J Biol Chem, 1998 Jan 30, 273(5), 2858 - 65 Recombinant human single chain Fv antibodies recognizing human interleukin-6 . Specific targeting of cytokine-secreting cells; Krebs B et al.; A human antibody library was displayed on the surface of filamentous bacteriophage and screened for binding to human interleukin-6 (IL-6) . Two antibody-bearing phages were selected that bound IL-6 . The complementary-determining region 3 loops of the variable heavy chains of these two antibodies differed in length and sequence and recognized two distinct epitopes . One of the single chain Fv fragments isolated (H1) was found to bind human (but not murine) IL-6 with an affinity comparable to that of the human IL-6 receptor . H1 also recognized newly synthesized human IL-6 intracellularly, as shown by indirect immunofluorescence . H1 did not neutralize human IL-6, and the H1 epitope was mapped to a region of IL-6 not involved in interactions with IL-6, IL-6 receptor, or the signal-transducing protein gp130 . To target IL-6-secreting cells, we then constructed a bispecific antibody fragment (a diabody) comprising H1 and the antigen binding site of the T-cell activating monoclonal antibody OKT3 . The diabody led to T-cell-mediated killing of cells secreting IL-6. Nucleic Acids Res, 1998 Jan 15, 26(2), 669 - 75 Recognition of DNA structure by 434 repressor; Koudelka GB; In complexes of bacteriophage 434 binding sites with 434 repressor the central 4 bp of the 14 bp site are not contacted by the protein, although changes in these bases alter binding site affinity for the repressor . Our previous data suggested that the ability of the non-contacted central bases to be overtwisted in repressor-DNA complexes governs affinity of the binding site for 434 repressor . This idea was tested by examining the affinity of two central sequence variant 434 binding sites for 434 repressor as a function of binding site average twist . The 434 repressor preferred the relatively overwound binding site to the two more underwound forms . The greatest affinity enhancement resulting from increasing twist was observed with a binding site that is relatively underwound and more resistant to twisting deformation . Consistent with the idea that 434 repressor overtwists its binding site upon DNA binding, we show that 434 repressor is capable of binding to sites bearing a single base insertion in their center (a 15mer), but binds poorly to binding sites bearing central base deletions (12mer and 13mer) . The N-terminal dimer interface plays a large role in determining 434 repressor central base preferences . Mutations in this interface eliminate central base discrimination and/or site size preferences . These mutations also lead to changes in the size of the repressor footprint on the various sized DNA sites that are consistent with their binding characteristics. Chem Res Toxicol, 1998 Jan, 11(1), 64 - 9 The major mitomycin C-DNA monoadduct is cytotoxic but not mutagenic in Escherichia coli; Ramos LA et al.; To determine the mutagenic and genotoxic properties of the major guanine N2-adduct formed by the antitumor drug mitomycin C, we have synthesized a decanucleotide, d(TTACG{MC}TATCT), containing the adduct, which was inserted into a gapped bacteriophage M13 genome . Analysis of the constructed genome indicated that 41% ligation of the adducted 10-mer occurred on both sides of the gap, whereas the control 10-mer ligated with 34% efficiency . After transfection of the adducted single-stranded M13 DNA into Escherichia coli, the adduct was found to be highly genotoxic . Viability of the adducted genome in a repair-competent strain was only 7%, which increased to 12% and 15% upon induction of SOS by irradiating the cells with 254-nm light at 20 and 50 J/m2, respectively . Even lower viability of 2%, 4.6%, and 0.2% was observed in uvrA, uvrB, and uvrC strains, respectively, which increased up to 10-fold with SOS . An examination of the surviving phage populations revealed that the adduct was not detectably mutagenic . No mutants from the repair-proficient strain were detected after analysis of more than 2500 progeny phage . Only 0.2% of the survivors were mutants in the uvrA strain . It is uncertain, however, if they were induced by the adduct, since all the mutants showed untargeted mutations . We conclude that the major guanine N2-adduct formed by mitomycin C is cytotoxic but not appreciably mutagenic in E . coli. Can J Microbiol, 1997 Dec, 43(12), 1147 - 56 Transcription termination by bacteriophage T3 and SP6 RNA polymerases at Rho-independent terminators; Jeng ST et al.; Transcription termination of T3 and SP6 DNA-dependent RNA polymerases have been studied on the DNA templates containing the threonine (thr) attenuator and its variants . The thr attenuator is from the regulatory region of the thr operon of Escherichia coli . The DNA template, encoding the thr attenuator, contains specific features of the rho-independent terminators . It comprises a dG + dC rich dyad symmetry, encoding a stem-and-loop RNA, which is followed by a poly(U) region at the 3'-end . Thirteen attenuator variants have been analyzed for their ability to terminate transcription and the results indicated that the structure as well as the sequence in the G + C rich region of RNA hairpin affect termination of both RNA polymerases . Also, a single base change in the A residues of the hairpin failed to influence termination, whereas changes in the poly(U) region significantly reduced the termination of both T3 and SP6 RNA polymerases . The requirement of a poly(U) region for termination by T3 and SP6 RNA polymerases was studied with nested deletion mutants in this region . The minimum number of U residues required for termination of SP6 and T3 RNA polymerases was five and three, respectively . However, both RNA polymerases needed at least eight U residues to reach a termination efficiency close to that achieved by wild-type thr attenuator encoding nine U residues . In addition, the orientation of the loop sequences of the RNA hairpin did not affect the transcription termination of either of the bacteriophage RNA polymerases. J Biotechnol, 1997 Dec 3, 58(3), 205 - 8 Amplification of pSC101 replicons in Escherichia coli during amino acid limitation; Wrobel B et al.; Amino acid starvation of Escherichia coli relA mutants was previously proposed as a method for efficient amplification of plasmids bearing origin of replication derived from ColE1-type plasmids and bacteriophage lambda . Here we demonstrate that plasmids derived from pSC101 replicon can be amplified in E . col relA+ and relA- strains during amino acid limitation but not during amino acid starvation . The amplification efficiency is dependent on temperature (37 degrees C was found to be the optimal temperature) . Under optimal conditions, up to 13-fold amplification of a pSC101-derived plasmid may be achieved. Biophys Chem, 1997 Oct, 68(1-3), 17 - 31 Mechanisms of virus assembly probed by Raman spectroscopy: the icosahedral bacteriophage P22; Tuma R et al.; A microdialysis flow cell has been developed for time-resolved Raman spectroscopy of biological macromolecules and their assemblies . The flow cell permits collection of Raman spectra concurrent with the efflux of small solute molecules into a solution of macromolecules and facilitates real-time spectroscopic detection of structural transitions induced by the effluent . Additionally, the flow cell is well suited to the investigation of hydrogen-isotope exchange phenomena that can be exploited as dynamic probes of viral protein folding and solvent accessibility along the assembly pathway . Here, we describe the application of the Raman dynamic probe to the maturation of the icosahedral capsid of bacteriophage P22, a double-stranded DNA virus . The P22 virion is constructed from a capsid precursor (procapsid) consisting of 420 coat subunits (gp5) in an outer shell and a few hundred scaffolding subunits (gp8) within . Capsid maturation involves expulsion of scaffolding subunits coupled with shell expansion at the time of DNA packaging . Raman static and dynamic probes reveal that the scaffolding subunit is highly alpha-helical and highly thermolabile, and lacks a typical hydrophobic core . When bound within the procapsid, the alpha-helical fold of gp8 is thermostabilized; however, this stabilization confers no apparent protection against peptide NH-->ND exchange . A molten globule model is proposed for the native scaffolding subunit that functions in procapsid assembly . Accompanying capsid expansion, a small conformational change (alpha-helix-->beta-strand) is also observed in the coat subunit . Domain movement mediated by hinge bending is proposed as the mechanism of capsid expansion . On the basis of these results, a molecular model is proposed for assembly of the P22 procapsid. DNA Cell Biol, 1998 Jan, 17(1), 61 - 8 The normal copy of the G0S19-3-associated, CpG island-containing, upstream sequence is downstream of G0S19-2/MIP1alpha in association with a TRE17 oncogene; Heximer SP et al.; The G0S19-1/MIP1alpha and G0S19-2/MIP1alpha genes locate to human chromosomes 17q and encode similar copies of the beta-chemokine G0S19/MIP1alpha . The G0S19-3 gene, present in 1 in 4 humans, is a 5' truncated version of G0S19-2; a CpG island-containing upstream sequence (CpG-US), rich in potential transcriptional activation motifs, replaces much of the first intron and the first exon . Sequences hybridizing with the CpG-US sequence, normally exist in all human genomes . Thus, it appears that there has been recombination between a duplicated G0S19 gene and a duplicated CpG-US-like sequence . We have isolated sequences hybridizing with the CpG-US sequence from a human genomic library in bacteriophage lambda . Restriction mapping and sequencing shows a CpG-US-like sequence approximately 8 kb downstream of G0S19-2 (hence, named CpG-DS sequence) . The sequence is contiguous with a TRE17 oncogene-associated sequence (GenBank locus HSTRE175) . Members of the TRE17 family are known to locate to chromosome 17q (Onno et al., 1993b), and have sequence characteristics suggestive of positive Darwinian selection . Linkage with a TRE17 oncogene may have arisen by recombination and imply no functional relationship . However, it is possible that the CpG-DS may normally regulate TRE17 expression . PCR and sequencing studies indicate the close proximity of other chemokine-related sequences in the 17q11.2 region. J Mol Biol, 1998 Jan 16, 275(2), 221 - 32 Mutational analysis of domain II beta of bacteriophage Mu transposase: domains II alpha and II beta belong to different catalytic complementation groups; Namgoong SY et al.; This study examines the contribution of domain II beta of bacteriophage Mu transposase (A protein), a subdomain of the central catalytic domain II, to the transposition reaction . The properties of several point mutations implicate a role for this domain in facilitating metal-assisted assembly of the synaptic complex, as well as in intramolecular DNA strand transfer . Point mutations as well as deletions in domain II beta can be complemented by those in domain II alpha but not those in domain III alpha . Thus, residues within subdomains II alpha and II beta belong to different catalytic complementation groups. Mol Microbiol, 1998 Jan, 27(1), 23 - 30 Bacteriophage T7 mRNA is polyadenylated; Johnson MD et al.; To determine whether the RNA of bacterial viruses is polyadenylated like bacterial mRNAs, pulse-labelled as well as the steady-state population of bacteriophage T7-specific transcripts were examined for the presence of poly(A) tracts by binding to oligo(dT) cellulose followed by hybridization with specific gene probes . Representatives of all classes of bacteriophage-specific mRNA--early, middle and late--were found to be polyadenylated . This conclusion was confirmed by screening the products of oligo(dT)-dependent cDNA synthesis . A cDNA library was prepared from RNA synthesized after bacteriophage T7 infection and the sequence of bacteriophage-specific clones was determined to define the sites of polyadenylation . About half of the clones were polyadenylated near the end of a protein-coding region, one of them at the site of post-transcriptional processing by RNase III . Other clones were polyadenylated within protein-coding regions . These observations suggest that polyadenylation occurs after the nucleolytic processing of primary transcripts and in some cases also after mRNA degradation has already begun. Appl Environ Microbiol, 1998 Feb, 64(2), 504 - 8 Immunomagnetic capture PCR for rapid concentration and detection of hepatitis A virus from environmental samples; Jothikumar N et al.; We studied the concentration of hepatitis A virus (HAV) from environmental samples by membrane filter-based urea-arginine phosphate buffer and its detection by using immunomagnetic capture (IC) reverse transcription (RT)-PCR (IC PCR) . Magnetic beads coated with anti-HAV rabbit antibodies were used for enrichment and concentration of HAV from environmental samples . IC PCR is sensitive enough to detect as few as 0.04 PFU of cell culture-adapted HAV in inoculated water and sewage samples . IC PCR is specific and does not yield positive reactions with poliovirus 1, HAV RNA, or selected bacteriophages . IC concentrates viruses suspended in small volumes to microliter volumes that can be used directly in RT-PCR . IC concentration of viruses from sewage samples without concentration of inhibitory substances is important for successful RT-PCR detection . In a field trial, 2 of 18 raw sewage samples tested by IC PCR were positive for HAV. Appl Environ Microbiol, 1998 Feb, 64(2), 411 - 8 Expression and regulation of the arsenic resistance operon of Acidiphilium multivorum AIU 301 plasmid pKW301 in Escherichia coli; Suzuki K et al.; The arsenic resistance (ars) operon from plasmid pKW301 