Genomics, 2000 Apr 15, 65(2), 129 - 36 A sequence-ready physical map of the region containing the human natural killer gene complex on chromosome 12p12.3-p13.2; Renedo M et al.; We developed a sequence-ready physical map of a part of human chromosome 12p12.3-p13.2 where the natural killer gene complex (NKC) is located . The NKC includes a cluster of genes with structure similar to that of the Ca(2+)-dependent lectin superfamily of glycoproteins that are expressed on the surface of most natural killer (NK) cells and a subset of T cells . These killer cell lectin-like receptors (KLR) are involved in NK target cell recognition, leading to activation or inhibition of NK cell function . We used a number of sequence-tagged site (STS) markers from this region to screen two large insert bacterial artificial chromosome (BAC) libraries and a bacteriophage P1-derived (PAC) chromosome library . The clones were assembled into contiguous sets by STS content analysis . The 72-BAC and 11-PAC contig covers nearly 2 Mb of DNA and provides an average marker resolution of 26 kb . We have precisely localized 17 genes, 5 expressed sequence tags, and 49 STSs within this contig . Of this total number of STS, 30 are newly developed by clone-end sequencing . We established the order of the genes as tel-M6PR-MAFAL (HGMW-approved symbol KLRG1)-A2M-PZP-A2MP-NKRP1A (HGMW-approved symbol KLRB1)-CD69-AICL (HGMW-approved symbol CLECSF2)-KLRF1-OLR1-CD94 (HGMW-approved symbol KLRD1)-NKG2D (HGMW-approved symbol D12S2489E)-PGFL-NKG2F (HGMW-approved symbol KLRC4)-NKG2E (HGMW-approved symbol KLRC3)-NKG2A (HGMW-approved symbol KLRC1)-LY49L (HGMW-approved symbol KLRA1)-cen . This map would facilitate the cloning of new KLR genes and the complete sequencing of this region . Biol Chem, 2000 Mar, 381(3), 255 - 8 Stability of bacteriophage T4 short tail fiber; Burda MR et al.; Adsorption of T-even bacteriophages to the E . coli host cell is mediated by long and short tail fibers . Bacteriophage T4 short tail fiber protein p12 was used to investigate the stability against thermal and chemical denaturation . Purified p12 is thermostable with a melting point of 78 degrees C . Guanidinium chloride-induced denaturation displayed strong hysteresis and an intermediate between 2 and 3 M denaturant . The transitions occur at 1.5 and 3.2 M denaturant as revealed by fluorescence spectroscopy and circular dichroism . The data suggest an equilibrium unfolding intermediate with a separate unfolding of the C-terminal knob domain and the shaft region. J Biol Chem, 2000 Apr 28, 275(17), 12598 - 602 A T4-phage deoxycytidylate deaminase mutant that no longer requires deoxycytidine 5'-triphosphate for activation; Keefe RG et al.; A deoxycytidylate (dCMP) deaminase encoded in T4-bacteriophage DNA that is induced on phage infection of Escherichia coli was shown earlier (Maley, G . F., Duceman, B . W., Wang, A . M., Martinez, J . M., and Maley, F . (1990) J . Biol . Chem . 265, 47-51) to be similar in size, properties, and amino acid composition to the T2-phage-induced deaminase . Neither enzyme is active in the absence of dCTP or its natural activator, 5-hydroxymethyl-dCTP . However, on changing the arginine (Arg) at residue 115 of the T4-deaminase to either a glutamate (R115E) or a glutamine (R115Q), the resulting mutant enzymes were active in the absence of dCTP, with each mutant possessing a turnover number or k(cat) that is about 15% that of the wild-type deaminase . When compared on the basis of specific activity, however, the mutants are about 40-50% of the wild-type (WT)-enzyme's specific activity . Molecular weight analysis on the wild-type and mutant deaminases using HPLC size exclusion chromatography revealed that the wild-type deaminase was basically a hexamer, particularly in the presence of dCTP, regardless of the extent of dilution . Under similar conditions, R115E remained a dimer, whereas R115Q and F112A varied from hexamers to dimers particularly at concentrations normally present in the assay solution . Activity measurements appear to support the conclusion that the hexameric form of the enzyme is activated by dCTP, while the dimer is not . Another feature emphasizing the difference between the WT and mutant deaminases was observed on their denaturation-renaturation in EDTA, which revealed the mutants to be restored to 50% of their original activities with the WT deaminase only marginally restored. Mikrobiologiia, 2000 Mar-Apr, 69(2), 261 - 5 {Coliphages inactivation using chitosan derivatives}; Kochkina ZM et al.; The effect of chitosan fragments with different degrees of polymerization and the chemical derivatives of chitosan differing in the number of amino groups and total molecule charge on phages T2, T4, and T7 was studied . The interaction of chitosan with bacteriophage particles inactivated them to the extent dependent on the chemical properties of chitosan and its concentration . Phage T2 was found to be most susceptible to inactivation by chitosan . The polycationic nature of chitosan plays an important role in the inactivation of phages . It is assumed that the abnormal rearrangement of the basal plate of phages, the loss of long tail fibers, and probably, modification of the receptor-recognizing phage proteins may be responsible for the inactivation of coliphages by chitosan. Blood Cells Mol Dis, 2000 Feb, 26(1), 37 - 46 Genomic structure of the human p47-phox (NCF1) gene; Chanock SJ et al.; The cytosolic factor p47-phox, encoded by the NCF1 gene, is an essential component of the phagocyte NADPH-oxidase system . Upon activation of this multicomponent system, p47-phox translocates to the membrane and participates in the electron transfer from NADPH to molecular oxygen . A deficiency or absence of p47-phox is the most common autosomal form of chronic granulomatous disease (CGD) . We have cloned and characterized the NCF1 gene from four bacteriophage clones, a P1 clone and genomic DNA from normal individuals . The gene is 15,236 base pairs long and includes 11 exons . It is 98.6% homologous in sequence to at least one pseudogene that maps to the same region of chromosome 7q11.23 . Slightly more than half (50.37%) of the wild-type NCF1 gene consists of repetitive elements . In particular, the density of Alu sequences is high (1.4 Alu/kb); there are 21 Alu repeats interspersed through 10 introns . These findings are consistent with the observation that recombination events between the wild-type gene and its highly homologous pseudogenes account for the majority of potentially lethal mutations in p47-phox-deficient chronic granulomatous disease . Analysis of 1.96 kb of sequence 5' of the start of translation revealed a high homology (99.6%) between wild-type and pseudogene clones . Characterization of NCF1 establishes a foundation for detailed molecular analysis of p47-phox-deficient CGD patients as well as for the study of the regulation of the NCF1 gene and pseudogenes, both of which are present as full-length transcripts in normal individuals . Copyright 2 Academic Press. J Biol Chem, 2000 Jun 23, 275(25), 18879 - 86 Thermodynamic and functional characterization of protein W from bacteriophage lambda . The three C-terminal residues are critical for activity; Maxwell KL et al.; Gene product W (gpW), the head-tail joining protein from bacteriophage lambda, provides a fascinating model for studying protein interactions . Composed of only 68 residues, it must interact with at least two other proteins in the phage, and probably with DNA . To study the structural and functional properties of gpW, plasmids were constructed expressing gpW with hexahistidine tag sequences at either the N or C terminus . The purified wild type fusion proteins were found to be stably folded and biologically active . The protein is monomeric as judged by equilibrium ultracentrifugation, and appears to unfold by a cooperative two-state mechanism . Circular dichroism studies indicate that the protein is 47% helical, with a T(m) of 71.3 degrees C, and a DeltaG(u) of 3.01 kcal/mol at 25 degrees C . Mutagenesis of the three hydrophobic C-terminal residues of gpW showed that they are critical for activity, even though they do not contribute to the thermodynamic stability of the protein . Using secondary structure prediction as a guide, we also designed destabilized gpW mutants . The hydrophobic nature of the gpW C terminus caused these mutants to be degraded by the ClpP-containing proteases in Escherichia coli. Antimicrob Agents Chemother, 2000 May, 44(5), 1132 - 9 A bacteriophage lambda-based genetic screen for characterization of the activity and phenotype of the human immunodeficiency virus type 1 protease; Martinez MA et al.; Human immunodeficiency virus type 1 (HIV-1) resistance to antiretroviral drugs is the main cause of patient treatment failure . Despite the problems associated with interpretation of HIV-1 resistance testing, resistance monitoring should help in the rational design of initial or rescue antiretroviral therapies . It has previously been shown that the activity of the HIV-1 protease can be monitored by using a bacteriophage lambda-based genetic assay . This genetic screening system is based on the bacteriophage lambda regulatory circuit in which the viral repressor cI is specifically cleaved to initiate the lysogenic to lytic switch . We have adapted this simple lambda-based genetic assay for the analysis of the activities and phenotypes of different HIV-1 proteases . Lambda phages that encode HIV-1 proteases either from laboratory strains (strain HXB2) or from clinical samples are inhibited in a dose-dependent manner by the HIV-1 protease inhibitors indinavir, ritonavir, saquinavir, and nelfinavir . Distinct susceptibilities to different drugs were also detected among phages that encode HIV-1 proteases carrying different resistance mutations, further demonstrating the specificity of this assay . Differences in proteolytic processing activity can also be directly monitored with this genetic screen system since two phage populations compete in culture with each other until one phage outgrows the other . In summary, we present here a simple, safe, and rapid genetic screening system that may be used to predict the activities and phenotypes of HIV-1 proteases in the course of viral infection and antiretroviral therapy . This assay responds appropriately to well-known HIV-1 protease inhibitors and can be used to search for new protease inhibitors. AIDS, 2000 Mar 10, 14(4), F55 - 62 Progressive specific immune attrition after primary, secondary and tertiary immunizations with bacteriophage phi X174 in asymptomatic HIV-1 infected patients; Rubinstein A et al.; BACKGROUND: Antibody responses to immunization are often compromised in patients infected by HIV-1, and the use of childhood immunization in affected children is controversial . We investigated whether multiple immunizations with a T cell-dependent neoantigen, bacteriophage phiX174, induce selective immune attrition and post-vaccination viremia . METHODS: Seventeen asymptomatic, antiretroviral therapy-naive HIV-1-infected patients with a CD4 cell count of 450 cells/microl or greater were immunized in 1990/1991 with three intravenous doses of bacteriophage phiX174 . Group 1 received zidovudine (ZDV) during the primary and secondary immunization . Group 2 received ZDV exclusively during the tertiary immunization . Bacteriophage-specific antibodies of the IgM and IgG class, lymphocyte phenotypes (CD4+, CD8+, CD4+DR+, CD8+DR+, CD4+CD45RO+ and CD4+45RA+, CD4+CD45RO+DR+) and HIV-1 plasma viremia were measured sequentially . RESULTS: In both patient groups the primary, secondary and tertiary antibody responses, as expressed by geometric mean antibody titres and IgM to IgG switch, were impaired . Booster immunizations resulted in a progressive attrition of specific antibody responses to bacteriophage . Antibodies to tetanus toxoid remained stable . The HIV-1 viral loads, which were evaluated in archived specimens from eight patients, increased after immunization but returned to baseline approximately 4 weeks later . The humoral immune attrition and increases in plasma viremia were blunted by concomitant short courses of ZDV . DISCUSSION: Multiple boosters of immunizations in asymptomatic treatment-naive HIV-1-infected patients may result in a specific immune attrition and vaccine-induced viremia . Short-term monotherapy with ZDV may have blunted these adverse effects . Hyperimmunization of HIV-1-infected patients may be detrimental unless accompanied by antiretroviral therapy. Hybridoma, 2000 Feb, 19(1), 89 - 94 Monoclonal antibodies against the major coat protein of filamentous phage as a useful analytical tool for bacteriophage peptide/protein display; Ibarra N et al.; Four mouse monoclonal antibodies (MAbs) that react with filamentous M13KO7 and R408 phage were obtained . Three of these MAbs (two IgG2a and one IgG3) recognize linear sequences of the p8 main structural coat protein, and one (IgG2a) identifies a putatively conformational epitope, as suggested by Western blot . These MAbs also react with recombinant phage expressing peptide antigens fused to p8, and are though useful reagents for peptide/protein phage display screening based methods . The latter was shown in an enzyme-linked immunoadsorbent assay (ELISA) and a visual immunoassay where one of the anti-p8 MAbs was used to capture recombinant phages displaying a peptide characteristic of the Hepatitis B virus surface antigen or a Dengue virus-related peptide antigen. Virus Genes, 2000, 20(1), 87 - 97 Expression of four genes of bacteriophage MB78 from contiguous open reading frames: the genomic organization as deduced by sequence analysis; Sharma R et