Phage

AIDS, 1992 Oct, 6(10), 1151 - 8
Progressive spastic myelopathy in a patient co-infected with HIV-1 and HTLV-II: autoantibodies to the human homologue of rig in blood and cerebrospinal fluid; Rosenblatt JD et al.; OBJECTIVE: Human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) are closely related human retroviruses . HTLV-I has been implicated in a chronic progressive myelopathy, known as tropical spastic paraparesis (TSP) or HTLV-I-associated myelopathy (HAM) . We sought to determine whether autoantibodies to brain antigens were present in the cerebrospinal fluid (CSF) of a patient with chronic progressive spastic myelopathy with evidence of both HIV-1 infection and HTLV-I/II seropositivity . DESIGN: A 54-year-old bisexual man with clinical features of HAM/TSP of over 20 years' duration was followed . METHODS: We applied discriminatory DNA amplification (polymerase chain reaction) to distinguish HTLV-I from HTLV-II and to verify co-infection with HIV-1 . The patient's CSF was used to screen a human brain cDNA expression library to identify antibodies directed against brain antigens . Autoreactive bacteriophage clones were isolated and sequenced . RESULTS: The patient was found to be co-infected with both HIV-1 and HTLV-II, but not with HTLV-I . HTLV-II proviral levels in the peripheral blood remained relatively constant, despite therapy with zidovudine . Prominent oligoclonal banding of immunoglobulins was present in the patient's CSF . A single repeatedly reactive cDNA clone was identified, by screening with CSF antibody, sequenced, and found to be the human homologue of the rat insulinoma gene, rig . CONCLUSIONS: HTLV-II infection may predispose to development of a HAM/TSP-like illness . Autoimmune mechanisms, such as autoantibody formation, may play a role in pathogenesis.

J Mol Biol, 1992 Oct 5, 227(3), 917 - 33
Structure of a hinge-bending bacteriophage T4 lysozyme mutant, Ile3-->Pro; Dixon MM et al.; The mutant T4 phage lysozyme in which isoleucine 3 is replaced by proline (I3P) crystallizes in an orthorhombic form with two independent molecules in the asymmetric unit . Relative to wild-type lysozyme, which crystallizes in a trigonal form, the two I3P molecules undergo large hinge-bending displacements with the alignments of the amino-terminal and carboxy-terminal domains changed by 28.9 degrees and 32.9 degrees, respectively . The introduction of the mutation, together with the hinge-bending displacement, is associated with repacking of the side-chains of Phe4, Phe67 and Phe104 . These aromatic residues are clustered close to the site of the mutation and are at the junction between the amino and carboxyl-terminal domains . As a result of this structural rearrangement the side-chain of Phe4 moves from a relatively solvent-exposed conformation to one that is largely buried . Mutant I3P also crystallizes in the same trigonal form as wild-type and, in this case, the observed structural changes are restricted to the immediate vicinity of the replacement . The main change is a shift of 0.3 to 0.5 A in the backbone of residues 1 to 5 . The ability to crystallize I3P under similar conditions but in substantially different conformations suggests that the molecule undergoes large-scale hinge-bending displacements in solution . It is also likely that these conformational excursions are associated with repacking at the junction of the N-terminal and C-terminal domains . On the other hand, the analysis is complicated by possible effects of crystal packing . The different I3P crystal structures show substantial differences in the binding of solvent, both at the site of the Ile3-->Pro replacement and at other internal sites.

J Mol Biol, 1992 Oct 5, 227(3), 711 - 8
Simplified methods for construction, assessment and rapid screening of peptide libraries in bacteriophage; Christian RB et al.; An efficient strategy has been devised for the construction of diverse peptide libraries in bacteriophage vectors . This strategy was used to generate a library of 4 x 10(8) random decapeptide inserts in the pIII protein of bacteriophage fd . A novel method for evaluating the genetic diversity of bacteriophage libraries based on colony hybridization with partially degenerate oligonucleotides has been developed . The decapeptide library was affinity-selected with a previously characterized monoclonal antibody specific for the V3 loop of the gp120 protein of HIV-1 . Immunological screening, an efficient technique for the rapid identification of putative binding bacteriophage, is described . Hexapeptide sequences similar to those obtained from affinity selection of a hexapeptide bacteriophage library were obtained from the decapeptide library in all five frames . Immunological screening of 20,000 clones from the two libraries after two rounds of affinity selection rapidly identified antibody-binding sequences; 93% and 86% of the sequences obtained from the hexapeptide and decapeptide libraries, respectively, had IC50 values < or = 10 mM as free peptides.

Biochim Biophys Acta, 1992 Oct 5, 1110(2), 218 - 24
Formation of non-bilayer structures induced by M13 coat protein depends on the conformation of the protein; Sanders JC et al.; A comparison is made of the interaction of the coat protein of bacteriophage M13 in a predominant alpha-helix conformation and in a predominant beta-sheet conformation . To perform a systematic study of the interaction between the protein in these two different forms of the surrounding lipid matrix, NMR spectra of 2H-nuclei of specific labelled phospholipid systems are measured . In addition 31P-NMR is employed to provide information about the morphological structure adopted by the reconstituted lipid/protein systems . From the 2H-NMR studies on specific headgroup and chain deuterium labelled phospholipids it is found that the protein in the predominant beta-sheet conformation causes a fraction of lipids to be trapped . By combining the results from the headgroup and acyl chains of the phospholipids, it is concluded that the trapped lipids are arranged in a non-bilayer structure, probably caused by a misfitting of the hydrophobic core of the protein and the membrane bilayer . The protein in the predominant alpha-helix conformation perfectly fits in the lipid bilayer and has only minor influences on the surrounding lipid matrix . A new model is proposed to explain the presence of the trapped lipids in the lipid/protein systems.

J Biol Chem, 1992 Oct 5, 267(28), 20153 - 8
Inhibition of T7 RNA polymerase by T7 lysozyme in vitro; Ikeda RA et al.; The in vivo observation that the expression of bacteriophage T7 gene 3.5 (T7 lysozyme) inactivates T7 class II transcription and the in vitro observation that T7 lysozyme inhibits T7 RNA polymerase lead to the hypothesis that T7 lysozyme might preferentially inhibit transcription from T7 class II promoters . T7 lysozyme was cloned into a lambda pL expression vector, overproduced in Escherichia coli, and purified . The ability of purified T7 lysozyme to inhibit transcription from T7 DNA, the cloned T7 class II promoters, phi 2.5 and phi 4.7, and the cloned class III promoter, phi 10, was measured in vitro . It was observed that the effectiveness of T7 lysozyme as an inhibitor of T7 RNA polymerase is inversely related to the concentration of Mg2+; T7 lysozyme inhibits T7 RNA polymerase most effectively at low Mg2+ concentrations . In addition, no preferential inhibition of transcription from cloned T7 class II promoters was observed, nor was a strong T7 class III promoter preferred when transcriptional capacity was reduced by T7 lysozyme . These observations contradict the hypotheses that the temporal control of T7 gene expression is either due to direct and selective inhibition of the T7 class II promoters by T7 lysozyme or to preferential transcription of the strong T7 class III promoters when transcriptional capacity is reduced by T7 lysozyme . It appears that alternative mechanisms such as the involvement of additional proteins and/or cellular conditions to enhance transcription from T7 class III promoters or to inhibit transcription from T7 class II promoter are necessary to explain the temporal control of transcription of bacteriophage T7.

Protein Eng, 1992 Oct, 5(7), 703 - 14
A molecular dynamics simulation of bacteriophage T4 lysozyme; Arnold GE et al.; An analysis of a 400 ps molecular dynamics simulation of the 164 amino acid enzyme T4 lysozyme is presented . The simulation was carried out with all hydrogen atoms modeled explicitly, the inclusion of all 152 crystallographic waters and at a temperature of 300 K . Temporal analysis of the trajectory versus energy, hydrogen bond stability, r.m.s . deviation from the starting crystal structure and radius of gyration, demonstrates that the simulation was both stable and representative of the average experimental structure . Average structural properties were calculated from the enzyme trajectory and compared with the crystal structure . The mean value of the C alpha displacements of the average simulated structure from the X-ray structure was 1.1 +/- 0.1 A; differences of the backbone phi and psi angles between the average simulated structure and the crystal structure were also examined . Thermal-B factors were calculated from the simulation for heavy and backbone atoms and both were in good agreement with experimental values . Relationships between protein secondary structure elements and internal motions were studied by examining the positional fluctuations of individual helix, sheet and turn structures . The structural integrity in the secondary structure units was preserved throughout the simulation; however, the A helix did show some unusually high atomic fluctuations . The largest backbone atom r.m.s . fluctuations were found in non-secondary structure regions; similar results were observed for r.m.s . fluctuations of non-secondary structure phi and psi angles . In general, the calculated values of r.m.s . fluctuations were quite small for the secondary structure elements . In contrast, surface loops and turns exhibited much larger values, being able to sample larger regions of conformational space . The C alpha difference distance matrix and super-positioning analyses comparing the X-ray structure with the average dynamics structure suggest that a 'hinge-bending' motion occurs between the N- and C-terminal domains.

Biotechniques, 1992 Oct, 13(4), 554 - 6, 558-60, 562
High-yield recovery of recombinant DNA from poorly growing cosmid and lambda genomic clones; Millar SJ et al.; Certain genomic sequences cannot be recovered efficiently in cosmid or lambda bacteriophage clones, presenting a barrier to efforts to construct a contiguous cloned library of a genome . We have encountered such sequences during our efforts to isolate cosmid and bacteriophage lambda clones carrying members of the human type 2 cystatin gene family . Several cosmid clones constructed in the pWE 15 vector did not survive purification, and using standard techniques, we were unable to obtain significant amounts of cosmid DNA from those clones we could purify . Similarly, several lambda bacteriophage clones constructed in the lambda DASH II vector could not be purified, and those lambda clones we were able to isolate gave low titers in liquid lysates . In this paper, we describe generally applicable methods for preparing high yields of recombinant DNA from such recalcitrant cosmid and lambda clones constructed in these vectors.

Curr Opin Genet Dev, 1992 Oct, 2(5), 727 - 38
Interaction between bacteriophage lambda and its Escherichia coli host; Friedman DI; Bacteriophage lambda relies to a large extent on processes requiring interactions between viral- and host-encoded proteins for its lytic growth, establishment of lysogeny, and release from the prophage state . Both biochemical and genetic studies of these interactions have yielded new information about important host and lambda functions . In particular, mutations in Escherichia coli that compromise lambda DNA replication, genome packaging, transcription elongation, and site-specific recombination have led to the identification of bacterial genes whose products are chaperones, transcription factors, or DNA-binding proteins.

J Protein Chem, 1992 Oct, 11(5), 467 - 73
Heterologous expression and purification of active human phosphoribosylglycinamide formyltransferase as a single domain; Kan CC et al.; We report here for the first time that the GART domain of the human trifunctional enzyme possessing GARS, AIRS, and GART activities can be expressed independently in Escherichia coli at high levels as a stable protein with enzymatic characteristics comparable to those of native trifunctional protein . Human trifunctional enzyme is involved in de novo purine biosynthesis, and has long been recognized as a target for antineoplastic intervention . The GART domain was expressed in E . coli under the control of bacteriophage T7 promotor and isolated by a three-step chromatographic procedure . Two residues, Asp 951 and His 915, were shown to be catalytically crucial by site-directed mutagenesis and subsequent characterization of purified mutant proteins . The active monofunctional GART protein produced in E . coli can serve as a valuable substitute of trifunctional enzyme for structural and functional studies which have been until now hindered because of insufficient quantity, instability, and size of the trifunctional GART protein.

J Bacteriol, 1992 Oct, 174(20), 6539 - 47
Sequence and characterization of the bacteriophage T4 comC alpha gene product, a possible transcription antitermination factor; Sanson B et al.; We have sequenced a 1,340-bp region of the bacteriophage T4 DNA spanning the comC alpha gene, a gene which has been implicated in transcription antitermination . We show that comC alpha, identified unambiguously by sequencing several missense and nonsense mutations within the gene, codes for an acidic polypeptide of 141 residues, with a predicted molecular weight of 16,680 . We have identified its product on one- and two-dimensional gel systems and found that it migrates abnormally as a protein with a molecular weight of 22,000 . One of the missense mutations (comC alpha 803) is a glycine-to-arginine change, and the resulting protein exhibits a substantially faster electrophoretic mobility . The ComC alpha protein appears immediately after infection . Its rate of synthesis is maximum around 2 to 3 min postinfection (at 37 degrees C) and then starts to decrease slowly . Some residual biosynthesis is still detectable during the late period of phage development.

J Bacteriol, 1992 Oct, 174(19), 6138 - 44
Bacteriophage P1 gene 10 is expressed from a promoter-operator sequence controlled by C1 and Bof proteins; Lehnherr H et al.; Gene 10 of bacteriophage P1 encodes a regulatory function required for the activation of P1 late promoter sequences . In this report cis and trans regulatory functions involved in the transcriptional control of gene 10 are identified . Plasmid-borne fusions of gene 10 to the indicator gene lacZ were constructed to monitor expression from the gene 10 promoter . Production of gp10-LacZ fusion protein became measurable at about 15 min after prophage induction, whereas no expression was observed during lysogenic growth . The activity of an Escherichia coli-like promoter, Pr94, upstream of gene 10, was confirmed by mapping the initiation site of transcription in primer extension reactions . Two phage-encoded proteins cooperate in the trans regulation of transcription from Pr94: C1 repressor and Bof modulator . Both proteins are necessary for complete repression of gene 10 expression during lysogeny . Under conditions that did not ensure repression by C1 and Bof, the expression of gp10-LacZ fusion proteins from Pr94 interfered with transformation efficiency and cell viability . Results of in vitro DNA-binding studies confirmed that C1 binds specifically to an operator sequence, Op94, which overlaps the -35 region of Pr94 . Although Bof alone does not bind to DNA, together with C1 it increases the efficiency of the repressor-operator interaction . These results are in line with the idea that gp10 plays the role of mediator between early and late gene transcription during lytic growth of bacteriophage P1.

EMBO J, 1992 Oct, 11(10), 3797 - 806
Characterization of the bacteriophage lambda excisionase (Xis) protein: the C-terminus is required for Xis-integrase cooperativity but not for DNA binding; Numrych TE et al.; We have performed a mutational analysis of the xis gene of bacteriophage lambda . The Xis protein is 72 amino acids in length and required for excisive recombination . Twenty-six mutants of Xis were isolated that were impaired or deficient in lambda excision . Mutant proteins that contained amino acid substitutions in the N-terminal 49 amino acids of Xis were defective in excisive recombination and were unable to bind DNA . In contrast, one mutant protein containing a leucine to proline substitution at position 60 and two truncated proteins containing either the N-terminal 53 or 64 amino acids continued to bind lambda DNA, interact cooperatively with FIS and promote excision . However, these three mutants were unable to bind DNA cooperatively with Int . Cooperativity between wild-type Xis and Int required the presence of FIS, but not the Int core-type binding sites . This study shows that Xis has at least two functional domains and also demonstrates the importance of the cooperativity in DNA binding of FIS, Xis and Int in lambda excision.

Curr Opin Biotechnol, 1992 Oct, 3(5), 474 - 80
Antibody expression in bacteriophage systems: the future of monoclonal antibodies?
Garrard LJ, Zhukovsky EA.
Bacteriophage systems have been utilized to express and isolate antibodies . This promising technology has been evolving rapidly and has the potential to revolutionize the way in which monoclonal antibodies are generated . This review focuses on the many recent advances that have been made in obtaining monoclonal antibodies from bacteriophage systems.

Eur J Clin Microbiol Infect Dis, 1992 Oct, 11(10), 901 - 7
Emergence during unsuccessful chemotherapy of multiple drug resistance in a strain of Mycobacterium tuberculosis; Rastogi N et al.; Serial isolates of Mycobacterium tuberculosis were cultured from a patient who failed to respond to standard antituberculous chemotherapy . Isolates were cultured in March 1989, July 1989, December 1989 and May 1990 . Each successive isolate was found to be resistant to a wider range of antituberculous drugs than its predecessors . The initial isolate was resistant to isoniazid and rifampin, the second isolate was also resistant to ethambutol, the third was also resistant to pyrazinamide, ansamycin (= rifabutin) and ofloxacin and the last isolate was also resistant to ciprofloxacin and sparfloxacin . All four isolates' bacteriophage typing profiles and DNA restriction fragment patterns determined by Southern blot hybridization using the IS6110/IS986 probes and the new probe pTBN12 were concordant . It was concluded that this patient was persistently infected with a single strain of Mycobacterium tuberculosis which developed resistance to a number of families of drugs but did not show any significant change in typing patterns . The problem of acquired multiple drug resistance, particularly to fluoroquinolones and rifamycins, represents a new challenge in tuberculosis therapy.

J Virol, 1992 Oct, 66(10), 6220 - 2
Protein P4 of the bacteriophage phi 6 procapsid has a nucleoside triphosphate-binding site with associated nucleoside triphosphate phosphohydrolase activity; Gottlieb P et al.; Bacteriophage phi 6 contains three segments of double-stranded RNA . The procapsid consists of proteins P1, P2, P4, and P7, which are encoded by the viral L segment . cDNA copies of this segment have been cloned into plasmids that direct the production of these proteins, which assemble into polyhedral procapsids . These procapsids are capable of packaging plus-sense phi 6 RNA in the presence of nucleoside triphosphate and synthesizing the complementary minus strand to form double-stranded RNA . In this article, we report the presence of a nucleotide-binding site in protein P4 . The viral procapsid and nucleocapsid exhibit a nucleoside triphosphate phosphohydrolase activity that converts nucleoside triphosphates into nucleoside diphosphates.

Virology, 1992 Oct, 190(2), 824 - 33
Role of exonuclease in the specificity of bacteriophage T7 DNA packaging; Son M et al.; During morphogenesis in vivo, bacteriophage T7 packages and cuts to mature size an end-to-end concatemer of its nonpermuted, terminally repetitious, double-stranded, mature DNA . Efficient production (90-100%) and packaging (20-35%) of concatemers has also been demonstrated in extracts of T7-infected cells (in vitro) (Son, M., Hayes, S . J., and Serwer, P . {1988} Virology 162, 38-46) . By use of both this procedure of in vitro DNA packaging and in-gel hybridization to packaged DNA fractionated by agarose gel electrophoresis, the specificity of packaging in vitro is found to depend on the presence of T7 gene 6 exonuclease (p6) . In the absence of p6 in vitro, no concatemerization is detected and packaging of DNA nonhomologous to T7 DNA (bacteriophage P22 DNA) is as efficient (0.05-1.1%) as the packaging of monomeric T7 DNA . Addition of p6 in vitro both stimulates the concatemerization-packaging of T7 DNA and suppresses the packaging of P22 DNA . The packaging efficiency for concatemeric T7 DNA is 29-611 x higher than that for monomeric T7 DNA . Inhibition of the packaging of P22 DNA by p6 is correlated with the formation of single-stranded P22 DNA ends . These data are explained by the hypothesis that a DNA molecule with a single-stranded end is packaged less efficiently than the same DNA without the single-stranded end . Testing this hypothesis in vivo reveals that both p6 and gene 3 endonuclease contribute to suppressing the packaging of host DNA.

J Biol Chem, 1992 Sep 25, 267(27), 19306 - 12
Transcription termination in vitro by bacteriophage T7 RNA polymerase . The role of sequence elements within and surrounding a rho-independent transcription terminator; Jeng ST et al.; rho-Independent transcription terminators in Escherichia coli contain a dG+dC-rich dyad-symmetrical structure that encodes an RNA hairpin structure and an adjacent, downstream dA+dT-rich region which encodes uridines at the 3'-end of the transcript . In the threonine (thr) attenuator, there are at least six sequence segments in the DNA that might affect termination: the sequence upstream of the attenuator, the deoxythymidine-rich stretch immediately preceding the G+C-rich region, the G+C-rich region itself and its hairpin loop-encoding region, the deoxyadenosine tract following the G+C-rich region, and the following downstream sequence . Our previous studies (Jeng, S.-T., Gardner, J.F., and Gumport, R.I . (1990) J . Biol . Chem . 265, 3823-3830) indicate that both the stability and sequence of the RNA hairpin formed by the G+C-rich region and the length of the uridine tract encoded by the deoxyadenosine stretch influence the termination of T7 RNA polymerase in vitro . In this report, we demonstrate that the template deoxythymidine run upstream of the G+C-rich region, the loop-encoding segment, and the sequences upstream and downstream of the thr attenuator also affect termination . These results indicate that: 1) a deoxythymidine tract is not absolutely required for termination, but increasing the number of deoxythymidines from one to nine base pairs causes T7 RNA polymerase to terminate more efficiently; 2) a template with the natural loop sequence reversed results in a higher termination efficiency than one encoded by the the wild-type attenuator; 3) the termination of T7 RNA polymerase is affected by sequences both proximal and distal to the thr attenuator.

J Biol Chem, 1992 Sep 25, 267(27), 19418 - 26
Host factor requirements for processive antitermination of transcription and suppression of pausing by the N protein of bacteriophage lambda; Mason SW et al.; The N protein of phage lambda prevents termination of transcription by Escherichia coli RNA polymerase at Rho-dependent and -independent terminators in the lambda early operons . The modification of RNA polymerase by N requires an N-utilization (nut) site, present in each lambda early operon, and involves the E . coli factors NusA, NusB, NusG, and ribosomal protein S10 . We show that, in the presence of NusA, N inhibits pausing by RNA polymerase and Rho-dependent termination in vitro at three sites in the lambda terminator tR1 which are located less than 100 base pairs downstream from nutR . NusA is also sufficient for partial antitermination at sites located farther downstream from nutL and nutR if there is a high concentration of N in the reaction . At low concentrations of N, the additional factors NusB, S10, and NusG are essential for antitermination at distal sites . In these conditions, the presence of NusA, NusB, S10, and NusG in the reaction enables N-modified RNA polymerase to elongate efficiently and processively through Rho-dependent and -independent terminators over distances as great as 7 kilobases downstream from the lambda nut sites . This substantial processivity of antitermination in vitro also occurs in vivo and probably reflects the stable association of N, NusA, NusB, S10, and NusG with RNA polymerase and nut site RNA in elongation complexes transcribing the lambda chromosome.

J Biol Chem, 1992 Sep 25, 267(27), 19101 - 6
Pyruvic acid is attached through its central carbon atom to the amino terminus of the recombinant DNA-derived DNA-binding protein Ner of bacteriophage Mu; Rose K et al.; Ner protein of bacteriophage Mu, produced by recombinant DNA techniques in Escherichia coli, has been found to possess a molecule of pyruvic acid attached covalently through carbon-2 to the amino-terminal cysteine residue . The intact protein and the amino-terminal chymotryptic peptide were found by mass spectrometry to be 70 mass units heavier than expected . The modified peptide was unstable under mildly acid or mildly basic conditions . Two-dimensional nuclear magnetic resonance spectroscopy of the modified and unmodified forms of the amino-terminal chymotryptic peptide was consistent with the presence of pyruvate linked through carbon-2 to the amino-terminal Cys residue . Treatment of the modified form with 2,4-dinitrophenylhydrazine in acid medium led to the expected hydrazone of pyruvic acid, which was identified by high pressure liquid chromatography . Of the two proteins known to be modified by pyruvate through its central carbon (the other being human adult hemoglobin, in which the modified form represents only a very minor fraction), Ner is the first protein found to be modified quantitatively . Given the instability of the modification, it may be more prevalent than recognized hitherto . Incubation with 2,4-dinitrophenylhydrazine may offer a useful means of detecting the presence of pyruvate linked to proteins in this way.

J Biol Chem, 1992 Sep 25, 267(27), 19521 - 8
Transmembrane signaling by the human insulin receptor kinase . Relationship between intramolecular beta subunit trans- and cis-autophosphorylation and substrate kinase activation; Frattali AL et al.; To examine the role of intramolecular beta subunit trans- and cis-autophosphorylation in signal transduction, the vaccinia virus/bacteriophage T7 expression system was used to generate insulin holoreceptors composed of a kinase-defective half-receptor precursor (alpha beta A/K or alpha beta A/K.delta CT) and a kinase-active half-receptor precursor (alpha beta delta CT or alpha beta WT) . In the alpha beta A/K-alpha beta delta CT hybrid insulin receptor, insulin stimulated a 20-fold increase in intramolecular beta subunit trans-phosphorylation, whereas cis-phosphorylation increased only 3-fold over the basal state . Similarly, in the alpha beta WT-alpha beta A/K.delta CT hybrid insulin receptor, insulin stimulated trans-phosphorylation approximately 30-fold and cis-phosphorylation only 3-fold over the basal state . Although cis-phosphorylation of the kinase-functional alpha beta half-receptor was observed within these hybrid receptor species, this was not sufficient to stimulate exogenous substrate kinase activity . These data demonstrate that insulin primarily activates an intramolecular beta subunit trans-phosphorylation reaction within the insulin holoreceptor and suggest that this reaction is necessary for activation of the holoreceptor . Furthermore, our results suggest a molecular basis for the dominant-negative phenotype observed in insulin-resistant patients possessing one kinase-defective insulin receptor allele.

Biochemistry, 1992 Sep 22, 31(37), 9073 - 80
In vivo and in vitro activities of point mutants of the bacteriophage T7 RNA polymerase promoter; Ikeda RA et al.; Two compatible plasmids were recently reported {Ikeda et al . (1992) Nucleic Acids Res . 20, 2517-2524} that together can be used to determine whether a mutant T7 RNA polymerase promoter is active or inactive in vivo . The first plasmid, pKGP1-1, carries T7 gene 1 (the gene encoding T7 RNA polymerase) ligated to a tac promoter, while the second plasmid, pCM-X#, carries the gene encoding chloramphenicol acetyltransferase (CAT) ligated to potential T7 promoters . If the pCM-X# plasmid carries a potential T7 promoter that can be utilized by T7 RNA polymerase, then CAT is produced from transcripts generated by T7 RNA polymerase from the potential promoter on the pCM-X# plasmid . To determine whether Escherichia coli growth characteristics and chloramphenicol (cam) resistance produced by the plasmids pKGP1-1 and pCM-X# reflect the T7 promoter activity of the possible promoters carried by the pCM-X# plasmids, the in vivo and in vitro strengths of the potential T7 promoters were compared and correlated . In vivo promoter strength was determined by measuring the relative amounts of CAT present in E . coli extracts, while relative in vitro promoter strength was measured in transcription assays . The in vivo and in vitro strengths of 22 point mutants of the consensus T7 promoter were shown to correlate with the growth characteristics and cam resistance conferred to E . coli harboring the plasmid pKGP1-1 and the respective pCM-X# plasmid.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1992 Sep 20, 227(2), 381 - 8
By-passing immunisation . Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro; Hoogenboom HR et al.; By display of antibody repertoires on the surface of a filamentous bacteriophage and selection of the phage by binding to antigen, we can mimic immune selection . Recently, by tapping the repertoire of rearranged V-genes from the peripheral blood lymphocytes of unimmunised donors, we succeeded in making human antibody fragments with different specificities, including both haptens and proteins, from the same library of phage . Now we have built a repertoire of human VH genes from 49 human germline VH gene segments rearranged in vitro to create a synthetic third complementarity determining region (CDR) of five or eight residues . The rearranged VH genes were cloned with a human V lambda 3 light chain as single chain Fv fragments for phage display, and the library of phage panned by binding to each of two haptens, 2-phenyl-5-oxazolone (phOx) or 3-iodo-4-hydroxy-5-nitrophenyl-acetate (NIP) coupled to bovine serum albumin (BSA) . Many different antibody fragments were isolated which bound specifically to hapten, some with affinities in the micromolar range . The in vitro "immune response" to the hapten NIP was dominated by the 9-1 segment (VH3 family), and that to phOx by the VH26 segment (VH3 family) with an invariant aromatic residue (Tyr, Phe, Trp) at residue 97 of CDR3 . However, the isolation of phage against protein antigens proved more elusive, with a single phage binding to human tumour necrosis factor, and none to bovine serum albumin, turkey egg-white lysozyme or human thyroglobulin . Nevertheless, the work shows that human antibody fragments with specific binding activities can be made entirely in vitro.

J Biol Chem, 1992 Sep 15, 267(26), 18623 - 30
Interaction of EcoRII endonuclease with DNA substrates containing single recognition sites; Gabbara S et al.; EcoRII is unusual among type II restriction enzymes in that, while it cleaves substrates such as pBR322 and bacteriophage lambda that contain several recognition sites for the enzyme efficiently, substrates such as the genomes of bacteriophages T3 and T7 which contain a small number of recognition sites are cut poorly by it . Interestingly, pBR322, or a short DNA duplex containing a single site for the enzyme, can activate the enzyme to cleave resistant substrates . We show here that, at low concentrations, activator short duplexes are themselves cleaved poorly by the enzyme . Further, the reaction shows substrate cooperativity, and at high concentrations, the duplexes are both activators and good substrates for the enzyme . This supports the model that the activation of EcoRII involves binding of more than one DNA molecule and provides a simple system to study the mechanism of activation . Using a gel mobility shift assay, we show that the enzyme forms sequence-specific, methylation-sensitive complexes with the duplexes in the absence of activating DNA . Therefore, resistance of the short duplexes to the enzyme at low concentrations cannot be due to an inability of the enzyme to bind the duplexes . Interestingly, these complexes are stable in the presence of Mg2+, the cofactor for the enzyme, and the complexes obtained in the presence of Mg2+ do not contain DNA that is cleaved by the enzyme . The inefficient step in the action of EcoRII on resistant substrates must occur subsequent to initial substrate binding and it is this step that the activating DNA must regulate.

Biochemistry, 1992 Sep 15, 31(36), 8619 - 28
Human hemoglobin expression in Escherichia coli: importance of optimal codon usage; Hernan RA et al.; The overexpression of a nonfusion product of human beta-globin in Escherichia coli from its cDNA sequence has been accomplished for the first time . Expression of beta-globin from its native cDNA required the use of the strong bacteriophage T7 promoter . In this system, beta-globin accumulated to approximately 10% of total E . coli proteins . alpha-Globin was not expressed in the T7 system using the native cDNA . For the expression of alpha-globin, synthetic genes containing optimal E . coli codons were constructed . Neither synthetic alpha- nor beta-globin gene alone was expressed from the lac or tac promoter . Globin expression was achieved when the two synthetic alpha- and beta-globin genes were combined as an operon downstream of the lac promoter . The two proteins combined intracellularly with endogenous heme, which was concomitantly overproduced to yield tetrameric hemoglobin as roughly 5-10% of total E . coli protein . Cloning the alpha- and beta-globin cDNAs in a construct identical with the lac promoter did not yield globin production, establishing the requirement for optimal codon usage . The recombinant beta-globin from the T7 expression system was purified and reconstituted in vitro with heme and native alpha chains . N-terminal analyses showed that the beta-globin produced in the T7 system and the tetrameric hemoglobin produced from the synthetic genes contained an additional beta 1 methionine residue . Two additional mutants, beta 1 Val----Met and beta 1 Val----Ala were produced using the T7 system . Functional and structural properties of the purified hemoglobins will be discussed in the following papers.

Biochemistry, 1992 Sep 15, 31(36), 8397 - 405
A small (58-nm) attached sphere perturbs the sieving of 40-80-kilobase DNA in 0.2-2.5% agarose gels: analysis of bacteriophage T7 capsid-DNA complexes by use of pulsed field electrophoresis; Serwer P et al.; Although the icosahedral bacteriophage T7 capsid has a diameter (58 nm) that is 234-fold smaller than the length of the linear, double-stranded T7 DNA, binding of a T7 capsid to T7 DNA is found here to have dramatic effects on the migration of the DNA during both pulsed field agarose gel electrophoresis (PFGE; the field inversion mode is used) and constant field agarose gel electrophoresis (CFGE) . For these studies, capsid-DNA complexes were obtained by expelling DNA from mature bacteriophage T7; this procedure yields DNA with capsids bound at a variable position on the DNA . When subjected to CFGE at 2-6 V/cm in 0.20-2.5% agarose gels, capsid-DNA complexes arrest at the electrophoretic origin . Progressively lowering the electrical potential gradient to 0.5 V/cm results in migration; most complexes form a single band . The elevated electrical potential gradient (3 V/cm) induced arrest of capsid-DNA complexes is reversed when PFGE is used instead of CFGE . For some conditions of PFGE, the mobility of capsid-DNA complexes is a function of the position of the capsid on the DNA . During either CFGE (0.5 V/cm) or PFGE, capsid-DNA complexes increasingly separate from capsid-free DNA as the percentage of agarose increases . During these studies, capsid-DNA complexes are identified by electron microscopy of enzymatically-digested pieces of agarose gel; this is apparently the first successful electron microscopy of DNA from an agarose gel.(ABSTRACT TRUNCATED AT 250 WORDS)

Proc Natl Acad Sci U S A, 1992 Sep 15, 89(18), 8562 - 6
DNA helicase from mammalian mitochondria; Hehman GL et al.; In spite of the fact that a DNA helicase is clearly required for the predominantly leading-strand synthesis occurring during mammalian mtDNA replication, no such activity has heretofore been identified . We report the characterization of a mammalian mitochondrial DNA helicase isolated from bovine brain tissue . The sucrose gradient-purified mitochondria in which the activity was detected had less than 1 part in 2500 nuclear contamination according to Western blot analysis using nuclear- and mitochondrial-specific probes . Mitochondrial protein fractionation by DEAE-Sephacel chromatography yielded a DNA helicase activity dependent upon hydrolysis of ATP or dATP but not other NTPs or dNTPs . The mitochondrial helicase unwound 15- and 20-base oligonucleotides but was unable to unwind 32-base or longer oligonucleotides, and the polarity of the unwinding is 3'-to-5' with respect to the single-stranded portion of the partial duplex DNA substrate . This direction of unwinding would place the bovine mitochondrial helicase on the template strand ahead of DNA polymerase gamma during mtDNA replication, a situation analogous to that of the Rep helicase of Escherichia coli during leading-strand DNA synthesis of certain bacteriophages.

Nucleic Acids Res, 1992 Sep 11, 20(17), 4451 - 5
Stoichiometry of the Cre recombinase bound to the lox recombining site; Mack A et al.; The site-specific recombinase Cre from bacteriophage P1 binds and carries out recombination at a 34 bp lox site . The lox site consists of two 13 bp inverted repeats, separated by an 8 bp spacer region . Both the palindromic nature of the site and the results of footprinting and band shift experiments suggest that a minimum of two Cre molecules bind to a lox site . We report here experiments that demonstrate the absolute stoichiometry of the Cre-lox complex to be one molecule of Cre bound per inverted repeat, or two molecules per lox site.

J Mol Biol, 1992 Sep 5, 227(1), 29 - 37
The Escherichia coli rpoB60 mutation blocks antitermination by coliphage HK022 Q-function; Atkinson BL et al.; The lambdoid bacteriophage regulate gene expression by suppressing transcription terminators . Although similar in sequence to lambda, HK022 lacks an analogue to the lambda N antitermination gene and a distinct nutR sequence . To define the HK022 antitermination system, we plated the phage on Escherichia coli nus mutants that inhibit lambda N function . Only rpoB60 (also called nusC60) blocked HK022 lytic growth . Analyses of HK022-lambda hybrid phage suggested that a HK022 function analogous to lambda Q was inhibited by rpoB60 . This result was confirmed with pR'-tR'-galK fusions . HK022 Q-protein suppressed tR' in wild-type but not in rpoB60 mutants . The lambda Q-protein, although inhibited by rpoB60, was more active than the HK022 analogue . A single amino acid difference between the two Q-proteins accounts for the phenotype . Changing the penultimate residue of HK022 Q from alanine to the lambda threonine generated a phage that could propagate on rpoB60 hosts . Host and phage mutations that permitted HK022 growth in rpoB60 strains were characterized . The bacterial suppressors were located in the Escherichia coli nusB gene . The phage suppressors represented recessive mutations in a HK022 b-region sequence encoding an open reading frame of 73 codons.

J Biol Chem, 1992 Sep 5, 267(25), 18080 - 4
Gene structure of semenogelin I and II . The predominant proteins in human semen are encoded by two homologous genes on chromosome 20; Ulvsback M et al.; The genes for semenogelin I and II, the major protein constituents of the human seminal fluid, have been characterized by three overlapping clones in bacteriophage lambda, encompassing 31.5 kilobases (kb) of genomic DNA . The two genes are located 11.5 kb apart in the region q12-q13.1 on chromosome 20 . Both genes are relatively compact, spanning only 2.7 and 3.1 kb, respectively . The transcription units are composed of three exons, of which the first encodes the signal peptide, the second encodes the secreted protein, while the third solely contains 3'-noncoding nucleotides . The nucleotide sequences exhibit a similarity of close to 90% in the exons and exceeding 80% in the introns and flanking nucleotides.

