Nucleic Acids Res, 1992 Dec 25, 20(24), 6487 - 93 Probing the conformations of eight cloned DNA dodecamers; CGCGAATTCGCG, CGCGTTAACGCG, CGCGTATACGCG, CGCGATATCGCG, CGCAAATTTGCG, CGCTTTAAAGCG, CGCGGATCCGCG and CGCGGTACCGCG; Fox KR; The self complementary DNA dodecamers d(CGCGAATTCGCG), d(CGCGTTAACGCG), d(CGCGTATACGCG), d(CGCGATATCGCG), d(CGCAAATTTGCG), d(CGCTTTAAAGCG), d(CGCGGATCCGCG) and d(CGCGGTACCGCG) have been cloned into the Smal site of plasmid pUC19 . Radiolabelled polylinker fragments containing these inserts have been digested with nucleases and chemical agents, probing the structure of the central AT base pairs . The sequences AATT and AAATTT are relatively resistant to digestion by DNase I, micrococcal nuclease and hydroxyl radicals, consistent with the suggestion that they possess a narrow minor groove . Nuclease digestion of TTAA is much more even, and comparable to that at mixed sequence DNA . TpA steps in ATAT, TATA and GTAC are cut less well by DNAse I than in TTAA . DNasel cleavage of surrounding bases, especially CpG is strongly influenced by the nature of the central sequence. Biochem J, 1992 Dec 15, 288 ( Pt 3), 953 - 8 Heparin increases chromatin accessibility by binding the trypsin-sensitive basic residues in histones; Villeponteau B; Recent evidence indicates that chromatin accessibility to transcription factors is of regulatory significance . The polyanion heparin is known to increase chromatin accessibility to DNAase I and to stimulate both RNA and DNA synthesis . In the present study, chromatin structure and its modification by polyanions were examined by using trypsin and micrococcal nuclease as probes . Both heparin and poly(glutamic acid) were found to be equivalent to trypsin digestion of histones in their ability to increase nuclease accessibility in chromatin . However, no increase in nuclease accessibility was observed when trypsin-digested chromatin was further treated with heparin, indicating that polyanions and trypsin are not additive in their effects on chromatin accessibility . Moreover, sucrose-gradient analysis demonstrated that heparin binds tightly to intact nucleosomes but not to trypsin-digested nucleosomes . These data suggest that polyanions interact predominantly with the trypsin-sensitive lysine and arginine residues in histone H1 and the N-terminal segments of the core histones . The possible relevance of these results to the chromatin structure of actively transcribed regions is discussed. Biochem Int, 1992 Dec, 28(4), 707 - 15 Purification and properties of chitinase from cabbage; Chang CT et al.; Chitinase has been purified from the extract of cabbage through successive steps of ammonium sulfate fractionation, chromatofocusing and Sephadex G-75 gel filtration . By these steps, the purity of the enzyme increased by 93.3 fold and the recovery of the enzyme activity was 20% . The purified enzyme had an optimal pH of 5.0, an optimal temperature between 40 to 50 degrees C and a Km of 76 microM for hydrolysis of ethylene glycol chitin . The molecular weight of the enzyme determined from filtration through Sephadex G-75 was 30,000 daltons . Heavy metal ions, Hg2+ (0.5 mM) and Ag+(2.5 mM) significantly inhibited the activity of the enzyme . NBSI1 (1.0 mM), DNFB (0.5 mM) and PMSF (0.5 mM) completely inhibited the activity of the enzyme . The enzyme also showed muramidase activity for hydrolysis of Micrococcus lysodeikticus cell wall . The presence of chitinase in cabbage may function as a defense enzyme against potential pathogens. Chem Biol Interact, 1992 Dec, 85(2-3), 265 - 81 Photochemical DNA modifications induced by 1,2-dioxetanes; Epe B et al.; 1,2-Dioxetanes are efficient sources of triplet excited carbonyl compounds, into which they decompose on thermal or photochemical activation . In the presence of DNA, the decomposition of dioxetanes gives rise to DNA modifications, which have been studied by means of specific repair endonucleases . Cyclobutane pyrimidine dimers, which are generated by triplet-triplet energy transfer, were detected by a UV endonuclease; they made up between 2% and 30% of the total modifications recognized by a crude repair endonuclease preparation from Micrococcus luteus . For various 1,2-dioxetanes, the yield of pyrimidine dimers was proportional to their triplet excitation flux . DNA strand breaks, sites of base loss (AP sites; recognized by exonuclease III and endonuclease IV) and dihydropyrimidines (recognized by endonuclease III) were found to represent only a small fraction of the modifications . The majority of the modifications detected were recognized by formamidopyrimidine-DNA glycosylase (FPG protein) and represent 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) residues or other yet not defined base modifications which are recognized by this enzyme . The modifications were generated in similar relative yields by thermal and photo-induced decomposition of the 1,2-dioxetanes and therefore emanate under both conditions from the excited carbonyl compounds . The formation of the FPG protein-sensitive modifications was efficiently quenched by azide anions; the Stern-Volmer quenching of these modifications was 150-fold more effective than that of the pyrimidine dimers . The relative amounts of the two types of modifications were strongly dependent on the structure of the 1,2-dioxetanes and on the concentration of molecular oxygen . Singlet oxygen appears to be involved only to some extent in the generation of the FPG protein-sensitive base modifications as their yield was only moderately (approximately 2-fold) increased in D2O as solvent . A mechanism is suggested in which oxidized guanine is predominantly formed by a single-electron-transfer reaction of the triplet excited carbonyl product derived from the 1,2-dioxetane, followed by unknown secondary oxidations, which involve molecular oxygen and/or undecomposed 1,2-dioxetane. J Clin Invest, 1992 Dec, 90(6), 2593 - 7 Endonuclease-induced DNA damage and cell death in oxidant injury to renal tubular epithelial cells; Ueda N et al.; Hydrogen peroxide (H2O2)-induced DNA damage and cell death have been attributed to the direct cytotoxicity of H2O2 and other oxidant species generated from H2O2 . We examined the possibility that oxidants activate endonucleases leading to DNA damage and cell death in renal tubular epithelial cells, similar to that described for apoptosis . Within minutes, H2O2 caused DNA strand breaks in a dose-dependent manner, followed by cell death . DNA fragmentation was demonstrated both by the release of {3H}thymidine in 27,000-g supernatant as well as the occurrence of low molecular weight DNA fragments on agarose gel electrophoresis, characteristic of endonuclease cleavage . Endonuclease inhibitors, aurintricarboxylic acid, Evans blue, and zinc ion prevented H2O2-induced DNA strand breaks, fragmentation, and cell death . Inhibitors of protein or mRNA synthesis had only minor protection against H2O2-induced DNA damage in contrast to complete protection reported in apoptotic thymocytes . Micrococcal endonuclease induced similar DNA strand breaks in LLC-PK1 cells, and the endonuclease inhibitors prevented the events confirming the ability of endonucleases to induce DNA damage . The protective effect of aurintricarboxylic acid was not due to the prevention of the rise in intracellular free calcium . We conclude that endonuclease activation occurs as an early event leading to DNA damage and cell death in renal tubular epithelial cells exposed to oxidant stress and, in contrast to apoptotic thymocytes, does not require macromolecular synthesis. J Biochem (Tokyo), 1992 Dec, 112(6), 743 - 9 Chain length distribution of the products formed in solanesyl diphosphate synthase reaction; Ohnuma S et al.; Factors that affect the termination of isoprenoid chain elongation catalyzed by prenyltransferase were investigated . The chain-length distribution of reaction products of solanesyl diphosphate synthase {EC 2.5.1.11} homogeneously purified from Micrococcus luteus changed dramatically according to the concentration of the complex formed between isopentenyl diphosphate and Mg2+ (IPP-Mg) in the reaction mixture . However, the concentration of the complex between farnesyl diphosphate and Mg2+ (FPP-Mg), the priming substrate for this synthase, did not affect the product distribution, provided that the concentration of IPP-Mg was maintained at a certain level . Thus, the level of IPP-Mg is decisive in affecting the chain length distribution of the products of the prenyltransferase reaction, and the Mg(2+)-dependent variability of product specificity so far observed can now be understood in terms of the effect of IPP-Mg concentration. Arch Biochem Biophys, 1992 Nov 15, 299(1), 172 - 8 Mechanism of lysozyme inactivation and degradation by iron; Sellak H et al.; The site-specific lysozyme damage by iron and by iron-catalysed oxygen radicals was investigated . A solution of purified lysozyme was inactivated by Fe(II) at pH 7.4 in phosphate buffer, as tested on cleavage of Micrococcus lysodeikticus cells; this inactivation was time- and iron concentration-dependent and was associated with a loss of tryptophan fluorescence . In addition, it was reversible at pH 4, as demonstrated by lysozyme reactivation and by the intensity of the 14.4-kD-band on SDS-PAGE . Desferal (1 mM) and Detapac (1 mM) added before iron, prevented lysozyme inactivation, while catalase (100 micrograms/ml), superoxide dismutase (100 micrograms/ml) and bovine serum albumin (100 micrograms/ml) gave about 30 to 40% protection by competing with lysozyme for iron binding . The denaturing effect of iron on lysozyme was studied in the presence of H2O2 (1 mM) and ascorbate (1 mM); under these conditions the enzyme underwent partly irreversible inactivation and degradation different to that produced by gamma radiolysis-generated .OH . Catalase almost fully protected lysozyme; in contrast, mannitol (10 mM), benzoate (10 mM), and formate (10 mM) provided no protection because of their inability to access the site at which damaging species are generated . In this system, radical species were formed in a site-specific manner, and they reacted essentially with lysozyme at the site of their formation, causing inactivation and degradation differently than the hydroxyl radical. FEBS Lett, 1992 Nov 9, 312(2-3), 127 - 31 Three-dimensional structure of catalase from Micrococcus lysodeikticus at 1.5 A resolution; Murshudov GN et al.; The three-dimensional crystal structure of catalase from Micrococcus lysodeikticus has been solved by multiple isomorphous replacement and refined at 1.5 A resolution . The subunit of the tetrameric molecule of 222 symmetry consists of a single polypeptide chain of about 500 amino acid residues and one haem group . The crystals belong to space group P4(2)2(1)2 with unit cell parameters a = b = 106.7 A, c = 106.3 A, and there is one subunit of the tetramer per asymmetric unit . The amino acid sequence has been tentatively determined by computer graphics model building and comparison with the known three-dimensional structure of beef liver catalase and sequences of several other catalases . The atomic model has been refined by Hendrickson and Konnert's least-squares minimisation against 94,315 reflections between 8 A and 1.5 A . The final model consists of 3,977 non-hydrogen atoms of the protein and haem group, 426 water molecules and one sulphate ion . The secondary and tertiary structures of the bacterial catalase have been analyzed and a comparison with the structure of beef liver catalase has been made. Clin Exp Rheumatol, 1992 Nov-Dec, 10(6), 589 - 94 Specificity analysis of antibodies to single-stranded micrococcal DNA in the sera of normal human subjects and patients with systemic lupus erythematosus; Robertson CR et al.; To evaluate the properties of antibodies to bacterial DNA in the sera of normal human subjects (NHS) and patients with systemic lupus erythematosus (SLE), the effects of ionic strength and pH on their binding to single-stranded DNA (ssDNA) from Micrococcus lysodeikticus (MC) were measured . By ELISA, antibodies to MC ssDNA in NHS showed greater activity at high ionic strength (0.2-1.0 M) than antibodies in lupus sera . Similarly, antibodies in NHS had higher activity at pH 9 than lupus anti-DNA . Competition binding assays indicated, moreover, that NHS anti-DNA showed greater inhibition by DNA than lupus anti-DNA at comparable inhibitor concentrations . Together, these results suggest that antibodies to MC ssDNA in NHS and SLE sera may differ in their mode of interaction with bacterial DNA and that NHS can generate high avidity antibodies to at least certain DNA determinants. Nucleic Acids Res, 1992 Oct 25, 20(20), 5413 - 22 In vivo stage- and tissue-specific DNA-protein interactions at the D . melanogaster alcohol dehydrogenase distal promoter and adult enhancer; Jackson JR et al.; We performed a high resolution analysis of the chromatin structure within the regions required for distal transcription of the Drosophila melanogaster alcohol dehydrogenase gene (Adh) . Using dimethyl sulfate, DNase I, and micrococcal nuclease as structural probes, and comparing chromatin structure in tissues isolated from several developmental stages, we have identified several sites of stage- and tissue-specific DNA-protein interactions that correlate with distal transcription initiation . Most were within previously identified cis-acting elements and/or in vitro protein binding sites of the adult enhancer (AAE) and distal promoter, including the TATA box . We also detected a novel stage-specific DNA-protein interaction at the Adf-2a binding site where a non-histone protein was bound to the DNA on the surface of a positioned nucleosome previously identified between the distal promoter and adult enhancer . In addition to footprints, we have also revealed stage- and tissue-specific DNA helix deformations between many of the non-histone protein binding sites . These helix distortions suggest there are interactions among the adjacently bound proteins that result in bending or kinking of the intervening DNA . The distal promoter and AAE have an accessible chromatin conformation in fat body prior to the third larval instar and many of the regulatory proteins that bind in these regions are also available before distal transcription