al.; Four proteins of bacteriophage MB78 having apparent molecular weights as 35, 14, 21 and 16 kDa are expressed from 3.9 kb SalI-HindIII fragment located almost in the middle of the phage genome . Analysis of the sequence supported by some experimental evidences suggest that these four proteins are expressed from polycistronic message without any intercistronic gap . Stop and start codons of consecutive ORFs overlap and rare initiation codons are used . Computer analysis of the sequence suggests the presence of two more open reading frames within the ORFs of 35 and 16 kDa proteins but in the opposite orientation, i.e . in the complementary strand. J Mol Biol, 2000 Apr 14, 297(5), 1195 - 202 Structure of the coat protein-binding domain of the scaffolding protein from a double-stranded DNA virus; Sun Y et al.; Scaffolding proteins are required for high fidelity assembly of most high T number dsDNA viruses such as the large bacteriophages, and the herpesvirus family . They function by transiently binding and positioning the coat protein subunits during capsid assembly . In both bacteriophage P22 and the herpesviruses the extreme scaffold C terminus is highly charged, is predicted to be an amphipathic alpha-helix, and is sufficient to bind the coat protein, suggesting a common mode of action . NMR studies show that the coat protein-binding domain of P22 scaffolding protein exhibits a helix-loop-helix motif stabilized by a hydrophobic core . One face of the motif is characterized by a high density of positive charges that could interact with the coat protein through electrostatic interactions . Results from previous studies with a truncation fragment and the observed salt sensitivity of the assembly process are explained by the NMR structure . J Mol Biol, 2000 Apr 14, 297(5), 1063 - 74 Endoribonuclease RegB from bacteriophage T4 is necessary for the degradation of early but not middle or late mRNAs; Sanson B et al.; The RegB endoribonuclease from bacteriophage T4 cleaves early mRNAs specifically in the middle of the sequence GGAG . We show here that RegB is required for the degradation of bulk T4 early mRNA . In the absence of RegB, the chemical half-life of early transcripts is increased nearly fourfold, whereas their functional half-life is increased twofold . RegB also regulates the translation of several prereplicative genes . The synthesis of several early proteins is down-regulated, probably as a consequence of RegB cleavages in the Shine-Dalgarno sequence of these genes . The synthesis of several other proteins is up-regulated, suggesting that processing by RegB might improve translation by changing the conformation of a transcript . In contrast, RegB does not affect the average half-life of middle and late mRNA . An analysis of the susceptibility to RegB of many GGAG motifs carried by these mRNA species showed that most middle and all late GGAG-carrying mRNAs escape RegB processing in spite of the fact that the enzyme is acting at least until ten minutes post-infection . The sensitivity or resistance to RegB observed during phage infection could be reproduced in uninfected Escherichia coli cells and in vitro . This shows that the GGAG-carrying RNAs that are uncut during T4 infection are not substrates, whatever the period of the T4 cycle when the transcripts are made . Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 4297 - 302 Genetic evidence that the bacteriophage phi X174 lysis protein inhibits cell wall synthesis; Bernhardt TG et al.; Protein E, a 91-residue membrane protein of phiX174, causes lysis of the host in a growth-dependent manner reminiscent of cell wall antibiotics, suggesting E acts by inhibiting peptidoglycan synthesis . In a search for the cellular target of E, we previously have isolated recessive mutations in the host gene slyD (sensitivity to lysis) that block the lytic effects of E . The role of slyD, which encodes a FK506 binding protein-type peptidyl-prolyl cis-trans isomerase, is not fully understood . However, E mutants referred to as Epos (plates on slyD) lack a slyD requirement, indicating that slyD is not crucial for lysis . To identify the gene encoding the cellular target, we selected for survivors of Epos . In this study, we describe the isolation of dominant mutations in the essential host gene mraY that result in a general lysis-defective phenotype . mraY encodes translocase I, which catalyzes the formation of the first lipid-linked intermediate in cell wall biosynthesis . The isolation of these lysis-defective mutants supports a model in which translocase I is the cellular target of E and that inhibition of cell wall synthesis is the mechanism of lysis. Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 3896 - 900 Leading and lagging strand DNA synthesis in vitro by a reconstituted herpes simplex virus type 1 replisome; Falkenberg M et al.; The synthesis of double-stranded DNA by a rolling circle mechanism was reconstituted in vitro with a replisome consisting of the DNA polymerase-UL42 complex and the heterotrimeric helicase-primase encoded by herpes simplex virus type 1 . Okazaki fragments 3 kilobases in length and leading strands that may exceed 10 kilobases are produced . Lagging strand synthesis is stimulated by ribonucleoside triphosphates . DNA replication appears to be processive because it resists competition with an excess of (dT)(150)/(dA)(20) . The single-strand DNA binding protein ICP8 is not required, and high concentrations of ICP8 can, in fact, inhibit lagging strand synthesis . The inhibition can, however, be overcome by the addition of an excess of the UL8 component of the helicase-primase . Rolling circle replication by the herpesvirus and bacteriophage T7 replisomes appears to proceed by a similar mechanism. Eur J Biochem, 2000 Apr, 267(8), 2397 - 408 Antitermination in bacteriophage lambda . The structure of the N36 peptide-boxB RNA complex; Scharpf M et al.; The solution structure of a 15-mer nutRboxB RNA hairpin complexed with the 36-mer N-terminal peptide of the N protein (N36) from bacteriophage lambda was determined by 2D and 3D homonuclear and heteronuclear magnetic resonance spectroscopy . These 36 amino acids include the arginine-rich motif of the N protein involved in transcriptional antitermination of phage lambda . Upon complex formation with boxB RNA, the synthetic N36 peptide binds tightly to the major groove of the boxB hairpin through hydrophobic and electrostatic interactions forming a bent alpha helix . Four nucleotides of the GAAAA pentaloop of the boxB RNA adopt a GNRA-like tetraloop fold in the complex . The formation of a GAAA tetraloop involves a loop-closing sheared base pair (G6-A10), base stacking of three adenines (A7, A8, and A10), and extrusion of one nucleotide (A9) from the loop, as observed previously for the complex of N(1-22) peptide and the nutLboxB RNA {Legault, P., Li, J., Mogridge, J., Kay, L.E . & Greenblatt, J . (1998) Cell 93, 289-299} . Stacking of the bases is extended by the indole-ring of Trp18 which also forms hydrophobic contacts to the side-chains of Leu24, Leu25, and Val26 . Based on the structure of the complex, three mutant peptides were synthesized and investigated by CD and NMR spectroscopy in order to determine the role of particular residues for complex formation . These studies revealed very distinct amino-acid requirements at positions 3, 4, and 8, while replacement of Trp18 with tyrosine did not result in any gross structural changes. Proc Natl Acad Sci U S A, 2000 Apr 25, 97(9), 4908 - 13 Identification with a recombinant antibody of an inner-ear cytokeratin, a marker for hair-cell differentiation; Cyr JL et al.; Extensive biochemical characterization of cells in the inner ear has been hampered by a lack of tools with which to identify inner-ear proteins . By using a single-chain antibody fragment isolated from a bacteriophage-displayed library, we have identified a cytokeratin that is abundant in nonsensory cells of the frog inner ear . Although the progenitors of hair cells exhibit strong immunoreactivity to this cytokeratin, the signal declines in immature hair cells and vanishes as the cells mature . The correlation between diminished immunoreactivity and hair-cell differentiation indicates that the cytokeratin is down-regulated during the transition from a nonsensory to a sensory cell and suggests that the marker is an early index of hair-cell differentiation. Biochemistry, 2000 Apr 18, 39(15), 4375 - 82 Nucleoside analogue substitutions in the trinucleotide DNA template recognition sequence 3'-(CTG)-5' and their effects on the activity of bacteriophage T7 primase; Searls T et al.; Bacteriophage T7 primase catalyzes the synthesis of the oligoribonucleotides pppACC(C/A) and pppACAC from the single-stranded DNA template sites 3'-d{CTGG(G/T)}-5' and 3'-(CTGTG)-5', respectively . The 3'-terminal deoxycytidine residue is conserved but noncoding . A series of nucleoside analogues have been prepared and incorporated into the conserved 3'-d(CTG)-5' site, and the effects of these analogue templates on T7 primase activity have been examined . The nucleosides employed include a novel pyrimidine derivative, 2-amino-5-(beta-2-deoxy-D-erythro-pentofuranosyl)pyridine (d2APy), whose synthesis is described . Template sites containing d2APy in place of the cryptic dC support oligoribonucleotide synthesis whereas those containing 3-deaza-2'-deoxycytidine (dc(3)C) and 5-methyl-6-oxo-2'-deoxycytidine (dm(5ox)C) substitutions do not, suggesting that the N3 nitrogen of cytidine is used for a critical interaction by the enzyme . Recognition sites containing 4-amino-1-(beta-2-deoxy-D-erythro-pentofuranosyl)-5-methyl-2,6{1H, 3H}-pyrimidione (dm(3)2P) or 2'-deoxyuridine (dU) substitutions for dT support oligoribonucleotide synthesis whereas those containing 5-methyl-4-pyrimidinone 2'-deoxyriboside (d(2H)T) substitutions do not, suggesting the importance of Watson-Crick interactions at this template residue . Template sites containing 7-deaza-2'-deoxyguanosine (dc(7)G) or 2'-deoxyinosine (dI) in place of dG support oligoribonucleotide synthesis . The reduced extent to which dc(7)G is successful within the template suggests a primase-DNA interaction . Inhibition studies suggest that the primase enzyme binds "null" substrates but cannot initiate RNA synthesis. Acta Microbiol Pol, 1999, 48(3), 233 - 42 Evidence of interactions between Gp27 and Gp28 constituents of the central part of bacteriophage T4 baseplate; Nieradko J et al.; The central part of the bacteriophage T4 baseplate consists of several proteins . However, for a number of the constituents the manner of incorporation are not convincingly established . Recently, we have presented evidence that gp28 is the structural component of the central part of the baseplate, which possesses a hydrophobic region and is membrane bound {Nieradko et al., 1998} . By utilizing extracts prepared from Escherichia coli cells that overexpressed genes 27 and 28 of phage T4, we proved that gp28 forms a complex with an another baseplate structural components: gp27 . This complex was located in the membrane fraction . Its affinity to the inner membrane indicates that the identified complex may function as an initiator of the central hub assembly . It was subsequently established that these products interact in the ratio 1:1 . We have also demonstrated that the particular components of the complex can be separated by action of SDS and to a lesser extent by Triton X-100. Nucleic Acids Res . 2000 May 1;28(9):E41. Retro-recombination screening of a mouse embryonic stem cell genomic library; Woltjen K et al.; Targeted gene disruption is an important tool in molecular medicine, allowing for the generation of animal models of human disease . Conventional methods of targeting vector (TV) construction are difficult and represent a rate limiting step in any targeting experiment . We previously demonstrated that bacteriophage are capable of acting as TVs directly, obviating the requirement for 'rolling out' plasmids from primary phage clones and thus eliminating an additional, time consuming step . We have also developed methods which facilitate the construction of TVs using recombination . In this approach, modification cassettes and point mutations are shuttled to specific sites in phage TVs using phage-plasmid recombination . Here, we report a further improvement in TV generation using a recombination screening-based approach deemed 'retro-recombination screening' (RRS) . We demonstrate that phage vectors containing specific genomic clones can be genetically isolated from a lambdaTK embryonic stem cell genomic library using a cycle of integrative recombination and condensation . By introducing the gam gene of bacteriophage lambda into the probe plasmid it is possible to select for positive clones which have excised the plasmid, thus returning to their native conformation following purification from the library . Rapid clone isolation using the RRS protocol provides another method by which the time required for TV construction may be further reduced. Trends Biochem Sci, 2000 Apr, 25(4), 165 - 73 Recombination-dependent DNA replication in phage T4; Kreuzer KN; Studies in the 1960s implied that bacteriophage T4 tightly couples DNA replication to genetic recombination . This contradicted the prevailing wisdom of the time, which staunchly supported recombination as a simple cut-and-paste process . More-recent investigations have shown how recombination triggers DNA synthesis and why the coupling of these two processes is important . Results from T4 were instrumental in our understanding of many important replication and recombination proteins, including the newly recognized replication/recombination