Eur J Biochem, 1990 Apr 30, 189(2), 307 - 12 Bacteriophage-associated glycan hydrolases specific for Escherichia coli capsular serotype K12; Altmann F et al.; Four bacteriophages were identified, which carry glycan hydrolases specific for the Escherichia coli K12 capsular polysaccharide . All these glycanases catalyze the hydrolysis of the alpha-L-rhamnosyl-1,5-beta-3-deoxy-D-manno-2-octulosonic acid linkage as demonstrated with a special thiobarbituric acid assay procedure, which discriminates between the C5 substituted and unsubstituted 3-deoxy-D-manno-2-octulosonic acid (dOclA) . This assay, together with gel filtration, 1H-NMR and 13C-NMR spectroscopy showed that depolymerization led to the dimer of the K12 repeating unit, (,5-beta-dOcl1Ap-2,3-alpha-LRhap-1,2-alpha LRhap-1,)2, as the primary degradation product . The phages (phi 12-W, phi 12-S, phi 82-W1, phi 82-W2) were tested for their ability to infect Escherichia coli strains Su65-42 (O4:K12:H-) and CDC63-57 {O139:K82(12):H1} . phi 12-W and phi 12-S, respectively, infected strain Su65-42 only, phi 82-W2 CDC63-57 only, and phi 82-W1 both bacterial strains . These distinct host specificities cannot be explained by differences in the action of the glycanases, which depolymerize the capsules of both strains. Nucleic Acids Res, 1990 Apr 25, 18 Suppl, 2549 - 87 Compilation of DNA sequences of Escherichia coli (update 1990); 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 second listing replacing and increasing the former listing roughly by one third . After deletion of all detected overlaps a total of 1 248 696 individual bp is found to be determined till the beginning of 1990 . This corresponds to a total of 26.46% of the entire E . coli chromosome consisting of about 4,720 kbp . This number may actually be higher by some extra 2% derived from the sequence of lysogenic bacteriophage lambda and various insertion sequences . This compilation is now available in machine readable form from the EMBL data library. Nucleic Acids Res, 1990 Apr 25, 18(8), 2061 - 4 FseI, a new type II restriction endonuclease that recognizes the octanucleotide sequence 5' GGCCGGCC 3'; Nelson JM et al.; A Type II restriction endonuclease, designated FseI, has been partially purified from a Frankia species (NRRL 18528) . This enzyme cleaves Adenovirus 2 DNA at three sites, but does not cleave the DNAs from bacteriophages lambda, T7, and phi X174, the animal virus SV40, pUC18 and pBR322 . FseI recognizes the octanucleotide sequence 5' GGCCGG decreases CC 3' and cleaves as indicated by the arrow . The frequency of occurrence of FseI sites within sequenced regions of the human genome is similar to that for NotI sites. Biochemistry, 1990 Apr 24, 29(16), 3828 - 34 Identification of trapped and boundary lipid binding sites in M13 coat protein/lipid complexes by deuterium NMR spectroscopy; Van Gorkom LC et al.; The major coat protein of M13 bacteriophage has been incorporated into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, deuterated in the trimethyl segments of the choline headgroup (DMPC-d9) . Two-component deuterium and phosphorus-31 NMR spectra have been observed from bilayer complexes containing the coat protein, indicating slow exchange (on the deuterium quadrupole anisotropy and phosphorus-31 chemical shift averaging time scales) of lipid molecules of less than 10(3) Hz between two motionally distinct environments in the complexes . The fraction of the isotropic spectral component increases with increasing M13 protein concentration, and this component is attributed to lipid headgroups, which are disordered relative to their order in protein-free bilayers . The activation energy of the fast local motions of the trimethyl groups of the choline residue in the headgroup decreases from 23 kJ mol-1 in the pure lipid bilayers to 20 kJ mol-1 for the protein-associated lipid headgroups . The chemical exchange rate of lipid molecules between the two motionally distinct environments has been estimated to be 20-50 Hz by steady-state line-shape simulations of the deuterium spectra of DMPC-d9/M13 coat protein complexes using exchange-coupled modified Bloch equations . The off-rate was, as expected from one-to-one exchange, independent of the L/P ratio; tau off -1 = 0.23 kHz . It is suggested that the protein-associated lipid may be trapped between closely packed parallel aggregates of M13 coat protein and that the high local concentration of protein in a one-dimensional arrangement in lipid bilayers may be required for the fast reassembly of phage