of Acidiphilium multivorum AIU 301 was cloned and sequenced . This DNA sequence contains five genes in the following order: arsR, arsD, arsA, arsB, arsC . The predicted amino acid sequences of all of the gene products are homologous to the amino acid sequences of the ars gene products of Escherichia coli plasmid R773 and IncN plasmid R46 . The ars operon cloned from A . multivorum conferred resistance to arsenate and arsenite on E . coli . Expression of the ars genes with the bacteriophage T7 RNA polymerase-promoter system allowed E . coli to overexpress ArsD, ArsA, and ArsC but not ArsR or ArsB . The apparent molecular weights of ArsD, ArsA, and ArsC were 13,000, 64,000, and 16,000, respectively . A primer extension analysis showed that the ars mRNA started at a position 19 nucleotides upstream from the arsR ATG in E . coli . Although the arsR gene of A . multivorum AIU 301 encodes a polypeptide of 84 amino acids that is smaller and less homologous than any of the other ArsR proteins, inactivation of the arsR gene resulted in constitutive expression of the ars genes, suggesting that ArsR of pKW301 controls the expression of this operon. Appl Environ Microbiol, 1998 Feb, 64(2), 405 - 10 Delineating the specific influence of virus isoelectric point and size on virus adsorption and transport through sandy soils; Dowd SE et al.; Many of the factors controlling viral transport and survival within the subsurface are still poorly understood . In order to identify the precise influence of viral isoelectric point on viral adsorption onto aquifer sediment material, we employed five different spherical bacteriophages (MS2, PRD1, Q beta, phi X174, and PM2) having differing isoelectric points (pI 3.9, 4.2, 5.3, 6.6, and 7.3 respectively) in laboratory viral transport studies . We employed conventional batch flowthrough columns, as well as a novel continuously recirculating column, in these studies . In a 0.78-m batch flowthrough column, the smaller phages (MS2, phi X174, and Q beta), which had similar diameters, exhibited maximum effluent concentration/initial concentration values that correlated exactly with their isoelectric points . In the continuously recirculating column, viral adsorption was negatively correlated with the isoelectric points of the viruses . A model of virus migration in the soil columns was created by using a one-dimensional transport model in which kinetic sorption was used . The data suggest that the isoelectric point of a virus is the predetermining factor controlling viral adsorption within aquifers . The data also suggest that when virus particles are more than 60 nm in diameter, viral dimensions become the overriding factor. Anal Chem, 1998 Jan 1, 70(1), 158 - 62 Microchip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing; Waters LC et al.; The steps of cell lysis, multiplex PCR amplification, and electrophoretic analysis are executed sequentially on a monolithic microchip device . The entire microchip is thermally cycled to lyse cells and to amplify DNA, and the products are then analyzed using a sieving medium for size separation and an intercalating dye for fluorescence detection . Using a standard PCR protocol, a 500-base pair (bp) region of bacteriophage lambda DNA and 154-, 264-, 346-, 410-, and 550-bp regions of E . coli genomic and plasmid DNAs are amplified . The electrophoretic analysis of the products is executed in <3 min following amplification using hydroxyethyl cellulose or poly(dimethylacrylamide) sieving gels . Product sizing is demonstrated by proportioning the amplified product with a DNA sizing ladder. Gene, 1998 Jan 5, 206(1), 49 - 52 Isolation, sequencing and expression of the gene encoding a major protein from the backteriophage associated with Bartonella henselae; Bowers TJ et al.; The gene encoding a 31-kDa major protein (Pap31) associated with the bacteriophage harbored in Bartonella henselae was cloned and sequenced . Analysis of the resulting sequence revealed an open reading frame of 837 nucleotides coding for a protein of 279 amino acids . pap31 was then subcloned downstream of the lacZ promoter in pUC19 . pap31 was amplified by polymerase chain reaction, and the linear amplicon was used as template for in-vitro transcription and translation . A protein with an apparent molecular mass of approximately 31 kDa was synthesized from this reaction . Upon analysis of the deduced aa sequence, a potential signal sequence and a consensus signal peptidase cleavage site were identified, indicative that Pap31 is modified posttranslationally, and the mature protein may be targeted to the host membrane. Nat Struct Biol, 1998 Feb, 5(2), 133 - 9 Crystal structure of an RNA aptamer-protein complex at 2.8 A resolution; Convery MA et al.; The crystal structure, at 2.8 A resolution, of an RNA aptamer bound to bacteriophage MS2 coat protein has been determined . It provides an opportunity to compare the interactions of MS2 coat protein and wild type operator with those of an aptamer, whose secondary structure differs from the wild type RNA in having a three-base loop (compared to a tetraloop) and an additional base pair between this loop and the sequence-specific recognition element in the stem . The RNA binds in the same location on the coat protein as the wild type operator and maintains many of the same RNA-protein interactions . In order to achieve this, the RNA stem loop undergoes a concerted rearrangement of the 3' side while leaving the 5' side and the loop interactions largely unchanged, illustrating the ability of RNA to present similar molecular recognition surfaces from distinct primary and secondary structures. Zh Mikrobiol Epidemiol Immunobiol, 1997 Nov-Dec, (6), 76 - 81 {The genetic typing of strains in the genus Francisella using universal probes}; Shaginian IA et al.; The determination of the genetic relationship of bacteria of the genus Francisella and their differentiation is one of the topical tasks of the epidemiology and infectology of F . tularensis, the causative agent of tularemia, belonging to this genus . To solve this task, investigation was carried out with a view to the determine the possibility of the genomic typing of Francisella . Genomic typing was based on the use of the hybridization of fragments of Francisella chromosomal DNA, split by restrictases EcoRI and Pstl, with DNA probes . As probes, "minisatellite" sequences of bacteriophage M13 DNA or Helicobacter pylori rDNA were used . The possibility of interspecific genomic typing of F . tularensis, F . novicida and F . philomiragia by the above-mentioned methods was established . The intraspecific typing of F . tularensis by the phenotypical sign of virulence was possible with the use of the hybridization of chromosomal DNA with bacteriophage M13 probe . The use of rDNA probe proved to be effective for the determination of subspecies of the causative agent of tularemia . The possibility of using the combination of these two methods for more complete characterization of the genomic polymorphism of Francisella, the determination of their genetic relationship and their differentiation is discussed. J Mol Biol, 1997 Nov 7, 273(4), 765 - 74 Interaction of the bacteriophage Mu transcriptional activator protein, C, with its target site in the mom promoter; Sun W et al.; The bacteriophage Mu C gene encodes a 16.5 kDa site-specific DNA binding protein that is a transcriptional activator of the four "late" promoters, Pmom, Plys, PI and PP . A symmetrical consensus C recognition sequence, TTAT{N5-6}ATAA, containing an inverted tetrad repeat separated by a spacer of five to six G+C-rich nucleotides, has been proposed . To investigate this, we used oligonucleotide mutagenesis to introduce random substitutions within and flanking the proposed C-target region; each variant site was tested for C recognition by an in vivo functional transactivation assay . We observed that all single mutations, in either tetrad, reduced C activation . Although two out of ten substitutions within the spacer reduced activation, the spacer region does not appear to make specific contact with C . We also used in vitro chemical-protection and -interference to study C contacts with Pmom . The results indicate that C contacts Pmom DNA on only one face of the helix through interactions within two adjacent major grooves; this conclusion was supported by gel shift analyses using synthetic oligonucleotide duplexes containing I.C or other base-pair substitutions . Evidence is also presented that C-Pmom contacts are asymmetrical, and that they extend two nucleotides 3' to the promoter-proximal tetrad . We also show that C binding induces a deformation, possibly a bend, in Pmom DNA. J Mol Biol, 1997 Nov 28, 274(2), 160 - 73 Assembly of the N-dependent antitermination complex of phage lambda: NusA and RNA bind independently to different unfolded domains of the N protein; Van Gilst MR et al.; The N protein of bacteriophage lambda activates expression of the delayed early genes of this phage by modifying RNA polymerase (RNAP) into a form that is resistant to termination signals . N binds to the boxB hairpin that forms in the nascent RNA transcript upon transcription of the nut regulatory element, and then interacts with RNAP by RNA looping . The binding of the N-boxB subassembly to the transcription complex is further stabilized by interaction with the Escherichia coli NusA protein . N, free in solution, exists as an unfolded protein that becomes partially structured upon binding specifically to boxB RNA . Because NusA does not assist in antitermination unless N is specifically bound to boxB, we have asked whether the structural change induced by binding to boxB affects the interaction of N with NusA . Using fluorescence spectroscopy, we have measured the affinity of N for NusA in the presence and absence of boxB RNA . We find that NusA binds to the unfolded N protein with a dissociation constant (Kd) of approximately 70 nM, and although N undergoes a significant structural change upon binding to boxB, the binding affinity of NusA for a N protein complexed with boxB is not altered . We have also shown that the boxA element of nut does not affect NusA binding to N-boxB . These results demonstrate that the interaction of N with NusA is independent of RNA binding, arguing that NusA must interact with an unfolded region of the polypeptide that remains unstructured even when N binds to boxB RNA . To further establish this point we isolated a truncated peptide containing the amino-terminal 36 residues of the N protein . Binding of boxB RNA to this peptide showed that all of the structural change in N that occurs upon binding to boxB RNA is localized within the amino-terminal 36 residues of N, therefore the C terminus of N, including the regions necessary for NusA binding and RNAP activation, remains unfolded when the full length N binds to boxB RNA . Thus it appears that N can be described as an unfolded multi-domain protein that becomes ordered in a modular fashion as it encounters its various binding partners within the N-dependent antitermination complex . J Biol Chem, 1997 Dec 12, 272(50), 31685 - 92 Structural analyses of gp45 sliding clamp interactions during assembly of the bacteriophage T4 DNA polymerase holoenzyme . III . The Gp43 DNA polymerase binds to the same face of the sliding clamp as the clamp loader; Latham GJ et al.; In the preceding paper (Latham, G . J., Bacheller, D . J., Pietroni, P . , and von Hippel, P . H . (1997) J . Biol . Chem . 272, 31677-31684), we demonstrated that the T4 gp44/62-ATP clamp loader binds to the C-terminal face of the gp45 sliding clamp . Here we extend these results by exploring the structural relationship between the gp43 polymerase and the gp45 sliding clamp . Using fluorescence intensity and polarization techniques, as well as photo-cross-linking methods, we present evidence that gp43, like gp44/62, binds to the C-terminal face of gp45 . In addition, we show that g43 binds to the gp45 clamp in two distinct interaction modes, depending on the presence or absence of template-primer DNA . When template-primer DNA is present, gp43 binds tightly to gp45 to form the highly processive DNA polymerase holoenzyme . Gp43 also binds to gp45 in the absence of template-primer DNA, but this interaction is more than 100 times weaker than gp43-gp45 binding on DNA . Specific interactions between gp43 and the C-terminal face of gp45 are maintained in both modes of binding . These results underscore the pivotal role of template-primer DNA in modulating the strength of protein-protein interactions during DNA synthesis and provide additional insight into the structural requirements of the replication process. J Biol Chem, 1997 Dec 12, 272(50), 31677 - 84 Structural analyses of gp45 sliding clamp interactions during assembly of the bacteriophage T4 DNA polymerase holoenzyme . II . The Gp44/62 clamp loader interacts with a single defined face of the sliding clamp ring; Latham GJ et al.; The phage T4 gp45 sliding clamp is a ring-shaped replication accessory protein that is mounted onto DNA in an ATP-dependent manner by the gp44/62 clamp loader . In the preceding paper (Pietroni, P., Young, M . C., Latham, G . J., and von Hippel, P . H . (1997) J . Biol . Chem . 