J Biol Chem, 1996 Nov 22, 271(47), 29624 - 31 Mechanism of tracking and cleavage of adduct-damaged DNA substrates by the mammalian 5'- to 3'-exonuclease/endonuclease RAD2 homologue 1 or flap endonuclease 1; Barnes CJ et al.; The mammalian 5'- to 3'-exonuclease/endonuclease, called RAD2 homologue 1 or flap endonuclease 1, has a unique cleavage activity, dependent on specific substrate structure . On a primer-template, in which the primer has an unannealed 5'-tail, endonucleolytic cleavage near the annealing point releases the tail intact . Entering at the 5'-end, the nuclease tracks along the entire tail to the point of cleavage . Genetic analyses suggest that this nuclease removes DNA adducts in vivo (Sommers, C . H., Miller, E . J., Dujon, B., Prakash, S., and Prakash, L . (1995) J . Biol . Chem . 270, 4193-4196) . Micrococcal nuclease footprinting shows that after tracking the nuclease protects a region of the tail 25 nucleotides long, adjacent to the cleavage site . Substrates with adducts at specific locations were used to assess the mechanism of RAD2 homologue 1 nuclease tracking and its ability to cleave modified DNA . Either a conventional cis-diamminedichloroplatinum (II) (CDDP) or a bulky CDDP derivative was placed within or beyond the region protected by the nuclease . The nuclease cleaved the tail of both substrates . In contrast, a CDDP adduct just adjacent to the expected cleavage point was inhibitory . A CDDP adduct at the very 5'-end of the tail was also cleaved . The nuclease could remove tails containing adducts on the sugar-phosphate backbone . Apparently, the nuclease is designed to slide over various types of damage on single stranded DNA and then cut past the damaged site. Biochem Biophys Res Commun, 1996 Nov 21, 228(3), 852 - 8 In vitro translation and translocation of apolipoprotein B in a cell-free system from HepG2 cells; Mohammadi A et al.; An mRNA-dependent cell-free system has been developed from HepG2 cells by hydrolysis of endogenous mRNA with micrococcal nuclease . When supplied with RNA extracted from HepG2 cells, the system synthesized liver specific proteins such as albumin and apolipoprotein B100 . Significant amounts of microsomes were also detected in the lysate by measuring NADH-cytochrome c reductase activity and ultracentrifugation . Protease protection assays showed the capability of the HepG2 lysate to translocate newly-synthesized proteins such as apolipoprotein Al, albumin, and apoB into the microsomes as they were protected from digestion with exogenously added protease K, but not protected in the presence of protease K and Triton X-100 . The system also proved to be very active toward translation of exogenous mRNAs as evidenced by efficient translation of brome mosaic virus RNA . The HepG2 translation-translocation system appears to provide a unique homologous system for studies on the biogenesis of liver specific proteins, particulary apoB100. Biochem Biophys Res Commun, 1996 Nov 21, 228(3), 846 - 51 Interaction between polyanions and cell nuclei: mechanism of gelatination of nuclei; Nakashima A et al.; We investigated the mechanism by which polyanions gelatinized nuclei of some mouse lymphocytes and ruptured these cells . The gel produced by the addition of dextran sulfate (DS) to mouse lymphocyte nuclei was composed of histones (H1, H2A, H2B, H3, and H4), DS, and DNA . Adding DS to the chromatin obtained from nuclei by micrococcal nuclease (MNase) digestion also produced a gel containing a complex of DS-histones-DNA . When this mixture was further digested by MNase, DNA debris of random sizes was observed instead of the 150-bp repeating units of DNA usually observed when normal nucleosomes are digested with MNase . Removal of DS from the chromatin-DS gel resulted in the regeneration of nucleosomes . These results suggest the following: After entering the cells with damaged cellular membrane, DS extracts histones from nucleosomes to form DS-histone complexes, which then aggregate with the liberated DNA to form a macromolecular gel . Finally, the swelling pressure of the gel destroys the cells. J Biol Chem, 1996 Nov 8, 271(45), 28667 - 76 In vivo footprinting analysis of the hepatic control region of the human apolipoprotein E/C-I/C-IV/C-II gene locus; Dang Q et al.; Expression of both the apolipoprotein (apo)E and apoC-I genes in the liver is specified by a 319-nucleotide hepatic control region (HCR-1) that is located 15 kilobase pairs downstream of the apoE gene and 5 kilobase pairs downstream of the apoC-I gene . In vivo footprint analysis of HCR-1 in intact nuclei revealed several liver-specific protein-binding sites that were not detectable by in vitro methods . In addition to three previously identified in vitro footprints, four in vivo footprints were identified in a region of HCR-1 that is required for directing gene expression to hepatocytes . Prominent liver-specific DNase I-hypersensitive sites were associated with these footprints . Liver-specific nuclear protein binding to these sites was confirmed by oligonucleotide gel-retention assays . The in vivo analysis also identified a cluster of nuclear protein-binding sites in the Alu family repeat segment adjacent to the domain required for liver expression . Micrococcal nuclease digestion indicated the presence of a nucleosome in the central domain of HCR-1 in liver chromatin that was in phase with the nucleosome location in tissues that did not express the transgene . These results suggest that HCR-1 functions in a highly structured chromatin environment requiring a complex interaction of liver-enriched transcription factors. J Biol Chem, 1996 Nov 8, 271(45), 28294 - 9 Apobec-1 interacts with a 65-kDa complementing protein to edit apolipoprotein-B mRNA in vitro; Mehta A et al.; The editing of apolipoprotein-B (apoB) mRNA involves the deamination of cytidine at nucleotide 6666 to uridine . The catalytic subunit of the editing enzyme, apobec-1, is a cytidine deaminase that requires other unidentified proteins to edit apoB mRNA in vitro . We partially purified an activity from baboon kidney that functionally complements apobec-1 . The complementing activity was protease-sensitive and micrococcal nuclease-resistant, had a native molecular mass of 65 +/- 10 kDa on size exclusion chromatography, and sedimented at 4.5 S in glycerol gradients . Purified recombinant His6-tagged apobec-1 immobilized on beads depleted >90% of the complementing activity from partially purified extracts . These beads edited apoB mRNA in vitro in the absence of exogenous apobec-1 or complementing activity . A functional holoenzyme containing apobec-1 and the complementing activity was eluted from the apobec-1-affinity resin using 0.5 M imidazole, whereas buffer containing 0.4 M KCl eluted only the complementing activity . The carboxyl-terminal 59 amino acids of apobec-1 were not required for interaction with the complementing activity in vitro . Our results demonstrate that the complementing protein interacts directly with apobec-1 in the absence of apoB mRNA. EMBO J, 1996 Nov 1, 15(21), 5928 - 35 Reconstitution of human telomerase activity and identification of a minimal functional region of the human telomerase RNA; Autexier C et al.; Telomerase is a ribonucleoprotein that catalyzes telomere elongation through the addition of TTAGGG repeats in humans . Activation of telomerase is often associated with immortalization of human cells and cancer . To dissect the human telomerase enzyme mechanism, we developed a functional in vitro reconstitution assay . After removal of the essential 445 nucleotide human telomerase RNA (hTR) by micrococcal nuclease digestion of partially purified human telomerase, the addition of in vitro transcribed hTR reconstituted telomerase activity . The activity was dependent upon and specific to hTR . Using this assay, truncations at the 5' and 3' ends of hTR identified a functional region of hTR, similar in size to the full-length telomerase RNAs from ciliates . This region is located between positions 1-203 . Furthermore, we found that residues 1-44, 5' to the template region (residues 46-56) are not essential for activity, indicating a minimal functional region is located between residues 44-203 . Mutagenesis of full-length hTR between residues 170-179, 180-189 or 190-199 almost completely abolished the ability of the hTR to function in the reconstitution of telomerase activity, suggesting that sequences or structures within this 30 nucleotide region are required for activity, perhaps by binding telomerase protein components. Biochemistry, 1996 Oct 22, 35(42), 13562 - 7 Origin of carbohydrate recognition specificity of human lysozyme revealed by affinity labeling; Muraki M et al.; In order to reveal the origin of carbohydrate recognition specificity of human lysozyme by clarifying the difference in the binding mode of ligands in the active site, the inactivation of human lysozyme by 2',3'-epoxypropyl beta-glycoside derivatives of the disaccharides, N,N'-diacetylchitobiose {GlcNAc-beta-(1-->4)-GlcNAc} and N-acetyllactosamine {Gal-beta-(1-->4)-GlcNAc}, was investigated and the three-dimensional structures of the affinity-labeled enzymes were determined by X-ray crystallography at 1.7 A resolution . Under the conditions comprising 2.0 x 10(-3) M labeling reagent and 1.0 x 10(-5) M human lysozyme at pH 5.4, 37 degrees C, the reaction time required to reduce the lytic activity against Micrococcus luteus cells to 50% of its initial activity was lengthened by 3.7 times through the substitution of the nonreducing end sugar residue, GlcNAc to Gal . The refined structure of human lysozyme labeled by 2',3'-epoxypropyl beta-glycoside derivatives of N,N'-diacetylchitobiose (HL/NAG-NAG-EPO complex) indicated that the interaction mode of the N,N'-diacetylchitobiose moiety in substites B and C in this study was essentially the same as in the case of the complex of human lysozyme with the free ligand . On the other hand, the hydrogen-bonding pattern and the stacking interaction at subsite B were remarkably different between the HL/NAG-NAG-EPO complex and human lysozyme labeled by the 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine (HL/GAL-NAG-EPO complex) . The reduced number of possible hydrogen bonds as well as the less favorable stacking between the side chain of Tyr63 in human lysozyme and the galactose residue in the HL/GAL-NAG-EPO complex reasonably explained the less efficient ability of the 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine as compared to that of N,N'-diacetylchitobiose as an affinity labeling reagent toward human lysozyme. J Biochem Biophys Methods, 1996 Oct 15, 33(1), 59 - 64 An improved method to distinguish micrococcal nuclease sensitivity of chromatin; Hamid QA et al.; The resolution of the standard micrococcal nuclease assay for sensitivity of active chromatin has been enhanced by the inclusion of an additional step of digesting nuclease-digested DNA with a suitable restriction enzyme prior to Southern hybridization . The improved assay has been used to analyze the chromatin structure of the lamin A, albumin and alpha-fetoprotein genes during rat liver development. Proc Natl Acad Sci U S A, 1996 Oct 15, 93(21), 11510 - 5 Increased histone H1 phosphorylation and relaxed chromatin structure in Rb-deficient fibroblasts; Herrera RE et al.; Fibroblasts derived from embryos homozygous for a disruption of the retinoblastoma gene (Rb) exhibit a shorter G1 than their wild-type counterparts, apparently due to highly elevated levels of cyclin E protein and deregulated cyclin-dependent kinase 2 (CDK2) activity . Here we demonstrate that the Rb-/- fibroblasts display higher levels of phosphorylated H1 throughout G1 with the maximum being 10-fold higher than that of the Rb+/+ fibroblasts . This profile of intracellular H1 phosphorylation corresponds with deregulated CDK2 activity observed in in vitro assays, suggesting that CDK2 may be directly responsible for the in vivo phosphorylation of H1 . H1 phosphorylation has been proposed to lead to a relaxation of chromatin structure due to a decreased affinity of this protein for chromatin after phosphorylation . In accord with this, chromatin from the Rb-/- cells is more susceptible to micrococcal nuclease digestion than that from Rb+/+ fibroblasts . Increased H1 phosphorylation and relaxed chromatin structure have also been observed in cells expressing several oncogenes, suggesting a common mechanism in oncogene and tumor suppressor gene function. Biochemistry, 1996 Oct 8, 35(40), 13107 - 11 EPR detection and characterization of lignin peroxidase porphyrin pi-cation radical; Khindaria A et al.; Lignin peroxidase (LiP) from Phanerochaete chrysosporium catalyzes the H2O2 dependent one- and two-electron oxidations of substrates . The catalytic cycle involves the oxidation of ferric-LiP by H2O2 by two electrons to compound I, which is an oxoferryl heme and a free radical . It has been speculated that the unpaired electron is in a pi delocalized porphyrin radical . However, no direct evidence for the presence of the free radical has been reported . We present electron paramagnetic resonance (EPR) detection and characterization of compound I of LiP . The LiP compound I EPR signal is different than those reported previously for compound I of horseradish peroxidase and chloroperoxidase . However, the EPR signal of compound I of LiP (axial g tensor extending from gperpendicular = 3.42 to gparallel approximately 2) is very similar to the EPR signals of compound I of ascorbate peroxidase and catalase from Micrococcus lysodeikticus, in which the radical has been identified as a porphyrin pi-cation radical . On the basis of the analysis of our data and comparison with the earlier published results for compounds I of other peroxidases, we interpret the LiP compound I signal by a model for exchange coupling between an S = 1 oxyferryl {Fe = O}2+ moiety and a porphyrin pi-cation radical (S = 1/2) {Schulz, C.E., et al . (1979) FEBS Lett . 