particles before release from an infected cell. Biochemistry, 1990 Apr 24, 29(16), 3817 - 21 In vitro and in vivo activities of T4 endonuclease V mutants altered in the C-terminal aromatic region; Ishida M et al.; Genes encoding mutants of the thymine photodimer repair enzyme from bacteriophage T4 (T4 endonuclease V) having an amino acid substitution (T127M, W128A, W128S, Y129A, K130L, Y131A, Y132A) were constructed by use of a previously obtained synthetic gene and expressed in Escherichia coli under the control of the E . coli tryptophan promoter . An in vitro assay of partially fractionated mutant proteins for glycosylase activity was performed with chemically synthesized substrates containing a thymine photodimer . T127M and K130L showed almost the same activity as the wild-type protein . Although W128S, Y131A, and Y132A were slightly active, W128A and Y129A lost activity . The results indicated that the aromatic amino acids around position 130 may be important for the glycosylase activity . Mutant T127M was purified, and the Km value was found to be of the same order as that of the wild type (10(-8) M) . In vivo activities for all mutants were characterized with UV-sensitive E . coli . The results showed that substitution of Thr-127 with Met or Lys-130 with Leu did not have an effect on the survival of the bacteria but substitution of aromatic amino acids (128-132) had various effects on survival. J Mol Biol, 1990 Apr 20, 212(4), 723 - 35 Cleavage specificity of bacteriophage T4 endonuclease VII and bacteriophage T7 endonuclease I on synthetic branch migratable Holliday junctions; Picksley SM et al.; Holliday junctions are intermediate structures that are formed and resolved during the process of genetic recombination . To investigate the interaction of junction-resolving nucleases with synthetic Holliday junctions that contain homologous arm sequences, we constructed substrates in which the junction point was free to branch migrate through 26 base-pairs of homology . In the absence of divalent cations, we found that both phage T4 endonuclease VII and phage T7 endonuclease I bound the synthetic junctions to form specific protein-DNA complexes . Such complexes were not observed in the presence of Mg2+, since the Holliday junctions were resolved by the introduction of symmetrical cuts in strands of like polarity . The major sites of cleavage were identified and found to occur within the boundaries of homology . T4 endonuclease VII showed a cleavage preference for the 3' side of thymine bases, whereas T7 endonuclease I preferentially cut the DNA between two pyrimidine residues . However, cleavage was not observed at all the available sites, indicating that in addition to their structural requirements, the endonucleases show strong site preferences. J Biol Chem, 1990 Apr 15, 265(11), 6427 - 35 High level bacterial expression of uteroglobin, a dimeric eukaryotic protein with two interchain disulfide bridges, in its natural quaternary structure; Miele L et al.; Bacterial expression of eukaryotic proteins is a tool of ever-increasing importance in biochemistry and molecular biology . However, the majority of the recombinant eukaryotic proteins that have been expressed in bacteria are produced as fusion proteins and not in their native conformation . In particular, correct formation of quaternary structures by recombinant proteins in bacterial hosts has been reported very rarely . To our knowledge, correct intracellular formation of multimeric structures containing more than one interchain disulfide bridge has not been reported so far . We have constructed three plasmids which are able to direct expression of recombinant rabbit uteroglobin, a homodimeric protein with two interchain disulfide bridges, in Escherichia coli . Among these, the plasmid pLE103-1, in which the expression of recombinant uteroglobin is controlled by a bacteriophage T7 late promoter, is by far the most efficient . With pLE103-1, recombinant uteroglobin production reached about 10% of total bacterial soluble proteins . This protein accumulated in bacterial cells in dimeric form, as it is naturally found in the rabbit uterus . Recombinant uteroglobin was purified to near-homogeneity and its NH2-terminal amino acid sequence was confirmed to be identical to that of its natural counterpart, except for 2 Ala residues the codons for which were added during the plasmid construction . This protein was found to be as active a phospholipase A2 inhibitor as natural uteroglobin on a molar basis . To our knowledge, this is the