272, 31666-31676), two gp45 mutants were exploited to probe interactions of the sliding clamp ring with the gp44/62 loading machinery at various steps during the clamp loading process . In this report, these studies are extended to examine the polarity of the binding interaction between gp45 and gp44/62 . Three different gp45 mutants containing a single cysteine in three topographically distinct positions were used . Several different reporter groups, including extrinsic fluorophores, a photo-cross-linker, and a biotin linker for use in a novel "streptavidin interference assay," were covalently attached to these cysteine residues . Since gp45 is a trimeric protein, these three different mutations allowed us to probe up to nine distinct local environments along the surface of the sliding clamp in the presence and absence of other replication proteins . The results show that the gp44/62-ATP clamp loader complex binds exclusively to the C-terminal (S19C) face of the gp45 ring. J Biol Chem, 1997 Dec 12, 272(50), 31666 - 76 Structural analyses of gp45 sliding clamp interactions during assembly of the bacteriophage T4 DNA polymerase holoenzyme . I . Conformational changes within the gp44/62-gp45-ATP complex during clamp loading; Pietroni P et al.; A multisubunit ring-shaped protein complex is used to tether the polymerase to the DNA at the primer-template junction in most DNA replication systems . This "sliding clamp" interacts with the polymerase, completely encircles the DNA duplex, and is assembled onto the DNA by a specific clamp loading complex in an ATP-driven process . Site-specific mutagenesis has been used to introduce single cysteine residues as reactive sites for adduct formation within each of the three subunits of the bacteriophage T4-coded sliding clamp complex (gp45) . Two such mutants, gp45S19C and gp45K81C, are reacted with the cysteine-specific photoactivable cross-linker TFPAM-3 and used to track the changes in the relative positioning of the gp45 subunits with one another and with the other components of the clamp loading complex (gp44/62) in the various stages of the loading process . Cross-linking interactions performed in the presence of nucleotide cofactors show that ATP binding and hydrolysis, interaction with primer-template DNA, and release of ADP all result in significant conformational changes within the clamp loading cycle . A structural model is presented to account for the observed rearrangements of intersubunit contacts within the complex during the loading process. Mol Microbiol, 1997 Oct, 26(2), 237 - 49 Reduced polysialic acid capsule expression in Escherichia coli K1 mutants with chromosomal defects in kpsF; Cieslewicz M et al.; Neuroinvasive Escherichia coli K1 synthesizes and assembles a polysialic acid capsule virulence factor on the external leaflet of the outer membrane . This capsule functions in pathogenesis by blocking non-immune host defence mechanisms and acting as a relatively non-immunogenic molecular mimic of the polysialic acid chains found in high concentrations on neural cell adhesion molecules of the human embryo and neonate . The synthetic, regulatory and export components for capsule expression are encoded in three functionally distinct gene blocks or regions of the 20 kb kps 'pathogenicity island' . These regions are organized as two convergently transcribed operons inserted into the monocistronic tRNA gene, pheV . The six genes of the so-called region 1 operon are transcribed in the same direction as pheV, and at least four of these genes are required for polysialic acid export . Expression of this operon is thermoregulated by transcriptional control of its first gene, kpsF . To investigate the function of region 1 further, two independent chromosomal disruptions were engineered by inserting promoterless, terminatorless kanamycin or chloramphenicol resistance cassettes into the HindIII site of the kpsF coding sequence . The chromosomal insertions were regulated by temperature in the same way as the wild-type operon, demonstrating that this control mechanism remained intact in these mutants . Chemical, immunological and ultrastructural microscopical methods demonstrated that full-length polysialic acid chains were synthesized but not exported by the kpsF mutants . This phenotype was correlated with decreased plaque diameter when the mutants were infected with the capsule-specific bacteriophage K1F . The export defect could not be complemented in trans with kpsF+ containing its cis-regulatory region because of titration of an apparent positive regulator of region 1 expression, whereas complementation was observed with a plasmid expressing kpsF from a physiologically irrelevant promoter . An N-terminal polyhistidine peptide was attached to KpsF and used to purify the overproduced polypeptide . Antibodies raised against KpsF identified at least one of its paralogues in E . coli, GutQ, suggesting that KpsF and its homologues are membrane associated . The results indicate the requirement for a precise balance between region 1 components of the capsule export machinery, and that KpsF plays a positive role in the assembly, operation or regulation of this apparatus. Nucleic Acids Res, 1997 Nov 15, 25(22), 4650 - 7 Non-homologous DNA end joining in plant cells is associated with deletions and filler DNA insertions; Gorbunova V et al.; Double strand DNA breaks in plants are primarily repaired via non-homologous end joining . However, little is known about the molecular events underlying this process . We have studied non-homologous end joining of linearized plasmid DNA with different termini configurations following transformation into tobacco cells . A variety of sequences were found at novel end junctions . Joining with no sequence alterations was rare . In most cases, deletions were found at both ends, and rejoining usually occurred at short repeats . A distinct feature of plant junctions was the presence of relatively large, up to 1.2 kb long, insertions (filler DNA), in approximately 30% of the analyzed clones . The filler DNA originated either from internal regions of the plasmid or from tobacco genomic DNA . Some insertions had a complex structure consisting of several reshuffled plasmid-related regions . These data suggest that double strand break repair in plants involves extensive end degradation, DNA synthesis following invasion of ectopic templates and multiple template switches . Such a mechanism is reminiscent of the synthesis-dependent recombination in bacteriophage T4 . It can also explain the frequent 'DNA scrambling' associated with illegitimate recombination in plants. Nucleic Acids Res, 1997 Nov 15, 25(22), 4626 - 38 Statistical modeling and analysis of the LAGLIDADG family of site-specific endonucleases and identification of an intein that encodes a site-specific endonuclease of the HNH family; Dalgaard JZ et al.; The LAGLIDADG and HNH families of site-specific DNA endonucleases encoded by viruses, bacteriophages as well as archaeal, eucaryotic nuclear and organellar genomes are characterized by the sequence motifs 'LAGLIDADG' and 'HNH', respectively . These endonucleases have been shown to occur in different environments: LAGLIDADG endonucleases are found in inteins, archaeal and group I introns and as free standing open reading frames (ORFs); HNH endonucleases occur in group I and group II introns and as ORFs . Here, statistical models (hidden Markov models, HMMs) that encompass both the conserved motifs and more variable regions of these families have been created and employed to characterize known and potential new family members . A number of new, putative LAGLIDADG and HNH endonucleases have been identified including an intein-encoded HNH sequence . Analysis of an HMM-generated multiple alignment of 130 LAGLIDADG family members and the three-dimensional structure of the I- Cre I endonuclease has enabled definition of the core elements of the repeated domain (approximately 90 residues) that is present in this family of proteins . A conserved negatively charged residue is proposed to be involved in catalysis . Phylogenetic analysis of the two families indicates a lack of exchange of endonucleases between different mobile elements (environments) and between hosts from different phylogenetic kingdoms . However, there does appear to have been considerable exchange of endonuclease domains amongst elements of the same type . Such events are suggested to be important for the formation of elements of new specficity. Nucleic Acids Res, 1997 Nov 15, 25(22), 4464 - 73 Functional recognition of fragmented operator sites by R17/MS2 coat protein, a translational repressor; Fouts DE et al.; The R17/MS2 coat protein serves as a translational repressor of replicase by binding to a 19 nt RNA hairpin containing the Shine-Dalgarno sequence and the initiation codon of the replicase gene . We have explored the structural features of the RNA operator site that are necessary for efficient translational repression by the R17/MS2 coat protein in vivo . The R17/MS2 coat protein efficiently directs lysogen formation for P22 R17 , a bacteriophage P22 derivative that carries the R17/MS2 RNA operator site within the P22 phage ant mRNA . Phages were constructed that contain fragmented operator sites such that the Shine-Dalgarno sequence and the initiation codon of the affected gene are not located within the RNA hairpin . The wild-type coat protein directs efficient lysogen formation for P22 phages that carry several fragmented RNA operator sites, including one in which the Shine-Dalgarno sequence is positioned 4 nt outside the coat protein binding site . Neither the wild-type R17/MS2 coat protein nor super-repressor mutants induce lysogen formation for a P22 phage encoding an RNA hairpin at a distance of 9 nt from the Shine-Dalgarno sequence, implying that a discrete region of biological repression is defined by the coat protein-RNA hairpin interaction . The assembly of RNA species into capsid structures is not an efficient means whereby the coat protein achieves translational repression of target mRNA transcripts . The R17/MS2 coat protein exerts translational regulation that extends considerably beyond the natural biological RNA operator site structure; however, the coat protein still mediates repression in these constructs by preventing ribosome access to linear sequence determinants of the translational initiation region by the formation of a stable RNA secondary structure . An efficient translational regulatory mechanism in bacteria appears to reside in the ability of proteins to regulate RNA folding states for host cell and phage mRNAs. J Bacteriol, 1998 Feb, 180(3), 464 - 72 Translation limits synthesis of an assembly-initiating coat protein of filamentous phage IKe; Madison-Antenucci S et al.; Translation is shown to be downregulated sharply between genes V and VII of IKe, a filamentous bacteriophage classed with the Ff group (phages f1, M13, and fd) but having only 55% DNA sequence identity to it . Genes V and VII encode the following proteins which are used in very different amounts: pV, used to coat the large number of viral DNA molecules prior to assembly, and pVII, used to serve as a cap with pIX in 3 to 5 copies on the end of the phage particle that emerges first from Escherichia coli . The genes are immediately adjacent to each other and are represented in the same amounts on the Ff and IKe mRNAs . Ff gene VII has an initiation site that lacks detectable intrinsic activity yet through coupling is translated at a level 10-fold lower than that of upstream gene V . The experiments reported reveal that by contrast, the IKe gene VII initiation site had detectable activity but was coupled only marginally to upstream translation . The IKe gene V and VII initiation sites both showed higher activities than the Ff sites, but the drop in translation at the IKe V-VII junction was unexpectedly severe, approximately 75-fold . As a result, gene VII is translated at similarly low levels in IKe- and Ff-infected hosts, suggesting that selection to limit its expression has occurred. J Biol Chem, 1998 Jan 2, 273(1), 524 - 7 PinA inhibits ATP hydrolysis and energy-dependent protein degradation by Lon protease; Hilliard JJ et al.; The bacteriophage T4 PinA protein inhibited degradation of {3H}alpha-methyl casein by purified Lon protease from Escherichia coli, but inhibition was noncompetitive with respect to casein . PinA did not inhibit cleavage of the fluorogenic peptide, N-glutaryl-alanylalanylphenylalanyl-3-methoxynaphthylamide and, moreover, did not block the ability of protein substrates, such as casein, to activate cleavage of fluorogenic peptides by Lon . Thus, PinA does not block the proteolytic active site or the allosteric protein-binding site on Lon . Inhibition of basal ATPase activity was variable (50-90%), whereas inhibition of protein-activated ATPase activity was usually 80-95% . Inhibition was noncompetitive with respect to ATP . PinA did not block activation of peptide cleavage by nonhydrolyzable analogs of ATP . These data suggest that PinA does not bind at the ATPase active site of Lon and does not interfere with nucleotide binding to the enzyme . PinA inhibited cleavage of the 72-amino acid protein, CcdA, degradation of which requires ATP hydrolysis, but did not inhibit cleavage of the carboxyl-terminal 41-amino acid fragment of CcdA, degradation of which does not require ATP hydrolysis . PinA thus appears to interact at a novel regulatory or enzymatic site involved in the coupling between ATP hydrolysis and proteolysis, possibly blocking the protein unfolding or remodeling step essential for degradation of high molecular weight protein substrates by Lon. J Biol Chem, 1998 Jan 2, 273(1), 518 - 23 Isolation and characterization of the phage T4 PinA protein, an inhibitor of the ATP-dependent lon protease of Escherichia coli; Hilliard JJ et al.; The bacteriophage T4 PinA protein, expression of which leads to inhibition of protein degradation in Escherichia coli cells, has been purified from cells carrying multiple copies of the pinA gene . PinA is a heat-stable protein with a subunit Mr of 18,800 and an isoelectric point of 4.6 . Under nondenaturing conditions on a gel filtration column, PinA migrated in two peaks corresponding to a dimer and a tetramer . Purified PinA inhibited ATP-dependent protein degradation by Lon protease in vitro; it did not inhibit the activity of other E . coli ATP-dependent proteases, ClpAP or ClpYQ . Furthermore, PinA did not inhibit ATP-independent