103, 102-105} . The exchange coupling is characterized by ferromagnetic rather than an antiferromagnetic interaction between the two species . The ferric-Lip EPR signal suggests that the iron in the heme is in near perfect orthogonal symmetry and provides additional evidence of the ferromagnetic interaction between the oxoferryl iron center and the porphyrin pi-cation radical. J Biol Chem, 1996 Oct 4, 271(40), 24922 - 6 The C-group heterogeneous nuclear ribonucleoprotein proteins bind to the 5' stem-loop of the U2 small nuclear ribonucleoprotein particle; Temsamani J et al.; The C-group heterogeneous nuclear ribonucleoprotein (hnRNP) proteins bind to nascent pre-messenger RNA . In vitro studies have indicated that the C hnRNP proteins bind particularly strongly to the intron polypyrimidine tract of pre-mRNA and may be important for pre-mRNA splicing . In addition, there is evidence that the interaction of the C hnRNP proteins with pre-mRNA is facilitated by the U1 and U2 small nuclear RNPs (snRNPs) . In the present study, we have uncovered another feature of the C hnRNP proteins that may provide a unifying framework for these previous observations; the C hnRNP proteins bind to the 5' stem-loop of the U2 snRNP . This was detected by incubating human 32P-labeled U2 snRNP in micrococcal nuclease-treated HeLa nuclear extracts, followed by UV-mediated protein-RNA cross-linking, which revealed that C hnRNP proteins were cross-linked to 32P-nucleotides in the U2 snRNP . In similar experiments, no cross-linking of C hnRNP proteins to 32P-labeled U1 or U4 snRNPs was observed . The observed cross-linking of C hnRNP proteins to U2 snRNP was efficiently competed by excess U2 RNA and by poly(U) but not by poly(A) . No competition was observed with an RNA molecule comprising U2 nucleotides 105-189, indicating that the C hnRNP protein interactive regions of U2 RNA reside solely in the 5' half of the molecule . Oligodeoxynucleotide-mediated RNase H cleavage experiments revealed that a 5' region of U2 RNA including nucleotides 15-28 is essential for the observed C hnRNP protein cross-linking . C hnRNP protein cross-linking to U2 snRNP was efficiently competed by a mini-RNA corresponding to the first 29 nucleotides of U2 RNA, whereas no competition was observed with a variant of this mini-RNA in which the UUUU loop of stem-loop I was mutationally configured into a single-stranded RNA by replacing the stem with non-pairing nucleotides . Competition experiments with another mutant mini-U2 RNA in which the UUUU loop was replaced by AAAA indicated that both the UUUU loop and the stem are important for C hnRNP protein cross-linking, a finding consistent with other recent data on the RNA sequence specificity of C hnRNP protein binding. Biochem Biophys Res Commun, 1996 Oct 3, 227(1), 221 - 6 In vivo characteristics and localisation of carotenoid pigments in psychrotrophic and mesophilic Micrococcus roseus using photoacoustic spectroscopy; Jagannadham MV et al.; Photoacoustic spectra of cells of M . roseus were recorded to study the in vivo characteristics and localisation of the carotenoid pigments in these cells . The PA spectra indicated that both the psychrotrophic and mesophilic strains had similar chromophores . The cis carotenoids were prominent in the psychrotrophic M.roseus whereas shorter polyenes were more prominent in mesophilic M.roseus . Further, depth profiling photoacoustic studies revealed that in both the strains of M.roseus the bulk of the chromophore was associated with the cell membrane. Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 10881 - 6 RIGS (repeat-induced gene silencing) in Arabidopsis is transcriptional and alters chromatin configuration; Ye F et al.; We have previously reported repeat-induced gene silencing (RIGS) in Arabidopsis, in which transgene expression may be silenced epigenetically when repeated sequences are present . Among an allelic series of lines comprising a primary transformant and various recombinant progeny carrying different numbers of drug resistance gene copies at the same locus, silencing was found to depend strictly on repeated sequences and to correlate with an absence of steady-state mRNA . We now report characterization, in nuclei isolated from the same transgenic lines, of gene expression by nuclear run-on assay and of chromatin structure by nuclease protection assay . We find that silencing is correlated with absence of run-on transcripts, indicating that expression is silenced at the level of transcription . We find further that silencing is also correlated with increased resistance to both DNase I and micrococcal nuclease, indicating that the silenced state reflects a change in chromatin configuration . We propose that silencing results when a locally paired region of homologous repeated nucleotide sequences is flanked by unpaired heterologous DNA, which leads chromatin to adopt a local configuration that is difficult to transcribe, and possibly akin to heterochromatin. Proc R Soc Lond B Biol Sci, 1996 Sep 22, 263(1374), 1205 - 10 Phenotypic segregation of Aedes aegypti for immune antibacterial activity and resistance to filariae; Ham PJ et al.; We report the phenotypic selection of two lines of Aedes aegypti, from a filariae susceptible parental stock (Refm) . This selection was based upon the level of inducible anti-gram negative Escherichia coli activity within the haemolymph following E . coli infection . These lines, denoted 'high' and 'low', demonstrated significant differences in anti-E . coli responses throughout . However no difference was observed in the anti-gram positive Micrococcus luteus response following E . coli challenge . F4, F6 and F9 mosquitoes were experimentally infected with Brugia pahangi microfilariae . Reductions of between 53 and 82% in the mean number of larvae completing development in the 'high' compared with the 'low' responding line were observed . Corresponding reductions of between 30 and 50% in the mosquito infection rates also occurred between these lines . These reductions were significant for trials on the F6 and F9 generations . We have therefore selected for Ae . aegypti possessing significant refractoriness to filaria infection . Importantly, this 'high' line has not been exposed to selection pressure using filariae development as a phenotypic selective marker. EMBO J, 1996 Sep 16, 15(18), 4959 - 69 Differential association of HMG1 and linker histones B4 and H1 with dinucleosomal DNA: structural transitions and transcriptional repression; Ura K et al.; We examined the structural and functional consequences of incorporating either histone H1, histone B4 or HMG1 into a synthetic dinucleosome containing two 5S rRNA genes . We found that all three proteins bind to linker DNA, stabilizing an additional 20 bp from micrococcal nuclease digestion and restrict nucleosome mobility . Histone H1 has the highest-affinity interaction with the dinucleosome; histone B4 and HMG1 associate with significantly reduced affinities . We found that histone H1 binds to the dinucleosome template with a dissociation constant (KD) of 7.4 nM, whereas the KD is 45 nM for histone B4 and 300 nM for HMG1 . The KDs for the interaction of these proteins with naked DNA are 18 nM for H1, 80 nM for B4 and 300 nM for HMG1 . The differences in association of these proteins with the dinucleosome are reflected in the efficiency with which the different proteins repress transcription from the 5S rRNA genes . Thus, although all three proteins can contribute to the organization of chromatin, the stability of the structures they assemble will vary . Our results provide a molecular explanation for the transcriptional promiscuity of Xenopus early embryonic chromatin, which is enriched in HMG1 and linker histone B4, but deficient in histone H1. Nucleic Acids Res, 1996 Sep 15, 24(18), 3583 - 9 Metabolism of pre-messenger RNA splicing cofactors: modification of U6 RNA is dependent on its interaction with U4 RNA; Zerby DB et al.; The requirements for the formation of pseudouridine (psi) in U4 and U6 RNAs, cofactors in the splicing of pre-messenger RNA, were investigated in vitro using HeLa nuclear (NE) and cytoplasmic (S100) extracts . Maximal psi formation for both RNAs was extract order-dependent . Maximal psi formation in U4 RNA required incubation in S100 followed by the addition of NE, paralleling the in vivo maturation pathway of U4 RNA . In contrast, maximal formation of psi in U6 RNA required incubation in NE followed by the addition of S100 extract . Since U6 RNA does not exit the nucleus in vivo the contribution of S100 was investigated . In experiments where the extracts were treated with micrococcal nuclease to digest endogenous snRNAs, the efficient formation of psi in U6 RNA was dependent on the presence of U4 RNA, but not in U5 RNA or tRNA . When mutant U4 RNAs that inhibit or strengthen the interaction between U4 RNA, and U6 RNA were substituted for wild-type U4 RNA, the results confirmed the need for the interaction between these two RNAs for psi formation in U6 RNA . U6 RNA isolated from glycerol gradients after incubation in extracts had four times as much psi when associated with U4 RNA. Biochem Biophys Res Commun, 1996 Sep 13, 226(2), 356 - 61 Age-related changes in the ovalbumin gene of the Japanese quail; Upadhyay R et al.; Northern hybridization studies showed that the level of ovalbumin mRNA decreases in the oviduct of the Japanese quail after adulthood . In order to find out if this is due to changes in the conformation of the chromatin containing the promoter region of the gene, nuclei of the oviduct of young, adult and old birds were digested by DNaseI and micrococcal nuclease (MNase) . Southern hybridization with the labelled promoter showed that this region is less sensitive to the two enzymes in the old birds . Both the endonucleases recognized the same hypersensitive sites . The results indicate that the chromatin containing the promoter is present in an open conformation in adult birds . Gel mobility shift assay using a 20-mer dsDNA containing the CAAT-box and nuclear extract of oviduct shows the presence of trans-acting factors that bind to this region . The levels of these factors are lower in old birds . This may be the reason for the lower expression of the gene in old birds. J Biol Chem, 1996 Sep 6, 271(36), 21803 - 7 Isolation and sequencing of the rho gene from Streptomyces lividans ZX7 and characterization of the RNA-dependent NTPase activity of the overexpressed protein; Ingham CJ et al.; The gene for transcription termination factor Rho was isolated from Streptomyces lividans ZX7 . It encoded a 77-kDa polypeptide (Rho 77) with considerable homology to known Rho factors . An atypical hydrophilic region of 228 residues was found within the N-terminal RNA-binding domain . Only Rho from Micrococcus luteus and Mycobacterium leprae (closely related GC-rich Gram-positive bacteria) had an analogous sequence . Rho 77 was overexpressed in Escherichia coli and purified using an N-terminal hexahistidine-tag . Rho 77 displayed a broad RNA-dependent ATPase activity, with poly(C) RNA being no more than 4-fold more effective than poly(A) . This contrasts with the ATPase activity of Rho from E . coli which is stimulated primarily by poly(C) RNA . Rho 77 was a general RNA-dependent NTPase, apparent Km values for NTPs were: GTP 0.13 mM, ATP 0.17 mM, UTP 1.1 mM, and CTP >2 mM . Rho 77 poly(C)-dependent ATPase activity was inhibited by heparin, unlike the E . coli Rho . The antibiotic bicyclomycin inhibited the in vitro RNA-dependent ATPase activity of Rho 77, did not inhibit growth of streptomycetes but delayed the development of aerial mycelia . N-terminal deletion analysis to express a truncated form of Rho (Rho 72, 72 kDa) indicated that the first 42 residues of Rho 77 were not essential for RNA-dependent NTPase activity and were not the targets of inhibition by heparin or bicyclomycin. Anticancer Res, 1996 Sep-Oct, 16(5A), 2559 - 64 Antimetastatic action and lymphocyte activation by the modified lysozyme mPEG-Lyso in mice with MCa mammary carcinoma; Pacor S et al.; The effects of Lysozyme (hen egg-white lysozyme) and of its modified derivative mPEG-Lyso, (Lysozyme coupled with monomethoxypolyethylenglycol) were tested on CBA mice bearing MCa mammary carcinoma . mPEG-Lyso, given by the oral route at a dose comparable to 100 mg/kg/day of native Lysozyme, is at least as active as Lysozyme for the activation of lymphocytes obtained from different districts along the axis GALT-spleen . These effects were evidenced by measuring the in vitro response of lymphocytes of animals treated in vivo with ConA and LPS using the SRB test, and measuring the content of nucleic acids by cytofluorimetric analysis . Lymphocytes obtained from the mesenteric lymph nodes of animals treated with mPEG-Lyso, show a response to ConA and to LPS at early stages of treatment, when tumor growth reduces the response to controls . mPEG-Lyso, was also effective on lung metastasis formation . Considering that mPEG-Lyso,, compared to the native Lysozyme, completely lost its enzymatic action on Micrococcus lysodehycticus cell walls, this data suggest that the effects of lysozyme on immunity and on tumour growth are unrelated to the production of immunoactive peptidoglycans in the gut. J Virol, 1996 Sep, 70(9), 6227 - 34 Complete replication in vitro of tobacco mosaic virus RNA by a template-dependent, membrane-bound RNA polymerase; Osman TA et al.; A crude membrane-bound RNA polymerase, obtained by differential centrifugation of extracts of tomato leaves infected with tobacco mosaic tobamovirus (tomato strain L) TMV-L), was purified by sucrose density gradient centrifugation . Removal of the endogenous RNA template with micrococcal nuclease rendered the polymerase template dependent and template specific . The polymerase was primer independent and able to initiate RNA synthesis on templates containing the 3'-terminal sequences of the TMV-L positive or negative strands . TMV-vulgare RNA was a less efficient template, while RNAs of cucumber mosaic cucumovirus and red clover necrotic mosaic dianthovirus, or 5'-terminal sequences of TMV-L positive or negative strands, did not act as templates for the polymerase . A main product of the reaction with TMV-L genomic RNA as a template, carried out in the presence of {alpha-32P}UTP, was genomic-length single-stranded RNA . This was shown to be the positive strand and uniformly labelled along its length, demonstrating complete replication of