first report of high level bacterial expression of a full length eukaryotic homodimeric protein with two interchain disulfide bridges in its natural, biologically active form . The plasmid pLE103-1 may be useful to explore structure-function relationships of rabbit uteroglobin . In addition, this plasmid may be useful in obtaining high level bacterial expression of other eukaryotic proteins with quaternary structure, as well as for other general applications requiring efficient bacterial expression of cDNAs. Nucleic Acids Res, 1990 Apr 11, 18(7), 1719 - 23 The initiation of translation in E . coli: apparent base pairing between the 16srRNA and downstream sequences of the mRNA; Sprengart ML et al.; Bacteriophage T7's gene 0.3, coding for an antirestriction protein, possesses one of the strongest translation initiation regions (TIR) in E . coli . It was isolated on DNA fragments of differing length and cloned upstream of the mouse dihydrofolate reductase gene in an expression vector to control the translation of this gene's sequence . The TIR's efficiency was highly dependent on nucleotides +15 to +26 downstream of the gene's AUG . This sequence is complementary to nucleotides 1471-1482 of the 16srRNA . Similar sequences complementary to this rRNA region are present in other efficient TIRs of the E . coli genome and those of its bacteriophages . There seems to be a correlation between this sequence homology and the efficiency of the initiation signals . We propose that this region specifies a stimulatory interaction between the mRNA and 16srRNA besides the Shine-Dalgarno interaction during the translation initiation step. Nature, 1990 Apr 5, 344(6266), 559 - 62 Specific interaction of the murine transcription termination factor TTF I with class-I RNA polymerases; Kuhn A et al.; The 18-base-pair sequence element AGGTCGACCAGTACTCCG (the Sal box) signals termination of mouse ribosomal gene transcription . This sequence is recognized by a sequence-specific DNA-binding protein, TTF I, which mediates the termination of transcription by RNA polymerase I (pol I) . Subsequently, the ends of the primary transcripts are trimmed by 10 nucleotides in a sequence-dependent 3'-terminal processing reaction . We have now investigated whether TTF I bound to its target sequence will block elongation by any RNA polymerase by steric hindrance, or whether it is specific for elongation by pol I . The results demonstrate that TTF I directs transcription termination with RNA polymerase I from species as divergent as mouse and yeast, but fails to affect elongation by heterologous polymerases (eukaryotic RNA polymerases II and III, Escherichia coli or bacteriophage T3 RNA polymerase) . By contrast, purified lac repressor bound to its operator sequence stops elongation by both RNA polymerase I and II. J Biol Chem, 1990 Apr 5, 265(10), 5684 - 9 Translational repression by bacteriophage MS2 coat protein expressed from a plasmid . A system for genetic analysis of a protein-RNA interaction; Peabody DS; The coat protein of bacteriophage MS2 is a translational repressor . It inhibits the synthesis of the viral replicase by binding a specific RNA structure that contains the replicase translation initiation region . In order to begin a genetic dissection of the repressor activity of coat protein, a two-plasmid system has been constructed that expresses coat protein and a replicase-beta-galactosidase fusion protein from different, compatible plasmids containing different antibiotic-resistant determinants . The coat protein expressed from the first plasmid (pCT1) represses synthesis of a replicase-beta-galactosidase fusion protein encoded on the other plasmid (pRZ5) . Mutations in the translational operator or in coat protein result in constitutive synthesis of the enzyme . This permits the straightforward isolation of mutations in the coat sequence that affect repressor function . Because of the potential importance of cysteine residues for RNA binding, mutations were constructed that substitute serines for the cysteine residues normally present at positions 46 and 101 . Both of these mutations result in translational repressor defects . Chromatographic and electron microscopic analyses indicate that the plasmid-encoded wild-type coat protein forms capsids in vivo . The ability of the mutants to adopt and/or maintain the appropriate conformation was assayed by comparing them to the wild-type protein for their ability to form capsids . Both mutants exhibited evidence of improper folding and/or instability as indicated