proteolysis in E . coli cell extracts . PinA binds with high affinity to Lon protease (Kd approximately 10 nM for dimer binding), and a complex with approximately 1 dimer of PinA per tetramer of Lon protease could be isolated by gel filtration . Lon activity was partially restored upon dilution of the PinA-Lon complex to subnanomolar concentrations, indicating that inhibition was reversible and that PinA did not covalently modify Lon protease . PinA was not cleaved by Lon protease, and heating the Lon-PinA complex at 65 degrees C denatured Lon protease and released active PinA . The properties of PinA in vitro suggest that PinA inhibits protein degradation in vivo by forming a tight, reversible complex with Lon protease. J Biol Chem, 1998 Jan 2, 273(1), 459 - 65 Versatile action of Escherichia coli ClpXP as protease or molecular chaperone for bacteriophage Mu transposition; Jones JM et al.; The molecular chaperone ClpX of Escherichia coli plays two distinct functions for bacteriophage Mu DNA replication by transposition . As specificity component of a chaperone-linked protease, it recognizes the Mu immunity repressor for degradation by the peptidase component ClpP, thus derepressing Mu transposition functions . After strand exchange has been promoted by MuA transposase, ClpX alone can alter the conformation of the transpososome (the complex of MuA with Mu ends), and the remodeled MuA promotes transition to replisome assembly . Although ClpXP can degrade MuA, the presence of both ClpP and ClpX in the reconstituted transposition system did not destroy MuA essential for initiation of DNA replication by specific host replication enzymes . Levels of ClpXP needed to overcome inhibition by the repressor did not prevent MuA from promoting strand transfer, and ClpP stimulated alteration of the transpososome by ClpX . Apparently intact MuA was still present in the resulting transpososome, promoting initiation of Mu DNA replication by specific replication enzymes . The results indicate that ClpXP can discriminate repressor and MuA in the transpososome as substrates of the protease or the molecular chaperone alone, degrading repressor while remodeling MuA for its next critical function. Genetics, 1997 Nov, 147(3), 1401 - 9 UV- and gamma-radiation sensitive mutants of Arabidopsis thaliana; Jiang CZ et al.; Arabidopsis seedlings repair UV-induced DNA damage via light-dependent and independent pathways . The mechanism of the "dark repair" pathway is still unknown . To determine the number of genes required for dark repair and to investigate the substrate-specificity of this process we isolated mutants with enhanced sensitivity to UV radiation in the absence of photoreactivating light . Seven independently derived UV sensitive mutants were isolated from an EMS-mutagenized population . These fell into six complementation groups, two of which (UVR1 and UVH1) have previously been defined . Four of these mutants are defective in the dark repair of UV-induced pyrimidine {6-4}pyrimidinone dimers . These four mutant lines are sensitive to the growth-inhibitory effects of gamma radiation, suggesting that this repair pathway is also involved in the repair of some type of gamma-induced DNA damage product . The requirement for the coordinate action of several different gene products for effective repair of pyrimidine dimers, as well as the nonspecific nature of the repair activity, is consistent with nucleotide excision repair mechanisms previously described in Saccharomyces cerevisiae and nonplant higher eukaryotes and inconsistent with substrate-specific base excision repair mechanisms found in some bacteria, bacteriophage, and fungi. Genetics, 1997 Nov, 147(3), 953 - 9 The advantage of sex in the RNA virus phi6; Chao L et al.; When laboratory populations of the RNA bacteriophage phi6 are subjected to intensified genetic drift, they experience a decline in fitness . These experiments demonstrate that the average effect of mutations is deleterious, and they are used to suggest that Muller's ratchet can operate in these viruses . However, the operation of Muller's ratchet does not alone guarantee an advantage of sex . When phi6 populations were subjected to a series of bottlenecks of one individual and then crossed, the measured advantage of sex was not significant . To determine whether a small sample size, as opposed to allelism or another explanation, can account for the negative result, we repeated the phi6 experiments by crossing a larger set of populations . We found that bottlenecked populations of phi6 could recover fitness through mutations . However, hybrids produced by crossing the populations recovered an additional amount over the contribution of mutations . This additional amount, which represents an advantage of sex to phi6, was determined to be significantly greater than zero . These results provide indirect support for an advantage of sex through Muller's ratchet . However, we also use our experimental design and results to propose an alternative to Muller's ratchet as a model for the evolution of sex. Biochimie, 1997 Sep, 79(8), 527 - 31 Regulation of protein synthesis by minigene expression; Hernandez J et al.; Peptidyl-tRNA hydrolase (Pth), an enzyme essential for Escherichia coli viability, scavenges peptidyl-tRNA released during abortive polypeptide chain elongation . Bacterial strains of E coli partially defective in Pth activity are unable to maintain bacteriophage lambda growth . Phage mutations that overcome the bacterial defect have been located to several regions in the lambda genome named bar . Plasmid constructs expressing just the bar region are toxic and cause a general arrest of protein synthesis in Pth-defective cells . Inspection of the nucleotide sequence from two bar regions reveals the short coding sequence AUG AUA Stop, spaced by an AT-rich segment from a Shine Dalgarno-like sequence (S-D) . These sequences have been named minigenes . Base changes altering the putative S-D, the two sense codons, or the stop codon have been found to reduce Bar-toxicity . Transcripts containing bar function as mRNA . Upon expression in pth mutants, wild-type (bar+) transcripts are found associated with ribosomes . In addition, bar+ RNA forms ternary complexes with the 30S ribosomal subunit and the initiator tRNA and can be released upon run-off translation in the same way as an authentic mRNA . A cell free system for protein synthesis reproduces the in vivo effects: bar+ expression inhibits protein synthesis, bar+ RNA sequences are associated with ribosomes in the inhibited extracts, addition of purified Pth restores synthesis, and excess of tRNA(Lys), specific for the last sense codon in a mutant toxic minigene, prevents protein synthesis inhibition . Also, bar expression promotes association of methionine with ribosomes possibly in a translation complex . These results are consistent with a model proposing tRNA starvation to explain the behaviour of a pth mutant, thermosensitive for protein synthesis. Carcinogenesis, 1997 Dec, 18(12), 2403 - 14 Specificity of mutagenesis by 4-aminobiphenyl: mutations at G residues in bacteriophage M13 DNA and G-->C transversions at a unique dG(8-ABP) lesion in single-stranded DNA; Verghis SB et al.; Mutagenesis by the human bladder carcinogen 4-aminobiphenyl (ABP) was studied in single-stranded DNA from a bacteriophage M13 cloning vector . In comparison to ABP lesions in double-stranded DNA, lesions in single-stranded DNA were approximately 70-fold more mutagenic and 50-fold more genotoxic . Sequencing analysis of ABP-induced mutations in the lacZ gene revealed exclusively base-pair substitutions, with over 80% of the mutations occurring at G sites; the G at position 6310 accounted for 25% of the observed mutations . Among the sequence changes at G sites, G-->T transversions predominated, followed by G-->C transversions and G-->A transitions . In order to further elucidate the mutagenic mechanism of ABP, an oligonucleotide containing the major DNA adduct, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG(8-ABP)), was situated within the PstI site of a single-stranded M13 genome . After in vivo replication of the adduct containing ABP-modified and control (unadducted) genomes, the mutational frequency and mutational specificity of the dG(8-ABP) lesion were determined . The targeted mutational efficiency was approximately 0.01%, and the primary mutation observed was the G-->C transversion . Thus dG(8-ABP), albeit weakly mutagenic at the PstI site, can contribute to the mutational spectrum of ABP lesions. Biophys J, 1998 Jan, 74(1), 559 - 68 Role of the scaffolding protein in P22 procapsid size determination suggested by T = 4 and T = 7 procapsid structures; Thuman-Commike PA et al.; Assembly of bacteriophage P22 procapsids requires the participation of approximately 300 molecules of scaffolding protein in addition to the 420 coat protein subunits . In the absence of the scaffolding, the P22 coat protein can assemble both wild-type-size and smaller size closed capsids . Both sizes of procapsid assembled in the absence of the scaffolding protein have been studied by electron cryomicroscopy . These structural studies show that the larger capsids have T = 7 icosahedral lattices and appear the same as wild-type procapsids . The smaller capsids possess T = 4 icosahedral symmetry . The two procapsids consist of very similar penton and hexon clusters, except for an increased curvature present in the T = 4 hexon . In particular, the pronounced skewing of the hexons is conserved in both sizes of capsid . The T = 7 procapsid has a local non-icosahedral twofold axis in the center of the hexon and thus contains four unique quasi-equivalent coat protein conformations that are the same as those in the T = 4 procapsid . Models of how the scaffolding protein may direct these four coat subunit types into a T = 7 rather than a T = 4 procapsid are presented. Annu Rev Entomol, 1998, 43, 421 - 47 Higher-order predators and the regulation of insect herbivore populations; Rosenheim JA; Empirical research has not supported the prediction that populations of terrestrial herbivorous arthropods are regulated solely by their natural enemies . Instead, both natural enemies (top-down effects) and resources (bottom-up effects) may play important regulatory roles . This review evaluates the hypothesis that higher-order predators may constrain the top-down control of herbivore populations . Natural enemies of herbivorous arthropods generally are not top predators within terrestrial food webs . Insect pathogens and entomopathogenic nematodes inhabiting the soil may be attacked by diverse micro- and mesofauna . Predatory and parasitic insects are attacked by their own suite of predators, parasitoids, and pathogens . The view of natural enemy ecology that has emerged from laboratory studies, where natural enemies are often isolated from all elements of the biotic community except for their hosts or prey, may be an unreliable guide to field dynamics . Experimental work suggests that interactions of biological control agents with their own natural enemies can disrupt the effective control of herbivore populations . Disruption has been observed experimentally in interactions of bacteria with bacteriophages, nematodes with nematophagous fungi, parasitoids with predators, parasitoids with hyperparasitoids, and predators with other predators . Higher-order predators have been little studied; manipulative field experiments will be especially valuable in furthering our understanding of their roles in arthropod communities. J Biochem (Tokyo), 1997 Nov, 122(5), 1046 - 51 Discovery of a novel thrombopoietin mimic agonist peptide; Kimura T et al.; A random phage peptide library was constructed for the filamentous bacteriophage fuse5 . The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein . This library, containing 1x10(9) different phages, was screened with a human immunoglobulin fusion protein containing the extracellular region of human thrombopoietin receptor . Several phages were isolated following four cycles of enrichment and amplification . These phages specifically bound to the fusion protein . One phage peptide acted as an agonist of the thrombopoietin receptor, since it stimulated the proliferation of thrombopoietin-dependent cells and the differentiation of mouse bone marrow cells to megakaryocytes . The amino acid sequence of this peptide is not present in the primary amino acid sequence of thrombopoietin . This discovery may lead to the design of a small-molecular mimic of thrombopoietin. J Biochem (Tokyo), 1997 Nov, 122(5), 977 - 82 Efficiency of recombination by Cre transient expression in embryonic stem cells: comparison of various promoters; Araki K et al.; The Cre-loxP recombination system of bacteriophage P1 is frequently utilized in genetic manipulation in embryonic stem (ES) cells . The level of Cre expression is critical to induce loxP site-specific recombination in ES cells . To compare the efficiency of recombination, we constructed four Cre expression vectors driven by different promoters: cytomegarovirus/chicken beta-actin (CAG) promoter, human polypeptide chain elongation factor 1alpha (hEF-1alpha) promoter, mouse phosphoglycerate kinase-1 (mPGK) promoter, and polyoma enhancer/herpes simplex virus thymidine kinase (MC1) promoter . We introduced these Cre expression vectors by electroporation into three ES cell lines carrying a single copy of CAG-loxP-chloramphenicol acetyltransferase (CAT) gene-loxP-beta-galactosidase (beta-gal) gene construct . Since the Cre-mediated recombination leads to excision of the CAT gene, the efficiency of recombination can be monitored as beta-gal expression . No selection system was used in the experiments . The maximum recombination frequency was obtained when the CAG promoter was used, followed by the hEF-1alpha promoter, the mPGK