J Biol Chem, 1992 Sep 5, 267(25), 17748 - 52
Critical functional role of the COOH-terminal ends of longitudinal hydrophobic strips in alpha-helices of T4 lysozyme; Rennell D et al.; The sensitivity of bacteriophage T4 lysozyme function to amino acid substitutions at defined positions in and around the longitudinal, hydrophobic strips of 9 alpha-helices was assessed after systematic replacement of each residue in the protein with a series of 13 amino acids . The hydrophobic strips were defined by identifying the longitudinal sectors in the helices with the highest mean residue hydrophobicities . Sensitivity to mutation (the percentage of replacements leading to loss of function) was calculated for each residue in the following positions: whole protein, helices, hydrophobic strips, other positions within the helices, and various positions within the hydrophobic strips as well as their extensions beyond the helices . Substitutions at positions in the hydrophobic strips led more frequently to loss of function than substitutions in the protein as a whole . One subset, the COOH-terminal hydrophobic strip residues, is apparently critical; substitutions of these residues (but not of their NH2-terminal counterparts) led at least as frequently to loss of function as substitutions of solvent-inaccessible residues, and nearly as frequently as substitutions of the most highly conserved residues.

J Biol Chem, 1992 Sep 5, 267(25), 17827 - 35
Integration host factor activates the Ner-repressed early promoter of transposable Mu-like phage D108; Kukolj G et al.; The lytic-lysogenic switch in transposable, Mu-like bacteriophage D108 is governed by two divergent and slightly overlapping transcription units originating from the Pe and Pc promoters . DNase I footprinting and in vivo mutational analysis suggest that lysogeny is maintained by c-repressor occupancy of the O2 operator, which precludes RNA polymerase from binding to Pe . Lytic development is controlled by the Ner repressor, which binds to a site symmetrically situated between the converging promoters and, in the absence of other factors, prevents RNA polymerase from binding to either Pc or Pe . DNase I protection and potassium permanganate hypersensitivity in the presence of integration host factor (IHF), which binds and alters the DNA structure upstream of Pe, revealed that RNA polymerase was able to bind Pe irrespective of the Ner.DNA-bound complex, and partially unwind the Pe "-10 region." Ner repression of Pe transcription in vitro was significantly more effective in the absence of IHF . Using a cloned D108 early region-lacZ fusion in IHF-deficient and -proficient backgrounds, we also demonstrate this host factor's affect on ner-repressed Pe in vivo, and generate a system for isolating mutants in the regulatory genes and sites controlling this genetic switch . D108 lytic growth is proposed to occur through IHF-mediated activation of the phage Ner-repressed early operon.

Proc Natl Acad Sci U S A, 1992 Sep 1, 89(17), 8145 - 9
The p50 subunit of NF-kappa B associates with the NF-IL6 transcription factor; LeClair KP et al.; The NF-kappa B-p50 polypeptide, a member of the Rel family of transcription factors, was produced as a fusion protein containing amino-terminal peptide additions that facilitate purification and detection with a monoclonal antibody and specific radiolabeling by phosphorylation in vitro . The 32P-labeled NK-kappa B-p50 fusion polypeptide was used as the probe in Western blotting experiments and in screenings of a bacteriophage expression library to isolate cDNAs encoding interacting protein domains . As expected, cDNAs encoding proteins of the Rel family were identified . Surprisingly, the 32P-labeled NF-kappa B protein also specifically bound to proteins encoded by cDNAs for the human NF-IL6 transcription factor . The NF-kappa B-p50 and NF-IL6 proteins directly interact, and the Rel homology domain and leucine-zipper motif, respectively, are important for this interaction . Since induction of the NF-kappa B and NF-IL6 factors are important events in immune and acute-phase responses, this interaction could permit coregulation of genes.

J Bacteriol, 1992 Sep, 174(17), 5740 - 4
Tandem cloning of bacteriophage T4 nrdA and nrdB genes and overproduction of ribonucleoside diphosphate reductase (alpha 2 beta 2) and a mutationally altered form (alpha 2 beta 2(93)); Tseng MJ et al.; To investigate the role of ribonucleoside diphosphate reductase in the deoxyribonucleoside triphosphate synthetase multienzyme complex induced by bacteriophage T4 infection and to study the expression of the T4 nrdA and nrdB genes, we have constructed separate plasmid expression strains overproducing their respective alpha 2 and beta 2 protein products . Because complementation of the two proteins to form an active alpha 2 beta 2 enzyme presented complications, nrdA and nrdB, each with its own tac promoter, were also cloned in tandem into a single expression vector . The resulting plasmid (pnrdAB) overproduces ribonucleoside diphosphate reductase . Phage T4 nrdB93, described by Wirak et al . (D . O . Wirak, K . S . Cook, and G . R . Greenberg, J . Biol . Chem . 263:6193-6201, 1988) contains a lesion in exon II of the gene . The mutation causes not only a temperature-sensitive inactivation of the catalytic structure of the beta 2(93) protein and of its ability to interact with alpha 2 protein to form the alpha 2 beta 2(93) enzyme but also a profound non-temperature-sensitive decrease in the formation of the beta 2(93) protein . An expression vector overproducing active alpha 2 beta 2(93) was constructed by site-directed mutagenesis of the nrdB gene.

Gene, 1992 Sep 1, 118(1), 5 - 11
Purification and characterization of the M.RsrI DNA methyltransferase from Escherichia coli; Kaszubska W et al.; The gene (rsrIM) encoding the RsrI DNA methyltransferase (M.RsrI) from Rhodobacter sphaeroides was cloned and expressed in Escherichia coli . Under the control of a bacteriophage T7 promoter, 2% of the total protein in a crude extract was M.RsrI . This level of expression represents an approximately 50-fold increase over that present in the natural host . Chromatography using DNA cellulose and the S-adenosylmethionine analogue, sinefungin, was useful in purifying the enzyme to homogeneity . The purification yielded 100 times more enzyme than was obtained from the same quantity of R . sphaeroides cell paste . M.RsrI deposits one methyl group per productive DNA-binding event, as does its functional but sequence-nonhomologous analogue, M.EcoRI . Unlike M.EcoRI, the R . sphaeroides enzyme is a dimer at micromolar concentrations.

Gene, 1992 Sep 1, 118(1), 103 - 7
A vector for controlled, high-yield production of specifically mutated proteins in Escherichia coli: test of a putative cytidine-binding domain in Rho factor and its Thr16----Ala mutant; Richardson LV et al.; A derivative of the plasmid vector, pET-3a {Rosenberg et al., Gene 56 (1987) 125-135}, is described that contains the origin of replication from bacteriophage f1 . This plasmid is well-suited for oligodeoxyribonucleotide mutagenesis and controlled production of mutant proteins from a single vector . Its utility is demonstrated by the preparation of a mutational alteration of Thr16----Ala (T16A) of the Escherichia coli transcription termination factor, Rho . The altered protein (T16A Rho) binds oligo(C)7 with the same affinity as wild-type (wt) Rho, thus indicating that Thr16 is not critical for binding cytidine residues in RNA, in spite of its being part of a sequence that is similar to a sequence in the CTP-binding domain of aspartate transcarbamoylase . However, T16A Rho was less efficient in terminating transcription than was wt Rho and had a lowered kcat for ATP hydrolysis with cro RNA as co-factor . Thus, the change affects the coupling of ATP hydrolysis by Rho to actions on RNA that cause termination.

Biochemistry, 1992 Sep 1, 31(34), 7948 - 56
Tryptophan contributions to the unusual circular dichroism of fd bacteriophage; Arnold GE et al.; The circular dichroism (CD) spectrum of fd bacteriophage has a deep minimum at 222 nm characteristic of highly alpha-helical protein, but there is a shoulder at 208 nm rather than a minimum, with a 222/208-nm amplitude ratio near 1.5 rather than near 1 . Oxidation of fd phage with the tryptophan reagent N-bromosuccinimide (NBS) changes the ratio . In this report, the NBS titration of fd is followed by CD and three other spectroscopies, the results of which yield an explanation of the unusual CD spectrum . Absorbance, fluorescence, and Raman data show the oxidation to have two phases, the first of which involves the destruction of tryptophan and the second, tryptophan and tyrosine . Raman spectra reveal the invariance of an environmentally-sensitive tyrosine Fermi resonance doublet during the first oxidative phase . Raman spectra also show that little or no change of alpha-helicity occurs in the first or second oxidation phase, although very slight changes in the helix parameters might be occurring . Concurrent with the destruction of tryptophan during the first phase is the appearance in CD difference spectra ({theta}NBS-treated fd - {theta}native fd) of positive maxima at 208-210 nm and negative maxima at 224 nm, with crossovers at 217 nm . Enormous difference ellipticities, per oxidized subunit of 50 amino acids, of +490,000 +/- 80,000 deg cm2 dmol-1 at 208 nm and -520,000 +/- 110,000 deg cm2 dmol-1 at 224 nm have been derived from the data.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Biol (Mosk), 1992 Sep-Oct, 26(5), 1022 - 35
{Site-specific mutagenesis of residue Lys-172 of phage T7 RNA polymerase: characterization of transcription properties of mutant proteins}; Liakhov DL et al.; Lys-172 residue of bacteriophage T7 RNA polymerase (T7RP) was substituted for Leu and Gly and Lys-172, Arg-173 were deleted by the site-directed mutagenesis using synthetic oligonucleotides . The specific activity of all mutant enzymes did not differ significantly from that of the wild-type (w.t.) T7RP while for Gly-172 mutant (G172) it was somewhat lower . Leu-172 (L172) and deletion (DEL172-3) mutants were able to direct RNA synthesis on the templates lacking the T7 promoter . DEL172-3 was not able to synthesize extraneous RNA sequences in addition to the expected run-off transcripts . L172 and DEL172-3 mutants revealed altered template specificity toward various DNA templates and showed the lower stability of enzyme-promoter complexes . The possible role of Lys-172 likely belonging to an interdomain "stretch" is discussed.

Mol Microbiol, 1992 Sep, 6(18), 2557 - 63
The role of integration host factor in gene expression in Escherichia coli; Freundlich M et al.; Integration host factor is a sequence-specific, histone-like, multifunctional DNA-binding and -bending protein of Escherichia coli . The characterization and functional analysis of this protein has been done mainly in bacteriophage lambda and other mobile genetic elements . Less is known concerning the role of integration host factor (IHF) in E . coli, although it has been implicated in a number of processes in this organism including DNA replication, site-specific recombination, and gene expression . This review presents recent work which suggests that IHF alters the activity of an unusually large number of operons in E . coli . We discuss the possible physiological relevance of the involvement of IHF in gene expression and the hypothesis that IHF is a member of a class of functionally redundant proteins that participate in chromosome structure and multiple processes involving DNA.

Appl Environ Microbiol, 1992 Sep, 58(9), 3180 - 2
A simple method to test condoms for penetration by viruses; Lytle CD et al.; A method by which virus penetration through condoms can be tested with simple, inexpensive equipment is described . The method uses chi X174 bacteriophage as the challenge virus and physiologically relevant pressure . Penetration by 0.1 microliters (or less) of challenge suspension can be readily detected . As examples, latex and natural-membrane condoms were examined.

J Reprod Fertil, 1992 Sep, 96(1), 135 - 9
Rapid transport of seminal immunoglobulin to distal parts of the female reproductive tract in rabbits; Jappel D; Bucks immunized with the bacteriophage-cloning vector fd-tet display strong humoral seminal immunity of 1.5 x 10(6) fd-tet neutralizing units (nu) ml-1 . We used this model to study the distribution of seminal immunoglobulin in the female reproductive tract after copulation and artificial insemination . In contrast to artificial insemination, significant (P < 0.01) amounts (147 nu ml-1, i.e . about 1.25 x 10(-4) input ejaculate) of neutralizing activity reached the uterus within 20 min of a single copulation, which is evidence for rapid transport of seminal plasma to distal parts of the tract . Oviducts were also quickly impregnated with antibodies to fd-tet, which persist in the distal compartments for at least 24 h.

Microbiol Rev, 1992 Sep, 56(3), 430 - 81
Bacteriophage lysis: mechanism and regulation; Young R; Bacteriophage lysis involves at least two fundamentally different strategies . Most phages elaborate at least two proteins, one of which is a murein hydrolase, or lysin, and the other is a membrane protein, which is given the designation holin in this review . The function of the holin is to create a lesion in the cytoplasmic membrane through which the murein hydrolase passes to gain access to the murein layer . This is necessary because phage-encoded lysins never have secretory signal sequences and are thus incapable of unassisted escape from the cytoplasm . The holins, whose prototype is the lambda S protein, share a common organization in terms of the arrangement of charged and hydrophobic residues, and they may all contain at least two transmembrane helical domains . The available evidence suggests that holins oligomerize to form nonspecific holes and that this hole-forming step is the regulated step in phage lysis . The correct scheduling of the lysis event is as much an essential feature of holin function as is the hole formation itself . In the second strategy of lysis, used by the small single-stranded DNA phage phi X174 and the single-stranded RNA phage MS2, no murein hydrolase activity is synthesized . Instead, there is a single species of small membrane protein, unlike the holins in primary structure, which somehow causes disruption of the envelope . These lysis proteins function by activation of cellular autolysins . A host locus is required for the lytic function of the phi X174 lysis gene E.

J Gen Virol, 1992 Sep, 73 ( Pt 9), 2235 - 44
Mutagenesis of the L protein encoded by Bunyamwera virus and production of monospecific antibodies; Jin H et al.; Bacterial fusion proteins containing portions of the Bunyamwera virus L protein were used as immunogens to prepare antisera in rabbits . Of five fusion proteins injected into rabbits, three yielded sera that reacted with the Bunyamwera virus L protein, detected by Western blotting or immunoprecipitation . Two of these antisera were specific for either the amino- or carboxy-terminal regions of the L protein . The specificity of these antisera was confirmed by their pattern of reactivity with full-length and truncated forms of the L protein . Plasmids containing the L gene cDNA under control of a bacteriophage T7 promoter were transfected into CV-1 cells which had previously been infected with a recombinant vaccinia virus, vTF7-3, that expresses T7 RNA polymerase . Antigenically authentic L protein was expressed . Using a nucleocapsid transfection assay developed previously, we showed that the transiently expressed L protein had RNA synthesis activity . Site-specific mutations were made in the L cDNA-containing plasmid to change certain amino acids in the putative polymerase domain of the L protein . The effects of these amino acid substitutions on the RNA synthesis activity of the L protein were monitored using the nucleocapsid transfection assay . These experiments showed that residues strictly conserved between the L proteins of different viruses in the family Bunyaviridae were obligatorily required for activity, whereas non-conserved residues could be substituted without abolishing RNA synthesis capability . Our results provide direct evidence for the functional significance of particular amino acids in the polymerase domain of a negative-strand virus RNA polymerase.

Biotechniques, 1992 Sep, 13(3), 422 - 8
Multipurpose vectors for peptide expression on the M13 viral surface; Fowlkes DM et al.; We have developed a set of three cloning vectors for the expression of polypeptides on the surface of the M13 viral coat . The M13mp8 genome has been engineered for expression of foreign protein sequences near the NH2-terminus of the mature pIII protein, which is present in five copies on the outside of each M13 viral particle . All three of the vectors carry the same two useful restriction sites for directed cloning of inserts in the pIII coding region; in addition, one vector carries the bacterial gene conferring resistance to the antibiotic tetracycline, and another expresses the lacZ' polypeptide that allows functional complementation of beta-galactosidase activity within the host bacterial cell . All of these vectors propagate well in E . coli DH5 alpha F' cells and do not require helper phage . We demonstrate that a bacteriophage, expressing an eleven amino acid epitope (from human c-myc) at the NH2-terminus of pIII in one of our vectors, can be purified from a vast mixture of other M13 phage through panning techniques . In particular, we find that the c-myc-expressing viral particles can be easily recovered from phage mixtures with the biotinylated form of the monoclonal antibody, 9E10, and streptavidin-coated MagneSphere beads.

Mutat Res, 1992 Sep, 275(3-6), 377 - 86
Interaction of singlet oxygen with DNA and biological consequences; Lutgerink JT et al.; To study the interaction of singlet oxygen (1O2) with DNA and the biological consequences of 1O2-induced DNA damage, we used the thermodissociable endoperoxide of 3,3'-(1,4 naphthalidene) dipropionate (NDPO2) as a generator of free 1O2 in reactions with (1) 2'-deoxynucleoside 3'-monophosphates (dNps), (2) an oligonucleotide (16-mer) having one deoxyguanine (dG), (3) native and denaturated rat kidney DNA and (4) single-stranded (ss) and double-stranded (ds) bacteriophage M13mp10 DNA . Using both anion exchange and reversed phase HPLC and 32P-postlabeling analyses, it was found that exposure of the various dNps to chemically generated 1O2 led to a detectable reaction with dGp and not with dAp, dCp, d5mCp or Tp . The reaction with dGp led to degradation of this nucleotide and the formation of a large number of reaction products, one of which could be identified as 7-hydro-8-oxo-2'-deoxyguanosine 3'-monophosphate (8-oxo-dGp) . A second product could tentatively be identified as a formamido pyrimidine derivative of dGp (Fapy-dGp) . When ss DNA, ds DNA or the oligonucleotide were exposed to 1O2, the formation of 8-oxo-dG could also be demonstrated . With the oligonucleotide, we found a so far unidentified reaction product . Under the same reaction conditions the yield of 8-oxo-dG was about 8-fold higher in ss DNA than in ds DNA . In ss DNA 8-oxo-dG seemed to be a more prominent product than in the case of reaction of 1O2 with free dGp . Reaction of 1O2 with ss or ds M13mp10 DNA led to biological inactivation of these DNAs, ss DNA being at least 100-fold more sensitive than ds DNA . It could be concluded that inactivation of the ss DNA must be largely due to 1O2-induced DNA lesions other than 8-oxo-dG . In agreement with the observed preferential reaction of 1O2 with dG most of the so far sequenced mutations, induced by 1O2 in a 144 bp mutation target sequence inserted in the lacZ alpha gene of ss or ds M13mp10 DNA, occurred at a G or G/C base pair respectively . A preference for G(C) to T(A) transversions can be observed for which 8-oxo-dG might have been responsible . In ss DNA a significant number of the mutations are characterized by the fact that a G is deleted.

Mutat Res, 1992 Sep, 275(3-6), 367 - 75
Singlet oxygen induced DNA damage; Sies H et al.; Singlet oxygen generated by photoexcitation and by chemiexcitation selectively reacts with the guanine moiety in nucleosides (kq + kr about 5 x 10(6) M-1s-1) and in DNA . The oxidation products include 8-oxo-7-hydro-deoxyguanosine (8-oxodG; also called 8-hydroxydeoxyguanosine) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) . Singlet oxygen also causes alkali-labile sites and single-strand breaks in DNA . The biological consequences include a loss of transforming activity as studied with plasmids and bacteriophage DNA, and mutagenicity and genotoxicity . Employing shuttle vectors, it was shown that double-stranded vectors carrying singlet oxygen induced lesions seem to be processed in mammalian cells by DNA repair mechanisms efficient in preserving the biological activity of the plasmid but highly mutagenic in mammalian cells . Biological protection against singlet oxygen is afforded by quenchers, notably carotenoids and tocopherols . Major repair occurs by excision of the oxidized deoxyguanosine moieties by the Fpg protein, preventing mismatch of 8-oxodG with dA, which would generate G:C to T:A transversions.

Mutat Res, 1992 Sep, 274(3), 163 - 76
Cell cycle progression in denV-transfected murine fibroblasts exposed to ultraviolet radiation; Kusewitt DF et al.; Repair-proficient murine fibroblasts transfected with the denV gene of bacteriophage T4 repaired 70-80% of pyrimidine dimers within 24 h after exposure to 150 J/m2 ultraviolet radiation (UVR) from an FS-40 sunlamp . Under the same conditions, control cells repaired only about 20% of UVR-induced pyrimidine dimers . After UVR exposure, both control and denV-transfected cells exhibited some degree of DNA-synthesis inhibition, as determined by flow cytometric analysis of cell-cycle kinetics in propidium iodide-stained cells . DenV-transfected cells had a longer and more profound S phase arrest than control cells, but both control and denV-transfected cells had largely recovered from UVR effects on cell-cycle kinetics by 48 h after UVR exposure . Inhibition of DNA synthesis by UVR was also measured by determining post-UVR incorporation of bromodeoxyuridine (BrdU) . The amount of BrdU incorporated was quantitated by determining with flow cytometry the quenching of Hoechst dye 33342 by BrdU incorporated in cellular DNA . DenV-transfected cells showed more marked inhibition of BrdU incorporation after low fluences of UVR than control cells . Differences between denV-transfected and control cells in cell-cycle kinetics following UVR exposure may be related to differences in mechanisms of repair when excision repair of pyrimidine dimers is initiated by endonuclease V instead of cellular repair enzymes.

Mol Gen Mikrobiol Virusol, 1992 Sep-Oct, (9-10), 5 - 8
{Use of non-radioactive biotin-labelled probes for detecting hepatitis A virus}; Budiak EV et al.; The biotin-labeled DNA probes were constructed on the basis of the hybrid bacteriophage M13nip 9 single-stranded DNA containing the fragments of the hepatitis A viral cDNA . The probes were biotin treated by chemical modification of the DNA by the peraminating reagent or photochemically . The labeled DNA probes were used in molecular hybridization experiments with the nuclear acids fixed on the nitrocellulose filters . The biotin treated DNA was determined by the avidin-gold colloid conjugate with the subsequent physical silver amplification or by the streptavidin-alkaline phosphatase conjugate . The sensitivity of both probes was identical and permitted the determination of 5 x 10(-11)-5 x 10(-12) g of the control DNA and 10(-9) g of the hepatitis A virus . The developed test systems were used for detection of the viral RNA in blood from patients.

Izv Akad Nauk SSSR Biol, 1992 Sep-Oct, (5), 744 - 52
{3'-->5'-exonucleases of the rat liver and the correction of replication errors}; Beliakova NV et al.; Mammalian nuclear DNA polymerases alpha and beta are lack of the proofreading 3'-->5' exonucleolytic activity . 40 and 50 kDa 3'-->5' exonucleases were isolated from rat liver . The exonucleases were shown to excise mismatched nucleotides from poly{d(A--T)} template 10 and 2 fold faster than matched ones . The addition of either exonuclease to DNA polymerase alpha from rat liver or calf thymus 5-10 times increased the accuracy of reproduction of primed DNA from bacteriophage phi X174 amber 3, values of exonuclease and DNA polymerase activities being approximately equal . The exonuclease activity surpasses the DNA polymerase one by an order of magnitude in chromatin and nuclear membrane . These data, taken together, are indicative of potent proofreading into hepatocytes.

Microb Pathog, 1992 Sep, 13(3), 225 - 36
Cloning and nucleotide sequence of a variant Shiga-like toxin II gene from Escherichia coli OX3:H21 isolated from a case of sudden infant death syndrome; Paton AW et al.; Escherichia coli OX3:H21 expressing a toxin related to Shiga-like toxin (SLT) was isolated from the small bowel contents of a case of Sudden Infant Death Syndrome (SIDS) . This strain was lysogenic for a lambdoid bacteriophage, but this did not encode the toxin . Southern hybridization analysis of chromosomal DNA revealed that the SLT-related gene was located on a 4.6 kb PstI fragment, which was cloned into E . coli JM109 in both orientations, using the vector pUC19, to generate plasmids pJCP501 and pJCP502 . JM109 cells harbouring the recombinant plasmid produced SLT, as judged by cytotoxicity for Vero cells . Nucleotide sequence analysis revealed that the SLT gene was related to, but distinct from, previously reported variants of Shiga-like toxin type II, produced by E . coli from both human and animal sources . The A subunit of the SLT gene from OX3:H21 exhibited 95.9% homology (at both the DNA and derived amino acid sequence level) to the A subunit of the most closely related SLT-II variant . The B subunit was less similar, exhibiting 88.6 and 88.8% homology to the related gene at the DNA and amino acid level, respectively.

Biochemistry, 1992 Aug 25, 31(33), 7587 - 94
Inhibition of T7 RNA polymerase initiation by triple-helical DNA complexes: a model for artificial gene repression; Maher LJ 3rd; An experimental approach is presented for the creation of an artificial and functional repressor/operator interaction that does not involve polypeptides . This in vitro approach confers oligonucleotide regulation upon a bacteriophage T7 RNA polymerase promoter by introducing an overlapping homopurine operator that can be recognized by oligonucleotide-directed DNA triple-helix formation . Recognition of optimized operator sequences in either of two triple-helix motifs is shown to efficiently inhibit T7 RNA polymerase transcription initiation in both a promoter- and oligonucleotide-specific manner . Inhibition due to triple helices of the pyrimidine motif is pH-dependent, as expected . Inhibition by purine motif triple helices is not pH-dependent and occurs efficiently under optimum T7 RNA polymerase transcription conditions . Repression by triple-helix formation can be observed rapidly after addition of purine motif repressor oligonucleotides, even when polymerase has been given prior access to the promoter . The mechanism of repression is shown to be occlusion of polymerase from the promoter rather than trapping of the polymerase in unproductive preinitiation or initiation complexes . In contrast to their inhibition of T7 RNA polymerase initiation, the triple-helical complexes studied here do not detectably inhibit transcription elongation.

Biochim Biophys Acta, 1992 Aug 17, 1132(1), 17 - 25
Enriched sources of Escherichia coli replication proteins . The dnaG primase is a zinc metalloprotein; Stamford NP et al.; Primase, the product of the Escherichia coli dnaG gene, is the enzyme responsible for RNA primer synthesis on both template strands at replication forks during chromosomal DNA synthesis . The dnaG gene was modified by replacement of the natural ribosome-binding site with one complementary to the 3' end of 16S rRNA, and then inserted downstream of tandem bacteriophage lambda PR and PL promoters in the pUC9-derived vector pCE30 . Following thermal induction of transcription, the resulting plasmid pPL195 directed synthesis of primase activity to levels corresponding to approx . 120,000 molecules per cell . The overproduced protein was soluble and was readily purified in high yield (31 mg per 1 of culture) . Purified primase was monomeric, was fully active in priming replication at the bacteriophage G4 complementary strand origin, and was shown to contain 0.92 +/- 0.08 g atom of tightly-bound zinc per mol of protein . Potential zinc-binding amino-acid residues near the N-terminus of the protein were identified . Although a mutant primase lacking 27 amino acid residues from the N-terminus was partly soluble, it was completely inactive.

Biochem Biophys Res Commun, 1992 Aug 14, 186(3), 1663 - 8
Delimitation of cohesive ends site (cos) of Streptomyces temperate bacteriophage R4; Mitsui H et al.; The cohesive ends site (cos) of actinophage R4 was delimitated by assaying the in vivo packaging activity of cosmid derivatives in Streptomyces lividans . A region of 66 bp from -30 to +36 from the center of cohesive ends was required for the basal level of packaging activity . Two additional regions outside the basal sequences from -39 to -31 and from +37 to +97 were necessary for the high level of activity, defined as the accessory sequences . Direct- or inverted-repeat sequences were found within the delimitated region, which might be involved in the recognition by the terminase of actinophage R4.

Nucleic Acids Res, 1992 Aug 11, 20(15), 3971 - 6
In vitro replication of bacteriophage PRD1 DNA . Metal activation of protein-primed initiation and DNA elongation; Caldentey J et al.; Bacteriophage PRD1 replicates its DNA by means of a protein-primed replication mechanism . Compared to Mg2+, the use of Mn2+ as the metal activator of the phage DNA polymerase results in a great stimulation of the initiation reaction . The molecular basis of the observed stimulatory effect is an increase in the velocity of the reaction . The phage DNA polymerase is also able to catalyze the formation of the initiation complex in the absence of DNA template . Although the presence of Mn2+ does not affect either the polymerization activity or the processivity of the DNA polymerase, this metal is unable to activate the overall replication of the phage genome . This can be explained by a deleterious effect of Mn2+ on the 3'-5'-exonucleolytic and/or the strand-displacement activity, the latter being an intrinsic function of the viral DNA polymerase required for protein-primed DNA replication.

J Mol Biol, 1992 Aug 5, 226(3), 889 - 96
Selection of phage antibodies by binding affinity . Mimicking affinity maturation; Hawkins RE et al.; We describe a process, based on display of antibodies on the surface of filamentous bacteriophage, for selecting antibodies either by their affinity for antigen or by their kinetics of dissociation (off-rate) from antigen . For affinity selection, phage are mixed with small amounts of soluble biotinylated antigen (less than 1 microgram) such that the antigen is in excess over phage but with the concentration of antigen lower than the dissociation constant (Kd) of the antibody . Those phage bound to antigen are then selected using streptavidin-coated paramagnetic beads . The process can distinguish between antibodies with closely related affinities . For off-rate selection, antibodies are preloaded with biotinylated antigen and diluted into excess unlabelled antigen for variable times prior to capture on streptavidin-coated paramagnetic beads . To mimic the affinity maturation process of the immune system, we introduced random mutations into the antibody genes in vitro using an error-prone polymerase, and used affinity selection to isolate mutants with improved affinity . Starting with a small library (40,000 clones) of mutants (average 1.7 base changes per VH gene) of the mouse antibody B1.8, and using several rounds of affinity selection, we isolated a mutant with a fourfold improved affinity to the hapten 4-hydroxy-5-iodo-3-nitrophenacetyl-(NIP)-caproic acid (mutant Kd = 9.4(+/- 0.3) nM compared with B1.8 Kd = 41.9(+/- 1.6) nm) . The relative increase in affinity of the mutant is comparable to the increase seen in the anti-4-hydroxy-3-nitrophenylacetyl/NIP-caproic acid murine secondary immune response.

J Mol Biol, 1992 Aug 5, 226(3), 661 - 73
Cre-lox recombination in Escherichia coli cells . Mechanistic differences from the in vitro reaction; Adams DE et al.; The mechanism of the Cre recombinase of bacteriophage P1 in Escherichia coli cells was analyzed by topological methods in order to determine the important features of the in vivo reaction . Lambda infection was used to introduce the cre gene into cells containing plasmid substrates . The products of Cre resolution on substrates with directly repeated sites were predominantly free circles, even though decatenation by DNA gyrase was blocked by the drug norfloxacin . Recombination by Cre was greatly stimulated by negative supercoiling, and inversion occurred inefficiently . These results are strikingly different from those found with purified enzyme in vitro . Our data imply that Cre recombination in vivo is much more tightly controlled than it is in vitro, and that Cre acts predominantly as a resolvase in vivo . We suggest a role for Cre-mediated recombination in P1 plasmid amplification that is consistent with the selectivity of the enzyme in vivo.

Gene, 1992 Aug 1, 117(1), 113 - 7
A T7 expression vector optimized for site-directed mutagenesis using oligodeoxyribonucleotide cassettes; Tanhauser SM et al.; Site-directed mutagenesis is widely used to examine structure/function relationships in proteins . We have designed a bacterial expression vector series which is optimized for efficient site-directed mutagenesis and subsequent protein synthesis without intervening subcloning steps . The vectors, derived from the T7 expression vectors of Studier and his collaborators {Studier et al., Methods Enzymol . 185 (1990) 60-89}, are small and have a bacteriophage f1 origin of replication for production of single-stranded (ss) DNA . Both single-site mutants {using ssDNA and mutating oligodeoxyribonucleotides (oligos)} and cassette mutants (mutagenesis of a short region by inserting double-stranded oligos into unique restriction sites) are rapidly synthesized and expressed with these vectors . Vector construction and use are detailed with examples showing the expression of the sequences encoding human carbonic anhydrases II and III . Production levels of greater than 60 mg of protein per liter of culture have been obtained.

Genes Dev, 1992 Aug, 6(8), 1373 - 85
Activation of the catalytic core of a group I intron by a remote 3' splice junction; Michel F et al.; Over 1000 nucleotides may separate the ribozyme core of some group I introns from their 3' splice junctions . Using the sunY intron of bacteriophage T4 as a model system, we have investigated the mechanisms by which proximal splicing events are suppressed in vitro, as well as in vivo . Exon ligation as well as cleavage at the 5' splice site are shown to require long-range pairing between one of the peripheral components of the ribozyme core and some of the nucleotides preceding the authentic 3' splice junction . Consistent with our three-dimensional modeling of the entire sunY ribozyme, we propose that this novel interaction is necessary to drive 5' exon-core transcripts into an active conformation . A requirement for additional stabilizing interactions, either RNA-based or mediated by proteins, appears to be a general feature of group I self-splicing . A role for these interactions in mediating putative alternative splicing events is discussed.

Virology, 1992 Aug, 189(2), 474 - 82
Bacteriophage T4 gene 21 encodes two proteins essential for phage maturation; Hintermann E et al.; The T4 prohead protease (T4 PPase) is the key enzyme in the morphopoietic pathway of the T4 phage head . It is responsible for the proteolytic processing of all head proteins allowing protein rearrangement and head expansion . To study its biochemistry and gene regulation, T4 gene 21 was cloned into an expression vector under the control of the inducible tac promoter . Two proteins of apparent molecular weights of 21.5 and 27.5 kDa were detected after induction . These proteins are synthesized using two different start codons in the same reading frame . Destruction of either start codon resulted in the loss of the respective protein . Complementation experiments with bacteriophage T4 21(-)-infected cells showed that both proteins are functional in vivo and essential for T4 phage assembly.

J Virol, 1992 Aug, 66(8), 5013 - 7
Dependence of minus-strand synthesis on complete genomic packaging in the double-stranded RNA bacteriophage phi 6; Frilander M et al.; Bacteriophage phi 6 has a segmented genome consisting of three pieces of double-stranded RNA (dsRNA) . The viral procapsid is the structure that packages plus strands, synthesizes the complementary negative strands to form dsRNA, and then transcribes dsRNA to form plus-strand message . The minus-strand synthesis of a particular genomic segment is dependent on prior packaging of the other segments . The 5' end of the plus strand is necessary and sufficient for packaging, while the normal 3' end is necessary for synthesis of the negative strand . We have now investigated the ability of truncated RNA segments which lack the normal 3' end of the molecules to stimulate the synthesis of minus strands of the other segments . Fragments missing the normal 3' ends were able to stimulate the minus-strand synthesis of intact heterologous segments . Minus-strand synthesis of one intact segment could be stimulated by the presence of two truncated nonreplicating segments . The 5' fragments of each single-stranded genomic segment can compete with homologous full-length single-stranded genomic segments in minus-strand synthesis reactions, suggesting that there is a specific binding site in the procapsid for each segment.

Genetics, 1992 Aug, 131(4), 769 - 81
Genetic recombination in bacteriophage T4: single-burst analysis of cosegregants and evidence in favor of a splice/patch coupling model; Shcherbakov VP et al.; To reveal the structure of penultimate DNA intermediates in T4 bacteriophage recombination, resolution of which produces free recombinant molecules, a single-burst analysis of the recombinant progeny was made in multifactor crosses, enabling one to determine quantitatively the different recombinants generated by one or two exchanges within the same chromosome segment . It was found that double and single exchanges are highly correlated in T4 recombination . These results were interpreted as evidence for simultaneous formation of a splice/patch pair as the primary recombination products . A recombination model called here the "splice/patch coupling model" is presented according to which resolution of a single DNA intermediate results in two linear heterozygous molecules containing a patch and a splice, respectively, in homologous positions.

Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 6866 - 70
Mutations induced by methylene blue plus light in single-stranded M13mp2; McBride TJ et al.; Reactive oxygen species are generated by a variety of cellular processes . These endogenously generated, reactive intermediates produce a multiplicity of DNA alterations and mutations and have been implicated in the pathogenesis of several human diseases . We report here that treatment of single-stranded M13mp2 bacteriophage DNA with methylene blue and white light generates increased levels of 8-hydroxydeoxyguanosine and that mutagenesis is both highly specific and dependent on the SOS response . Lesions produced block the progression of DNA synthesis one base preceding template guanines . In SOS-induced Escherichia coli, 97% of all methylene blue-induced mutations in the lacZ alpha gene of M13mp2 DNA are single-base substitutions opposite template guanines . The most frequent mutations are G----C transversions . The G----T transversions expected from the presence of 8-hydroxydeoxyguanosine in the template strand occur, but at a lower frequency . Sequence data together with SOS dependency and the presence of replication blockage demonstrate that while 8-hydroxydeoxyguanosine may serve as an important marker to monitor oxygen-induced DNA damage in humans, it does not account for either the observed blockage to replication or the mutagenesis by methylene blue plus light in SOS-induced E . coli . Instead, an as yet unidentified lesion generated by active oxygen species is a more potent mutagenic event.

Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 6861 - 5
Tissue- and site-specific DNA recombination in transgenic mice; Orban PC et al.; We have developed a method of specifically modifying the mammalian genome in vivo . This procedure comprises heritable tissue-specific and site-specific DNA recombination as a function of recombinase expression in transgenic mice . Transgenes encoding the bacteriophage P1 Cre recombinase and the loxP-flanked beta-galactosidase gene were used to generate transgenic mice . Genomic DNA from doubly transgenic mice exhibited tissue-specific DNA recombination as a result of Cre expression . Further characterization revealed that this process was highly efficient at distinct chromosomal integration sites . These studies also imply that Cre-mediated recombination provides a heritable marker for mitoses following the loss of Cre expression . This transgene-recombination system permits unique approaches to in vivo studies of gene function within experimentally defined spatial and temporal boundaries.