mediator proteins . Recombination-dependent DNA replication is crucial to the T4 life cycle as it is the major mode of DNA replication and is also central to the repair of DNA breaks and other damage. Nat Biotechnol, 2000 Apr, 18(4), 442 - 5 Intrachromosomal recombination between attP regions as a tool to remove selectable marker genes from tobacco transgenes; Zubko E et al.; Recombinant genes conferring resistance to antibiotics or herbicides are widely used as selectable markers in plant transformation . Once transgenic material has been selected, the marker gene is dispensable . We report a novel strategy to remove undesirable parts of a transgene after integration into the tobacco genome . This approach is based on the transfer of a vector containing a NPTII gene flanked by two 352 bp attachment P (attP) regions of bacteriophage lambda, and the identification of somatic tissue with deletion events following intrachromosomal recombination between the attP regions . This system was used to delete a 5.9 kb region from a recombinant vector that had been inserted into two different genomic regions . As the attP system does not require the expression of helper proteins to induce deletion events, or a genetic segregation step to remove recombinase genes, it should provide a useful tool to remove undesirable transgene regions, especially in vegetatively propagated species. J Biol Chem, 2000 Jun 30, 275(26), 19449 - 55 Cysteines involved in radical generation and catalysis of class III anaerobic ribonucleotide reductase . A protein engineering study of bacteriophage T4 NrdD; Andersson J et al.; Class III ribonucleotide reductase (RNR) is an anaerobic glycyl radical enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides . We have investigated the importance in the reaction mechanism of nine conserved cysteine residues in class III RNR from bacteriophage T4 . By using site-directed mutagenesis, we show that two of the cysteines, Cys-79 and Cys-290, are directly involved in the reaction mechanism . Based on the positioning of these two residues in the active site region of the known three-dimensional structure of the phage T4 enzyme, and their structural equivalence to two cysteine residues in the active site region of the aerobic class I RNR, we suggest that Cys-290 participates in the reaction mechanism by forming a transient thiyl radical and that Cys-79 participates in the actual reduction of the substrate . Our results provide strong experimental evidence for a similar radical-based reaction mechanism in all classes of RNR but also identify important differences between class III RNR and the other classes of RNR as regards the reduction per se . We also identify a cluster of four cysteines (Cys-543, Cys-546, Cys-561, and Cys-564) in the C-terminal part of the class III enzyme, which are essential for formation of the glycyl radical . These cysteines make up a CX(2)C-CX(2)C motif in the vicinity of the stable radical at Gly-580 . We propose that the four cysteines are involved in radical transfer between Gly-580 and the cofactor S-adenosylmethionine of the activating NrdG enzyme needed for glycyl radical generation. J Biol Chem, 2000 May 26, 275(21), 16155 - 9 Peptide mimics of the M13 coat protein transmembrane segment . Retention of helix-helix interaction motifs; Wang C et al.; Sequence-specific noncovalent helix-helix interactions between transmembrane (TM) segments in proteins are investigated by incorporating selected TM sequences into synthetic peptides using the construct CKKK-TM-KKK . The peptides are of suitable hydrophobicity for spontaneous membrane insertion, whereas formation of an N-terminal S-S bond can bring pairs of TM helices into proximity and promote their parallel orientation . Using the propensity of the protein to undergo thermally induced alpha-helix --> beta-sheet transitions as a parameter for helix stability, we compared the wild type and mutant (V29A and V31A) bacteriophage M13 coat proteins with their corresponding TM peptide constructs (M13 residues 24-42) . Our results demonstrated that the relevant helix-helix tertiary contacts found in the intact proteins persist in the peptide mimics . Molecular dynamics simulations support the tight "two in-two out" dimerization motif for V31A consistent with mutagenesis data . The overall results reinforce the notion of TM segments as autonomous folding domains and suggest that the generic peptide construct provides a viable reductionist system for membrane protein structural and computational analysis. Biochemistry, 2000 Apr 11, 39(14), 4136 - 44 Factors determining mutagenic potential for individual cis and trans opened benzo{c}phenanthrene diol epoxide-deoxyadenosine adducts; Ponten I et al.; Four adducts that would result from trans opening at C-1 of benzo{c}phenanthrene 3,4-diol 1,2-epoxide (B{c}PhDE) isomers (i.e., DE-1 enantiomers, where the epoxide oxygen and benzylic hydroxyl group are cis, and DE-2 enantiomers, where they are trans) by the N(6)-amino group of dAdo, together with the two cis opened N(6)-dAdo adducts of B{c}PhDE-1, were incorporated into two oligonucleotides at the underlined site in 5'-TTTAGAGTCTGCTCCC {context I(A)} and 5'-CAGATTTAGAGTCTGC {context II(A)} . After ligation of these, and the corresponding unsubstituted oligonucleotides, into single-stranded M13mp7L2 bacteriophage and transfection into SOS-induced Escherichia coli SMH77, base substitution mutations induced by the different B{c}PhDE-dAdo adducts were determined . These findings were compared with data {Ponten et al . (1999) Biochemistry 38, 1144-1152} for cis opened B{c}PhDE-2-dAdo adducts in the same sequence contexts . In most cases, adducts with S absolute configuration at the site of attachment of the nucleoside to the hydrocarbon had higher mutation frequencies (1.9-56.5%) than the corresponding adducts with R configuration (0.05-5.6%) . For adducts derived from B{c}PhDE-1, the predominant mutations were A-->T transversions in context I(A) and A-->G transitions for most of these adducts in context II(A) . For adducts derived from B{c}PhDE-2, A-->T base substitutions predominated for most of the trans adducts, but A-->G mutations were favored by the cis adduct with S configuration in either context . Thus, the structural feature that most dramatically affected mutagenic activity was the configuration of the carbon at the attachment point, with S configuration mostly being associated with greater mutagenicity than the R configuration . However, other structural variations and sequence context also affected mutagenicity, indicating that a combination of structure and context effects define mutagenicity. Microbiology, 2000 Mar, 146 ( Pt 3), 599 - 609 A bacteriophage-like particle from Bartonella bacilliformis; Barbian KD et al.; Bartonella bacilliformis and Bartonella henselae, the respective agents of Oroya fever and cat-scratch disease in humans, are known to produce bacteriophage-like particles (BLPs) that package 14 kbp segments of the host chromosome . Data from this study suggest that other Bartonella species including Bartonella quintana, Bartonella doshiae and Bartonella grahamii also contain similar BLPs, as evidenced by the presence of a 14 kbp extrachromosomal DNA element in their genomes, whereas Bartonella elizabethae and Bartonella clarridgeiae do not . A purification scheme utilizing chloroform, DNase I and centrifugation was devised to isolate BLPs from B . bacilliformis . Intact BLPs were observed by transmission electron microscopy and were round to icosahedral in shape and approximately 80 nm in diameter . RFLP and Southern blot analysis of BLP DNA from B . bacilliformis suggest that packaging, while non-selective, is less than the near-random packaging previously reported for the B . henselae phage . Data also suggest that the linear, double-stranded BLP DNA molecules have blunt ends with noncovalently closed termini . Packaging of the BLP DNA molecules into a protein coat appears to be closely related to nucleic acid synthesis, as unpackaged phage DNA is not detectable within the host cell . SDS-PAGE analysis of purified BLPs from B . bacilliformis showed three major proteins with apparent molecular masses of 32, 34 and 36 kDa; values that closely correspond to proteins found in B . henselae BLPs . Western blot analysis performed with patient convalescent serum showed that BLP proteins are slightly immunogenic in humans . To determine if BLPs contribute to horizontal gene transfer, mutants of B . bacilliformis were generated by allelic exchange with an internal fragment of the 16S-23S rDNA intergenic spacer region and a suicide vector construct, termed pKB1 . BLPs from one of the resultant strains were able to package the mutagenized region containing the kanamycin-resistance cassette; however, numerous approaches and attempts at intraspecies transduction using these BLPs were unsuccessful. Biol Pharm Bull, 1999 Dec, 22(12), 1372 - 5 Escherichia coli O157 strains which caused Japanese outbreaks have residues of bacteriophage sequences; Miyahara M et al.; Twelve strains of Escherichia coli O157 which caused outbreaks in Japan were used as DNA sources . The sequences of the gene encoding the Shiga toxin 2 in all 12 strains were almost identical and the sequences downstream of this gene were similar to that of bacteriophage 933W. Curr Biol, 2000 Mar 23, 10(6), 337 - 40 The human Rad52 protein exists as a heptameric ring; Stasiak AZ et al.; The RAD52 epistasis group was identified in yeast as a group of genes required to repair DNA damaged by ionizing radiation {1} . Genetic evidence indicates that Rad52 functions in Rad51-dependent and Rad51-independent recombination pathways {2} {3} {4} . Consistent with this, purified yeast and human Rad52 proteins have been shown to promote single-strand DNA annealing {5} {6} {7} and to stimulate Rad51-mediated homologous pairing {8} {9} {10} {11} . Electron microscopic examinations of the yeast {12} and human {13} Rad52 proteins have revealed their assembly into ring-like structures in vitro . Using both conventional transmission electron microscopy and scanning transmission electron microscopy (STEM), we found that the human Rad52 protein forms heptameric rings . A three-dimensional (3D) reconstruction revealed that the heptamer has a large central channel . Like the hexameric helicases such as Escherichia coli DnaB {14} {15}, bacteriophage T7 gp4b {16} {17}, simian virus 40 (SV40) large T antigen {18} and papilloma virus E1 {19}, the Rad52 rings show a distinctly chiral arrangement of subunits . Thus, the structures formed by the hexameric helicases may be a more general property of other proteins involved in DNA metabolism, including those, such as Rad52, that do not bind and hydrolyze ATP. J Bacteriol, 2000 Apr, 182(8), 2336 - 40 Genetic requirements of phage lambda red-mediated gene replacement in Escherichia coli K-12; Poteete AR et al.; Recombination between short linear double-stranded DNA molecules and Escherichia coli chromosomes bearing the red genes of bacteriophage lambda in place of recBCD was tested in strains bearing mutations in genes known to affect recombination in other cellular pathways . The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell; formation of Lac(-) chloramphenicol-resistant bacterial progeny was measured . Recombinant formation was found to be reduced in ruvAB and recQ strains . In this genetic background, mutations in recF, recO, and recR had large effects on both cell viability and on recombination . In these cases, deletion of the sulA gene improved viability and strain stability, without improving recombination ability . Expression of a gene(s) from the nin region of phage lambda partially complemented both the viability and recombination defects of the recF, recO, and recR mutants and the recombination defect of ruvC but not of ruvAB or recQ mutants. Genes Genet Syst, 1999 Oct, 74(5), 227 - 39 Complete nucleotide sequence of the prophage VT2-Sakai carrying the verotoxin 2 genes of the enterohemorrhagic Escherichia coli O157:H7 derived from the Sakai outbreak; Makino K et al.; The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain RIMD 0509952, derived from an outbreak in Sakai city, Japan, in 1996, produces two kinds of verotoxins, VT1 and VT2, encoded by the stx1 and stx2 genes . In the EHEC strains, as well as in other VT-producing E . coli strains, the toxins are encoded by lysogenic bacteriophages . The EHEC O157:H7 strain RIMD 0509952 did not produce plaque-forming phage particles upon inducing treatments . We have determined the complete nucleotide sequence of a prophage, VT2-Sakai, carrying the stx2A and stx2B genes on the chromosome, and presumed the putative functions of the encoded proteins and the cis-acting DNA elements based on sequence homology data . To our surprise, the sequences in the regions of VT2-Sakai corresponding to the early gene regulators and replication proteins, and the DNA sequences recognized by the regulators share very limited homology to those of the VT2-encoding 933W phage carried by the EHEC O157:H7 strain EDL933 reported by Plunkett et al . (J . Bacteriol., p1767-1778, 181, 1999), although the sequences corresponding to the structural components are almost identical . These data suggest that these two phages were derived from a common ancestral phage and that either or both of them underwent multiple genetic rearrangements . An IS629 insertion was found downstream of the stx2B gene and upstream of the lysis gene S, and this might be responsible for the absence of plaque-forming activity in the lysate obtained after inducing treatments. Protein Expr Purif, 2000 Apr, 18(3), 316 - 9 Expression of human