by their aberrant elution behavior on a column of Sepharose CL-4B . Methods were developed for the rapid purification of plasmid-encoded coat protein, facilitating future biochemical analyses of mutant coat proteins. J Biol Chem, 1990 Apr 5, 265(10), 5434 - 9 A critical arginine in the large subunit of ribulose bisphosphate carboxylase/oxygenase identified by site-directed mutagenesis; Haining RL et al.; Rapid inactivation by phenylglyoxal of ribulose bisphosphate carboxylase/oxygenase (ribulose-P2 carboxylase) from the cyanobacterium Anacystis nidulans suggests the presence of an essential arginine, the modification of which is reduced in the presence of the substrate ribulose bisphosphate . Arginine 292 in the large subunit of ribulose-P2 carboxylase from A . nidulans was chosen for site-directed mutagenesis studies on the basis of the complete conservation of this residue in corresponding sequences of ribulose-P2 carboxylase from divergent organisms . Arginine 292 was changed to leucine and to lysine by directed mutagenesis using suitable plasmids and the bacteriophage M13 . Both substitutions resulted in the production of purifiable holoenzyme with no activity after expression in Escherichia coli. Genome, 1990 Apr, 33(2), 164 - 9 Genomic arrangement of repeated PS700 elements in the nematode Panagrellus silusiae; Retterath MA et al.; When genomic DNA from the free-living nematode Panagrellus silusiae is digested with the restriction endonuclease BamHI and separated by electrophoresis, a band in the 700 base pair size range is evident after ethidium bromide staining . One of the 0.7-kilobase fragments (PS700-1) was characterized and found to be a member of a moderately repetitive DNA family (T . Warren and J.J . Pasternak . 1988 . Nucleic Acids Res . 16: 10,833-10,847) . In the current study, DNA sequence analyses of three independently isolated copies of the PS700 DNA family showed the same nucleotide sequence and greater than 98% similarity to PS700-1 . Four EMBL-4 bacteriophage clones were isolated from a Panagrellus genomic DNA library with PS700-1 as the probe and were analyzed by restriction endonuclease site mapping and Southern blot DNA hybridization . These clones contain 31 copies of the PS700 DNA family . In each case, the units are arranged in head-to-tail arrays . One of the EMBL-4 clones contains copies of a novel variant of the PS700 elements . The maintenance of both nucleotide sequence and restriction endonuclease restriction site homogeneity among members of the dispersed PS700 DNA family may denote a functional role for these sequences. Genomics, 1990 Apr, 6(4), 626 - 34 Development of an automated procedure for fluorescent DNA sequencing; Wilson RK et al.; We describe here the development of a procedure for complete automation of the dideoxynucleotide DNA sequencing chemistry using fluorescent dye-labeled oligonucleotide primers . This procedure combines rapid preparation of template DNA using a modification of the polymerase chain reaction, automation of the DNA sequencing reactions using a robotic laboratory workstation, and subsequent analysis of the fluorescent-labeled reaction products on a commercial automated fluorescent sequencer . Using this procedure, we were able to produce sufficient quantities of template DNA directly from bacterial colonies or bacteriophage plaques, perform the DNA sequencing reactions on these templates, and load the reaction products on the fluorescent DNA sequencer in a single work day . This scheme for automation of the fluorescent DNA sequencing method allows the fluorescent sequencer to be run at its full capacity every day and eliminates much of the labor required to obtain a high level of data output . Currently, we are able to perform and analyze 16 fluorescent-labeled reactions every day, with an average output of over 7000 bp per sequencer run. J Electron Microsc Tech, 1990 Apr, 14(4), 313 - 23 Small colloidal gold conjugated to Fab fragments or to immunoglobulin G as high-resolution labels for electron microscopy: a technical overview; Baschong W et al.; Fab-colloidal gold labelling in conjunction with negative staining and high-resolution electron microscopy was used for targeting single protein units in regular arrays . These were bacteriophage T4 polyheads with Fab-Au2.5, and a specific antibody binding site on the haemagglutinin polypeptide of influenza virus with Fab-Au3, Fab-Au2.5, and Fab-Au1-2 . For the latter, IgG-Au3 was also used . Experimental details are summarized to provide generally applicable methods