Acta Crystallogr B, 1992 Aug 1, 48 ( Pt 4), 499 - 511
Structure determination of the bacteriophage phiX174; McKenna R et al.; The structure of the single-stranded DNA phage phiX174 has been determined to 3.4 A resolution . The crystal space group was P2(1) with one icosahedral particle per asymmetric unit, giving 60-fold noncrystallographic redundancy . Oscillation diffraction photographs were collected using synchrotron radiation at various wavelengths . The particle orientations in the unit cell were determined with a rotation function . Because cowpea mosaic virus has a similar external envelope to phiX174, it was used as a search model to find the approximately positions of the phiX174 particles in the unit cell relative to the crystallographic symmetry axes . An initial phase set to 12 A resolution was then based on the cowpea mosaic virus atomic structure . These phases were improved by 20 cycles of real-space molecular replacement averaging . The phase information was gradually extended to 3.4 A resolution by molecular replacement electron density averaging . One reciprocal lattice point was used for each extension followed by four cycles of averaging . The unusual particle capsid, with its 12 pentameric spikes, required the careful determination of a precise molecular envelope . This was redetermined at regular intervals, as was the particle center . The resultant electron density map was readily interpreted in terms of the F, G and J polypeptides in the capsid . A difference electron density map between full and partially empty particles showed some ordered DNA structure.

Virus Genes, 1992 Aug, 6(3), 229 - 46
Expression of biologically active HIV glycoproteins using a T7 RNA polymerase-based eucaryotic vector system; Wilk T et al.; Bacteriophage T7 RNA polymerase and a derivative containing a nuclear localization signal were transiently expressed in CV-1 cells and were shown to localize to the cytoplasm and nucleus, respectively . A vector was constructed containing T7 promoter and transcription terminator sequences flanking a picornaviral 5' untranslated sequence for cap-independent translation and a polyA signal . Expression of the HIV-1 envelope glycoproteins in this vector system gave high levels of specific transcripts and translation products, independent of the subcellular localization of T7 RNA polymerase . The synthesis of HIV glycoproteins was also completely independent of the coexpression of the HIV rev protein, which is normally required for the expression of HIV structural proteins . In addition, a polyA signal was not required, whereas the presence of the picornaviral 5' untranslated region was necessary for efficient expression . Different possibilities to account for these findings are discussed . The HIV glycoproteins synthesized in this system were normally processed and assembled; they could induce syncytium formation and complement an env-deletion mutant of HIV-1.

Comput Appl Biosci, 1992 Aug, 8(4), 401 - 4
DNABIND: an interactive microcomputer program searching for nucleotide sequences that may code for conserved DNA-binding protein motifs; Mrazek J et al.; This paper presents a simple program for interactive searching for nucleotide sequences that may code for the helix-turn-helix, zinc finger or leucine zipper motifs in proteins . The helix-turn-helix motifs are predicted using the recently published method of Dodd and Egan, while zinc fingers and leucine zippers are searched for by our original methods . DNABIND is shown to detect all four known helix-turn-helix motifs in bacteriophage lambda genes and both zinc fingers of the adr1 gene of yeast.

Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 7139 - 43
Partial loss of function mutations in DnaK, the Escherichia coli homologue of the 70-kDa heat shock proteins, affect highly conserved amino acids implicated in ATP binding and hydrolysis; Wild J et al.; A set of 37 mutations in DnaK, the Escherichia coli homologue of the 70-kDa heat shock proteins, was isolated using a selection for high constitutive expression of heat shock proteins . Of these, 11 mutants were able to carry out some but not all functions of DnaK . These partial function mutants were divided into two classes . Class I mutants are recessive and permit replication of bacteriophage lambda and growth of cells up to 40 degrees C . Class II mutants are dominant, do not permit growth of lambda, and are temperature-sensitive for growth above 34 degrees C . Mutations in both classes alter amino acids that are highly conserved in the 70-kDa heat shock protein family . The dominant negative mutations provide strong genetic evidence that at least one form of DnaK is multimeric . Moreover, every dominant negative mutation occurs at an amino acid that has been hypothesized to be intimately involved in the process of ATP binding and hydrolysis . Our findings provide strong support for the hypothesis that such mutations are excellent tools for identifying amino acids that play critical roles in protein function.

Mutat Res, 1992 Aug, 282(4), 247 - 52
UV resistance of E . coli K-12 deficient in cAMP/CRP regulation; Puyo MF et al.; Deletion of genes for adenylate cyclase (delta cya) or cAMP receptor protein (delta crp) in E . coli K-12 confers a phenotype that includes resistance to UV radiation (254 nm) . Such mutations lead to UV resistance of uvr+, uvrA, lexA and recA strains which could partly be abolished by the addition of cAMP to delta cya but not to delta crp strain culture medium . This effect was not related to either inducibility of major DNA repair genes or growth rate of the bacteria . Enhanced survival was also observed for UV-irradiated lambda bacteriophage indicating that a repair mechanism of UV lesions was involved in this phenomenon.

PCR Methods Appl, 1992 Aug, 2(1), 21 - 7
Ligation-mediated PCR of restriction fragments from large DNA molecules; Smith DR; A general method is described for PCR amplification of single restriction fragments from large DNA molecules . The method involves sequence-specific ligation of synthetic oligonucleotides to ambiguous 4-base 5' overhangs produced by type IIS restriction endonucleases . Such "adapter-tags" provide one target for primer annealing in subsequent PCR reactions . The second target for primer annealing is provided by a universal "bubble-tag" ligated to blunt ends produced with another endonuclease . The key advantage of this approach is that specific fragments can be isolated without any prior knowledge of the nucleotide sequence of the target . Using bacteriophage lambda DNA as a test system, unique PCR products could be generated consistently . Conditions of temperature, ionic strength, and substrate concentration in the adapter-tag ligations--which affect sequence specificity--were found to have a major influence on the purity of PCR-generated fragments . In principle, the method permits the amplification of virtually any sequence from purified cosmid or YAC DNA using a library of only 240 adapter-tags.

Genomics, 1992 Aug, 13(4), 1133 - 42
Isolation of 1001 new markers from human chromosome 11, excluding the region of 11p13-p15.5, and their sublocalization by a new series of radiation-reduced somatic cell hybrids; Gerhard DS et al.; The determination of the physical map of human chromosome 11 will require more clones than are currently available . We have isolated an additional 1001 new markers in a bacteriophage vector from a somatic cell hybrid cell line that contains most of chromosome 11, except the middle of the short arm . These markers were localized to five different regions, 11p15-pter, 11p12-cen, 11q11-q14, 11q14-q23, and 11q23-qter, by a panel of previously characterized somatic cell hybrids . The region 11q11-14 harbors genes that have been shown to be important in breast cancer, B-cell lymphomas, centrocytic lymphomas, asthma, and multiple endocrine neoplasia, type 1 (MEN1) . To determine the positions of the recombinant clones located there, we developed a new series of radiation-reduced somatic cell hybrids . These hybrids, together with those previously characterized, allowed us to map the 11q11-q14 markers into 11 separate segregation groups.

Infect Control Hosp Epidemiol, 1992 Aug, 13(8), 463 - 71
Rapid inactivation of infectious pathogens by chlorhexidine-coated gloves; Modak S et al.; OBJECTIVE: Gloves containing chlorhexidine gluconate in an instant-release matrix on their inner surface (CHG gloves) were tested to determine their ability to rapidly inactivate infectious pathogens that may permeate or leak through the latex surface . DESIGN: CHG gloves were exposed for 1 to 10 minutes to blood or media containing infectious pathogens (e.g., bacteria, fungi, parasites, and viruses) as well as to lymphocytes and macrophages that are known to be the primary carriers of human immunodeficiency virus (HIV) . Inactivation of pathogens was determined either by in vitro assay or in vivo infectivity . Stressed control and CHG glove fingers were submerged in a viral pool (retrovirus or bacteriophage) and after a set time, the glove interiors were checked for presence of permeated virions . RESULTS: CHG gloves rapidly inactivate all the pathogens tested including retrovirus and hepatitis B virus (90% to 100%) . In the stressed glove fingers, live virus was detected in 26% of the control group but not in any of the CHG group . CONCLUSIONS: The use of CHG gloves may reduce the risk of exposure to infectious fluid-borne pathogens should the integrity of the latex barrier be compromised by overt failure or by permeation of viruses . Rapid destruction of lymphocytes and macrophages may facilitate inactivation of HIV associated with these cells . Tests have shown that CHG coating does not alter physical properties of the glove, and, furthermore, CHG gloves do not show potential for dermal irritation or sensitization.

FEBS Lett, 1992 Jul 28, 307(2), 181 - 4
Raman spectroscopic study on the conformation of a peptide fragment representing the DNA-binding domain of filamentous virus Pf3 coat protein; Miura T et al.; Raman spectra have been measured of a nonapeptide which has an amino acid sequence identical to that of the C-terminal region of the major coat protein subunit of filamentous bacteriophage Pf3 . The peptide shows a strong tendency to form a beta-sheet structure in aqueous solution . The beta-sheet formation is significantly promoted by complexation with single-stranded DNA but not with double-stranded DNA . It is suggested that the C-terminal region of the Pf3 coat protein binds to the single-stranded DNA genome in the virion with a beta-sheet conformation, in sharp contrast with the alpha-helical binding in other filamentous bacteriophages.

FEBS Lett, 1992 Jul 27, 307(1), 66 - 70
Peptide display on filamentous phage capsids . A new powerful tool to study protein-ligand interaction; Cesareni G; Peptides can be displayed on the surface of filamentous bacteriophages by fusion to phage coat proteins . It was recently shown that vast (10(8)) collections of phages, each exposing a variant of the original peptide, can be constructed and utilized as a general source of peptide ligands . By panning these libraries on a target molecule linked to a solid support it is possible to select, out of the hundreds of millions of clones, those few phages that display a peptide that binds the target molecule . Searching these libraries is a powerful tool to be applied in many areas of fundamental and applied biology.

Nucleic Acids Res, 1992 Jul 25, 20(14), 3591 - 8
Transcription by an immobilized RNA polymerase from bacteriophage T7 and the topology of transcription; Cook PR et al.; It is often assumed that a polymerase moves along the template as it synthesizes RNA . However, a polymerase that tracks along a helical strand will generate a transcript that is entwined about the template . No such interlocking results if the polymerase is immobile and the template moves past it . Therefore we investigated whether immobilization inhibits the RNA polymerase of T7 bacteriophage using a hybrid protein, in which the polymerase is connected through a peptide linker to an immobilizing domain, which in turn was attached through an antibody to protein A covalently linked to plastic beads . Polymerase could be released by cleaving the linker with a protease, factor Xa . Comparison of the activity of the bound and free enzymes showed that immobilization reduced the rate of initiation about fivefold . However, when re-initiation was eliminated by removing excess template, immobilization was found to have little effect on the rate of elongation . Perhaps the untwining problem is sidestepped in vivo by immobilizing the polymerase.

J Biol Chem, 1992 Jul 25, 267(21), 15032 - 40
Interactions of gene 2.5 protein and DNA polymerase of bacteriophage T7; Kim YT et al.; Bacteriophage T7 gene 2.5 protein has been shown to interact with T7 DNA polymerase (the complex of T7 gene 5 protein and Escherichia coli thioredoxin) by affinity chromatography and fluorescence emission anisotropy . T7 DNA polymerase binds specifically to a resin coupled to gene 2.5 protein and elutes from the resin when the ionic strength of the buffer is raised to 250 mM NaCl . In contrast, T7 gene 5 protein alone binds more weakly to gene 2.5 protein, eluting when the ionic strength of the buffer is 50 mM NaCl . Thioredoxin does not bind to gene 2.5 protein . Steady-state fluorescence emission anisotropy gives a dissociation constant of 1.1 +/- 0.2 microM for the complex of gene 2.5 protein and T7 DNA polymerase, with a ratio of gene 2.5 protein to T7 DNA polymerase in the complex of 1:1 . Nanosecond emission anisotropic analysis suggests that the complex contains one monomer each of gene 2.5 protein, gene 5 protein, and thioredoxin . The ability of T7 gene 2.5 protein to stimulate the activity and processivity of T7 DNA polymerase is compared with the ability of three other single-stranded DNA-binding proteins: E . coli single-stranded DNA-binding protein, T4 gene 32 protein, and E . coli recA protein . All except E . coli recA protein stimulate the activity and processivity of T7 DNA polymerase; E . coli recA protein inhibits these activities.

J Biol Chem, 1992 Jul 25, 267(21), 15022 - 31
Purification and characterization of the bacteriophage T7 gene 2.5 protein . A single-stranded DNA-binding protein; Kim YT et al.; Bacteriophage T7 gene 2.5 protein has been purified to homogeneity from cells overexpressing its gene . Native gene 2.5 protein consists of a dimer of two identical subunits of molecular weight 25,562 . Gene 2.5 protein binds specifically to single-stranded DNA with a stoichiometry of approximately 7 nucleotides bound per monomer of gene 2.5 protein; binding appears to be noncooperative . Electron microscopic analysis shows that gene 2.5 protein is able to disrupt the secondary structure of single-stranded DNA . The single-stranded DNA is extended into a chain of gene 2.5 protein dimers bound along the DNA . In fluorescence quenching and nitrocellulose filter binding assays, the binding constants of gene 2.5 protein to single-stranded DNA are 1.2 x 10(6) M-1 and 3.8 x 10(6) M-1, respectively . Escherichia coli single-stranded DNA-binding protein and phage T4 gene 32 protein bind to single-stranded DNA more tightly by a factor of 25 . Fluorescence spectroscopy suggests that tyrosine residue(s), but not tryptophan residues, on gene 2.5 protein interacts with single-stranded DNA.

J Biol Chem, 1992 Jul 25, 267(21), 15005 - 12
Cloning and expression of gene 4 of bacteriophage T7 and creation and analysis of T7 mutants lacking the 4A primase/helicase or the 4B helicase; Rosenberg AH et al.; T7 gene 4, which is required for DNA replication, specifies two proteins whose coding sequences overlap in the same reading frame: the 4A protein, a 566-amino acid primase/helicase, and the 4B protein, a 503-amino acid helicase whose initiation codon is the 64th codon of the 4A protein . To study better the individual functions of these two overlapping proteins, we made clones that express both 4A and 4B proteins, only 4B protein, or only what we refer to as the 4A' protein, in which methionine 64 is replaced by leucine, thereby eliminating the 4B initiation codon . These clones provide considerably more gene 4 protein for biochemical analysis than do infected cells . They can also be used to isolate and propagate T7 gene 4 deletion mutants, and we have made T7 mutants which lack all gene 4 coding sequences, or which express 4A' protein but no 4B protein, or 4B protein but no 4A protein . Analysis of these phage mutants shows that 4A' protein without any 4B protein can support essentially normal replication and growth, whereas 4B protein without any 4A protein supports little replication or growth . Apparently, the primase activity of the 4A protein is essential for replication, but the 4B protein is dispensable, presumably because the 4A protein also supplies helicase activity . The mutation at amino acid 64 of 4A' appears to have little effect on 4A function . The rate of replication during normal T7 infection appears to be limited by the amount of gene 4 protein, but too high a level of either 4A or 4B protein is inhibitory to growth.

J Mol Biol, 1992 Jul 20, 226(2), 455 - 70
Three-dimensional structure of a cloning vector . X-ray diffraction studies of filamentous bacteriophage M13 at 7 A resolution; Glucksman MJ et al.; Filamentous bacteriophage M13 is a single-stranded DNA phage about 65 A in diameter and 9300 A long . X-ray diffraction studies of magnetically oriented fibers of native, mercury and iodine-labeled phage particles have been used to determine the arrangement of the major coat protein, the gene 8 product, in the virion . The coat protein is made up of a single gently curving alpha-helix extending from approximately Pro6 to near the carboxyl terminus . The axis of the alpha-helix is tilted about 20 degrees from the viral axis and wraps around the axis in a right-handed helical sense . The surface of the virus is made up largely of polar residues in the amino-terminal half of the protein including the segment of alpha-helix extending from Pro6 to Tyr24 . The interior surface of the protein coat faces the DNA and consists of an amphipathic helical segment extending from Thr36 to Ser50 . The alpha-helices form a tightly packed 15 to 20 A thick cylindrical coat around the DNA . This structural model provides insight into the potential sites for incorporating foreign protein domains that may act as functional binding sites on the surface of M13.

J Mol Biol, 1992 Jul 20, 226(2), 311 - 7
Formation of the right before the left mature DNA end during packaging-cleavage of bacteriophage T7 DNA concatemers; Serwer P et al.; During bacteriophage T7 morphogenesis in a T7-infected cell, mature length T7 DNA molecules join end-to-end to form concatemers that are subsequently both packaged in the T7 capsid and cut to mature size . In the present study, the kinetics of the appearance in vivo of the mature right and left T7 DNA ends have been analyzed . To perform this analysis, the intercalating dye proflavine is used to interrupt DNA packaging . When used at 0.5 to 8.0 micrograms/ml, proflavine progressively inhibits events in the T7 DNA packaging pathway, without either altering protein synthesis or degrading intracellular T7 DNA . Restriction endonuclease kinetic analysis reveals that proflavine (8 micrograms/ml) completely blocks formation of the mature T7 DNA left end, but only partially blocks formation of the mature T7 DNA right end . Both these and other observations are explained by the hypothesis that, in the T7 DNA packaging pathway, events occur in the following sequence: (1) formation of a mature right end; (2) packaging of at least some of the genome; (3) formation of the mature left end.

FEBS Lett, 1992 Jul 20, 306(2-3), 129 - 32
On the functional role of the Tyr-639 residue of bacteriophage T7 RNA polymerase; Rechinsky VO et al.; Substitution of Asp for a Tyr residue normally present at position 639 of the bacteriophage T7 RNA polymerase leads to a drastic drop in the enzymatic activity . This mutation does not affect the enzyme-promoter interaction but decreases the ability of the RNA polymerase to discriminate between GTP and ATP molecules, resulting in a decrease in the rate of the incorporation of the nucleotide into the RNA chain.

Proc Natl Acad Sci U S A, 1992 Jul 15, 89(14), 6658 - 62
Identification of a family of bacteriophage T4 genes encoding proteins similar to those present in group I introns of fungi and phage; Sharma M et al.; The bacteriophage T4 segA gene lies in a genetically unmapped region between the gene beta gt (beta-glucosyltransferase) and uvsX (recombination protein) and encodes a protein of 221 amino acids . We have found that the first 100 amino acids of the SegA protein are highly similar to the N termini of four other predicted T4 proteins, also of unknown function . Together these five proteins, SegA-E (similar to endonucleases of group I introns), contain regions of similarity to the endonuclease I-Tev I, which is encoded by the mobile group I intron of the T4 td gene, and to putative endonucleases of group I introns present in the mitochondria of Neurospora crassa, Podospora anserina, and Saccharomyces douglasii . Intron-encoded endonucleases are required for the movement (homing) of the intron DNA into an intronless gene, cutting at or near the site of intron insertion . Our in vitro assays indicate that SegA, like I-Tev I, is a Mg(2+)-dependent DNA endonuclease that has preferred sites for cutting . Unlike the I-Tev I gene, however, there is no evidence that segA (or the other seg genes) resides within introns . Thus, it is possible that segA encodes an endonuclease that is involved in the movement of the endonuclease-encoding DNA rather than in the homing of an intron.

J Biol Chem, 1992 Jul 15, 267(20), 14157 - 66
Processive proofreading is intrinsic to T4 DNA polymerase; Reddy MK et al.; DNA replication occurs in vivo with very high processivity, meaning that the replication complex assembles at the origin(s) of replication and then performs template-directed synthesis of DNA over virtually the entire genome without dissociation . Such processivity also characterizes reconstituted replication holoenzyme complexes in vitro . However, the isolated DNA polymerases are much less processive, especially under physiological conditions . In this paper we monitor the degree of processivity displayed by the bacteriophage T4-coded DNA polymerase while in its proofreading mode by asking whether an isolated polymerase can "edit-out" the 3'-terminal nucleotide from the primer (using the 3'----5'-exonuclease activity of the polymerase) and then switch into the synthesis mode without dissociating from the DNA template . This "switch experiment" is accomplished by using mismatched primer/template substrates as an experimental tool to mimic the situation that T4 DNA polymerase encounters after a misincorporation event has occurred . By performing experiments under single-turnover conditions (obtained using a heparin trap), we demonstrate that T4 DNA polymerase, upon encountering a misincorporated base, neither synthesizes the next base nor dissociates into solution . Instead, with a greater than 80% probability, it removes the misincorporated base and then continues synthesis in a fully processive manner . We also show that the removal of a doubly mispaired sequence from the 3'-terminus of the primer, followed by synthesis, is comparably processive . In contrast, the apparent processivity of removing a triply mispaired terminus is much reduced . Taken together, these observations are consistent with the notion that the "editing active site" of the T4 enzyme optimally accommodates only two unpaired nucleotide residues . Our results do not support the idea that the exonuclease activity of T4 DNA polymerase is highly selective for mismatched termini; they suggest instead that the dwell time at a misincorporated base determines overall editing efficiency . The integrated results of this study provide additional insight into the structure of the T4 DNA polymerase, as well as into the interactions between the polymerase and the polymerase accessory proteins that are required to provide the holoenzyme complex with full processivity.

Gene, 1992 Jul 15, 116(2), 195 - 203
DNA-binding activity of the murine homeodomain protein Hox-2.3 produced by a hybrid phage T7/vaccinia virus system; de Jong R et al.; Homeobox-containing genes encode transcription factors that, via the homeodomain, bind specifically to DNA . To study the DNA-binding properties of the murine homeodomain-containing protein, Hox-2.3, a hybrid expression system was used, combining gene expression by recombinant vaccinia virus (reVV) with bacteriophage T7 transcription . Expression was achieved by co-infecting HeLa cells with two reVVs, one expressing the T7-RNA polymerase-encoding gene directed by the VV promoter, P7.5, and another containing the Hox-2.3 coding sequence under control of a T7 promoter {Fuerst et al., Mol . Cell . Biol . 7 (1987) 2538-2544} . Co-infected HeLa cells produced large amounts of full-length Hox-2.3 protein . Cytoplasmic and nuclear extracts from these cells were used to examine DNA-binding specificity in vitro . reVV-produced Hox-2.3 protein bound to oligos that contained one or several copies of the common homeodomain-binding site, 5'-TCA-ATTAAAT, and to a lesser extent to multiple (TAA) repeats . Using Southwestern blot analysis, no Hox-2.3-binding sites were detected in a region of the Hox-2 cluster containing the Hox-2.3, Hox-2.4 and Hox-2.5 genes.

J Mol Biol, 1992 Jul 5, 226(1), 37 - 45
Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity; Osumi-Davis PA et al.; To define catalytically essential residues of bacteriophage T7 RNA polymerase, we have generated five mutants of the polymerase, D537N, K631M, Y639F, H811Q and D812N, by site-directed mutagenesis and purified them to homogeneity . The choice of specific amino acids for mutagenesis was based upon photoaffinity-labeling studies with 8-azido-ATP and homology comparisons with the Klenow fragment and other DNA/RNA polymerases . Secondary structural analysis by circular dichroism indicates that the protein folding is intact in these mutants . The mutants D537N and D812N are totally inactive . The mutant K631M has 1% activity, confined to short oligonucleotide synthesis . The mutant H811Q has 25% activity for synthesis of both short and long oligonucleotides . The mutant Y639F retains full enzymatic activity although individual kinetic parameters are somewhat different . Kinetic parameters, (kcat)app and (Km)app for the nucleotides, reveal that the mutation of Lys to Met has a much more drastic effect on (kcat)app than on (Km)app, indicating the involvement of K631 primarily in phosphodiester bond formation . The mutation of His to Gln has effects on both (kcat)app and (Km)app; namely, three- to fivefold reduction in (kcat)app and two- to threefold increase in (Km)app, implying that His811 may be involved in both nucleotide binding and phosphodiester bond formation . The ability of the mutant T7 RNA polymerases to bind template has not been greatly impaired . We have shown that amino acids D537 and D812 are essential, that amino acids K631 and H811 play significant roles in catalysis, and that the active site of T7 RNA polymerase is composed of different regions of the polypeptide chain . Possible roles for these catalytically significant residues in the polymerase mechanism are discussed.

J Biol Chem, 1992 Jul 5, 267(19), 13154 - 9
Identification of potential active-site residues in the Escherichia coli leader peptidase; Sung M et al.; Leader peptidase of Escherichia coli cleaves the leader sequence from the amino terminus of membrane and secreted proteins after these proteins insert across the membrane . Despite considerable research, the mechanism of catalysis of leader peptidase remains unknown . This peptidase cannot be classified using protease inhibitors to the serine, cysteine, aspartic acid, or metallo- classes of proteases (Zwizinski, C., Date, T., and Wickner, W . (1981) J . Biol . Chem . 256, 3593-3597) . Using site-directed mutagenesis, we have attempted to place leader peptidase in one of these groups . We found that leader peptidase, lacking all of the cysteine residues, can cleave the leader peptide from procoat, the precursor to bacteriophage M13 coat protein . Replacement of each histidine residue with an alanyl residue was without effect on catalysis . Among all the serine and aspartic acid residues, serine 90 and serine 185 as well as aspartic acid 99, 153, 273, and 276 are necessary to cleave procoat in a detergent extract . However, only serine 90 and aspartic acid 153 were required for processing using a highly sensitive in vivo assay . In addition to the residues directly affecting catalysis, aspartic acid 99 plays a role in maintaining the structure of leader peptidase . Replacement of this residue with alanine results in a very unstable leader peptidase protein . This study thus defines two critical residues, serine 90 and aspartic acid 153, that may be directly involved in catalysis and provides evidence that leader peptidase belongs to a novel class of serine proteases.

Gene, 1992 Jul 1, 116(1), 43 - 9
Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp; Bierman M et al.; We have constructed cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp . All vectors contain the 760-bp oriT fragment from the IncP plasmid, RK2 . Transfer functions need to be supplied in trans by the E . coli donor strain . We have incorporated into these vectors selectable antibiotic-resistance markers (AmR, ThR, SpR) that function in Streptomyces spp . and other features that should allow for: (i) integration via homologous recombination between cloned DNA and the Streptomyces spp . chromosome, (ii) autonomous replication, or (iii) site-specific integration at the bacteriophage phi C31 attachment site . Shuttle cosmids for constructing genomic libraries and bacteriophage P1 cloning vector capable of accepting approx . 100-kb fragments are also described . A simple mating procedure has been developed for the conjugal transfer of these vectors from E . coli to Streptomyces spp . that involves plating of the donor strain and either germinated spores or mycelial fragments of the recipient strain . We have shown that several of these vectors can be introduced into Streptomyces fradiae, a strain that is notoriously difficult to transform by PEG-mediated protoplast transformation.

Virology, 1992 Jul, 189(1), 21 - 30
Mutations abolishing the endonuclease activity of bacteriophage lambda terminase lie in two distinct regions of the A gene, one of which may encode a "leucine zipper" DNA-binding domain; Davidson AR et al.; Bacteriophage lambda terminase is a multifunctional enzyme composed of two subunits which are the products of the phage-encoded Nu1 and A genes . The enzyme catalyzes the endonucleolytic cleavage of lambda DNA at a site known as cosN and mediates packaging of the phage DNA into empty heads . This work describes the characterization of mutations within the A gene which lead to the loss of terminase endonuclease activity without affecting the ability of the enzyme to package monomeric mature (cut) lambda DNA . The residues changed by these mutations lie in two distinct regions within the carboxy half of the A protein . One of these regions has sequence homology with a conserved region of DNA polymerases . The other region resembles the "leucine zipper" DNA binding domain (bZIP) found in eukaryotic transcription factors in that both a basic region and leucine heptad-repeat are present . This terminase domain may be involved in the recognition and/or cleavage of cosN.

J Gen Microbiol, 1992 Jul, 138 ( Pt 7), 1393 - 7
Molecular characterization of two bacteriophages isolated from Desulfovibrio vulgaris NCIMB 8303 (Hildenborough); Seyedirashti S et al.; A preliminary endonuclease restriction map of a bacteriophage isolated from Desulfovibrio vulgaris has been established . BamHI cleaved whole phage DNA into four fragments while HindIII cut the same DNA into seven fragments . Mapping studies succeeded in linking the four BamHI fragments into two DNA segments; however, no linkage between the two segments was detected . These data imply that two phages were induced from cultures of D . vulgaris and that the two segments represented the DNA from these phages . Support for this hypothesis came from size approximation of restriction enzyme fragments, electron micrographs, and density gradients.

Ultramicroscopy, 1992 Jul, 42-44 ( Pt B), 1113 - 7
Atomic force microscopy imaging of T4 bacteriophages on silicon substrates; Kolbe WF et al.; A new atomic force microscope incorporating microfabricated cantilevers and employing laser beam deflection for force detection has been constructed and is being applied to studies of biological material . In this study, T4 bacteriophage virus particles were deposited from solution onto electronic-grade flat silicon wafers and imaged in air with the microscope . Microliter droplets of the solution were deposited and either allowed to dry or removed with blotting paper . The images show both isolated viruses and aggregates of various sizes . The external structure as well as strands believed to be DNA streaming out of the virus could be observed . The construction of the microscope and its performance are also described.

Biopolymers, 1992 Jul, 32(7), 795 - 810
Flow linear dichroism spectra of four filamentous bacteriophages: DNA and coat protein contributions; Clack BA et al.; In this study, we have separated the contributions of DNA and protein to the absorption and linear dichroism (LD) of each of four phages: fd, IKe, Pf1, and Pf3 . We have found that the DNA packaged in each of the phages is hypochromic relative to the purified single-stranded DNA, suggesting that bases are stacked in all of the phages . We have oriented the phages by flow and for the first time report the intrinsic LD from 320 to 190 nm for each of these phages . From the intrinsic LD of the phages and the isotropic absorption of the individual components, we have determined the reduced dichroism of the DNA within the phages and, subsequently, the maximum angle of inclination of the DNA bases (from the helix axis) for the packaged DNA . The maximum angles were 63 degrees and 64 degrees for the DNAs of class I phages fd and IKe, respectively . The angles were significantly less, 51 degrees and 49 degrees, for the DNAs of the class II phages Pf1 and Pf3, respectively . Thus, the two classes of phage differ in the structures of their packaged DNA, the DNA bases of the class II phages being more parallel to the long axis of the phage than are the DNA bases of the class I phages.

Plasmid, 1992 Jul, 28(1), 14 - 24
New cloning vectors for integration in the lambda attachment site attB of the Escherichia coli chromosome; Diederich L et al.; A set of plasmid cloning vectors has been constructed, allowing the integration of any DNA fragment into the bacteriophage lambda attachment site attB of the Escherichia coli chromosome . The system is based upon two components: (i) a number of cloning vectors containing the lambda attachment site attP and (ii) a helper plasmid, bearing the lambda int gene, transcribed from the lambda PR promoter under the control of the temperature-sensitive repressor cI857 . The DNA fragment of interest is cloned into the multicloning site of one of the attP-harboring plasmids . Subsequently, the origin of the plasmid, located on a cloning cassette, is cut out and the DNA becomes newly ligated, resulting in a circular DNA molecule without replication ability . The strain of choice, containing the int gene carrying helper plasmid, is transformed with this DNA molecule and incubated at 42 degrees C to induce int gene expression . Additionally, the temperature shift leads to the loss of the helper plasmid after a few cell generations, because the replication ability of its replicon is blocked at 42 degrees C . These vectors have been successfully used for integration of several promoter-lacZ fusions into the chromosome . The ratio between integration due to homologous recombination and Int protein-mediated integration has been determined.

Photochem Photobiol, 1992 Jul, 56(1), 35 - 42
DNA as a solar dosimeter in the ocean; Regan JD et al.; Stratospheric ozone depletion may result in increased solar UV-B radiation to the ocean's upper layers and may cause deleterious effects on marine organisms . The primary UV-B damage induced in biological systems is to DNA . While physical measurements of solar UV-B penetration into the sea have been made, the effective depth and magnitude of actual DNA damage have not been determined . In the experiments reported here, UV-B-induced photoproducts (cyclobutane pyrimidine dimers) have been quantified in DNA molecules exposed to solar UV at the surface and at various depths in clear, tropical marine waters off Lee Stocking Island (23 degrees 45' N, 76 degrees 0.7' W), Exuma Cays, Bahamas . (14C)thymidine-labeled DNA or unlabeled bacteriophage phi X174 DNA was placed in specially designed quartz tubes at various depths for up to five days . Following exposure, DNA samples were removed to the laboratory where UV-B-induced pyrimidine dimers were quantified using a radiochromatographic assay, and bacteriophage DNA inactivation by solar UV-B was assayed by plaque formation in spheroplasts of Escherichia coli . Pyrimidine dimer induction was linear with time but the accumulation of dimers in DNA with time varied greatly with depth . Attenuation of dimer formation with depth of water was exponential . DNA at 3 m depth had only 17% of the pyrimidine dimers found at the surface . Bacteriophage phi X174 DNA, while reduced 96% in plaque-forming ability by a one day exposure to solar UV at the surface of the water, showed no effect on plaque formation after a similar exposure at 3 m . The data collected at the water's surface showed a "surface-enhanced dose" in that DNA damages at the real surface were greater than at the imaginary surface, which was obtained by extrapolating the data at depth to the surface . These results show the sensitivity of both the biochemical (dimers) and biological (phage plaques) DNA dosimeters . DNA dosimeters offer a sensitive, convenient and relatively inexpensive monitoring system, having both biochemical and biological endpoints for monitoring the biologically effective UV-B flux in the marine environment . Unlike physical dosimeters, DNA dosimeters do not have to be adjusted for biological effectiveness since they are sensitive only to DNA-mediated biologically effective UV-B radiation . Results of pyrimidine dimer induction in DNA by solar UV accurately predicted UV doses to the phage DNA.

Genomics, 1992 Jul, 13(3), 565 - 74
Genomic organization, chromosomal localization, and independent expression of human cyclin D genes; Inaba T et al.; Murine cDNA clones for three cyclin D genes that are normally expressed during the G1 phase of the cell cycle were used to clone the cognate human genes . Bacteriophage and cosmid clones encompassing five independent genomic loci were partially sequenced and chromosomally assigned by an analysis of somatic cell hybrids containing different human chromosomes and by fluorescence in situ hybridization to metaphase spreads from normal peripheral blood lymphocytes . The human cyclin D1 gene (approved gene symbol, CCND1) was assigned to chromosome band 11q13, cyclin D2 (CCND2) to chromosome band 12p13, and cyclin D3 (CCND3) to chromosome band 6p21 . Pseudogenes containing sequences related to cyclin D2 and cyclin D3 mapped to chromosome bands 11q13 and 6p21, respectively . Partial nucleotide sequence analysis of exons within each gene revealed that the authentic human cyclin D genes are more related to their mouse counterparts than to each other . These genes are ubiquitously transcribed in human tumor cell lines derived from different cell lineages, but are independently and, in many cases, redundantly expressed . The complex patterns of expression of individual cyclin D genes and their evolutionary conservation across species suggest that each family member may play a distinct role in cell cycle progression.

Genetics, 1992 Jul, 131(3), 541 - 50
Meiotic recombination on artificial chromosomes in yeast; Ross LO et al.; We have examined the meiotic recombination characteristics of artificial chromosomes in Saccharomyces cerevisiae . Our experiments were carried out using minichromosome derivatives of yeast chromosome III and yeast artificial chromosomes composed primarily of bacteriophage lambda DNA . Tetrad analysis revealed that the artificial chromosomes exhibit very low levels of meiotic recombination . However, when a 12.5-kbp fragment from yeast chromosome VIII was inserted into the right arm of the artificial chromosome, recombination within that arm mimicked the recombination characteristics of the fragment in its natural context including the ability of crossovers to ensure meiotic disjunction . Both crossing over and gene conversion (within the ARG4 gene contained within the fragment) were measured in the experiments . Similarly, a 55-kbp region from chromosome III carried on a minichromosome showed crossover behavior indistinguishable from that seen when it is carried on chromosome III . We discuss the notion that, in yeast, meiotic recombination behavior is determined locally by small chromosomal regions that function free of the influence of the chromosome as a whole.

Trends Biochem Sci, 1992 Jul, 17(7), 241 - 5
Discovering peptide ligands using epitope libraries; Scott JK; Epitope libraries are large collections of peptides . Each peptide is displayed on the surface of a bacteriophage particle and is encoded by a randomly mutated region of the phage genome, thus associating each unique peptide with the DNA molecule encoding it . Antibodies and other binding proteins are used to select specifically for rare, phage-bearing peptide ligands; sequencing of the corresponding viral DNA will reveal their amino acid sequences . Relatively high-affinity peptides for a variety of peptide- and non-peptide-binding ligates have been affinity-isolated from epitope libraries . This technology has been used to map epitopes on proteins and to find peptide mimics for non-peptide-binding ligates . The current challenge lies in developing epitope library technology so that tight-binding peptide ligands can be detected for a wider variety of ligates, including those that recognize folded proteins . Should this be accomplished, many powerful applications can be envisioned in the areas of drug design and the development of diagnostic markers and vaccines.

Biotechnology (N Y), 1992 Jul, 10(7), 779 - 83
By-passing immunization: building high affinity human antibodies by chain shuffling; Marks JD et al.; Diverse antibody libraries can be displayed on the surface of filamentous bacteriophage, and selected by panning of the phage with antigen . This allows human antibodies to be made directly in vitro without prior immunization, thus mimicking the primary immune response . Here we have improved the affinity of one such "primary" antibody by sequentially replacing the heavy and light chain variable (V) region genes with repertoires of V-genes (chain shuffling) obtained from unimmunized donors . For a human phage antibody for the hapten 2-phenyloxazol-5-one (phOx) (Kd = 3.2 x 10(-7) M), we shuffled the light chains and isolated an antibody with a 20 fold improved affinity . By shuffling the first two hypervariable loops of the heavy chain, we isolated an antibody with a further 15-fold improved affinity . The reshuffled antibody differed in five of the six hypervariable loops from the original antibody and the affinity for phOx (Kd = 1.1 x 10(-9) M) was comparable to that of mouse hybridomas from the tertiary immune response . Reshuffling offers an alternative to random point mutation for affinity maturation of human antibodies in vitro.