cardiac-specific homeobox protein in Escherichia coli; Zhao JH et al.; Human cardiac-specific homeobox protein cDNA (hCsx) was cloned into expression plasmid pET32a and fused with Escherichia coli thioredoxin (Trx) . The Trx-Csx fusion protein was under the control of bacteriophage T7 promoter . When expressed in E . coli BL21(DE3), about half of the recombinant Trx-Csx products existed in the form of insoluble inclusion bodies . When coexpressed with human protein disulfide isomerase, more than 90% of Trx-Csx products accumulated in the soluble form in the cell lysate . The recombinant Csx fusion protein was purified by one-step metal-chelating affinity chromatography . J Biochem (Tokyo), 2000 Mar, 127(3), 367 - 72 Synthesis of a new Cre recombinase gene based on optimal codon usage for mammalian systems; Koresawa Y et al.; The origin of the Cre recombinase gene is bacteriophage P1, and thus the codon usages are different from in mammals . In order to adapt this codon usage for mammals, we synthesized a "mammalian Cre recombinase gene" and examined its expression in Chinese hamster ovarian tumor (CHO) cells . Significant increases in protein production as well as mRNA levels were observed . When the recombination efficiency was compared using CHO cell transfectants having a cDNA containing loxP sites, the "mammalian Cre recombinase gene" recombined the loxP sites much more efficiently than the wild-type Cre recombinase gene. J Mol Biol, 2000 Mar 31, 297(3), 615 - 26 Visualization of the maturation transition in bacteriophage P22 by electron cryomicroscopy; Zhang Z et al.; Large-scale conformational transitions are involved in the life-cycle of many types of virus . The dsDNA phages, herpesviruses, and adenoviruses must undergo a maturation transition in the course of DNA packaging to convert a scaffolding-containing precursor capsid to the DNA-containing mature virion . This conformational transition converts the procapsid, which is smaller, rounder, and displays a distinctive skewing of the hexameric capsomeres, to the mature virion, which is larger and more angular, with regular hexons . We have used electron cryomicroscopy and image reconstruction to obtain 15 A structures of both bacteriophage P22 procapsids and mature phage . The maturation transition from the procapsid to the phage results in several changes in both the conformations of the individual coat protein subunits and the interactions between neighboring subunits . The most extensive conformational transformation among these is the outward movement of the trimer clusters present at all strict and local 3-fold axes on the procapsid inner surface . As the trimer tips are the sites of scaffolding binding, this helps to explain the role of scaffolding protein in regulating assembly and maturation . We also observe DNA within the capsid packed in a manner consistent with the spool model . These structures allow us to suggest how the binding interactions of scaffolding and DNA with the coat shell may act to control the packaging of the DNA into the expanding procapsids . J Virol, 2000 Apr, 74(8), 3871 - 3 Identification of additional coat-scaffolding interactions in a bacteriophage P22 mutant defective in maturation; Thuman-Commike PA et al.; Scaffolding proteins play a critical role in the assembly of certain viruses by directing the formation and maturation of a precursor capsid . Using electron cryomicroscopy difference mapping, we have identified an altered arrangement of a mutant scaffolding within the bacteriophage P22 procapsid . This mutant scaffolding allows us to directly visualize scaffolding density within the P22 procapsid . Based on these observations we propose a model for why the mutant prevents scaffolding release and capsid maturation. J Virol, 2000 Apr, 74(8), 3464 - 9 Molecular characterization of a bacteriophage (Chp2) from Chlamydia psittaci; Liu BL et al.; Comparisons of the proteome of abortifacient Chlamydia psittaci isolates from sheep by two-dimensional gel electrophoresis identified a novel abundant protein with a molecular mass of 61.4 kDa and an isoelectric point of 6.41 . C-terminal sequence analysis of this protein yielded a short peptide sequence that had an identical match to the viral coat protein (VP1) of the avian chlamydiaphage Chp1 . Electron microscope studies revealed the presence of a 25-nm-diameter bacteriophage (Chp2) with no apparent spike structures . Thin sections of chlamydia-infected cells showed that Chp2 particles were located to membranous structures surrounding reticulate bodies (RBs), suggesting that Chp2 is cytopathic for ovine C . psittaci RBs . Chp2 double-stranded circular replicative-form DNA was purified and used as a template for DNA sequence analysis . The Chp2 genome is 4,567 bp and encodes up to eight open reading frames (ORFs); it is similar in overall organization to the Chp1 genome . Seven of the ORFs (1 to 5, 7, and 8) have sequence homologies with Chp1 . However, ORF 6 has a different spatial location and no cognate partner within the Chp1 genome . Chlamydiaphages have three viral structural proteins, VP1, VP2, and VP3, encoded by ORFs 1 to 3, respectively . Amino acid residues in the phiX174 procapsid known to mediate interactions between the viral coat protein and internal scaffolding proteins are conserved in the Chp2 VP1 and VP3 proteins . We suggest that VP3 performs a scaffolding-like function but has evolved into a structural protein. Biochemistry, 2000 Mar 28, 39(12), 3377 - 83 DNA sequence dependent and independent conformational changes in multipartite operator recognition by lambda-repressor; Deb S et al.; Binding of regulatory proteins to multipartite DNA binding sites often occurs with protein-protein interaction, resulting in cooperative binding . The operators of bacteriophage lambda have several pairs of repressor binding sites (O(R)1-O(R)2, O(R)2-O(R)3, O(L)1-O(L)2, and O(L)2-O(L)3) separated by a variable number of base pairs, and thus, bacteriophage lambda is a model system for studying multipartite operator recognition by DNA-binding proteins . Near-UV circular dichroism spectra show that the DNA is distorted in O(R)1-O(R)2 and O(L)2-O(L)3 but much less so in O(R)2-O(R)3 . Upon titration of lambda-repressor with single-operator sites O(R)1, O(R)2, and O(R)3, it was observed that the tryptophan fluorescence quenches to different degrees, suggesting different conformations of the protein in the three DNA-protein complexes . Acrylamide quenching of tryptophan fluorescence of lambda-repressor bound to these single operators also shows different Stern-Volmer constants, supporting the above conclusions . Titration of lambda-repressor with oligonucleotides containing pairs of operator sites also causes different degrees of fluorescence quenching . In particular, fluorescence quenching induced by O(R)1-O(R)2 binding is less than the quenching induced by either of the single operators alone, suggesting additional conformational changes upon establishment of protein-protein contact . Stern-Volmer constants obtained from acrylamide quenching of tryptophan fluorescence of lambda-repressor bound cooperatively to pairs of operator sites are different from those of the single-operator-site-bound repressors . For example, O(R)2-O(R)3-bound repressor has significantly higher acrylamide quenchable components than either of the O(R)2- or O(R)3-bound proteins, again suggesting additional conformational changes upon establishment of protein-protein contact . We conclude that the strategy of recognition of multipartite operator by lambda-repressor is complex and varied, involving conformational changes in both DNA and protein that are determined by the separation of the binding sites as well as the nucleic acid sequence. J Biomol NMR, 2000 Feb, 16(2), 165 - 9 Resolution of the 1H-1H NOE spectrum of RNA into three dimensions using 15N-1H two-bond couplings; Hoffman DW; The feasibility of using two-bond 15N-1H couplings to resolve the 1H-1H nuclear Overhauser effect spectrum of RNA into a third dimension was investigated, using the 36-nucleotide gene 32 messenger RNA pseudoknot of bacteriophage T2 as an example . The two-bond 15N-1H couplings present in adenosine and guanosine were found to be suitable for generating a three-dimensional 1H-1H-15N NOESY-HSQC spectrum with reasonably good sensitivity, as well as favorable chemical shift dispersion in the nitrogen dimension . The described NMR experiment provides a tool that can be used to complement other heteronuclear methods in the analysis of RNA structure. Antimicrob Agents Chemother, 2000 Apr, 44(4), 1097 - 9 Toward antiviral strategies that resist viral escape; Endy D et al.; We studied the effect on viral growth of drugs targeting different virus functions using a computer simulation for the intracellular growth of bacteriophage T7 . We found that drugs targeting components of negative-feedback loops gain effectiveness against mutant viruses that attenuate the drug-target interaction . The greater inhibition of such mutants than of the wild type suggests a drug design strategy that would hinder the development of drug resistance. Biochim Biophys Acta, 2000 Mar 16, 1478(1), 113 - 24 Mechanisms for the enhanced thermal stability of a mutant of transcription factor 1 as explained by (1)H, (15)N and (13)C NMR chemical shifts and secondary structure analysis; Vu HM et al.; A variant of the bacteriophage SPO1-encoded transcription factor 1 (TF1) with two site-specific mutations (E15G and T32I) was shown to be more thermally stable and bind DNA more tightly compared to the wild-type protein . In order to understand the biochemical mechanisms underlying these properties, we are engaged in determining the solution structures of this mutant alone and in complex with DNA using nuclear magnetic resonance (NMR) spectroscopy . The first phase of this project is reported here, as we have completed most of the backbone and sidechain sequential NMR assignments of the mutant protein, TF1-G15/I32 . Insights derived from the (1)H, (15)N and (13)C chemical shifts and from the secondary structure analysis provide us with an explanation for the noted increase in thermal stability of TF1-G15/I32 . Compared to the structure of the wild-type protein, the beta-sheet and the C-terminal helix remain largely unaffected whereas the mutations cause great changes in the first two helices and their enclosed loop . Specifically, we have found that the second helix is extended by one residue at its N-terminus and rotated in a way that allows Ala-37 to interact with Tyr-94 of the C-terminal helix . The loop has been found to become more rigid as a result of hydrophobic interactions between the flanking second and first helices and also between the second helix and the loop itself . Furthermore, the T32I mutation allows tighter packing between the second helix and the beta-sheet . Collectively, these changes contribute to a more tightly associated dimer and hence, to a greater thermal stability. J Virol Methods, 2000 Mar, 85(1-2), 1 - 10 Induction of recombinant gene expression in stably transfected cell lines using attenuated vaccinia virus MVA expressing T7 RNA polymerase with a nuclear localisation signal; Huemer HP et al.; There are major drawbacks using vaccinia virus (VV) expressing T7 polymerase for eukaryotic expression . VV is infectious for humans and due to cytosolic replication of Poxviridae, transient transfection of T7 promoter containing plasmids is necessary, which varies in efficiency . Several improvements have been introduced to this system to enhance expression of herpes viral glycoproteins . Stably transfected cell lines were generated with an EBV-based episomal plasmid vector which can be pushed to increasing copy numbers under selective pressure . The avirulent vaccine MVA strain was adopted to generate a safe laboratory vector for inserting the bacteriophage T7 RNA polymerase gene with (+) or without (-) a nuclear localisation signal . Constructs were designed for recombination into the VV haemagglutinin gene as recombinants could not be isolated successfully when inserting into the MVA thymidine kinase locus . Both T7 MVA recombinants induced foreign protein expression in transiently transfected cells but only the T7-/+ MVA induced target protein expression in stably transfected cells . The level of protein expression by this induction mechanism was comparable to, or superior to levels obtained with VV recombinants expressing the gene under control of the VV 11 k IE promoter . The results suggests that the T7+ MVA virus can be used to induce gene expression in stable recombinant cell lines and offers an attractive and safe alternative to other inducible eucaryotic expression systems. Biochemistry, 2000 Mar 21, 39(11), 3076 - 90 Tracking sliding clamp opening and closing during bacteriophage T4 DNA polymerase holoenzyme assembly; Alley SC et al.; The bacteriophage T4 DNA polymerase holoenzyme, consisting of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62), is loaded onto DNA in an ATP-dependent, multistep reaction . The trimeric, ring-shaped gp45 is loaded onto DNA such that the DNA passes through the center of the ring . gp43 binds to this complex, thereby forming a topological link with the DNA and increasing its processivity . Using stopped-flow fluorescence-resonance energy transfer, we have investigated opening and closing of the gp45 ring during the holoenzyme assembly process . Two amino acids that lie on opposite sides of the gp45 subunit interface, W91 and V162C labeled with coumarin, were used as the fluorescence donor and acceptor, respectively . Free in solution, gp45 has two closed subunit interfaces with W91 to V162-coumarin