begins . Nevertheless, the timing of DNA-protein interactions in the distal promoter and AAE suggest these proteins do not bind individually or assemble progressively as they and their binding sites become available . Instead, there appears to be a coordinated assembly of a large cooperative complex of proteins interacting with the distal promoter, the positioned nucleosome, the enhancer of the distal promoter (the AAE), and each other. J Biol Chem, 1992 Oct 15, 267(29), 21265 - 72 Evolution of intestinal apolipoprotein B mRNA editing . Chicken apolipoprotein B mRNA is not edited, but chicken enterocytes contain in vitro editing enhancement factor(s); Teng B et al.; Mammalian intestinal apolipoprotein B (apoB) messenger RNA (mRNA) undergoes posttranscriptional editing, changing codon 2153 from CAA in apoB100 mRNA to an in-frame translational stop codon (UAA) in apoB48 mRNA . By contrast, chicken intestinal apoB cDNA contains a CAA codon at the corresponding site and apoB mRNA from chicken enterocytes, kidney, and liver is unedited . The cDNA sequence of chicken apoB spanning the edited base is divergent from mammalian apoB cDNA sequence, with 70% homology over the conserved 29-nucleotide sequence (6662-6690) flanking codon 2153 . Efficient in vitro editing of both human and rat, but not chicken, synthetic apoB RNA was achieved using rat enterocyte S-100 extracts . By contrast, chicken enterocyte S-100 extracts failed to edit chicken, rat, or human synthetic apoB RNA . Mixing experiments, however, revealed that chicken enterocyte S-100 extracts enhance the in vitro editing activity of rat, pig, and human enterocyte S-100 extracts upon homologous RNAs . The editing enhancement activity of chicken enterocyte S-100 extracts is tissue-specific, heat-sensitive, substrate-saturable, and sensitive to proteinase K, but resistant to micrococcal nuclease . The activity was partially purified by Q-Sepharose chromatography and has an average molecular mass of 49 kDa when analyzed by gel filtration chromatography . We conclude that the evolutionary adaptation of intestinal apoB mRNA editing requires both a requisite RNA motif and tissue-specific factors which mediate the site-specific modification. Nucleic Acids Res, 1992 Oct 11, 20(19), 4997 - 5002 Nutritional regulation of nucleosomal structure at the chicken malic enzyme promoter in liver; Ma XJ et al.; Transcription of the chicken malic enzyme gene in the liver is stimulated by feeding and inhibited by starvation . Concomitant with the increase in transcription caused by refeeding, chromatin structure around the transcription start site of the malic enzyme gene is modified in the liver . Digestion of chromatin in isolated nuclei with DNase I revealed four feeding-induced DNase I hypersensitive sites (-220, -170, -130 and -70 bp) near the malic enzyme promoter . Similarly, digestion of chromatin with restriction endonucleases detected enhanced cleavage within this region when birds were refed . Micrococcal nuclease detected the presence of nucleosomes over this region in the starved state, but not in the fed state . After food was withdrawn from fed birds, nucleosomes were reformed in this region within 6 h . The speed and magnitude of the changes in nucleosomal structure in this region suggest that they did not require DNA replication. Nucleic Acids Res, 1992 Oct 11, 20(19), 4987 - 95 Plasmid cDNA-directed protein synthesis in a coupled eukaryotic in vitro transcription-translation system; Craig D et al.; A system is described in which transcription of cDNA clones by bacteriophage T7 RNA polymerase is coupled to translation in the micrococcal nuclease treated rabbit reticulocyte lysate in a single reaction of coupled transcription-translation . The monovalent and divalent cation requirements for translation are dominant for optimum expression in this coupled system, so that transcription is relatively inefficient . Nevertheless, the use of appropriate DNA concentrations leads to the synthesis of sufficient RNA to saturate the protein synthesis capacity of the system . The fidelity and efficiency of expression in this coupled system are high, and the degree of purification of the plasmid DNA is relatively uncritical . The system therefore offers very considerable advantages for rapid screening of 'mini-preparations' of cDNA plasmid constructs for retention of the correct reading frame and expression of the desired protein product. Int J Pancreatol, 1992 Oct, 12(2), 121 - 5 Influence of feeding regimen and postnatal developmental stages on antibacterial activity of pancreatic juice; Pierzynowski S et al.; Antibacterial activity of pancreatic juice in the pig (n = 8) was investigated during early postnatal development and in cattle (n = 6) receiving a different feeding regimen . For pancreatic juice collection, a catheter was surgically implanted in the pancreatic duct . Reintroduction of pancreatic juice was achieved through a T-shaped cannula in the duodenum . Pancreatic juice was collected for 30 min in all cases . In piglets, collections were carried out at 2, 5-6, and 7-10 wk of age, and in cattle, after a standard meal, 48 h starvation, and following 24 h intraduodenal glucose infusion . Antibacterial activity was tested on Micrococcus Pyogenes strain ATTC 6538P by disc agar diffusion technique using nonactivated pancreatic juice, before and after heat treatment for 15 min at 65 and 100 degrees C, respectively . Piglets showed a significant rise in antibacterial activity of pancreatic juice after weaning . In cattle, 48 h of starvation resulted in a marked suppression of antibacterial activity . This activity was found to be normal after a standard meal and comparable to that after 24-h intraduodenal glucose infusion . Heating of pancreatic juice to 65 degrees C caused a 35% increase in the antibacterial potency, whereas heating to 100 degrees C completely abolished it . Additionally, dilution of pancreatic juice to 1:10 did not affect antibacterial potency. Anal Biochem, 1992 Oct, 206(1), 183 - 8 Localization of in vivo ribosome pause sites; Kim JK et al.; A protocol for the localization of the 5' boundaries of in vivo ribosomal pausing sites has been developed . These mapping experiments combine two basic techniques . The first is the isolation of polysomal transcripts via centrifugation of tissue extracts through a sucrose cushion in the presence of translational elongation inhibitors . The second technique involves a micrococcal nuclease protection assay first developed by Wolin and Walter for in vitro-bound ribosomes (EMBO J . 7, 3559-3569, 1988) . Using this method, the 5' boundaries of in vivo ribosomal pause sites were localized on spinach chloroplast mRNAs derived from the atpA gene . This method is easily adaptable to the identification of in vivo ribosomal pause sites from any organism . It could also be adapted to the localization of in vivo binding sites for other nucleic acid binding proteins. Exp Cell Res, 1992 Oct, 202(2), 224 - 32 Reactivation of DNA replication in erythrocyte nuclei by Xenopus egg extract involves energy-dependent chromatin decondensation and changes in histone phosphorylation; Blank T et al.; Reactivation of chicken erythrocyte nuclei for DNA replication in Xenopus egg extracts involves two phases of chromatin remodelling: a fast decondensation leading to a small volume increase and chromatin dispersion occurring within a few minutes (termed stage I decondensation), followed by a slower membrane-dependent decondensation and enlargement of up to 40-fold from the initial volume (stage II decondensation) . Chromatin decondensation as measured by nuclear swelling and micrococcal nuclease digestion required ATP . We observed a characteristic change in the phosphorylation pattern of erythrocyte proteins upon incubation in egg extract . While histones H5, H2A, and H4 became selectively phosphorylated during decondensation, the phosphorylation of histone H3 and of several nonhistone proteins was prevented . Furthermore, histone H5 was selectively released from erythrocyte nuclei in an energy-dependent reaction . These molecular changes already occurred during stage I decondensation and they persisted during stage II decondensation . DNA replication was confined to nuclei of stage II decondensation which incorporated lamin LIII from the egg extract . These results show that initiation of DNA replication in chicken erythrocytes requires in addition to ATP-dependent chromatin remodelling (stage I), further changes in chromatin structure that correlates with lamin LIII incorporation, and stage II decondensation. J Biol Chem, 1992 Sep 15, 267(26), 18735 - 43 Nuclease-hypersensitive sites define a region with enhancer activity in the third intron of the human apolipoprotein B gene; Levy-Wilson B et al.; The positions of several DNase I-hypersensitive (DH) sites have been mapped in the second and third introns of the human apolipoprotein B gene . Two such DH sites, I and V, are present both in human hepatoma (HepG2) and colon carcinoma (CaCo-2) cells that express the gene but absent from HeLa cells that do not express the gene . These DH sites map near sequence elements that have been highly conserved between the human and mouse genes . A PvuII-EcoRI fragment (+1064 to +2977) from the hypersensitive region exhibited enhancer activity, which was further localized by means of deletion experiments to a 155-base pair segment located entirely within the third intron and flanked by two DH sites . Three DNase I footprints were observed within this core enhancer, one of which contains putative binding sites for three liver specific nuclear proteins . Experiments are presented that suggest that this enhancer operates by a similar mechanism as that described previously for the strong second intron enhancer, involving an interaction with the basal transcriptional machinery . Digestions with low levels of micrococcal nuclease were performed to ascertain whether nucleosomes were present in the DNase I sensitive enhancer region . Nine different micrococcal nuclease-hypersensitive (MH) sites were detected in HepG2 cells but not in HeLa cells; one MH site was common to both cell types, and HeLa cells exhibited three unique MH sites . The first six MH sites (I-VI) are spaced approximately 200 base pairs apart, suggesting the presence of positioned nucleosomes in that region . MH sites VI-X are more closely spaced, suggesting either additional cutting sites within the core particle or the absence of one or two nucleosomes in this segment of the third intron enhancer. Nucleic Acids Res, 1992 Sep 11, 20(17), 4525 - 31 Ligand dependence of estrogen receptor induced changes in chromatin structure; Gilbert DM et al.; To determine whether the human estrogen receptor requires ligand to bind to its cognate estrogen receptor element (ERE) in vivo, we have examined the structure of chromatin at a chromosomally integrated ERE-URA3 reporter gene in yeast, and the influence of ligand bound and ligand free estrogen receptors on that structure . Using indirect end-labelling to map DNaseI and micrococcal nuclease sensitive sites, we found that receptor induced alterations in chromatin structure were completely dependent upon the presence of estradiol . These same alterations in chromatin structure were induced by a truncated estrogen receptor with both TAF-1 and TAF-2 transactivation functions deleted, suggesting that DNA binding per se disrupts chromatin structure . These results support models in which the estrogen receptor requires ligand to bind to the ERE in vivo. Mol Biol (Mosk), 1992 Sep-Oct, 26(5), 1036 - 46 {Enzymatic cleavage of superhelical DNA in a liquid crystal state}; Salianov VI et al.; Superhelical pBR322 DNA molecules form liquid-crystalline dispersions in water-salt solutions containing poly(ethyleneglycol) . The formation of the liquid-crystalline dispersions from superhelical DNA molecules results in the appearance of two sites inside the DNA molecules that are split by Micrococcal nuclease . The first site of digestion does not differ from the standard site split by this enzyme in water-salt solutions, whereas the second one represents a new site specific only for the DNA molecules forming liquid-crystalline dispersions . Splitting of the DNA molecule through the first site is accompanied by formation of its linear form; splitting of a new site results in the formation of two linear DNA fragments with molecular masses equal to half of the initial DNA molecules . Enzyme digestion of superhelical DNA molecules forming liquid-crystalline dispersions induces a reformation of the "nonspecific" space organization of dispersions to the cholesteric one . A hypothetic model for packing of the superhelical DNA molecules inside liquid-crystalline dispersions and its transformation under enzyme action is suggested. Int J Biochem, 1992 Sep, 24(9), 1385 - 90 Nuclear glycoproteins of hamster liver and Kirkman-Robbins hepatoma recognized by concanavalin A; Lipinska A et al.; 1 . Glycoproteins recognized by Concanavalin A (ConA) have been identified in nuclei and nuclear fractions differing in sensitivity to micrococcal nuclease digestion from hamster liver and Kirkman-Robbins hepatoma . 2 . The major ConA binding proteins from hamster liver and Kirkman-Robbins hepatoma nuclei have molecular weights about 27,000 and 57,000, and 38,000 and 49,000, respectively . 3 . A distinct distribution of glycoproteins between fractions differing in sensitivity to nuclease digestion has not been observed. Virology, 1992 Aug, 189(2), 800 - 2 Abutilon mosaic geminivirus double-stranded DNA is packed into minichromosomes; Pilartz M et al.; An intermediate form of Abutilon mosaic geminivirus, a complex of viral double-stranded DNA (dsDNA) and protein, was characterized by two different techniques . Cesium sulphate gradient centrifugation was used to show that the majority of this form comigrates with host chromatin . Micrococcus nuclease digestion experiments with isolated nuclei demonstrated that the viral dsDNA is organized in a manner comparable to that of host nucleosomes . Results from a previous electron microscopical work (Abouzid, A . M., Frischmuth, T., and H . Jeske, 1988, Mol . Gen . Genet . 