TMV-L RNA . Genomic-length double-stranded RNA, labelled in both strands, and small amounts of RNAs corresponding to the single- and double-stranded forms of the coat protein subgenomic mRNA were also formed . Antibodies to N-terminal and C-terminal portions of the 126-kDa protein detected the 126-kDa protein and the 183-kDa readthrough protein in purified RNA polymerase preparations, whereas antibodies to the readthrough portion of the 183-kDa protein detected only the 183-kDa protein . All three antibodies inhibited the template-dependent RNA polymerase, but none of them had any effect on the template-bound enzyme. Eur J Biochem, 1996 Aug 15, 240(1), 186 - 94 Dissociation of protamine-DNA complexes by Xenopus nucleoplasmin and minichromosome assembly in vitro; Ruiz-Lara SA et al.; Nucleoplasmin, an acidic thermostable protein abundant in the nucleus of Xenopus laevis oocytes, has been found to dissociate complexes of pUC19 DNA and protein phi 1, an intermediate protamine present in ripe sperm from the mollusc Mytilus edulis . Cruder preparations of nucleoplasmin, such as the amphibian oocyte S150 extract and its thermostable fraction, also dissociate the heterologous DNA-phi 1 complexes and, in addition, promote the assembly of plasmid DNA into a minichromosome displaying regular nucleosomal periodicity, as revealed by micrococcal nuclease digestion . In contrast, purified nucleoplasmin complemented with rat hepatocyte core histone octamers in the presence of DNA topoisomerase I, although capable of inducing nucleoprotein formation onto the complexed DNA, fails to position nucleosomes at the native spacings seen in chromatin in vivo . These data favour the existence of a general mechanism to bring about, in a concerted manner, removal of sperm-specific nuclear proteins and reconstitution of somatic chromatin following fertilization. Nucleic Acids Res, 1996 Aug 15, 24(16), 3158 - 66 An RNase P RNA subunit mutation affects ribosomal RNA processing; Chamberlain JR et al.; RNase P is a ribonucleoprotein endoribonuclease responsible for the 5' maturation of precursor tRNAs in all organisms . While analyzing mutations in conserved positions of the yeast nuclear RNase P RNA subunit, significant accumulation of an aberrant RNA of approximately 193 nucleotides was observed . This abundant RNA was identified as a 3'extended form of the 5.8S rRNA . This strain also displays a slightly elevated level of other rRNA processing intermediates with 5-ends at processing site A2 in the internal transcribed spacer 1 (ITS1) region of the rRNA primary transcript . To test whether pre-rRNA in the region of ITS1/5.8S/ITS2 is a substrate for RNase P in vitro, nuclear RNase P was partially purified to remove contaminating nucleases . Cleavage assays were performed using an rRNA substrate transcribed in vitro which includes the 5.8S region and its surrounding processing sites in ITS1 and ITS2 . Discrete cleavages of this rRNA substrate were coincident with the peak fractions of nuclear RNase P, but not with fractions corresponding to mitochondrial RNase P or ribonuclease MRP RNA . The cleavage activity is sensitive to treatment with micrococcal nuclease, also consistent with an activity attributable to RNase R The strong RNase P cleavage sites were mapped and their possible relationships to steps in the rRNA processing pathway are considered . These observations suggest an intimate relationship between the processes of tRNA and rRNA maturation in the eukaryotic nucleus. Carcinogenesis, 1996 Aug, 17(8), 1549 - 52 Nucleotide level detection of cyclobutane pyrimidine dimers using oligonucleotides and magnetic beads to facilitate labelling of DNA fragments incised at the dimers and chemical sequencing reference ladders; Li S et al.; We present a method for detecting cyclobutane pyrimidine dimers (CPDs) at the nucleotide level and an adaptation of Maxam-Gilbert sequencing for generating sequence reference ladders . UV irradiated genomic DNA from Escherichia coli was digested with restriction enzyme(s) and incised at the CPDs with Micrococcus luteus UV endonuclease . The subsequent specific fragments were separated using a biotin labelled oligonucleotide containing a sequence complementary to the fragments of interest and streptavidin magnetic beads . These fragments were then radiolabelled on the beads just prior to the running of the sequencing gel . For generating sequence reference ladders, the unlabelled DNA fragments of interest were base-specifically modified and subsequently cleaved at the A+G or C+T sites using the rapid Maxam-Gilbert sequencing treatments . These chemically cleaved fragments can be stored almost indefinitely . Whenever the sequence reference ladders are required, the chemically cleaved fragments can be labelled alongside the CPD-specifically incised DNA fragments using the same procedure . The adaptation of the method to detect other types of DNA damage is also discussed. Biochem J, 1996 Aug 1, 317 ( Pt 3), 865 - 70 Calcium-dependent ADP-ribosylation of high-mobility-group I (HMGI) proteins; Giancotti V et al.; Micrococcal nuclease digestion of nuclei from mouse Lewis lung carcinoma cells releases a protein mixture into the supernatant that lacks histone H1 and contains a full complement of high-mobility-group I (HMGI) proteins (i.e . I, Y and I-C) . This implies that all three HMGI proteins are localized at the nuclease-sensitive regions of active chromatin . It is also shown that if Ca2+ ions are present in the nuclear incubation buffer (with or without exogenous nuclease), all three HMGI proteins become ADP-ribosylated . We propose that this modification of HMGI family proteins is part of the general poly(ADP-ribosyl)ation that accompanies DNA damage in apoptosis and other processes. Arzneimittelforschung, 1996 Jul, 46(7), 716 - 9 Use of rats chronically cannulated in the jugular vein and the duodenum in pharmacokinetic studies . Effect of ether anesthesia on absorption of amoxicillin; Torres-Molina F et al.; In order to be able to use unanesthetized rats in pharmacokinetic studies it is necessary to find methods of drug administration and repeated blood sampling that do not stress the animals and therefore avoid possible alterations in the pharmacokinetic parameters caused by stress . Two surgical procedures that permit chronic cannulation of the jugular vein and duodenum of the rat are described in this paper, and the influence of ether (CAS 60-29-7) anesthesia on the intestinal absorption of amoxicillin (CAS 26787-78-0) is evaluated . Amoxicillin (2.2 mg) was administered to the rats by the oral and intraduodenal routes . Intraduodenal administration was performed in conscious rats, whereas oral administration was performed under light and heavy ether anesthesia . In each animal, 10 blood samples were collected through the jugular cannula at fixed times after drug administration and the plasma antibiotic concentration was determined by a microbiological diffusion procedure, with Micrococcus luteus as the test organism . Plasma concentration versus time curves of amoxicillin after intraduodenal and oral administration under light anesthesia were similar, with a slight delay in the tmax value for the oral administration . However, after oral administration of amoxicillin to rats under heavy anesthesia, antibiotic absorption was very irregular, with a decreased Cmax value and an increased tmax value as compared with the other two administration methods . Furthermore, the amount of amoxicillin absorbed in rats given the drug under heavy anesthesia was smaller than in the other two groups . These results suggest that drugs can be administered to rats under light ether anaesthesia without altering their pharmacokinetic features, but heavy anaesthesia can significantly alter their absorption. J Biol Chem, 1996 Jun 14, 271(24), 14150 - 5 DNA knotting abolishes in vitro chromatin assembly; Rodriguez-Campos A; Topological knots can be formed in vitro by incubating covalently closed double stranded DNA and purified topoisomerase II from the yeast Saccharomyces cerevisiae in an ATP-dependent reaction . Knotting production requires a starting enzyme/DNA mass ratio of 1 . Analysis of knotted DNA was carried out by using both one- and two-dimensional agarose gel electrophoresis . The knots generated are efficiently untied, and give relaxed DNA rings, by catalytic amounts of topoisomerase II, but not by topoisomerase I . Time course analysis shows the knotting formation over relaxed and supercoiled DNA . When supercoiled DNA was used as a susbtrate, knots appear immediately whereas no transient relaxed rings were observed . The cell-free extract from Xenopus oocytes S-150 cannot assemble nucleosomes on knotted DNA templates as revealed by topological and micrococcal nuclease analysis . Nevertheless, the presence of knotted DNA templates does not inhibit the assembly over the relaxed plasmid . Finally, a pretreatment of knotted DNA with trace amounts of topoisomerase II before the addition of the S-150 yields a canonical minichromosome assembled in vitro . Taking into account these results, I suggest a mechanism of chromatin assembly regulation directed by topoisomerase II. Virus Res, 1996 Jun, 42(1-2), 149 - 58 Influenza A virus RNA-dependent RNA polymerase cleaves influenza mRNA in vitro; Peng Q et al.; We have investigated the endonuclease activity of the influenza A virus RNA polymerase in an in vitro assay with an artificial influenza-like mRNA containing a cap structure at its 5' terminus, followed by a 10 nt beta-globin mRNA sequence, and the 5' and 3' conserved termini of a truncated nucleoprotein (NP) cRNA influenza sequence . Results showed that partially purified virion ribonucleoprotein complexes (RNPs) and micrococcal nuclease treated RNPs cleaved the artificial influenza-like mRNA substrate specifically at positions near the 5' terminus to generate capped 14 and 15 nucleotide long RNA fragments which subsequently served as primers to initiate transcription . The endonuclease activity was completely blocked by addition of cap analog and competitively inhibited by added globin mRNA . Furthermore, an in vitro reconstituted influenza RNA transcription reaction containing a truncated NP vRNA as template, micrococcal nuclease treated RNPs and globin mRNA as primer, synthesized capped and uncapped full length (+) sense products . Enzyme kinetics showed that capped RNA was made earlier in the reaction; it reached a peak at 120 min and then declined . However, uncapped cRNA synthesis appeared later and remained as the dominant product later in the reaction . The nature of these products was confirmed by ribonuclease protection assays and by primer extension. J Bacteriol, 1996 Jun, 178(11), 3238 - 45 The Streptomyces peucetius drrC gene encodes a UvrA-like protein involved in daunorubicin resistance and production; Lomovskaya N et al.; The drrC gene, cloned from the daunorubicin (DNR)- and doxorubicin-producing strain of Streptomyces peucetius ATCC 29050, encodes a 764-amino-acid protein with a strong sequence similarity to the Escherichia coli and Micrococcus luteus UvrA proteins involved in excision repair of DNA . Expression of drrC was correlated with the timing of DNR production in the growth medium tested and was not dependent on the presence of DNR . Since introduction of drrC into Streptomyces lividans imparted a DNR resistance phenotype, this gene is believed to be a DNR resistance gene . The drrC gene could be disrupted in the non-DNR-producing S . peucetius dnrJ mutant but not in the wild-type strain, and the resulting dnrJ drrC double mutant was significantly more sensitive to DNR in efficiency-of-plating experiments . Expression of drrC in an E . coli uvrA strain conferred significant DNR resistance to this highly DNR-sensitive mutant . However, the DrrC protein did not complement the uvrA mutation to protect the mutant from the lethal effects of UV or mitomycin even though it enhanced the UV resistance of a uvrA+ strain . We speculate that the DrrC protein mediates a novel type of DNR resistance, possibly different from the mechanism of DNR resistance governed by the S . peucetius drrAB genes, which are believed to encode a DNR antiporter. J Biol Chem, 1996 May 17, 271(20), 12009 - 16 High mobility group protein 14 and 17 can prevent the close packing of nucleosomes by increasing the strength of protein contacts in the linker DNA; Tremethick DJ et al.; High mobility group (HMG) proteins 14 and 17 are abundant chromatin-associated proteins found in all higher eukaryotic nuclei . This observation demonstrates that HMGs 14 and 17 must have an important and universal function with regard to the structure and function of chromatin . What this function is, including how they interact with a nucleosomal array in vivo, is not known . Recently, we have demonstrated that HMGs 14 and 17 can organize nucleosomes into a regular array and increase the repeat length from 145 to about 160-165 base pairs in vitro . In addition, they can increase the apparent repeat length of chromatin deficient in histones H2A/H2B from 125 to approximately 145 base pairs . Importantly, this template was transcriptionally active . In this study, we report five new observations that begin to address the mechanism by which HMGs 14 and 17 space nucleosomal particles . First, we demonstrate that both human placenta HMG 14 and HMG 17 can space nucleosomes to produce a chromatin template with a repeat length around 160 base pairs . This result further highlights the similarity between these proteins in terms of protein structure and perhaps function . Second, we show that digestion of HMG containing chromatin with micrococcal nuclease produces DNA fragments that were approximately 10 and 20 base pairs longer than nucleosome core-particle DNA . This suggests that HMG 14 or HMG 17 can protect, directly or indirectly, at least an additional 10 base pairs of linker DNA from micrococcal digestion . However, this HMG-containing particle does not produce a strong kinetic block, and further digestion results in the eventual accumulation of DNA fragments 145 base pairs in length . Third, by comparing the full-length protein with different domains, we demonstrate that the acidic carboxyl-terminal