for the preparation of small gold colloids Fab-Au and of labelling . The putative mechanism of protein-gold complex formation and adsorption to preferred sites on Fab and IgG, most probably to sulphur-rich regions, is discussed . The influence of pH during complex formation was found to be of minor importance in the samples investigated . Reported experimental details and our own experiences suggest that the importance of a protein's pI relative to its optimum gold complexing pH critically depends on the nature of the protein in question rather than being of general importance for protein-gold complex stability. Virology, 1990 Apr, 175(2), 500 - 7 The sim gene of Escherichia coli phage P1: nucleotide sequence and purification of the processed protein; Maillou J et al.; The sim gene of bacteriophage P1 causes exclusion of a superinfecting P1 phage . We determined the nucleotide sequence of a 1.9-kb DNA fragment that, in plasmids, causes Sim phenotype . There are two open reading frames within this region for proteins of 82 and 259 amino acids . A 1.3-kb fragment containing the larger open reading frame was inserted into an expression vector . Induced cells carrying the hybrid plasmid, termed pBD5, were not infected by phage P1 and produced a 24-kDa protein and, to a smaller extent, a 25-kDa protein . The 24-kDa protein was purified . Comparison of its amino-terminal amino acid sequence with the nucleotide sequence indicated that it is processed from a precursor protein by removal of a hydrophobic leader peptide of 20 amino acids . In vivo processing depends on secA gene function and is necessary for Sim interference with P1 infection . The data are discussed with respect to the function of the sim gene in superinfection exclusion. Anal Biochem, 1990 Apr, 186(1), 121 - 6 Labeled antigen capture assay: a method for detecting monoclonal antibodies to cloned gene products; Urban RG et al.; We report here a relatively easy and highly sensitive assay for detecting monoclonal antibodies to the product of virtually any cloned gene . The protocol, termed labeled antigen capture assay (LACA), is a solid-phase type radioimmunoassay which uses a bacteriophage T7 expression system to generate exclusively radiolabeled antigen . Thus, to generate radiolabeled antigen for screening, the gene encoding the protein of interest need only be subcloned downstream of a T7 promoter, and the new construct transformed into an Escherichia coli strain harboring a compatible plasmid which encodes a thermal inducible copy of the T7 RNA polymerase . Expression of the T7-promoted gene in the presence of rifampicin and {35S}cysteine (or methionine) yields labeled antigen, which is then "captured" by specific monoclonal antibody and detected by autoradiography . Our results indicate that as little as 30 ng of specific monoclonal antibody can be detected using the LACA protocol . The protocol is applicable to the product of any cloned gene but is particularly useful in the case where the biochemical properties of the gene product of interest are unavailable . In this report we use the LACA protocol to screen a hybridoma library for monoclonal antibodies to the STb heat-stable enterotoxin (STb) of E . coli . Mice were immunized with a genetically constructed, affinity-purified Protein A-STb hybrid protein, and following spleen cell fusion and HAT selection, hybridomas were screened by the described LACA protocol for production of STb-specific monoclonal antibody . Of over 1500 hybridomas tested 138 were positive, by primary LACA screening, for STb-specific IgG monoclonal antibodies. Antimicrob Agents Chemother, 1990 Apr, 34(4), 628 - 31 Influence of beta-lactam antibiotics on serum resistance of K1-positive blood culture isolates of Escherichia coli; Suerbaum S et al.; The K1-positive strains of Escherichia coli are a group with considerable clinical importance, serum resistance being a common virulence factor of these strains . In the present paper, the influences of cephaloridine, imipenem, and ceftazidime on the serum resistance of eight serum-resistant K1-positive E . coli blood culture isolates with smooth-type lipopolysaccharide were studied . All strains were rendered more serum sensitive by treatment with subinhibitory concentrations of antibiotics . The amount of the reduction of serum resistance was dependent on the concentration of the antibiotic . Amounts of K1 produced under the influence of the antibiotics were measured and were found to be reduced for almost all strains tested . To further test the