distances of 19 A and one open subunit interface with a W91 to V162C-coumarin distance of 40 A . Making the assumption that the distance across the two closed subunit interfaces is unchanged during the holoenzyme assembly process, we have found that the distance across the open subunit interface is first increased to greater than 45 A and is then decreased to 30 A during a 10-step assembly mechanism . The gp45 ring is not completely closed in the holoenzyme complex, consistent with previous evidence suggesting that the C-terminus of gp43 is inserted into the gp45 subunit interface . Unexpectedly, ATP-hydrolysis events are coupled to only a fraction of the total distance change, with conformational changes linked to binding DNA and gp43 coupled to the majority of the total distance change . Using the nonhydrolyzable ATP analogue ATP-gamma-S results in formation of a nonproductive gp45 x gp44/62 complex; however, adding an excess of ATP to this nonproductive complex results in rapid ATP/ATP-gamma-S exchange to yield a productive gp45 x gp44/62 complex within seconds. J Bacteriol, 2000 Apr, 182(7), 1978 - 86 Characterization of the uup locus and its role in transposon excisions and tandem repeat deletions in Escherichia coli; Reddy M et al.; Null mutations in the Escherichia coli uup locus (at 21.8 min) serve to increase the frequency of RecA-independent precise excision of transposable elements such as Tn10 and to reduce the plaque size of bacteriophage Mu (Uup(-) phenotype) . By the combined approaches of physical mapping of the mutations, complementation analyses, and protein overexpression from cloned gene fragments, we have demonstrated in this study that the Uup(-) phenotype is the consequence of the absence of expression of the downstream gene (uup) of a two-gene operon, caused either directly by insertions in uup or indirectly by the polar effect of insertions in the upstream gene (ycbY) . The promoter for uup was mapped upstream of ycbY by primer extension analysis on cellular RNA, and assays of reporter gene expression indicated that it is a moderately active, constitutive promoter . The uup mutations were also shown to increase, in a RecA-independent manner, the frequencies of nearly precise excision of Tn10 derivatives and of the deletion of one copy of a chromosomal tandem repeat, suggesting the existence of a shared step or intermediate in the pathways of these latter events and that of precise excision . Finally, we found that mutations that increase the frequency of precise excision of Tn10 are divisible into two categories, depending upon whether they did (uup, ssb, polA, and topA) or did not (mutHLS, dam, and uvrD) also increase precise excision frequency of the mini-Tn10 derivatives . It is suggested that the differential response of mini-Tn10 and Tn10 to the second category of mutations is related to the presence, respectively, of perfect and of imperfect terminal inverted repeats in them. Mutat Res, 2000 Feb 16, 459(1), 43 - 53 AP lyases and dRPases: commonality of mechanism; Piersen CE et al.; Enzymes that release 5'-deoxyribose-5-phosphate (dRP) residues from preincised apurinic/apyrimidinic (AP) DNA have been collectively termed DNA deoxyribophosphodiesterases (dRPases), but they fall into two distinct categories: the hydrolytic dRPases and AP lyases . In order to resolve a number of conflicting reports in the dRPase literature, we examined two putative hydrolytic dRPases (Escherichia coli exonuclease I (exo I) and RecJ) and four AP lyases (E . coli 2, 6-dihydroxy-5N-formamidopyrimidine (Fapy) DNA glycosylase (Fpg) and endonuclease III (endo III), bacteriophage T4 endonuclease V (endo V), and rat polymerase beta (beta-pol)) for their abilities to (i) excise dRP from preincised AP DNA and (ii) incise AP DNA . Although exo I and RecJ exhibited robust 3' to 5' and 5' to 3' exonucleolytic activities, respectively, on appropriate substrates, they failed to demonstrate detectable dRPase activity . All four AP lyases possessed both dRPase and traditional AP lyase activities, albeit to varying degrees . Moreover, as best illustrated with Fpg, AP lyase enzymes could be trapped on both preincised and unincised AP DNA using NaBH(4) as the reducing agent . These results further support the assertion that the catalytic mechanism of the AP lyases, the beta-elimination reaction, does proceed through an imine enzyme-DNA intermediate and that the active site residues responsible for dRP release must contain primary amines . Further, these data indicate a biological significance for the beta-elimination reaction of DNA glycosylase/AP lyases in that they, in concert with hydrolytic AP endonucleases, can create appropriate gapped substrates for short patch base excision repair (BER) synthesis to occur efficiently. Mol Microbiol, 2000 Mar, 35(5), 1180 - 91 The bacteriophage T4 anti-sigma factor AsiA is not necessary for the inhibition of early promoters in vivo; Pene C et al.; Bacteriophage T4 early promoters are utilized immediately after infection and are abruptly turned off 2-3 min later (at 30 degrees C) when the middle promoters are activated . The viral early protein AsiA has been suspected to bring about this transcriptional switch: not only does it activate transcription at middle promoters in vivo and in vitro but it also shows potent anti-sigma70 activity in vitro, suggesting that it is responsible for the shut-off of early transcription . We show here that after infection with a phage deleted for the asiA gene the inhibition of early transcription occurs to the same extent and with the same kinetics as in a wild-type infection . Thus, another AsiA-independent circuit efficiently turns off early transcription . The association of a mutation in asiA with a mutation in mod, rpbA, motA or motB has no effect on the inhibition of early promoters, showing that none of these phage-encoded transcriptional regulators is necessary for AsiA-independent shut-off . It is not known whether AsiA is able to inhibit early promoters in vivo, but host transcription is strongly inhibited in vivo upon induction of AsiA from a multicopy plasmid. Proteins, 2000 Mar 1, 38(4), 393 - 406 Transcriptional repressor CopR: structure model-based localization of the deoxyribonucleic acid binding motif; Steinmetzer K et al.; The plasmid pIP501 encoded transcriptional repressor CopR is one of the two regulators of plasmid copy number . CopR binds as a dimer to a nearly palindromic operator with the consensus sequence 5'-CGTG . Intermediate sequence searches revealed a significant structural relationship between CopR and the bacteriophage P22 c2 and the 434 c1 repressors . In this report we describe the experimental verification of a CopR homology model, which is based on a fairly low-sequence identity of 13.8% to P22 c2 repressor . A model for the complex of CopR with the deoxyribonucleic acid (DNA) target was built on the basis of experimental footprinting data, the above-mentioned CopR homology model, and the crystal structure of the 434 c1 repressor-DNA complex . Site-directed mutagenesis was used to test the function of amino acids involved in sequence and nonsequence-specific DNA recognition and amino acids important for correct protein folding . CD measurements were performed to detect structural changes caused by the mutations . Exchanges of residues responsible for sequence-specific DNA recognition reduced binding to a nonspecific level . Mutations of amino acids involved in nonspecific DNA binding lead to decreased binding affinity while maintaining selectivity . Substitution of amino acids necessary for proper folding caused dramatic structural changes . The experimental data support the model of CopR as a helix-turn-helix protein belonging to the lambda repressor superfamily. Curr Microbiol, 2000 May, 40(5), 341 - 3 A new Mesorhizobium loti HAMBI 1129 phage isolated from Polish soil; Turska-Szewczuk A et al.; Phage A1 isolated from the rhizosphere of Lotus corniculatus was studied . It had a very narrow host range, as it was active only against Mesorhizobium loti HAMBI 1129 . Phage A1 was classified as belonging to C Bradley's group bacteriophages . The latent period of A1 was 120-130 min and a burst size 13-17 particles per cell . The nature of the phage receptor was examined . Lipopolysaccharide from the phage-sensitive strain inactivated phage A1 in contrast to LPS from the phage-resistant bacteria . Purified LPS obtained from M . loti HAMBI 1129 had a high receptor activity with PhI(50) value of 0 . 025 microgram/ml. J Mol Biol, 2000 Mar 17, 297(1), 99 - 117 Pulse-chase analysis of the in vivo assembly of the bacteriophage T4 tail; Ferguson PL et al.; The in vivo assembly pathway of the complex tail of bacteriophage T4 virus was determined using pulse-chase analysis as a non-invasive alternative to the in vitro experiments previously used to map assembly . Bacteriophage T4 mutants defective in head assembly were used to infect cultures of Escherichia coli in order to study tail assembly in isolation . Beginning with the onset of late protein synthesis, the cultures were labeled continuously with {(3)H}leucine to normalize against subsequent sample losses . After completed tails had begun to accumulate at a constant rate, the cultures were pulsed with {(35)S}methionine, and then chased . Completed tails were purified at one minute intervals for the next 30 minutes and their proteins separated electrophoretically and counted by liquid scintillation . Total (35)S incorporation into each protein rose and then leveled off as the chase of unlabeled methionine flushed the label through the pools of soluble proteins and assembly intermediates and into completed tails . The inflection point in the sigmoidal (35)S-incorporation curve of each protein marks the maximal uptake of (35)S within that pool just before the effect of the chase becomes apparent and the curve begins to level off . The length of the delay in the apparent chase time reflects the position of that protein in the pathway . The closer the assembly point to the end of the pathway, the sooner the chase appears, revealing the relative order of assembly . As predicted, tail completion proteins such as gp18 (tail sheath) and 19 (tail tube) show the earliest inflection, while those earlier in the pathway take longer to chase . Of the 17 tail proteins analyzed, 14 are in agreement with the established in vitro pathway . The other three, gp15, gp10 and gp53, have helped us to develop a model that offers a plausible explanation for their altered chase times . Nat Struct Biol, 2000 Mar, 7(3), 230 - 7 Novel fold and capsid-binding properties of the lambda-phage display platform protein gpD; Yang F et al.; The crystal structure of gpD, the capsid-stabilizing protein of bacteriophage lambda, was solved at 1.1 A resolution . Data were obtained from twinned crystals in space group P21 and refined with anisotropic temperature factors to an R-factor of 0.098 (Rfree = 0 . 132) . GpD (109 residues) has a novel fold with an unusually low content of regular secondary structure . Noncrystallographic trimers with substantial intersubunit interfaces were observed . The C-termini are well ordered and located on one side of the trimer, relatively far from its three-fold axis . The N-termini are disordered up to Ser 15, which is close to the three-fold axis and on the same side as the C-termini . A density map of the icosahedral viral capsid at 15 A resolution, obtained by cryo-electron microscopy and image reconstruction, reveals gpD trimers, seemingly indistinguishable from the ones seen in the crystals, at all three-fold sites . The map further reveals that the side of the trimer that binds to the capsid is the side on which both termini reside . Despite this orientation of the gpD trimer, fusion proteins connected by linker peptides to either terminus bind to the capsid, allowing protein and peptide display. J Hum Genet, 2000, 45(1), 6 - 11 Genomic organization and chromosomal mapping of ELKS, a gene rearranged in a papillary thyroid carcinoma; Yokota T et al.; We recently isolated a novel cDNA, designated ELKS, that was fused to RET cDNA in a papillary thyroid carcinoma . Its encoded polypeptide sequence was rich in glutamic acid (E), leucine (L), lysine (K), and serine (S), and was characterized by the presence of nine alpha-helical coiled-coil domains consisting of periodic heptad repeats . We have now cloned the entire structure of the human ELKS gene from within a 700-kb genomic region represented by overlapping bacteriophage P1-derived artificial chromosome (PAC) and bacterial artificial chromosome (BAC) clones, and localized it to chromosomal band 12p13.3 by fluorescence in situ hybridization . The gene is approximately 500 kb long, with 19 exons and 18 introns; the transcription initiation site within exon 1 is separate from the initiation codon (in exon 2) . Analysis of the exon/intron structure revealed that introns interrupt the coding sequence in such a way that many functional segments of the protein are encoded by distinct exons . Exon 1 encodes the 5' non-coding region; exons 2, 3, 6, 7, 8, 9, 11, 14, and 15 encode the nine coiled-coil domains . Exons 17-19 constitute the 3' non-coding region . Analysis of the region immediately upstream of exon 1 showed that it was extremely rich in G/C nucleotides and contained multiple Sp-1 and AP2 binding sequences . The ELKS-RET gene fusion rearrangement we had observed in a papillary thyroid carcinoma occurred between intron 10 of the ELKS gene and intron 11 of RET. Prog Nucleic Acid Res Mol Biol, 2000, 64, 65 - 96 DNA polymerase of the T4-related bacteriophages; Karam JD et al.; The DNA polymerase of bacteriophage T4, product