212, 252-258) suggested to us that a part of the viral dsDNA must be free of nucleosomes . Whether this nucleosome-free space corresponds to the intergenic region which contains most of the promotor sequences and the putative origin of replication is discussed. Nucleic Acids Res, 1992 Jul 25, 20(14), 3671 - 8 hnRNP I, the polypyrimidine tract-binding protein: distinct nuclear localization and association with hnRNAs; Ghetti A et al.; Many hnRNP proteins and snRNPs interact with hnRNA in the nucleus of eukaryotic cells and affect the fate of hnRNA and its processing into mRNA . There are at least 20 abundant proteins in vertebrate cell hnRNP complexes and their structure and arrangement on specific hnRNAs is likely to be important for the processing of pre-mRNAs . hnRNP I, a basic protein of ca . 58,000 daltons by SDS-PAGE, is one of the abundant hnRNA-binding proteins . Monoclonal antibodies to hnRNP I were produced and full length cDNA clones for hnRNP I were isolated and sequenced . The sequence of hnRNP I (59,632 daltons and pI 9.86) demonstrates that it is identical to the previously described polypyrimidine tract-binding protein (PTB) and shows that it is highly related to hnRNP L . The sequences of these two proteins, I and L, define a new family of hnRNP proteins within the large superfamily of the RNP consensus RNA-binding proteins . Here we describe experiments which reveal new and unique properties on the association of hnRNP I/PTB with hnRNP complexes and on its cellular localization . Micrococcal nuclease digestions show that hnRNP I, along with hnRNP S and P, is released from hnRNP complexes by nuclease digestion more readily than most other hnRNP proteins . This nuclease hypersensitivity suggests that hnRNP I is bound to hnRNA regions that are particularly exposed in the complexes . Immunofluorescence microscopy shows that hnRNP I is found in the nucleoplasm but in addition high concentrations are detected in a discrete perinucleolar structure . Thus, the PTB is one of the major proteins that bind pre-mRNAs; it is bound to nuclease-hypersensitive regions of the hnRNA-protein complexes and shows a novel pattern of nuclear localization. J Biol Chem, 1992 Jul 25, 267(21), 14622 - 8 S(T)PXX motifs promote the interaction between the extended N-terminal tails of histone H2B with "linker" DNA; Lindsey GG et al.; The assembly of hybrid core particles onto long chicken DNA with histone H2B in the chicken histone octamer replaced with either wheat histone H2B(2) or sea urchin sperm histone H2B(1) or H2B(2) is described . All these histone H2B variants have N-terminal extensions of between 18 and 20 amino acids, although only those from sea urchin sperm have S(T)PXX motifs present . Whereas chicken histone octamers protected 167 base pairs (bp) (representing two full turns) of DNA against micrococcal nuclease digestion (Lindsey, G . G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D . L . (1991) J . Mol . Biol . 218, 805-813), all the hybrid histone octamers protected an additional 17-bp DNA against nuclease digestion . This protection was more marked in the case of hybrid octamers containing sea urchin sperm histone H2B variants and similar to that described previously (Lindsey, G . G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D . L . (1991) J . Mol . Biol . 218, 805-813) for hybrid histone octamers containing wheat histone H2A variants all of which also have S(T)PXX motifs present . Continued micrococcal nuclease digestion reduced the length of DNA associated with the core particle via 172-, 162-, and 152-bp intermediates until the 146-bp core particle was obtained . These DNA lengths were approximately 5 bp or half a helical turn longer than those reported previously for stripped chicken chromatin and for core particles containing histone octamers reconstituted using "normal" length histone H2B variants . This protection pattern was also found in stripped sea urchin sperm chromatin, demonstrating that the assembly/digestion methodology reflects the in vivo situation . The interaction between the N-terminal histone H2B extension and DNA of the "linker" region was confirmed by demonstrating that stripped sea urchin sperm chromatin precipitated between 120 and 500 mM NaCl in a manner analogous to unstripped chromatin whereas stripped chicken chromatin did not . Tryptic digestion to remove all the histone tails abolished this precipitation as well as the protection of DNA outside of the 167-bp core particle against nuclease digestion. J Mol Biol, 1992 Jul 5, 226(1), 47 - 58 Formation of open and elongating transcription complexes by RNA polymerase III; Kassavetis GA et al.; The Saccharomyces cerevisiae transcription factors (TF) IIIB and IIIC assemble onto their respective DNA-binding sites on the SUP4 tRNA(Tyr) gene at 0 degrees C . RNA polymerase III specifically associates at 0 degrees C with this TFIIIC-TFIIIB-DNA complex to form a stable "closed" promoter complex in which the DNA surrounding the transcriptional start retains its duplex form . Promoter "opening" is a temperature-dependent and readily reversible process that involves up to 22 unwound base-pairs of DNA, and can be followed by analyzing the hyperreactivity of thymine to KMnO4 oxidation . This promoter opening increases progressively from 10 degrees C to 40 degrees C, with at least two regions within the transcription bubble appearing to melt independently . In contrast, the temperature dependence of forming an initiated transcription complex containing a 17 nucleotide nascent RNA chain displays a sharp transition between 10 degrees C and 15 degrees C . When RNA polymerase initiates transcription under conditions that limit the nascent RNA chain to less than six nucleotides, there is no displacement of the transcription bubble . These transcription complexes are distinguishable from "open" promoter complexes in their maintenance of the transcription bubble at 0 degrees C, and from transcription complexes with more extended (17 nucleotide) RNA chains in their sensitivity to disruption by heparin . In light of recent results by others that demonstrate a requirement for an RNA transcription factor in a Bombyx mori-based in vitro RNA polymerase III transcription system, we have searched for a comparable component in the S . cerevisiae-derived system . We show that if an RNA component is required in the yeast-derived system, it is not susceptible to inactivation by massive amounts of micrococcal nuclease, RNase A, or RNase T1. Gen Comp Endocrinol, 1992 Jul, 87(1), 105 - 10 Tissue-specific sensitivity of chromatin and the vitellogenin gene to micrococcal nuclease after continuous exposure of salmon (Salmo salar) to 17 beta-estradiol; Waters S et al.; Smoltified Atlantic salmon (Salmo salar), 2 years old and weighing 217 +/- 13 g, were treated for 2 weeks with 17 beta-estradiol containing silastic capsules implanted intraperitoneally . Control fish received empty capsules . Vitellogenin, present in the blood of both groups of fish, was enhanced by estradiol treatment . Nuclei were isolated from liver, blood cells, and brain and incubated with increasing concentrations of micrococcal nuclease (EC 3.1.31.1) . In liver there were more mononucleosomes as a percentage of total chromatin in hormone-treated than in control fish . Using vitellogenin cDNA as a probe the highest hybridization signals were seen when 2 to 4% of the chromatin was digested to mononucleosomes . In blood cell and brain nuclei independent of the extent of the chromatin released the hybridization signals remained low . The expression of the vitellogenin gene in immature females was potentiated by exogenous estradiol to give increased micrococcal nuclease sensitivity of the chromatin without enhancement of the hybridization level . Micrococcal nuclease digestion and hybridization of the vitellogenin gene demonstrated that the hepatic specificity of vitellogenin synthesis is manifested as structural modulations of the chromatin containing the vitellogenin gene. Arch Biochem Biophys, 1992 Jul, 296(1), 264 - 70 Characterization of ribonuclease P isolated from rat liver cytosol; Jayanthi GP et al.; Rat liver ribonuclease P was isolated from a cytosolic fraction and shown to have optimal activity in the presence of 1 mM MgCl2 and 150-200 mM KCl using Escherchia coli pre-tRNA(Tyr) as substrate . In cesium sulfate isopycnic density gradients, the enzyme had a buoyant density of 1.36 g/ml, indicating that it is a ribonucleoprotein complex . Analysis of the RNAs in the enzyme sample purified through two successive Cs2SO4 density gradient steps revealed the copurification of two major species of RNA (RRP1 and RRP2) along with several less abundant RNAs . Rat liver ribonuclease P activity was insensitive to micrococcal nuclease pretreatment . However, the nuclease-treated preparations contained several incompletely degraded RNA species that may have been sufficient to support the ribonuclease P activity . When RNase A was substituted for micrococcal nuclease, the ribonuclease P activity was diminished by greater than 90%, suggesting the requirement for an RNA subunit for activity. Proc Natl Acad Sci U S A, 1992 Jul 1, 89(13), 6119 - 23 Vitamin D-responsive protein-DNA interactions at multiple promoter regulatory elements that contribute to the level of rat osteocalcin gene expression; Bortell R et al.; The observation that vitamin D-mediated enhancement of osteocalcin (OC) gene expression is dependent on and reciprocally related to the level of basal gene expression suggests that an interaction of the vitamin D responsive element (VDRE) with basal regulatory elements of the OC gene promoter contributes to both basal and vitamin D-enhanced transcription . Protein-DNA interactions at the VDRE of the rat OC gene (nucleotides -466 to -437) are reflected by direct sequence-specific and antibody-sensitive binding of the endogenous vitamin D receptor present in ROS 17/2.8 osteosarcoma nuclear protein extracts . In addition, a vitamin D-responsive increase in OC gene transcription is accompanied by enhanced non-vitamin D receptor-mediated protein-DNA interactions in the "TATA" box region (nucleotides -44 to +23), which also contains a potential glucocorticoid responsive element . Evidence for proximity of the VDRE with the basal regulatory elements is provided by two features of nuclear architecture . (i) Nuclear matrix attachment elements in the rat OC gene promoter that bind nuclear matrix proteins with sequence specificity may impose structural constraints on promoter conformation . (ii) Limited micrococcal nuclease digestion and Southern blot analysis indicate that three nucleosomes can be accommodated in the sequence spanning the OC gene VDRE, the OC/CCAAT box (nucleotides -99 to -76), and the TATA/glucocorticoid responsive element, and thereby the potential distance between the VDRE and the basal regulatory elements can be reduced . A model is presented for the contribution of both the VDRE and proximal promoter elements to the enhancement of OC gene transcription in response to vitamin D . The vitamin D receptor plus accessory proteins may function cooperatively with basal regulatory factors to modulate the extent to which the OC gene is transcribed. Neurochem Int, 1992 Jul, 21(1), 21 - 7 Isolation and characterization of hamster brain polyribosome-cytomatrix complexes; Desjardins P et al.; We have developed a method for the isolation of a brain subcellular fraction enriched in both highly aggregated polyribosomes and cytoskeletal proteins . This method is based on gentle dispersion of brain tissue and low speed centrifugation . This fraction is enriched in typical cytoskeletal proteins as glial fibrillary protein, neurofilament proteins and actin . Messenger RNA did not seem to be involved in the polyribosome association to the cytomatrix as shown by the effect of exposure to micrococcal nuclease . On the other hand, in vivo disruption of protein synthesis by acute experimental phenylketonuria, hypothermia or heat-shock did not cause the release of ribosomes from the cytomatrix. Biochim Biophys Acta, 1992 Jun 15, 1131(2), 139 - 44 Histone H4 mRNA levels are down-regulated by 3' RNA processing during terminal differentiation of myoblasts; Larson DE et al.; The capacity for 3' processing of the histone H4 pre-mRNA is lost following differentiation of rat L6 myoblasts to myotubes . Nuclear extracts prepared from proliferating myoblasts, but not differentiated myotubes, actively process histone H4 pre-mRNA in vitro . The activity of two factors required for 3' processing, the heat-labile factor and U7 snRNP, also changes during the differentiation period, concurrent with the loss of 3' processing activity . During myotube formation, the activity of the heat-labile factor decreases significantly while the 5' sequences of the U7 snRNA become progressively resistant to micrococcal nuclease digestion . Thus, the dramatic down-shift in histone H4 mRNA levels which occurs during myoblast differentiation is controlled at both the transcriptional and posttranscriptional level. Chem Pharm Bull (Tokyo), 1992 Jun, 40(6), 1523 - 6 Colorimetric assay for lysozyme using Micrococcus luteus labeled with a blue dye, Remazol brilliant blue R, as a substrate; Ito Y et al.; Micrococcus luteus (M . lysodeikticus) labeled with Remazol brilliant blue R (blue ML) was prepared as a novel substrate for the colorimetric assay of lysozyme . The treatment of the labeled substrate with lysozyme resulted in the release of soluble blue products which can be easily measured spectrophotometrically at 600 nm . The blue color was most efficiently released at pH 7 and ionic strength of 0.2 on incubation with hen lysozyme at 40 degrees C . A new colorimetric method for the assay of lysozyme using this substrate was developed . The assay system gave a linear dose-response curve, and as little as 0.1 microgram of human lysozyme (1 microgram/ml, 100 microliters) can be detected . The present method is more convenient and reproducible than the conventional lysozyme assay with bacterial cells . Application of the system to the determination of lysozyme in human serum is described. J Appl Bacteriol, 1992 May, 72(5), 429 - 34 The effect of dilution rate and pH on biomass and proteinase production by Micrococcus sedentarius grown in continuous culture; Holland KT et al.; Micrococcus sedentarius, an organism associated with pitted keratolysis, produced two proteinases in