promoter and the MC1 promoter in order . These results indicate that the efficiency of recombination in transient expression system correlates with the promoter activity of Cre expression vector . Thus, it is important to choose the promoter for effective recombination by Cre. Nature, 1998 Jan 15, 391(6664), 251 - 8 Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 A resolution; Doublie S et al.; DNA polymerases change their specificity for nucleotide substrates with each catalytic cycle, while achieving error frequencies in the range of 10(-5) to 10(-6) . Here we present a 2.2 A crystal structure of the replicative DNA polymerase from bacteriophage T7 complexed with a primer-template and a nucleoside triphosphate in the polymerase active site . The structure illustrates how nucleotides are selected in a template-directed manner, and provides a structural basis for a metal-assisted mechanism of phosphoryl transfer by a large group of related polymerases. J Basic Microbiol, 1997, 37(6), 451 - 63 Replication of plasmids derived from P1, F, R1, R6K and RK2 replicons in amino acid-starved Escherichia coli stringent and relaxed strains; Wrobel B et al.; Replication of mini-plasmids derived from bacteriophage P1 and naturally existing plasmids F, R1, R6K and RK2 in otherwise isogenic relA+ and relA- Escherichia coli strains during amino acid starvation and limitation was investigated . Since it was previously demonstrated that inhibition of DNA synthesis or amplification of plasmid DNA may depend on the nature of deprived amino acid, we starved bacteria for five different amino acids . We found differential replication of all these plasmids but RK2 (which did not replicate at all in amino acid-starved bacteria) during the stringent and relaxed response . While in almost all cases plasmid DNA replication was inhibited during the stringent response irrespective of the nature of deprived amino acid, wild-type or copy-up mini-P1, mini-F and mini-R1 plasmids replicated in relA- bacteria depending on the kind of starvation . R6K-derived plasmids harbouring ori beta and gamma (but not those containing ori alpha, beta and gamma or only ori gamma) were able to replicate in relA- bacteria starved for all tested amino acids . Possible explanations for the mechanisms of regulation of replication of plasmids derived from P1, F, R1, R6K and RK2 during amino acid starvation are discussed . Our results also indicate that, like in the case of some other replicons, appropriate amino acid starvation or limitation may be used as a method for efficient amplification of plasmids derived from P1, F, R1 and R6K. Toxicol Appl Pharmacol, 1997 Dec, 147(2), 459 - 64 Genotoxicity of acyl glucuronide metabolites formed from clofibric acid and gemfibrozil: a novel role for phase-II-mediated bioactivation in the hepatocarcinogenicity of the parent aglycones? Sallustio BC, Harkin LA, Mann MC, Krivickas SJ, Burcham PC. Glucuronides formed from carboxylate-containing xenobiotics are more chemically reactive than most Phase II conjugates . However, while they have been shown to form protein adducts, their reactions with DNA have received little attention . We thus used the M13 forward mutational assay to assess the genotoxicity of acyl glucuronides formed from two widely used fibrate hypolipidemics, clofibric acid and gemfibrozil . Single-stranded M13mp19 bacteriophage DNA was incubated in pH 7.4 buffer for 16 h in the presence of 0, 1, 2.5, and 5 mM concentrations of each glucuronide as well as the respective aglycones . The modified DNA was then transfected into SOS-induced competent Escherichia coli JM105 cells and the transfection efficiency was determined after phage growth overnight at 37 degrees C . Significantly, both acyl glucuronides, but not the aglycones, caused a concentration-dependent decrease in the transfection efficiency of the DNA, with a greater than 80% decrease in phage survival produced by the 5 mM concentrations of the glucuronides . No increase in lacZa mutations accompanied the loss of phage survival . We propose that these genotoxic effects involve reactions with nucleophilic centers in DNA via a Schiff base mechanism that is analogous to the glycosylation of DNA by endogenous sugars . Since strand nicking is known to accompany such damage, we also analyzed glucuronide-treated pSP189 plasmids for strand breakages via agarose gel electrophoresis . Both clofibric acid and gemfibrozil glucuronides produced significant concentration-related strand nicking and exhibited over 10-fold greater reactivity than the endogenous glycosylating agent, glucose 6-phosphate . On the basis of these findings, the possibility that this novel bioactivation route participates in the carcinogenicity of the fibrate hypolipidemics deserves investigation. Biochem Biophys Res Commun, 1998 Jan 6, 242(1), 10 - 5 Isolation and characterization of EMS induced splicing defective point mutations within the intron of the nrdB gene of bacteriophage T4; Khan AU et al.; The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598-base-pair self-splicing intron which is closely related to other group I introns of T4 and eukaryotes . The screening, isolation, and mapping of 31 nrdB intron mutations were conducted by the strategic usage of the white halo phenotype exhibited by T4 mutants defective in dyhydrofolate reductase or thymidylate synthase . These intron mutations cluster towards the ends, mainly the 3' end, and show a defect in self-splicing . These mutations map in regions of conserved structural elements, thus supporting secondary structure predictions . A distinct pattern of clustering is observed with the highest number of mutations mapping within three of the smaller regions (A, C, and D) of the nrdB intron and no mutations mapping in the largest (B) region . The highest density of mutations mapped in the smallest region (C) of the intron, containing only 96 bases, thus showing a distinct pattern of clustering within the catalytic core. J Biol Chem, 1997 Dec 19, 272(51), 32267 - 73 Asymmetric interactions of hexameric bacteriophage T7 DNA helicase with the 5'- and 3'-tails of the forked DNA substrate; Ahnert P et al.; Bacteriophage T7 DNA helicase requires two noncomplementary single-stranded DNA (ssDNA) tails next to a double-stranded DNA (dsDNA) region to initiate DNA unwinding . The interactions of the helicase with the DNA were investigated using a series of forked DNAs . Our results show that the helicase interacts asymmetrically with the two tails of the forked DNA . When the helicase was preassembled on the forked DNA before the start of unwinding, a DNA with 15-nucleotide (nt) 3'-tail and 35-nt 5'-tail was unwound with optimal rates close to 60 base pairs/s at 18 degrees C . When the helicase was not preassembled on the DNA, a >65-nt long 5'-tail was required for maximal unwinding rates of 12 base pairs/s . We show that the helicase interacts specifically with the ssDNA region and maintains contact with both ssDNA strands during DNA unwinding, since conversion of the two ssDNA tails to dsDNA structures greatly inhibited unwinding, and the helicase was unable to unwind past a nick in the dsDNA region . These studies have provided new insights into the mechanism of DNA unwinding . We propose an exclusion model of DNA unwinding in which T7 helicase hexamer interacts mainly with the ssDNA strands during DNA unwinding, encircling the 5'-strand and excluding the 3'-strand from the hole. Appl Environ Microbiol, 1998 Jan, 64(1), 304 - 9 Role of the air-water-solid interface in bacteriophage sorption experiments; Thompson SS et al.; Batch sorption experiments were carried out with the bacteriophages MS2 and phi X174 . Two types of reactor vessels, polypropylene and glass, were used . Consistently lower concentrations of MS2 were found in the liquid phase in the absence of soil (control blanks) than in the presence of soil after mixing . High levels of MS2 inactivation (approximately 99.9%) were observed in control tubes made of polypropylene (PP), with comparatively little loss of virus seen in PP tubes when soil was present . Minimal inactivation of MS2 was observed when the air-water interface was completely eliminated from PP control blanks during mixing . All batch experiments performed with reactor tubes made of glass demonstrated no substantial inactivation of MS2 . In similar experiments, bacteriophage phi X174 did not undergo inactivation in either PP or glass control blanks, implying that this virus is not affected by the same factors which led to inactivation of MS2 in the PP control tubes . When possible, phage adsorption to soil was calculated by the Freundlich isotherm . Our data suggest that forces associated with the air-water-solid interface (where the solid is a hydrophobic surface) are responsible for inactivation of MS2 in the PP control tubes . The influence of air-water interfacial forces should be carefully considered when batch sorption experiments are conducted with certain viruses. Acta Cient Venez, 1996, 47(2), 121 - 6 {In vivo cloning of genes of Escherichia coli K12 by means of homologue recombination}; Ruiz O et al.; Plasmid pT153, a stable recombinant between pBR322 plasmid and M13 bacteriophage, and Tn10 transposon were employed for in vivo cloning of a chromosomal segment of Escherichia coli including the nar locus . The strategy consisted in creating an homology between pT153 and the E . coli chromosome, incorporating a Tn10 transposon close to the nar locus of a polA12 temperature-sensitive strain . The selection of clones carrying the plasmid into the chromosome was realized at 42 degrees C, with ampiciline, taken advantage that the replicon of pT153 requires the ADN Polymerase I for its functioning . The plasmid integrated in the polA12 strain has the opportunity of excising and carrying part of bacterial genome and autonomically replicating after a thermal change from 42 degrees C to 30 degrees C . The stable recombinant plasmids could be efficiently transduced with the M13 bacteriophage to a E . coli strain (delta narGHJI), obtaining Nit+ Apr transductant clones . The versatility of this method of E . coli gene cloning is the facility to create the homologue region anywhere in the bacterial genome and the efficient transduction of pT153 plasmid by M13 bacteriophage. Gene, 1997 Nov 20, 202(1-2), 193 - 201 cDNA cloning and chromosomal mapping of genes encoding novel protein kinases termed PKU-alpha and PKU-beta, which have nuclear localization signal; Yamakawa A et al.; We have cloned cDNAs for novel serine/threonine protein kinases (PK), termed PKU-alpha and PKU-beta, by screening a bacteriophage expression library for kinase activity . Sequence analysis of PKU-alpha and PKU-beta genes revealed that their open reading frames (ORF) were 2151 and 2361 nucleotides (nt) encoding polypeptides of 717 and 787 amino acid (aa) residues, respectively . The deduced aa sequences of PKU-alpha and PKU-beta contained typical serine/threonine PK domains at the C-terminal region and were 86% identical to each other, indicating that they belong to the same PK family . Northern analysis reveals that they are expressed in nearly all human tissues and in cultured cells . The genes for PKU-alpha and PKU-beta were mapped to chromosome 17q23 and 8p12-p22, respectively, by fluorescence in situ hybridization . The proteins encoded by both cDNAs contain a putative nuclear localization signal (NLS) in their N-terminal region . These signals are likely to function in nuclear localization . Glutathione S-transferase (GST)-fusions to regions of PKU-alpha and beta containing the NLS were efficiently localized to the nucleus . In addition, PKU-beta transiently expressed in COS-1 cells was predominantly nuclear . PKU-alpha and PKU-beta differ: a consensus sequence for a nt binding motif is present near the NLS of PKU-beta . These results suggest that PKU-alpha and beta may phosphorylate serine and/or threonine residues on similar proteins, but their activities are regulated through distinct interactions with a nuclear component. Gene, 1997 Dec 12, 203(2), 209 - 16 Genomic targeting of a bicistronic DNA fragment by Cre-mediated site-specific recombination; Kolb AF et al.; The Cre-recombinase of bacteriophage P1 catalyses site-specific recombination between DNA fragments containing loxP sites . Targeting of predefined genomic loci can be achieved by Cre-mediated linkage of a promoterless resistance marker gene to a floxed promoter pre-existing in the genome . In order to avoid the introduction of plasmid sequences into the host genome, we have constructed a series of plasmids in which the DNA segment to be integrated is flanked by two loxP sites . We show here that this floxed targeting fragment is reliably and effectively separated from the vector backbone and integrated into genomic loxP sites by Cre-mediated site-specific recombination in mammalian cells . We also demonstrate that by using this approach two convergent, promoterless coding regions can simultaneously be linked to two independent promoter elements at a pre-existing genomic loxP site . This methodology will be particularly useful for genomic targeting experiments in transgenic animals. Protein Expr Purif, 1997 Dec, 11(3), 233 - 40 Balancing the production of two recombinant proteins in Escherichia coli by manipulating plasmid copy number: high-level expression of heterodimeric Ras farnesyltransferase; Tsao KL et al.; The native Ras farnesyltransferase heterodimer (alpha beta) and a heterodimer with a truncated alpha subunit (alpha' beta) were overproduced at a high level and in a soluble form in Escherichia coli . The alpha, alpha', and beta subunits were synthesized from individual plasmid vectors under the control of bacteriophage T7 promoters . Although each subunit could be expressed at a high level by itself, when either the alpha or alpha' and the