hypothesis that antibiotic-induced reduction of serum resistance is mediated by inhibition of K1 expression, isogenic mutants of one strain were produced by selection for resistance against infection with K1-specific bacteriophages . These mutants were found to be highly serum sensitive . We conclude from this study that beta-lactam antibiotics can render K1-positive serum-resistant strains of E . coli highly serum sensitive and that this effect is mediated by inhibition of K1 expression. Virology, 1990 Apr, 175(2), 525 - 34 Protein kinase of bacteriophage T7 induces the phosphorylation of only a small number of proteins in the infected cell; Robertson ES et al.; Bacteriophage T7 expresses a serine/threonine-specific, cAMP-independent protein kinase activity encoded by the early gene 0.7 . The phosphoproteins specifically resulting from gp0.7 protein kinase expression in T7-infected Escherichia coli have been examined by one-dimensional, SDS-polyacrylamide gel electrophoresis . Only seven major, stable phosphoproteins dependent on gp0.7 protein kinase expression are observed . Two of the gp0.7 protein kinase-specific phosphoproteins observed have been previously identified: the beta' subunit of RNA polymerase and the RNA processing enzyme RNase III . The gp0.7-catalyzed protein phosphorylation activity appears at 9-11 min postinfection at 30 degrees . The new phosphoproteins have a metabolic stability comparable to that of uninfected cell phosphoproteins . T7 protein kinase expression causes the phosphorylation of the same, limited set of proteins in B, C, or K strains of E . coli . Expression of the T3 and BA14 phage protein kinase activities also produces the same phosphoproteins. Proc Natl Acad Sci U S A, 1990 Apr, 87(8), 3166 - 9 Improved genetic selection for screening bacteriophage libraries by homologous recombination in vivo; Kurnit DM et al.; Three major difficulties have hindered the general application of in vivo recombination techniques to library screening: (i) the original selection could not be applied to libraries prepared in phage vectors lacking amber mutations, (ii) nonirradiated packaging extracts gave high backgrounds even when amber mutated vectors were used, and (iii) most red- vectors lacked rap, a recently discovered phage gene promoting phage-plasmid recombination . Here we describe a selection scheme for phage bearing suppressor tRNA plasmids, which relies upon an Escherichia coli host bearing an amber mutation in the dnaB gene . The selection is tight enough to allow library screening by recombination, is applicable to almost every phage vector in common use, and overcomes the background associated with nonirradiated packaging extracts . We also describe an ancillary plasmid that supplies the rap gene function in trans, permitting the recombination level to be raised fruitfully in phage libraries lacking endogenous rap . Finally, we demonstrate simple procedures for preparing and detecting phages that have lost integrated suppressor tRNA plasmids by homologous recombination. Proc Natl Acad Sci U S A, 1990 Apr, 87(7), 2690 - 4 Three Escherichia coli heat shock proteins are required for P1 plasmid DNA replication: formation of an active complex between E . coli DnaJ protein and the P1 initiator protein; Wickner SH; DNA containing the plasmid origin of bacteriophage P1 is replicated in vitro by a protein fraction prepared from uninfected Escherichia coli supplemented with purified P1 RepA protein . It has previously been shown that the reaction required the E . coli DnaA initiator protein, the DnaB helicase, DnaC protein, RNA polymerase, and DNA gyrase . I show here that three E . coli heat shock proteins, DnaJ, DnaK, and GrpE, are directly involved in P1 plasmid replication . Purified DnaJ, DnaK, and GrpE proteins were required to stimulate P1 plasmid ori DNA-dependent replication in in vitro complementation assays in which the host protein fractions were prepared from cells mutated in the corresponding gene . I have also found that the DnaJ and RepA proteins form a complex . This complex exists in crude cell extracts and can be isolated as a molecular species of about 160,000 Da containing one dimer of DnaJ protein and one dimer of RepA . The complex can also be reconstituted by mixing purified DnaJ and RepA proteins . These results imply that the DnaJ-RepA complex, DnaK, and GrpE are directly involved in P1 plasmid replication. J Bacteriol, 1990 Apr, 172(4), 2105 - 12 SOS-dependent replication past a single trans-syn T-T cyclobutane dimer gives a