Genetika, 1992 Jul, 28(7), 66 - 76
{Isolation, characterization and mapping of the N15 phasmid insertion mutations}; San'kova TP et al.; Prophage of N15 temperate bacteriophage is stably maintained in Escherichia coli lysogens as a 46.33 kb linear plasmid . Using different transposons we obtained 18 insertion mutants of the N15 plasmid prophage . They were analysed for plaque formation ability, stability of the plasmid state and lysogenic conversion . Restriction mapping of the insertions allowed us to localize on the map the regions necessary for lytic growth and to map the lysogenic conversion gene . A recombinant phage encoding two antibiotic resistance genes was obtained . The phage contains an additional 4.77 kb DNA fragment (over 10% of the N15 genome).

Proc Natl Acad Sci U S A, 1992 Jul 1, 89(13), 6015 - 9
Rapid fitness losses in mammalian RNA virus clones due to Muller's ratchet; Duarte E et al.; Muller's ratchet is an important concept in population genetics . It predicts that when mutation rates are high and a significant proportion of mutations are deleterious, a kind of irreversible ratchet mechanism will gradually decrease the mean fitness of small populations of asexual organisms . In contrast, sexual recombination may stop or reverse this mutational ratchet by recombinational repair of genetic damage . Experimental support for Muller's ratchet has previously been obtained in protozoa and in a tripartite RNA bacteriophage . We now show clear evidence that Muller's ratchet can operate on a nonsegmented nonrecombining pathogenic RNA virus of animals and humans . We did genetic bottleneck passages (plaque-to-plaque transfers) of vesicular somatitis virus (VSV) and then quantitated relative fitness of the bottleneck clones by allowing direct replication competition in mixed infections in cell culture . We document variable fitness drops (some severe) following only 20 plaque-to-plaque transfers of VSV . In some clones no fitness changes (or only insignificant changes) were observed . Surprisingly, the most regular and severe fitness losses occurred during virus passages on a new host cell type . These results again demonstrate the extreme genetic and biological variability of RNA virus populations . Muller's ratchet could have significant implications for variability of disease severity during virus outbreaks, since genetic bottlenecks must often occur during respiratory droplet transmissions and during spread of low-yield RNA viruses from one body site to another (as with human immunodeficiency virus) . Likewise, the lower-probability generation of increased-fitness clones during repeated genetic bottleneck transfers of RNA viruses in nature might also affect disease pathogenesis in infected individuals and in host populations . Whenever genetic bottlenecks of RNA viruses occur, enhanced biological differences among viral subpopulations may result.

Biochem Cell Biol, 1992 Jul, 70(7), 605 - 8
Development of T7 phage and T7 phage containing apurinic sites in an exonuclease III, endonuclease IV double mutant of Escherichia coli; Sanchez G et al.; The development of bacteriophage T7 was examined in an Escherichia coli double mutant defective for the two major apurinic, apyrimidinic endonucleases (exonuclease III and endonuclease IV, xth nfo) . In cells infected with phages containing apurinic sites, the defect in repair enzymes led to a decrease of phage survival and a total absence of bacterial DNA degradation and of phage DNA synthesis . These results directly demonstrate the toxic action of apurinic sites on bacteriophage T7 at the intracellular level and its alleviation by DNA repair . In addition, untreated T7 phage unexpectedly displayed reduced plating efficiency and decreased DNA synthesis in the xth nfo double mutant.

Cell, 1992 Jun 26, 69(7), 1199 - 212
Suppressor of Hairless, the Drosophila homolog of the mouse recombination signal-binding protein gene, controls sensory organ cell fates; Schweisguth F et al.; Suppressor of Hairless (Su(H)) is required at two stages of adult sensory organ development in Drosophila . Complete loss of Su(H) function results in a "neurogenic" phenotype in imaginal discs, in which too many cells adopt the sensory organ precursor cell fate . Su(H) is also involved in controlling the fates of sensillum accessory cells and is specifically expressed in two of these cells . Su(H) is the Drosophila homolog of the mouse J kappa RBP gene, whose product binds specifically to the recombination signal sequence of immunoglobulin J kappa segments . The Su(H) and J kappa RBP proteins are 82% identical over most of their length, and share with bacteriophage integrates and yeast recombinases a motif that includes residues directly involved in catalyzing recombination.

Cell, 1992 Jun 26, 69(7), 1181 - 9
The phage lambda gene Q transcription antiterminator binds DNA in the late gene promoter as it modifies RNA polymerase; Yarnell WS et al.; The bacteriophage lambda gene Q transcription antiterminator modifies RNA polymerase during an extended pause in elongation at nt +16 and +17 of the phage late gene promoter transcript . We show here that Q binds a specific DNA site between the -10 and -35 elements of the promoter as it interacts with the enzyme . We show that the pause must reflect a specialized elongation structure that is receptive to modification by Q, because Q does not bind to RNA polymerase stopped artificially after transcribing 16 nt of mutant DNA that does not encode the natural pause . Footprinting shows that RNA polymerase in the paused complex makes distinctive interactions with DNA in the region where Q binds; binding of Q, in turn, changes the footprint both at the Q-binding site and in the transcription bubble . Binding of Q to the paused transcription complex is stabilized by the transcription factor NusA, as expected from the dependence of lambda Q-mediated antitermination on NusA.

Nucleic Acids Res, 1992 Jun 25, 20(12), 3085 - 90
The c4 repressor of bacteriophage P1 is a processed 77 base antisense RNA; Citron M et al.; The c4 repressors of the temperate bacteriophages P1 and P7 inhibit antirepressor synthesis and are essential for establishment and maintenance of lysogeny . Using in vivo complementation tests we have previously shown that c4 is an antisense RNA acting on a target, ant mRNA, which is transcribed from the same promoter . Here we identify the c4 repressor molecule of P1 as a 77 +/- 1 base RNA by mapping its termini and show that the c4 RNA in P7 lysogens has the same or a similar size . P1 c4 RNA is encoded in a region shown to be sufficient for c4 complementation . It covers exactly the 74 bases previously suggested to fold into a stem-loop secondary structure essential for c4 function . Furthermore, we demonstrate that the 5' end of c4 RNA is generated by processing . Thus, c4 is the first example of an antisense RNA to be processed . A possible mechanism of processing is discussed.

J Biol Chem, 1992 Jun 25, 267(18), 12660 - 7
Molecular cloning of rat prostate transglutaminase complementary DNA . The major androgen-regulated protein DP1 of rat dorsal prostate and coagulating gland; Ho KC et al.; Complementary DNA (cDNA) that codes for a major androgen-dependent secretory protein of rat coagulating gland and dorsal prostate, dorsal protein 1 (DP1), was isolated by molecular cloning . Recombinant DP1 cDNA clones were identified from a bacteriophage lambda gt11 rat coagulating gland expression library using an affinity purified polyclonal antibody . Amino acid sequence deduced from DNA contained sequences identical with several DP1 cyanogen bromide cleavage fragments . Northern blot hybridization of poly(A) RNA isolated from intact rat dorsal prostate and coagulating gland revealed a predominant messenger RNA (mRNA) species of approximately 3200 nucleotides . Tissue-specific expression of DP1 mRNA was indicated by the absence of DP1 mRNA in ventral prostate and other tissues of the rat . Expression of DP1 mRNA was androgen-dependent, decreasing approximately 80% 7 days after castration and increasing rapidly following androgen replacement . Southern blot analysis of restriction enzyme-digested rat DNA indicated that DP1 is encoded by a single gene and that no major genomic rearrangements accounted for its lack of expression in the dorsal prostate-derived rat Dunning tumor . Sequence comparisons revealed that rat prostate DP1 shares sequence identity with Factor XIIIa and tissue transglutaminase, including the active center, GQCWVF, indicating that DP1 is a member of the transglutaminase gene family.

Ann N Y Acad Sci, 1992 Jun 16, 653, 122 - 30
Screening of a Babesia bigemina cDNA library with monoclonal antibodies directed to surface antigens; Figueroa JV et al.; A Babesia bigemina cDNA library prepared in lambda ZAP bacteriophage vector was immunoscreened to detect clones expressing surface-exposed epitopes of B . bigemina . A nonradioactive indirect plaque-lift immunoassay was used to detect the positive clones . The primary antibody consisted of a pooled sample of six monoclonal antibodies (mAb) specific for B . bigemina that recognizes various parasite surface antigens of different molecular mass . Screening of approximately 300,000 plaque-forming units from the lambda ZAP cDNA expression library resulted in the identification of five positive clones . The five recombinant clones were immunoscreened individually with each of the six mAb . All five independently obtained clones consisted of lambda ZAP recombinants expressing B . bigemina components recognized by mAb C2F3G3 and B1B3C4 . Restriction enzyme digests of rescued recombinant phagemids showed that only four clones contained B . bigemina cDNA . One clone (lambda ZAP Bbi1) contained an insert of approximately 0.6 kBp whereas the other three clones (lambda ZAP Bbi2, lambda ZAP Bbi3, and lambda ZAP Bbi5) carried a cDNA insert of approximately 1.7 kBp . Immunoblotting of protein extracts from recombinants lambda ZAP Bbi2, lambda ZAP Bbi3, and lambda ZAP Bbi5 with mAb C2F3G3 and B1B3C4 demonstrated the expression of a recombinant B . bigemina polypeptide of 55 kDa in E . coli.

Eur J Biochem, 1992 Jun 15, 206(3), 605 - 12
Fluorescence studies of the binding of bacteriophage M13 gene V mutant proteins to polynucleotides; Stassen AP et al.; This investigation describes how the binding characteristics of the single-stranded DNA-binding protein encoded by gene V of bacteriophage M13, are affected by single-site amino acid substitutions . The series of mutant proteins tested includes mutations in the purported monomer-monomer interaction region as well as mutations in the DNA-binding domain at positions which are thought to be functionally involved in monomer-monomer interaction or single-stranded DNA binding . The characteristics of the binding of the mutant proteins to the homopolynucleotides poly(dA), poly(dU) and poly(dT), were studied by means of fluorescence-titration experiments . The binding stoichiometry and fluorescence quenching of the mutant proteins are equal to, or lower than, the wild-type gene V protein values . In addition, all proteins measured bind a more-or-less co-operative manner to single-stranded DNA . The binding affinities for poly(dA) decrease in the following order: Y61H greater than wild-type greater than F68L and R16H greater than Y41F and Y41H greater than F73L greater than R21C greater than Y34H greater than G18D/Y56H . Possible explanations for the observed differences are discussed . The conservation of binding affinity, also for mutations in the single-stranded DNA-binding domain, suggests that the binding to homopolynucleotides is largely non-specific.

J Biol Chem, 1992 Jun 15, 267(17), 12174 - 81
The Bof protein of bacteriophage P1 exerts its modulating function by formation of a ternary complex with operator DNA and C1 repressor; Velleman M et al.; Bacteriophage P1 encodes several regulatory elements for the lytic or lysogenic response, which are located in the immC, immI, and immT regions . Their products are the C1 repressor of lytic functions with the C1 inactivator protein Coi, the C4 repressor of antirepressor synthesis and the modulator protein Bof, respectively . We have studied in vitro the interaction of the components of the immC and immT regions with C1-controlled operators using highly purified Bof, C1, and Coi proteins . Bof protein (M(r) = 9,800) does not interact with C1 repressor alone, but as shown by DNA mobility shift experiments, in the presence of C1 repressor Bof binds to all operators tested by forming a C1.Bof-operator DNA ternary complex . The effect of this complex formation was studied in more detail with the operator of the c1 gene . Here, Bof only marginally alters the C1 repressor footprint at Op99a,b, but nevertheless considerably influences the repressibility of the operator.promoter element: (i) the autoregulated c1 mRNA synthesis is further down-regulated and (ii) the ability of Coi protein to dissociate the C1.operator DNA complex is strongly inhibited . We suggest that Bof protein functions by modulating C1 repression of many widely dispersed operators on the prophage genome.

Cell, 1992 Jun 12, 69(6), 1011 - 20
Infectious defective interfering particles of VSV from transcripts of a cDNA clone; Pattnaik AK et al.; The generation of infectious defective interfering (DI) particles of vesicular stomatitis virus (VSV) entirely from cDNA clones is reported . Bacteriophage T7 RNA polymerase was used to direct the transcription of a complete negative-stranded genomic RNA from a cDNA clone of a VSV DI RNA in cells simultaneously expressing the five VSV proteins from separately transfected cDNA clones . The negative-stranded transcript was encapsidated with N protein, replicated by the VSV polymerase, and the replicated RNAs were assembled and budded to yield infectious DI virions . No helper VSV was required . Replication occurred at high levels and was assayed by direct biochemical means . An exact 3' terminus of the initial transcript, which was generated by autolytic cleavage using a ribozyme from hepatitis delta virus, was critical for replication.

J Biol Chem, 1992 Jun 5, 267(16), 11399 - 407
Purified MotA protein binds the -30 region of a bacteriophage T4 middle-mode promoter and activates transcription in vitro; Schmidt RP et al.; The bacteriophage T4-encoded MotA protein is critical for transcription from T4 middle-mode promoters . However, a direct interaction of this protein with a middle-mode promoter has not previously been demonstrated . We have cloned the motA gene and overexpressed the gene product using the T7 expression system . A simple procedure was then developed to purify the MotA protein to homogeneity . Using the purified protein we have demonstrated that MotA protein binds to the -30 region of the middle-mode promoter PuvsY . This promoter has previously been shown to be a necessary component of a T4 replication origin, and thus MotA is also a T4 origin-binding protein . Modified RNA polymerase purified from T4-infected cells was used to establish middle-mode transcription in vitro . Transcription from PuvsY was markedly enhanced by the addition of MotA protein, whether or not the template contained the cytosine modifications characteristic of T4 DNA . However, transcription from PuvsY was apparently independent of the MotA protein when unmodified RNA polymerase from uninfected cells was used.

J Biol Chem, 1992 Jun 5, 267(16), 11637 - 44
A mutation in GroEL interferes with protein folding by reducing the rate of discharge of sequestered polypeptides; Baneyx F et al.; GroEL140, a mutant Escherichia coli chaperonin unable to support bacteriophage lambda head assembly, was purified to near homogeneity and compared to wild type GroEL (cpn60) . GroEL140 exhibited a 1.5-fold lower ATPase activity relative to the wild type protein . The hydrolysis of ATP by both polypeptides was fully inhibited by an excess of ATP gamma S and partially inhibited by ADP and 5'-adenylylimidodiphosphate, suggesting that adenine nucleotides display different affinities for the ATP binding site of chaperonins . GroEL140 was more sensitive to trypsin digestion compared to wild type GroEL indicating that the mutation destabilized the conformation of the mutant . The proteolytic susceptibility of both chaperonins was similarly enhanced upon addition of ATP, ADP or non-hydrolyzable ATP analogs, providing evidence (i) of a conformational change in the chaperonin structure which is likely to drive the protein discharge process, and (ii) that hydrolysis of ATP is not required to achieve topological modifications . GroEL140 retained its ability to bind non-native ribulose bisphosphate carboxylase/oxygenase (Rbu-P2-carboxylase), but released bound proteins upon addition of ATP and GroES (cpn 10) 6-7-fold less efficiently compared to GroEL . This functional defect was shown to be related to a suboptimal, but not an absence of, interaction with GroES since (i) GroEL140 and GroES were unable to form a complex isolatable by size exclusion chromatography, and (ii) increasing the incubation time or the concentration of GroES enhanced the amount of refolded Rbu-P2-carboxylase discharged from GroEL140-Rbu-P2-carboxylase binary complexes . Pulse-chase experiments involving a double immunoabsorption technique confirmed that Rbu-P2-carboxylase remained associated two times longer with GroEL140 than with GroEL in vivo.

Mol Gen Genet, 1992 Jun, 233(3), 348 - 54
Survival of phage M13 with uracils on one or both DNA strands; Schunemann S et al.; The survival of M13 DNA was studied after partial replacement of thymine by uracil in the bacteriophage . Uracils carry the same genetic information as the thymines . Nevertheless in Escherichia coli wild-type cells, uracils in DNA are replaced by thymines by excision repair initiated by uracil-DNA glycosylase (UDG) . Thus inactivation of uracil-containing phage DNA is solely due to repair initiated by UDG . Incorporation of uracils was achieved in one or in both strands, either randomly or site-specifically using differently uracylated oligonucleotides . The results show that up to 580 uracils can be repaired without a significant decrease in survival if the uracils are localized in the (-) strand only . Incorporation of 246 uracils into the (+) strand leads to approximately 30% or approximately 10% survival when expressed in Escherichia coli strains CMK and JM103, respectively . However, when uracils are distributed over both strands a sharp decrease in survival occurs . This shows that the repair of two uracils localized in close proximity on opposite strands of the DNA by the excision repair mechanism is difficult, whereas uracils occurring in one strand are repaired more efficiently, irrespective of their number.

J Bacteriol, 1992 Jun, 174(12), 4094 - 100
Escherichia coli K-12 and B contain functional bacteriophage P2 ogr genes; Slettan A et al.; The bacteriophage P2 ogr gene encodes an essential 72-amino-acid protein which acts as a positive regulator of P2 late transcription . A P2 ogr deletion phage, which depends on the supply of Ogr protein in trans for lytic growth on Escherichia coli C, has previously been constructed . E . coli B and K-12 were found to support the growth of the ogr-defective P2 phage because of the presence of functional ogr genes located in cryptic P2-like prophages in these strains . The cryptic ogr genes were cloned and sequenced . Compared with the P2 wild-type ogr gene, the ogr genes in the B and K-12 strains are conserved, containing mostly silent base substitutions . One of the base substitutions in the K-12 ogr gene results in replacement of an alanine with valine at position 57 in the Ogr protein but does not seem to affect the function of Ogr as a transcriptional activator . The cryptic ogr genes are constitutively transcribed, apparently at a higher level than the wild-type ogr gene in a P2 lysogen.

J Bacteriol, 1992 Jun, 174(12), 4086 - 93
Attachment sites for bacteriophage P2 on the Escherichia coli chromosome: DNA sequences, localization on the physical map, and detection of a P2-like remnant in E . coli K-12 derivatives; Barreiro V et al.; Integration of bacteriophage P2 into the Escherichia coli genome involves recombination between two attachment sites, attP and attB, one on the phage and one on the host genome, respectively . At least 10 different attB sites have been identified over the years . In E . coli C, one site, called locI, is preferred, being occupied before any of the others . In E . coli K-12, no such preference is seen (reviewed in L . E . Bertani and E . W . Six, p . 73-143, in R . Calendar, ed., The Bacteriophages, vol . 2, 1988) . The DNA sequence of locI has been determined, and it shows a core sequence of 27 nucleotides identical to attP (A . Yu, L . E . Bertani, and E . Haggard-Ljungquist, Gene 80:1-12, 1989) . By inverse polymerase chain reactions, the prophage-host junctions of DNA extracted from P2 lysogenic strains have been amplified, cloned, and sequenced . By combining the attL and attR sequences, the attB sequences of locations II, III, and H have been deduced . The core sequence of location II had 20 matches to the 27-nucleotide core sequence of attP; the sequences of locations III and H had 17 matches . Thus, the P2 integrase accepts at least up to 37% mismatches within the core sequence . The E . coli K-12 strains examined all contain a 639-nucleotide-long cryptic remnant of P2 at a site with a sequence similar to that of locI but that may have a different map position . The P2 remnant consists of the C-terminal part of gene D, all of gene ogr, and attR . Locations II, III, and H have been located on Kohara's physical map to positions 3670, 1570 to 1575, and 2085, respectively.

Radiat Res, 1992 Jun, 130(3), 366 - 71
Increased resistance to ionizing and ultraviolet radiation in Escherichia coli JM83 is associated with a chromosomal rearrangement; McLean KM et al.; Cells cope with radiation damage through several mechanisms: (1) increased DNA repair activity, (2) scavenging and inactivation of radiation-induced radical molecules, and (3) entry into a G0-like quiescent state . We have investigated a chromosomal rearrangement to elucidate further the molecular and genetic mechanisms underlying these phenomena . A mutant of Escherichia coli JM83 (phi 80dlacZ delta M15) was isolated that demonstrated significantly increased resistance to both ionizing and ultraviolet radiation . Surviving fractions of mutant and wild-type cells were measured following exposure to standardized doses of radiation . Increased radioresistance was directly related to a chromosomal alteration near the bacteriophage phi 80 attachment site (attB), as initially detected by the LacZ- phenotype of the isolate . Southern hybridization of chromosomal DNA from the mutant and wild-type E . coli JM83 strains indicated that a deletion had occurred . We propose that the deletion near the attB locus produces the radioresistant phenotype of the E . coli JM83 LacZ- mutant, perhaps through the alteration or inactivation of a gene or its controlling element(s).

Proc Natl Acad Sci U S A, 1992 Jun 1, 89(11), 5053 - 7
Regions of bacteriophage T4 and RB69 RegA translational repressor proteins that determine RNA-binding specificity; Jozwik CE et al.; RegA protein of T4 and related bacteriophages is a highly conserved RNA-binding protein that represses the translation of many phage mRNAs that encode enzymes involved in DNA metabolism . RB69, a T4-related bacteriophage, has a unique regA gene, which we have cloned, sequenced, and expressed . The predicted amino acid sequence of RB69 RegA is 78% identical to that of T4 RegA . Plasmid-encoded RB69 RegA expressed in vivo represses the translation of T4 early mRNAs, including those of rIIA, rIIB, 44, 45, rpbA, and regA . Nucleotide sequences were determined for several T4 and RB69 regA mutations, and their corresponding repressor properties were characterized . All of the 10 missense mutations affect residues conserved between RB69 and T4 RegA . Two regions of RegA are especially sensitive to mutation: one between Val-15 and Ala-25 and another between Arg-70 and Ser-73 . Sequence alignments and mutational data suggest that the region from Val-15 to Ala-25 is similar to helix-turn-helix domains of DNA-binding proteins and confers RNA-binding specificity upon RegA . The RegA691 protein (Ile-24----Thr) has an in vivo phenotype that appears to distinguish site-specific and cooperative binding modes of hierarchical RegA-mediated translational repression.

Virology, 1992 Jun, 188(2), 887 - 9
Cloning, sequence, and overexpression of bacteriophage T4 gene 51; Nivinskas R et al.; The nucleotide sequence of the 907-bp XbaI-EcoRV T4 DNA fragment containing gene 51 is presented . The sequence of gene 51 predicts a 249 amino acid peptide with an M(r) of 29387 and a pl of 6.34 . We have cloned and overexpressed this gene in the T7 RNA polymerase system . The observed molecular mass of gp51 is in agreement with the sequence data . We also show that the low level of gene 51 expression usually seen is caused by an RNA stem-loop structure in the region between genes 26 and 51 . In addition, discrepancies with the sequence published by other authors are indicated.

Virology, 1992 Jun, 188(2), 831 - 9
The polarity suppression factor of bacteriophage P4 is also a decoration protein of the P4 capsid; Isaksen ML et al.; We show that the product of the polarity suppression (psu) gene from bacteriophage P4 associates with P4 capsids . This association can occur when Psu is (i) provided in vivo from the P4 genome or from a plasmid or (ii) provided in vitro by mixing viable phage particles with Psu protein . Psu is unable to associate with the larger capsid of P4's helper phage P2 . Discrimination of the P4 and P2 capsids by Psu appears to be independent of the presence of the P4 genome in the capsid, since P2 size capsids filled with P4 DNA cannot accommodate Psu association . P4 psu particles devoid of Psu are less stable than P4 particles carrying Psu . These results indicate that, in addition to its antitermination activity at Rho-dependent terminators, Psu is also a decoration protein that stabilizes the P4 capsids.

J Bacteriol, 1992 Jun, 174(12), 3936 - 44
DNA inversion regions Min of plasmid p15B and Cin of bacteriophage P1: evolution of bacteriophage tail fiber genes; Sandmeier H et al.; Plasmid p15B and the genome of bacteriophage P1 are closely related, but their site-specific DNA inversion systems, Min and Cin, respectively, do not have strict structural homology . Rather, the complex Min system represents a substitution of a Cin-like system into an ancestral p15B genome . The substituting sequences of both the min recombinase gene and the multiple invertible DNA segments of p15B are, respectively, homologous to the pin recombinase gene and to part of the invertible DNA of the Pin system on the defective viral element e14 of Escherichia coli K-12 . To map the sites of this substitution, the DNA sequence of a segment adjacent to the invertible segment in the P1 genome was determined . This, together with already available sequence data, indicated that both P1 and p15B had suffered various sequence acquisitions or deletions and sequence amplifications giving rise to mosaics of partially related repeated elements . Data base searches revealed segments of homology in the DNA inversion regions of p15B, e14, and P1 and in tail fiber genes of phages Mu, T4, P2, and lambda . This result suggest that the evolution of phage tail fiber genes involves horizontal gene transfer and that the Min and Pin regions encode tail fiber genes . A functional test proved that the p15B Min region carries a tail fiber operon and suggests that the alternative expression of six different gene variants by Min inversion offers extensive host range variation.

Mol Microbiol, 1992 Jun, 6(12), 1715 - 22
Stabilization of bacteriophage Mu repressor-operator complexes by the Escherichia coli integration host factor protein; Gama MJ et al.; All of the previously described effects of integration host factor (IHF) on bacteriophage Mu development have supported the view that IHF favours transposition-replication over the alternative state of lysogenic phage growth . In this report we show that, consistent with a model in which Mu repressor binding to its operators requires a particular topology of the operator DNA, IHF stimulates repressor binding to the O1 and O2 operators and enhances Mu repression . IHF would thus be one of the keys, besides supercoiling and the H-NS protein, that lock the operator region into the appropriate topological conformation for high-affinity binding not only of the phage transposase but also of the phage repressor.

Mol Microbiol, 1992 Jun, 6(12), 1707 - 14
Escherichia coli integration host factor stabilizes bacteriophage Mu repressor interactions with operator DNA in vitro; Alazard R et al.; Using gel retardation and DNase I protection techniques, we have demonstrated that the Escherichia coli integration host factor (IHF) stabilizes the interaction between Mu repressor and its cognate operator-binding sites in vitro . These results are discussed in terms of a model in which IHF may commit the phage to the lytic or lysogenic pathway depending on the occupancy of the operator sites by the repressor.

J Exp Med, 1992 Jun 1, 175(6), 1677 - 84
Role of a major autoepitope in forming the DNA binding site of the p70 (Ku) antigen; Chou CH et al.; The Ku antigen is a heterodimer consisting of 70- and 80-kD protein subunits that binds to termini of double-stranded DNA . DNA binding appears to be mediated partly by the 70-kD (p70) subunit, but the precise mechanism of its association with DNA is unclear . High-titer autoantibodies in sera from certain patients with systemic lupus erythematosus recognize at least eight distinct epitopes of Ku, and inhibit DNA binding . In the present studies, the binding of DNA to truncated p70 fusion proteins was determined in Southwestern blots and DNA immunoprecipitation assays . Appropriate folding of the p70 protein was crucial for efficient DNA binding . The minimal DNA binding site, amino acids 536-609, contains a major conformational autoepitope of p70 (amino acids 560-609) . Deletion of amino acids 601-609, or substitution of ala-ala-ala for lys-ser-gly at positions 591-593, eliminated DNA binding as well as autoantibody binding, suggesting that the same secondary or supersecondary structure is involved in both DNA binding and autoantibody recognition . Residues within the DNA binding site/autoepitope closely resemble the helix-turn-helix motif in bacteriophage lambda Cro protein and certain other DNA binding proteins, and mutations predicted to destabilize this structure eliminated DNA binding . Adjacent to the helix-turn-helix is a highly basic domain (positions 539-559) that was also required for DNA binding . The findings suggest that the DNA binding site of p70 consists of a basic domain adjacent to a helix-turn-helix structure that also forms a major autoepitope.

Virology, 1992 Jun, 188(2), 429 - 37
Biologically active cymbidium ringspot virus satellite RNA in transgenic plants suppresses accumulation of DI RNA; Rubino L et al.; A full-length DNA copy of cymbidium ringspot virus (CyRSV) satellite RNA was cloned downstream of the bacteriophage T7 RNA polymerase promoter . In vitro transcripts were biologically active in plants when coinoculated with the helper virus or its RNA . Although the transcripts contained 7 or 29 extra nucleotides at the 3' end, the proper 3' terminus was restored in the satRNA progeny . Full-length cDNA clones of CyRSV satRNA under the control of the cauliflower mosaic virus 35S promoter and terminator were used to transform Nicotiana benthamiana plants . Integration of CyRSV satRNA sequence in the plant genome was tested by PCR amplification of DNA extracts from transformed plants and by detection of satRNA-related transcripts in total RNA extracts . Inoculation of transgenic plants with the helper virus induced replication of satRNA of the same size as the native molecule . Sequence analysis of the satRNA progeny showed that it was identical to natural CyRSV satRNA . Infected transgenic plants were not protected from apical necrosis and death by the presence of satRNA sequences . Rather, replication of satRNA was found to suppress accumulation of defective interfering RNA, which acts in the absence of satRNA as an attenuator of virus replication and disease.

Genome, 1992 Jun, 35(3), 528 - 33
Genetic variation between strains of the Mediterranean fruit fly, Ceratitis capitata, detected by DNA fingerprinting; Haymer DS et al.; DNA fingerprinting has been used to detect genetic variation in the Mediterranean fruit fly, Ceratitis capitata . Three different probes have been identified that can be used to detect DNA restriction fragment length polymorphisms between strains of this species . The strains used in this study differ only in terms of their geographic origin or genetic background . One of the probes used is the bacteriophage vector M13, and the other two are repetitive sequences derived from the medfly genome based on a weak homology to M13 . Within a strain, each probe produces a consistent restriction fragment profile that is not affected by the method or timing of DNA extraction . Between strains, when M13 is used as a probe, an average of 10% of the observable bands are polymorphic . Use of the medfly genomic sequences as a probe increases the proportion of polymorphic bands between strains up to 30% . The fact that genetic differences between even such closely related strains can be reliably detected by this method holds great promise for studies of insect pests including the ability to monitor the movements of pest species, determining the extent of genetic variation in pest populations, and in making identifications from otherwise unidentifiable material.

J Bacteriol, 1992 Jun, 174(11), 3800 - 6
Localization of TraC, a protein involved in assembly of the F conjugative pilus; Schandel KA et al.; TraC is one of the proteins encoded by the F transfer region of the F conjugative plasmid which is required for the assembly of F pilin into the mature F pilus structure . Overproduction of this protein from the plasmid pKAS2, which carries only traC, resulted in the formation of inclusion bodies from which soluble TraC was purified . When small amounts of TraC were produced from pKAS2, the protein was localized to the cytoplasm by using anti-TraC antibodies . Similar analysis of a set of TraC-alkaline phosphatase fusion proteins localized all of these fusion proteins to the cytoplasm . However, when TraC was expressed from the F plasmid, much of it appeared associated with the bacterial membrane fraction . Under these conditions, TraC does not appear to be part of the tip of the F pilus, as neither anti-TraC antibodies nor purified TraC had any effect on the infection of F-containing bacteria by the filamentous bacteriophage f1 . These data suggest that TraC is normally associated with the membrane through interactions with other proteins specified by the tra region . This interaction may be via the carboxyl-terminal region of the TraC protein, as a mutant TraC protein containing an Arg-Cys substitution at amino acid 811 exhibits an interaction with the membrane weaker than that of the wild-type protein in the presence of the other Tra proteins.

Science, 1992 May 29, 256(5061), 1298 - 303
A transcriptional enhancer whose function imposes a requirement that proteins track along DNA; Herendeen DR et al.; Transcriptional regulation of the bacteriophage T4 late genes requires the participation of three DNA polymerase accessory proteins that are encoded by T4 genes 44, 62, and 45, and that act at an enhancer-like site . Transcriptional activation by these DNA replication proteins also requires the function of an RNA polymerase-bound coactivator protein that is encoded by T4 gene 33 and a promoter recognition protein that is encoded by T4 gene 55 . Transcriptional activation in DNA constructs, in which the enhancer and a T4 late promoter can be segregated on two rings of a DNA catenane, has now been analyzed . The ability of an interposed DNA-binding protein to affect communication between the enhancer and the promoter was also examined . Together, these experiments demonstrate that this transcription-activating signal is conveyed between its enhancer and a T4 late promoter by a DNA-tracking mechanism . Alternative activation mechanisms relying entirely on through-space interactions of enhancer-bound and promoter-bound proteins are excluded.

Biochemistry, 1992 May 26, 31(20), 4822 - 7
Phosphorylation of Escherichia coli translation initiation factors by the bacteriophage T7 protein kinase; Robertson ES et al.; The lytic coliphage T7 encodes a serine/threonine-specific protein kinase which supports viral reproduction under suboptimal growth conditions . Expression of the protein kinase in T7-infected Escherichia coli cells results in the phosphorylation of 30S ribosomal subunit protein S1, and initiation factors IF1, IF2, and IF3, as determined by high-resolution two-dimensional gel electrophoresis and specific immunoprecipitation analysis . Phosphorylation occurs either exclusively on threonine (IF1, IF3, S1) or on serine and threonine (IF2) . There is no phosphorylation of these proteins in uninfected cells or in cells infected with T7 lacking the protein kinase function . Phosphorylation of the initiation factors coincides with the onset of T7 late protein synthesis, occurring 9-12-min postinfection . T7 late protein synthesis, otherwise inhibited in ColIb plasmid-containing cells, is specifically supported by expression of the protein kinase . These results provide the first evidence for the functional involvement of protein phosphorylation in the control of bacterial translation.

J Biol Chem, 1992 May 25, 267(15), 10856 - 65
Sequencing, cloning, and expression of human red cell-type acid phosphatase, a cytoplasmic phosphotyrosyl protein phosphatase; Wo YY et al.; Low molecular weight phosphotyrosyl protein phosphatases of human placenta and human red cell were purified and sequenced by a combination of Edman degradation and tandem mass spectrometry . Screening of a human placental lambda gt11 cDNA library yielded overlapping cDNA clones coding for two distinct human cytoplasmic low molecular weight phosphotyrosyl protein phosphatases (HCPTPs) . The two longest clones, designated HCPTP1-1 and HCPTP2-1, were found to have identical nucleotide sequences, with the exception of a 108-base pair segment in the middle of the open reading frame . Polymerase chain reaction studies with human genomic DNA suggest that the difference between HCPTP1-1 and HCPTP2-1 does not result from alternative RNA splicing . Studies with a human chromosome 2-specific library confirmed that these sequences are located on chromosome 2, which is known to be the location of red cell acid phosphatase locus ACP1 . The coding sequences of HCPTP1-1 and HCPTP2-1 were placed downstream from a bacteriophage T7 promoter and the proteins were expressed in Escherichia coli . The resulting recombinant enzymes (designated HCPTP-A and HCPTP-B, respectively) showed molecular weights of 18,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and both of them exhibited immunoreactivity with antisera raised against authentic human placental and bovine heart enzymes . The expressed proteins were highly active towards the phosphatase substrates p-nitrophenyl phosphate, beta-naphthyl phosphate, and O-phospho-L-tyrosine, but not alpha-naphthyl phosphate, threonine phosphate, or O-phospho-L-serine . HCPTP-A and -B possessed effectively identical amino acid compositions, immunoreactivities, inhibition by formaldehyde, and kinetic properties when compared with two human red cell acid phosphatase isoenzymes . It is concluded that HCPTP-A and -B are the fast and slow forms of red cell acid phosphatase, respectively, and that this enzyme is not unique to the red cell but is instead expressed in all human tissues.

J Biol Chem, 1992 May 25, 267(15), 10786 - 90
Interactions between T4 phage-coded deoxycytidylate hydroxymethylase and thymidylate synthase as revealed with an anti-idiotypic antibody; Young JP et al.; Anti-idiotypic antibodies were used to mimic the binding surface of the T4 bacteriophage deoxycytidylate hydroxymethylase enzyme, providing an immunological probe for protein-protein interactions involving this enzyme . Polyclonal dCMP hydroxymethylase antibodies were affinity-purified and used to generate anti-idiotypic antibodies . The anti-idiotypic serum immunoprecipitated two native viral proteins, deoxycytidylate hydroxymethylase (EC 2.1.2.8) and thymidylate synthase (EC 2.1.1.45), from a sonicated detergent-treated extract of T4-infected Escherichia coli . The anti-anti-dCMP hydroxymethylase antibody was found to be specific in binding to the T4 dTMP synthase, with no detectable affinity for the host dTMP synthase . Previous work in our laboratory has demonstrated the viral dCMP hydroxymethylase and dTMP synthase to be associated in a deoxyribonucleotide synthetase enzyme complex . Our current approach, using anti-idiotypic antibodies as probes for protein-protein interactions, and complementary studies involving dCMP hydroxymethylase enzyme affinity columns indicate a direct association between bacteriophage T4 dCMP hydroxymethylase and dTMP synthase.