domain is absolutely required for nucleosome spacing, neither the nucleosome binding domain of HMG 14 or HMG 17 nor the amino-terminal domain plus the nucleosome binding domain of HMG 14 could space nucleosomes . Fourth, we demonstrate that extensive micrococcal nuclease digestion of chromatin deficient in histones H2A/H2B led to the accumulation of DNA fragments about 110 base pairs in length, which is presumably the length of DNA associated with a nucleosomal particle deficient in one H2A/H2B dimer . Incorporation of either HMG 14 or HMG 17 into this chromatin results in the disappearance of this band and increase in the accumulation of fragments around 140-150 base pairs in length . Finally, in contrast to spacing of complete nucleosomes, we find that the nucleosome binding domain of HMG 17 (but not the nucleosome binding of HMG 14) is the only domain required for spacing of H2A/H2B-deficient chromatin. Eur J Biochem, 1996 May 15, 238(1), 64 - 9 Enlarged scale chemical synthesis and range of activity of drosocin, an O-glycosylated antibacterial peptide of Drosophila; Bulet P et al.; Insects respond to a bacterial challenge by rapidly synthesizing a diverse range of antibacterial and antifungal peptides . One of them, drosocin, a 19-residue proline-rich antibacterial peptide, was isolated from Drosophila . This peptide carries a disaccharide moiety attached to a threonine residue in mid-chain position . The present report describes the enlarged-scale chemical synthesis of drosocin, glycosylated with Gal (beta 1 --> 3)GalNAc(alpha 1 --> O) . We have studied the range of activity of the synthetic glycopeptide, of two truncated glycosylated isoforms, and of the unglycosylated L and D enantiomers . Both isolated and chemically synthesized drosocins carrying the disaccharide display the same antibacterial activity . Using circular dichroic spectroscopy we demonstrated that the O-linked disaccharidic motif did not affect the backbone conformation of drosocin . The antibacterial activity of the synthetic glycopeptide was directed against gram-negative strains with the exception of the gram-positive bacteria Micrococcus luteus . Deletion of the first five N-terminal residues completely abolished the activity of drosocin . As a first approach to the study of the mode of action of drosocin, we have synthesized a non-glycosylated D enantiomer and, using this molecule, we have shown that drosocin may act on the gram-negative bacteria through a stereospecific target. EMBO J, 1996 May 15, 15(10), 2496 - 507 Tissue-specific factors additively increase the probability of the all-or-none formation of a hypersensitive site; Boyes J et al.; DNase I-hypersensitive sites lack a canonical nucleosome and have binding sites for various transcription factors . To understand how the hypersensitivity is generated and maintained, we studied the chicken erythroid-specific beta(A)/epsilon globin gene enhancer, a region where both tissue-specific and ubiquitous transcription factors can bind . Constructions containing mutations of this enhancer were stably introduced into a chicken erythroid cell line . We found that the hypersensitivity was determined primarily by the erythroid factors and that their binding additively increased the accessibility . The fraction of accessible sites in clonal cell lines was quantitated using restriction endonucleases; these data implied that the formation of each hypersensitive site was an all-or-none phenomenon . Use of DNase I and micrococcal nuclease probes further indicated that the size of the hypersensitive site was influenced by the binding of transcription factors which then determined the length of the nucleosome-free gap . Our data are consistent with a model in which hypersensitive sites are generated stochastically: mutations that reduce the number of bound factors reduce the probability that these factors will prevail over a nucleosome; thus, the fraction of sites in the population that are accessible is also diminished. Anticancer Res, 1996 May-Jun, 16(3A), 1225 - 30 Chromatin structure and endonuclease sensitivity in human leukemic cell lines; Kuribayashi N et al.; The relationship between chromatin structure and endonuclease sensitivity was investigated . The cells used in this study were a) human myelogenous leukemic cell lines (HL-60, ML-I, U-937, THP-I) (Group I), which produced internucleosomal DNA cleavage, and b) human T-cell leukemia (MOLT-4), erythroleukemia (K562), glioblastoma (T98G, U87MG) and glioma (KG-1-C) cell lines (Group II), which produced no internucleosomal DNA cleavage, upon treatment with various apoptosis-inducing agents . When the nuclei, isolated from these cells were digested with micrococcal nuclease, chromatin DNA was cleaved into oligonucleosomal units . Although sensitivity to micrococcal nuclease considerably differed from cell to cell, Group I cells were generally more sensitive to micrococcal nuclease digestion than Group II cells . Similar sensitivity to DNase I was observed in both groups of cells . Acid-urea polyacrylamide gel electrophoresis of histone fractions from control and apoptosing HL-60 cells (induced either by hydrogen peroxide or UV irradiation) revealed no significant change in the relative composition of five major histones, indicating the absence of selective degradation of histone HI, but rather the nonspecific degradation of many nuclear proteins . These data suggest a difference in a chromatin structure between Group I and II cells, which might result in the selective production of internucleosomal DNA cleavage only in Group I cells. Mol Cell Biol, 1996 May, 16(5), 2037 - 43 Relationship between nuclease-hypersensitive sites and meiotic recombination hot spot activity at the HIS4 locus of Saccharomyces cerevisiae; Fan QQ et al.; Meiotic double-strand DNA breaks (DSBs), the lesions that initiate meiotic recombination at the HIS4 recombination hot spot, occur in a region upstream of the coding sequence associated with multiple DNase I-hypersensitive sites . Mutations in transcription factors that lead to loss of the DSBs result in the loss of some but not all DNase I-hypersensitive sites in the upstream region . A meiosis-specific change in chromatin structure is detected in strains with the wild-type hot spot but not in strains with alterations that elevate or reduce hot spot activity . The position and intensity of micrococcal nuclease-hypersensitive sites correlate poorly with the sites of DSB formation. Mol Cell Biol, 1996 May, 16(5), 1978 - 88 Chromatin remodeling during Saccharomyces cerevisiae ADH2 gene activation; Verdone L et al.; We have analyzed at both low and high resolution the distribution of nucleosomes over the Saccharomyces cerevisiae ADH2 promoter region in its chromosomal location, both under repressing (high-glucose) conditions and during derepression . Enzymatic treatments (micrococcal nuclease and restriction endonucleases) were used to probe the in vivo chromatin structure during ADH2 gene activation . Under glucose-repressed conditions, the ADH2 promoter was bound by a precise array of nucleosomes, the principal ones positioned at the RNA initiation sites (nucleosome +1), at the TATA box (nucleosome -1), and upstream of the ADR1-binding site (UAS1) (nucleosome -2) . The UAS1 sequence and the adjacent UAS2 sequence constituted a nucleosome-free region . Nucleosomes -1 and +1 were destabilized soon after depletion of glucose and had become so before the appearance of ADH2 mRNA . When the transcription rate was high, nucleosomes -2 and +2 also underwent rearrangement . When spheroplasts were prepared from cells grown in minimal medium, detection of this chromatin remodeling required the addition of a small amount of glucose . Cells lacking the ADR1 protein did not display any of these chromatin modifications upon glucose depletion . Since the UAS1 sequence to which Adr1p binds is located immediately upstream of nucleosome -1, Adr1p is presumably required for destabilization of this nucleosome and for aiding the TATA-box accessibility to the transcription machinery. J Mol Biol, 1996 Apr 19, 257(5), 919 - 34 Chromatin structure of the yeast URA3 gene at high resolution provides insight into structure and positioning of nucleosomes in the chromosomal context; Tanaka S et al.; To characterize nucleosome structure and positioning in the chromosomal context, the chromatin structure of the whole URA3 gene was studied in the genome and in a minichromosome by testing the accessibility of DNA to micrococcal nuclease and DNase I . The cutting patterns and hence the chromatin structures were almost indistinguishable in the genome and in the minichromosomes . The only notable exception was enhanced cutting between nucleosomes U3/U4 and U4/U5 in the minichromosomes . The results demonstrate that there is no severe constraint acting from outside the URA3 gene in chromosomes and minichromosomes . While low-resolution mapping showed six regions with a positioned nucleosome (U1 to U6), each region resolved in a complex pattern consistent with multiple overlapping positions . Some regions (U1, U4, U5 and U6) showed multiple positions with a dominant rotational setting (DNase I pattern), while U2 showed positioning within 10 bp but with no defined rotational setting, demonstrating that nucleosome positions were not in phase and not coordinately regulated . Reduced DNase I cutting from about 50 bp form the 5' end towards 3' end was common to all nucleosome regions . This polarity has been observed on isolated core particles . The results demonstrate that the DNase I pattern observed in vitro indeed reflects a structural property of nucleosomes in the chromosomal context . It is emphasized that despite the local heterogeneity revealed by high-resolution mapping, the low-resolution map is a reasonably accurate representation of the chromatin structure. Biochemistry, 1996 Apr 16, 35(15), 5093 - 102 Changes in chromatin structure support constitutive and developmentally regulated transcription of the bone-specific osteocalcin gene in osteoblastic cells; Montecino M et al.; Transcription of the osteocalcin gene, which encodes a 10 kDa bone-specific protein, is controlled by modularly organized basal regulatory sequences and hormone-responsive enhancer elements . We have previously shown that in the ROS 17/2.8 rat osteosarcoma cell line, which continuously expresses the osteocalcin gene, key regulatory elements reside in two DNase I hypersensitive sites that are fucntionally correlated with transcriptional activity . We now report that a specific nucleosomal organization supports this constitutive expression in ROS 17/2.8 cells, and that chromatin remodeling directly correlates with the developmentally regulated transcriptional activation of the osteocalcin gene during differentiation of normal diploid rat osteoblasts . By combining DNase I, micrococcal nuclease, and specific restriction endonuclease digestion analysis, we observed that the presence of DNAse I hypersensitive sites (-170 to -70 and -600 to -400) and a selective nucleosome positioning over the OC gene promoter are directly associated with developmental stage-specific transcriptional activation in bone-derived cells. Biochem J, 1996 Apr 15, 315 ( Pt 2), 473 - 9 The effects of tyrosine nitration on the structure and function of hen egg-white lysozyme; Richards PG et al.; We have investigated the effects of tyrosine nitration (to form the weak acid, 3-nitrotyrosine) at positions 23 or 20 plus 23, on the structure and function of hen egg-white lysozyme . Enzyme activity against Micrococcus luteus cell-wall fragments or soluble substrates exhibits two phenomena . (a) A decrease in Km and kcat for the hydrolysis of soluble oligo- and poly-saccharides, resulting in only minor changes in the catalytic efficiency (kcat/Km) upon nitration . (b) The hydrolysis of M . luteus cell-wall fragments appeared to be dominated by electrostatic interactions with the protein, giving a decrease in enzyme activity as the 3-nitrotyrosyl group became ionized . Removal of the cell-wall anionic polymer, teichuronic acid, from M . luteus abolished this effect . The 3-nitrotyrosine group was also found to act as a fluorescence quencher of exposed tryptophan residues in lysozyme. FEBS Lett, 1996 Apr 15, 384(2), 107 - 11 The spatial distribution of phospholipids and glycolipids in the membrane of the bacterium Micrococcus luteus varies during the cell cycle; Welby M et al.; Recently, we have developed a photocrosslinking approach which uses anthracene as a photoactivatable group and which allows us to determine the lateral distribution of lipids in membranes quantitatively . In synchronous cultures of the gram-positive bacterium Micrococcus luteus, this approach shows that the spatial distribution of phosphatidylglycerol and dimannosyldiacylglycerol, the two major lipids in the bacterial membrane, varies greatly during the cell cycle . Minimum heterogeneity was observed during cell growth while maximum heterogeneity was detected during cell division. J Biol Chem, 1996 Apr 12, 271(15), 9129 - 34 Poly(ADP-ribosyl)ation of histone H1 correlates with internucleosomal DNA fragmentation during apoptosis; Yoon YS et al.; The biochemical role of poly(ADP-ribosyl)ation on internucleosomal DNA fragmentation associated with apoptosis was investigated in HL 60 human premyelocytic leukemia cells . It was found that UV light and chemotherapeutic drugs including adriamycin, mitomycin C, and cisplatin increased poly(ADP-ribosyl)ation of nuclear proteins, particularly histone H1 . A poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide, prevented both internucleosomal DNA fragmentation and histone H1 poly(ADP-ribosyl)ation in cells treated with the apoptosis inducers . When nuclear chromatin was made accessible to the exogenous nuclease in a permeabilized cell system, chromatin of UV-treated cells was more susceptible to micrococcal nuclease than the chromatin of control cells . Suppression of histone H1 poly(ADP-ribosyl)ation by 3-aminobenzamide reduced the micrococcal nuclease digestibility of internucleosomal chromatin in UV-treated cells . These results suggest that the poly(ADP-ribosyl)ation of histone H1 correlates with the internucleosomal