of phage gene 43 (gp43), has served as a model replicative DNA polymerase in nucleic acids research for nearly 40 years . The base-selection (polymerase, or Pol) and editing (3'-exonuclease, or Exo) functions of this multifunctional protein, which have counterparts in the replicative polymerases of other organisms, are primary determinants of the high fidelity of DNA synthesis in phage DNA replication . T4 gp43 is considered to be a member of the "B family" of DNA-dependent DNA polymerases (those resembling eukaryotic Pol alpha) because it exhibits striking similarities in primary structure to these enzymes . It has been extensively analyzed at the genetic, physiological, and biochemical levels; however, relationships between the in vivo properties of this enzyme and its physical structure have not always been easy to explain due to a paucity of structural data on the intact molecule . However, gp43 from phage RB69, a phylogenetic relative of T4, was crystallized and its structure solved in a complex with single-stranded DNA occupying the Exo site, as well as in the unliganded form . Analyses with these crystals, and crystals of a T4 gp43 proteolytic fragment harboring the Exo function, are opening new avenues to interpret existing biological and biochemical data on the intact T4 enzyme and are revealing new aspects of the microanatomy of gp43 that can now be explored further for functional significance . We summarize our current understanding of gp43 structure and review the physiological roles of this protein as an essential DNA-binding component of the multiprotein T4 DNA replication complex and as a nucleotide-sequence-specific RNA-binding translational repressor that controls its own biosynthesis and activity in vivo . We also contrast the properties of the T4 DNA replication complex to the functionally analogous complexes of other organisms, particularly Escherichia coli, and point out some of the unanswered questions about gp43 and T4 DNA replication. Prog Nucleic Acid Res Mol Biol, 2000, 64, 1 - 63 ATP-dependent restriction enzymes; Rao DN et al.; The phenomenon of restriction and modification (R-M) was first observed in the course of studies on bacteriophages in the early 1950s . It was only in the 1960s that work of Arber and colleagues provided a molecular explanation for the host specificity . DNA restriction and modification enzymes are responsible for the host-specific barriers to interstrain and interspecies transfer of genetic information that have been observed in a variety of bacterial cell types . R-M systems comprise an endonuclease and a methyltransferase activity . They serve to protect bacterial cells against bacteriophage infection, because incoming foreign DNA is specifically cleaved by the restriction enzyme if it contains the recognition sequence of the endonuclease . The DNA is protected from cleavage by a specific methylation within the recognition sequence, which is introduced by the methyltransferase . Classic R-M systems are now divided into three types on the basis of enzyme complexity, cofactor requirements, and position of DNA cleavage, although new systems are being discovered that do not fit readily into this classification . This review concentrates on multisubunit, multifunctional ATP-dependent restriction enzymes . A growing number of these enzymes are being subjected to biochemical and genetic studies that, when combined with ongoing structural analyses, promise to provide detailed models for mechanisms of DNA recognition and catalysis . It is now clear that DNA cleavage by these enzymes involves highly unusual modes of interaction between the enzymes and their substrates . These unique features of mechanism pose exciting questions and in addition have led to the suggestion that these enzymes may have biological functions beyond that of restriction and modification . The purpose of this review is to describe the exciting developments in our understanding of how the ATP-dependent restriction enzymes recognize specific DNA sequences and cleave or modify DNA. J Mol Biol, 2000 Mar 10, 296(5), 1215 - 23 Crystal structure of the DNA polymerase processivity factor of T4 bacteriophage; Moarefi I et al.; The protein encoded by gene 45 of T4 bacteriophage (gene 45 protein or gp45), is responsible for tethering the catalytic subunit of T4 DNA Polymerase to DNA during high-speed replication . Also referred to as a sliding DNA clamp, gp45 is similar in its function to the processivity factors of bacterial and eukaryotic DNA polymerases, the beta-clamp and PCNA, respectively . Crystallographic analysis has shown that the beta-clamp and PCNA form highly symmetrical ring-shaped structures through which duplex DNA can be threaded . Gp45 shares no sequence similarity with beta-clamp or PCNA, and sequence comparisons have not been able to establish whether it adopts a similar structure . We have determined the crystal structure of gp45 from T4 bacteriophage at 2.4 A resolution, using multiple isomorphous replacement . The protein forms a trimeric ring-shaped assembly with overall dimensions that are similar to those of the bacterial and eukaryotic processivity factors . Each monomer of gp45 contains two domains that are very similar in chain fold to those of beta-clamp and PCNA . Despite an overall negative charge, the inner surface of the ring is in a region of positive electrostatic potential, consistent with a mechanism in which DNA is threaded through the ring . J Bacteriol, 2000 Mar, 182(6), 1523 - 8 Molecular basis for the temperature sensitivity of Escherichia coli pth(Ts); Cruz-Vera LR et al.; The gene pth, encoding peptidyl-tRNA hydrolase (Pth), is essential for protein synthesis and viability of Escherichia coli . Two pth mutants have been studied in depth: a pth(Ts) mutant isolated as temperature sensitive and a pth(rap) mutant selected as nonpermissive for bacteriophage lambda vegetative growth . Here we show that each mutant protein is defective in a different way . The Pth(Ts) protein was very unstable in vivo, both at 43 degrees C and at permissive temperatures, but its specific activity was comparable to that of the wild-type enzyme, Pth(wt) . Conversely, the mutant Pth(rap) protein had the same stability as Pth(wt), but its specific activity was low . The thermosensitivity of the pth(Ts) mutant, presumably, ensues after Pth(Ts) protein levels are reduced at 43 degrees C . Conditions that increased the cellular Pth(Ts) concentration, a rise in gene copy number or diminished protein degradation, allowed cell growth at a nonpermissive temperature . Antibiotic-mediated inhibition of mRNA and protein synthesis, but not of peptidyl-tRNA drop-off, reduced pth(Ts) cell viability even at a permissive temperature . Based on these results, we suggest that Pth(Ts) protein, being unstable in vivo, supports cell viability only if its concentration is maintained above a threshold that allows general protein synthesis. Annu Rev Genet, 1999, 33, 565 - 602 Family values in the age of genomics: comparative analyses of temperate bacteriophage HK022; Weisberg RA et al.; HK022 is a temperate coliphage related to phage lambda . Its chromosome has been completely sequenced, and several aspects of its life cycle have been intensively studied . In the overall arrangement, expression, and function of most of its genes, HK022 broadly resembles lambda and other members of the lambda family . Upon closer view, significant differences emerge . The differences reveal alternative strategies used by related phages to cope with similar problems and illuminate previously unknown regulatory and structural motifs . HK022 prophages protect lysogens from superinfection by producing a sequence-specific RNA binding protein that prematurely terminates nascent transcripts of infecting phage . It uses a novel RNA-based mechanism to antiterminate its own early transcription . The HK022 protein shell is strengthened by a complex pattern of covalent subunit interlinking to form a unitary structure that resembles chain-mail armour . Its integrase and repressor proteins are similar to those of lambda, but the differences provide insights into the evolution of biological specificity and the elements needed for construction of a stable genetic switch. Gene, 2000 Feb 22, 244(1-2), 137 - 49 Molecular organization of the mouse gastrin-releasing peptide receptor gene and its promoter; Weber HC et al.; The murine gastrin-releasing peptide receptor (mGRP-R) is a member of the G protein-coupled receptor family and mediates important physiological actions of its specific ligand, the gastrointestinal hormone/neurotransmitter GRP, including mitogenic properties in the mouse Swiss 3T3 fibroblasts . Glucocorticoids and increases in intracellular cAMP are reported to alter GRP-R gene transcription, but the molecular basis for these effects is unknown . To begin to identify possible gene regulatory mechanisms that are responsible for modifying mGRP-R expression, we determined its structure and investigated its basal promoter activity . We isolated and characterized genomic bacteriophage P1 clones encoding the mouse gastrin-releasing peptide receptor (mGRP-R) . By DNA sequencing and Southern blot analyses, we determined the protein coding region to be contained in three exons interrupted by two introns 20 and 2kb in length . The open reading frame of the putative GRP-R gene encodes for a 384-amino-acid protein which demonstrates 48% identity with the mouse BRS-3 protein and 53% identity with the mouse NMB-R protein . The mGRP-R gene locus extends over 29kb and was mapped to the X-chromosome (DXMit20) utilizing a minisatellite polymorphism in the 5' UTR and by fluorescent in-situ hybridization (FISH) . In Swiss 3T3 cells, which natively express mGRP-R, two gene-specific mRNA species of 3 and 7kb can be detected by Northern blot analysis . With RNase protection assays, and independently with inverse PCR of 5' RACE clones, common mRNA initiation sites were identified clustered between 21 and 61bp downstream of a TTTAAA motif, which is located 450bp upstream of the ATG translation start site . However, different polyadenylation sites are utilized . A 2kb genomic DNA fragment extending from 2147 to 141 bases 5' to the ATG translation start was cloned into a luciferase reporter plasmid and shown to contain promoter activity in Swiss 3T3 and COS-7 cells . Progressive promoter truncations and mutations of a cyclic AMP response element (CRE) located 83bp upstream of the TTTAAA motif demonstrate that transcriptional mGRP-R activation in Swiss 3T3 cells only occurs when both the TTTAAA motif and the intact CRE site are retained . With the availability of the full structure of the mGRP-R gene and the minimal promoter sequences reported in this study, it will be possible in future studies to investigate the molecular basis for transcriptional regulation of the mGRP-R gene by glucocorticoids, cAMP and other factors. Science, 2000 Feb 18, 287(5456), 1279 - 83 Convergent solutions to binding at a protein-protein interface; DeLano WL et al.; The hinge region on the Fc fragment of human immunoglobulin G interacts with at least four different natural protein scaffolds that bind at a common site between the C(H2) and C(H3) domains . This "consensus" site was also dominant for binding of random peptides selected in vitro for high affinity (dissociation constant, about 25 nanomolar) by bacteriophage display . Thus, this site appears to be preferred owing to its intrinsic physiochemical properties, and not for biological function alone . A 2.7 angstrom crystal structure of a selected 13-amino acid peptide in complex with Fc demonstrated that the peptide adopts a compact structure radically different from that of the other Fc binding proteins . Nevertheless, the specific Fc binding interactions of the peptide strongly mimic those of the other proteins . Juxtaposition of the available Fc-complex crystal structures showed that the convergent binding surface is highly accessible, adaptive, and hydrophobic and contains relatively few sites for polar interactions . These are all properties that may promote cross-reactive binding, which is common to protein-protein interactions and especially hormone-receptor complexes. Plasmid, 2000 Mar, 43(2), 171 - 5 Transfer of conjugative plasmids and bacteriophage lambda occurs in the presence of antibiotics that prevent de novo gene expression; Cooper TF et al.; Plasmids transferred between bacteria prevented from expressing genes by the presence of bacteriostatic antibiotics . Whereas it has long been known that de novo gene expression is not required in donor cells for conjugation, the observations reported here extend the autonomy of plasmid transfer to the early events of establishment in recipients . In addition, this phenomenon was extended to bacteriophage lambda . Plasmid, 2000 Mar, 43(2), 166 - 70 Strand switching during rolling circle replication of plasmid-like DNA circles in the mitochondria of the higher plant Chenopodium album (L.); Backert S; The structure of sigma-like mitochondrial DNA molecules prepared from suspension cultured cells of Chenopodium album (L.) was studied by electron microscopy . These molecules were highly variable in size, ranging from about 1 to 104 kb, and had single- and double-stranded regions typical for rolling circle replicating intermediates . Partial denaturation studies confirmed that these structures constitute rolling circles . Close inspection of the circle-tail junctions of the replication fork at high magnification suggests that in circles with a double-stranded tail, both strands of the tail seem to be covalently attached to the circle in about 27% of the molecules . This