culture supernatant fluids, as shown by non-denaturing PAGE with overlaying with a casein substrate . A mixture had optimal activity at pH 10 with azocasein substrate . At pH 7.1 and 8.1 in continuous culture with varying dilution rates high proteinase production occurred at relative specific growth rates (mu rels) 0.39 and 0.77 and biomass concentrations decreased with increasing dilution rate . One proteinase was constitutive and varied little in production with different growth rates . The other proteinase was under control with high production at low growth rates and no production at high growth rates . With varying pH at mu rels 0.39 and 0.77 maximum biomass concentration and proteinase production occurred between pH 8.0 and 9.0 as did the highest specific growth rate . These results support the hypothesis that Mic . sedentarius produces pitting in the stratum corneum when the skin is hydrated and the pH rises above neutrality. J Gen Virol, 1992 May, 73 ( Pt 5), 1243 - 9 Detection of RNA-protein complex in vaccinia virus core in vitro transcription system; Damaso CR et al.; The incubation of vaccinia virus cores in appropriate conditions promotes the release of core proteins into a supernatant fraction . Under transcription assay conditions core mRNAs are extruded in association with viral core proteins, however the presence of these proteins within the core particle is not essential for RNA synthesis and extrusion . The RNA-protein complex is resistant to micrococcal nuclease . Five proteins of 60K, 43K, 28K, 18K and 14.5K with RNA-binding abilities have been identified by {32P}RNA overlay protein blot assays . These proteins are likely to be a component of the viral ribonucleoprotein complex since core basic proteins with similar MrS have been identified and at least one RNA-binding protein is predicted in the vaccinia virus genome. Mol Immunol, 1992 May, 29(5), 609 - 17 Immunogenicity of Z-DNA depends on the size of polynucleotide presented in complexes with methylated BSA; Edgington SM et al.; The importance of polynucleotide size for immunogenicity was tested with size-fractionated Z-DNA . High molecular weight Z-DNA, larger than 1000 bp, was fragmented by digestion with micrococcal nuclease . Fractions corresponding to less than 60, 60-120, 100-200, 200-400 and 400-900 bp were isolated by gel filtration on Sepharose 4B . These fractions and the greater than 1000 bp Z-DNA were mixed with methylated BSA and the complexes were injected into C57BL/6 mice with RIBI adjuvant . Only one of four mice responded to the less than 60 bp immunogen . All the fractions larger than 60 bp induced specific anti-Z-DNA antibodies, mostly of IgG isotype, in all animals injected . Fractions larger than 200 bp induced antisera of higher titer than did 60-120 or 100-200 bp fractions . All positive sera reacted with Z-DNA but not with B-DNA and only very weakly with denatured DNA . In competitive assays, similar concentrations of fragments larger than 60 bp inhibited binding to immobilized Z-DNA . A higher concentration of less than 60 bp fragments was required for competitive binding . Even for a highly immunogenic nucleic acid that differs from the B-DNA conformation, a polynucleotide larger than 100 bp is much more immunogenic than smaller fragments. Antimicrob Agents Chemother, 1992 May, 36(5), 955 - 61 Dissociation of the antimicrobial activity of bacitracin USP from its renovascular effects; Drapeau G et al.; Bacitracin is a nephrotoxic antibiotic that has recently been shown to induce contractile effects in aortas isolated from rabbits by stimulating receptors for 5-hydroxytryptamine (5-HT) . The possible renovascular actions of this antibiotic were investigated . Bacitracin USP increased the vascular resistance in a concentration-dependent manner (9 to 175 micrograms/ml) in rat kidneys perfused with a constant flow of Krebs solution . This was significantly inhibited by 5-HT antagonists, but only partially at the higher bacitracin concentration . An antagonist of the chemotactic peptide fMet-Leu-Phe failed to influence the pressor effect of bacitracin in rat kidneys . Indomethacin modestly reduced the effect of all potent pressor agents in the rat organ . Bacitracin USP was separated in several fractions by using C18 reverse-phase chromatography . Two distinct fractions were vasoconstrictive when infused in rat kidneys; both fractions were 5-HT mimetics . These peaks were different from the major antibiotic peak, bacitracin A, which was identified by using analytical high-pressure liquid chromatography, mass spectrometry, and inhibition of Micrococcus luteus growth . The less polar vasoactive peak corresponded to at least two minor peptides of the bacitracin family . The most abundant of these vasoactive peptides had no direct contractile effect on an aorta isolated from a rabbit, but a preliminary metabolic study in rat kidneys suggests that it is apparently transformed into a potent 5-HT agonist that is active on the aorta preparation . Bacitracin A, the major constituent of bacitracin with antimicrobial activity, had no vasoconstrictor effect in the test systems that we used; however, we did rule out the possibility that the renovascular stimulants found in the bacitracin mixture do not derive spontaneously or by biotransformation from the antibacterial forms of bacitracin. Nucleic Acids Res, 1992 Apr 25, 20(8), 1943 - 8 Chromatin structure and DNase I hypersensitivity in the transcriptionally active and inactive porcine tumor necrosis factor gene locus; Kuhnert P et al.; We have analyzed the chromatin structure of the porcine tumor necrosis factor gene locus (TNF-alpha and TNF-beta) . Nuclei from porcine peripheral blood mononuclear cells were digested with different nucleases . As assessed with micrococcal nuclease, the two TNF genes displayed slightly faster digestion kinetics than bulk DNA . Studies with DNaseI revealed distinct DNaseI hypersensitive sites (DH-sites) within the porcine TNF locus . Four DH-sites could be observed in the promoter and mRNA leader regions of the TNF-beta gene . Two DH-sites could be observed for the TNF-alpha gene, one located in the promoter region close to the TATA-box and the other site in intron 3 . This pattern of DH-sites was present independently of the activation state of the cells . Interestingly in a porcine macrophage-like cell line, we found that the TNF-alpha promoter DH-site disappeared and another DH-site appeared in the region of intron 1 . Additionally, the DH-site of intron 3 could be enhanced by PMA-stimulation in these cells . TNF-beta sites were not detected in this cell line . However, DH-sites were totally absent in fibroblasts (freshly isolated from testicles) and in porcine kidney cells (PK15 cell line) both of which do not transcribe the TNF genes . Therefore, the pattern of DH-sites corresponds to the transcriptional activity of analyzed cells. J Biol Chem, 1992 Apr 5, 267(10), 6782 - 8 A ribonuclease activity is activated by heparin or by digestion with proteinase K in mitochondrial extracts of Leishmania tarentolae; Simpson AM et al.; A ribonuclease activity in a 100,000 x g supernatant of a Triton lysate of a mitochondrial-kinetoplast fraction from Leishmania tarentolae is activated by incubation with heparin or by predigestion of the lysate with proteinase k or pronase . In vitro-transcribed pre-edited cytochrome b mRNA is cleaved at several sites . With time, complete degradation of the RNA occurs . All cleavages occurred within putative single-stranded regions of the RNA . No cleavage was observed with 9 S rRNA . The presence of a nonspecific nucleotide or nucleoside slows the rate of cleavage . The cleavage activity is inhibited by sodium dodecyl sulfate or phenol/chloroform extraction, is retained by a 10-kDa cutoff filter, and passes through a 30-kDa filter . Micrococcal nuclease inhibits the proteinase-induced activity but not the heparin-induced activity. J Biochem (Tokyo), 1992 Apr, 111(4), 496 - 9 Comparison of two reconstituted systems for in vitro transcription and replication of influenza virus; Seong BL et al.; The transcription and replication of influenza RNA can be studied in vitro by the reconstitution of functional ribonucleoprotein (RNP) complex from viral core proteins including the RNA polymerase (complex of three P protein subunits) and nucleoprotein (NP), and model templates . Here, two different core protein preparations, one based on CsCl centrifugation (CS enzyme) and the other on micrococcal nuclease treatment of viral cores (MN enzyme), were compared side-by-side . Short model RNA templates and their 3'-half molecules of both viral RNA (vRNA) and complementary RNA (cRNA) senses were reconstituted with the core protein preparations in parallel, and RNA polymerase activity was tested either in the presence or absence of ApG or globin mRNA as primers . Both enzyme preparations were active in the syntheses of short vRNA and cRNA transcripts using ApG as a primer, although the synthesis of cRNA was 2-10-fold higher (depending on the template used) than the synthesis of vRNA . The MN enzyme, however, was more active per weight of total protein than the CS enzyme, probably because of its higher content of RNA polymerase . Both enzymes failed to show primer-independent synthesis of vRNA . The differences observed in the synthesis of short transcripts using globin mRNA as a primer are discussed. Mol Cell Biol, 1992 Apr, 12(4), 1553 - 60 Nuclear processing of the 3'-terminal nucleotides of pre-U1 RNA in Xenopus laevis oocytes; Yang H et al.; U1 small nuclear RNA is synthesized as a precursor with several extra nucleotides at its 3' end . We show that in Xenopus laevis oocytes, removal of the terminal two nucleotides occurs after the RNA has transited through the cytoplasm and returned to the nucleus . The activity is controlled by an inhibitor of processing, which we call TPI, for 3'-terminal processing inhibitor . This inhibitor is sensitive to both micrococcal nuclease and trypsin treatment, indicating that it is a nucleoprotein . TPI inhibits the 3' processing of pre-U1 RNAs that have 5' ends containing m7G caps but not mature m2,2,7G caps; this finding suggests that TPI interacts directly or indirectly with the 5' end of pre-U1 RNA . The inhibition of processing by TPI, almost complete at 19 degrees C, is reversibly inactivated at slightly higher temperatures . TPI activity is solely in the soluble fraction of oocyte nuclear extracts, in contrast to the 3'-terminal processing activity, which is present in both the particulate and soluble fractions . We propose that the differential processing of the 3'-terminal nucleotides of pre-U1 RNA after its return from the cytoplasm, but not before its exit from the nucleus, may be due to the association of TPI with the m7G cap on the newly synthesized pre-U1 RNA. J Pharmacol Toxicol Methods, 1992 Apr, 27(2), 95 - 100 A rapid microtiter plate method for the detection of lysozyme release from human neutrophils; Moreira-Ludewig R et al.; An improved method was devised to measure lysozyme secreted from human neutrophils {polymorphonuclear leukocyte (PMN)} using a microtiter plate reader capable of analyzing enzyme kinetics . The assay is an adaptation of the classical photometric method which detects changes in the turbidity of a bacterial suspension, Micrococcus lysodeikticus, caused by the enzymatic activity of lysozyme . A standard curve using chicken egg white lysozyme was generated, and activity was detectable between the range of 1 and 100 ng/ml . Leukotriene B4 (LTB4)-induced lysozyme release from human PMN was comparable in both the standard assay and the microtiter plate adaptation with EC50 values of 6.5 and 7.2 nM, respectively . Other select stimuli and their receptor antagonists were also used to evaluate the method . Dose-response curves for chemotactic hexapeptide (CHP), recombinant human C5a (rhC5a), and platelet-activating factor (PAF) resulted in EC50 values of 0.14, 0.80, and 542.00 nM, respectively . Inhibition of lysozyme release was studied using receptor antagonists N-t-Boc-L-methionyl-L-leucyl-L-phenylalanine (N-t-Boc), LY223982, and protamine, which are putative inhibitors of formyl peptides (i.e., CHP), LTB4, and C5a, respectively . N-t-Boc inhibited CHP-induced (0.2 nM) enzyme release with an IC50 of 2 microM; LY223982 blocked LTB4-induced (20 nM) release resulting in an IC50 of 52 nM; and protamine inhibited rhC5a-induced (1.5 nM) release with an IC50 of 2 microM . Further studies revealed that CHP, LTB4, and rhC5a were selectively inhibited by their respective antagonists, albeit LY223982 and protamine were also weak inhibitors of CHP and LTB4, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1992 Mar 31, 31(12), 3084 - 91 Isolation and structure of an intrastrand cross-link adduct of mitomycin C and DNA; Bizanek R et al.; A new covalent mitomycin C-DNA adduct (4) was isolated from DNA exposed to reductively activated mitomycin C (MC) in vitro . The MC-treated DNA was hydrolyzed enzymatically under certain conditions, and the new adduct was isolated from the hydrolysate by HPLC . Its structure was determined by ultraviolet and circular dichroism spectroscopy and chemical and enzymatic transformations conducted on microscale . In the structure, a single 2" beta, 7"-diaminomitosene residue is linked bifunctionally to two guanines in the dinucleoside phosphate d(GpG) . The guanines are linked at their N2 atoms to the C1" and C10" positions of the mitosene, respectively . A key to the structure was a finding that removal of the mitosene from the adduct by hot piperidine yielded d(GpG); another was that the adduct was slowly converted to the known interstrand cross-link adduct 3 by snake venom diesterase and alkaline phosphatase . Adduct 4 represents an intrastrand cross-link in DNA formed by MC . Of the two possible strand-polarity isomers of 4, 4a in which the mitosene 1"-position is linked to the 3'-guanine of d(GpG) is designated as the proper structure, on the basis of the mechanism of the cross-linking reaction . The same adduct 4 was isolated from poly(dG).poly(dC), synthetic oligonucleotides containing the GpG sequence, and Micrococcus luteus and calf thymus DNAs . The relative yields of interstrand and intrastrand cross-links (3 and 4) were determined under first-order kinetic conditions; an average 3.6-fold preference for the formation of 3 over that of 4 was observed . An explanation for this preference is proposed.