Gene, 1992 May 15, 114(2), 223 - 7
Infectious in vitro transcripts from amplified cDNAs of the Y and Kin strains of cucumber mosaic virus; Boccard F et al.; Using a method based on the polymerase chain reaction (PCR) with primers that include the phi 10 promoter of bacteriophage T7, we obtained cDNA clones of the three RNA genomes of two different strains of cucumber mosaic virus (CMV; Kin and Y strains) from which infectious in vitro transcripts were generated, and demonstrated that the same primers could be used for amplification of at least two other strains of CMV (O and Py) . This method is rapid and requires only limited nucleotide (nt) sequence data (16-18 nt) from the termini of the RNA species . Either viral RNA or unpurified RNA samples from infected plants can be used as template for first-strand cDNA synthesis . For cDNAs of RNA1 and RNA2 of the Y strain, the transcription efficiency was substantially lower than with the Kin strain, unless the primer sequence included transcribed G residues on the 5' side of the viral cDNA, so that the promoter for T7 RNA polymerase resembled more closely the canonical sequence from the bacteriophage T7 phi 10 promoter . The lower specific infectivity of transcripts of the modified cDNAs was more than compensated for by increased transcription efficiency . The possibility that the PCR process may introduce deleterious mutations into the viral cDNA was investigated by re-amplification of a functional cloned cDNA of RNA2: all six cDNA clones of the re-amplified cDNA produced transcripts as infectious as those from the progenitor cDNA.

Science, 1992 May 15, 256(5059), 992 - 7
Site-specific modification of pre-mRNA: the 2'-hydroxyl groups at the splice sites; Moore MJ et al.; A simple and efficient method for synthesizing long, site-specifically modified RNA molecules was developed whereby segments of RNA were joined with the use of bacteriophage T4 DNA ligase . A single hydrogen or O-methyl group was substituted for the 2'-hydroxyl group at either splice site of a nuclear pre-messenger RNA substrate . Splicing of the modified pre-messenger RNA's in vitro revealed that, although a 2'-hydroxyl is not absolutely required at either splice site, the 2'-hydroxyl at the 3' splice site is important for the second step of splicing . These results are compared to previous studies of analogous 2'-hydroxyl groups in the self-splicing Tetrahymena group I intron.

Nucleic Acids Res, 1992 May 11, 20 Suppl, 2119 - 44
Compilation of DNA sequences of Escherichia coli (update 1992); Kroger M et al.; We have compiled the DNA sequence data for E . coli available from the GENBANK and EMBL data libraries and over a period of several years independently from the literature . This is the fourth listing replacing and increasing the former listings substantially . However, in order to save space this printed version contains DNA sequence information only, if they are publically available in electronic form . The complete compilation including a full set of genetic map data and the E . coli protein index can be obtained in machine readable form from the EMBL data library (ECD release 10) or from the CD-ROM version of this supplement issue directly . After deletion of all detected overlaps a total of 1,820,237 individual bp is found to be determined till the beginning of 1992 . This corresponds to a total of 38.56% of the entire E . coli chromosome consisting of about 4,720 kbp . This number may actually be higher by some extra 2.5% derived from lysogenic bacteriophage lambda and various DNA sequences already received for other strains of E . coli.

J Gen Microbiol, 1992 May, 138 ( Pt 5), 941 - 4
Bacterial ice nucleation activity after T4 bacteriophage infection; Kozloff LM et al.; The changes in ice nucleation activity of transformed Ina+ Escherichia coli K12 after infection with T4D bacteriophage have been examined . Within 2 min after infection class A nucleation activity (measured at -4 degrees C) fell about 100-1000-fold whilst class B (measured at -5.5 degrees C) and class C (measured at -9 degrees C) nucleation activities increased 50-100-fold and then rapidly decreased . These changes also occurred after interaction with T4D ghost particles or T4D 11-/12- particles . Since ghost particles lack DNA and 11-/12- particles lack short tail fibres, the T4D particles appear to be exerting their effect by the attachment of the phage long tail fibres to the cell . The changes were not influenced by the addition of chloramphenicol.

Mol Gen Genet, 1992 May, 233(1-2), 319 - 21
Activation and quantitative estimation of bacteriophage T4 late regulatory signal in cis- and transconditions; Noguchi T et al.; Activation of the bacteriophage T4 late gene 22 carried on the phage genome (ciscondition) or borne on a plasmid (transcondition) was quantitatively analyzed after phage infection using a lacZ reporter gene . Transcription from the late promoter was activated postreplicatively by concomitantly infecting T4 phage . A truncated T4-lacZ gene including only 15 bp of sequence upstream of the late promoter consensus of gene 22 TATAAATA, was fully activated in the ciscondition but induced to a reduced extent in the transcondition . A possible mechanism for trans-activation is discussed.

Proteins, 1992 May, 13(1), 38 - 40
Functional significance of conserved amino acid residues; Poteete AR et al.; A systemic study of single amino acid substitutions in bacteriophage T4 lysozyme permitted a test of the concept that conserved amino acid residues are more functionally important than nonconserved residues . Substitutions of amino acid residues that are conserved among five bacteriophage-encoded lysozymes were found to lead more frequently to loss of function than substitutions of nonconserved residues . Of 163 residues tested, only 74 (45%) are sensitive to at least one substitution; however, all 14 residues that are fully conserved are sensitive to substitutions.

Biochem J, 1992 May 1, 283 ( Pt 3), 863 - 70
Molecular cloning of a cDNA coding for mouse liver xanthine dehydrogenase . Regulation of its transcript by interferons in vivo; Terao M et al.; The cDNA coding for xanthine dehydrogenase (XD) is isolated from mouse liver mRNA by cross-hybridization with a DNA fragment of the Drosophila melanogaster homologue . Two lambda bacteriophage overlapping clones represent the copy of a 4538-nucleotide-residue-long transcript with an open reading frame of 4005 nucleotide residues, coding for a putative polypeptide of 1335 amino acid residues . Comparison of the deduced amino acid sequence of the mouse XD with those of the Drosophila and the rat homologues shows a high conservation of this protein (55% identity between mouse and Drosophila, and 94% identity between mouse and rat) . RNA blotting analysis demonstrates that interferon-alpha (IFN-alpha) and its inducers, i.e . poly(I).poly(C), bacterial lipopolysaccharide (LPS) and tilorone (2,7-bis-{2-(diethylamino)ethoxy}fluoren-9-one), increase the expression of XD mRNA in liver . Poly(I).poly(C) also induces XD mRNA in several other tissues in vivo . Protein synthesis de novo is not required for the elevation of XD mRNA after IFN-alpha treatment, since cycloheximide does not block the induction . The elevation of XD mRNA concentration is relatively fast and precedes the induction of both XD and xanthine oxidase (XO) enzymic activities.

Gene, 1992 May 1, 114(1), 85 - 90
Gene rIII is the nearest downstream neighbour of bacteriophage T4 gene 31; Raudonikiene A et al.; The nucleotide sequence of the 2218-bp T4 DNA fragment encompassing gene 31 and five complete open reading frames (ORFs) is presented . We show here that one of these ORFs, ORF31.-1, located downstream from gene 31, is the rIII gene . The position of the gene was established by comparison with the sequences of the rIII gene mutants, r67, rES40 and rBB9 . The ORF corresponding to the rIII gene encodes a basic protein of 82 amino acids with an M(r) of 9323 and a pI of 9.28 . According to the Chou and Fasman {Adv . Enzymol . 47 (1978) 45-148} secondary structure prediction, the rIII protein has a relatively high helical content . In addition, discrepancies with the overlapping sequences determined by other authors in this region are indicated.

Gene, 1992 May 1, 114(1), 115 - 9
Sequence of the bacteriophage SP01 gene 30; Scarlato V et al.; The bacteriophage SP01 gene 30, whose function is essential for DNA synthesis, has been analyzed for its primary structural features . Conditionally lethal mutations in the gene 30 locus have been mapped and sequenced, and the wild-type amino acid (aa) sequence has been deduced along with that of a co-transcribed and possibly co-translated upstream unidentified reading frame (URF) . The aa sequence deduced for gene 30 shares partial similarity with protein P of bacteriophage lambda, which participated in lambda DNA replication, and also with the exonuclease, gp46, of bacteriophage T4 . A lysine-rich region of the hypothetical product of the URF shares similarity with both the T4 DNA topoisomerase and the phi 29 gene 3-encoded protein; the latter codes for a terminal protein which participates in the priming of DNA elongation.

Carcinogenesis, 1992 May, 13(5), 751 - 8
N-acetylated and deacetylated 4'-fluoro-4-aminobiphenyl and 4-aminobiphenyl adducts differ in their ability to inhibit DNA replication of single-stranded M13 in vitro and of single-stranded phi X174 in Escherichia coli; van de Poll ML et al.; Calf thymus single-stranded (ss) DNA was modified with the N-sulfate conjugate of N-hydroxy-2-acetylaminofluorene (N-OH-AAF), N-hydroxy-4'-fluoro-4-acetylaminobiphenyl (N-OH-FAABP) or N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) to yield predominantly N-acetylated adducts of 2-aminofluorene, 4-aminobiphenyl and 4'-fluoro-4-amino-biphenyl respectively to C8 of deoxyguanosine (dG-C8-AAF, dG-C8-AABP and dG-C8-FAABP) . The modified DNAs were used as templates for in vitro DNA synthesis . DNA replication on the randomly primed template was inhibited as compared to control (unmodified) DNA to the same extent by all three types of adducts, irrespective of whether polymerization was performed by Escherichia coli DNA polymerase I, modified T7 DNA polymerase or Thermus aquaticus (Taq) DNA polymerase . In addition, all three types of adducts completely blocked replication of ss phi X174 in an E . coli host: on average one adduct per DNA molecule was sufficient to inactivate the bacteriophage . Polyacrylamide gel electrophoresis of DNA fragments synthesized by E . coli DNA polymerase I on FAABP- and AABP-modified ss M13mp9 DNA templates, showed that termination occurred predominantly one nucleotide before (and occasionally opposite) a modified deoxyguanosine in the template . However, the deacetylated adducts, dG-C8-AF, dG-C8-ABP and dG-C8-FABP (obtained by reacting DNA with their N-trifluoroacetyl-N-acetoxy esters) were frequently bypassed during replication of ss phi X174 in E . coli, though with different efficiencies: 1 out 7, 1 out of 2 and 1 out of 3 adducts on average respectively caused bacteriophage inactivation . Polyacrylamide gel electrophoresis showed that termination of DNA synthesis occurred at least as frequently opposite as 3' to a modified deoxyguanosine in the template.

Genomics, 1992 May, 13(1), 144 - 51
The gene for autosomal dominant polycystic kidney disease lies in a 750-kb CpG-rich region; Germino GG et al.; PKD1, the locus most commonly affected by mutations that produce autosomal dominant polycystic kidney disease (ADPKD), has previously been localized to chromosome 16p13.3 . Since no cytogenetic abnormalities have been found in association with ADPKD, flanking genetic markers have been required to define an interval--the PKD1 region--that contains the PKD1 gene . In this report we demonstrate, through the construction of a long-range restriction map that links the flanking genetic markers GGG1 (D16S84) and 26.6PROX (D16S125), that the PKD1 gene lies within an extremely CpG-rich 750-kb segment of chromosome 16p13.3 . Approximately 90% of this region has been cloned in three extensive cosmid/bacteriophage contigs . The cloned DNA is a valuable resource for identifying new closer flanking genetic markers and for isolating candidate genes from the region.

Proc Natl Acad Sci U S A, 1992 May 1, 89(9), 3962 - 6
Characterization of human 12-lipoxygenase genes; Funk CD et al.; Two human 12-lipoxygenase enzyme (arachidonate:oxygen 12-oxidoreductase, EC 1.13.11.31)-related genes were characterized from 13 distinct clones isolated from three genomic bacteriophage and cosmid libraries . A complete gene (12-lipoxygenase gene 1) spanning approximately 17 kilobases and consisting of 14 exons with sequence matching the cloned platelet/human erythroleukemia (HEL) cell cDNA sequence was identified . Several consensus sites for transcription factors and two potential transcription initiation sites within the 5' flanking region, encompassing the putative promoter region, were identified . A segment of a second, probable pseudogene (12-lipoxygenase gene 2), which displays approximately 85% identity to gene 1 within exon sequences, was also characterized . The presence of two 12-lipoxygenase genes was also substantiated by Southern blot analysis of total human genomic DNA . Exon-intron boundaries for the 12-lipoxygenase genes were located in the identical corresponding positions to the previously cloned human 5-lipoxygenase and rabbit 15-lipoxygenase genes, indicating a highly related gene family . Three lipoxygenase genes (12-lipoxygenase genes 1 and 2, 15-lipoxygenase) were localized to human chromosome 17, whereas the most unrelated lipoxygenase (5-lipoxygenase) was mapped to chromosome 10 by PCR analysis of a human-hamster somatic hybrid DNA panel . 12-Lipoxygenase gene 1 expression could be detected in human erythroleukemia cells, platelets, and human umbilical vein endothelial cells with certainty by reverse transcription-PCR analysis . There was no detectable 12-lipoxygenase gene 2 expression in several tissues and cell lines.

Proc Natl Acad Sci U S A, 1992 May 1, 89(9), 3751 - 5
Folding and function of a T4 lysozyme containing 10 consecutive alanines illustrate the redundancy of information in an amino acid sequence; Heinz DW et al.; Single and multiple Xaa----Ala substitutions were constructed in the alpha-helix comprising residues 39-50 in bacteriophage T4 lysozyme . The variant with alanines at 10 consecutive positions (A40-49) folds normally and has activity essentially the same as wild type, although it is less stable . The crystal structure of this polyalanine mutant displays no significant change in the main-chain atoms of the helix when compared with the wild-type structure . The individual substitutions of the solvent-exposed residues Asn-40, Ser-44, and Glu-45 with alanine tend to increase the thermostability of the protein, whereas replacements of the buried or partially buried residues Lys-43 and Leu-46 are destabilizing . The melting temperature of the lysozyme in which Lys-43 and Leu-46 are retained and positions 40, 44, 45, 47, and 48 are substituted with alanine (i.e., A40-42/44-45/47-49) is increased by 3.1 degrees C relative to wild type at pH 3.0, but reduced by 1.6 degrees C at pH 6.7 . In the case of the charged amino acids Glu-45 and Lys-48, the changes in melting temperature indicate that the putative salt bridge between these two residues contributes essentially nothing to the stability of the protein . The results clearly demonstrate that there is considerable redundancy in the sequence information in the polypeptide chain; not every amino acid is essential for folding . Also, further evidence is provided that the replacement of fully solvent-exposed residues within alpha-helices with alanines may be a general way to increase protein stability . The general approach may permit a simplification of the protein folding problem by retaining only amino acids proven to be essential for folding and replacing the remainder with alanine.

Hepatology, 1992 May, 15(5), 757 - 66
Genetic alterations in the gene encoding the major HBsAg: DNA and immunological analysis of recurrent HBsAg derived from monoclonal antibody-treated liver transplant patients; McMahon G et al.; A gene region encoding a segment of the major surface protein, HBsAg, of hepatitis B virus was analyzed from serum samples after orthotopic liver transplantation of three hepatitis B virus chronic carrier patients treated with a human anti-hepatitis B virus monoclonal antibody (SDZ OST 577) . Each of these three patients became HBsAg negative after transplantation and therapy with the human anti-hepatitis B virus monoclonal antibody but returned to HBsAg positivity (first detected 143,251 and 252 days after the transplantation) . Polymerase chain reaction DNA amplification was performed on DNA from serum samples showing low levels of recurrent HBsAg and reduced antigen reactivity with SDZ OST 577 antibody . Polymerase chain reaction DNA included a 230-bp highly conserved, major S gene region that was cloned into M13 bacteriophage; analysis of this DNA segment provided a consensus of DNA sequences for the serum samples exhibiting altered reactivity with the therapeutic monoclonal . Analysis of independent DNA clones from serum samples of patients exhibiting low but detectable recurrent serum levels of posttherapy HBsAg revealed the presence of S protein variant sequences when compared with polymerase chain reaction DNA derived from the original infected liver or pretherapy serum HBsAg . Genetic variation was predominant in a highly conserved peptide domain that has previously been implicated in antibody binding and neutralizing antibody epitopes . In independent patients infected with either adw or ayw hepatitis B virus subtypes, single nucleotide changes resulted in one to two amino acid differences for each variant allele (residues 124, 129, 131, 137, 140 and/or 145) when compared with pretherapy viral DNA . Administration of serum containing one of these variant viruses to a single hepatitis B-naive chimpanzee resulted in subclinical hepatitis and detectable levels of circulating anti-HBs and anti-HBc antibodies 49 and 70 days after virus administration, respectively . Hepatitis B virus DNA was recovered on liver biopsy between 6 and 8 wk after inoculation, although the animal remained persistently seronegative for HBsAg . DNA sequence analysis of both primate and patient liver hepatitis B virus confirmed the presence of the DNA encoding the S protein variant and associates this DNA with the predominant hepatotropic virus in liver infection.

Arch Biochem Biophys, 1992 May 1, 294(2), 735 - 40
Expression and characterization of human mitochondrial ferredoxin reductase in Escherichia coli; Brandt ME et al.; Ferredoxin reductase (Fd-reductase) supplies reducing equivalents obtained from NADPH to mitochondrial cytochrome P450 enzymes via the small iron-sulfur protein ferredoxin . Two cDNAs (differing by the presence or absence of an 18-bp insert in the coding region) for the human Fd-reductase were subcloned into a newly constructed general purpose expression vector, p delta blue; protein expression under control of the bacteriophage lambda pL promoter was then induced in Escherichia coli . Western blot analysis of subcellular fractions indicated that Fd-reductase protein expressed from both plasmids was present in both inclusion bodies and soluble fractions . However, only the form lacking the insert exhibited Fd-reductase activity . The active material was purified and was found to have electrophoretic, chromatographic, optical, and circular dichroism properties comparable to the bovine homologue . The apparent Km of the expressed protein for NADPH was determined to be 0.7 +/- 0.1 microM and the apparent Km for human ferredoxin was found to be 106 +/- 8 nM . While yields of active enzyme were relatively low (approximately 0.1 mg/liter of culture), the production of Fd-reductase in E . coli will allow structural and mechanistic studies of the enzyme and its interactions with ferredoxin.

J Virol, 1992 May, 66(5), 2611 - 16
In vitro packaging and replication of individual genomic segments of bacteriophage phi 6 RNA; Gottlieb P et al.; The genome of bacteriophage phi 6 contains three segments of double-stranded RNA . Procapsid structures whose formation was directed by cDNA copies of the large genomic segment are capable of packaging the three viral message sense RNAs in the presence of ATP . Addition of UTP, CTP, and GTP results in the synthesis of minus strands to form double-stranded RNA . In this report, we show that procapsids are capable of taking up any of the three plus-strand single-stranded RNA segments independently of the others . In manganese-containing buffers, synthesis of the corresponding minus strand takes place . In magnesium-containing buffers, individual message sense viral RNA segments were packaged, but minus-strand replication did not take place unless all three viral single-stranded RNA segments were packaged . Since the conditions of packaging in magnesium buffer more closely resemble those in vivo, these results indicated that there is no specific order or dependence in packaging and that replication is regulated so that it does not begin until all segments are in place.

J Virol, 1992 May, 66(5), 2605 - 10
Heterologous recombination in the double-stranded RNA bacteriophage phi 6; Mindich L et al.; Bacteriophage phi 6 contains three double-stranded RNA genomic segments . We have constructed a virus with an insertion of a kanamycin resistance gene in genomic RNA segment M . The virus forms small, turbid plaques, and its genome is unstable . Virus from a single plaque contained from about 0.1 to 10% large clear-plaque forms of the virus; these were usually missing the kanamycin resistance gene, and in many cases, the resulting segment M was larger or smaller than its normal size . Sequence analysis of the genomic RNA of the apparent deletions showed that they were formed by recombination events between segment M and either segment S or L . These heterologous recombination events resulted in the loss of the kanamycin resistance gene from segment M and the replacement of the 3' end of segment M with the 3' end of segment S or L . Although the 3' ends of the single-stranded RNA transcripts of the genomic segments appear to have extensive secondary structure, the sequences at the 3' ends are not involved in the specificity of genomic packaging.

Cell, 1992 May 1, 69(3), 483 - 94
The neurospora CYT-18 protein suppresses defects in the phage T4 td intron by stabilizing the catalytically active structure of the intron core; Mohr G et al.; The Neurospora CYT-18 protein, a tyrosyl-tRNA synthetase, which functions in splicing group I introns in mitochondria, promotes splicing of mutants of the distantly related bacteriophage T4 td intron . In an in vivo assay, wild-type CYT-18 protein expressed in E . coli suppressed mutations in the td intron's catalytic core . CYT-18-suppressible mutations were also suppressed by high Mg2+ or spermidine in vitro, suggesting they affect intron structure . Both the N- and C-terminal domains of CYT-18 are required for efficient splicing, but CYT-18 with a large C-terminal truncation retains some activity . Our results indicate that CYT-18 interacts with conserved structural features of group I introns, and they provide direct evidence that a protein promotes splicing by stabilizing the catalytically active structure of the intron RNA.

Biotechniques, 1992 May, 12(5), 722 - 7
Producing a P1 bacteriophage library from pine: isolation and cloning of very high molecular weight DNA; Gorman SW et al.; We have generated a genomic P1 bacteriophage library using Monterey pine (Pinus radiata) DNA . We first developed a method for isolating from pine tissue the very high molecular weight DNA necessary for the preparation of libraries requiring large inserts . The method involves protoplasting the cells, isolating nuclei and lysis in a high concentration of detergent . Fragments of greater than two megabases in size are produced in solution . Modifications introduced to the protocol for library preparation and for P1 plasmid isolation are described.

Mol Gen Mikrobiol Virusol, 1992 May-Jun, (5-6), 22 - 5
{Structure of a region in the genome of bacteriophage N15, necessary for formation of hairpins at ends of the linear plasmid prophage}; Malinin AIu et al.; A fragment containing telRL site of bacteriophage N15 has been cloned in the vector plasmid pUC19 . The nucleotide sequence of a small region from EcoRV-PstI fragment has been defined by Maxam-Gilbert technique . The analysis of the obtained sequence has shown the telRL site to be a nonideal palindrome (the size of 56 nucleotide ops) in which two nucleotide pairs differ in the positions 12 and 14 on both sides of the palindrome centre . The DNA region with alteration of purines and pyrimidines (GC) surrounded by AT-rich regions: 5'-ATTATACGCGCGTATAAT-3'--in the symmetry centre of palindrome is characteristic of the telRL site structure . This characteristic of the region may play a key role in recognition of the site by the specific enzyme at formation of linear prophage-plasmid during lysogenization.

Electrophoresis, 1992 May, 13(5), 286 - 94
Concave Ferguson plots of DNA fragments and convex Ferguson plots of bacteriophages: evaluation of molecular and fiber properties, using desktop computers; Tietz D et al.; A desktop computer program evaluating physical properties of DNA and bacteriophages is presented . The analysis is based on data obtained from capillary and submarine-type agarose electrophoresis . Native molecular/particle properties and properties of the gel (or polymer) medium can be derived from electrophoresis at several gel concentrations . This is done conveniently by a computerized evaluation of the semi-logarithmic plot of mobility vs . gel concentration, designated the Ferguson plot . In application to most proteins, this plot is linear and computer programs exist to evaluate it . However, nonlinear Ferguson plots have assumed great importance in view of the fact that the plots are concave for DNA . Similarly, convex plots are important since they prevail in the electrophoresis of large particles in agarose . The computer program reported here is the first to (i) address concave Ferguson plots and (ii) allow for the evaluation of both cases using a desktop computer . Program ELPHOFIT version 2.0, a Macintosh application, is available upon request.

J Gen Microbiol, 1992 May, 138 ( Pt 5), 871 - 7
The 23S/5S ribosomal RNA genes (rrl/rrf) are separate from the 16S ribosomal RNA gene (rrs) in Borrelia burgdorferi, the aetiological agent of Lyme disease; Fukunaga M et al.; DNA fragments containing the rRNA genes for Borrelia burgdorferi strain B31 were cloned in bacteriophage lambda EMBL3 . A restriction map of the fragments was constructed and the organization of the rRNA genes was determined by Southern hybridization . One genomic DNA fragment contained a single copy of the rrs sequence and another cloned fragment contained both rrl and rrf sequences . The results revealed that the rrs gene is located separately from the set of rrl/rrf genes, suggesting that these rRNA genes are expressed independently in B . burgdorferi.

Gene, 1992 May 1, 114(1), 81 - 3
Corrected nucleotide sequence of M13mp18 gene III; Ebright R et al.; There are seven differences between the actual nucleotide (nt) sequence of bacteriophage M13mp18 gene III and the previously reported nt sequence (which had been compiled based on the nt sequence of wild-type bacteriophage M13 gene III).

Mutat Res, 1992 May, 267(1), 139 - 51
Spontaneous transposition in the bacteriophage lambda cro gene residing on a plasmid; Skaliter R et al.; A new mutagenesis assay system based on the phage lambda cro repressor gene residing on a plasmid was developed . The assay detects mutations in cro that decrease the binding of the repressor to the OR operator in an OR PR-lacZ fusion present in a lambda prophage . Mutations arose spontaneously during growth of E . coli cells harboring cro plasmids at a frequency of 3-6 x 10(-6) . Analysis of some 200 cro mutants from several 'wild-type' strains revealed a substantial fraction of 25-70% insertion events caused by transposition of IS elements . Most of the insertions were caused by IS1, but IS5 insertions were observed too . In strains harboring Tn10, IS10 was responsible for most insertions . Restriction nuclease digestion analysis revealed a preference for insertion of IS10 into the C-terminal half of cro, despite the absence of sequences which are known hot spots for Tn10 insertions . The frequency of IS1 insertions into cro decreased 25-60-fold and that of IS10 insertions decreased 200-fold in cells carrying the recA56 mutation, suggesting that RecA is involved in transposition of these elements . During the logarithmic phase of growth, the mutation frequency was constant for at least 22 generations; however, upon continuous incubation at the stationary phase, the mutation frequency gradually increased, yielding a 3-fold increase in the frequency of insertion and a 4-5-fold increase in point mutation . Genomic Southern analysis of chromosomal IS elements in cells which underwent a transposition from the chromosome into the cro plasmid revealed that the number and distribution of IS1 and IS5 were usually unaltered compared to cells which did not undergo a transposition event . In contrast, essentially each IS10 transposition was accompanied by multiple events which led to changes in the number and distribution of chromosomal IS10 elements.

J Hosp Infect, 1992 May, 21(1), 39 - 50
Bacteriophage phi X-174 as an aerobiological marker for surgical plume generated by the electromagnetic field focusing system; Price JA et al.; The aerosol of surgical plume could be measured effectively with the use of bacteriophage phi X-174 as a biological marker, in contrast to previous methodologies reported by others . Recovery of virus plaque-forming units was highest from hydrophobic polytetrafluoro-ethylene membranes compared to hydrophobic polycarbonate screen filters or polyvinylidene difluoride depth filters, indicating that the method of virus recovery strongly affects the utility of a virus as an aerobiological marker . With this new method, surgical plume was indeed found in significant amounts when cutting tissue phantoms made with agar containing virus . The Electromagnetic Field Focusing System was used, which is a new thermal surgical device . The nominal power setting did not appear to be a factor in the amount of virus recovered . However, when pulse modulating the power by adjusting the crest factor from 1.4 to 4.3, a measure of the duty cycle for power delivery which adjusted the device from its cutting to haemostatic mode, a nearly five-fold increase in surgical plume, as evidence by the recovery of phi X-174 plaque forming units, was seen . The data indicate that bacteriophage phi X-174 can be used effectively as an aerobiological marker for aerosols generated during clinical procedures, and reinforce the need to use a safety vacuum during aerosol generating procedures . The availability of a safe and economical biological marker for aerosols from clinical procedures, which may lead to acquired infections in hospital personnel, makes evaluation of procedures and containment systems markedly easier . The data also indicates that surgical plume biohazard may be present in other techniques that employ pulse modulation including surgical lasers and electrocauteries.

Cell, 1992 May 1, 69(3), 425 - 37
Three-dimensional structure of the beta subunit of E . coli DNA polymerase III holoenzyme: a sliding DNA clamp; Kong XP et al.; The crystal structure of the beta subunit (processivity factor) of DNA polymerase III holoenzyme has been determined at 2.5 A resolution . A dimer of the beta subunit (M(r) = 2 x 40.6 kd, 2 x 366 amino acid residues) forms a ring-shaped structure lined by 12 alpha helices that can encircle duplex DNA . The structure is highly symmetrical, with each monomer containing three domains of identical topology . The charge distribution and orientation of the helices indicate that the molecule functions by forming a tight clamp that can slide on DNA, as shown biochemically . A potential structural relationship is suggested between the beta subunit and proliferating cell nuclear antigen (PCNA, the eukaryotic polymerase delta {and epsilon} processivity factor), and the gene 45 protein of the bacteriophage T4 DNA polymerase.

Genomics, 1992 May, 13(1), 25 - 34
Characterization of radiation/fusion hybrids containing parts of human chromosome 10 and their use in mapping chromosome 10-specific probes; Rothschild CB et al.; We have characterized a panel of somatic cell hybrid cell lines which contain different portions of human chromosome 10 . Genomic DNA from the somatic cell hybrids was tested for hybridization with each of an ordered set of probes used previously to construct a genetic map of chromosome 10, as well as several additional probes, previously localized by in situ hybridization . Hybridization of an unmapped probe to the cell line DNAs can be used to determine its most likely position on the chromosome relative to the mapped set of probes . Genomic DNA from two of the cell lines has been used to construct region-specific cosmid and bacteriophage libraries, and clones derived from these libraries were localized by hybridization to the panel of hybrid cell lines . Several of these probes reveal restriction fragment length polymorphisms which have been genetically mapped . Three of the probes map near the locus for multiple endocrine neoplasia type 2A, and one of these probes, BG-JC353 (D10S167), maps between RBP3 and TB14.34 (D10S34) . Another probe, CRI-J282 (D10S104), is close to the FNRB locus . The panel of hybrid cell lines is thus useful for rapidly localizing unmapped probes and as a source of DNA for the construction of recombinant libraries derived from specific regions of the chromosome.

Mol Microbiol, 1992 May, 6(10), 1289 - 96
A system of transposon mutagenesis for bacteriophage T4; Woodworth DL et al.; We have developed a system of transposon mutagenesis for bacteriophage T4 . The transposon is a plasmid derivative of Tn5 which contains the essential T4 gene 24, permitting a direct selection for transposition events into a gene 24-deleted phage . The transposition occurred at a frequency of only 10(-7) per progeny phage, even though a dam- host was used to increase transposition frequency . Phage strains with a transposon insert were distinguished from most pseudorevertants of the gene 24 deletion by plaque hybridization using a transposon-specific probe . Mapping analysis showed that the transposon inserts into a large number of sites in the T4 genome, probably with a preference for certain regions . The transposon insertions in four strains were analysed by DNA sequencing using primers that hybridize to each end of the transposon and read out into the T4 genome . In each case, a 9 bp T4 target sequence had been duplicated and the insertions had occurred exactly at the IS50 ends of the transposon, demonstrating that bona fide transposition had occurred . Finally, the transposon insert strains were screened on the TabG Escherichia coli strain, which inhibits the growth of T4 motA mutants, and a motA transposon insert strain was found.

Acta Endocrinol (Copenh), 1992 May, 126(5), 460 - 6
Effect of serum thyrotropin levels on the concentration of messenger RNA for thyroid peroxidase in the rat; Magnusson RP et al.; The effect of serum TSH on rat thyroid peroxidase mRNA levels was studied in order to investigate the regulation of thyroid peroxidase gene expression in vivo . A nearly full-length rat thyroid peroxidase cDNA clone was isolated from a bacteriophage cDNA library synthesized using poly A+ RNA isolated from the thyroids of propylthiouracil-treated rats . cDNA probes derived from this clone were used to study rat thyroid peroxidase mRNA levels in response to the level of serum TSH . Two major rat thyroid peroxidase mRNA bands were detected on Northern blots of total cellular RNA (at 3.2 kb and at 3.7 kb) . Injection of thyroxine, which lowered the levels of serum TSH, also lowered the steady-state levels of both rat thyroid peroxidase mRNAs, whereas treatment with methimazole, which increased serum TSH, increased both rat thyroid peroxidase mRNA levels . In hypophysectomized rats 10 days postoperative, very low levels of thyroid peroxidase mRNA were observed . Injection of bovine TSH (1 IU/day) increased rat thyroid peroxidase mRNA expression, preferentially in the 3.2 kb band . These results clearly demonstrate that TSH regulates rat thyroid peroxidase mRNA levels in vivo.

Biochemistry, 1992 Apr 28, 31(16), 3975 - 90
Replication inhibition and translesion synthesis on templates containing site-specifically placed cis-diamminedichloroplatinum(II) DNA adducts; Comess KM et al.; A series of site-specifically plantinated, covalently closed circular M13 genomes (7250 bp) was constructed in order to evaluate the consequences of DNA template damage induced by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP) . Here are reported the synthesis and characterization of genomes containing the intrastrand cross-linked adducts cis-{Pt(NH3)2{d(ApG)-N7(1),-N7(2)}}, cis-{Pt-(NH3)2{d(GpCpG)-N7(1),-N7(3)}}, and trans-{Pt(NH3)2{d(CpGpCpG)-N3(1),-N7(4)}} . These constructs, as well as the previously reported M13 genome containing a site-specifically placed cis-{Pt(NH3)2{d-(GpG)-N7(1),-N7(2)}} adduct, were used to study replication in vitro . DNA synthesis was initiated from a position approximately 177 nucleotides 3' to the individual adducts, and was terminated either by the adducts or by the end of the template, located approximately 25 nucleotides on the 5' side of the adducts . Analysis of the products of these reactions by gel electrophoresis revealed that, on average, bypass of the cis-DDP adducts occurred approximately 10% of the time and that the cis-{Pt(NH3)2{d(GpG)-N7(1),-N7(2)}} intrastrand cross-link is the most inhibitory lesion . The cis-{Pt(NH3)2{(GpCpG)-N7(1),-N7(3)}} adduct allowed a higher frequency of such translesion synthesis (ca . 25%) for two of the polymerases studied, modified bacteriophage T7 polymerase and Escherichia coli DNA polymerase I (Klenow fragment) . These enzymes have either low (Klenow) or no (T7) associated 3' to 5' exonuclease activity . Bacteriophage T4 DNA polymerase, which has a very active 3' to 5' exonuclease, was the most strongly inhibited by all three types of cis-DDP adducts, permitting only 2% translesion synthesis . This enzyme is therefore recommended for replication mapping studies to detect the location of cis-DDP-DNA adducts in a heterologous population . The major replicative enzyme of E . coli, the DNA polymerase III holoenzyme, allowed less than 10% adduct bypass . Postreplication restriction enzyme cleavage studies established that the templates upon which translesion synthesis was observed contained platinum adducts, ruling out the possibility that the observed products were due to a small amount of contamination with unplatinated DNA . The effects on in vitro replication of a recently characterized adduct of trans-DDP {Comess, K . M., Costello, C . E., & Lippard, S . J . (1990) Biochemistry 29, 2102-2110} were also evaluated . This adduct provided a poor block both to DNA polymerases and to restriction enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

FEBS Lett, 1992 Apr 27, 301(3), 322 - 4
Inserting foreign peptides into the major coat protein of bacteriophage M13; Ilyichev AA et al.; Foreign DNA fragments were inserted into filamentous phage gene VIII to create hybrid B-proteins with foreign sequences in the amino terminus . The hybrid proteins are incorporated into the virions which retain viability and infectivity . Virions with hybrid B-proteins have the same contour length and the same number of B-protein molecules as virions with natural B-proteins . It was shown that for one of hybrid B-proteins the position of the processing site had changed.

J Mol Biol, 1992 Apr 20, 224(4), 1143 - 59
Design and structural analysis of alternative hydrophobic core packing arrangements in bacteriophage T4 lysozyme; Hurley JH et al.; An attempt has been made to design modified core-packing arrangements in bacteriophage T4 lysozyme . Alternative replacements of the buried residues Leu99, Met102, Val111 and Phe153 were selected using packing calculations and energy minimization . To test the design procedure, a series of multiple mutants was constructed culminating in the replacement L99F/M102L/V111I/F153L . These variants decrease the stability of T4 lysozyme by approximately 0 to 2 kcal/mol . The crystal structures of a number of the variants were determined . In the variant in which Val111 was replaced by Ile, alpha-helix 107-114 moved by approximately 1.5 A, breaking the hydrogen bond between the backbone carbonyl group of Thr109 and the backbone amide group of Gly113 . This conformational change was not anticipated by the design procedure . Compensating interactions of magnitude up to 1.1 kcal/mol occur for some sets of mutations, while other sets display nearly additive stability changes . Within experimental error, the stability of the double mutant V111F/F153L is additive, with delta delta G different by only 0.1 kcal/mol from the sum of the two single mutants . The quadruple mutant L99F/M102L/V111I/F153L is destabilized by 0.5 kcal/mol, compared to delta delta G = -1.6 kcal/mol for the sum of the four single mutants . Multiple mutants show smaller overall structural changes from wild-type than M102L or V111I alone . Co-operative changes in structure and stability can be rationalized in terms of specific structural differences between single and multiple mutants . Genuine repacking of the hydrophobic core of T4 lysozyme with minimal effects on structure, stability and activity thus appears to have been achieved.