DNA fragmentation during apoptosis mediated by DNA damaging agents . This suggestion is supported by the finding that xeroderma pigmentosum cells which are defective in introducing incision at the site of DNA damage, failed to induce DNA fragmentation as well as histone H1 poly(ADP-ribosyl)ation after UV irradiation . We propose that poly(ADP-ribosyl)ation of histone H1 protein in the early stage of apoptosis facilitates internucleosomal DNA fragmentation by increasing the susceptibility of chromatin to cellular endonuclease. J Virol, 1996 Apr, 70(4), 2360 - 8 Influenza A virus RNA-dependent RNA polymerase: analysis of RNA synthesis in vitro; Galarza JM et al.; Influenza A virus RNA-dependent RNA polymerase, purified from virion ribonucleoprotein particles and from which endogenous genomic RNA (vRNA) has been depleted by treatment with micrococcal nuclease, was used to study transcription initiation, elongation, and termination in vitro . Templates that contained either minus- or plus-sense influenza virus nucleoprotein minigenes with conserved 5' and 3' termini and the uridylate tract were constructed . The dinucleotide ApG and alfalfa mosaic virus RNA4 (AlMV4) were used as primers . ApG primed the synthesis of full-length positive-strand or cRNA products and shorter transcripts, depending upon the molar ratio between the nucleoprotein and the vRNA template . Sequence analysis of the ends of these transcripts demonstrated that the 5' termini of both transcripts and the 3' terminus of the full-length product were complementary to the 3' and 5' termini of the vRNA template, respectively, whereas the 3' terminus of the incomplete product corresponded to a sequence located 40 bases downstream from the 5' terminus of the template and was about 20 nucleotides downstream from the uridylate tract, which is the putative signal for polyadenylation . Binding of the cap structure of AlMV4 by the polymerase activated RNA synthesis by ligation-elongation of small genomic RNA fragments which were likely derived from a genome segment protected by the polymerase from micrococcal nuclease digestion . The sequence of these fragments mapped to a region 14 to 28 nucleotides upstream of the 3' terminus of the viral genome . Polymerase subunit involvement in transcription initiation with ApG or AlMV4 was characterized by studying the effect of purified polyclonal antisubunit immunoglobulins of the G class (IgGs) in transcription assays . These results showed that anti-PB2 IgG inhibited transcription initiation in both ApG- and AlMV4-primed reactions, whereas anti-PB1 antibodies also blocked transcription initiated with AlMV4 . The differences observed in product size, product sequence, and differential inhibition by antisubunit IgGs are discussed . These observations would support the notion that the influenza virus RNA-dependent RNA polymerase undergoes a conformational change after the binding of the cap structure of host cell heterogeneous nuclear RNA by PB2, which then usually leads to endonucleolytic cleavage of the capped primer 13 nucleotides downstream from the cap. Biochem Biophys Res Commun, 1996 Mar 27, 220(3), 724 - 8 The major carotenoid pigment of a psychrotrophic Micrococcus roseus strain: fluorescence properties of the pigment and its binding to membranes; Jagannadham MV et al.; The fluorescence excitation and emission spectra are reported for P-3 (bis-dehydro-B-carotene-2-carboxylic acid), the major carotenoid pigment of psychrotrophic M . roseus . The excitation spectrum and the absorption spectrum showed good agreement with respect to the position of their peak maxima . The study also demonstrates that P-3 binds to liposomes prepared from synthetic lipids (PC, DOPG, or CL) or the total lipids of a mutant colourless M . roseus . Binding of P-3 to the membranes was accompanied by a decrease in the fluorescence emission intensity and a blue shift in lambda(em) maximum by 15 to 20 nm . The quantum yield of P-3 was observed to be low (1.7 x 10(-5)). Biochem Biophys Res Commun, 1996 Mar 27, 220(3), 594 - 9 Nucleotide sequence of 5'-upstream region and expression of a silkworm gene encoding a new member of the attacin family; Taniai K et al.; A genomic clone encoding a new member of attacin, an insect antibacterial protein, was isolated from a genomic library of the silkworm, Bombyx mori, and the nucleotide sequence of the 5'-upstream region was determined . The region contained Bm 1, a highly repetitive element of B . mori and a lipopolysaccharide (LPS) response element (RE)(NF-kappaB binding site), CAAT box and TATA box . Northern blot analysis showed that the attacin gene expression was rapidly induced by bacterial cell wall components such as LPS from Escherichia coli and peptidoglycan (PG) from Micrococcus luteus, suggesting that attacin plays an important role in an early phase of the self-defense system upon bacterial infection. Genes Dev, 1996 Mar 15, 10(6), 686 - 99 Yeast histone H3 and H4 amino termini are important for nucleosome assembly in vivo and in vitro: redundant and position-independent functions in assembly but not in gene regulation; Ling X et al.; The hydrophilic amino-terminal sequences of histones H3 and H4 extend from the highly structured nucleosome core . Here we examine the importance of the amino termini and their position in the nucleosome with regard to both nucleosome assembly and gene regulation . Despite previous conclusions based on nonphysiological nucleosome reconstitution experiments, we find that the histone amino termini are important for nucleosome assembly in vivo and in vitro . Deletion of both tails, a lethal event, alters micrococcal nuclease-generated nucleosomal ladders, plasmid superhelicity in whole cells, and nucleosome assembly in cell extracts . The H3 and H4 amino-terminal tails have redundant functions in this regard because the presence of either tail allows assembly and cellular viability . Moreover, the tails need not be attached to their native carboxy-terminal core . Their exchange re-establishes both cellular viability and nucleosome assembly . In contrast, the regulation of GAL1 and the silent mating loci by the H3 and H4 tails is highly disrupted by exchange of the histone amino termini. Microbiology, 1996 Mar, 142 ( Pt 3), 561 - 73 The application of serological techniques to the taxonomy of Arthrobacter and related organisms; Gray TR et al.; Antisera were raised against rods of 17 named Arthrobacter and Aureobacterium strains . Antigenic relationships between these strains, other soil bacteria and new Arthrobacter isolates from several soils were studied, using agglutination, immunodiffusion, immunofluorescence and ELISA techniques . Many of the named Arthrobacter species had common antigens, and there were also common antigens amongst named Arthrobacter strains and many fresh Arthrobacter isolates . Agglutination, ELISA and immunofluorescence tests revealed greater antigenic differences between the named strains than did immunodiffusion tests . Serological similarities between the 17 named strains and the fresh Arthrobacter isolates were calculated using SJ coefficients . The occurrence of named strains in serogroups based on immunodiffusion data supported the taxonomic scheme for arthrobacters in Bergey's Manual of Systematic Bacteriology . The distribution of soil isolates in serogroups resembled that in groups based on numerical analysis of diverse characters . This makes it possible to use serological tests to locate particular species in soil samples . Arthrobacter atrocyaneus was serologically distinct from other members of the Ar . globiformis/Ar . citreus group and did not cluster with them phenetically . Serological data suggest that Ar . aurescens, Ar . ureafaciens and Ar . histidinolovorans constitute a single species . Although A . simplex and A . tumescens have been placed in the genus Pimelobacter, they are serologically distinct from one another and more closely resemble Ar . globiformis . Aureobacterium strains were serologically distinct from arthrobacters . Micrococcus roseus showed many cross-reactions with several antisera, supporting the placement of micrococci in the same family as arthrobacters. Mol Cell Biol, 1996 Mar, 16(3), 1241 - 6 A discrete 3' region of U6 small nuclear RNA modulates the phosphorylation cycle of the C1 heterogeneous nuclear ribonucleoprotein particle protein; Mayrand SH et al.; The C heterogeneous ribonucleoprotein particle (hnRNP) protein bind to nascent pre-mRNA and may participate in assembly of the early prespliceosome . Ser/Thr phosphorylation of the C1 hnRNP protein in HeLa nuclear extracts regulates its binding to pre-mRNA (S . H . Mayrand, P . Dwen, and T . Pederson, Proc . Natl . Acad . Sci . USA 90:7764-7768, 1993) . We have now further investigated the phosphorylation cycle of the C1 hnRNP protein, with emphasis on its regulation . Pretreatment of nuclear extracts with micrococcal nuclease eliminated the phosphorylation of C1 hnRNP protein, but pretreatment with DNase did not, suggesting a dependence on RNA . Oligodeoxynucleotide-targeted RNase H cleavage of U1, U2, and U4 small nuclear RNAs did not affect the phosphorylation of C1 hnRNP protein . However, cleavage of nucleotides 78 to 95, but not other regions, of U6 small nuclear RNA resulted in an inhibition of the dephosphorylation step of the C1 hnRNP protein phosphorylation cycle . This inhibition was as pronounced as that seen with the serine/threonine protein phosphatase inhibitor okadaic acid . C1 hnRNP protein dephosphorylation could be completely restored by the addition of intact U6 RNA . Add-back experiments with mutant RNAs further delineated the minimal region essential for C1 protein dephosphorylation as residing in nucleotides 85 to 92 of U6 RNA . These results illuminate a hitherto unanticipated function of U6 RNA: the modulation of a phosphorylation-dephosphorylation cycle of C1 hnRNP protein that influences the binding affinity of this protein for pre-mRNA . This newly revealed function of U6 RNA is likely to play a very early role in the prespliceosome assembly pathway, prior to U6 RNA's entry into the mature spliceosome's active center. Biochemistry, 1996 Feb 20, 35(7), 2146 - 56 Efficient extraction and partial purification of the polyribosome-associated stem-loop binding protein bound to the 3' end of histone mRNA; Hanson RJ et al.; Replication-dependent histone mRNAs end in a highly conserved stem-loop sequence rather than a polyA sequence . A 45-kDa stem-loop binding protein (SLBP), which specifically binds the stem-loop of histone mRNA, is present in both polyribosomes and nuclei . An identical 45-kDa protein, as determined by partial protease digestion, is cross-linked to a 30 nt RNA containing the 3' stem-loop from both nuclei and polyribosomes . The SLBP can also be detected by a Northwestern blot procedure using the 30 nt RNA as a probe . As judged from the Northwestern assay, more than 90% of the SLBP in the cell is found in the polyribosomes with the remaining SLBP localized to the nucleus . Only 5-10% of the SLBP could be extracted from the polyribosomes with salt . Treatment of the polyribosomes with micrococcal nuclease prior to salt extraction solubilized 5-10 times more SLBP as an RNA-protein complex . The SLBP could be subsequently partially purified from this complex. Biochemistry, 1996 Feb 13, 35(6), 1890 - 6 Synergistic contributions of asparagine 46 and aspartate 52 to the catalytic mechanism of chicken egg white lysozyme; Matsumura I et al.; The X-ray structure of a chicken egg white lysozyme (ChEWL) complex with a peptidoglycan-derived inhibitor suggests that interactions of Asn46 and Asp52 with the D-subsite N-acetylmuramic acid residue help to distort that pyranose ring into the reactive half-chair conformation and that a hydrogen bond is formed between Asn46 and Asp52 {Strynadka, N . C . J., & James, M . N . G . (1991) J . Mol . Biol . 220, 401-424} . These hypotheses were investigated through the D52A, N46A, and D52A/N46A mutants of ChEWL . The Michaelis constants of the D52A and D52A/N46A ChEWL complexes with Micrococcus luteus cells are 3- and 4-fold higher, respectively, than the wild-type KM; the corresponding kcat values are 25- and 50-fold lower, respectively, than the wild-type kcat . These results support the proposal of Strynadka and James . The velocities of reactions catalyzed by the N46A and D52A mutants are approximately equal to each other for all classes of substrate, suggesting that the respective roles of Asn46 and Asp52 in transition state stabilization do not vary . The mutation of either Asn46 or Asp52 to Ala apparently disrupts the interactions of the other (nonmutated) residue with the substrate, supporting the crystallographic evidence of a hydrogen-bond interaction between the two residues . The mutations do not change the values of the dissociation constants of complexes with (carboxymethyl)chitin complexes, suggesting that ground state complexes of ChEWL with chitin-derived substrates differ in conformation from complexes with bacterial peptidoglycans. Biochemistry, 1996 Feb 13, 35(6), 1881 - 9 Is aspartate 52 essential for catalysis by chicken egg white lysozyme? The role of natural substrate-assisted hydrolysis; Matsumura I et al.; The chicken and goose egg white lysozymes (ChEWL and GoEWL) are homologues, but differ in substrate specificity . ChEWL catalyzes the hydrolysis of the glycosidic bonds of bacterial peptidoglycans and chitin-derived substrates, while GoEWL is specific for bacterial peptidoglycans . The active-site aspartate 52 residue of ChEWL, which is postulated to stabilize the oxocarbenium ion intermediate, has no counterpart in GoEWL . The substrate specificity of the D52A ChEWL mutant was compared with those of wild-type ChEWL and GoEWL . D52A ChEWL retains approximately 4% of the wild-type catalytic activity in reactions with three different bacterial cell suspensions . Asp52 therefore is not essential to the catalytic mechanism, accounting for only a 2 kcal/mol decrease in delta G++ . The function of Asp52 in D52A ChEWL- and GoEWL-catalyzed cleavage of (carboxymethyl)chitin may be partially fulfilled by an appropriately positioned carboxyl group on the substrate (substrate-assisted