmentosum group C and D cells was analyzed by sequential digestion of repaired DNA with Micrococcus luteus UV-endonuclease and Escherichia coli DNA polymerase I . Although this enzymatic digestion removed one-third of the pyrimidine dimers, less than 3% of the label associated with repair patches and a similar amount of uniformly labeled DNA were removed . The repair patches therefore appear to be similarly distant from persistent dimers in all cell types, and, in particular, are not adjacent to unexcised dimers in xeroderma pigmentosum group D cells . A previous model that suggested that patches are inserted adjacent to dimers in xeroderma pigmentosum group D cells receives no support from these results. Biotechniques . 1988 Nov-Dec;6(10):936, 938, 940. An economical large-scale procedure to purify Micrococcus plasmid DNA; Verma V et al.; A reproducible and economical procedure for obtaining a large yield of highly purified covalently closed circular (ccc) plasmid DNA from an industrially important strain of Micrococcus is described . The procedure adopted here departs in several ways from commonly used protocols for isolation of plasmid DNA from Gram (positive) and Gram (negative) bacteria . The plasmid DNA prepared by this procedure is free of contaminants and is pure enough to be used for electron microscopy, DNA transformation, sequencing, in vitro transcription and mutagenesis. J Biol Chem, 1988 Oct 25, 263(30), 15643 - 51 Histone H3 thiol reactivity and acetyltransferases in chicken erythrocyte nuclei; Chan S et al.; Chicken erythrocyte nuclei previously incubated separately with two novel mercury compounds (N-chloromercuribenzoyl)-biocytin and bis(p-(chloromercuribenzoyl))-{3H}lysine diamide) were digested with micrococcal nuclease and the digest products fractionated according to their solubility in 0.15 M NaCl and molecular size . The identity and quantitation of the chromatin fractions and proteins containing covalently bound mercury were determined by Western blotting, autoradiography, and scintillation counting . The most highly acetylated species of histone H3 in the 0.15 M NaCl-soluble polynucleosome fraction also contained the highest proportion of bound mercury . This fraction contains hyperacetylated core histones, is depleted in linker histones, and enriched in nonhistone proteins . Histone H3 in the 0.15 M NaCl-soluble mononucleosomes, which are unacetylated and lack linker histones, was 45% less labeled than histone H3 in the 0.15 M NaCl-soluble polynucleosome fraction . In the 0.15 M NaCl-insoluble polynucleosomes, which contain unacetylated histones and molar proportions of linker histones, histone H3 was 63% less labeled . Allowing for the differential abundance of these subfractions in the nucleus, the relative H3 reactivities are 50, 7, and 1 for 0.15 M NaCl-soluble polynucleosomes, mononucleosomes, and 0.15 M NaCl-insoluble polynucleosomes, respectively . Thus a gradation of reactivities exists which correlates with increasing hyperacetylation and linker histone depletion . High mobility group proteins 1 and 2, found in subnucleosome particles in the 0.15 M NaCl-soluble fraction, are extensively mercury-labeled . Distribution of histone acetyltransferase activity among salt- and size-resolved micrococcal nuclease produced fractions was almost 5-fold greater in the 0.15 M NaCl-soluble supernatant than in the 0.15 M NaCl-insoluble pellet . Furthermore, the acetyltransferase activity, which is tightly bound to undigested chromatin, is rapidly released by both micrococcal nuclease and DNase I . For short digestion times the enzyme is associated with the salt-soluble polynucleosomes, but at longer times of digestion the enzyme appears to be free from intact nucleosomes . The enzyme may be localized in the globin domai