beta plasmid were present in cells at the same time, the alpha and alpha' subunits were preferentially expressed to such a degree that little or none of the beta subunit accumulated . A satisfactory balance between both combinations of subunits (alpha beta and alpha' beta) was achieved by making incremental adjustments in the copy number of the beta-encoding plasmid . As the copy number of the beta plasmid increased, so did the ratio of beta:alpha or beta:alpha', but there was little difference in the total amount of recombinant protein (alpha + beta or alpha' + beta) that was produced . This may be a generally useful method for balancing the production of two recombinant polypeptides in E . coli . A noteworthy advantage of this approach is that it can be undertaken without first determining the cause of the imbalance. J Mol Biol, 1997 Nov 21, 274(1), 1 - 7 Activation of P2 late transcription by P2 Ogr protein requires a discrete contact site on the C terminus of the alpha subunit of Escherichia coli RNA polymerase; Wood LF et al.; Bacteriophage P2 late transcription requires the product of the P2 ogr gene . Ogr-dependent transcription from P2 late promoters is blocked by certain point mutations affecting the alpha subunits of the host RNA polymerase . An alanine scan spanning the putative activation target in the alpha C-terminal domain (alphaCTD) was carried out to identify individual residues essential for Ogr-dependent transcription from P2 late promoters . In addition, the effects of alanine substitutions in the regions of the alphaCTD previously reported to affect CAP-dependent activation of the lac promoter and UP-element DNA binding were examined . Residues E286, V287, L289 and L290 in helix 3, and residue L300 at the beginning of helix 4, define a surface-exposed patch on the alphaCTD important for Ogr-dependent activation . These residues, adjacent to the recently identified DNA-binding determinants, constitute a newly identified activation surface for protein:protein contact . Alanine substitutions at some of the residues that affect UP-element DNA binding also impaired activation . This suggests that upstream DNA-alpha contacts, in addition to alpha-Ogr contacts, may be important in P2 late transcription . Other residues implicated in the interaction of alpha with CAP are not required for activation by Ogr, consistent with previous genetic evidence suggesting that these activators contact different sites on the alphaCTD . Photochem Photobiol, 1997 Nov, 66(5), 672 - 5 Solar UVB dosimetry by amplification of short and long segments in phage lambda DNA; Yoshida H et al.; DNA UVB dosimeters, consisting of minidots of dried bacteriophage DNA placed on a UV-transparent polymer film, were analyzed by polymerase chain reactions (PCR) . Ultraviolet-B dosimetry obtained with amplification of phage lambda DNA of segments of 1.08 kilobase pairs (kbp) and 2.24 kbp was compared with that obtained with amplification of a 0.5 kbp segment (H . Yoshida and J . D . Regan, 66, 82-88, Photochem . Photobiol . 1997) . The number of lesions in each segment induced by UV radiation is proportional to the size of the amplified segments; thus, the average lesion frequency per unit dose was greatest in the 2.24 kbp and least in the 0.5 kbp segment . The average lesion frequency per unit dose was approximately 3.5 x 10(-2) and approximately 11.9 x 10(-2) m2 kJ-1 for 1.08 kbp and 2.24 kbp, respectively, compared to that for 0.5 kbp of approximately 1.7 x 10(-2) m2 kJ-1 at 50 ng DNA . Dependability of DNA dosimeters, containing 50 ng and 100 ng, was tested by placing the DNA dosimeters for a time period of either 1 or 2 days outdoors on 8-12 January at Melbourne, FL . The daily dose was obtained directly with amplification of the 2.24 kbp segment and the 2 day continuous dose was obtained with amplification of the 1.08 kbp segment . Although the average lesion frequencies were different, both 50 ng and 100 ng DNA dosimeters provided about the same UVB dose, equivalent to the dose applied with a solar UVB simulator . The total UVB dose for 4 days obtained by amplification of the 1.08 and 2.24 kbp segments was 19.4-20.8 kJ m-2, which is within experimental error with the 4 day continuous dose obtained with 0.5 kbp segments . The average daily dose obtained by 0.5 kbp and 1.08 kbp agreed with the average daily dose directly obtained with 2.24 kbp. EMBO J, 1997 Nov 17, 16(22), 6886 - 95 The phiX174-type primosome promotes replisome assembly at the site of recombination in bacteriophage Mu transposition; Jones JM et al.; Initiation of Escherichia coli DNA synthesis primed by homologous recombination is believed to require the phiX174-type primosome, a mobile priming apparatus assembled without the initiator protein DnaA . We show that this primosome plays an essential role in bacteriophage Mu DNA replication by transposition . Upon promoting transfer of Mu ends to target DNA, the Mu transpososome undergoes transition to a pre-replisome that permits initiation of DNA synthesis only in the presence of primosome assembly proteins PriA, DnaT, DnaB and DnaC . These assembly proteins promote the engagement of primase and DNA polymerase III holoenzyme, initiating semi-discontinuous replication preferentially at the Mu left end . The results indicate that these proteins play a crucial role in promoting replisome assembly on a recombination intermediate. J Bacteriol, 1998 Jan, 180(1), 46 - 51 Endonuclease V (nfi) mutant of Escherichia coli K-12; Guo G et al.; Endonuclease V (deoxyinosine 3' endonuclease), the product of the nfi gene, has a specificity that encompasses DNAs containing dIMP, abasic sites, base mismatches, uracil, and even untreated single-stranded DNA . To determine its importance in DNA repair pathways, nfi insertion mutants and overproducers (strains bearing nfi plasmids) were constructed . The mutants displayed a twofold increase in spontaneous mutations for several markers and an increased sensitivity to killing by bleomycin and nitrofurantoin . An nfi mutation increased both cellular resistance to and mutability by nitrous acid . This agent should generate potential cleavage sites for the enzyme by deaminating dAMP and dCMP in DNA to dIMP and dUMP, respectively . Relative to that of a wild-type strain, an nfi mutant displayed a 12- to 1,000-fold increase in the frequency of nitrite-induced mutations to streptomycin resistance, which are known to occur in A x T base pairs . An nfi mutation also enhanced the lethality caused by a combined deficiency of exonuclease III and dUTPase, which has been attributed to unrepaired abasic sites . However, neither the deficiency nor the overproduction of endonuclease V affected the growth of the single-stranded DNA phages M13 or phiX174 nor of Uracil-containing bacteriophage lambda . These results suggest that endonuclease V has a significant role in the repair of deaminated deoxyadenosine (deoxyinosine) and abasic sites in DNA, but there was no evidence for its cleavage in vivo of single-stranded or uracil-containing DNA. J Virol, 1998 Jan, 72(1), 717 - 25 Inhibition of human cytomegalovirus DNA maturation by a benzimidazole ribonucleoside is mediated through the UL89 gene product; Underwood MR et al.; 2-Bromo-5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (BDCRB) is a member of a new class of benzimidazole ribonucleosides which inhibit human cytomegalovirus (HCMV) late in the replication cycle without inhibiting viral DNA synthesis . We show here that polygenomic concatemeric HCMV DNA does not mature to unit genome length in the presence of BDCRB . To discover the locus of action, virus resistant to BDCRB was selected by serial passage in the presence of the compound . Genetic mapping experiments with BDCRB-resistant virus demonstrated that the resistance phenotype mapped to one amino acid (Asp344Glu; low resistance) or two amino acids (Asp344Glu and Ala355Thr; high resistance) within the product of exon 2 of the HCMV U(L)89 open reading frame . The HCMV U(L)89 open reading frame and its homologs are among the most conserved open reading frames in the herpesviruses, and their products have sequence similarities to a known ATP-dependent endonuclease from the double-stranded DNA bacteriophage T4 . These findings strongly suggest that BDCRB prevents viral DNA maturation by interacting with a U(L)89 gene product and that the U(L)89 open reading frame may encode an endonucleolytic subunit of the putative HCMV terminase . Further, since mammalian cell DNA replication does not involve a DNA maturation step, compounds which inhibit viral DNA maturation should be selective and safe. Genomics, 1997 Dec 1, 46(2), 183 - 90 Construction of a 780-kb PAC, BAC, and cosmid contig encompassing the minimal critical deletion involved in B cell chronic lymphocytic leukemia at 13q14.3; Bouyge-Moreau I et al.; A putative tumor suppressor gene involved in B cell chronic lymphocytic leukemia (B-CLL) was mapped to human chromosome 13q14.3 close to the genetic markers D13S25 and D13S319 . We constructed a 780-kb-long contig composed of cosmids, bacterial artificial chromosomes, and bacteriophage P1-derived artificial chromosomes that provides essential information and tools for the positional cloning of this gene . The conting contains both flanking markers as well as several additional genetic markers, three ESTs, and one potential CpG island . In addition, using one B-CLL patient, we characterized a small internal deleted region of 550 kb . Comparing this deletion with other recently published deletions narrows the minimally deleted area to less than 100 kb in our physical map . This deletion core region should contain all or part of the disrupted in B cell malignancies tumor suppressor gene. J Mol Biol, 1997 Nov 14, 273(5), 958 - 77 Role of open complex instability in kinetic promoter selection by bacteriophage T7 RNA polymerase; Villemain J et al.; By measuring steady-state rates of dinucleotide synthesis on double-stranded (d.s.) and partially single-stranded (p.s.s.) promoters, and topological unwinding due to open complex formation on plasmids, we have obtained evidence that open complex formation in bacteriophage T7 RNA polymerase:promoter binary complexes is thermodynamically disfavored and that the rate of collapse of the open complex is competitive with the rate of transcription initiation . It is suggested that open complex instability is a kinetic mechanism that allows T7 RNA polymerase (RNAP) to achieve promoter specificity while still allowing for efficient promoter release . Open complex instability could also provide a mechanism for modulating the KM for the initiating NTPs so as to allow different promoters to respond differently to physiological changes in NTP concentration . Mol Med, 1997 Nov, 3(11), 740 - 9 Molecular characterization of a mouse genomic element mobilized by advanced glycation endproduct modified-DNA (AGE-DNA); Pushkarsky T et al.; BACKGROUND: DNA modified by advanced glycation endproducts (AGEs) undergoes a high frequency of insertional mutagenesis . In mouse lymphoid cells, these mutations are due in part to the transposition of host genomic elements that contain a DNA region homologous to the Alu family of repetitive elements . One particular 853 bp insertion, designated INS-1, was identified previously as a DNA element common to plasmids recovered from multiple, independent lymphoid cell transfections . MATERIALS AND METHODS: To characterize the genomic origin of this element, we used a 281-bp region of non-Alu-containing INS-1 sequence, designated . CORE, as a probe in Southern hybridization and for screening a bacteriophage mouse genomic DNA library . The resultant clones were sequenced and localized within the mouse genome . RESULTS: Two distinct genomic clones of 15 kB and 17 kB in size were isolated . A 522-bp unique region common to INS-1 and corresponding to the CORE sequence was identified in each clone . In both cases, CORE was found to be surrounded by repetitive DNA sequences: a 339-bp MT repeat at the 5' end, and a 150-bp B1 repeat at the 3' end . The CORE sequence was localized to mouse chromosome 1 . CONCLUSIONS: These studies revealed that the CORE region of INS is present in low copy number but is associated with known repetitive DNA elements . The presence of these repetitive elements may facilitate the transposition of CORE by recombination or other, more complex rearrangement events, and explain in part the origin of AGE-induced insertional mutations. Histochem J, 1997 Sep, 29(9), 685 - 93 Optimization of PCR/lambda exonuclease-mediated synthesis of sense and antisense DNA probes for in situ hybridization; Michel D et al.; In situ hybridization experiments are stringently dependent on the quality of the probes, which should be single-stranded when efficient comparison of signals obtained with antisense and control sense probes are needed . In this report, we describe an optimized synthesis of radioactive single-stranded DNA probes, without vector cloning and requiring a unique polymerization step . The sequence region selected as probe is amplified by polymerase chain reaction in the presence of radiolabelled nucleotides . The sense and antisense probes are then yielded by the action of the lambda bacteriophage exonuclease, which can specifically eliminate one out of the two strands of the amplified fragments . In this way, sense and antisense probes with identical length and specific activity can be generated by selecting the primer to be phosphorylated . We have verified the efficiency of our probes for in situ hybridization of the clusterin transcripts within the peripheral olfactory system, after surgical lesion of its synaptic target. Biotechnol Prog, 1997 Nov-Dec, 13(6), 837 - 43 Thermal unfolding of bacteriophage T4 short tail fibers; Jayaraman A et al.; The short tail fibers of bacteriophage T4 are composed of a