catalysis) . D52A ChEWL and GoEWL, unlike wild-type ChEWL, exhibit biphasic kinetics in the clearing of Micrococcus luteus cell suspensions, suggesting preferences for subsets of the linkages in the M . luteus peptidoglycan . These subsets do not exist in the peptidoglycans of Escherichia coli or Sarcina lutea, since neither D52A ChEWL nor GoEWL exhibits initial bursts in reactions with suspensions of these bacteria . We propose that substrate-assisted catalysis occurs in reactions of D52A ChEWL and GoEWL with M . luteus peptidoglycans, with the glycine carboxyl group of un-cross-linked peptides attached to N-acetylmuramic acid partially substituting the function of the missing Asp52. EMBO J, 1996 Feb 1, 15(3), 548 - 61 Evidence for a shared structural role for HMG1 and linker histones B4 and H1 in organizing chromatin; Nightingale K et al.; The high mobility group proteins 1 and 2 (HMG1/2) and histone B4 are major components of chromatin within the nuclei assembled during the incubation of Xenopus sperm chromatin in Xenopus egg extract . To investigate their potential structural and functional roles, we have cloned and expressed Xenopus HMG1 and histone B4 . Purified histone B4 and HMG1 form stable complexes with nucleosomes including Xenopus 5S DNA . Both proteins associate with linker DNA and stabilize it against digestion with micrococcal nuclease, in a similar manner to histone H1 . However, neither histone B4 nor HMG1 influence the DNase I or hydroxyl radical digestion of DNA within the nucleosome core . We suggest that HMG1/2 and histone B4 have a shared structural role in organizing linker DNA in the nucleosome. J Biol Chem, 1996 Jan 12, 271(2), 742 - 7 Characterization of an unusual Rho factor from the high G + C gram-positive bacterium Micrococcus luteus; Nowatzke WL et al.; A transcription termination factor (Rho) was purified from the Gram-positive bacterium Micrococcus luteus, and the complete gene sequence was determined . The M . luteus Rho polypeptide has 690 residues, which is 271 residues more than its homolog from Escherichia coli . Most of the additional residues compose a highly charged, hydrophilic segment that is inserted in a non-conserved region between two conserved regions of the RNA-binding domain of the known Rho homolog proteins . This segment extends from residues 49 to 311 and includes a stretch of 238 residues that contain no hydrophobic side chains . Biochemical studies indicate that the M . luteus protein is very similar to E . coli Rho in terms of its RNA-dependent NTPase activity and its sensitivity to the Rho-specific inhibitor bicyclomycin . However, the M . luteus protein has a less stringent RNA cofactor specificity . It also acts to terminate RNA transcription with E . coli RNA polymerase on the lambda cro DNA template, but at much earlier termination stop points than those recognized by E . coli Rho . Thus, the M . luteus protein functions as a true Rho factor, but with a different specificity than that of E . coli Rho . We propose that this altered specificity is consistent with its need to function on transcripts that have a high content of G + C residues. Biol Res, 1996, 29(1), 31 - 46 Photoaffinity labeling and photoaffinity crosslinking of enzymes; Schafer HJ et al.; Photoaffinity labeling is a special type of chemical modification, where the label is activated by the action of light . This article presents the general principles and limitations of this technique, its application to the study of Micrococcus luteus ATPase and the use of photoaffinity crosslinking to probe the structure of this enzyme. Scand J Infect Dis, 1996, 28(1), 83 - 5 Self-inflicted bacteraemia and fungaemia in Vietnamese migrants; Fung KS et al.; From early 1993, we received a number of blood cultures, all from Vietnamese inmates of the Whitehead Detention Centre, the biggest detention camp in Hong Kong, which grew unusual organisms . Upon follow-up, the majority of the patients were found to abscond from the hospital a few days after having been admitted with a clinical picture of septic shock . During the period from March 1993 to late 1994, we noted a total of 25 positive blood cultures from 21 previously healthy Vietnamese migrants with this syndrome . The mean age was 27.8 years and 20 were males . The organisms isolated from the blood cultures were of low pathogenicity such as Saccharomyces cerevisiae, Trichosporon spp., Baciflus spp . and Micrococcus spp . Fourteen of the patients complained of abdominal pain and 3 others had apical pneumothoraces . Eighteen had shock requiring resuscitation . Multiple puncture or needle marks were found in 11 patients, mainly involving the lower limbs . One patient admitted inducing illness by self-injection, and it was suspected that all these infections were self-inflicted . The range of hospitalization was 1-13 days (mean 3.05 days) . Eighteen of the 21 patients absconded. Eisei Shikenjo Hokoku, 1996, (114), 128 - 9 {Lysozyme Reference Standard (Control 951) of the National Institute of Health Sciences}; Kitajima A et al.; The "Lysozyme Reference Standard (Control 951)" of the National Institute of Health Sciences was prepared . The lysozyme potency of the standard material was assayed against the Lysozyme Reference Standard (Control 915) by two turbidimetric methods using the drycells of Micrococcus luteus as the substrate . The potency of the standard material was in satisfactory agreement with that of Lysozyme Reference Standard (Control 915) and was defined as 1 mg {potency} per mg. Chromosoma, 1996, 104(5), 386 - 92 A male-specific nuclease-resistant chromatin fraction in the mealybug Planococcus lilacinus; Khosla S et al.; In mealybugs, chromatin condensation is related to both genomic imprinting and sex determination . The paternal chromosomal complement is condensed and genetically inactive in sons but not in daughters . During a study of chromatin organization in Planococcus lilacinus, digestion with micrococcal nuclease showed that 3% to 5% of the male genome is resistant to the enzyme . This Nuclease Resistant Chromatin (NRC) apparently has a nucleosomal organization . Southern hybridization of genomic DNA suggests that NRC sequences are present in both sexes and occur throughout the genome . Cloned NRC DNA is A+T-rich with stretches of adenines similar to those present in mouse alpha-satellite sequences . NRC DNA also contains sequence motifs that are typically associated with the nuclear matrix . Salt-fractionation experiments showed that NRC sequences are matrix associated . These observations are discussed in relation to the unusual cytological features of mealybug chromosomes, including the possible existence of multiple centres of inactivation. Arch Biochem Biophys, 1995 Dec 20, 324(2), 357 - 66 Rigidity of a B-Z region incorporated into a plasmid as monitored by electron paramagnetic resonance; Strobel OK et al.; Electron paramagnetic resonance spectroscopy was employed to monitor the dynamics associated with a B-Z transition in both a linear (dG-dC)n and a modified pUC8 plasmid . A spin label consisting of cytidine substituted in position C5 with an 11-atom-tethered 5-membered ring nitroxide (DCAVAP) was incorporated into linear (dG-dC)n with Micrococcus luteus DNA polymerase or into a specific 34-bp Z-DNA-forming region of the 2.7-kb plasmid pRDZ8 with Thermus aquaticus DNA polymerase (Stoffel fragment) . Although DCA-VAP is a modified nucleotide, it was an excellent substrate for both enzymes . The Z conformation was induced by changing the salt concentration from 0.01 to 4.5 M . The EPR line shape changed in response to the switch in DNA conformation . The degree of change was quantitatively similar for both the linear polymer and the plasmid with its Z-DNA-forming region . A motional analysis which focuses on local dynamics indicates that the order parameter S for the spin-labeled systems increases upon conversion from B-DNA to Z-DNA . This decrease in motional freedom is consistent with the observation that Z-DNA is more rigid than B-DNA. Biochemistry, 1995 Dec 5, 34(48), 15768 - 76 Ionizing radiation activates nuclear factor kappa B but fails to produce an increase in human immunodeficiency virus gene expression in stably transfected human cells; Valerie K et al.; We have investigated the differential effects of ultraviolet light(UV) and ionizing radiation (IR) on human immunodeficiency virus type 1 (HIV) and c-jun expression in HIVcat/HeLa cells . This cell line harbors integrated copies of the chloramphenicol acetyltransferase (cat) gene under control of the HIV promoter . Both UV and IR increased the binding of nuclear proteins to an oligonucleotide spanning the HIV enhancer region nuclear factor kappa B sites, but only UV increased HIVcat steady-state mRNA and CAT activity . By comparison, transcription of the cellular c-jun gene increased after both types of radiation, but UV was at least 5-fold more effective than IR despite the fact that protein binding to an activator protein 1 oligonucleotide increased similarly after both UV and IR . The lack of HIVcat transcriptional response after IR does not appear to be the result of the repressor binding to upstream promoter elements since cells stably transfected with different HIV promoter deletions showed a lack of response to IR distinguishable from that of the intact promoter . While our findings indicate no correlation between increased binding of transcription factors to upstream promoter elements and increased expression of these genes after radiation, we did observe major differences in how UV and IR affected chromatin structure . UV produced extensive global chromatin decondensation, whereas IR did not, as seen in the microscope and determined by the increased susceptibility of chromatin to micrococcal nuclease digestion.(ABSTRACT TRUNCATED AT 250 WORDS) Clin Ter, 1995 Dec, 146(12), 843 - 56 {Magnetic resonance in AIDS-related encephalopathy}; Gualdi GF et al.; Fifty-eight patients with AIDS disease were studied with MR imaging in the aim of detecting the grade of brain involvement . The examinations were performed with a 1.5 Tesla magnet . Thirty-seven showed white matter lesion (63.5%), twenty-five patients showed cerebral atrophy (43%), in eight patients the MR appearance was consistent with toxoplasmosis infection (13.5%), two patients showed a linfoma (3.4%) and two patients micrococcosis (3.4%) . Seventeen out of the thirty-seven patients with white matter disease showed focal well circumscribed lesion (46%), while twenty showed diffuse involvement . Between the twenty-five patients with cerebral atrophy, twelve showed a prevalence of the cortical involvement and eight a subcortical atrophy . In five patients a concomitant, cortical and subcortical atrophy was found . Between the eight patients with neurotoxolesion and two of them a widespread encephalitis picture . The MR appearance of the two limphomas was that of periventricular, space occupying, masses . In two patients with micrococcis a nodular aspect of leptomeningeal lesions was found. Arch Microbiol, 1995 Dec, 164(6), 428 - 34 Formation of polyhydroxylated isoflavones from the soybean seed isoflavones daidzein and glycitein by bacteria isolated from tempe; Klus K et al.; Five tempe-derived bacterial strains identified as Micrococcus or Arthrobacter species were shown to transform the soybean isoflavones daidzein and glycitein to polyhydroxylated isoflavones by different hydroxylation reactions . All strains converted glycitein and daidzein to 6,7,4'-trihydroxyisoflavone (factor 2) and the latter substrate also to 7,8,4'-trihydroxyisoflavone . Three strains transformed daidzein to 7,8,3',4'-tetrahydroxyisoflavone and 6,7,3',4'-tetrahydroxyisoflavone . In addition, two strains formed 6,7,8,4'-tetrahydroxyisoflavone from daidzein . Conversion of glycitein by these two strains led to the formation of factor 2 and 6,7,3',4'-tetrahydroxyisoflavone . The structures of these transformation products were elucidated by spectroscopic techniques and chemical degradation. Chem Phys Lipids, 1995 Nov 17, 78(2), 137 - 47 Reconstitution of bacteriorhodopsin and ATP synthase from Micrococcus luteus into liposomes of the purified main tetraether lipid from Thermoplasma acidophilum: proton conductance and light-driven ATP synthesis; Freisleben HJ et al.; The archaebacterium Thermoplasma acidophilum is cultivated at 59 degrees C in a medium containing sulfuric acid of pH 2 . The purified bipolar membrane spanning main phospholipid (MPL) of this organism can be used to produce stable liposomes of 100-500 nm in diameter either using a French pressure cell detergent dialysis or sonication . Despite a potassium diffusion potential of 186 mV very low ionic permeability of sonicated MPL liposomes was measured using the potassium binding fluorescent indicator benzofuran isophthalate PBF1, which measures net K+ uptake . The latter also remained very low, in the presence of the K(+) ionophore valinomycin and palmitic acid . Addition of valinomycin and the potent uncoupler carbonylcyanid-p-trifluormehoxyphenyl-hydrazone (FCCP), led to a stimulation in potassium uptake . The rate of proton flux can be calculated from the net K(+) uptake . Under these conditions MPL liposomes are 1-2 orders of magnitude less permeable than egg yolk lecithin vesicles . The difference in proton permeability becomes even more pronounced with increasing temperature, examined using the fluorescent pH indicator pyranine . Purified bacteriorhodopsin from Halobacterium halobium was reconstituted into MPL liposomes in order to study the light-driven proton uptake in 150 mM KCl following addition of valinomycin, gramicidin, FCCP and Triton X-100 . The light-driven proton transport into the liposomes was increased 30-fold by addition of valinomycin decreased by gramicidin and FCCP, and abolished by Triton X-100 . Co-reconstituted MPL proteoliposomes containing bacteriorhodopsin and ATP synthase from Micrococcus luteus were capable of light-driven ATP synthesis demonstrating the functional coupling of proton transport and nucleotide generation in liposomal MPL membranes. Toxicol Lett, 1995 Nov 15, 81(2-3), 141 - 9 Inhibition of NAD(H)/NADP(H)--requiring enzymes by aurintricarboxylic acid; Thompson DC et al.; Aurintricarboxylic acid (ATA), an inhibitor of Ca(2+)-dependent endonuclease activity, is often used to implicate a role for increased intracellular calcium in mechanistic toxicology studies . We report here on the ability of ATA to inhibit the activity of several NAD(H)/NADP(H)-requiring enzymes (purified or cellular homogenates), including lactic dehydrogenase, alcohol dehydrogenase, cytochrome c reductase, ethoxycoumarin o-dealkylase, isocitric dehydrogenase, glutathione reductase and glucose-6-phosphate dehydrogenase . These results were compared with the ability of ATA to inhibit micrococcal nuclease and rat liver Ca(2+)-dependent endonuclease activity in similar incubations . With the exception of alcohol dehydrogenase, ATA was a potent inhibitor of each of the purified enzymes, with IC50s ranging from 0.5 to 82 microM . In cell homogenates, however, ATA was from 10 to 100-fold less potent at inhibiting these enzymes . When exogenous protein was added to purified enzyme incubations, the effect of ATA was similarly diminished . Our results demonstrate that ATA inhibits a wide range of NAD(H)/NADP(H)-requiring enzymes in in vitro incubations using purified enzymes, but that the inhibitory effects are markedly reduced in incubations which more closely resemble a cellular milieu. J Biol Chem, 1995 Nov 10, 270(45), 27000 - 6 Inactivation of chloramphenicol by O-phosphorylation . A novel resistance mechanism in Streptomyces venezuelae ISP5230, a chloramphenicol producer; Mosher RH et al.; Plasmid pJV4, containing a 2.4-kilobase pair insert of genomic DNA from the chloramphenicol (Cm) producer Streptomyces venezuelae ISP5230, confers resistance when introduced by transformation into the Cm-sensitive host Streptomyces lividans M252 (Mosher, R . H . Ranade, N . P., Schrempf, H., and Vining, L . C . (1990) J . Gen . Microbiol . 