different mutation spectrum and increased error rate compared with replication past this lesion in uninduced cells; Banerjee SK et al.; We have transfected SOS-induced and uninduced cells of a uvrA6 strain of Escherichia coli with single-stranded M13mp7-based vectors that carried a single trans-syn T-T cyclobutane dimer at a unique site . Unlike constructs carrying the cis-syn isomer of this lesion, these vectors could be replicated with modest efficiency (14%) in the absence of SOS induction and therefore provided an opportunity to measure directly the influence of such induction on error rate and mutation spectrum . We found that translesion synthesis in the absence of SOS induction was remarkably accurate; only 4% of the replicated bacteriophage contained mutations, which were exclusively targeted single T deletions . In SOS-induced cells, error frequency increased to 11% and the resulting mutations included targeted substitutions and near-targeted single base additions, as well as the T deletions . Replication efficiency was 29% in these conditions . SOS induction therefore leads not only to an enhanced capacity to replicate damaged DNA but also to a marked change in mutation frequency and spectrum. J Bacteriol, 1990 Apr, 172(4), 2055 - 64 Identification and characterization of a new Escherichia coli gene that is a dosage-dependent suppressor of a dnaK deletion mutation; Kang PJ et al.; We report the isolation and characterization of a previously unidentified Escherichia coli gene that suppresses the temperature-sensitive growth and filamentation of a dnaK deletion mutant strain . Introduction of a multicopy plasmid carrying this wild-type gene into a dnaK deletion mutant strain rescued the temperature-sensitive growth of the dnaK deletion mutant strain at 40.5 degrees C and the filamentation, fully at 37 degrees C and partially at 40.5 degrees C . However, the inability of dnaK mutant cells to support bacteriophage lambda growth was not suppressed . This gene was also able to suppress the temperature-sensitive growth of a grpE280 mutant strain at 41 degrees C . Filamentation of the grpE280 mutant strain was suppressed at 37 degrees C but not at 41 degrees C . The dnaK suppressor gene, designated dksA, maps near the mrcB gene (3.7 min on the E . coli chromosome) . DNA sequence analysis and in vivo experiments showed that dksA encodes a 17,500-Mr polypeptide . Gene disruption experiments indicated that dksA is not an essential gene. J Bacteriol, 1990 Apr, 172(4), 1889 - 98 Nucleotide sequence and transcription of the right early region of bacteriophage PRD1; Gerendasy D et al.; We have sequenced the rightmost 1,700 base pairs of bacteriophage PRD1 . This region encompasses the right early region and completes the sequence of all PRD1 early functions . We have also mapped the 5' initiation site of right early transcripts in vivo and in vitro . This has allowed us to assign gene XII to an open reading frame and suggests that another open reading frame may also be expressed . Gene XII, which has been implicated in the replication process and the regulation of gene expression, is predicted to encode a protein with a molecular mass of 16.7 kilodaltons . Data base searches have revealed no significant homology between the product of this gene and other proteins . Transcription mapping studies have revealed that right early transcripts elongate from right to left and have enabled us to identify the right early promoter . This promoter behaves identically in vivo and in vitro . We also demonstrate that this promoter directs the transcription of two RNAs of different sizes in vitro. J Bacteriol, 1990 Apr, 172(4), 1877 - 88 Characterization of the genetic elements required for site-specific integration of plasmid pSE211 in Saccharopolyspora erythraea; Brown DP et al.; The 18.1-kilobase plasmid pSE211 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site . Restriction analysis of the integrated plasmid, pSE211int, and adjacent chromosomal sequences allowed identification of attP, the plasmid attachment site . Nucleotide sequencing of attP, attB, attL, and attR revealed a 57-base-pair sequence common to all sites with no duplications of adjacent plasmid or chromosomal sequences in the integrated state, indicating that integration takes place through conservative, reciprocal strand exchange . An analysis of the sequences indicated the presence of a putative gene for Phe-tRNA at attB which is preserved at attL after integration has occurred . A comparison of the attB site for a number of