homotrimer of the product of gene 12 (P12) with a molecular weight of 165,000 . P12 is capable of reconstituting defective phage particles lacking gene 12 . After heating to 75 degrees C, P12 was able to retain 90% of its ability to reconstitute T412- particles . When heated above 75 degrees C, P12 was no longer capable of fully reconstituting defective phage particles . By 95 degrees C, the reconstitution efficiency of the P12 preparation was reduced by 4 orders of magnitude . Thermal unfolding was also monitored by heating the protein in the presence of SDS to freeze partially unfolded states; by protease hydrolysis; and by intrinsic fluorescence changes . Exposure to SDS had little effect for temperatures up to 55 degrees C, but by 65 degrees C, the reconstitution efficiency of P12 treated with 0.01% SDS dropped to less than 1% of the original titer . Thermolysin digestion of P12 heated to various temperatures showed that treated P12 started to inactivate before 45 degrees C and inactivation was essentially complete by 55 degrees C . Intrinsic fluorescence data of heated P12 indicated that the protein begins to unfold by 45 degrees C and exhibits distinct peak shifts at 60 degrees C and above 80 degrees C . We conclude that, in the absence of SDS or proteases, P12 heated to 75 degrees C can refold back to an active conformation . Trimeric P12 undergoes some irreversible denaturation between 75 and 85 degrees C, and heating between 85 and 95 degrees C results in dissociation of P12 into monomers. Mol Gen Mikrobiol Virusol, 1997, (4), 25 - 9 {Use of a phage peptide library in mapping the group-specific hemagglutinating domain of alpha-virus glycoprotein E2}; Kuz'micheva GA et al.; Phage display peptide library f88-4/15 (G . P . Smith, USA) was used for mapping the hemagglutination activity domain of glycoprotein E2 of alphaviruses . Using affinity selection and ELISA, we selected the clones binding monoclonal antibody 4H5 to Venezuelan equine encephalomyelitis virus and inhibiting alphavirus hemagglutinating activity . Analysis of the similarity between the peptides amino acid sequences with the alphavirus glycoprotein E2 sequences revealed a structural motive of 4 amino acid residues (HTSR) which was identified in the 85-88 region . Bacteriophages F36 and F19 contained motives corresponding to 102-SXXM-105 and 109-AXXP-112 regions in alphavirus proteins E2 . These data permit us to propose that the detected regions are fragments of a group-specific alphavirus hemagglutination domain. Genetics, 1997 Dec, 147(4), 1497 - 507 Exceptional convergent evolution in a virus; Bull JJ et al.; Replicate lineages of the bacteriophage phiX 174 adapted to growth at high temperature on either of two hosts exhibited high rates of identical, independent substitutions . Typically, a dozen or more substitutions accumulated in the 5.4-kilobase genome during propagation . Across the entire data set of nine lineages, 119 independent substitutions occurred at 68 nucleotide sites . Over half of these substitutions, accounting for one third of the sites, were identical with substitutions in other lineages . Some convergent substitutions were specific to the host used for phage propagation, but others occurred across both hosts . Continued adaptation of an evolved phage at high temperature, but on the other host, led to additional changes that included reversions of previous substitutions . Phylogenetic reconstruction using the complete genome sequence not only failed to recover the correct evolutionary history because of these convergent changes, but the true history was rejected as being a significantly inferior fit to the data . Replicate lineages subjected to similar environmental challenges showed similar rates of substitution and similar rates of fitness improvement across corresponding times of adaptation . Substitution rates and fitness improvements were higher during the initial period of adaptation than during a later period, except when the host was changed. J Biol Chem, 1997 Nov 28, 272(48), 30147 - 53 Kinetic mechanism of GTP binding and RNA synthesis during transcription initiation by bacteriophage T7 RNA polymerase; Jia Y et al.; We have used stopped-flow and rapid chemical quench-flow methods to investigate the kinetics of the early steps during transcription initiation by bacteriophage T7 RNA polymerase . Most promoters of T7 RNA polymerase initiate with two GTPs . The kinetics of GTP binding was investigated by monitoring the fluorescence changes resulting from GTP binding to polymerase and fluorescent 2-aminopurine-containing promoter DNA complex . Scheme 1 was determined from studies of T7 Phi10 promoter at 25 degrees C, where (E.D)n represents the polymerase.DNA complex in different conformations . GTPE and GTPI represent the elongating and initiating GTP molecules incorporated at the +2 and +1 positions, respectively . Our studies show that GTP at the elongation site binds with at least 10-fold tighter affinity than the GTP at the initiation site . Two conformational changes were revealed upon GTP binding to the polymerase.2-aminopurine DNA complex . The first conformational change occurred upon GTP binding to the elongation site . This conformational change was reversible, and studies with partially melted DNA and incorrect NTPs suggested that it may represent a DNA melting and/or base pairing step . A second rate-limiting conformational change whose rate was same as the maximum rate of pppGpG synthesis occurred after two GTPs were bound . As with DNA polymerases, this rate-limiting conformational change probably occurs at each NMP incorporation event and may be involved in proper positioning of the initiation and the elongating GTPs within the polymerase active site to achieve efficient and accurate RNA synthesis. Science, 1997 Nov 28, 278(5343), 1635 - 8 The filamentous phage pIV multimer visualized by scanning transmission electron microscopy; Linderoth NA et al.; A family of homomultimeric outer-membrane proteins termed secretins mediates the secretion of large macromolecules such as enzymes and filamentous bacteriophages across bacterial outer membranes to the extracellular milieu . The secretin encoded by filamentous phage f1 was purified . Mass determination of individual molecules by scanning transmission electron microscopy revealed two forms, a unit multimer composed of about 14 subunits and a multimer dimer . The secretin is roughly cylindrical and has an internal diameter of about 80 angstroms, which is large enough to accommodate filamentous phage (diameter of 65 angstroms). Mutat Res, 1997 Oct, 385(1), 59 - 74 Incomplete complementation of the DNA repair defect in cockayne syndrome cells by the denV gene from bacteriophage T4 suggests a deficiency in base excision repair; Francis MA et al.; Endonuclease V (denV) from bacteriophage T4 has been examined for its ability to complement the repair defect in Cockayne syndrome (CS) cells of complementation groups A and B . CS is an autosomal recessive disorder characterized by hypersensitivity to UV light and a defect in the preferential repair of UV-induced lesions in transcriptionally active DNA by the nucleotide excision repair (NER) pathway . The denV gene was introduced into non-transformed normal and CS fibroblasts transiently via a recombinant adenovirus (Ad) vector and into SV40-transformed normal and CS cells via a retroviral vector . Expression of denV in CS-A cells resulted in partial correction of the UV-sensitive phenotype in assays of gene-specific repair and cell viability, while correction of CS-B cells by expression of denV in the same assays was minimal or non-existent . In contrast, denV expression led to enhanced host cell reactivation (HCR) of viral DNA synthesis in both CS complementation groups to near normal levels . DenV is a glycosylase which is specific for cyclobutane-pyrimidine dimers (CPDs) but does not recognize other UV-induced lesions . Previous work has indicated that CS cells can efficiently repair all non-CPD UV-induced transcription blocking lesions (S.F . Barrett et al. . Mutation Res . 255 (1991) 281-291 {1}) and that denV incised lesions are believed to be processed via the base excision repair (BER) pathway . The inability of denV to complement the NER defect in CS cells to normal levels implies an impaired ability to process denV incised lesions by the BER pathway, and suggests a role for the CS genes, particularly the CS-B gene, in BER. Biochemistry, 1997 Nov 18, 36(46), 14080 - 7 A hexameric helicase encircles one DNA strand and excludes the other during DNA unwinding; Hacker KJ et al.; The bacteriophage T7 DNA helicase/primase (gene 4 protein) is a ring-like hexamer that encircles ssDNA and requires forked DNA to catalyze DNA unwinding . We report that optimal rates of unwinding of forked DNA require ssDNA tails of 55 nucleotides on the 5'-to-3' strand and 15 nucleotides on the 3'-to-5' strand . Surprisingly, streptavidin bound to a biotinylated 3'-end fully substitutes for the 3'-to-5' ssDNA tail . This suggests that excluding the 3'-to-5' DNA strand from the center of the helicase is an essential aspect of the mechanism of hexameric helicase-catalyzed DNA unwinding . We also report that streptavidin bound to a biotinylated dT within the 5'-to-3' strand of the duplexed region abolishes DNA unwinding; whereas, streptavidin bound to a biotinylated dT within the duplexed region of the other strand has no effect . These results unambiguously demonstrate that the T7 gene 4 protein is a 5'-to-3' helicase and imply that during DNA unwinding the 5'-to-3' strand transverses the center of the ring while the 3'-to-5' strand is excluded from the center of the ring . Implications for collisions between a helicase and other protein-DNA complexes are discussed. J Biol Chem, 1997 Nov 21, 272(47), 29752 - 8 Human apolipoprotein B transgenic mice generated with 207- and 145-kilobase pair bacterial artificial chromosomes . Evidence that a distant 5'-element confers appropriate transgene expression in the intestine; Nielsen LB et al.; We reported previously that approximately 80-kilobase pair (kb) P1 bacteriophage clones spanning either the human or mouse apoB gene (clones p158 and p649, respectively) confer apoB expression in the liver of transgenic mice, but not in the intestine . We hypothesized that the absence of intestinal expression was due to the fact that these clones lacked a distant DNA element controlling intestinal expression . To test this possibility, transgenic mice were generated with 145- and 207-kb bacterial artificial chromosomes (BACs) that contained the human apoB gene and more extensive 5'- and 3'-flanking sequences . RNase protection, in situ hybridization, immunohistochemical, and genetic complementation studies revealed that the BAC transgenic mice manifested appropriate apoB gene expression in both the intestine and the liver, indicating that both BACs contained the distant intestinal element . To determine whether the regulatory element was located 5' or 3' to the apoB gene, transgenic mice were generated by co-microinjecting embryos with p158 and either the 5'- or 3'-sequences from the 145-kb BAC . Analysis of these mice indicated that the apoB gene's intestinal element is located 5' to the structural gene . Cumulatively, the transgenic mouse studies suggest that the intestinal element is located between -33 and -70 kb 5' to the apoB gene. DNA Res, 1997 Aug 31, 4(4), 281 - 9 A 3-Mb sequence-ready contig map encompassing the multiple disease gene cluster on chromosome 11q13.1-q13.3; Kitamura E et al.; Despite the presence of several human disease genes on chromosome 11q13, few of them have been molecularly cloned . Here, we report the construction of a contig map encompassing 11q13.1-q13.3 using bacteriophage P1 (P1), bacterial artificial chromosome (BAC), and P1-derived artificial chromosome (PAC) . The contig map comprises 32 P1 clones, 27 BAC clones, 6 PAC clones, and 1 YAC clone and spans a 3-Mb region from D11S480 to D11S913 . The map encompasses all the candidate loci of Bardet-Biedle syndrome type I (BBS1) and spinocerebellar ataxia type 5 (SCA5), one-third of the distal region for hereditary paraganglioma 2 (PGL2), and one-third of the central region for insulin-dependent diabetes mellitus 4 (IDDM4) . In the process of map construction, 61 new sequence-tagged site (STS) markers were developed from the Not I linking clones and the termini of clone inserts . We have also mapped 30 ESTs on this map . This contig map will facilitate the isolation of polymorphic markers for a more refined analysis of the disease gene region and identification of candidate genes by direct cDNA selection, as well as prediction of gene function from sequence information of these bacterial clones. Genomics, 1997 Nov 15, 46(1), 120 - 6 Mouse connexin40: gene structure and promoter analysis; Seul KH et al.; A family of related connexin genes encodes the subunit gap junction proteins that form intercellular channels in different tissues . Connexin40 (Cx40) is one of these proteins, and it exhibits limited expression only in a few cells of the cardiovascular system . To begin to analyze Cx40 expression, we isolated a 3.3-kb rat Cx40 cDNA by hybridization screening of a bacteriophage library prepared from BWEM cells and isolated corresponding mouse genomic clones from a bacterial artificial chromosome library . Restriction mapping, sequencing, and comparison to the rat cDNA showed that the mouse Cx40 gene contained a short first exon, an 11.4-kb intron, and a second exon containing the complete coding region and 3'-UTR . Exon I contained only 1 base that differed between rat and mouse . Primer extension experiments yielded a single band and confirmed the position of the transcriptional start site . We obtained 1.2 kb of sequence 5' of the transcriptional start site and 400 bp 3' of exon I . Exon I was