(ABSTRACT TRUNCATED AT 250 WORDS) Biochem J, 1992 Mar 15, 282 ( Pt 3), 723 - 7 Effect of polyamine depletion on chromatin structure in U-87 MG human brain tumour cells; Basu HS et al.; The chromatin structure of polyamine-depleted U-87 MG human brain tumour cells was studied by following the kinetics of digestion of cell nuclei by micrococcal nuclease and bovine pancreatic DNAase I . Cells growing in monolayers were treated with either alpha-difluoromethylornithine (DFMO), to deplete putrescine and spermidine, or N1,N14-bis(ethyl)homospermine (BE-4-4-4), to deplete putrescine, spermidine and spermine . BE-4-4-4 increased the initial rates of digestion and the magnitudes of limit digest by both enzymes; DFMO increased the limit digests without affecting initial digestion rates . Addition of 1 mM-putrescine 1 day after addition of DFMO reversed the effect of DFMO on limit digests . (Because polyamine uptake is low in cells treated with BE-4-4-4, and because putrescine does not reverse the growth-inhibitory effects of BE-4-4-4, reversal of the effects of BE-4-4-4 with putrescine was not attempted.) The increases in initial rates and limit digests did not result from changes in the lengths of nucleosomal or linker DNA, from blocks in cell-cycle progression, or from growth inhibition caused by DFMO or BE-4-4-4 . Thus, because the limit digest is highest in cells with the lowest polyamine levels, it seems clear that the enhanced enzymic digestion of nuclei is caused by polyamine depletion and its possible effect on chromatin structure. Biochim Biophys Acta, 1992 Mar 13, 1099(3), 219 - 25 Significant quantities of endogenous GDP and ADP are present on catalytic sites of the F1-ATPase isolated from M . lysodeikticus in the absence of added nucleotides; Mileykovskaya EI et al.; The F1-ATPase from Micrococcus lysodeikticus is isolated in the absence of exogenous nucleotides . After removing loosely bound nucleotides from the isolated enzyme by gel permeation chromatography, analysis for tightly bound nucleotides revealed in 14 experiments 0.4 +/- 0.1 mol ADP, 0.5 +/- 0.2 mol GDP, and 0.8 +/- 0.2 mol ATP per mol of F1 . Incubation of the isolated enzyme with Mg2+ or Ca2+ did not alter the endogenous nucleotide composition of the enzyme, indicating that endogenous ATP is not bound to a catalytic site . Incubation of the enzyme with P(i) decreased the amount of tightly bound ADP and GDP but did not effect the ATP content . Hydrolysis of MgATP in the presence of sulfite raised the tightly bound ADP and lowered tightly bound GDP on the enzyme . In the reciprocal experiment, hydrolysis of MgGTP in the presence of sulfite raised tightly bound GDP and lowered tightly bound ADP . Turnover did not affect the content of tightly bound ATP on the enzyme . These results suggest that endogenous ADP and GDP are bound to exchangeable catalytic sites, whereas endogenous ATP is bound to noncatalytic sites which do not exchange . The presence of endogenous GDP on catalytic sites of isolated F1 suggests that the F0F1-ATP synthase of M . lysodeikticus might synthesize both GTP and ATP under physiological conditions . In support of this hypothesis, we have found that plasma membrane vesicles derived from M . lysodeikticus synthesize {32P}GTP from {32P}P(i) using malate as electron donor for oxidative phosphorylation. Nucleic Acids Res, 1992 Mar 11, 20(5), 1031 - 8 Activation of the weakly regulated PHO8 promoter in S . cerevisiae: chromatin transition and binding sites for the positive regulatory protein PHO4; Barbaric S et al.; PHO8 encodes an alkaline phosphatase in Saccharomyces cerevisiae whose transcription is regulated by the phosphate concentration in the medium . This occurs through the action of several positive and negative regulatory proteins, also involved in the regulation of other members of the phosphatase gene family . A central role is played by PHO4, the gene encoding a DNA binding regulatory protein . Digestion experiments with DNasel, micrococcal nuclease and 20 different restriction nucleases show that under conditions of PHO8 repression, there is a highly ordered chromatin structure at the promoter consisting of three hypersensitive regions, approximately 820 to 690, 540 to 510, and 230 to 160 bp upstream of the initiation codon . These hypersensitive sites are surrounded by DNA organized in nucleosomes . Gel shift analysis and in vitro footprinting revealed the presence of two PHO4 binding sites at the PHO8 promoter: a low affinity site at -728 and a high affinity site at -532 . Each one is located within a hypersensitive site . Upon derepression of PHO8, the chromatin structure changes significantly: The two upstream hypersensitive sites containing the PHO4 binding sites merge, resulting in a long region of hypersensitivity . This transition is PHO4 dependent . However, not all of the promoter becomes nucleosome free . Instead, as a novel feature, regions of intermediate accessibility are generated upstream and downstream of the third hypersensitive site, the latter region encompassing the TATA-box . The available data fit best into a concept that these regions are organized in unstable or partly unfolded nucleosomes. Biochem Int, 1992 Mar, 26(3), 509 - 19 Structure of a Micrococcus lysodeikticus cell wall fragment containing phosphorylated sugars; Siddiqui M et al.; A polysaccharide-peptidoglycan complex containing different phosphorylated sugars from Micrococcus lysodeikticus cell wall has been isolated and purified . The peptidoglycan contained muramic acid 6-phosphate and N-acetylglucosamine 6-phosphate as phosphorylated sugars in addition to other sugar residues . Mild acid hydrolysis of the peptidoglycan and subsequent reduction of the released polysaccharide showed therein the presence of glucose and N-acetyl-glucosamine in the linkage of the external polysaccharide residues to the peptidoglycan through phosphodiester linkage . These data suggest the presence of polysaccharide chains linked to a peptidoglycan core through two phosphorylated sugars via two different terminal carbohydrate residues of the external polysaccharide chains in a same polymer. Cell Signal, 1992 Mar, 4(2), 145 - 51 Importance of substrate conformation in the phosphorylation of chromatin-associated proteins by exogenous protein kinase C; Testori A et al.; Protein kinase C (PKC)-mediated phosphorylation of chromatin-associated proteins was studied in vitro . HL-60 and HeLa nuclear proteins were notably unresponsive to exogenously added brain PKC . In contrast, 3T3 fibroblasts and lymphocytes from primary cultures exhibited PKC-dependent phosphorylation of chromatin-associated proteins when chromatin was induced to expand . Unexpanded nuclei in all cell lines were unresponsive . Responsiveness was particularly obvious in the decondensed chromatin of primary lymphocytes, where a large number of proteins were phosphorylated in response to exogenous PKC . DNAase-I and micrococcal nuclease strongly modulated these phosphorylation patterns indicating that the substrates were DNA-associated . It was concluded that although substrate conformation, i.e . condensation state, was the primary determining factor in control of PKC-dependent nuclear protein phosphorylation, different cell lines greatly differ in their overall responsiveness to exogenous PKC. Leukemia, 1992 Mar, 6(3), 224 - 6 Pneumonia due to Micrococcus spp . in a patient with acute myeloid leukaemia; Adang RP et al.; A 26-year-old female patient with acute myeloid leukaemia was hospitalized for the second cycle of remission induction chemotherapy . While neutropenic she developed progressive pulmonary infiltrate, with Micrococcus spp . cultured from two consecutive bronchoalveolar lavage fluids, resulting in respiratory insufficiency . The patient died after an unsuccessful cardiopulmonary resuscitation . This report of micrococcal pneumonia emphasizes that the pathogenicity of this skin commensal is not limited to infections in tissues surrounding prosthetic devices or indwelling intravenous catheters . Especially in immunocompromised patients, Micrococcus spp . from bronchoalveolar lavage fluids cannot be lightly dismissed as non-pathogenic when pneumonia is considered. Clin Infect Dis, 1992 Mar, 14(3), 641 - 6 Stomatococcus mucilaginosus: an emerging pathogen in neutropenic patients; McWhinney PH et al.; Stomatococcus mucilaginosus was isolated from eight neutropenic patients during nine febrile episodes over a 13-month period . Five of these isolates were from definite infections, including one case of fatal meningitis . This slime-producing, catalase-variable, gram-positive coccus is a component of the normal oral flora of humans . Its biochemical profile may result in misidentification; however, unlike most micrococci, it characteristically fails to grow on media containing 5% NaCl . All but one of our isolates were sensitive to benzylpenicillin, and all were sensitive to vancomycin . S . mucilaginosus may prove to be an important pathogen in severely immunocompromised patients. Proc Soc Exp Biol Med, 1992 Mar, 199(3), 282 - 6 Altered chromatin structure of cerebral nuclei in experimental diabetes mellitus; Mooradian AD; To determine whether diabetes alters chromatin structure in vivo, micrococcal nuclease digestion kinetics were analyzed in cerebral cortical and hepatic nuclei of streptozotocin-induced diabetic rats . Cerebral nuclei of diabetic rats maintained for 6 weeks were less susceptible to micrococcal nuclease digestion compared with control rats . Insulin treatment reversed diabetes-related changes in nuclease digestion kinetics . There were no changes in the kinetics of digestion in hepatic nuclei . The reduced digestibility of cerebral DNA in diabetes could not be attributed to altered DNA fluorescence spectra, or altered distribution of most abundant chromatin proteins that were either solubilized or that remained insoluble immediately following nuclease digestion . It is concluded that chronic, uncontrolled hyperglycemia can alter chromatin structure of some tissues in vivo, and this change is probably related to subtle alterations in DNA-protein interactions. Nucleic Acids Res, 1992 Feb 25, 20(4), 865 - 70 Evidence that nucleosomes on the mouse mammary tumor virus promoter adopt specific translational positions; Bresnick EH et al.; We have previously demonstrated that an array of six nucleosomes are phased on the mouse mammary tumor virus (MMTV) long terminal repeat (1,2) . In this study, we devised a new assay to measure the translational positions of specific nucleosomes on the MMTV promoter . Nucleosome core particles were purified and shown to contain A and B nucleosomal DNA by Taq polymerase primer extension with nucleosome-specific primers . The 5' and 3' boundaries of A and B nucleosomes were measured by extending to the end of the core DNA with internal primers . This approach yielded results consistent with major translational positions of -23 to +123 and -221 to -75 for A and B nucleosomes, respectively . The micrococcal nuclease cleavage patterns of A and B nucleosome regions in isolated nuclei are conserved at base-pair resolution in multiple murine cell lines containing either stable MMTV-reporter chimeras or endogenous proviruses . As the refined nucleosome positions place important transcription factor binding sites at the 3' edge of the B nucleosome and in the nucleosome A/B linker, we propose that linker histone depletion and chromatin unfolding may be required to expose these cis-elements during steroid hormone-induced transcription initiation. FEBS Lett, 1992 Feb 24, 298(2-3), 195 - 8 Interaction of calmodulin with lactoferrin; de Lillo A et al.; Calmodulin, as a major intracellular calcium-binding protein, regulates many Ca(2+)-dependent enzymes and plays an important role in a wide spectrum of cellular functions of the eukaryotes . Interaction between calmodulin and human lactoferrin, a 78 kDa protein with antibacterial properties, was found in the presence of Ca2+ using (i) a method for the detection of calmodulin binding proteins with biotinylated calmodulin, (ii) affinity chromatography on an agarose-calmodulin column with subsequent detection by an enzyme-linked immunosorbent assay (ELISA) . The binding of calmodulin to lactoferrin blocked the ability of lactoferrin to agglutinate Micrococcus lysodeikticus. Mol Pharmacol, 1992 Feb, 41(2), 245 - 51 Incorporation of the carbocyclic analog of 2'-deoxyguanosine into the DNA of herpes simplex virus and of HEp-2 cells infected with herpes simplex virus; Parker WB et al.; The carbocyclic analog of 2'-deoxyguanosine (CdG) is active against herpes simplex virus (HSV), human cytomegalovirus, and human hepatitis-B virus . In order to understand the mechanism of action of this compound against HSV, we have evaluated (a) the incorporation of {3H}CdG into viral and host DNA in HEp-2 cells infected with HSV and (b) the interaction of the 5'-triphosphate of CdG (CdG-TP) with the HSV DNA polymerase and human DNA polymerases alpha, beta, and gamma (EC 2.7.7.7) . Incubation of HSV-1-infected HEp-2 cells with {3H}CdG resulted in the incorporation of CdG into both the HSV and the host cell DNA . These results indicated that CdG-TP was used as a substrate for HSV DNA polymerase and for at least one of the cellular DNA polymerases . Degradation of both viral and host DNA with micrococcal nuclease and spleen phosphodiesterase indicated that CdG was incorporated primarily into internal positions in both DNAs . The viral DNA containing CdG sedimented in neutral and alkaline sucrose gradients in the same way as did viral DNA labeled with {3H}thymidine, indicating that the HSV DNA containing CdG was similar in size to untreated HSV DNA . CdG-TP was a competitive inhibitor of the incorporation of dGTP into DNA by the HSV DNA polymerase (Ki of 0.35 microM) and the human DNA polymerase alpha (Ki of 1 microM) . CdG-TP was not a potent inhibitor of either DNA polymerase beta or gamma . Using DNA-sequencing technology, CdG-TP was found to be an efficient substrate for HSV DNA polymerase . Incorporation of CdG monophosphate (CdG-MP) into the DNA by HSV DNA polymerase did not interfere with subsequent chain extension . These results suggested that the antiviral activity of CdG was due to its incorporation into the DNA and subsequent disruption of viral functions . In contrast, CdG-TP was not as good as dGTP as a substrate for DNA synthesis by DNA polymerase alpha, and incorporation of CdG-MP by DNA polymerase alpha inhibited further DNA chain elongation. J Mol Biol, 1992 Feb 5, 223(3), 651 - 9 Site-directed mutagenesis studies on the binding of the globular domain of linker histone H5 to the nucleosome; Buckle RS et al.; The globular domain of the linker histone H5 has been expressed in Escherichia coli . The purified peptide is functional as it permits chromatosome protection during micrococcal nuclease digestion of chromatin reconstituted with the peptide, indicating that it binds correctly at the dyad axis of the nucleosomal core particle . The globular domain residue lysine 64 is highly conserved within the linker histone family, and site-directed mutagenesis has been used to assess the importance of this residue in the binding of the globular domain of linker histone H5 to the nucleosome . Recombinant peptides mutated at lysine 64 are unable to elicit chromatosome protection to the same degree as the wild-type peptide, and since they appear to be fully folded, these observations confirm a major role for this residue in determining the effective interaction between the globular domain of histone H5 and the nucleosome. Genes Dev, 1992 Feb, 6(2), 197 - 210 Saccharomyces telomeres assume a non-nucleosomal chromatin structure; Wright JH et al.; The chromatin structures of the telomeric and subtelomeric regions on chromosomal DNA molecules in Saccharomyces cerevisiae were analyzed using micrococcal nuclease and DNAse I . The subtelomeric repeats X and Y' were assembled in nucleosomes . However, the terminal tracts of C1-3A repeats were protein protected in a particle larger than a nucleosome herein called a telosome . The proximal boundary of the telosome was a DNase I hypersensitive site . This boundary between the telosome and adjacent nucleosomes was completely accessible to Escherichia coli dam methylase when this enzyme was expressed in yeast, whereas a site 250 bp internal to the telomeric repeats was relatively inaccessible . Telosomes could be cleaved from chromosome ends with nuclease and solubilized as protein-DNA complexes . Immunoprecipitation of chromosomal telosomes with antiserum to the RAP1 protein indicated that RAP1 was one component of isolated telosomes . Thus, the termini of chromosomal DNA molecules in yeast are assembled in a non-nucleosomal structure encompassing the entire terminal C1-3A tract . This structure is separated from adjacent nucleosomes by a region of DNA that is highly accessible to enzymes. Blood, 1992 Feb 1, 79(3), 760 - 4 Specific detection of monocytic lysozyme within normal and leukemic cells; Leculier C et al.; A murine monoclonal antibody against human lysozyme (AHL MoAb) was produced and tested on normal and leukemic monocytes using flow cytometry . The antibody gave a positive reactivity on normal monocytes permeabilized by saponin (82% to 98% of positive cells) and a negative reactivity on normal permeabilized neutrophils . This monocyte-specific reactivity had not been observed using a polyclonal antibody . Nevertheless, immunoblotting detected lysozyme in both monocyte and polymorphonuclear leukocyte (PMNL) lysates . The AHL MoAb, in the presence of lysozyme substrate (Micrococcus lysodeikticus cell walls), strongly inhibited the enzymatic activity . Flow cytometric analysis of leukemic cells isolated from patients suffering from different subtypes of acute myeloid leukemia (French-American-British {FAB} classification FAB M1-5) showed a highly significant positivity of FAB M5 for lysozyme compared with the other subtypes . The present results were consistent with the detection of a lysozyme epitope by AHL MoAb located near the catalytic site in monocytes . The same epitope was probably masked in PMNL granules. Proc Natl Acad Sci U S A, 1992 Feb 1, 89(3), 1040 - 4 An in vitro system for the editing of ATP synthase subunit 9 mRNA using wheat mitochondrial extracts; Araya A et al.; A posttranscriptional modification (C-to-U) at specific positions of plant mitochondrial mRNA leads to changes in the amino acid sequence as well as to the emergence of novel initiation or termination sites . This phenomenon, named RNA editing, has been described for several mitochondrial genes from different plant sources . We have found recently that RNA editing of the ATP synthase subunit 9 (atp9) mRNA involves eight changes including the creation of a new stop codon . In this article, we describe an in vitro system devised to follow the editing of wheat mitochondrial atp9 mRNA . Nonedited mRNA was obtained to serve as substrate for this reaction by in vitro transcription of the corresponding gene with T7 RNA polymerase . The source of conversion factor(s) was a soluble fraction obtained from purified wheat mitochondria lysed with salt and detergent . Edited RNA molecules were detected by hybridization with an end-labeled synthetic oligodeoxynucleotide probe complementary to a short region containing four editing events . Optimal conditions for the in vitro RNA editing reaction were determined . The reaction is sensitive to high temperature and protease digestion . Pretreatment with micrococcal nuclease decreased RNA editing activity in the mitochondrial extract, suggesting that a nucleic acid is necessary for the enzymatic reactions . Analysis of the edited mRNA showed that the in vitro reaction led to the same products as those observed in vivo. J Biochem (Tokyo), 1992 Feb, 111(2), 259 - 64 Inducibility of protein-reactive antibodies by peptide immunization: comparison of three epitope peptides of hen egg-white lysozyme; Seki J et al.; Three epitope peptides of hen egg-white lysozyme (HEL) were tested for ability to induce antibodies reactive with native HEL . Each peptide was coupled to bovine gamma-globulin (B gamma G) and 4 rabbits were immunized with each peptide-B gamma G conjugate in complete Freund's adjuvant . The mean association constants (K0s) of HEL-reactive antibodies (HEL-R-Abs) from each immunizing group to {3H}acetyl HEL or to {3H}acetyl-peptide were measured in solution by a double antibody method . Only peptide loop I.II (sequences 57-107 containing Cys64-Cys80 and Cys76-Cys94) induced high-affinity antibodies to HEL (K0 = 2.5 x 10(6)-2.3 x 10(7) M-1) among the three epitope peptides tested . The association constants of antipeptide loop I.II to {3H}acetyl peptide loop I.II were always one to two orders of magnitude higher than those to HEL . In addition, 50 to 80% of the anti-peptide loop I.II antibodies were reactive with native HEL . The specificity of anti-peptide loop I.II was directed to a conformational feature of the peptide rather than to native HEL and reactivity of the antibody to HEL was interpreted as a kind of cross-reaction . The HEL-R-Abs from anti-Ploop I.II antisera also manifested neutralizing activities against the enzymic activity of HEL when Micrococcus luteus was used as the substrate. Mol Cell Biochem, 1992 Jan 15, 109(1), 17 - 24 Polyamines in liver and their influence on chromatin condensation after 17-beta estradiol treatment of Atlantic salmon; Waters S et al.; Atlantic salmon (Salmo salar) were treated with 17-beta estradiol to induce vitellogenin synthesis in liver . This led to an increase in liver wet weight and total DNA . After incubation with micrococcal nuclease (EC 3.1.31.1) less soluble chromatin was obtained from nuclei of the estradiol treated than the control fish, but active gene regions were solubilized by the nuclease . Thus, in the estradiol treated fish soluble mononucleosomes contained hybridizable vitellogenin gene sequences . As a result of estradiol treatment the content in total liver of putrescine rose 3-fold, that of spermidine 2-fold, while spermine was unchanged . In muscle no significant changes were observed . The regulatory functions of polyamines during gene expression were investigated by binding (14C)spermine to isolated liver nuclei depleted of endogenous polyamines . The number of binding sites was higher in nuclei of estradiol treated than control fish . (14C)spermine associated preferentially with micrococcal nuclease insensitive chromatin . Thus, the high content of putrescine and spermidine in liver supported the view of polyamine accumulation in proliferating tissues . The preferential binding to condensed chromatin indicated a stabilizing effect of polyamines on the organization of inactive chromatin structures. Clin Immunol Immunopathol, 1992 Jan, 62(1 Pt 1), 25 - 32 Patterns of heavy and light chain utilization in the antibody response to single-stranded bacterial DNA in normal human subjects and patients with systemic lupus erythematosus; Robertson CR et al.; Although anti-DNA antibodies are generally considered to be specific markers for systemic lupus erythematosus (SLE), antibodies binding DNA from certain bacterial species can be found in the sera of normal subjects . To characterize the immunochemical properties of these antibodies, the IgG subclass and light chain profile of antibodies to single-stranded micrococcal DNA (MC DNA) in the sera of normal subjects and patients with SLE was determined . The anti-MC DNA response in normal sera was predominantly of the IgG2 subclass with a marked predominance of kappa light chains . In contrast, anti-MC DNA antibodies in SLE sera exhibited all IgG subclasses with a predominance of the IgG1 subclass and both kappa and lambda light chains were represented . These results suggest that antibodies to bacterial DNA in the sera of normal subjects and patients with SLE differ in patterns of immunoglobulin gene expression; the restricted response of normal subjects may be related to the binding to a discrete DNA determinant. Vopr Pitan, 1992 Jan-Feb, (1), 56 - 60 {Hydrolysis and absorption of lysozyme in the small intestine}; Basova NA et al.; Preparations of chicken small intestine were used in the experiment in vitro simulating processes of membranous digestion (inverted intestinal segments) and absorption (inverted intestinal myasis) . It was established that lysozyme was hydrolyzed on the internal mucosa surface regardless of its concentration in the gastro-intestinal tract, and only insignificant quantity of lysozyme (0.027%) penetrates the intestinal wall . The method of lysozyme determination through its action on the cellular wall of Micrococcus lisodeicticus, and highly efficient liquid chromatography were used to study the transport process . The data presented have evidenced that lysozyme is well hydrolyzed under the action of intestinal peptide hydrolyses, and only insignificant amounts of non-splitted lysozyme can penetrate the blood. Chem Res Toxicol, 1992 Jan-Feb, 5(1), 26 - 33 32P-postlabeling analysis of DNA adduction in mouse skin following topical administration of (+)-7,8-dihydroxy-7,8-dihydrobenzo{a}pyrene; Reddy AP et al.; 32P-Postlabeling was employed for analysis of DNA adducts produced in mouse skin following topical administration of enantiomers of 7,8-dihydroxy-7,8-dihydrobenzo{a}pyrene (BP-7,8-diol) . Deoxynucleoside 3'-monophosphates were isolated by digestion of epidermal DNA with micrococcal endonuclease and spleen phosphodiesterase and phosphorylated with {gamma-32P}ATP . 32P-Labeled deoxynucleoside 3',5'-bisphosphate adducts to diastereomeric benzo{a}pyrene dihydrodiol epoxides (BPDE) were separated by four-directional thin-layer chromatography on poly(ethylenimine)-cellulose plates using a recently described solvent system {Reddy, A . P., Pruess-Schwartz, D., and Marnett, L . J . (1992) Chem . Res . Toxicol . (preceding paper in this issue)} . When (+)-BP-7,8-diol was topically administered, a major adduct spot was detected that cochromatographed with a standard produced by reaction of 7(S),8(R)-dihydroxy-9-(S),10(R)-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene {(+)-syn-BPDE} with DNA . The level of this adduct increased in a dose- and time-dependent fashion and was elevated in animals pretreated with beta-naphthoflavone . Relatively small amounts of radioactivity cochromatographed with standards of deoxynucleoside 3',5'-bisphosphate adducts derived from 7(S),8(R)-dihydroxy-9(R),10(S)-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene {(-)-anti-BPDE} . Following topical administration of (-)-BP-7,8-diol, a single adduct spot was detected that cochromatographed with a standard of the major deoxyguanosine adduct derived from 7(R),8(S)-dihydroxy-9-(S),10(R)-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene {(+)-anti-BPDE} . The stereochemistry of epoxidation of the enantiomers of BP-7,8-diol indicates that cytochrome P-450 catalyzes the terminal activation step of benzo{a}pyrene activation to an ultimate carcinogen in mouse skin, a target organ for its carcinogenic activity. Neurobiol Aging, 1992 Jan-Feb, 13(1), 115 - 21 Nuclear compartmentalization of aluminum in Alzheimer's disease (AD); Lukiw WJ et al.; Senile dementia of the Alzheimer type (AD) is a fatal encephalopathy of uncertain etiology . Whether the neurotoxin aluminum plays any role in the AD process in unknown . Here we report an increased amount of aluminum in a chromatin subcompartment, the micrococcal nuclease (MN; EC 3.1.31.1) accessible dinucleosome fraction, in neocortical nuclei isolated from 17 control and 21 AD-affected brains . At these MN-accessible loci we also observe an increase in H1 zero linker histone proteins, DNA-binding proteins which are thought to act as regulators of chromatin compaction . These data support the hypothesis that one deleterious effect of aluminum upon nuclear structure in AD-afflicted brain may be to condense brain chromatin nonrandomly through an interaction with H1 zero linker protein and thereby alter the ability of brain DNA to be effectively transcribed. Cancer Chemother Pharmacol, 1992, 31(1), 61 - 70 Mechanistic implications of alterations in HL-60 cell nascent DNA after exposure to 1-beta-D-arabinofuranosylcytosine; Ross DD et al.; To improve our understanding of the mechanism of 1-beta-D-arabinofuranosylcytosine (ara-C) incorporation into DNA, we investigated the physical properties (size, position of nucleoside incorporation) of