J Mol Biol, 1992 Apr 20, 224(4), 1055 - 74
Bacteriophage P22 portal protein is part of the gauge that regulates packing density of intravirion DNA; Casjens S et al.; The complex double-stranded DNA bacteriophages assemble DNA-free protein shells (procapsids) that subsequently package DNA . In the case of several double-stranded DNA bacteriophages, including P22, packaging is associated with cutting of DNA from the concatemeric molecule that results from replication . The mature intravirion P22 DNA has both non-unique (circularly permuted) ends and a length that is determined by the procapsid . In all known cases, procapsids consist of an outer coat protein, an interior scaffolding protein that assists in the assembly of the coat protein shell, and a ring of 12 identical portal protein subunits through which the DNA is presumed to enter the procapsid . To investigate the role of the portal protein in cutting permuted DNA from concatemers, we have characterized P22 portal protein mutants . The effects of several single amino acid changes in the P22 portal protein on the length of the DNA packaged, the density to which DNA is condensed within the virion, and the outer radius of the capsid have been determined . The results obtained with one mutant (NT5/1a) indicate no change (+/- 0.5%) in the radius of the capsid, but mature DNA that is 4.7% longer and a packing density that is commensurately higher than those of wild-type P22 . Thus, the portal protein is part of the gauge that regulates the length and packaging density of DNA in bacteriophage P22 . We argue that these findings make models for DNA packaging less likely in which the packing density is a property solely of the coat protein shell or of the DNA itself.

J Mol Biol, 1992 Apr 20, 224(4), 937 - 48
Supercoiling, integration host factor, and a dual promoter system, participate in the control of the bacteriophage lambda pL promoter; Giladi H et al.; The high level of efficiency of the bacteriophage lambda pL promoter is dependent upon the topological state of the promoter DNA and the binding of a DNA-bending protein, IHF, to a site centered -86 base-pairs upstream from the pL transcription start site . Abortive initiation assays indicate that DNA supercoiling stimulates open complex formation, whereas IHF enhances promoter recognition . IHF stimulates promoter recognition to the same extent on linear and supercoiled templates . We found that the pL region contains a second promoter, pL2, that initiates transcription 42 base-pairs upstream from pL . Although competitive with pL and inhibited by IHF, mutations in pL2 do not affect the regulation of pL . Stimulation by IHF is helix-face-dependent . IHF inhibits pL when the IHF binding site is displaced a helical half-turn upstream . The pL sequences protected against DNase I digestion by bound IHF and RNA polymerase do not overlap . However, DNase I-hypersensitive sites appear in the region between the two bound proteins . In addition, IHF enhances RNA polymerase binding to pL . These data suggest that stimulation of pL by IHF involves the interaction of IHF and RNA polymerase to form a loop or otherwise distort the DNA between their binding sites.

J Biol Chem, 1992 Apr 15, 267(11), 7664 - 70
Specific associations of T4 bacteriophage proteins with immobilized deoxycytidylate hydroxymethylase; Wheeler L et al.; Is the enzymatic machinery for DNA precursor biosynthesis linked to the DNA replication apparatus? To identify intermolecular associations among deoxyribonucleotide biosynthetic enzymes and to ask whether these enzymes are linked to replication proteins, we analyzed radiolabeled T4 bacteriophage proteins that bind specifically to a column of immobilized T4 deoxycytidylate hydroxymethylase . More than a dozen T4 proteins and a few Escherichia coli proteins are adsorbed specifically by this column . Several of the T4 proteins were identified by two-dimensional gel electrophoresis and radioautography . These include five enzymes involved in DNA precursor biosynthesis, dCMP hydroxymethylase, thymidylate synthase, dihydrofolate reductase, dCTPase-dUTPase, and ribonucleotide reductase large and small subunits, plus several proteins of DNA metabolism and replication . Analysis of extracts of cells infected with phage amber mutants defective in specific proteins suggested a specific association involving thymidylate synthase and the gene 32 single-strand DNA-binding protein.

Biochemistry, 1992 Apr 14, 31(14), 3703 - 8
A novel, sensitive, and specific assay for abasic sites, the most commonly produced DNA lesion; Kubo K et al.; Free radicals produce a wide spectrum of damages; among these are DNA base damages and abasic (AP) sites . Although several methods have been used to detect and quantify AP sites, they either are relatively laborious or require the use of radioactivity . A novel reagent for detecting abasic sites in DNA was prepared by reacting O-(carboxymethyl)hydroxylamine with biotin hydrazide in the presence of carbodiimide . This reagent, called Aldehyde Reactive Probe (ARP), specifically tagged AP sites in DNA with biotin residues . The number of biotin-tagged AP sites was then determined colorimetrically by an ELISA-like assay using avidin/biotin complex conjugated to horseradish peroxidase as the indicator enzyme . With heat/acid-depurinated calf thymus or bacteriophage f1 DNA, ARP detected femtomoles of AP sites in DNA . Using this assay, DNA damages generated in calf thymus, phi X174 RF, and f1 single-stranded DNA, X-irradiated in phosphate buffer, were easily detectable at 10 rad (0.1 Gy) . Furthermore, ARP sites were detectable in DNA isolated from heat-inactivated X-irradiated (10 Gy) and methyl methanesulfonate (MMS)-treated (5 microM) Escherichia coli cells . The rate of production of ARP sites was proportional to the X-ray dose as well as to the concentration of MMS . Thus, the sensitivity and simplicity of the ARP assay should provide a potentially powerful method for the quantitation of AP sites or other DNA lesions containing an aldehyde group.

Nucleic Acids Res, 1992 Apr 11, 20(7), 1733 - 7
DnaG-dependent priming signals can substitute for the two essential DNA initiation signals in oriV of the broad host-range plasmid RSF1010; Honda Y et al.; Broad host-range plasmid RSF1010 contains in the oriV region two DNA initiation signals, ssiA(RSF1010) and ssiB(RSF1010), which are essential for plasmid replication . Each of ssiA and ssiB could be substituted functionally by either of the two G4-type (DnaG-dependent) priming signals, the oric of bacteriophage G4 and an ssi signal from plasmid pSY343 (an R1 plasmid derivative) . Functions of the chimeric oriVs of RSF1010 thus constructed were dependent on the RSF1010-specific replication proteins, RepA, RepB' and RepC . When both of ssiA and ssiB were replaced by the G4-type ssi signals, functions of the chimeric oriVs were no longer dependent on RepB' (RSF1010-specific DNA primase) . The replication activities of the chimeric oriVs of RSF1010 were not influenced markedly by the type of heterologous priming signals they contained . It is conceivable that DNA replication of RSF1010 does not need the priming mechanism for lagging strand synthesis and proceeds by the strand displacement mechanism.

Nucleic Acids Res, 1992 Apr 11, 20(7), 1649 - 55
Control of translational repression by protein-protein interactions; Peabody DS et al.; The coat protein of the RNA bacteriophage MS2 is a translational repressor and interacts with a specific RNA stem-loop to inhibit translation of the viral replicase gene . As part of an effort to dissect genetically its RNA binding function, mutations were identified in the coat protein sequence that suppress mutational defects in the translational operator . Each of the mutants displayed a super-repressor phenotype, repressing translation from the wild-type and a variety of mutant operators better than did the wild-type coat protein . At least one mutant probably binds RNA more tightly than wild-type . The other mutants, however, were defective for assembly of virus-like particles, and self-associated predominantly as dimers . It is proposed that this assembly defect accounts for their super-repressor characteristics, since failure to assemble into virus-like particles elevates the effective concentration of repressor dimers . This hypothesis is supported by the observation that deletion of thirteen amino acids known to be important for assembly of dimers into capsids also resulted in the same assembly defect and in super-repressor activity . A second class of assembly defects is also described . Deletion of two amino acids from the C-terminus of coat protein resulted in failure to form capsids, most of the coat protein having the apparent molecular weight expected of trimers . This mutant (dl-8) was completely defective for repressor activity, probably because of an inability to form dimers . These results point out the inter-dependence of the structural and regulatory functions of coat protein.

Science, 1992 Apr 10, 256(5054), 198 - 203
Lambda Int protein bridges between higher order complexes at two distant chromosomal loci attL and attR; Kim S et al.; The excisive recombination reaction of bacteriophage lambda involves a specific and efficient juxtaposition of two distant higher order protein-DNA complexes on the chromosome of Escherichia coli . These complexes, which mediate synapsis and strand exchange, consist of two DNA sequences, attL and attR, the bivalent DNA binding protein Int, and the sequence-specific DNA bending proteins, IHF, Xis, and Fis . The protein-protein and protein-DNA interactions within, and between, these complexes were studied by various biochemical techniques and the patterns of synergism among pairs of mutants with marginally impaired recombination function were analyzed . The DNA bending proteins facilitated long-range tethering of high- and low-affinity DNA sites by the bivalent Int protein, and a specific map is proposed for the resulting Int bridges . These structural motifs provide a basis for postulating the mechanism of site-specific recombination and may also be relevant to other pathways in which two distant chromosomal sites become associated.

Biochim Biophys Acta, 1992 Apr 6, 1130(3), 277 - 88
Nucleotide sequence of the genome of the bacteriophage alpha 3: interrelationship of the genome structure and the gene products with those of the phages, phi X174, G4 and phi K; Kodaira K et al.; The complete nucleotide sequence of the genome of the circular single-stranded DNA (isometric) phage alpha 3 has been determined and compared with that of the related phages phi X174 and G4 . The alpha 3 genome consists of 6087 nucleotides, which is 701 nucleotides longer than the nucleotide sequence of the phi X174 genome and 510 nucleotides more than that of the G4 genome . The results demonstrated that the three phage species have 11 homologous genes (A, A*, B, C, K, D, E, J, F, G and H), the order of which is fundamentally identical, suggesting that they have evolved from a common ancestor . The sequence of some genes and untranslated intergenic regions, however, differs significantly from phage to phage: for example, the degree of amino acid sequence homology of the gene product is averaged at 47.7% between alpha 3 and phi X174 and 46.9% between alpha 3 and G4, and alpha 3 has a remarkable longer intergenic region composed of 758 nucleotides between the genes H and A compared with the counterparts of phi X174 and G4 . Meanwhile, in vivo experiments of genetic complementation showed that alpha 3 can use none of the gene products of phi X174 and G4, whereas the related phage phi K can rescue alpha 3 nonsense mutants of the genes B, C, D and J . These sequencing and in vivo rescue results indicated that alpha 3 is closely related to phi K, but distantly remote from phi X174 or G4, and supported an evolutional hypothesis which has been so far proposed that the isometric phages are classified into three main groups: the generic representatives are phi X174, G4 and alpha 3.

J Mol Biol, 1992 Apr 5, 224(3), 601 - 11
Mutations that eliminate the requirement for the vertex protein in bacteriophage T4 capsid assembly; Johnson K et al.; The capsid of bacteriophage T4 is composed of two essential structural proteins, gp23, the major constituent of the capsid, and gp24, a less prevalent protein that is located in the pentameric vertices of the capsid . gp24 is required both to stabilize the capsid and to allow it to be further matured . This requirement can be eliminated by bypass-24 (byp24) mutations within g23 . We have isolated, cloned and sequenced several new byp24 mutations . These mutations are cold-sensitive in the absence of gp24, and are located in regions of g23 not known to contain any other mutations affecting capsid assembly . The cold-sensitivity of the byp24 mutations can be reduced by further mutations within g23 (trb mutations) . Cloning and sequencing of these trb mutations has revealed that they lie in regions of g23 that contain clusters of mutations that cause the production of high levels of petite and giant phage (ptg mutations) . Despite the proximity of the trb mutations to the ptg mutations, none of the ptg mutations has a Trb phenotype . The mutation ptE920g, which is also located near one of the ptg clusters, and which produces only petite and wild-type phage, has been shown to confer a Trb but not a Byp24 phenotype . The relevance of these observations to our understanding of capsid assembly is discussed.

Appl Environ Microbiol, 1992 Apr, 58(4), 1371 - 3
Survival and replication of male-specific bacteriophages in molluscan shellfish; Burkhardt W 3rd et al.; The survival and replication of male-specific bacteriophages in hard-shelled clams (Mercenaria mercenaria) and their homogenates were examined to further assess their potential utility as indicator organisms . Trials were conducted in the presence and absence of a suitable bacterial host, Escherichia coli HS{pFamp}R . Results of this study demonstrated that male-specific bacteriophages were unable to replicate in hard-shelled clams, with or without added host cells . In addition, the densities of these bacteriophages were stable for up to 7 days in shellfish held at ambient seawater temperatures (less than 25 degrees C) . Evidence of replication, although not observed in live shellfish, was found to occur in temperature-abused shellfish homogenates and supernatants, but only when a suitable bacterial host was present.

Mol Cell Biochem, 1992 Apr, 111(1-2), 3 - 9
Strand scission in DNA induced by dietary flavonoids: role of Cu(I) and oxygen free radicals and biological consequences of scission; Rahman A et al.; The naturally occurring flavonoid, quercetin, in the presence of Cu(II) and molecular oxygen caused breakage of calf thymus DNA, supercoiled pBR322 plasmid DNA and single stranded M13 phage DNA . In the case of the plasmid, the product(s) were relaxed circles or a mixture of these and linear molecules depending upon the conditions . For the breakage reaction, Cu(II) could be replaced by Fe(III) but not by other ions tested {Fe(II), Co(II), Ni(II), Mn(II) and Ca(II)} . Structurally related flavonoids, rutin, galangin, apigenin and fisetin were effective or less effective than quercetin in causing DNA breakage . In the case of the quercetin-Cu(II) reaction, Cu(I) was shown to be essential intermediate by using the Cu(I)-sequestering reagent, bathocuproine . By using Job plots we established that, in the absence of DNA, five Cu(II) ions were reduced by one quercetin molecule; in contrast two ions were reduced per quercetin molecule in the DNA breakage reaction . Equally neocuproine inhibited the DNA breakage reaction . The involvement of active oxygen in the reaction was established by the inhibition of DNA breakage by superoxide dismutase, iodide, mannitol, formate and catalase (the inhibition was complete in the last case) . The strand scission reaction was shown to account for the biological activity of quercetin as assayed by bacteriophage inactivation . From these data we propose a mechanism for the DNA strand scission reaction of quercetin and related flavonoids.

Mol Biochem Parasitol, 1992 Apr, 51(2), 229 - 38
A stage-specific calcium-binding protein expressed in eggs of Schistosoma mansoni; Moser D et al.; A cDNA sequence encoding a Schistosoma mansoni egg antigen SmE16 was cloned in Escherichia coli . The 16-kDa polypeptide deduced from the nucleotide sequence is related to the calmodulin and troponin C gene families of calcium-binding proteins, and the most significant homology is displayed around the four calcium-binding sites . The antigen was expressed as a hybrid protein of the bacteriophage MS2 polymerase . The MS2-SmE16 fusion protein binds calcium, as demonstrated via ligand blotting with 45Calcium . The detection of antibodies to the purified recombinant egg antigen in sera of schistosomiasis patients opens up the possibility that it may be a useful candidate for the development of serodiagnostic assays . The function of the protein in the egg is presently unclear.

Gene, 1992 Apr 1, 113(1), 17 - 23
A rapid polymerase-chain-reaction-directed sequencing strategy using a thermostable DNA polymerase from Thermus flavus; Rao VB et al.; We have developed a polymerase chain reaction (PCR)-directed sequencing strategy for rapid sequencing of DNA from crude viral or cell preparations . The basic strategy consists of two phases . In the first phase, the target DNA is amplified by symmetric PCR with low concentrations of deoxyribonucleotide triphosphate (dNTP) and oligodeoxyribonucleotide primers . This results in exponential amplification of DNA in the initial cycles, reaching a plateau by 25 cycles due to limiting concentrations of dNTP and primers . In the second phase, a small aliquot of the PCR mixture is amplified without any purification, by asymmetric PCR in the presence of a 5'-labeled primer and one of the four dideoxyribonucleotide triphosphates . This results in the accumulation of single-stranded DNA products that are terminated at specific points by incorporation of the appropriate dideoxyribonucleotide monophosphate . The products are then analyzed by electrophoresis on a sequencing gel followed by autoradiography . The PCR conditions are optimized to generate sequence ladders of several hundred nucleotides starting from as low as 100 copies of bacteriophage or bacterial genome in one to two days.

Gene, 1992 Apr 1, 113(1), 113 - 7
Cloning a replication function from the streptomycete bacteriophage FP43; Howell M et al.; Streptomyces griseofuscus cells carrying a 4.4-kb SphI DNA fragment from bacteriophage FP43 inhibited plaque formation (Pin) by FP43, and the Pin function was localized to a 0.96-kb SacII fragment . The same 4.4-kb SphI fragment was able to replicate freely in several streptomycetes, including S . griseofuscus, and the replication (Rep) function was localized to a 1.2-kb SphI-FspI fragment . Plasmids with FP43 Rep function are unstable and are present at about 20-50 copies per chromosome . Plasmids with FP43 Rep function are compatible with SCP2* plasmids.

J Bacteriol, 1992 Apr, 174(8), 2493 - 500
Dissection of IncP conjugative plasmid transfer: definition of the transfer region Tra2 by mobilization of the Tra1 region in trans; Lessl M et al.; We constructed a transfer system consisting of two compatible multicopy plasmids carrying the transfer regions Tra1 and Tra2 of the broad-host-range IncP plasmid RP4 . In this system, the plasmid containing the Tra1 region with the origin of transfer (oriT) was transferred, whereas additional functions essential for the conjugative process were provided from the Tra2 plasmid in trans . The Tra2 region, as determined for matings between Escherichia coli cells, maps between coordinates 18.03 and 29.26 kb of the RP4 standard map . The section of Tra2 required for mobilization of the plasmid RSF1010 (IncQ) and the propagation of bacteriophages Pf3 and PRD1 appears to be the same as that needed for RP4 transfer . Tra2 regions of RP4 (IncP alpha) and R751 (IncP beta) are interchangeable, facilitating mobilization of the plasmid carrying the RP4 Tra1 region . The transfer frequencies of both systems are similar . Transcription of Tra2 proceeds clockwise relative to the standard map of RP4 and is probably initiated at a promoter region located upstream of trbB (kilB) . From this promoter region the trfA operon and the Tra2 operon are likely to be transcribed divergently . A second potential promoter has been located immediately upstream of trbB (kilB) . Plasmids encoding the functional Tra2 region can only be maintained stably in host cells in the presence of the RP4 regulation region carrying the korA-korB operon or part of it . This indicates the involvement of RP4 key regulatory functions that apparently are active not only in the control of replication but also in conjugation.

J Bacteriol, 1992 Apr, 174(8), 2460 - 5
Membrane localization and topology of a viral assembly protein; Guy-Caffey JK et al.; The gene I protein (pI) of the filamentous bacteriophage f1 is required for the assembly of this virus . Antibodies specific to either the amino or carboxyl terminus of this protein were used to determine the location and topology of the gene I protein in f1-infected bacteria . pI is anchored in the inner membrane of Escherichia coli cells via a 20-amino-acid hydrophobic stretch, with its carboxyl-terminal 75 residues located in the periplasm and its amino-terminal 253 amino acids residing in the cytoplasm . By using the carboxyl-terminal pI antibody, a smaller protein, pI*, is also detected in f1-infected cells at a ratio of one to two molecules per molecule of pI . Analysis of proteins produced from a gene I amber mutant plasmid or bacteriophage suggests that pI* is most likely the result of an in-frame internal translational initiation event at methionine 241 of the 348-amino-acid pI . pI* is shown to be an integral inner membrane protein inserted in the same orientation as pI . The relation of the cellular locations of pI and pI* to some of the proposed functions of pI is discussed.

J Virol, 1992 Apr, 66(4), 2335 - 45
Cellular expression of a functional nodavirus RNA replicon from vaccinia virus vectors; Ball LA; RNA replication provides a powerful means for the amplification of RNA, but to date it has been found to occur naturally only among RNA viruses . In an attempt to harness this process for the amplification of heterologous mRNAs, both an RNA replicase and its corresponding RNA templates have been expressed in functional form, using vaccinia virus-bacteriophage T7 RNA polymerase vectors . Plasmids were constructed which contained in 5'-to-3' order (i) a bacteriophage T7 promoter; (ii) a full-length cDNA encoding either the RNA replicase (RNA 1) or the coat protein (RNA 2) of flock house virus (FHV), (iii) a cDNA sequence that encoded the self-cleaving ribozyme of satellite tobacco ringspot virus, and (iv) a T7 transcriptional terminator . Both in vitro and in vivo, circular plasmids of this structure were transcribed by T7 RNA polymerase to produce RNAs with sizes that closely resembled those of the two authentic FHV genomic RNAs, RNA 1 and RNA 2 . In baby hamster kidney cells that expressed authentic FHV RNA replicase, the RNA 2 (coat protein) transcripts were accurately replicated . Moreover, the RNA 1 (replicase) transcripts directed the synthesis of an enzyme that could replicate not only authentic virion-derived FHV RNA but also the plasmid-derived transcripts themselves . Under the latter conditions, replicative amplification of the RNA transcripts ensued and resulted in a high rate of synthesis of the encoded proteins . This successful expression from a DNA vector of the complex biological process of RNA replication will greatly facilitate studies of its mechanism and is a major step towards the goal of harnessing RNA replication for mRNA amplification.

Virology, 1992 Apr, 187(2), 788 - 95
DNA sequences necessary for packaging bacteriophage T3 DNA; Hashimoto C et al.; A recombinant plasmid, pUCE1-TR, carrying a target for processing of the concatemer joint (TR) and sequences to the left of the target (E1), is efficiently packaged into transducing particles during T3 phage infection . Using this plasmid packaging/transduction system, the minimal sequences necessary for packaging of T3 DNA were determined . The TR sequence contains the targets for initiation cleavage and termination cleavage of concatemer processing (pacCR and pacCL, respectively) . A plasmid lacking pacCL was packaged as efficiently as pUCE1-TR but one deleted for pacCR was packaged at a very low efficiency, showing that pacCR is essential for production of transducers but that pacCL is dispensable . DNA from transducing particles carrying a recombinant plasmid lacking pacCL or pacCR had the same right or left end as T3 DNA, respectively, but its other end was not unique . In the absence of pacCL, packaging is initiated from the DNA end created by cleavage at the pacCR and terminated at any sequence after packaging a headful of DNA . In the absence of pacCR, packaging is initiated from the DNA end created by nonspecific, inefficient cleavage and terminated by cleavage at the pacCL after packaging a headful of DNA . A 23-bp segment flanking the site where the mature right end is formed was found to support efficient formation of transducing particles . A 53-bp sequence, including a consensus sequence for the promoter for T3 RNA polymerase, was a responsible element in the E1 sequence for packaging of plasmid DNA . Deletions of the 5'-upstream sequence of the promoter sequence from the left decreased the promoter and packaging activities in parallel, but with those of the 3'-downstream sequence from the right, the packaging activity was impaired before the promoter activity, indicating that transcription from the promoter is necessary but not sufficient for T3 DNA packaging.

Virology, 1992 Apr, 187(2), 548 - 54
Bacteriophage P2 and P4 morphogenesis: protein processing and capsid size determination; Rishovd S et al.; An interesting feature of the bacteriophage P2-P4 system is the switch in size between a large P2 (60 nm) and a small P4 (45 nm) capsid . We have investigated whether the protein processing reactions cleaving the primary translation product gpN to several capsid proteins (h1, h2, and N*) are involved in this switch . Using antibodies specific against gpN and its derivatives we have identified all the structural components of mature P2 and P4 particles that are derived from gpN . Our estimate of the relative amounts of gpN derivatives suggests that the previously identified minor capsid proteins h1 and h2 can only be essential structural components of the P4, and not the P2, capsid . Nevertheless, the relative amounts are similar in vivo during a P2 and a P4 infection . This indicates that the switch in head size is not caused by the presence of elevated amounts of h1 and h2 during P4 morphogenesis . We have also identified the sites where gpN is cleaved to its derivatives h1, h2, and N*, ascertaining that the cleavage sites are the same in P2 and P4 . Our results indicate that the processing reactions are not directly involved in the head size determination mechanism.

Anal Biochem, 1992 Apr, 202(1), 46 - 9
In vitro amplification of DNA fragments greater than 10 kb; Kainz P et al.; The synthesis of a 10.9-kb DNA fragment from a bacteriophage lambda template was used in the search for conditions to extend the range for the polymerase chain reaction (PCR) . Using the same primer sequences and conditions (denaturation at 94 degrees C, 1 min; annealing at 57 degrees C, 1 min; polymerization at 70 degrees C, 20 to 30 min) as published by W . Rychlik, W . J . Spencer, and R . E . Rhoads {(1990) Nucleic Acids Res . 18, 6409-6412}, unsatisfactory results were obtained with AmpliTaq and native Taq polymerase (poor reproducibility, low product yield, nonspecific products), whereas Tub polymerase completely failed to amplify this fragment . Only after changes in the following parameters were reliable results obtained but only with Tub polymerase: A two-step PCR procedure with primer annealing and extension at 65 degrees C followed by DNA denaturation at 94 degrees C for 1.5 min was performed . The DNA fragment desired was specifically amplified when the enzyme concentration was reduced to 0.4 U/50 microliters and extension times as low as 4 to 12 min with an optimum at 8 min were used . A prolongation to 20 min or more resulted in an accumulation of unspecific products with a concomitant reduction in the yield of the fragment . Under the conditions described above it was also possible to amplify a DNA fragment even significantly longer (15.6 kb).

J Bacteriol, 1992 Apr, 174(8), 2717 - 9
Functional relationship between the J proteins of bacteriophages phi X174 and G4 during phage morphogenesis; Fane BA et al.; The functions of the small DNA-binding protein, gpJ, of bacteriophages phi X174 and G4 were examined by in vivo cross-complementation and sucrose gradient sedimentation . The morphogenetic roles of the two proteins may differ . The phi X174 J protein may be required for the formation or stabilization of the phi X174 prohead.

J Bacteriol, 1992 Apr, 174(7), 2404 - 6
Proteolysis of bacteriophage phi X174 prohead accessory protein gpB by Escherichia coli OmpT protease is not essential for phage maturation in vivo; Dalphin ME et al.; To examine whether cleavage of the phi X174 prohead accessory protein, gpB, by the OmpT protease is required for phage development in vivo, a phage mutant lacking the OmpT cleavage site and an Escherichia coli C delta ompT strain were constructed . The results of burst size experiments suggest that neither the cleavage site nor the OmpT protein is required for phi X174 development.

J Virol, 1992 Apr, 66(4), 2226 - 31
Ar+ plasma-induced damage to DNA in bacteriophage lambda: implications for the arrangement of DNA in the phage head; Mendelson EC et al.; Bacteriophage lambda was bombarded with low-energy Ar+ ions with the goal of determining whether particular regions of the DNA genome are found preferentially in the outer portion of the packaged DNA mass . The strategy was to fragment the DNA selectively near the surface of the virus by exposing intact phage to Ar+ ions energetic enough to break covalent chemical bonds in DNA but not energetic enough to penetrate deeply beneath the viral capsid shell . Broken DNA was then isolated, and its genomic origin was identified by Southern hybridization to mapped restriction fragments of lambda DNA . Analysis of such Southern blots revealed that all regions of the lambda genome were represented among the small DNA fragments generated during all times of Ar+ bombardment examined . Depending on the duration of exposure, however, particular regions of the genome were found to be enriched in the small-fragment population . After short periods of exposure, sequences from the leftmost 10% and from the right half of the standard genetic map were enriched in the broken-DNA fraction . Among sequences in the right half of the genome, the enrichment was progressively more pronounced beginning in the middle of the genetic map and proceeding toward the right end . In phage bombarded for longer periods of time, rightward sequences were preferentially depleted in the small-fragment population . In contrast, when Ar+ bombardment was carried out with free lambda DNA rather than intact phage, small DNA fragments arose uniformly from all regions of the genome at all times of exposure examined . The results indicate that in the intact phage, DNA sequences from the right half and from the very leftmost regions of the genome have a tendency to lie closer to the capsid than does the remainder of the genome . Since DNA is packaged into the prohead beginning at the left end, our results suggest that packaging occurs in such a way that newly entering DNA tends to be disposed externally to that packaged at earlier times.

J Biomol Struct Dyn, 1992 Apr, 9(5), 911 - 20
Dynamics of double stranded DNA reptation from bacteriophage; Gabashvili IS et al.; The dynamics of dsDNA release process from a phage head has been analyzed theoretically . The process was considered as dsDNA reptation through the phage tail . The driving force is assumed to be the decrease of the DNA globule free energy on its releasing from the head in the surrounding medium . The results of the equilibrium theory on an intraphage DNA globule were applied . Three possible sources of friction were examined . The first one is in the inner channel of the tail . The second is the friction of DNA segments in the whole globule volume, which is essential when the globule decondensation is a collective process, at simultaneous moving of all the turns (mechanism 1) . The third is the globule friction with the capsid inner surface, that is most important when decondensation proceeds via the globule rotation as a whole spool (mechanism 2) . Mechanism 1 would require a lot of time for ejection . Mechanism 2 would lead to different ejection dynamics of short- and long-tailed phages . Comparison of the theoretical results with the published experimental data argues in favor of mechanism 2.

Genet Anal Tech Appl, 1992 Apr, 9(2), 64 - 7
Detection of individual human chromosomes by chromosome in situ suppression hybridization using PCR-amplified bacteriophage library probes; Burde S et al.; We used the polymerase chain reaction (PCR) to prepare chromosome-specific probes from the bacteriophage lambda library LAO1NS01, prepared at the Los Alamos National Laboratory from flow sorted human chromosome 1 . By using oligonucleotide primers flanking the EcoRI insertion site of the Charon 21A vector, we were able to amplify the human sequences preferentially in the library up to 9.1 kb (maximum insert size) . The product of the PCR reaction was nick translated with incorporation of biotinylated residues and used with fluorescence in situ hybridization to observe metaphase chromosomes by fluorescence microscopy . This technique allows for a relatively easy method for preparation of chromosome-specific library probes for "chromosome painting." The quality of the results obtained by this method compares favorably to those obtained by using bulk-purified library inserts . This method offers potential advantages in terms of cost and ease of use.

Biochimie, 1992 Apr, 74(4), 363 - 71
Identification of the Escherichia coli 30S ribosomal subunit protein neighboring mRNA during initiation of translation; Dontsova OA et al.; To identify the proteins of the 30S ribosomal subunit of E coli that neighbor mRNA in the ternary initiation complex (mRNA*30S subunit*tRNA(fMet), we used an affinity cross-linking approach in which photoactivated groups were attached to different positions along the mRNA chain . A series of mini-genes originating from the 5'-end region of the cro gene of lambda bacteriophage were constructed as templates for mini-mRNA synthesis . Two strategies were used to introduce photo-reactive agents into the message . According to the first, two transcripts were isolated from E coli and chemically derivatized at their 5'-ends with a photoinducible diaziril group . One of these messages allowed for localization of the 5'-end of the Shine-Dalgarno sequence while the other one allowed for labeling of the ribosome at the 5'-end side of the initiation AUG codon in the P site . According to the second approach, 5-azidouridine (5N3U) was randomly incorporated into mRNA transcripts during a T7 RNA polymerase catalyzed reaction by using a mixture of 5N3UTP and UTP . A message that had U residues at either -4, -3, -1, +2 and +14, +19, +20 positions was used (A from cro AUG is +1) . Whereas cross-links with the 5N3U transcripts were essentially 'zero-length', the 5'-derivatized transcripts were covalently attached to ribosomal components about 14 A from the 5'-end . We found that proteins S1, S7, S5, S3 and S4 compose, or were close to, the ribosomal mRNA-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biotechnol, 1992 Apr, 23(2), 153 - 65
Tac promoter vectors incorporating the bacteriophage T7 gene 10 translational enhancer sequence for improved expression of cloned genes in Escherichia coli; Lehmeier B et al.; Two new plasmid expression vectors, pTacT7 and pTacT7L, have been constructed, which incorporate between the tac promoter and a downstream NcoI-HindIII polylinker sequence a synthetic sequence derived from the region upstream from gene 10 of bacteriophage T7 (g10-L) . This sequence was recently shown to act as a translational enhancer (Olins et al., 1988) and was termed "Epsilon" (Enhancer of Protein Synthesis Initiation) element (Olins and Rangwala, 1989) . In this communication we describe in detail the construction of ptacT7 and ptacT7L . Furthermore, we present evidence that the "Epsilon" element is able to enhance 3 to 20-fold the expression levels of two poorly expressed test genes encoding the human placental proteins PP9 and PP15 . On the other hand, the expression levels of two highly expressed test genes encoding the human placental proteins PP4 and FXIIIa could not be further enhanced by the presence of the "Epsilon" element . These experiments show that the T7 gene 10 leader sequence can be utilized to improve the expression yields of otherwise poorly expressed heterologous genes in Escherichia coli.

J Gen Virol, 1992 Apr, 73 ( Pt 4), 989 - 93
Vaccinia virus encodes four putative DNA and/or RNA helicases distantly related to each other; Koonin EV et al.; Computer-assisted analysis of the amino acid sequences of vaccinia virus proteins containing the purine NTP-binding pattern revealed the seven motifs typical of the DNA (RNA) helicase superfamily II in the proteins I8R and A18R . Together with the previously described putative helicases D6R and D11L, the number of putative helicases of this superfamily encoded by the genome of vaccinia virus now reaches four . Aside from the helicase motifs, the sequences of I8R and A18R showed no strong similarity to each other, nor to D6R and D11L . Statistically significant similarity was demonstrated between I8R and the putative RNA helicases involved in pre-mRNA splicing in yeast, PRP2, PRP16 and PRP22, whereas A18R appeared to be related to the putative helicases encoded by the human DNA repair gene ERCC3 and the D10 gene of bacteriophage T5 . These findings suggest that I8R may be an RNA helicase . Based on the known properties of the virion NTPases of vaccinia virus, it is possible that the I8R protein may be identical to the previously characterized virion NTPase II . A18R is likely to possess DNA and/or RNA helicase activity . Circumstantial evidence suggests that this activity might be involved in melting duplex structures in late mRNAs . The possibility of independent acquisition of the putative helicases I8R, A18R and a common progenitor to D6R and D11L by an ancestral poxvirus is discussed.

Virus Genes, 1992 Apr, 6(2), 187 - 96
Evolution of thymidine and thymidylate kinases: the possibility of independent capture of TK genes by different groups of viruses; Koonin EV et al.; Phylogenetic analysis of viral and cellular thymidine and thymidylate kinases was performed using computer-assisted methods . Multiple alignments and tentative phylogenetic trees were generated for the two families of these enzymes, which include a) thymidine kinases (TK) of mammals, poxviruses, African swine fever virus, E . coli, and bacteriophage T4; and b) thymidylate kinases (ThyK) of yeast and poxviruses and distantly related herpesvirus proteins with both enzymatic activities . Analysis of the alignment of the TKs of the first family highlighted three strongly conserved segments . Two of these corresponded to the A and B motifs of the purine NTP-binding pattern . The third, C-terminal segment, showing the highest conservation, encompassed a modified Zn finger motif . It is speculated that this motif might be involved in TK oligomerization . Phylogenetic trees constructed by three different methods suggested that cellular TK genes could be captured independently by T4 bacteriophage, African swine fever virus, fowlpox virus, and the other poxviruses . The observed tree topologies appear to contradict the popular virus-host coevolution schemes and to imply that different subdivisions of poxviruses diverged at earlier stages of evolution than their hosts did . It was shown that deoxynucleoside monophosphate kinase of bacteriophage T4 is related to the ThyK family . Phylogenetic analysis suggested that ThyK genes probably have been acquired independently by phage T4, poxviruses, and herpes-viruses.

Gene, 1992 Apr 1, 113(1), 25 - 33
A phage T4 in vitro packaging system for cloning long DNA molecules; Rao VB et al.; Recombinant plasmid DNAs containing long DNA inserts that can be propagated in Escherichia coli would be useful in the analysis of complex genomes . We tested a bacteriophage T4 in vitro DNA packaging system that has the capacity to package about 170 kb of DNA into its capsid for cloning long DNA fragments . We first asked whether the T4 in vitro system can package foreign DNA such as concatemerized lambda imm434 DNA and phage P1-pBR322 hybrid DNA . The data suggest that the T4 system can package foreign DNA as efficiently as the mature phage T4 DNA . We then tested the system for its ability to clone foreign DNA fragments using the P1-pBR322 hybrid vectors constructed by Sternberg {Proc . Natl . Acad . Sci . USA 87 (1990) 103-107} . E . coli genomic DNA fragments were ligated with the P1 vectors containing two directly oriented loxP sites, and the ligated DNA was packaged by the T4 in vitro system . The packaged DNA was then transduced into E . coli expressing the phage P1 cyclization recombination protein recombinase to circularize the DNA by recombination between the loxP sites situated at the ends of the transduced DNA molecule . Clones with long DNA inserts were obtained by using this approach, and these were maintained as single-copy plasmids under the control of the P1 plasmid replicon . Clones with up to about 122-kb size inserts were recovered using this approach.