136, 293-301) . Transformants rapidly metabolized Cm to one major product, which was isolated and purified by reversed phase chromatography . The metabolite was identified by nuclear magnetic resonance spectroscopy and mass spectrometry as 3'-O-phospho-Cm, and was shown to have negligible inhibitory activity against Cm-sensitive Micrococcus luteus . The nucleotide sequence of the S . venezuelae DNA insert in pJV4 contains an open reading frame (ORF) that encodes a polypeptide (19 kDa) with a consensus motif at its NH2 terminus corresponding to a nucleotide-binding amino acid sequence (motif A or P-loop; Walker, J . E., Saraste, M., Runswick, M . J., and Gay, N . J . (1982) EMBO J . 1, 945-951) . When a recombinant vector containing this ORF as a 1.6-kilobase pair SmaI-SmaI fragment was used to transform S . lividans M252, uniformly Cm-resistant transformants were obtained . A strain of S . lividans transformed by a vector in which the ORF had been disrupted by an internal deletion yielded clones that were unable to phosphorylate Cm, and exhibited normal susceptibility to the antibiotic . The results implicate the product of the ORF from S . venezuelae as an enzymic effector of Cm resistance in the producing organism by 3'-O-phosphorylation . We suggest the trivial name chloramphenicol 3'-O-phosphotransferase for the enzyme. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 813 - 23 Preribosomal RNA processing in archaea: characterization of the RNP endonuclease mediated processing of precursor 16S rRNA in the thermoacidophile Sulfolobus acidocaldarius; Potter S et al.; The hyperthermophilic archaeon Sulfolobus acidocaldarius uses a novel RNA-containing endonuclease to excise and mature 16S rRNA from the precursor (pre) rRNA transcript . A cell-free processing system has been developed using an in vitro transcribed RNA substrate containing the entire 144 nucleotide 5' external transcribed spacer (5'ETS) and the first 72 nucleotides of 16S rRNA . The cell-free extract cleaves in the 5'ETS at positions -99, -31, and +1 (i.e., the 5'ETS-16S junction) . These positions are at or near the positions cleaved in vivo during processing of the pre rRNA transcript . The processing activity has been purified between 100 and 200-fold and appears to contain five or six polypeptide components and perhaps as many as 10 different small RNA components . Using combined reverse transcription-PCR amplification, full or partial cDNA copies of two of the RNA components have been obtained . One of the RNAs exhibits sequence and structural similarities to eukaryotic U3 snoRNA . The processing activity has been shown to be inactivated by micrococcal nuclease . It can be reactivated by reconstituting using bulk RNA from S.acidocaldarius but not bulk RNA from distantly related organisms . The activity is also abolished by RNase H digestion in the presence of oligonucleotides complementary to the U3-like RNA . These results demonstrate that the U3-like RNA is an essential component of the pre rRNA processing RNP endonuclease . Furthermore, this RNP endonuclease is not a derived eukaryotic feature, instead its existence predates the divergence of archaea and eukaryotes. Pharmazie, 1995 Nov, 50(11), 762 - 6 Influence of physical parameters on the germ-reducing effect of microwave irradiation on medicinal plants; Brantner A et al.; Plant material is to a different extent contaminated by microorganisms . As DAB 10 demands limiting values for aerobic bacteria, for yeasts and moulds, a reduction of microorganisms of the crude herbal drug is necessary . An effective method is the application of microwave irradiation . The microorganisms isolated and identified on different herbal drugs (leaves, flowers, herbs) were mainly spore-forming aerobic bacteria, Micrococcus luteus, and moulds of the genera Aspergillus and Penicillium . Various parameters such as the use of different microwave powers and the influence of humidity on the total count of microorganisms were tested according to the method described in DAB 10 . It can be concluded from the results that microwave treatment allows careful drying with a germ-reducing effect, provided testing takes place under optimized and accurately defined conditions. Proc Natl Acad Sci U S A, 1995 Oct 10, 92(21), 9732 - 6 Human white blood cells contain cyclobutyl pyrimidine dimer photolyase; Sutherland BM et al.; Although enzymatic photoreactivation of cyclobutyl pyrimidine dimers in DNA is present in almost all organisms, its presence in placental mammals is controversial . We tested human white blood cells for photolyase by using three defined DNAs (supercoiled pET-2, nonsupercoiled bacteriophage lambda, and a defined-sequence 287-bp oligonucleotide), two dimer-specific endonucleases (T4 endonuclease V and UV endonuclease from Micrococcus luteus), and three assay methods . We show that human white blood cells contain photolyase that can photorepair pyrimidine dimers in defined supercoiled and linear DNAs and in a 287-bp oligonucleotide and that human photolyase is active on genomic DNA in intact human cells. J Biol Chem, 1995 Oct 6, 270(40), 23475 - 84 Purification and cloning of Micrococcus luteus ultraviolet endonuclease, an N-glycosylase/abasic lyase that proceeds via an imino enzyme-DNA intermediate; Piersen CE et al.; Although Micrococcus luteus UV endonuclease has been reported to be an 18-kDa enzyme with possible homology to the 16-kDa endonuclease V from bacteriophage T4 (Gordon, L . K., and Haseltine, W . A . (1980) J . Biol . Chem . 255, 12047-12050; Grafstrom, R . H., Park, L., and Grossman, L . (1982) J . Biol . Chem . 257, 13465-13474), this study describes three independent purification schemes in which M . luteus UV damage-specific or pyrimidine dimer-specific nicking activity was associated with two proteins of apparent molecular masses of 31 and 32 kDa . An 18-kDa contaminant copurified with the doublet through many of the chromatographic steps, but it was determined to be a homolog of Escherichia coli ribosomal protein L6 . Edman degradation analyses of the active proteins yielded identical NH2-terminal amino acid sequences . The corresponding gene (pdg, pyrimidine dimer glycosylase) was cloned . The protein bears strong sequence similarities to the E . coli repair proteins endonuclease III and MutY . Nonetheless, traditionally purified M . luteus protein acted exclusively on cis-syn thymine dimers; it was unable to cleave site-specific oligonucleotide substrates containing a trans-syn -I, (6-4), or Dewar thymine dimer, a 5,6-dihydrouracil lesion, or an A:G or A:C mismatch . The UV endonuclease incised cis-syn dimer-containing DNA in a dose-dependent manner and exhibited linear kinetics within that dose range . Enzyme activity was inhibited by the presence of NaCN or NaBH4 with NaBH4 additionally being able to trap a covalent enzyme-substrate product . These last findings confirm that the catalytic mechanism of M . luteus UV endonuclease, like those of other glycosylase/AP lyases, involves an imino intermediate. J Cell Biochem, 1995 Oct, 59(2), 161 - 7 Sea urchin zygote chromatin exhibit an unfolded nucleosomal array during the first S phase; Imschenetzky M et al.; To investigate changes in chromatin organization associated with DNA replication during the first stages of development of the sea urchin Tetrapygus niger, we compared micrococcal nuclease (MNase) digestion patterns of chromatin from zygotes harvested during the first S phase and from unfertilized eggs . We observed that the majority of DNA fragments derived from MNase digested zygote nuclei were similar to or smaller than a mononucleosome, while those derived from unfertilized egg nuclei were larger (1,500 to 410 bp) . This result indicates that in zygotes, where active DNA replication is occurring, the major chromatin fraction is represented as unfolded nucleosomes . In contrast, in unfertilized eggs chromatin appears to be organized into polynucleosomes . To determine if the unfolded structure of nucleosomes observed during S phase is related to the level of poly (ADP-ribosylation) of cleavage stage (CS) histone variants, zygotes were treated with 20 mM 3-Amino Benzamide (3 ABA) during the interval between 3 and 30 min post-insemination (p.i.) . This treatment with 3 ABA decreases the poly (ADP-ribosylation) of CS histone variants and inhibits the first S phase in zygotes {Imschenetzky et al . (1991): J Cell Biochem 46:234-241; Imschenetzky et al . (1993): J Cell Biochem 51:198-205} . When the MNase digested patterns of chromatin from these 3 ABA treated and control zygotes were compared, we found that the unfolded structure of the nucleosomes remains unaltered by the inhibition of the poly(ADP-ribose) synthetase with 3 ABA . This result indicates that the unfolded nucleosomal structure, particular to the chromatin of S phase zygotes, is not contemporaneous to DNA replication and is independent of the normal level of poly(ADP-ribosylation) of CS histone variants. Int J Syst Bacteriol, 1995 Oct, 45(4), 837 - 9 Reclassification of Micrococcus agilis (Ali-Cohen 1889) to the genus Arthrobacter as Arthrobacter agilis comb . nov . and emendation of the genus Arthrobacter; Koch C et al.; Phylogenetic evidence derived from a 16S ribosomal DNA analysis indicated that the type strain of Micrococcus agilis, DSM 20550 (= ATCC 966 = CCM 2390), is less closely related to the type species of the genus Micrococcus, Micrococcus luteus, than to the type species of the genus Arthrobacter, Arthrobacter globiformis, and related Arthrobacter species . The phylogenetic position of M . agilis is supported by the presence of peptidoglycan variation A3 alpha and by the presence of MK-9(H2) as the major isoprenolog, a characteristic also found in strains of A . globiformis, Arthrobacter crystallopoietes, Arthrobacter atrocyaneus, Arthrobacter citreus, Arthrobacter aurescens, Arthrobacter ilicis, Arthrobacter ureafaciens, Arthrobacter oxydans, Arthrobacter histidinolovorans, and Arthrobacter nicotinovorans . The last six species and M . agilis are characterized by the presence of threonine in the interpeptide bridge of the peptidoglycan . Threonine has not been found in the peptidoglycans of other Arthrobacter species or in members of the genus Micrococcus . Despite the fact that a morphological life cycle is not known, these data support the proposal that M . agilis should be transferred to the genus Arthrobacter as Arthrobacter agilis comb . nov. Int J Syst Bacteriol, 1995 Oct, 45(4), 682 - 92 Taxonomic dissection of the genus Micrococcus: Kocuria gen . nov., Nesterenkonia gen . nov., Kytococcus gen . nov., Dermacoccus gen . nov., and Micrococcus Cohn 1872 gen . emend; Stackebrandt E et al.; The results of a phylogenetic and chemotaxonomic analysis of the genus Micrococcus indicated that it is significantly heterogeneous . Except for Micrococcus lylae, no species groups phylogenetically with the type species of the genus, Micrococcus luteus . The other members of the genus form three separate phylogenetic lines which on the basis of chemotaxonomic properties can be assigned to four genera . These genera are the genus Kocuria gen . nov . for Micrococcus roseus, Micrococcus varians, and Micrococcus kristinae, described as Kocuria rosea comb . nov., Kocuria varians comb . nov., and Kocuria kristinae comb . nov., respectively; the genus Nesterenkonia gen . nov . for Micrococcus halobius, described as Nesterenkonia halobia comb . nov.; the genus Nesterenkonia gen . nov . for Micrococcus halobius, described as Nesterenkonia halobia comb . nov.; the genus Dermacoccus gen . nov . for Micrococcus nishinomiyaensis, described as Dermacoccus nishinomiyaensis comb . nov.; and the genus Kytocossus gen . nov . for Micrococcus sedentarius, described as Kytococcus sedentarius comb . nov . M . luteus and M . lylae, which are closely related phylogenetically but differ in some chemotaxonomic properties, are the only species that remain in the genus Micrococcus Cohn 1872 . An emended description of the genus Micrococcus is given {corrected}. RNA, 1995 Oct, 1(8), 794 - 806 Titration of serine/arginine (SR) splicing factors during adenoviral infection modulates E1A pre-mRNA alternative splicing; Himmelspach M et al.; Alternative splicing of the adenovirus-2 E1A pre-mRNA involves the use of three 5' splice sites and is modulated during infection because the 13S mRNA and 9S