observation can be explained by a phenomenon called strand switching or strand splippage during rolling circle replication, similar to a mechanism proposed for bacterial replicons or in vitro replicating constructs harboring bacteriophage T4 replication origins . J Mol Biol, 2000 Mar 3, 296(4), 989 - 99 Structural and functional comparative study of the complexes formed by viral ø29, Nf and GA-1 SSB proteins with DNA; Gascon I et al.; Single-stranded DNA-binding proteins have in common their crucial roles in DNA metabolism, although they exhibit significant differences in their single-stranded DNA binding properties . To evaluate the correlation between the structure of different nucleoprotein complexes and their function, we have carried out a comparative study of the complexes that the single-stranded DNA-binding proteins of three related bacteriophages, o29, Nf and GA-1, form with single-stranded DNA . Under the experimental conditions used, o29 and Nf single-stranded DNA-binding proteins are stable monomers in solution, while GA-1 single-stranded DNA-binding protein presents a hexameric state, as determined in glycerol gradients . The thermodynamic parameters derived from quenching measurements of the intrinsic protein fluorescence upon single-stranded DNA binding revealed (i) that GA-1 single-stranded DNA-binding protein occludes a larger binding site (n=51 nt/oligomer) than o29 and Nf SSBs (n=3.4 and 4.7 nt/monomer, respectively); and (ii) that it shows a higher global affinity for single-stranded DNA (GA-1 SSB, K(eff)=18.6 x 10(5) M(-1); o29 SSB, K(eff)=2.2 x 10(5) M(-1); Nf SSB, K(eff)=2.9 x 10(5) M(-1)) . Altogether, these parameters justify the differences displayed by the GA-1 single-stranded DNA-binding protein and single-stranded DNA complex under the electron microscope, and the requirement of higher amounts of o29 and Nf single-stranded DNA-binding proteins than of GA-1 SSB in gel mobility shift assays to produce a similar effect . The structural differences of the nucleoprotein complexes formed by the three single-stranded DNA-binding proteins with single-stranded DNA correlate with their different functional stimulatory effects in o29 DNA amplification . Nucleic Acids Res, 2000 Mar 15, 28(6), 1397 - 406 Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39; Read TD et al.; The genome sequences of Chlamydia trachomatis mouse pneumonitis (MoPn) strain Nigg (1 069 412 nt) and Chlamydia pneumoniae strain AR39 (1 229 853 nt) were determined using a random shotgun strategy . The MoPn genome exhibited a general conservation of gene order and content with the previously sequenced C.trachomatis serovar D . Differences between C.trachomatis strains were focused on an approximately 50 kb 'plasticity zone' near the termination origins . In this region MoPn contained three copies of a novel gene encoding a >3000 amino acid toxin homologous to a predicted toxin from Escherichia coli O157:H7 but had apparently lost the tryptophan biosyntheis genes found in serovar D in this region . The C . pneumoniae AR39 chromosome was >99.9% identical to the previously sequenced C.pneumoniae CWL029 genome, however, comparative analysis identified an invertible DNA segment upstream of the uridine kinase gene which was in different orientations in the two genomes . AR39 also contained a novel 4524 nt circular single-stranded (ss)DNA bacteriophage, the first time a virus has been reported infecting C . pneumoniae . Although the chlamydial genomes were highly conserved, there were intriguing differences in key nucleotide salvage pathways: C.pneumoniae has a uridine kinase gene for dUTP production, MoPn has a uracil phosphororibosyl transferase, while C.trachomatis serovar D contains neither gene . Chromosomal comparison revealed that there had been multiple large inversion events since the species divergence of C.trachomatis and C.pneumoniae, apparently oriented around the axis of the origin of replication and the termination region . The striking synteny of the Chlamydia genomes and prevalence of tandemly duplicated genes are evidence of minimal chromosome rearrangement and foreign gene uptake, presumably owing to the ecological isolation of the obligate intracellular parasites . In the absence of genetic analysis, comparative genomics will continue to provide insight into the virulence mechanisms of these important human pathogens. Hua Xi Yi Ke Da Xue Xue Bao, 1997 Mar, 28(1), 18 - 22 {Construction of genomic library of L . interrogans serovar lai using lambda gt11 as the vector and a study of recombiant plasmid pDL121}; Liu H et al.; A genomic library of L . interrogans serovar lai strain 017 has been constructed using lambda gt11 as the vector . DNA was partially digested by two blunt-end restriction enzymes, then methylated with EcoR I methylase; after EcoR I linker was added to the DNA, the linker-ended DNA was ligated to the dephosphorylated EcoR I digested lambda gt11 arms . The recombined DNA was packaged in vitro, and used to transduct E . coli Y1090 for amplification . There were 2.1 x 10(6) recombinant bacteriophages as recognized by their ability to form white plaques plated on Lac host in the presence of both IPTG and X-Ga1 . A positive clone, designated lambda DL12, was screened with a rabbit anti-serum against L . interrogans serovar lai from the genomic library . The DNA from lambda DL12 was subcloned into plasmid pUC18 . A recombinant (designated as pDL121) was obtained . SDS-PAGE analysis indicated that a 23 kd was expressed in E . coli JM 103 harboring pDL121 . Western blotting analysis showed that a specific protein band molecular weight of 23 kd could be recognized by the rabbit antiserum against L . interrogans serovar lai strain 017. Proc Natl Acad Sci U S A, 2000 Feb 29, 97(5), 2075 - 80 Noise-based switches and amplifiers for gene expression; Hasty J et al.; The regulation of cellular function is often controlled at the level of gene transcription . Such genetic regulation usually consists of interacting networks, whereby gene products from a single network can act to control their own expression or the production of protein in another network . Engineered control of cellular function through the design and manipulation of such networks lies within the constraints of current technology . Here we develop a model describing the regulation of gene expression and elucidate the effects of noise on the formulation . We consider a single network derived from bacteriophage lambda and construct a two-parameter deterministic model describing the temporal evolution of the concentration of lambda repressor protein . Bistability in the steady-state protein concentration arises naturally, and we show how the bistable regime is enhanced with the addition of the first operator site in the promotor region . We then show how additive and multiplicative external noise can be used to regulate expression . In the additive case, we demonstrate the utility of such control through the construction of a protein switch, whereby protein production is turned "on" and "off" by using short noise pulses . In the multiplicative case, we show that small deviations in the transcription rate can lead to large fluctuations in the production of protein, and we describe how these fluctuations can be used to amplify protein production significantly . These results suggest that an external noise source could be used as a switch and/or amplifier for gene expression . Such a development could have important implications for gene therapy. Proc Natl Acad Sci U S A, 2000 Feb 29, 97(5), 2276 - 81 A library of bacteriophage-displayed antibody fragments directed against proteins of the inner ear; Cyr JL et al.; Bacteriophage display of antibodies provides a method for the generation of immunological reagents against rare and uncharacterized antigens . To ascertain the usefulness of this approach for the characterization of inner-ear proteins, we produced a bacteriophage-displayed antibody-fragment library directed against proteins from the bullfrog's sacculus . This library was probed for bacteriophage that bound to proteins present in a lysate of hair cells, the sensory receptors of the inner ear . The predominant bacteriophage clone after selection expressed an antibody fragment that recognized a single protein in the inner ear . This antigen occurred in both the nonsensory and sensory epithelia of the sacculus . The specificity of the antibody fragment indicates that our bacteriophage-displayed library provides a useful source of immunological tools that should facilitate the identification and biochemical characterization of novel proteins in the inner ear. Biochem Biophys Res Commun, 2000 Feb 16, 268(2), 359 - 64 Restoration of mRNA splicing by a second-site intragenic suppressor in the T4 ribonucleotide reductase (small subunit) self-splicing intron; Khan AU et al.; The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598-base self-splicing intron which is closely related to other group I introns of T4 and eukaryotes . Thirty-one mutants causing splicing defects in the nrdB intron were isolated . Twenty-three EMS-induced revertants for these 31 primary mutants were isolated by the strategic usage of the white halo plaque phenotype . We mapped these revertants by marker rescue using subclones of the nrdB gene . Some of these second-site mutations mapped to regions currently predicted by the secondary structure model of the nrdB intron . One of these suppressor mutants (nrdB753R) was found to be intragenic by marker rescue with the whole nrdB gene . However, this mutation failed to map within the nrdB intron . Splicing assays showed that this pseudorevertant restored splicing proficiency of the nrdB primary mutation to almost wild-type conditions . This is the first example of a mutation within the exons of a gene containing a self-splicing intron that is capable of restoring a self-splicing defect caused by a primary mutation within the intron . In addition, two other suppressor mutations are of interest (nrdB429R and nrdB399R) . These suppressors were able to restore their primary 5' defect but in turn create a 3' splicing defect . Both of these revertants mapped in different regions of the intron with respect to their primary mutations . J Gen Virol, 2000 Mar, 81(Pt 3), 557 - 66 Identification of a pathogenicity determinant of Plum pox virus in the sequence encoding the C-terminal region of protein P3+6K(1); Saenz P et al.; A full-length genomic cDNA clone of a plum pox potyvirus (PPV) isolate belonging to the M strain (PPV-PS) has been cloned downstream from a bacteriophage T7 polymerase promoter and sequenced . Transcripts from the resulting plasmid, pGPPVPS, were infectious and, in herbaceous hosts, produced symptoms that differed from those of virus progeny of pGPPV, a full-length genomic cDNA clone of the D strain PPV-R . Viable PPV-R/-PS chimeric viruses were constructed by recombination of the cDNA clones in vitro . Analysis of plants infected with the different chimeras indicated that sequences encoding the most variable regions of the potyvirus genome, the P1 and capsid protein coding sequences, were not responsible for symptom differences between the two PPV isolates in herbaceous hosts . On the contrary, complex symptomatology determinants seem to be located in the central region of the PPV genome . The results indicate that a genomic fragment that encodes 173 aa from the C-terminal part of the P3+6K(1) coding region is enough to confer, on a PPV-R background, a PS phenotype in Nicotiana clevelandii . This pathogenicity determinant also participates in symptom induction in Pisum sativum, although the region defining the PS phenotype in this host is probably restricted to 74 aa. Biochim Biophys Acta, 1999 Dec 23, 1489(2-3), 374 - 82 The stem hairpin loop structure of p2Sp1 RNA is required for RNA-cleaving activity; Hosono K et al.; We studied the hairpin-loop structure of an RNA fragment (GUUUCGUACAAAC) (R13) with the sequence corresponding to the self-cleavage domain in the precursor of an RNA molecule from bacteriophage T4-infected Escherichia coli cells (p2Sp1 RNA) . In order to determine the influence of the hairpin-loop structure on these sequence-specific cleavage reactions, we have synthesized oligoribonucleotides containing hairpin-loop, double-helical stem-loop, and single-stranded RNA structures . The cleavage was affected by the hairpin-loop structure . Furthermore, the helix-stem, which retains the thermodynamically extrastable stem hairpin-loop structures, is also important for the cleavage activity . However, the thermodynamically extrastable helix-stem structure reduced the cleavage activity of the adjacent UA and CA sequences at the helix-stem site . For the cleavage reactions of the RNA cleavage products, the R6 (ACAAAC), R7 (GUUUCGU), and R9 (GUUUCGUAC) mers from the parent RNA, R13 (GUUUCGUACAAAC), a very slight amount of cleavage product (2%) from the RNA 9 was observed, but no reaction occurred for the R6 and R7 . We also describe the influences of the sequences (UA and CA) on the cleavage activity. J Mol Biol, 2000 Feb 18, 296(2), 597 - 612 Bacteriophage T4 gene 59 helicase assembly protein binds replication fork DNA . The 1.45 A resolution crystal structure reveals a novel alpha-helical two-domain fold; Mueser TC et al.; The bacteriophage T4 gene 59 helicase assembly protein is required for recombination-dependent DNA replication, which is the predominant mode of DNA replication in the late stage of T4 infection . T4 gene 59 helicase assembly protein accelerates the loading of the T4 gene 41 helicase during DNA synthesis by the T4 replication system in vitro . T4 gene 59 helicase assembly protein binds