small fragments of nascent DNA (nDNA) obtained by pH-step alkaline elution of intact HL-60 cells following their exposure to ara-C . In the pH-step alkaline elution procedure, the smallest fragments of nDNA elute at pH 11 . Anion-exchange high-performance liquid chromatography (HPLC) of nDNA obtained by 1 h elution at pH 11.0 of lysed HL-60 cells revealed a preponderance of nDNA fragments ranging from 0.5 to 40 kb in control ({3H}-dThd-labeled) cells . Exposure of cells to ara-C (0.8-1 microM) resulted in a loss of the preponderance of radiolabel in fragments of 0.5-40 kb along with redistribution of the radiolabel (from {3H}-dThd or {3H}-ara-C) into smaller nDNA fragments (predominantly < 100 bases in length) as determined by HPLC . We used the ability of pH-step alkaline elution to provide these small nDNA fragments produced by ara-C to investigate the paradoxical behavior of ara-C as a chain terminator in cell-free DNA synthetic systems while being incorporated into an internucleotide position in intact cells . Following the digestion of purified nDNA with micrococcal nuclease and spleen phosphodiesterase II, the proportion of radiolabel in 3'-dNMP (indicating an internucleotide position) or free nucleoside (indicating a chain terminus position) was determined by reverse-phase HPLC . In digests of prelabeled genomic DNA, as expected, > 90% of the radiolabel from {14C}-dThd or {3H}-ara-C was found to exist in an internucleotide position (as determined by co-chromatography with authentic 3'-dTMP or 3'-ara-CMP) . In contrast, digests of nDNA that eluted at pH 11.0 revealed a significantly higher proportion of radiolabel in the chain terminus position (29%-35%) when the nDNA was obtained from cells exposed to 1 microM {3H}-ara-C as compared with cells exposed to {3H}-dThd or {3H}-dCyd alone (< 10%) . These data obtained from pH-step alkaline elution of intact cells suggest that by causing the inhibition of chain elongation while failing to inhibit the formation of new nDNA replication intermediates, ara-C exposure leads to the production of very small nDNA fragments . This relative chain-terminating effect of ara-C is most apparent in the small nDNA replication fragments that elute at pH 11.0.(ABSTRACT TRUNCATED AT 400 WORDS) Gesnerus, 1992, 49 Pt 2, 151 - 60 {The research of Theodor Kocher on the etiology of osteomyelitis and acute struma}; Benaroyo L; In 1879, Kocher demonstrated experimentally that osteomyelitis is an infectious disease which is caused by a non-specific microorganism (the "micrococcus") . He showed that the infection of the bone marrow is the consequence of a hematogenous dissemination of a local infection, either cutaneous or mucous . The clinical and pathological form of the disease depends not only on the virulence of the microorganism, but also on the state of the tissue in which it develops . In the same year, Kocher suggested that acute strumitis (inflammation of the goitre) has the same aetiology; he did not, however, produce any experimental evidence for this . Thus, Kocher defined in an original way infection as the result of an interaction between a microorganism and his host. Biokhimiia, 1992 Jan, 57(1), 69 - 76 {The role of terminal DNA groups in the activation of (ADP-ribose)polymerase}; Karabashian LV et al.; Some peculiarities of activation of (ADP-ribose) polymerase by DNA fragments were studied . DNA fragments were produced by the digestion of calf thymus DNA by micrococcal nuclease and with a subsequent enzymatic modification of their end groups by nuclease S1, polynucleotide kinase of phage T4 and alkaline phosphatase . The dependence of the activating effect of DNA on the chemical structure of its end groups was established . It was shown that the terminal phosphate groups are involved in the formation of a catalytically active complex of (ADP-ribose) polymerase with DNA. Mutat Res, 1992 Jan, 273(1), 43 - 8 UV endonuclease-mediated enhancement of UV survival in Micrococcus luteus: evidence revealed by deficiency in the Uvr homolog; Nakayama H et al.; Unlike its phage T4 counterpart (also known as endonuclease V), Micrococcus luteus UV endonuclease (pyrimidine dimer DNA glycosylase/apurinic-apyrimidinic endonuclease) has suffered from lack of genetic evidence to implicate it in the promotion of UV survival of the cell, i.e., mutants with its deficiency are no more UV-sensitive than the wild type . On the assumption that the contribution of UV endonuclease is obscured by the presence of a homolog of Escherichia coli UvrABC endonuclease, which has recently been identified in this bacterium, survival studies were carried out in its absence . With 254-nm UV irradiation, which generates not only pyrimidine dimers but also 6-4 photoproducts as lethal lesions, a double mutant defective in both UV endonuclease and the Uvr homolog was shown to be more sensitive than a single mutant defective only in the latter, with a dose reduction factor of approximately 2 at the survival level of 37% . Furthermore, molecular photosensitization, which produces only pyrimidine dimers, revealed an even greater difference in sensitivity, the dose reduction factor being about 3.4 . These results indicate that the contribution to cell survival of UV endonuclease, an enzyme specific for pyrimidine dimers, is manifest if the backup by the Uvr homolog is absent. Eur J Histochem, 1992, 36(3), 263 - 70 Methyl green and its analogues bind selectively to AT-rich regions of native DNA; Fox KR et al.; Methyl green has long been used as a DNA stain in histochemistry . The sequence selective binding of the cationic triphenylmethane dyes methyl green, crystal violet and Malachite green to DNA was investigated by DNAase 1 and micrococcal nuclease footprinting . At low concentrations the ligands showed similar footprinting patterns which centred around AT-rich regions with a mild preference for hompolymeric A and T . At higher concentrations the dyes bound to almost all available DNA sites . Models, with and without intercalation are discussed to account for the specific binding. Nucleic Acids Res, 1991 Dec 25, 19(24), 6719 - 24 Interaction of echinomycin with An.Tn . and (AT)n regions flanking its CG binding site; Waterloh K et al.; We have prepared DNA fragments containing the sequences A15CGT15, T15CGA15 and T(AT)8CG(AT)15 cloned within the SmaI site of the pUC19 polylinker . These have been used as substrates in footprinting experiments with DNase I and diethylpyrocarbonate probing the effects of echinomycin, binding to the central CG, on the structure of the surrounding sequences . No clear DNase I footprints are seen with T15CGA15 though alterations in the nuclease susceptibility of surrounding regions suggest that the ligand is binding, albeit weakly at this site . All the other fragments show the expected footprints around the CG site . Regions of An and Tn are rendered much more reactive to DNase I and adenines on the 3'-side of the CG become hyperreactive to diethylpyrocarbonate . Regions of alternating AT show unusual changes in the presence of the ligand . At low concentrations (5 microM) cleavage of TpA is enhanced, whereas at higher concentrations a cleavage pattern with a four base pair repeat is evident . A similar pattern is seen with micrococcal nuclease . Modification by diethylpyrocarbonate is strongest at alternate adenines which are staggered in the 5'-direction across the two strands . We interpret these changes by suggesting secondary drug binding within regions of alternating AT, possibly to the dinucleotide ApT . DNase I footprinting experiments performed at 4 degrees C revealed neither enhancements nor footprints for flanking regions of homopolymeric A and T suggesting that the conformational changes are necessary consequence of drug binding. J Biol Chem, 1991 Dec 15, 266(35), 23706 - 13 Purification of solanesyl-diphosphate synthase from Micrococcus luteus . A new class of prenyltransferase; Ohnuma S et al.; The activity of solanesyl-diphosphate synthase from Micrococcus luteus is stimulated by a high molecular mass fraction (HMF) which is separated from cell-free extracts of the same bacterium by DEAE-Toyopearl chromatography followed by Sephadex G-100 chromatography . By employing HMF in the assay procedure, solanesyl-diphosphate synthase was able to be purified to homogeneity and was found to be a homodimer with a monomeric molecular mass of 34 kDa . In contrast to hexaprenyl- and heptaprenyl-diphosphate synthases, which are composed of two easily dissociable components that are inactive unless combined, the homogeneously purified solanesyl-diphosphate synthase itself showed a catalytic activity, though weak, catalyzing the synthesis of both (all-E)-nonaprenyl-(solanesyl-) and (all-E)-octaprenyl diphosphate . HMF does not affect the stability of solanesyl-diphosphate synthase or Km values for isopentenyl diphosphate and farnesyl diphosphate, but it markedly increases Vmax values in a time-dependent manner . Several lines of evidence indicate that HMF contains a factor which binds to polyprenyl products and removes them out of the active site of enzyme to facilitate and maintain the turnover of catalysis. Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10596 - 600 Nucleosome positioning is determined by the (H3-H4)2 tetramer; Dong F et al.; It is demonstrated that the histone (H3-H4)2 tetramer can find specific positions on DNA, even in the absence of other histones . Purified histone (H3-H4)2 tetramers were reconstituted onto 208-base-pair (bp) DNA molecules containing a nucleosome-positioning sequence by using salt-gradient dialysis . The stoichiometry of histone tetramer to DNA was shown to be 1:1 . Digestion with micrococcal nuclease led to formation of protected DNA fragments of approximately 73 bp . Cleavage of the 73-bp DNA with restriction enzymes produced a small set of defined bands, demonstrating positioning of the (H3-H4)2 tetramer on DNA . Analysis of the restriction digests shows that the 73-bp DNA corresponds mainly to two fragments, one lying on either side of the pseudo-dyad axis of the major position adopted by complete histone octamers on this DNA . This result means that a single (H3-H4)2 histone tetramer can fold approximately 146 bp of DNA with the same positioning as the complete octamer but that a region near the pseudo-dyad is only weakly protected against micrococcal nuclease attack in the absence of histones H2A and H2B. J Bacteriol, 1991 Dec, 173(24), 7911 - 7 The major carotenoid pigment of a psychrotrophic Micrococcus roseus strain: purification, structure, and interaction with synthetic membranes; Jagannadham MV et al.; The major carotenoid pigment of a psychrotrophic Micrococcus roseus strain was purified to homogeneity from methanol extracts of dried cells by reverse-phase liquid chromatography and was designated P-3 . On the basis of the UV-visible, infrared, mass, and 1H nuclear magnetic resonance spectra of P-3, it was identified as bisdehydro-beta-carotene-2-carboxylic acid . The pigment interacted with synthetic membranes of phosphatidylcholine and dimyristoyl phosphatidylcholine and stabilized the membranes . These results also indicate that P-3 is different from canthaxanthin, the major carotenoid pigment from a mesophilic M . roseus strain. Mycopathologia, 1991 Dec, 116(3), 203 - 8 Clinical isolates of yeast produce a gliotoxin-like substance; Shah DT et al.; Candida infections are major causes of morbidity in compromised human hosts, but our understanding of the virulence of Candida remains incomplete . The possibility that toxic fungal metabolites belonging to the chemical class epipolythiodioxopiperzine (ETP), which are reported to possess immunomodulating and antiphagocytic properties may be produced by Candida species was investigated . Reversed phase HPLC analysis of flash evaporated chloroform extracts of 7 day cultures of clinical Candida isolates grown in Minimal Essential Medium (MEM) with 5% fetal calf serum revealed the presence of a compound which eluted at the same time as the ETP, gliotoxin . Of 50 strains of yeast tested, 32 produced this gliotoxin-like material . This material was tested for other properties of ETP type toxins including the presence of mercaptans (Ellman reaction), ultraviolet absorbance spectrum and antibacterial activity against Micrococcus lutea . These tests revealed gliotoxin-like material from yeast cultures to be similar to commercially supplied gliotoxin . This represents the first report of the presence of ETP-like compounds in yeast and raises the possibility that ETP's may contribute to the virulence of the organism. DNA Cell Biol, 1991 Dec, 10(10), 751 - 6 The effect of the simple repeating d(CG.GC)n, d(CA.GT)n, and d(A.T)n DNA sequences on the nucleosomal organization of SV40 minichromosomes; Casasnovas JM et al.; The effect of several simple repeating DNA sequences--d(CG.GC)5, d(CA.GT)30, and d(A.T)60--on the nucleosomal organization of the SV40 minichromosome is analyzed . These three different sequences were cloned at the Hpa II site of SV40 (position 346) which occurs at the 3' border of the nucleosome-free SV40 control region . Our results show that neither the d(A.T)60 sequence nor the d(CG.GC)5 sequence appear to have any relevant effect on the nucleosomal organization of the region of the minichromosome surrounding the inserted repeated sequence . Both sequences are hypersensitive to micrococcal nuclease cleavage in the minichromosome, indicating that they are not organized into nucleosomes . On the other hand, the d(CA.GT)30 sequence is found organized as nucleosomes and causes the delocation of nucleosomes in the minichromosomal region close to the inserted repeated sequence. Nucleic Acids Res, 1991 Nov 11, 19(21), 5821 - 9 DNA-sequence specific recognition by a thiazole analogue of netropsin: a comparative footprinting study; Plouvier B et al.; Four different footprinting techniques have been used to probe the DNA sequence selectivity of Thia-Net, a bis-cationic analogue of the minor groove binder netropsin in which the N-methylpyrrole moieties are replaced by thiazole groups . In Thia-Net the ring nitrogen atoms are directed into the minor groove where they could accept hydrogen bonds from the exocyclic 2-amino group of guanine . Three nucleases (DNAase I, DNAase II, and micrococcal nuclease) were employed to