closely preceded by a consensus TATA box . The flanking sequences contained a number of potential transcription factor binding sites (including AP-1, AP-2, SP1, TRE, and p53) . To identify transcriptional regulatory elements in the Cx40 promoter region, a series of DNA deletion fragments flanking exon I was prepared, subcloned adjacent to a luciferase reporter gene, and used for transient transfections of BWEM, SHM, and N2A cells . The resulting luciferase activity determinations suggested that an area of 300 bp 5' of the transcription start site acted as a basal promoter for Cx40 and that there was a strong negative regulatory element in the region from +100 to +297. J Bacteriol, 1997 Dec, 179(24), 7827 - 33 A Mycobacterium smegmatis mutant with a defective inositol monophosphate phosphatase gene homolog has altered cell envelope permeability; Parish T et al.; A bacteriophage infection mutant (strain LIMP7) of Mycobacterium smegmatis was isolated following transposon mutagenesis . The mutant showed an unusual phenotype, in that all phages tested produced larger plaques on this strain compared to the parent strain . Other phenotypic characteristics of the mutant were slower growth, increased clumping in liquid culture, increased resistance to chloramphenicol and erythromycin, and increased sensitivity to isoniazid and several beta-lactam antibiotics . Permeability studies showed decreases in the accumulation of lipophilic molecules (norfloxacin and chenodeoxycholate) and a small increase with hydrophilic molecules (cephaloridine); taken together, these characteristics indicate an altered cell envelope . The DNA adjacent to the transposon in LIMP7 was cloned and was shown to be highly similar to genes encoding bacterial and mammalian inositol monophosphate phosphatases . Inositol is important in mycobacteria as a component of the major thiol mycothiol and also in the cell wall, with phosphatidylinositol anchoring lipoarabinomannan (LAM) in the cell envelope . In LIMP7, levels of phosphatidylinositol dimannoside, the precursor of LAM, were less than half of those in the wild-type strain, confirming that the mutation had affected the synthesis of inositol-containing molecules . The impA gene is located within the histidine biosynthesis operon in both M . smegmatis and Mycobacterium tuberculosis, lying between the hisA and hisF genes. Virology, 1997 Nov 24, 238(2), 283 - 90 A glycine to alanine substitution in the paramyxovirus SV5 fusion peptide increases the initial rate of fusion; Bagai S et al.; Simian virus 5 fusion (F) protein mutant F-G3A, which contains a glycine-to-alanine substitution at position 3 in the conserved hydrophobic fusion peptide at the N-terminus of the F1 subunit, has been shown previously to cause increased syncytium formation compared to wild-type (wt) F protein, when expressed using an SV40 recombinant virus vector system (C . M . Horvath and R . A . Lamb (1992) J . Virol . 66, 2443-2455) . The wt F and the F-G3A proteins were expressed in eukaryotic cells using the vaccinia virus-bacteriophage T7 RNA polymerase (vac-T7) expression system, and they showed similar cell surface expression levels as determined by flow cytometry . The final extent of fusion when the vac-T7 expression system was used was not found to be greatly different when examined with a reporter gene activation assay . However, the initial rate of fusion was found to be five- to sixfold higher for the F-G3A mutant protein than the wt F protein, when examined using a quantitative assay for lipid mixing based on relief of self-quenching of fluorescence of the lipid probe octadecyl rhodamine (R18) . A microscopic fluorescent dye transfer assay also showed a much earlier spread of dye from R18-labeled red blood cells to the cells expressing the mutant F-G3A protein than the wt F protein . Thus, these data indicate that a single gly-to-ala mutation in the fusion peptide domain, although not affecting the final extent of fusion, significantly increased the rate of fusion . Possible mechanisms for the increased rate of fusion are discussed. Biochemistry, 1997 Nov 25, 36(47), 14409 - 17 Dissociation of bacteriophage T4 DNA polymerase and its processivity clamp after completion of Okazaki fragment synthesis; Carver TE Jr et al.; The mechanism of bacteriophage T4 DNA polymerase (gp43) and clamp (gp45) protein dissociation from the holoenzyme DNA complex was investigated under conditions simulating the environment encountered upon completion of an Okazaki fragment . Lagging strand DNA synthesis was approximated using a synthetic construct comprised of a doubly biotinylated, streptavidin-bound 62-mer DNA template, paired with complementary primers to generate an internal 12-base gap where the 5'-end primer contained either a 5'-OH (DNA primer) or a 5'-triphosphate (RNA primer) group . Rapid kinetic measurements revealed that upon encountering the blocking primer, the holoenzyme either dissociates from DNA (approximately 40%) or strand-displaces the blocking strand (approximately 60%) . The two blocking oligonucleotides (DNA or RNA) induce a 30-50-fold increase in the rate of holoenzyme dissociation, with both polymerase and clamp proteins dissociating simultaneously . Inhibition of ATP hydrolysis by ATP-gamma-S did not have a measurable effect upon holoenzyme dissociation from DNA . The presence of gp32, the single-strand binding protein, caused a small (3-fold) increase in the rate constant for dissociation. J Electron Microsc (Tokyo), 1997, 46(5), 425 - 30 Electron microscopy of biological specimens by the plasma-polymerization rapid-freeze replica method; Yamaguchi M et al.; The plasma-polymerization replica method is a unique replica technique for transmission electron microscopy . In the present study, we used this method in combination with a rapid-freeze technique to observe T4 bacteriophages and hepatitis B virus core particles . The heads of T4 bacteriophages appeared hexagonal and measured approximately 110 nm in length . Striations in their tails were also visible, indicating that the resolution of the present method is better than 4 nm . The images corresponded well with those obtained by ice-embedding and negative staining methods, with respect to both morphology and size of the phage particle . Hepatitis B virus core particles observed by the present method appeared round, approximately 30 nm in diameter, with hollow centres . Again, the morphology and size of the particles corresponded well with those obtained by ice-embedding, negative staining, and ultrathin sectioning . From these results, we conclude that the plasma-polymerization rapid-freeze replica method provides a useful technique for observation of biological specimens in a natural state and at high resolution. Infect Immun, 1997 Dec, 65(12), 4951 - 7 MTC28, a novel 28-kilodalton proline-rich secreted antigen specific for the Mycobacterium tuberculosis complex; Manca C et al.; Proteins that are actively secreted by Mycobacterium tuberculosis serve as major targets of immune responses in the infected host . To identify and purify novel proteins in the filtrates of M . tuberculosis cultures, a bacteriophage lambda library of M . tuberculosis H37Rv DNA was immunoscreened by using an anti-culture filtrate rabbit antiserum . Of 20 positive clones isolated, 6 were analyzed and found to express the genes for two known components of the early culture filtrate, the secreted 45/47-kDa antigen complex and the KatG protein, and two novel genes . Here we report the molecular cloning and nucleotide sequence of one of the new genes encoding a culture filtrate protein of 310 amino acid (aa) residues . We called this gene mtc28 . The deduced polypeptide sequence contained an NH2-terminal, highly hydrophobic 32-aa region having properties of a secretion signal peptide . The putative 278-aa mature MTC28 protein was characterized at its NH2 and COOH termini by a high content of proline and alanine residues organized in an (AP)n motif . Thus, MTC28 is a new member of a group of proline-rich antigens found in M . tuberculosis and Mycobacterium leprae . As shown by DNA hybridization experiments, the mtc28 gene was present only in species of the M . tuberculosis complex . Purified recombinant MTC28 antigen evoked strong delayed-type hypersensitivity and antibody responses in guinea pigs immunized with Mycobacterium bovis BCG, but not in guinea pigs immunized with Mycobacterium avium . The strong immunological activity of MTC28 and the absence of B- and T-cell epitopes cross-reactive with a common environmental mycobacterial species, such as M . avium, make this novel antigen an attractive reagent for immunodiagnosis of tuberculosis. Pac Symp Biocomput . 1996;:126-41. How similar must a template protein be for homology modeling by side-chain packing methods? Chung SY, Subbiah S. Given the correct backbone coordinates of a globular protein, side-chain packing methods can be generally expected to predict the side-chain coordinates of the buried core residues accurately . In the context of a study in modeling a family of bacteriophage DNA-binding proteins, we observed that when the coordinates of the actual perfect backbone are not available, the side-chain packing methods are still of predictive value using homologous but imperfect backbones . This is the situation in practical homology modeling where a target protein sequence is modeled from template structures of known protein homologs . In order to assess the quality and degree of accuracy of such predictions and their dependence on the extent of homology, we have now extended these studies to a well characterized family of globin structures that span a much wider range of sequence-structure similarity . The collective results show a clear relationship that is independent of protein family between side-chain prediction accuracy and the level of similarity between the template and target proteins . We judge this similarity in terms of sequence identity and the backbone r.m.s . deviation of the template structure used for modeling and the actual target structure in cases where the target structures are available . In summary, as sequence identity drops from 100% to about 50%, or when the backbone r.m.s . deviation between template and target structures increases from 0 A to about 1 A, the overall average r.m.s . error for the buried-core residues rises from 1.2 A to 1.5 A while the chi 1 prediction accuracy drops from 85% to 70-75% and the chi 2 prediction accuracy drops from 80% to 60-65% . When the sequence identity drops below 50% or the backbone r.m.s . deviation rises above 1 A, all 3 measures of prediction accuracy decrease rapidly . When the sequence identity edges to the so-called twilight zone of sequence similarity at around 22%, or when the backbone r.m.s . deviation exceeds 2 A, the prediction accuracy approaches the values to be expected for random predictions, namely, 3.1 A for average r.m.s . error, 22% and 29% for accuracy of chi 1 and chi 2 prediction . These observations provide a practical evaluation of the side-chain packing methods and are of value to the homology-modeler . The extent and degree to which the backbone topology of a protein fold can constrain internal side-chain orientation gives insight into the plasticity of the sequence-structure relationship found in the architecture of proteins. J Long Term Eff Med Implants, 1998, 8(3-4), 241 - 8 Influence of latex glove hydration on bacteriophage penetration; Rodeheaver GT et al.; The purpose of this study was to determine whether glove hydration influenced bacteriophage penetration . Using an electronic glove hole-detection device, one brand of latex glove was identified that hydrated rapidly (3.25 min +/- 0.71 min), while another brand was selected that resisted hydration (120 min +/- 0 min) . Using a standard bacteriophage penetration model, the amount of bacteriophage penetration in both the rapidly hydrating gloves and the gloves that resisted hydration was extremely small and did not differ significantly from each other. Microbiology, 1997 Nov, 143 ( Pt 11), 3661 - 9 Increased and controlled expression of the Rickettsia prowazekii ATP/ADP translocase and analysis of cysteine-less mutant translocase; Dunbar SA et al.; Detailed molecular analysis of the Rickettsia prowazekii ATP/ADP translocase, an obligate exchange transport system that is specific for ATP and ADP, has been extremely difficult due to limited quantities of material available from these obligate intracytoplasmic bacteria and by the toxicity and poor expression in recombinant Escherichia coli expression systems . In this study, a stable and controllable system for the increased expression of the rickettsial ATP/ADP translocase was developed in E . coli where the expression of translocase from the bacteriophage T7 promoter in the pET11a vector led to a 26-fold increase in ATP transport activity and a 34-fold increase in translocase protein as compared to the expression with the native rickettsial promoter in E . coli . When compared to R . prowazekii, ATP transport activity was increased sixfold and membrane translocase was increased threefold . Approximately 24% of the translocase protein produced was localized in an inclusion body fraction . This expression system was then used to determine whether the two cysteine residues in the ATP/ADP translocase were essential for activity or expression . The translocase was modified by oligonucleotide-directed site-specific mutagenesis such that the two cysteines were converted to alanines . The ATP transport properties and ATP/ADP translocase production kinetics, translocase protein concentration and subcellular localization were indistinguishable in the wild-type and mutant strains, proving that cysteines play no functional role in the R . prowazekii ATP/ADP translocase and providing a system suitable for cysteine-scanni