to both T4 gene 41 helicase and T4 gene 32 single-stranded DNA binding protein, and to single and double-stranded DNA . We show here that T4 gene 59 helicase assembly protein binds most tightly to fork DNA substrates, with either single or almost entirely double-stranded arms . Our studies suggest that the helicase assembly protein is responsible for loading T4 gene 41 helicase specifically at replication forks, and that its binding sites for each arm must hold more than six, but not more than 12 nucleotides . The 1.45 A resolution crystal structure of the full-length 217-residue monomeric T4 gene 59 helicase assembly protein reveals a novel alpha-helical bundle fold with two domains of similar size . Surface residues are predominantly basic (pI 9.37) with clusters of acidic residues but exposed hydrophobic residues suggest sites for potential contact with DNA and with other protein molecules . The N-terminal domain has structural similarity to the double-stranded DNA binding domain of rat HMG1A . We propose a speculative model of how the T4 gene 59 helicase assembly protein might bind to fork DNA based on the similarity to HMG1, the location of the basic and hydrophobic regions, and the site size of the fork arms needed for tight fork DNA binding . The fork-binding model suggests putative binding sites for the T4 gene 32 single-stranded DNA binding protein and for the hexameric T4 gene 41 helicase assembly. Chin J Biotechnol, 1999, 15(1), 29 - 35 Hirudin display on the surface of bacteriophage M13; Zhou Y et al.; Hirudin was fused to the N terminus of M13 minor protein gp3 (197-406) through a linker GGGS by inserting both the hirudin gene and the gp8 signal sequence into the modified phagemid vector pCANTAB 5V to construct pCANTAB 5G8-Hir . The expressed fusion protein was directed by gp8 signal peptide into the periplasm and assembled to the phage particle to form the hirudin-phage . The fusion protein and fusion phage were detected with biotin-thrombin by Western blotting analysis . Antithrombin activity analysis confirmed that the hirudin portion in the fusion protein and fusion phage bear similar native conformation . The successful display of hirudin on the surface of M13 phage laid a sound foundation for the further study on directed evolution of antithrombotic proteins with altered properties. Acta Crystallogr D Biol Crystallogr, 2000 Jan, 56 ( Pt 1), 95 - 7 Crystallization and preliminary X-ray analysis of a bacteriophage T4 primase fragment; Korndorfer IP et al.; The primase from bacteriophage T4 is a single-stranded DNA-dependent RNA polymerase that is one of the seven proteins that constitute the DNA-replication machinery of bacteriophage T4 . In an attempt to crystallize the protein, a number of variants were generated . One such construct, which includes the C-terminal region (residues 196-340), gave four different crystal forms which diffract in the 3 . 5-6.0 A resolution range. Acta Crystallogr D Biol Crystallogr, 2000 Feb, 56 ( Pt 2), 137 - 50 The molecular structure and structural transition of the alpha-helical capsid in filamentous bacteriophage Pf1; Welsh LC et al.; The major coat protein in the capsid of Pf1 filamentous bacteriophage (Inovirus) forms a helical assembly of about 7000 identical protein subunits, each of which contains 46 amino-acid residues and can be closely approximated by a single gently curved alpha-helix . Since the viral DNA occupies the core of the tubular capsid and appears to make no significant specific interactions with the capsid proteins, the capsid is a simple model system for the study of the static and dynamic properties of alpha-helix assembly . The capsid undergoes a reversible temperature-induced structural transition at about 283 K between two slightly different helix forms . The two forms can coexist without an intermediate state, consistent with a first-order structural phase transition . The molecular model of the higher temperature form was refined using improved X-ray fibre diffraction data and new refinement and validation methods . The refinement indicates that the two forms are related by a change in the orientation of the capsid subunits within the virion, without a significant change in local conformation of the subunits . On the higher temperature diffraction pattern there is a region of observed intensity that is not consistent with a simple helix of identical subunits; it is proposed that the structure involves groups of three subunits which each have a slightly different orientation within the group . The grouping of subunits suggests that a change in subunit libration frequency could be the basis of the Pf1 structural transition; calculations from the model are used to explore this idea. Pharmacol Ther, 1999 Dec, 84(3), 367 - 88 Unusual transcriptional and translational regulation of the bacteriophage Mu mom operon; Hattman S; The bacteriophage Mu mom gene encodes a novel DNA modification that protects the viral genome against a wide variety of restriction endonucleases . Expression of mom is subject to a series of unusual regulatory controls . Transcription requires the action of a phage-encoded protein, C, which binds (probably as a dimer) the mom promoter from -33 to -52 (with respect to the transcription start site) in two adjacent DNA major grooves on one face of the helix . No apparent direct interaction between C and the host RNA polymerase (RNAP) is evident; however, C binding alters mom DNA conformation . In the absence of C, RNAP binds the mom promoter at a site that results in transcription in a direction away from the mom gene . The function of this transcription is unknown . An additional layer of transcriptional regulation complexity is due to the fact that the host Dam DNA-(N6-adenine)methyltransferase is required . Dam methylation of three closely spaced upstream GATC sequences is necessary to prevent binding by the host protein, OxyR, which acts as a repressor . Repression is not mediated by inhibition of C binding, but rather through interference with C-mediated recruitment of RNAP to the correct site . Translation of mom is regulated by the phage Com protein . Com is only 62 amino acids long and contains a zinc finger-like structure (coordinated by four cysteine residues) in the amino terminal domain . Com binds mom mRNA 5' to the mom open reading frame, whose translation start signals are contained in a stem-loop translation-inhibition-structure . Com binding to its target site (5' to and adjacent to the translation-inhibition-structure) results in a stable change in RNA secondary structure that exposes the translation start signals. Cell, 2000 Jan 21, 100(2), 253 - 63 Maturation dynamics of a viral capsid: visualization of transitional intermediate states; Lata R et al.; Typical of DNA bacteriophages and herpesviruses, HK97 assembles in two stages: polymerization and maturation . First, capsid protein polymerizes into closed shells; then, these precursors mature into larger, stabler particles . Maturation is initiated by proteolysis, producing a metastable particle primed for expansion-the major structural transition . We induced expansion in vitro by acidic pH and monitored the resulting changes by time-resolved X-ray diffraction and cryo-electron microscopy . The transition, which is not synchronized over the population, proceeds in a series of stochastically triggered subtransitions . Three distinct intermediates were identified, which are comparable to transitional states in protein folding . The intermediates' structures reveal the molecular events occurring during expansion . Integrated into a movie (see Dynamic Visualization below), they show capsid maturation as a dynamic process. Nature, 2000 Jan 20, 403(6767), 339 - 42 Construction of a genetic toggle switch in Escherichia coli; Gardner TS et al.; It has been proposed' that gene-regulatory circuits with virtually any desired property can be constructed from networks of simple regulatory elements . These properties, which include multistability and oscillations, have been found in specialized gene circuits such as the bacteriophage lambda switch and the Cyanobacteria circadian oscillator . However, these behaviours have not been demonstrated in networks of non-specialized regulatory components . Here we present the construction of a genetic toggle switch-a synthetic, bistable gene-regulatory network-in Escherichia coli and provide a simple theory that predicts the conditions necessary for bistability . The toggle is constructed from any two repressible promoters arranged in a mutually inhibitory network . It is flipped between stable states using transient chemical or thermal induction and exhibits a nearly ideal switching threshold . As a practical device, the toggle switch forms a synthetic, addressable cellular memory unit and has implications for biotechnology, biocomputing and gene therapy. Bioorg Med Chem, 1999 Dec, 7(12), 2931 - 6 Substrate/inhibition studies of bacteriophage T7 RNA polymerase with the 5'-triphosphate derivative of a ring-expanded ('fat') nucleoside possessing potent antiviral and anticancer activities; Bretner M et al.; As part of an effort to explore the mechanism of potent, broad spectrum antiviral and anticancer activities of a number of ring-expanded ('fat') nucleosides that we recently reported, a representative 'fat' nucleoside 4,6-diamino-8-imino-8H-1-beta-D-ribofuranosylimidazo{4,5-e}{1,3}di azepine (1) was converted to its 5'-triphosphate derivative (2), and biochemically screened for possible inhibition of nucleic acid polymerase activity, employing synthetic DNA templates and the bacteriophage T7 RNA polymerase as a representative polymerase . Our results suggest that 2 is a moderate inhibitor of T7 RNA polymerase, and that the 5'-triphosphate moiety of 2 appears to be essential for inhibition as nucleoside 1 alone failed to inhibit the polymerase reaction. J Immunol, 2000 Feb 15, 164(4), 1977 - 85 Transfer of immune complexes from erythrocyte CR1 to mouse macrophages; Reinagel ML et al.; We are developing a potential therapeutic approach for removing pathogens from the circulation of primates in which the pathogen is bound to the complement receptor (CR1) on E using a bispecific mAb complex, a heteropolymer (HP) . We have used mAb this approach to demonstrate that cleared prototype pathogens are localized to, phagocytosed in, and destroyed in the liver . Extension of this work to a clinical setting will require a detailed understanding of the mechanism by which the E-bound immune complex substrates are transferred to fixed tissue macrophages in the liver, the transfer reaction . Therefore, we examined an in vitro system to study this process using bacteriophage phiX174 as a model pathogen . E containing phiX174 (bound via an anti-CR1/anti-phiX174 HP) were incubated with P388D1 murine macrophages, and the two cell types were separated by centrifugation through Ficoll . Both E and macrophages were then probed and analyzed by RIA or flow cytometry . The results indicate that all three components of the E-bound IC (phiX174, HP, and CR1) were removed from the E and internalized by the macrophages . We found that transfer requires the Fc portion of IgG, because little transfer of phiX174 occurs when it is bound to E CR1 using a HP containing only Fab fragments . These findings, taken in the context of other studies, suggest a general mechanism for the transfer reaction in which Fc receptors facilitate close juxtaposition of the macrophage to the E-bound IC which then allows a macrophage-associated protease to cleave CR1 . The released IC are then internalized and processed by the macrophages. J Mol Evol, 2000 Jan, 50(1), 82 - 92 Intron conservation in a UV-specific DNA repair gene encoded by chlorella viruses; Sun L et al.; Large dsDNA-containing chlorella viruses encode a pyrimidine dimer-specific glycosylase (PDG) that initiates repair of UV-induced pyrimidine dimers . The PDG enzyme is a homologue of the bacteriophage T4-encoded endonuclease V . The pdg gene was cloned and sequenced from 42 chlorella viruses isolated over a 12-year period from diverse geographic regions . Surprisingly, the pdg gene from 15 of these 42 viruses contain a 98-nucleotide intron that is 100% conserved among the viruses and another 4 viruses contain an 81-nucleotide intron, in the same position, that is nearly 100% identical (one virus differed by one base) . In contrast, the nucleotides in the pdg coding regions (exons) from the intron-containing viruses are 84 to 100% identical . The introns in the pdg gene have 5'-AG/GTATGT and 3'-TTGCAG/AA splice site sequences which are characteristic of nuclear-located, spliceosomal processed pre-mRNA introns . The 100% identity of the 98-nucleotide intron sequence in the 15 viruses and the near-perfect identity of an 81-nucleotide intron sequence in another 4 viruses imply strong selective pressure to maintain the DNA sequence of the intron when it is in the pdg gene . However, the ability of intron-plus and intron-minus viruses to repair UV-damaged DNA in the dark was nearly identical . These findings contradict the widely accepted dogma that intron sequences are more variable than exon sequences. Biochemistry, 2000 Feb 8, 39(5), 1142 - 51 Folding defects caused by single amino acid substitutions in a subunit are not alleviated by assembly; Capen CM et al.; Significant stabilization of a protein often occurs when it is assembled into an oligomer . Bacteriophage P22 contains 420 monomers of coat protein that are stabilized by the assembly and maturation processes . The effects of eight single amino acid substitutions in coat protein that each cause a temperature-sensitive-folding defect were investigated to