detect binding sites on the 160bp tyr T fragment obtained from plasmid pKM delta-98, and further experiments were performed with 117mer and 253mer fragments cut out of the plasmid pBS . MPE.Fe(II) was used to footprint binding sites on an EcoRI/HindIII fragment from pBR322 . Thia-Net binds to sites in the minor groove containing 4 or 5 base pairs which are predominantly composed of alternating A and T residues, but with significant acceptance of intrusive GC base pairs . Unlike the parent antibiotic netropsin, Thia-Net discriminates against homooligomeric runs of A and T . The evident preference of Thia-Net for AT-rich sites, despite its containing thiazole nitrogens capable of accepting GC sites by hydrogen bonding, supports the view that the biscationic nature of the ligand imposes a bias due to the electrostatic potential differences in the receptor which favour the ligand reading alternating AT sequences. J Mol Biol, 1991 Nov 5, 222(1), 45 - 57 Conserved chromatin structure in c-myc 5'flanking DNA after viral transduction; Kumar S et al.; The role of local sequence information in establishing the chromatin structure of the human c-myc upstream region (MUR) was investigated . Adeno-associated virus (AAV)-mediated gene transduction was used to introduce an additional unrearranged copy of the 2.4 kb HindIII-XhoI fragment of the MUR into a novel location in the genome in each of two cloned HeLa cell lines . The AAV-based rep- cap- viral vector SKMA used to transduce the MUR retained only 1.4 kb (24%) of the AAV genome and could accommodate inserts as large as 2.4 kb . SKMA was capable of infecting HeLa cells and integrating into the host genome at single copy number . Integration may have occurred at a preferred site in the HeLa genome, but this site was apparently distinct from the previously identified preferred AAV integration site on human chromosome 19 . Indirect end-labelling was used to map DNase I and micrococcal nuclease (MNase) cleavage sites over the transduced c-myc sequences and the endogenous c-myc loci in infected HeLa cells . A similarly ordered chromatin domain, extending 5' from c-myc promoter P0, was found to exist at the transduced c-myc locus in each clone . The position and relative sensitivity of 13 MNase cleavage sites and five DNase I hypersensitive sites, originally identified at the endogenous MUR in non-transduced cells, were shown to be conserved when this DNA was moved to a new chromosome site . A conserved DNase I hypersensitive site also was mapped to the region between the left AAV terminal repeat and AAV promoter P5 . These results suggest that the information required to establish the particular chromatin structure of the MUR resides within the local DNA sequence of that region. Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9675 - 9 Positive DNA supercoiling generates a chromatin conformation characteristic of highly active genes; Lee MS et al.; During transcription, positive DNA supercoils generated ahead of RNA polymerase could theoretically uncoil the negative DNA supercoils associated with nucleosomes and thereby decondense the chromatin fiber in preparation for RNA polymerase passage . Here we examine the effect of positive DNA supercoiling on the structure of yeast 2-microns minichromosomes . We utilized a conditional topoisomerase mutant expressing Escherichia coli topoisomerase I to convert the DNA supercoiling state from negative to positive in vivo . Minichromosomes containing positively supercoiled DNA exhibited a striking increase in DNase I sensitivity . They also displayed additional micrococcal nuclease cleavage sites but yielded nearly typical nucleosomal ladders after extensive digestion . Upon in vitro relaxation with eukaryotic topoisomerase I, the minichromosomes remained DNase I sensitive but were converted to negative DNA supercoiling with a slightly increased linking number compared to typical minichromosomes, thus indicating the presence of bound histones . Therefore, positive DNA supercoiling provides a mechanism for generating, but is not required for maintaining, a conformation in chromatin characteristic of highly transcribed genes. J Biochem (Tokyo), 1991 Nov, 110(5), 719 - 25 Preparation and properties of a lysozyme derivative in which two domains are cross-linked intramolecularly between Trp62 and Asp101; Ueda T et al.; A lysozyme derivative in which two domains were cross-linked intramolecularly was newly prepared by means of a two-step reaction . First, the beta-carboxyl group of Asp101 in lysozyme was selectively modified with 2-(2-pyridyldithio)ethylamine in the presence of 1-ethyl-3-{3-(dimethylamino)propyl}carbodiimide hydrochloride . After reduction of the pyridyldithio moiety of Asp101 modified lysozyme at pH 4.5 with dithiothreitol, the derivative was allowed to cross-link intramolecularly by reaction with 1,3-dichloroacetone at pH 7 . Intramolecularly cross-linked lysozyme thus formed was purified by gel chromatography followed by ion-exchange chromatography . Based on the results of 1H-NMR and peptide analyses, it was concluded that Asp101 was cross-linked to Trp62 with a -CH2COCH2SCH2CH2NH-bridge in this derivative . The derivative showed minor but distinct activity against Micrococcus lysodeikticus and glycol chitin . Its melting temperature for thermal denaturation was higher by 7.3 degrees than that of native lysozyme at pH 3. J Biol Chem, 1991 Oct 25, 266(30), 20356 - 62 The U2 small nuclear ribonucleoprotein particle associates with nuclear factors in a pre-mRNA independent reaction; Temsamani J et al.; Northern blot analysis of HeLa cell nuclear extract following electrophoresis in nondenaturing gels revealed that a small proportion of U2 small nuclear ribonucleoprotein (snRNP) displays a low mobility, in confirmation of previous reports . This low mobility form of U2 snRNP (termed LMC, for low mobility complex) also formed in vitro when U2 snRNP present in HeLa cytoplasmic S100 was added to a micrococcal nuclease-treated nuclear extract . Of greater experimental value, we found that the LMC also formed when a T7 U2 RNA transcript was assembled into U2 snRNP in a HeLa cytoplasmic S100 system, followed by its incubation in micrococcal nuclease-treated nuclear extract . LMC formation was ATP-dependent and was specific for U2 snRNP since it was not observed with S100-assembled U1 or U4 snRNPs . RNase H cleavage of U2 snRNP in the nuclear extract with an oligonucleotide complementary to nucleotides 28-42 of U2 RNA, as opposed to micrococcal nuclease treatment, rendered the extract competent to form the LMC, indicating that the nuclear factors responsible for LMC formation reside on endogenous U2 snRNP . LMC formation was not competed by excess U2 RNA but was competed by partially purified native U2 snRNP, providing further evidence that the LMC represents an interaction of nuclear factors with already assembled U2 snRNP . LMC formation did not take place on a mutant U2 snRNP lacking the binding site for the two U2-specific proteins, A' and B", nor on mutant U2 snRNPs lacking nucleotides 34-37 or nucleotides 46-49 . Further results revealed that nucleotides 35 and 36 of U2 RNA, but not 34 and 37, are required for LMC formation . These experiments demonstrate a nucleotide sequence-specific interaction of U2 snRNP with nuclear factors in the absence of pre-mRNA . Among the U2 RNA nucleotides involved in the formation of this complex are ones previously implicated in base pairing between U2 RNA and the pre-mRNA lariat branch site . These findings are discussed in the context of the possibility that the LMC is on the spliceosome assembly pathway. Genes Dev, 1991 Oct, 5(10), 1847 - 58 Modulation of alternative splicing of adenoviral E1A transcripts: factors involved in the early-to-late transition; Gattoni R et al.; The E1A pre-mRNA of adenovirus is spliced into three mRNA species (13S, 12S, and 9S mRNAs) by the use of three alternative 5'-splice sites . The 13S and 9S mRNAs predominate during the early and late periods of infection, respectively . With HeLa nuclear extracts isolated in early and late periods of infection, we were able to reproduce a 13S-9S modulation that resembles that occurring in infected cells . An in vitro analysis of the cis-acting parameters involved in the 13S-9S switch indicates that the 13S mRNA splicing inhibition is one of the first events of the late period and leads to the subsequent stimulation of the 9S mRNA reaction . The new abilities of the late nuclear extract for the 9S mRNA reaction were also confirmed by analyzing splicing of a major late transcript containing leaders 1 and 2 separated by the wild-type intervening sequence (IVS) of 1021 nucleotides . Complementation experiments show that the trans-acting factor(s) are micrococcal nuclease sensitive . They were partially characterized by induction experiments, and we show that the primary factors responsible for the 13S-9S modulation in vitro are viral RNAs of high molecular weight that accumulate late in infection . We postulate that the splicing modulation of E1A pre-mRNA results from an indirect mode of action for these viral RNAs, based on a sequestration of common splicing factors that are not present in vast excess in HeLa cells. Mol Cell Biol, 1991 Oct, 11(10), 5259 - 65 Inhibition of chromatin assembly in Xenopus oocytes correlates with derepression of the mouse mammary tumor virus promoter; Perlmann T et al.; The mouse mammary tumor virus (MMTV) promoter is positively regulated by glucocorticoid hormone via binding of glucocorticoid receptor to a specific response element . Upon addition of hormone, a nucleosome containing the glucocorticoid response element is removed or structurally altered, suggesting that the nucleosome interferes with transcription . Accordingly, inhibition of chromatin assembly should relieve the repression and result in an increased constitutive activity . We have tested this hypothesis by injecting nonspecific competitor DNA into Xenopus laevis oocytes to titrate endogenous histones . The coinjection of competitor DNA altered chromatin structure: nucleosomal ladders produced by micrococcal nuclease were disrupted, and the unique helical setting of the MMTV promoter in a nucleosome was lost, as shown by in situ DNase I footprinting . Basal MMTV transcription was drastically increased by competitor DNA, whereas a coinjected, constitutively active adenovirus 2 major late promoter was not stimulated . These results show that the uninduced MMTV promoter is under negative control and provide direct support for the theory that the nucleosomal organization maintains the repression of this promoter in its uninduced state. Biokhimiia, 1991 Sep, 56(9), 1599 - 606 {Activation of (ADP-ribose) polymerase by DNA fragmented by micrococcal nuclease and DNAase I}; Karabashian LV et al.; Activation of (ADP-ribose) polymerase by DNA fragments obtained by digestion of calf thymus DNA with micrococcal nuclease and DNAase I was studied . It was found that activation of the enzyme is due to its interaction with the terminal parts of double-stranded DNA fragments, the level of activation being independent of the size of DNA fragments. Infect Immun, 1991 Sep, 59(9), 3303 - 8 Trypanosoma cruzi but not Trypanosoma brucei fails to induce a chemiluminescent signal in a macrophage hybridoma cell line; Vray B et al.; Macrophage-Trypanosoma cruzi interactions were studied by using a newly generated macrophage hybridoma cell line (2C11-12) that was selected for its capacity to produce high levels of reactive oxygen intermediates . This cell line was found to be a suitable host cell for T . cruzi, and intracellular parasitic development could be inhibited by activation with gamma interferon . When exposed to opsonized Trypanosoma brucei, Micrococcus lysodeikticus, or Legionella pneumophila, the activated macrophage cell line produces a high chemiluminescent signal, indicating the release of reactive oxygen intermediates . Alternatively, when opsonized T . cruzi was added to these activated macrophages, this parasite failed to stimulate a chemiluminescent response, suggesting an impairment in the triggering of the respiratory burst. Biochim Biophys Acta, 1991 Aug 27, 1090(1), 38 - 42 Histones and DNA methylation in mammalian chromatin . Differential inhibition by histone H1; Caiafa P et al.; Histones (from calf thymus or from human placenta), if renatured in the presence of EDTA, caused a severe inhibition of in vitro methylation of double-stranded DNA (from Micrococcus luteus) by human placenta DNA methyltransferase . The absence of EDTA during the histone renaturation procedure abolished--at least in the 'physiological' range of the histones/DNA ratio--the inhibition . The H1 component was responsible for this inhibition, no effect being exerted by the other histones . H1 preparations were more effective if renatured in the presence of EDTA--90% inhibition being reached at a 0.3:1 (w/w) H1/DNA ratio . It seems likely that the requirement for the presence of EDTA during the renaturation process is correlated to its ability to induce a fairly stable ordered conformation of the histones, although this effect could also be shown with the 'inactive' H2a, H2b and H3 components, and was instead less evident with histone H1 . The restriction to histone H1 of the ability to inhibit enzymic DNA methylation may account for the lower methylation levels present in the internucleosomal DNA of mammalian chromatin. J Biol Chem, 1991 Aug 25, 266(24), 16141 - 8 Replication of the simian virus 40 chromosome with purified proteins; Ishimi Y et al.; SV40 chromosomes prepared from infected CV-1 cells were replicated with the purified proteins of SV40 T antigen, HeLa DNA polymerase alpha-primase complex, single-stranded DNA-binding protein, and topoisomerases I and II, all of which have been shown to be essential for SV40 DNA replication in vitro . Replication started near the origin and proceeded bidirectionally . The maximum speed of replication fork movement was 200-300 nucleotides/min, which was similar to the rate of SV40 DNA replication with the same set of proteins . When replication products were digested with micrococcal nuclease, DNA fragments of 160-180 base pairs, which is the typical size of mononucleosomal DNA, were protected . This result indicates that replicated DNA was rec