Proc Natl Acad Sci U S A, 1992 Apr 1, 89(7), 2516 - 20
Sequence and expression in Escherichia coli of the 40-kDa subunit of activator 1 (replication factor C) of HeLa cells; Chen M et al.; Activator 1 (A1; also called replication factor C), in conjunction with proliferating-cell nuclear antigen (PCNA), is essential for the elongation of primed DNA templates by DNA polymerases delta and epsilon . A1 contains five distinct subunits of 145, 40, 38, 37, and 36.5 kDa . Here we describe the isolation, sequence, and bacterial expression of a cDNA coding for the 40-kDa subunit . In keeping with the presence of an ATP-binding motif, the bacterially expressed 40-kDa subunit binds ATP . The interaction between the 40-kDa subunit and ATP was reduced by the addition of PCNA . In addition, antibodies raised against the 40-kDa subunit abolished the A1- and PCNA-dependent synthesis of DNA catalyzed by polymerase delta . The putative amino acid sequence of the 40-kDa subunit of A1 revealed significantly homology with the bacteriophage T4 gene 44 protein and, to a lesser degree, with the tau and gamma subunits of Escherichia coli DNA polymerase III holoenzyme.

J Virol, 1992 Apr, 66(4), 2000 - 7
Inhibition of human immunodeficiency virus type 1 Tat activity by coexpression of heterologous trans activators; Carroll R et al.; We examined the mechanism of Tat-mediated trans activation through competition experiments employing Tat proteins of human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) . EIAV Tat, as well as chimeric EIAV/HIV-1 Tat proteins, inhibited HIV-1 Tat-mediated trans activation in a cell-type-dependent fashion . Furthermore, these proteins inhibited trans activation by Tat-bacteriophage R17 coat protein chimeras . Inhibition resulted from competition between activation domains of effectors and competitors for a limiting cellular cofactor . The context in which competitor activation domains were expressed contributed to the extent of inhibition . In transfected cells, EIAV Tat and all chimeric competitors were located primarily in the cytoplasm, whereas HIV-1 Tat was primarily located in the nucleus . These data are consistent with a model for trans activation in which the activation domain of Tat associates with and conveys a cellular factor to the transcription complex via the trans-acting-responsive element (TAR).

FEBS Lett, 1992 Mar 30, 300(2), 141 - 4
Improved band shift assay for the simultaneous analysis of protein-DNA interactions and enzymatic functions of DNA polymerases; Strick R et al.; A simple method to assay the major properties of DNA polymerases such as template binding, polymerase and exonuclease activities in one step is exemplified with the DNA polymerases of E . coli, bacteriophage T4 and herpes simplex virus . Combining the advantages of the band-shift assay with the resolving power of polyacrylamide gradient gel electrophoresis, the procedure is particularly useful for a rapid functional analysis of mutant polymerases as well as inhibitors of DNA replication.

Nucleic Acids Res, 1992 Mar 25, 20(6), 1363 - 70
Statistical analyses of counts and distributions of restriction sites in DNA sequences; Karlin S et al.; Counts and spacings of all 4- and 6-bp palindromes in DNA sequences from a broad range of organisms were investigated . Both 4- and 6-bp average palindrome counts were significantly low in all bacteriophages except one, probably as a means of avoiding restriction enzyme cleavage . The exception, T4 of normal 4- and 6-palindrome counts, putatively derives protection from modification of cytosine to hydroxymethylcytosine plus glycosylation . The counts and distributions of 4-bp and of 6-bp restriction sites in bacterial species are variable . Bacterial cells with multiple restriction systems for 4-bp or 6-bp target specificities are low in aggregate 4- or 6-bp palindrome counts/kb, respectively, but bacterial cells lacking exact 4-cutter enzymes generally show normal or high counts of 4-bp palindromes when compared with random control sequences of comparable nucleotide frequencies . For example, E . coli, apparently without an exact 4-bp target restriction endonuclease (see text), contains normal aggregate 4-palindrome counts/kb, while B . subtilis, which abounds with 4-bp restriction systems, shows a significant under-representation of 4-palindrome counts . Both E . coli and B . subtilis have many 6-bp restriction enzymes and concomitantly diminished aggregate 6-palindrome counts/kb . Eukaryote, viral, and organelle sequences generally have aggregate 4- and 6-palindromic counts/kb in the normal range . Interpretations of these results are given in terms of restriction/methylation regimes, recombination and transcription processes, and possible structural and regulatory roles of 4- and 6-bp palindromes.

J Biol Chem, 1992 Mar 25, 267(9), 6063 - 73
Four different DNA helicases from calf thymus; Thommes P et al.; Using a strand displacement assay we have followed DNA helicase activities during the simultaneous isolation of several enzymes from calf thymus such as DNA polymerases alpha, delta, and epsilon, proliferating cell nuclear antigen, and replication factor A . Thus we were able to discriminate and isolate four different DNA helicases called A, B, C, and D . DNA helicase A is identical with the enzyme described earlier (Thommes, P., and Hubscher, U . (1990) J . Biol . Chem . 265, 14347-14354) . The four enzymes can be distinguished by (i) their putative molecular weights after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (ii) glycerol gradient sedimentation under low and high salt conditions, (iii) sensitivity to salt, (iv) binding to DNA, (v) nucleoside- and deoxynucleoside 5'-triphosphate requirements, and (vi) by their direction of movement . DNA helicase A unwinds in the 3'----5' direction on the DNA it was bound to, while DNA helicases B, C, and D do so in the 5'----3' direction . DNA helicase D, and to some extent DNA helicases B and C, are able to unwind long substrates of more than 400 nucleotides . Replication factor A, a single-stranded heterotrimeric DNA binding protein involved in cellular DNA replication and DNA repair stimulates the DNA helicases . The stimulatory effect is most pronounced on DNA helicase A, where replication factor A enables this helicase to unwind longer substrates . DNA helicases B, C, and D are also stimulated by replication factor A . The effect of replication factor A appears to be specific since corresponding single-stranded DNA binding proteins from Escherichia coli and bacteriophage T4 have no or even a negative effect on the four DNA helicases . Heterologous human replication factor A has no stimulatory effect on any of the four DNA helicases suggesting a species specificity of these interactions . Thus it appears that mammalian cells possess, as does E . coli, a variety of different enzymes that can transiently abolish the double helical DNA structure in the cell.

J Mol Biol, 1992 Mar 20, 224(2), 319 - 34
Model for the mechanism of bacteriophage T7 RNAP transcription initiation and termination; Sousa R et al.; Characterization of a mutant T7 RNA polymerase (RNAP) that is active on non-promoter templates but has lost the ability to selectively utilize the T7 promoter led to the finding that wild-type T7 RNAP initiates transcription at a high rate on non-promoter templates but that most (approximately 90%) of these initiation events lead to synthesis of dinucleotides only . The anomalously high activity of T7 RNAP on poly(dC) templates (relative to other non-promoter templates) is due to a reduction in the rate of transcription abortion after dinucleotide synthesis rather than an increase in initiation . Evidence is presented that the transition from abortive to processive transcription is associated with a conformational change in T7 RNAP . The stability of the nascent chain in a ternary complex is shown to increase with increasing chain length in the 2 to 14 base range even when the size of the complementary RNA-DNA hybrid remains constant and small (2 to 3 base-pairs) . Two mutant polymerases that show increased release of transcripts during abortive transcription and a proteolytically nicked polymerase that exhibits reduced RNA binding are shown to have reduced ability to read-through a T7 RNAP hairpin U-stretch transcription terminator . Single-stranded nucleic acids are shown to bind more tightly than double-stranded nucleic acids to T7 RNAP . These observations and a large set of published studies on T7 RNAP structure and mechanism are accommodated in a relatively simple model of T7 RNAP transcription initiation and termination in which a T7 RNAP that has initiated transcription is proposed to be capable of assuming two functionally distinct conformations: an abortive conformer characterized by a loose association with the nascent RNA and an inability to translocate along the template; and a processive conformer characterized by the stable retention of the nascent RNA and the ability to process stably along the template . The equilibrium between these two conformations is shifted towards the processive form when the nascent chain binds at a site located at least partly on the T7 RNAP N-terminal domain . The interaction requires that the RNA be more than approximately nine bases and this RNAP-RNA interaction plays a primary role in retaining the RNA within the ternary complex.(ABSTRACT TRUNCATED AT 400 WORDS)

J Mol Biol, 1992 Mar 20, 224(2), 307 - 18
Isolation and characterization of mutant bacteriophage T7 RNA polymerases; Patra D et al.; We have isolated and characterized a number of bacteriophage T7 RNAP (RNA polymerase) null mutants . Most of the mutants found to be completely inactive in vitro map to one of the well-conserved blocks of residues in the family of RNAPs homologous to T7 RNAP . The in vitro phenotypes of a smaller number of partially active T7 RNAP mutants, mapping outside these well-conserved regions, support the following assignment of functions in T7 RNAP: (1) the N-terminal region of T7 RNAP contains a nascent RNA binding site that functions to retain the nascent chain within the ternary complex; (2) the region surrounding residue 240 is involved in binding the initiating NTP; (3) residues at the very C terminus of T7 RNAP are involved in binding the elongating NTP.

J Mol Biol, 1992 Mar 20, 224(2), 395 - 412
Cryoelectron microscopic visualization of functional subassemblies of the bacteriophage T4 DNA replication complex; Gogol EP et al.; A specific complex of proteins involved in bacteriophage T4 replication has been visualized by cryoelectron microscopy as distinctive structures in association with DNA . Formation of these structures, which we term "hash-marks" for their characteristic appearance in association with DNA, requires the presence of the T4 polymerase accessory proteins (the products of T4 genes 44, 45 and 62), ATP and appropriate DNA cofactors . ATP hydrolysis by the DNA-stimulated ATPase activity of the accessory proteins is required for visualization of the hash-mark structures . If ATP hydrolysis is stopped by chelation of Mg2+, by dilution with a non-hydrolyzable ATP analogue, or by exhaustion of the ATP supply, the DNA-associated structures disappear within seconds to minutes, indicating that they have a finite and relatively short lifetime . The labile nature of the structures makes their study by more conventional methods of electron microscopy, as well as by most other structural approaches, difficult if not impossible . Addition of T4 gene 32 protein increases the number of hash-mark structures, as well as increasing the rate of ATP hydrolysis . Using plasmid DNA in either a native (supercoiled) or enzymatically modified state, we have shown that nicked or gapped DNA is required as a cofactor for hash-mark formation . Stimulation of the ATPase activity of the accessory proteins has a similar cofactor requirement . These conditions for the formation and visualization of the structures parallel those required for the action of these complexes in promoting the enzymatic activity of the T4 DNA polymerase, as well as the transcription of late T4 genes . Substructure in the hash-marks has been examined by image analysis, which reveals a variation in the projected density of the subunits comprising the structures . The three-dimensional size of the hash-marks, modeled as a solid ellipsoid, is consistent with that of the gene 44/62 protein subcomplex . Density variations suggest an arrangement of subunits, either tetragonal or trigonal, viewed from a variety of angles about the DNA axis . The hash-mark structures often appear in clusters, even in DNA that has a single nick . We interpret this distribution as the result of one-dimensional translocation of the hash-marks along the DNA after their ATP-dependent initial association with, and injection into, the DNA at nicks or gaps.

Biochemistry, 1992 Mar 17, 31(10), 2670 - 7
Stoichiometry, selectivity, and exchange dynamics of lipid-protein interaction with bacteriophage M13 coat protein studied by spin label electron spin resonance . Effects of protein secondary structure; Peelen SJ et al.; Bacteriophage M13 major coat protein has been isolated with cholate and reconstituted in dimyristoyl- and dioleoylphosphatidylcholine (DMPC and DOPC, respectively) bilayers by dialysis . Fourier transform infrared spectra of DMPC/coat protein recombinants confirmed that, whereas the protein isolated by phenol extraction was predominantly in a beta-sheet conformation, the cholate-isolated coat protein contained a higher proportion of the alpha-helical conformation {cf . Spruijt, R . B., Wolfs, C . J . A . M., & Hemminga, M . A . (1989) Biochemistry 28, 9158-9165} . The cholate-isolated coat protein/lipid recombinants gave different electron spin resonance (ESR) spectral line shapes of incorporated lipid spin labels, as compared with those from recombinants with the phenol-extracted protein that were studied previously {Wolfs, C . J . A . M., Horvath, L . I., Marsh, D., Watts, A., & Hemminga, M . A . (1989) Biochemistry 28, 9995-10001} . Plots of the ratio of the fluid/motionally restricted components in the ESR spectra of spin-labeled phosphatidylglycerol were linear with respect to the lipid/protein ratio in the recombinants up to 20 mol/mol . The corresponding values of the relative association constants, Kr, and number of association sites, N1, on the protein were Kr approximately 1 and N1 approximately 4 for DMPC recombinants and Kr approximately 1 and N1 approximately 5 for DOPC recombinants . Simulation of the two-component lipid spin label ESR spectra with the exchange-coupled Bloch equations gave values for the off-rate of the lipids leaving the protein surface of 2.0 x 10(7) s-1 at 27 degrees C in DMPC recombinants and 3.0 x 10(7) s-1 at 24 degrees C in DOPC recombinants.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1992 Mar 16, 183(2), 842 - 8
2'-Amino-2'-deoxyguanosine is a cofactor for self-splicing in group I catalytic RNA; Stromberg R et al.; In investigations on self-splicing in the group I intron of the pre-mRNA from the nrdB gene of bacteriophage T4 it was found that 2'-amino-2'-deoxyguanosine can replace guanosine as cofactor . This is the first guanosine-analogue with a modification in the 2'-position and substantial activity in a group I self-splicing reaction . The results suggest that the 2'-amino and 2'-hydroxy groups of the cosubstrates have some properties in common, which are important for binding as well as for catalysis.

Biochem Biophys Res Commun, 1992 Mar 16, 183(2), 797 - 802
Production of antibodies specific for double stranded antigen DNA cloned from immune complexes in plasma of a SLE patient; Terada K et al.; The antigenicity of antigen DNA isolated from immune complexes in plasma of a SLE patient was examined . DDY mice were immunized with the cloned antigen DNA carrying a sequence homologous with a part of bacteriophage f1 (KS8 DNA) by the coupling method, and the antibody response was estimated by radioimmunoassay . Antibodies specific to double stranded DNA were elicited . Moreover, the antibodies showed preferential binding to KS8 DNA than other DNA derived from Escherichia coli . These results suggest that KS8 DNA has a significant antigenicity in mice.

Eur J Biochem, 1992 Mar 15, 204(3), 1003 - 4
Selection and characterization of randomly produced mutants of gene V protein of bacteriophage M13; Stassen AP et al.; Gene V protein of bacteriophage Ff (M13, f1, fd) is a master regulator of phage DNA replication and phage mRNA translation . It exerts these two functions by binding to single-stranded viral DNA or to specific sequences in the 5' ends of its target mRNAs, respectively . To study the structure/function relationship of gene V protein, M13 gene V was inserted in a phagemid expression vector and a library of missense and nonsense mutants was constructed by random chemical mutagenesis . Phagemids encoding gene V proteins with decreased biological activities were selected and the nucleotide sequences of their gene V fragments were determined . Furthermore, the mutant proteins were characterized both with respect to their ability to inhibit the production of phagemid DNA transducing particles and their ability to repress the translation of a chimeric lacZ reporter gene whose expression is controlled by the promoter and translational initiation signals of M13 gene II . From the data obtained, it can be deduced that the mechanism by which gene V protein binds to single-stranded DNA differs from the mechanism by which it binds to its target sequence in the gene II mRNA.

J Biol Chem, 1992 Mar 15, 267(8), 5296 - 300
Transmembrane region of wild-type and mutant M13 coat proteins . Conformational role of beta-branched residues; Deber CM et al.; Although transmembrane (TM) segments of integral membrane proteins are putatively alpha-helical in conformation, beta-sheet promoters (Val, Ile, Thr) often account for approximately 40% of TM residue composition . We are examining the conformational role(s) of these residues, using as a model system the major coat protein of the filamentous bacteriophage M13 . This 50-residue protein, which is located at the Escherichia coli host membrane during phage reproduction, contains a prototypic 19-residue hydrophobic midregion (residues 21-39: YIGYAWAMVVVIVGATIGI) . Using "Eckstein" site-directed mutagenesis, we have generated several viable M13 coat protein mutants with beta-branched amino acid substitutions within their TM region . Mutant coat proteins, including Ile32----Val (I32V) and Ala27----Thr (A27T), were obtained in milligram quantities by growing M13 mutant phages in liter preparations, confirming that these coat proteins are capable of assuming their normal biological function(s) in phage reproduction . Circular dichroism spectroscopy performed in the membrane-mimetic medium of deoxycholate micelles indicated comparable alpha-helical contents of mutants I32V and A27T to wild-type protein . 13C nuclear magnetic resonance experiments with mutant A27T demonstrated that the combination of additional beta-branched content and introduction of an -OH substituent induced chemical shift and temperature-dependent changes and influenced the local protein environment at sites up to 12 residues remote from the mutation site . In contrast, mutant I32V (of which a salient feature is a mid-TM pentavaline segment) behaved very similarly to wild-type coat . These findings are interpreted in terms of the range of TM secondary structure and stability which can be accommodated by viable M13 coat protein mutants.

J Mol Biol, 1992 Mar 5, 224(1), 103 - 12
Three-dimensional structure of T3 connector purified from overexpressing bacteria; Valpuesta JM et al.; The bacteriophage T3 connector has been purified from overexpressed protein in Escherichia coli, harboring a plasmid containing the gene encoding p8 protein . The connector, which is composed of 12 copies of p8, has been crystallized in two-dimensional sheets and studied by electron microscopy from negatively stained specimens . A two-dimensional Fourier filtering and averaging procedure was performed with crystalline specimens . In addition, single particle averaging techniques were used with other preparations . The average images obtained from these two approaches gave similar results . A three-dimensional reconstruction from two-dimensional crystals of T3 connectors was obtained by collecting several sets of tilted views and using standard Fourier procedures . The resolution of the three-dimensional map was 1.65 nm . The reconstructed connector shows two main domains: a wider one with 12 small units in the periphery and with an external diameter of 14.9 nm, and a smaller one with 8.5 nm diameter . The height of the reconstructed connector has been determined to be around 8.5 nm . The reconstruction clearly shows an internal open channel running along the longitudinal axis of the particle and having an average diameter of 3.7 nm.

FEMS Microbiol Lett, 1992 Mar 1, 70(2), 97 - 100
A natural mutant of plasmid RP4 that confers phage resistance and reduced conjugative transfer; Kornstein LB et al.; A natural isolate of RP4 (PRC#116) acquired from the Stanford University Plasmid Reference Center differed from the wild-type Incompatibility Group P plasmid in several respects . Cells of Escherichia coli harboring PRC#116 were resistant to the IncP pili-specific bacteriophage PRD1 and GU5, and transferred this plasmid at a lower efficiency than the wild-type RP4 . Phage sensitivity was restored, and transfer considerably improved in PRC#116+ bacteria transformed with plasmid constructs containing the origin of transfer (oriT region) of RP4 . Mutant RP4 plasmids equivalent to PRC#116 were selected at a high frequency from an RP4+ E . coli population infected with PRD1 indicating that this RP4 variant may be the product of a very common mutation of the wild-type plasmid.

Appl Environ Microbiol, 1992 Mar, 58(3), 900 - 4
Model system using coliphage phi X174 for testing virus removal by air filters; Rapp ML et al.; Short-term (15-min-duration) and long-term (5- to 6-day-duration) test procedures have been developed for determining the efficiency of the removal of bacteriophage phi X174 by air-sterilizing filters . These procedures were sensitive enough to measure a 10(8)-fold reduction in the number of bacteriophage . A filter commonly used in industrial air sterilizations (Domnick-Hunter Bio-X borosilicate glass) effected a 10(8)-fold removal of viable phage in both short-term and long-term tests . A prototype low-flux, hollow-fiber membrane gave similar results; however, a prototype high-flux, hollow-fiber membrane removed only about 99.999% of the bacteriophage in short-term tests.

Mol Gen Genet, 1992 Mar, 232(2), 257 - 61
An internal AUU codon initiates a smaller peptide encoded by bacteriophage T4 baseplate gene 26; Nivinskas R et al.; Bacteriophage T4 baseplate gene 26 codes for two in-frame overlapping peptides with identical C-terminal regions . By site-directed mutagenesis we have now determined that an internal AUU, codon 114 of gene 26, is used as the initiation codon for the synthesis of a smaller peptide (gp26*) . Thus gene 26* gives rise to a peptide of 95 amino acid residues with an Mr of 10,873, while the complete gene 26 encodes a peptide of 208 amino acid residues of M(r) 23,880 . Expression of gene 26* is shown to depend on the RNA secondary structure in the translational initiation region of this gene.

EMBO J, 1992 Mar, 11(3), 839 - 46
Image reconstruction from cryo-electron micrographs reveals the morphopoietic mechanism in the P2-P4 bacteriophage system; Dokland T et al.; The satellite bacteriophage P4 does not have genes coding for any major structural proteins, but assembles a capsid from the gene products of bacteriophage P2 . The capsid assembled under control of P4 is smaller (45 nm) than the normal P2 capsid (60 nm) . The low resolution (4.5 nm) structures of P2 and P4 capsids were determined by cryo-electron microscopy and image processing . The capsid of P2 shows T = 7 symmetry with most of the mass clustered as 12 pentamers and 60 hexamers . The P4 capsid has T = 4 symmetry with a similar distribution of mass to P2, but the hexamer geometry has changed . The major capsid protein has a two-domain structure . The major domains form the capsomers proper, while connecting domains form trivalent contacts between the capsomers . The size determination by P4 appears to function by altering hexamer geometry rather than by affecting the interdomain angle alone.

Arch Biochem Biophys, 1992 Mar, 293(2), 391 - 400
Prostaglandin endoperoxide synthase gene structure: identification of the transcriptional start site and 5'-flanking regulatory sequences; Kraemer SA et al.; The gene for the murine prostaglandin endoperoxide (PGH) synthase (8, 11, 14-eicosatrienoate, hydrogen-donor:oxygen oxidoreductase, EC 1.14.99.1) has been cloned . The gene was isolated from a mouse NIH 3T3 cell genomic library and is contained in four overlapping lambda FIXII bacteriophage clones . The gene spans approximately 22 kb and consists of 11 exons . Primer extension and RNAse protection assays indicate that transcription of the gene begins at an initiation site 63 nucleotides 5' to the ATG translation initiation codon . Neither TATA or CAAT boxes are present immediately upstream of the transcriptional start site, but SP1 binding sites are present at positions -47 to -42 and -30 to -25, relative to the transcription initiation site . Examination of the 5'-end and 2400 bp of the 5'-flanking sequence of the gene revealed sequences with homology to several transcriptional regulatory sequences . Three putative AP-1 binding sites were found, two within the first exon and intron and another at position -2097 to -2090 . The AP-1 site at position -2097 is adjacent to a sequence with similarity to a negative glucocorticoid regulatory element (nGRE) (position -2123 to -2009) . The presence of AP sites by themselves, or in conjunction with an nGRE sequence, suggests a possible interplay between jun/fos regulatory proteins and the glucocorticoid receptor for positive and negative regulation of the PGH synthase gene . An unexpected finding was the presence at position -403 to -385 of a putative dioxin responsive element, a sequence found to be responsible for the induction of transcription of the cytochrome P450IA1 gene (CYPIA1) and other genes involved in detoxification/activation of polycyclic aromatic hydrocarbons.

J Bacteriol, 1992 Mar, 174(5), 1462 - 77
DNA sequences of the tail fiber genes of bacteriophage P2: evidence for horizontal transfer of tail fiber genes among unrelated bacteriophages; Haggard-Ljungquist E et al.; We have determined the DNA sequence of the bacteriophage P2 tail genes G and H, which code for polypeptides of 175 and 669 residues, respectively . Gene H probably codes for the distal part of the P2 tail fiber, since the deduced sequence of its product contains regions similar to tail fiber proteins from phages Mu, P1, lambda, K3, and T2 . The similarities of the carboxy-terminal portions of the P2, Mu, ann P1 tail fiber proteins may explain the observation that these phages in general have the same host range . The P2 H gene product is similar to the products of both lambda open reading frame (ORF) 401 (stf, side tail fiber) and its downstream ORF, ORF 314 . If 1 bp is inserted near the end of ORF 401, this reading frame becomes fused with ORF 314, creating an ORF that may represent the complete stf gene that encodes a 774-amino-acid-long side tail fiber protein . Thus, a frameshift mutation seems to be present in the common laboratory strain of lambda . Gene G of P2 probably codes for a protein required for assembly of the tail fibers of the virion . The entire G gene product is very similar to the products of genes U and U' of phage Mu; a region of these proteins is also found in the tail fiber assembly proteins of phages TuIa, TuIb, T4, and lambda . The similarities in the tail fiber genes of phages of different families provide evidence that illegitimate recombination occurs at previously unappreciated levels and that phages are taking advantage of the gene pool available to them to alter their host ranges under selective pressures.

Biophys J, 1992 Mar, 61(3), 725 - 35
Structural polymorphism correlated to surface charge in filamentous bacteriophages; Bhattacharjee S et al.; Fiber diffraction studies are used to demonstrate that changes in the helical symmetry of the protein coat of filamentous bacterial viruses fd and M13 are correlated with changes in the surface charge . Comparison of the structure of M13 and fd at pH 2 and 8 indicate that surface charge affects both the helical symmetry and flexibility of the virions . The changes in helical symmetry are similar in magnitude to that observed in the Pseudomanas phage Pf1 and probably reflect an inocuous side effect of the particle flexibility required for protection of the virus particles from damage due to shear . The magnitude of the observed changes in helical symmetry appears to be limited to that which can occur without repacking of the interfaces between the alpha-helices making up the viral protein coat.

J Struct Biol, 1992 Mar-Apr, 108(2), 168 - 75
The three-dimensional structure of frozen-hydrated bacteriophage phi X174; Olson NH et al.; The three-dimensional structure of bacteriophage phi X174 (phi X174) was determined to approximately 2.6 nm resolution from images of frozen-hydrated 114 S particles . The outer surface of phi X174 is characterized by several prominent features: (i) 12 mushroom-shaped caps (approximately 7.1 nm wide x 3.8 nm high) are situated at each of the vertices of the icosahedral virion and extend to a maximum radius of 16.8 nm; (ii) a "collar" of density surrounds the base of each apical cap; and (iii) 20 conical protrusions (approximately 2.3 nm high) lie along the three-fold symmetry axes . The caps have a pentagonal morphology composed of five globular "subunits" and appear to be loosely connected to the underlying capsid . The distribution of the four gene products present in virions (60 copies each of gpF, gpG, and gpJ, and 12 copies of gpH), and the single-stranded DNA (ssDNA) genome cannot be directly discerned in the reconstructed density map, although plausible assignments can be made on the basis of solvent-excluded volume estimates and previous biochemical data . Thus, gpG accounts for most of the mass in the caps; gpH, a presumed cap protein, cannot be identified in part due to the symmetry-averaging procedures, but may be partially located within the interior of the capsid; and gpF and gpJ make up the remainder of the capsid . The genome appears to be less densely packaged inside the capsid compared to many dsDNA viruses whose nucleic acid is arranged in a liquid-crystalline state.

J Pharm Pharmacol, 1992 Mar, 44(3), 266 - 8
Increase in susceptibility to EcoRII restriction of bacteriophage lambda produced by propagation on host cells growing in 5-azacytidine: a new in-vivo method for demonstration of DNA-methylation inhibition; Radnedge L et al.; The efficiency of plating on EcoRII-restricting cells of bacteriophage lambda vir propagated on an Escherichia coli K-12 dcm+ host decreased with increase in concentration of 5-azacytidine (5-azaC) in the propagating medium . This illustrates, in-vivo, the inhibition of DNA-cytosine methylation induced by 5-azaC and provides a simple system for the detection of DNA-methylation inhibitors.

J Med Microbiol, 1992 Mar, 36(3), 209 - 14
Identification of an endoflagellar associated protein in Borrelia burgdorferi; Eiffert H et al.; DNA of Borrelia burgdorferi was cleaved by the endonuclease EcoRI and ligated with the bacteriophage expression vector lambda gt11 . After infection of the Escherichia coli strain Y1089, the plaques of recombinant phages were screened with a B . burgdorferi antiserum (human) for fusion proteins containing borrelia antigen.s A positive clone produced a hybrid protein (p200) of c . 200 Kda . The corresponding native borrelia protein (p97) was identified as having an Mr of 97 Kda . To localise protein p97 in the B . burgdorferi cell, immunoelectronmicroscopy and a Western blot of isolated flagella were used . Antibodies directed against proteins p200 and p97 recognised epitopes associated with the flagella.

Genes Dev, 1992 Mar, 6(3), 497 - 510
The Rex system of bacteriophage lambda: tolerance and altruistic cell death; Parma DH et al.; The rexA and rexB genes of bacteriophage lambda encode a two-component system that aborts lytic growth of bacterial viruses . Rex exclusion is characterized by termination of macromolecular synthesis, loss of active transport, the hydrolysis of ATP, and cell death . By analogy to colicins E1 and K, these results can be explained by depolarization of the cytoplasmic membrane . We have fractionated cells to determine the intracellular location of the RexB protein and made RexB-alkaline phosphatase fusions to analyze its membrane topology . The RexB protein appears to be a polytopic transmembrane protein . We suggest that RexB proteins form ion channels that, in response to lytic growth of bacteriophages, depolarize the cytoplasmic membrane . The Rex system requires a mechanism to prevent lambda itself from being excluded during lytic growth . We have determined that overexpression of RexB in lambda lysogens prevents the exclusion of both T4 rII mutants and lambda ren mutants . We suspect that overexpression of RexB is the basis for preventing self-exclusion following the induction of a lambda lysogen and that RexB overexpression is accomplished through transcriptional regulation.

Trends Biotechnol, 1992 Mar, 10(3), 80 - 4
Phage antibodies: will new 'coliclonal' antibodies replace monoclonal antibodies?
Chiswell DJ, McCafferty J.
Antibodies can now be rapidly isolated from large and diverse recombinant libraries by displaying functional antibody fragments on the surface of bacteriophage particles and directly selecting with antigen . This method has been used to isolate antibodies, including human antibodies, with and without immunization, and to improve the affinity and specificity of antigen binding.

Biochim Biophys Acta, 1992 Feb 28, 1130(1), 52 - 62
Defective DNA injection by alkylated and nonalkylated bacteriophage T7; Czaika G et al.; DNA injection by alkylated and nonalkylated bacteriophage T7 has been analyzed by a physical method which involved Southern hybridization to identify noninjected regions of DNA . Treatment of phage with methyl methanesulfonate reduced the amount of DNA injected into wild-type Escherichia coli cells . This reduction was correlated with a decreased injection of DNA segments located on the right-hand third of the T7 genome . An essentially identical injection defect was observed when alkylated phage infected E . coli mutant cells unable to repair 3-methyladenine . Furthermore, untreated phage particles were discovered to be naturally injection-defective . Some injected all their DNA except those segments located in the rightmost 15% of the T7 genome, while other injected no DNA at all . In the presence of rifampicin, untreated phages injected only segments from the left end of the genome . These results provide direct physical evidence that T7 DNA injection is strictly unidirectional, starting from the left end of the T7 genome . The injection defect quantified here for alkylated phage is probably partially, if not totally, responsible for phage inactivation, when that inactivation is measured in wild-type E . coli cells . Since alkylated phage injected the same DNA sequences into both wild-type and repair-deficient cells, we conclude that DNA injection is independent of the host-cell's capacity for repair of 3-methyladenine residues.

Nucleic Acids Res, 1992 Feb 25, 20(4), 711 - 7
Isolation of dominant negative mutants and inhibitory antisense RNA sequences by expression selection of random DNA fragments; Holzmayer TA et al.; Selective inhibition of specific genes can be accomplished using genetic suppressor elements (GSEs) that encode antisense RNA, dominant negative mutant proteins, or other regulatory products . GSEs may correspond to partial sequences of target genes, usually identified by trial and error . We have used bacteriophage lambda as a model system to test a concept that biologically active GSEs may be generated by random DNA fragmentation and identified by expression selection . Fragments from eleven different regions of lambda genome, encoding specific peptides or antisense RNA sequences, rendered E . coli resistant to the phage . Analysis of these GSEs revealed some previously unknown functions of phage lambda, including suppression of the cellular lambda receptor by an 'accessory' gene of the phage . The random fragment selection strategy provides a general approach to the generation of efficient GSEs and elucidation of novel gene functions.

J Biol Chem, 1992 Feb 25, 267(6), 4054 - 63
Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork . III . A polymerase-primase interaction governs primer size; Zechner EL et al.; Studies with a rolling-circle DNA replication system reconstituted in vitro with a tailed form II DNA template, the DNA polymerase III holoenzyme (Pol III HE), the Escherichia coli single-stranded DNA binding protein, and the primosome, showed that within the context of a replication fork, the oligoribonucleotide primers that were formed were limited to a length in the range of 9 to 14 nucleotides, regardless of whether they were subsequently elongated by the lagging-strand DNA polymerase . This is in contrast to the 8-60-nucleotide-long primers synthesized by the primosome in the absence of DNA replication on a bacteriophage phi X174 DNA template, although when primer synthesis and DNA replication were catalyzed concurrently in this system, the extent of RNA polymerization decreased . As described in this report, we therefore examined the effect of the DNA Pol III HE on the length of primers synthesized by primase in vitro in the absence of DNA replication . When primer synthesis was catalyzed either: i) by the primosome on a phi X174 DNA template, ii) by primase on naked DNA with the aid of the DnaB protein (general priming), or iii) by primase alone at the bacteriophage G4 origin, the presence of the DNA Pol III HE in the reaction mixtures resulted in a universal reduction in the length of the heterogeneous RNA products to a uniform size of approximately 10 nucleotides . dNTPs were not required, and the addition of dGMP, an inhibitor of the 3'----5' exonuclease of the DNA Pol III HE, did not alter the effect; therefore, neither the 5'----3' DNA polymerase activity nor the 3'----5' exonuclease activity of the DNA Pol III HE was involved . E . coli DNA polymerase I, and the DNA polymerases of bacteriophages T4 and T7 could not substitute for the DNA Pol III HE . The Pol III core plays a crucial role in mediating this effect, although other subunits of the DNA Pol III HE are also required . These observations suggest that the association of primase with the DNA Pol III HE during primer synthesis regulates its catalytic activity and that this regulatory interaction occurs independently of, and prior to, formation of a preinitiation complex of the DNA Pol III HE on the primer terminus.

Nucleic Acids Res, 1992 Feb 25, 20(4), 903 - 7
Immunoprecipitation of SV40 replicating minichromosomes complexed with bacteriophage T4 gene 32 protein; Miranda EI et al.; Simian Virus 40 (SV40) DNA replication is a useful model to study eukaryotic cell DNA replication because it encodes only one replication protein and its genome has a nucleoprotein structure ('minichromosome') indistinguishable from cellular chromatin . Late after infection SV40 replicating DNA molecules represent about 5% of total viral minichromosomes . Since gene 32 protein (P32) from bacteriophage T4 interacts with single-stranded DNA and SV40 replication complexes are expected to contain single-stranded regions at the replication forks, we asked whether P32 might be used to isolate replicating SV40 minichromosomes . When nuclear extracts from SV40 infected cells were treated sequentially with P32 and anti-P32 antibodies, pulse-labeled minichromosomes were selectively immunoprecipitated . Agarose gel electrophoresis analysis confirmed that immunoprecipitated material corresponded to SV40 replicative intermediates . Protein analysis of the pelleted material revealed several proteins of viral and cellular origin . Among them, T antigen and histones were found to be complexed with at least other three proteins from cellular origin, to the replicative complexes . Additionally, anti-P32 antibodies were able to detect three cellular proteins of approximately 70, 32 and 13 kDa in western blots . These proteins could correspond to those found as part of an eukaryotic multisubunit single-stranded DNA binding protein . The use of P32 and anti-P32 antibodies thus allows the separation of replicating from mature SV40 minichromosomes and can constitute a novel method to enrich and to study replicative active chromatin.

J Mol Biol, 1992 Feb 20, 223(4), 999 - 1011
Conformation of DNA packaged in bacteriophage T7 . Analysis by use of ultraviolet light-induced DNA-capsid cross-linking; Serwer P et al.; The conformation of the linear, double-stranded, 39,936 kilobase-pair DNA packaged in the protein capsid of bacteriophage T7 is investigated here by use of short wavelength ultraviolet light-induced DNA-capsid cross-linking . To detect both DNA-capsid and DNA-DNA cross-links, DNA is expelled from the T7 capsid and the products of expulsion are analyzed by use of Nycodenz buoyant density centrifugation, followed by either pulsed field gel electrophoresis or invariant field gel electrophoresis . Short wavelength ultraviolet light is found to progressively induce both DNA-DNA and DNA-protein cross-links in intact bacteriophage T7, but not in T7 from which DNA had been expelled before exposure to ultraviolet light . Protein-protein cross-links are not induced . When DNA expelled from previously cross-linked T7 is cleaved with restriction endonuclease (1 to 3 sites cleaved), analysis of the resulting fragments reveals no regions on T7 DNA that are excluded from cross-linking to the capsid . However, the efficiency of cross-linking decreases as the distance from the left end (last end packaged) of the packaged DNA increases . Electron microscopy of negatively stained capsid-DNA complexes reveals no DNA-retaining structure other than the outer shell of the capsid . Together with previously reported data that indicate lack of protein-based specificity for ultraviolet light-induced cross-linking, these observations are interpreted by the assumptions that, within the limits of resolution of these experiments: (1) no region of packaged T7 DNA is excluded from contact with the outer shell of the T7 capsid; (2) the probability of contacting the outer shell decreases as the distance from the left end of packaged T7 DNA increases . Thus, T7 DNA packaging concentrates the last end packaged near the inner surface of the outer shell of the T7 capsid.