mRNA reactions are predominant during the early and late periods, respectively . We had previously reproduced in vitro the 13S to 9S modulation with nuclear extracts isolated from infected HeLa cells and shown that high molecular weight viral RNAs are involved in this modulation, most likely by sequestering or titrating general splicing factors . To further test this hypothesis, we titrated splicing factors from an uninfected nuclear extract using competitor RNA or by progressive inactivation of splicing factors with monoclonal antibodies . We found that the 13S to 9S modulation occurs when titrating only with certain RNAs (essentially adenoviral RNAs), and also by progressively inactivating the 9G8 SR splicing factor . The demonstration that late nuclear extracts contain levels of active SR splicing factors limiting for the 13S reaction has been made by complementation experiments . We show that late nuclear extracts do not complement SR factor-deficient extracts, whereas late extracts treated with micrococcal nuclease complement them . Furthermore, complementation of late nuclear extracts with each of the three 30-35-kDa SR factors (9G8, SC35, and SF2/ASF) restores an efficient 13S mRNA reaction . Thus, our results provide evidence that the 13S to 9S modulation is triggered through a titration of SR factors required for the 13S mRNA reaction by major late transcripts that accumulate in nuclei late in infection. Nucleic Acids Res, 1995 Sep 25, 23(18), 3756 - 63 The uni chromosome of Chlamydomonas: histone genes and nucleosome structure; Walther Z et al.; The uni linkage group (ULG) of Chlamydomonas reinhardtii contains many genes involved in the basal body-flagellar system . Recent evidence suggests that the corresponding uni chromosome is located in close proximity to the basal body complex . In the course of studies into its molecular organization, we have found a cluster of four histone genes on the ULG . The genes are arranged as divergently-transcribed pairs: H3-H4 and H2B-H2A . Genomic sequencing reveals that these genes lack introns and contain characteristic 3' palindromes similar to those of animals . The predicted amino acid sequences are highly conserved across species, with greatest similarities to the histone genes of Volvox . Southern analysis shows that each histone gene is present in 15-20 copies in Chlamydomonas and suggests a dispersed genomic organization . Northern analysis of mitotically-synchronized cells shows that, like the replication-dependent histones of higher eukaryotes, Chlamydomonas histone genes are expressed during S-phase . Using a gene-specific probe on Northern blots, we provide evidence that the ULG H4 gene is regulated in the same manner as other Chlamydomonas histone genes . Finally, micrococcal nuclease protection experiments show that the uni chromosome itself associates with histone proteins and displays a conventional nucleosomal banding pattern. Nucleic Acids Res, 1995 Sep 25, 23(18), 3742 - 9 Enzymatic synthesis of polymers containing nicotinamide mononucleotide; Liu R et al.; Nicotinamide mononucleoside 5'-diphosphate in its reduced form is an excellent substrate for polynucleotide phosphorylase from Micrococcus luteus both in de novo polymerization reactions and in primer extension reactions . The oxidized form of the diphosphate is a much less efficient substrate; it can be used to extend primers but does not oligomerize in the absence of a primer . The cyanide adduct of the oxidized substrate, like the reduced substrate, polymerizes efficiently . Loss of cyanide yields high molecular weight polymers of the oxidized form . Terminal transferase from calf thymus accepts nicotinamide mononucleoside 5'-triphosphate as a substrate and efficiently adds one residue to the 3'-end of an oligodeoxynucleotide . T4 polynucleotide kinase accepts oligomers of nicotinamide mononucleotide as substrates . However, RNA polymerases do not incorporate nicotinamide mononucleoside 5'-triphosphate into products on any of the templates that we used. Gene, 1995 Sep 22, 163(1), 87 - 92 Cloning and sequencing of a secY homolog from Streptomyces scabies; Hale VA et al.; The complete DNA sequence of the Streptomyces scabies (Ss) secY homolog and partial sequences of adjacent upstream and downstream open reading frames (ORFs) have been determined . The nucleotide sequence of a 2-kb region predicts a polypeptide of 437 amino acids in length with homology to the SecY protein family . The Ss secY homolog lies upstream from a sequence that has homology to the adenylate kinase gene (adk) family . The translational stop codon of the putative SecY ORF overlaps the predicted start codon for the Adk ORF . Another ORF that lies upstream from the secY homolog has sequence similarity to the genes that code for the L15 r-protein . Within the 243-bp intergenic region between the L15 and SecY coding sequences, the presence of a streptomycete-like promoter sequence and an 18-bp inverted repeat suggests that the secY homolog and the adjacent downstream sequences may be transcribed independently of the L15 coding sequence . Transcript analysis indicates that the secY homolog is expressed in both Ss and Streptomyces lividans . The proposed gene and transcript organization of the L15-SecY-Adk coding regions in the Ss clone resembles that of Micrococcus luteus which, like the streptomycetes, has a G+C-rich genome. Nucleic Acids Res, 1995 Sep 11, 23(17), 3524 - 30 Double stranded scission of DNA directed through sequence-specific R-loop formation; Landgraf R et al.; R-loop formation with short (100 nt) RNAs provides a highly flexible and stringent method to achieve sequence-specific separation of target DNA at any given sequence . After stabilization of R-loops with glyoxal and removal of the RNA through RNase treatment the remaining single-stranded DNA bubble provides a highly favorable substrate for attenuated micrococcal nuclease . We investigated this method for sequence-specific scission of double-stranded DNA and achieved quantitative scission of 3-5 kb plasmids . The applicability to larger size DNA is demonstrated through specific excision of the intervening segment between two R-loops from a P1 plasmid of approximately 120 kb. J Biochem (Tokyo), 1995 Sep, 118(3), 546 - 51 Insect lysozyme from house fly (Musca domestica) larvae: possible digestive function based on sequence and enzymatic properties; Ito Y et al.; Lysozyme was purified from the homogenate of the whole body of house fly (Musca domestica) larvae by standard chromatographic techniques . The purified lysozyme was sequenced and its enzymatic properties were examined . This lysozyme was a chicken-type lysozyme composed of 122 amino acids, showing about 75% identity with fruit fly lysozymes and 38% with human lysozyme . This enzyme was inactive towards Micrococcus luteus and under the physiological conditions of PH 7.0 and ionic strength 0.1, but was as active toward glycol chitin as was hen lysozyme . The pH-dependent profile of lytic activity towards M . luteus showed that house fly lysozyme has an acidic pH optimum and shows no enzymatic activity above Ph 7 . These features are analogous with those of ruminant stomach lysozymes which have evolved for the digestive function, suggesting that this lysozyme does not function as a self-defense protein, like hen and human lysozyme, but as a digestive enzyme, probably in the gut of the insect body . Although a similar functional conversion to digestive enzyme was reported in fruit fly, phylogenetic tree analysis indicates that the evolutionary change of lysozyme to a digestive enzyme occurred similarly in fruit fly and house fly, but the events are not related and occurred independently in each strain . This observation is in contrast with the case of ruminant stomach lysozymes, which were recruited before the divergence of each species of ruminants. J Cell Physiol, 1995 Sep, 164(3), 571 - 8 Two nuclear proteins in tracheal epithelial cells are recognized by antibodies specific to a squamous differentiation marker, sprI; Tesfaigzi J et al.; In cell-free translations of RNA from primary cultures of pig trachea surface epithelial cells we observed that a mRNA encoding a 20 kDa proline-rich protein (sPRP) was dramatically induced during culturing (Tesfaigzi et al., 1990, Biochem . Biophys . Res . Commun., 172:M1304-1309) . This mRNA was not detected in tracheal tissue or in epithelial cells prior to culturing . Antisera were raised to synthetic peptide sequences corresponding to 23 amino acids on the C-terminus (C23-antiserum) and 29 amino acids on the N-terminus (N29 antiserum) of sPRP . On Western blot analysis, C23 antiserum reacted with a 20 kDa protein in cytosolic extracts from pig tracheal cells maintained in culture for 4 days . The reaction with the 20 kDa protein was inhibited by adding C23 peptide . Two nuclear proteins (66 and 70 kDa) obtained by micrococcal nuclease treatment of tracheal cell nuclei were detected on Western blots with C23 antiserum . These proteins were present in cells both before and after culturing . Sucrose gradient fractionation indicated that these nuclear proteins are associated with chromatin . Small amounts of the 66 and 70 kDa proteins were obtained from nuclear matrix fractions . These nuclear proteins also reacted with N29 antiserum . Since these proteins share similar epitopes with the N- and C-termini of sPRP, it is likely that the 20 kDa protein (sPRP) is part of these proteins . However, purification of the nuclear proteins followed by an amino acid sequence analysis is necessary to clarify whether sPRP is part of these proteins. J Gen Virol, 1995 Sep, 76 ( Pt 9), 2205 - 13 In vitro transcription and polymerase binding studies of the termini of influenza A virus cRNA: evidence for a cRNA panhandle; Pritlove DC et al.; An in vitro transcription assay was used to study transcription from synthetic RNA corresponding to the 3' terminus of influenza A virus cRNA . Micrococcal nuclease-treated influenza virus ribonucleoprotein was used as a source of active polymerase complex . Mutations at two regions of the 13 nucleotide-long conserved cRNA 3' terminus were shown to reduce transcription templated by the short added model RNAs . The first region, at positions 1 and 2 from the 3' terminus, was shown to be affected by the exact nature of the dinucleotide primer used in the in vitro transcription reactions and may not be relevant in vivo . The second region, centred on positions 11 and 12, may be involved in base pairing with conserved nucleotides at the 5' terminus of the cRNA . Evidence for this comes from the finding that RNA corresponding to 5' conserved sequences, but mutated to restore the postulated base pairing with the mutated 3' ends, could partly restore transcription . Binding of the influenza virus polymerase complex to a set of 5'-mutated RNAs was investigated using a photochemical cross-linking assay . Specific binding to two regions of the cRNA 5' terminus was demonstrated, at positions 1 to 3 and positions 8 to 10 . Together, these observations suggest that a panhandle forms from the termini of the cRNA molecule and that this structure may play a role in transcription to produce virion RNA. J Med Entomol, 1995 Sep, 32(5), 578 - 82 Bacteriocidal qualities of ixodid tick (Acarina: Ixodidae) salivary cement plugs and their changes under the influence of a viral tick-borne pathogen; Alekseev AN et al.; The abundance of bacteriocidal compounds contained in the salivary cement plug of ixodid ticks was changed because of the reproduction of tick-borne encephalitis virus (TBEV) in their bodies . The size of TBEV-infected Ixodes persulcatus Shulze lytic zone surrounding the cement plug enlarged to that of naive ticks, whereas Micrococcus lysodeikticus (Cohn) lytic zones induced by the cement plugs of TBEV-infected Amblyomma hebraeum Koch nymphs or Rhipicephalus appendiculatus Neumann females were reduced, compared with those produced by noninfected specimens . It is possible that an increase of lysozyme production by the primary TBEV vectors (Ixodinae) infected salivary gland cells, compared with suppression of the bacteriocidal qualities of saliva of ticks that are not TBEV vectors in nature (Amblyomminae), is an indication of the specificity of a I . persulcatus-TBEV interface. Proc R Soc Lond B Biol Sci, 1995 Aug 22, 261(1361), 217 - 21 Full sequence and characterization of two insect defensins: immune peptides from the mosquito Aedes aegypti; Chalk R et al.; We report the complete amino acid sequence and biological activity of two immune peptides, from the yellow fever mosquito Aedes aegypti, that are induced in response to infection . Both peptides display biological activity against the Gram positive microbe Micrococcus luteus and substantial sequence homology to insect defensins, small heat-stable, antibiotic peptides previously described from several non-vector insects . These mosquito peptides, designated Ae . aegypti defensins A and B, are isoforms . Defensin B is the most abundant antibacterial peptide in this species whereas defensin A is much less abundant and carries two amino acid substitutions compared to defensin B, making it more basic in character . Apparent convergence between isoforms from Ae . aegypti and the fleshfly Phormia terranovae is discussed . The synergistic activity previously described between Ae . aegypti immune haemolymph and lysozyme is not caused by these peptides because synergy occurred only at concentrations far outside the physiological range seen in Ae . aegypti. Eur J Biochem, 1995 Aug 1, 231(3), 831 - 8 Purification and molecular cloning of a major antibacterial protein of the protozoan parasite Entamoeba histolytica with lysozyme-like properties; Jacobs T et al.; A protein with potent antibacterial activity was purified to apparent homogeneity from pathogenic Entamoeba histolytica . It resembles lysozyme in that it is a basic protein which degrades cell walls of Micrococcus luteus, displays optimal activity at acidic pH, and shows a preference for Gram-positive bacteria . The protein has a molecular mass of approximately 23 kDa upon SDS/PAGE and is localized inside the cytoplasmic granules of the amoebae . The primary structure was elucidated by pr