J Mol Biol, 1992 Feb 20, 223(4), 977 - 89
Bacteriophage P1 genes involved in the recognition and cleavage of the phage packaging site (pac); Skorupski K et al.; The packaging of bacteriophage P1 DNA is initiated by cleavage of the viral DNA at a specific site, designated pac . The proteins necessary for that cleavage, and the genes that encode those proteins, are described in this report . By sequencing wild-type P1 DNA and DNA derived from various P1 amber mutants that are deficient in pac cleavage, two distinct genes, referred to as pacA and pacB, were identified . These genes appear to be coordinately transcribed with an upstream P1 gene that encodes a regulator of late P1 gene expression (gene 10) . pacA is located upstream from pacB and contains the 161 base-pair pac cleavage site . The predicted sizes of the PacA and PacB proteins are 45 kDa and 56 kDa, respectively . These proteins have been identified on SDS-polyacrylamide gels using extracts derived from Escherichia coli cells that express these genes under the control of a bacteriophage T7 promoter . Extracts prepared from cells expressing both PacA and PacB are proficient for site-specific cleavage of the P1 packaging site, whereas those lacking either protein are not . However, the two defective extracts can complement each other to restore functional pac cleavage activity . Thus, PacA and PacB are two essential bacteriophage proteins required for recognition and cleavage of the P1 packaging site . PacB extracts also contain a second P1 protein that is encoded within the pacB gene . We have identified this protein on SDS-polyacrylamide gels and have shown that it is translated in the same reading frame as is PacB . Its role, if any, in pac cleavage is yet to be determined.

J Mol Biol, 1992 Feb 20, 223(4), 831 - 5
Preformed ribozyme destroys tumour necrosis factor mRNA in human cells; Sioud M et al.; Maintaining RNA stability is a major problem in the delivery of preformed inhibitory RNA to target cells . In this study, we delivered a hammerhead ribozyme directed against tumour necrosis factor alpha into human promyelocytic leukaemia cells by cationic liposome-mediated transfection . Delivering a ribozyme in this manner reduced by 90% and 85% tumour necrosis factor alpha mRNA and protein, respectively . A modified ribozyme with a bacteriophage T7 transcription terminator at its 3' end was more stable than one lacking this sequence . This indicates that ribozyme stability can be improved by the addition of terminal sequences expected to protect against cellular nucleases.

Biochemistry, 1992 Feb 18, 31(6), 1712 - 21
Cloning, expression, and catalytic mechanism of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart; Wo YY et al.; The first representative of a group of mammalian, low molecular weight phosphotyrosyl protein phosphatases was cloned, sequenced and expressed in Escherichia coli . Using a 61-mer oligonucleotide probe based on the amino acid sequence of the purified enzyme, several overlapping cDNA clones were isolated from a bovine heart cDNA library . A full-length clone was obtained consisting of a 27-bp 5' noncoding region, an open reading frame encoding the expected 157 amino acid protein, and an extensive 3' nontranslated sequence . The identification of the clone as full length was consistent with results obtained in mRNA blotting experiments using poly(A)+ mRNA from bovine heart . The coding sequence was placed downstream of a bacteriophage T7 promoter, and protein was expressed in E . coli . The expressed enzyme was soluble, and catalytically active and was readily isolated and purified . The recombinant protein had the expected Mr of 18,000 (estimated by SDS-PAGE), and it showed cross-reactivity with antisera that had been raised against both the bovine heart and the human placenta enzymes . The amino acid sequence of the N-terminal region of the expressed protein showed that methionine had been removed, resulting in a sequence identical to that of the enzyme isolated from the bovine tissue, with the exception that the N-terminal alanine of the protein from tissue is acetylated . A kinetically competent phosphoenzyme intermediate was trapped from a phosphatase-catalyzed reaction . Using 31P NMR, the covalent intermediate was identified as a cysteinyl phosphate . By analogy with the nomenclature used for serine esterases, these enzymes may be called cysteine phosphatases.

Nucleic Acids Res, 1992 Feb 11, 20(3), 587 - 94
In vitro DNA replication implicates O2-ethyldeoxythymidine in transversion mutagenesis by ethylating agents; Bhanot OS et al.; A 36-nucleotide oligomer containing a single O2-ethyldeoxythymidine (O2-Et-dT) adduct at a specific site was synthesized . The oligomer, which corresponds to a specific DNA sequence in gene G of bacteriophage phi X174, was used as a template by T7 DNA polymerase to investigate the in vitro mutagenic specificity of O2-Et-dT . At 10 microM dNTP and 5 mM Mg++, the progress of T7 DNA polymerase was interrupted by O2-Et-dT: 80% 3' to O2-Et-dT and 14% after incorporating a nucleotide opposite O2-Et-dT (incorporation-dependent blocked product) . DNA synthesis past the lesion was low (6%) . Incorporation of a nucleotide opposite O2-Et-dT and subsequent postlesion synthesis were enhanced by increasing the dNTP concentration, with postlesion synthesis reaching 30% at 200 microM . Postlesion synthesis was further increased to 45% by addition of 10 mM dAMP to the polymerization reactions . DNA sequencing revealed that both dA and dT were incorporated opposite O2-Et-dT with dA incorporation impeding the progress of DNA synthesis . dT incorporation was efficiently extended implicating O2-Et-dT in transversion mutagenesis in vivo . These studies provide a basis for understanding the molecular mechanisms by which ethylating agents contribute to cytotoxicity, A.T transversion mutagenesis and activation of the oncogene neu by an A.T----T.A transversion event in rat neuroblastomas.

Nucleic Acids Res, 1992 Feb 11, 20(3), 495 - 500
Highly efficient generation of recombinant baculoviruses by enzymatically medicated site-specific in vitro recombination; Peakman TC et al.; We have used the Cre-lox system of bacteriophage P1 to develop a highly efficient in vitrosystem for construction of recombinant baculoviruses . A positive visual selection has been included to make identification of recombinant viral progeny rapid and straightforward . We report recombination frequencies as high as 5 x 10(7) recombinants/micrograms starting plasmid DNA and under certain conditions, up to 50% of the viral progeny are recombinants . Genes inserted into the baculovirus genome can be readily recovered in a simple one step process and re-inserted after manipulation if required . We have confirmed the structure of recovered plasmids by diagnostic restriction endonuclease digestion and the structure of recombinant viral genomes by Southern analysis . Possible uses and the significance of the system are discussed and experiments currently being done to improve it are described.

Nucleic Acids Res, 1992 Feb 11, 20(3), 563 - 71
Unusual ribosome binding properties of mRNA encoding bacteriophage lambda repressor; Balakin AG et al.; The mRNA encoding repressor cI of phage lambda is the only known E . coli message which starts directly with the initiation AUG codon . The ability of in vitro synthesized cI mRNA fragments (150 or 400 nts) to form ternary initiation complexes has been studied using the toeprint method . In the presence of tRNA(Met)f, these fragments are capable of forming the ternary complexes at the 5'-terminal AUG codon not only with 30S subunits but also with undissociated 70S ribosomes (70S tight couples) . In the latter case, no binding at other positions of cI mRNA can be detected at all . The starting region of cI mRNA has a single stranded conformation and is highly enriched in A-residues . This feature of cI mRNA RBS is suggested to be the main factor which allows cI mRNA to form the initiation complex with the ribosome . Unlike 30S subunits, the binding to 70S tight couples is not affected by any of the initiation factors, although it is as efficient as that to 30S subunits supplemented with the factors . 30S subunits prefer to associate with the internal RBSs of the preformed mRNA molecules, provided that they are not sequestered by the secondary structure . In contrast, 70S tight couples tend to avoid extra sequences upstream of the codon directed to the P site and occupy a position as close as possible to the 5'-end of the message . This has been found to be the case both for tRNA(Met)f and for elongator tRNA(Glu)2 . The structural features of mRNA RBSs which influence their different binding for 30S subunits and 70S ribosomes are discussed.

J Biol Chem, 1992 Feb 5, 267(4), 2594 - 9
The bacteriophage phi 29 DNA polymerase, a proofreading enzyme; Garmendia C et al.; The bacteriophage phi 29 DNA polymerase, involved both in the protein-primed initiation and elongation steps of the viral DNA replication, displays a very processive 3',5'-exonuclease activity acting preferentially on single-stranded DNA . This exonucleolytic activity showed a marked preference for excision of a mismatched versus a correctly paired 3' terminus . These characteristics enable the phi 29 DNA polymerase to act as a proofreading enzyme . A comparative analysis of the wild-type phi 29 DNA polymerase and a mutant lacking 3',5'-exonuclease activity indicated that a productive coupling between the exonuclease and polymerase activities is necessary to prevent fixation of polymerization errors . Based on these data, the phi 29 DNA polymerase, a model enzyme for protein-primed DNA replication, appears to share the same mechanism for the editing function as that first proposed for T4 DNA polymerase and Escherichia coli DNA polymerase I on the basis of functional and structural studies.

J Mol Biol, 1992 Feb 5, 223(3), 595 - 600
Fidelity of human immunodeficiency virus type I reverse transcriptase in copying natural RNA; Hubner A et al.; The in vitro fidelity of reverse transcriptase from human immunodeficiency virus type I (HIV-1 RT) upon copying an RNA template was measured using the phi Xam 16 reversion assay . A phi X174 sequence harboring the amber 16 codon was cloned into a transcription vector . RNA obtained from transcription by bacteriophage T7 RNA polymerase was used as a template for RNA-directed DNA synthesis by HIV-1 RT . An imbalance of dNTP concentrations during the reverse transcription step served to distinguish between errors that arose from the transcription step and errors from reverse transcription . The frequency of dGTP.U mismatches was determined to be 1/360, while dGTP.rA mismatches formed at a rate of 1/4600 . These are 20-fold and sevenfold higher, respectively, than the error rates determined for the same sequence with a DNA template . Due to a high background of errors in the RNA template originating from the transcription step only upper limits for the frequency of three other mismatches can be given . The data indicate that the reverse transcription step of the HIV-1 replication cycle contributes significantly to the generation of mutant viruses.

Biochemistry, 1992 Feb 4, 31(4), 1254 - 62
Assignment of the 1H NMR spectrum and secondary structure elucidation of the single-stranded DNA binding protein encoded by the filamentous bacteriophage IKe; van Duynhoven JP et al.; By means of 2D NMR techniques, all backbone resonances in the 1H NMR spectrum of the single-stranded DNA binding protein encoded by gene V of the filamentous phage IKe have been assigned sequence specifically (at pH 4.6, T = 298 K) . In addition, a major part of the side chain resonances could be assigned as well . Analysis of NOESY data permitted the elucidation of the secondary structure of IKe gene V protein . The major part of this secondary structure is present as an antiparallel beta-sheet, i.e., as two beta-loops which partly combine into a triple-stranded beta-sheet structure, one beta-loop and one triple-stranded beta-sheet structure . It is shown that a high degree of homology exists with the secondary structure of the single-stranded DNA binding protein encoded by gene V of the distantly related filamentous phage M13.

Genomics, 1992 Feb, 12(2), 319 - 25
A polymorphic (CA)n repeat element maps the human glucokinase gene (GCK) to chromosome 7p; Matsutani A et al.; A compound imperfect dinucleotide repeat element, {CA}4TTTGT{CT}7{CA}9AA{CA}4CCACATA{CA}3, was found approximately 10 kb 3' to the human glucokinase gene (GCK) from analysis of contiguous genomic DNA obtained from a bacteriophage lambda chromosome walk . Direct human genomic sequencing revealed the source of polymorphism to be variable numbers of CT and CA repeats . Altogether six alleles that range in length from +10 to -15 nucleotides compared to the most common (Z) allele have been identified . Alleles Z, Z + 2, and Z + 4 were present in American Blacks, Pima Indians, and Caucasians, with somewhat varied frequencies among the groups . Two alleles, Z + 10 and Z - 15, appear to be unique to American Blacks, while a Z + 6 allele was observed only in the Caucasian population studied . Observed heterozygosity of the polymorphism in the CEPH reference pedigree collection is 44% and the PIC 0.44 . The polymorphism is assayed by PCR amplification and resolution of 32P-end-labeled products (ranging in length from 180 to 205 bp) on denaturing polyacrylamide sequencing gels . Using the PCR assay, the human glucokinase gene was physically localized to chromosome 7 in a panel of rodent/human somatic cell lines . Genetic analysis in CEPH pedigrees placed the dinucleotide repeat element, and thereby the human glucokinase gene, on chromosome 7p between TCRG and a RFLP locus D7S57 . The glucokinase dinucleotide repeat genetic marker can now be used to assess the role of the glucokinase gene in diabetes by population association studies . In addition, this repeat marker and others flanking it on chromosome 7 can be used in linkage studies with families segregating the disorder.

Biochim Biophys Acta, 1992 Feb 1, 1118(3), 211 - 7
Effect of thermoinduced changes in T4 bacteriophage structure on the process of molecular recognition of 'host' cells; Khusainov AA et al.; By means of high-precision acoustic measurements and by methods of fluorescent and electron microscopy, investigations have been performed of thermoinduced conformational changes in T4 bacteriophage and its thermolabile mutants altered in baseplate proteins (gene products 7, 8, 10) . A relationship was found between the conformational changes in T4 bacteriophage structure in the temperature range of 33-45 degrees C and the efficiency of bacteriophage adsorption and the changes in the orientation of long tail fibers . The possibility of heat regulation of 'recognition' of 'host' cells by bacterial viruses is suggested.

Proc Natl Acad Sci U S A, 1992 Feb 1, 89(3), 1050 - 4
Structural basis for the nucleic acid binding cooperativity of bacteriophage T4 gene 32 protein: the (Lys/Arg)3(Ser/Thr)2 (LAST) motif; Casas-Finet JR et al.; To identify the functional residues of the N-terminal B region of bacteriophage T4 gene 32 protein involved in its cooperative binding to single-stranded nucleic acids, a process dependent on homotypic protein-protein interaction, we have studied the interaction of the protein with synthetic peptides containing different portions of this domain . Gel-permeation chromatography showed that a 6-acryloyl-2-dimethylaminonaphthalene (acrylodan)-labeled fluorescent peptide corresponding to the first 17 residues of gene 32 protein formed a complex with whole protein . The fluorescence was blue-shifted 14 nm upon interaction with intact protein, and somewhat less so (7-11 nm) with cleavage products of the protein lacking B domains . The intrinsic tryptophan fluorescence of whole and truncated protein was quenched by this peptide and by the nonderivatized peptide . The peptide bound tightly to truncated protein at both 0.015 and 0.44 M Na+, with a stoichiometry of 1:1 . Similar tryptophan quenching or acrylodan blue shifts were obtained with peptides corresponding to residues 1-9 and 3-8, but not residues 1-4, 5-9, or 5-17, indicating that the essential amino acids are contained within positions 3-8, Lys-Arg-Lys-Ser-Thr-Ala . Several other DNA binding proteins contain a LAST motif with documented involvement of these residues in nucleic acid interaction . The amino acid and coding sequence of residues 110-114, a region proposed to be involved in nucleic acid binding, is virtually identical to that of residues 3-7 . Based on these observations, we have formulated a model for the cooperative interactions of gene 32 protein with single-stranded nucleic acids.

Virology, 1992 Feb, 186(2), 693 - 700
A frog virus 3 gene codes for a protein containing the motif characteristic of the INT family of integrases; Rohozinski J et al.; The integrase (INT) family of bacteriophage coded integrase-recombinase proteins are responsible for catalyzing strand exchange between DNA molecules and play an important role in the DNA replication of many bacteriophages . Within the frog virus 3 (FV3) genome we have identified an open reading frame (ORF) of which the deduced amino acid sequence contains a motif characteristic of the INT family of integrases-recombinases . The ORF consists of 825 bp which codes for a protein of 275 amino acids with a predicted Mr of 29,945 . RNA transcribed from this ORF during virus infection was detected by Northern blot analysis and it is a delayed early message of approximately 1100 bases . The 5' and 3' ends of the putative FV3 integrase-recombinase transcript were mapped . The transcriptional start site is preceded by a presumptive TATA box, and a region of hyphenated dyad symmetry is present at the 3' end of the message . A protein with an Mr of approximately 30,500 was synthesized by a rabbit reticulocyte lysate programmed with capped runoff transcripts from the cloned gene, indicating that the ORF can be transcribed into a message coding for a viral protein . In the FV3 life cycle, DNA replication occurs in a large complex formed through the recombination of small viral DNA molecules . Thus, at this stage, DNA replication and recombination are interlinked . Resolution of concatameric DNA is required for the packaging of genomes into virus particles . The putative FV3 INT gene may be involved in one or more of these functions.

J Exp Med, 1992 Feb 1, 175(2), 537 - 43
Structural differences between the two human complement C4 isotypes affect the humoral immune response; Finco O et al.; An animal model has been used to address the question of the biological importance of the known structural difference between the two isotypes of human C4, i.e., C4A and C4B . Guinea pigs deficient in C4 were reconstituted transiently with either human C4A or C4B protein and immunized with the bacteriophage phi X174 . Results from this study showed that C4A-reconstituted animals made a secondary response, i.e., switch from IgM to IgG; whereas the C4B-reconstituted animals did not.

Appl Environ Microbiol, 1992 Feb, 58(2), 747 - 9
Filtration sizes of human immunodeficiency virus type 1 and surrogate viruses used to test barrier materials; Lytle CD et al.; Filters with well-defined holes were used to determine the effective diameters in buffer of human immunodeficiency virus type 1, herpes simplex virus type 1, and four bacteriophages (phi X174, T7, PRD1, and phi 6), which may serve as surrogate viruses for testing barrier materials . Bacteriophages phi 6 and PRD1 most closely model human immunodeficiency virus type 1 in filtration size.

J Mol Evol, 1992 Feb, 34(2), 141 - 52
Nucleotide sequence of the genome of the filamentous bacteriophage I2-2: module evolution of the filamentous phage genome; Stassen AP et al.; The nucleotide sequence of the circular single-stranded genome of the filamentous Escherichia coli phage I2-2 has been determined and compared with those of the filamentous E . coli phages Ff(M13, fl, or fd) and IKe . The I2-2 DNA sequence comprises 6744 nucleotides; 139 nucleotides less than that of the N- and I2-plasmid-specific phage IKe, and 337 (336) nucleotides more than that of the F-plasmid-specific phage Ff . Nucleotide sequence comparisons have indicated that I2-2, IKe, and Ff have a similar genetic organization, and that the genomes of I2-2 and IKe are evolutionarily more closely related than those of I2-2 and Ff . The studies have further demonstrated that the I2-2 genome is a composite replicon, composed of only two-thirds of the ancestral genome of IKe . Only a contiguous I2-2 DNA sequence of 4615 nucleotides encompassing not only the coat protein and phage assembly genes, but also the signal required for efficient phage morphogenesis, was found to be significantly homologous to sequences in the genomes of IKe and Ff . No homology was observed between the consecutive DNA sequence that contains the origins for viral and complementary strand replication and the replication genes . Although other explanations cannot be ruled out, our data strongly suggest that the ancestor filamentous phage genome of phages I2-2 and IKe has exchanged its replication module during evolution with that of another replicon, e.g., a plasmid that also replicates via the so-called rolling circle mechanism.

Epidemiol Infect, 1992 Feb, 108(1), 123 - 34
Removal of micro-organisms by filtration through unwoven cloth coated with a pyridinium-type polymer; Kawabata N et al.; Unwoven cloth coated with 32 mg/g of a copolymer of N-benzyl-4-vinyl-pyridinium chloride and styrene was found to be effective in removing micro-organisms from water . In experiments demonstrating removal of Escherichia coli by filtration through ten sheets of the unwoven cloth, the rate of removal was 99.99% at a filtration rate of 2.6 cm/h, and remained at 99% even at a high filtration rate of 300 cm/h and a low influent concentration of the bacterial cells such as 10(3) cells/ml . The rate of removal tended to increase with a decrease in the influent bacterial concentration . Seven other bacteria and two yeasts were effectively removed by filtration through the unwoven cloth . Filtration through the unwoven cloth was also effective in removing spores of fungi from water but was not very effective in removing bacteriophage T4 from aqueous solution.

Vet Microbiol, 1992 Feb, 30(2-3), 203 - 12
Characteristics and diffusion in the rabbit of a phage for Escherichia coli 0103 . Attempts to use this phage for therapy; Reynaud A et al.; A bacteriophage for Escherichia coli 0103 was isolated during a study on E . coli diarrhoea in intensive breeding units of rabbits . The phage had an isometric head and a short tail and resembled coliphage N4 (Podoviridae) . It had a very narrow host range and seemed to be specific for serogroup 0103, suggesting that it might be used for preliminary identification of E . coli strains of this serogroup instead of the usual slide agglutination . In view of its possible use as a therapeutic phage, we investigated its dissemination in rabbit organs after oral administration . The phage persisted in the spleen for at least 12 days . However, in vivo studies showed that this phage and a mixture of more virulent phages for E . coli 0103 were ineffective in preventing disease in rabbits inoculated with an enteropathogenic strain of E . coli 0103.

Mol Gen Genet, 1992 Feb, 231(3), 480 - 4
RexAB proteins of bacteriophage lambda enhance the effect of photolyase-dimer complexes on lacZ gene expression in Escherichia coli; Li BH et al.; Expression of the lacZ gene in Escherichia coli is inactivated by exposure to ultraviolet light (UV) . Inactivation is exceptionally effective when cells contain amplified levels of DNA photolyase (which forms complexes with pyrimidine dimers in the absence of light for actual photoreversal) and a lambda prophage . Without amplified photolyase, the lambda prophage or both, inactivation rates are similar and much lower . UV-inactivation of lacZ gene expression in the presence of both amplified photolyase and lambda is even more effective if lambda cI857 is used in place of the wildtype prophage but is wholly unexceptional if the prophage carries defects in the lambda genes rexA or rexB . When Rex AB proteins are provided by expression from a plasmid and the cell also contains amplified photolyase, exceptional inactivation rates again obtain; in fact inactivation is most effective under these conditions . The data are considered to reveal a role for Rex AB proteins, which mediate superinfection exclusion, in the exceptional inactivation of gene expression by photolyase bound to pyrimidine dimers in DNA . Photolyase-dimer complexes may mimic the structure of certain complexes that arise during phage development and thus influence Rex A and/or B proteins, thereby shutting down cell metabolism.

EMBO J, 1992 Feb, 11(2), 551 - 7
Random mutagenesis of CSF-1 receptor (FMS) reveals multiple sites for activating mutations within the extracellular domain; van Daalen Wetters T et al.; Retroviral vectors containing human FMS protooncogene cDNA were reconfigured to allow single-step excision and reinsertion of restriction fragments encoding short segments of the extracellular domain of the colony-stimulating factor 1 receptor (CSF-1R) . Fragments ligated into M13 bacteriophages were subjected to random chemical mutagenesis on both strands and recloned into the parental vector to create libraries of FMS genes containing mutations restricted to predefined target cassettes . Transfection of retroviral vector libraries into NIH/3T3 cells gave rise to transformed foci from which cellular DNA was amplified by the polymerase chain reaction (PCR), using primers flanking the mutagenized target sequences . Amplified fragments from individual primary transformants were recloned into intact FMS vector plasmids, and those with transforming activity were subjected to nucleotide sequence analysis . Alternatively, retroviruses rescued from transformed cells by superinfection with helper virus were used to generate secondary transformants containing unique copies of proviral DNA, whose sequences were determined after PCR amplification . Novel activating mutations were identified within sequences separating the third and fourth immunoglobulin-like loops, as well as within non-covalently stabilized loop 4 of the CSF-1R extracellular domain . Thus, FMS mutations able to convert human CSF-1R to an active oncoprotein are not restricted to those previously identified at codon 301 . This approach should be generally applicable for defining activating mutations in related growth factor receptors, including those for platelet-derived growth factor and Steel factor (KIT ligand), in which ligand-independent oncoprotein variants have not been identified.

J Bacteriol, 1992 Feb, 174(4), 1339 - 44
Effect of Escherichia coli nusG function on lambda N-mediated transcription antitermination; Sullivan SL et al.; The Escherichia coli Nus factors act in conjunction with the bacteriophage lambda N protein to suppress transcription termination on the lambda chromosome . NusA binds both N and RNA polymerase and may also interact with other Nus factors . To search for additional components of the N antitermination system, we isolated host revertants that restored N activity in nusA1 mutants . One revertant, nusG4, was mapped to the rif region of the E . coli chromosome and shown to represent a point mutation near the 3' end of the nusG gene . The nusG4 mutation also suppressed nusE71 but not nusASal, nusB5, nusC60 (rpoB60), or nusD026 (rho026) . However, nusG+ expressed from a multicopy plasmid suppressed nusD026 and related rho mutants for both lambda and phage T4 growth . These results suggest that NusG may act as a component of the N antitermination complex . In addition, the data imply a role for NusG in Rho-dependent termination.

J Bacteriol, 1992 Feb, 174(3), 850 - 6
Analysis of a mutation affecting the specificity domain for prohead binding of the bacteriophage lambda terminase; Sippy J et al.; Genetic studies have identified a specificity domain for prohead binding in the C-terminal 32 amino acids of gpA, the large subunit of bacteriophage lambda terminase (S . Frackman, D . A . Siegele, and M . Feiss, J . Mol . Biol . 180:283-300, 1984) . In the present work, an amber mutation, Aam42, in the fifth-to-last codon of the A gene was found to be lethal in nonsuppressing hosts . The mutation, expected to generate gpA lacking the last five amino acids, caused the production of a terminase that cut cos efficiently both in vivo and in vitro but was defective in DNA packaging . lambda Aam42 lysates contained unused proheads, consistent with a defect in prohead binding . Aam42 terminase was more strongly dependent than wild-type terminase on gpFI, the catalyst of prohead binding . Like wild-type terminase, Aam42 terminase did not cut cos in vivo when prohead assembly was blocked by a mutation in one of the genes encoding the prohead.

Mutat Res, 1992 Feb, 281(2), 81 - 7
Frameshift mutagenesis in bacteriophage T7; Pierce JC et al.; We have undertaken an initial characterization of frameshift mutagenesis in bacteriophage T7 and have identified a subset of very low reversion frameshift mutations in the T7 ligase gene (gene 1.3) . We used this information to construct bacteriophage T7 strains that contain one extra or one less base pair in gene 1.3 such that a frameshift event restores the reading frame of that gene . These events can be quantified and the frameshift mutation isolated within a localized region of the ligase gene . We have also identified a portion of the T7 ligase protein that will accept tracts of nonsense amino acids yet still give a ligase positive phenotype . This allows flexibility in the design of the target DNA sequence with which to study frameshift mutagenesis . These assays for frameshift mutagenesis performed in E . coli cells infected with the appropriate T7 strain, were used to measure the frequency of both plus and minus frameshifts in vivo.

J Neurocytol, 1992 Feb, 21(2), 129 - 36
Detection of migrated allogeneic oligodendrocytes throughout the central nervous system of the galactocerebrosidase-deficient twitcher mouse; Huppes W et al.; Galactocerebrosidase-deficient oligodendrocytes of 'twitcher' (twi/twi) mice degenerate prematurely . Transplantation of normal bone marrow cells has been shown to alleviate symptoms and to prolong survival time . However, characteristic ataxia ('twitching') is not cured . In an attempt to improve further the condition of twitcher mice, allogeneic foetal liver cells were transplanted as a source of normal haemopoietic stem cells and supplemented with intracerebral transplantation of foetal brain cells . A reliable method was developed to detect donor-type cells in brain tissue . Bacteriophage lambda transgenic foetal mice were used as donors of both foetal liver and brain cells . Integrated copies of lambda DNA in donor cells were detected by in situ hybridization with biotinylated probes, which were then stained using streptavidin alkaline phosphatase . This technique was combined with immunohistochemistry to distinguish donor-type oligodendrocytes from macrophages . Immunoperoxidase staining with an antiserum to carbonic anhydrase-II produced dark perikarya of oligodendrocytes . The results demonstrated that local foetal brain cell grafts resulted in a wide dissemination of donor-type oligodendrocytes throughout the twitcher brain . The addition of a foetal brain cell graft to haemopoietic cell transplantation resulted in significantly prolonged survival of twitcher mice.

Comput Appl Biosci, 1992 Feb, 8(1), 29 - 34
GLOBIC: a very fast microcomputer program for fingerprinting, characterization and comparison of long nucleotide sequences; Mrazek J et al.; This paper describes the program GLOBIC, which compares, characterizes and fingerprints even 0.1 Mbase sequences in a few minutes with the aid of an IBM-AT microcomputer . Instead of the nucleotide sequences themselves, GLOBIC compares the local nucleotide or short oligonucleotide compositions . GLOBIC presents two-dimensional maps of contour lines depicting the similarity of two different sequences, a sequence compared to itself, to its complementary sequence or to a random sequence . A vocabulary is presented to translate the typical patterns appearing in the two-dimensional maps into their meanings as relationships between the compared sequences . The application of GLOBIC is demonstrated using several examples from the genomic nucleotide sequences of bacteriophage T7, adenovirus type-2 and Epstein-Barr virus.

Gene, 1992 Feb 1, 111(1), 21 - 6
TnblaM: a transposon for directly tagging bacterial genes encoding cell envelope and secreted proteins; Tadayyon M et al.; A transposon, TnblaM, designed for the direct selection of bacterial mutants with insertions in genes encoding cell envelope and secreted proteins, was constructed and subcloned into plasmid and bacteriophage lambda delivery vectors . TnblaM is a spectinomycin-resistant derivative of Tn5 with an unexpressed open reading frame encoding mature beta-lactamase (BlaM) at its left end . Therefore, when it inserts into genes in the correct orientation and reading frame, gene fusions encoding hybrid proteins are generated . By introducing TnblaM into bacterial cells and selecting ampicillin-resistant (ApR) colonies, the subset of isolates producing extracytoplasmic BlaM, and hence containing TnblaM inserted in genes encoding secreted proteins and cell envelope proteins, can be directly selected . TnblaM, like TnphoA, can therefore be used to preferentially mutagenise genes encoding extracytoplasmic proteins, but it has the advantage over TnphoA that the desired mutants can be isolated by direct selection (as ApR colonies) rather than by phenotypic screening . Isolates in which TnblaM occupies sites in the chromosome from which it can transpose at high frequency are readily identifiable, and constitute TnblaM donors, with which to simply and efficiently generate rare types of insertion mutants . Moreover, the ApR selection that is used with TnblaM can be fine-tuned to obtain blaM fusions to poorly or well-expressed genes.

FASEB J, 1992 Feb 1, 6(3), 871 - 8
Protein-protein interactions at a DNA replication fork: bacteriophage T4 as a model; Nossal NG; The DNA replication system of bacteriophage T4 serves as a relatively simple model for the types of reactions and protein-protein interactions needed to carry out and coordinate the synthesis of the leading and lagging strands of a DNA replication fork . At least 10 phage-encoded proteins are required for this synthesis: T4 DNA polymerase, the genes 44/62 and 45 polymerase accessory proteins, gene 32 single-stranded DNA binding protein, the genes 61, 41, and 59 primase-helicase, RNase H, and DNA ligase . Assembly of the polymerase and the accessory proteins on the primed template is a stepwise process that requires ATP hydrolysis and is strongly stimulated by 32 protein . The 41 protein helicase is essential to unwind the duplex ahead of polymerase on the leading strand, and to interact with the 61 protein to synthesize the RNA primers that initiate each discontinuous fragment on the lagging strand . An interaction between the 44/62 and 45 polymerase accessory proteins and the primase-helicase is required for primer synthesis on 32 protein-covered DNA . Thus it is possible that the signal for the initiation of a new fragment by the primase-helicase is the release of the polymerase accessory proteins from the completed adjacent fragment.

Virology, 1992 Feb, 186(2), 452 - 62
Molecular cloning and expression of the bacteriophage T7 0.7(protein kinase) gene; Michalewicz J et al.; The bacteriophage T7 0.7 gene encodes a protein which supports viral reproduction under specific suboptimal growth conditions . The 0.7 protein (gp0.7) shuts off host RNA polymerase-catalyzed transcription and also expresses a serine/threonine-specific, cAMP-independent protein kinase (PK) activity . To determine the role of the gp0.7 PK in viral reproduction, the 0.7 gene of the T7(JS78) mutant phage--whose gp0.7 expresses only the PK activity--was cloned in the plasmid expression vector pET-11a . Cells containing the recombinant plasmid were viable, and upon IPTG induction produced a 30-kDa polypeptide, similar in size to the gp0.7-related polypeptide seen in T7(JS78)-infected cells . Extracts of cells containing this polypeptide can phosphorylate the exogenous substrate lysozyme . Expression of plasmid-encoded gp0.7(JS78) in vivo results in phosphorylation of the same proteins which are phosphorylated in T7(JS78)-infected cells; moreover, the plasmid-encoded gp0.7(JS78) is itself phosphorylated . The JS78 mutation changes Gln243 in gp0.7 to an amber codon, which explains the production of the truncated, 30-kDa gp0.7-related polypeptide, and implicates the 11-kDa C-terminal domain in host transcription shut-off . The T7(A23) 0.7 point mutant fails to express PK activity in infected cells . However, the truncated T7(A23)-related polypeptide, expressed from a plasmid, exhibits PK activity in vivo and in vitro, but with an altered specificity . Thus, the A23 mutation, which changes Asp100 to Asn, may identify a substrate recognition determinant.

Science, 1992 Jan 31, 255(5044), 589 - 92
Experimental phylogenetics: generation of a known phylogeny; Hillis DM et al.; Although methods of phylogenetic estimation are used routinely in comparative biology, direct tests of these methods are hampered by the lack of known phylogenies . Here a system based on serial propagation of bacteriophage T7 in the presence of a mutagen was used to create the first completely known phylogeny . Restriction-site maps of the terminal lineages were used to infer the evolutionary history of the experimental lines for comparison to the known history and actual ancestors . The five methods used to reconstruct branching pattern all predicted the correct topology but varied in their predictions of branch lengths; one method also predicts ancestral restriction maps and was found to be greater than 98 percent accurate.

Biochemistry, 1992 Jan 28, 31(3), 765 - 74
Zn(II) coordination domain mutants of T4 gene 32 protein; Giedroc DP et al.; Gene 32 protein (g32P), the replication accessory single-stranded nucleic acid binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol of protein . Zinc coordination provides structural stability to the DNA-binding core domain of the molecule, termed g32P-(A+B) (residues 22-253) . Optical absorption studies with the Co(II)-substituted protein and 113Cd NMR spectroscopy of 113Cd(II)-substituted g32P-(A+B) show that the metal coordination sphere in g32P is characterized by approximately tetrahedral ligand symmetry and ligation by the Cys-S- atoms of Cys77, Cys87, and Cys90 . These studies predicted the involvement of a fourth protein-derived non-thiol ligand to complete the tetrahedral complex, postulated to be His81 on the basis of primary structure prediction and modeling {Giedroc, D.P., Johnson, B.A., Armitage, I.M., & Coleman, J.E . (1989) Biochemistry 28, 2410-2418} . To test this model, we have employed site-directed mutagenesis to substitute each of the two histidine residues in g32P (His64 and His81), accompanied by purification and structural characterization of these single-site mutant proteins . We show that g32P's containing any of three substitutions at residue 64 (H64Q, H64N, and H64L) are isolated from Escherichia coli in a Zn(II)-free form {less than or equal to 0.03 g.atom Zn(II)} . All derivatives show extremely weak affinity for the ssDNA homopolymer poly(dT) . All are characterized by a far-UV-CD spectrum reduced in negative intensity relative to the wild-type protein . These structural features parallel those found for the known metal ligand mutant Cys87----Ser87 (C87S) g32P . In contrast, g32P-(A+B) containing a substitution of His81 with glutamine (H81Q), alanine (H81A) or cysteine (H81C), contains stoichiometric Zn(II) as isolated and binds to polynucleotides with an affinity comparable to the wild-type g32P-(A+B) . Spin-echo 1H NMR spectra recorded for wild-type and H81Q g32P-(A+B) as a function of pH allow the assignment of His81 ring proteins to delta = 6.81 and 6.57 ppm, respectively, at pH 7.8, corresponding to the C and D histidyl protons of 1H-His-g32P-(A+B) {Pan, T., Giedroc, D.P., & Coleman, J.E . (1989) Biochemistry 28, 8828-8832} . These resonances shift downfield as the pH is reduced from 7.8 to 6.6 without metal dissociation, a result incompatible with His81 donating a ligand to the Zn(II) in wild-type g32P . Likewise, Cys81 in Zn(II) H81C g32P is readily reactive with 5,5'-dithiobis(2-nitrobenzoic acid), unlike metal ligands Cys77, Cys87, and Cys90.(ABSTRACT TRUNCATED AT 400 WORDS)

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