actinomycete plasmids is presented . Integration at attB was also observed when a 2.4-kilobase segment of pSE211 containing attP and the adjacent plasmid sequence was used to transform a pSE211- host . Nucleotide sequencing of this segment revealed the presence of two complete open reading frames (ORFs) and a segment of a third ORF . The ORF adjacent to attP encodes a putative polypeptide 437 amino acids in length that shows similarity, at its C-terminal domain, to sequences of site-specific recombinases of the integrase family . The adjacent ORF encodes a putative 98-amino-acid basic polypeptide that contains a helix-turn-helix motif at its N terminus which corresponds to domains in the Xis proteins of a number of bacteriophages . A proposal for the function of this polypeptide is presented . The deduced amino acid sequence of the third ORF did not reveal similarities to polypeptide sequences in the current data banks. Sex Transm Dis, 1990 Apr-Jun, 17(2), 58 - 62 Virus leakage through natural membrane condoms; Lytle CD et al.; The authors determined virus leakage from condoms made from processed sheep caecum using two viral probes simultaneously . They poured a mixture of two viruses, the bacteriophage, phi X174 (4 X 10(7) pfu/ml), and the human pathogen, herpes simplex virus (about 1 X 10(6) pfu/ml), in a buffered solution into condoms, which were suspended into beakers also containing buffered solution . The authors then assayed aliquots from the beakers to measure the extent of virus leakage from the condoms . With one brand of condom, 10 out of 24 samples leaked small amounts of phi X174; with the other brand of condom, 13 out of 24 samples gave similar leakage . The extent of leakage varied over two orders of magnitude from condom to condom within each brand . Of the 23 condoms that leaked the smaller virus, phi X174 (27 nm in diameter), only two also leaked the larger herpesvirus (120-150 nm in diameter) . These data demonstrate that (1) large and small viruses can leak from natural membrane condoms; (2) there is considerable variation from condom to condom in allowing leakage of the viruses; and (3) leakage of a small virus does not necessarily indicate that a larger virus will leak from that particular condom . The authors explain some inconsistencies in the published literaturePIP: 24 natural membrane condoms of 2 brand were tested in a static bath for leakage of a small virus, PhiX174, 27 nm in diameter, and a larger virus, Herpes Simplex Virus Type I, 120-150 nm in diameter, in a 4-hour experiment . These viruses were chosen because the bacteriophage PhiX174 is slightly smaller than Hepatitis B and is easy and safe to assay, and Herpes virus is close in size and chemical composition to HIV, and is relatively easy to assay on mouse kidney cells . For the test 40 million plaque forming units (pfu/ml of PhiX174 and 1 million herpes simplex pfu/ml were incubated in a condom suspended in a beaker containing Dulbecco's phosphate buffer, with magnetic stirring . 10 to 24 condoms of Brand A and 13 of 24 Brand B leaked some phage . 2 condoms leaked some herpes virus . The results were computed into an index of barrier function, the barrier ratio . There was a variation in leakage over 2 orders of magnitude between condoms . The results in this status situation were similar to those obtained by others in a simulated active coitus experiment, in that greater amounts of the smaller viruses leaked through natural condoms . J Bacteriol, 1990 Apr, 172(4), 1861 - 9 Insertion mutagenesis of the gene encoding the ferrichrome-iron receptor of Escherichia coli K-12; Carmel G et al.; The ferrichrome-iron receptor of Escherichia coli K-12 encoded by the fhuA gene is a multifunctional outer membrane receptor with an Mr of 78,000 . It is required for the binding and uptake of ferrichrome and is the receptor for bacteriophages T5, T1, phi 80, and UC-1 as well as for colicin M . The fhuA gene was cloned into pBR322, and the recombinant plasmid pGC01 was mutagenized by the insertion of 6-base-pair TAB (two amino acid Barany) linkers into CfoI and HpaII restriction sites distributed throughout the coding region . A library of 18 TAB linker insertions in fhuA was generated; 8 of the mutations were at CfoI sites and 10 were at HpaII sites . All mutations inserted a hexamer that encoded a unique SacI site . A large deletion in fhuA was also isolated by TAB linker mutagenesis . Except for the deletion mutant, all of the linker insertion mutant FhuA proteins were found in the outer membrane in amounts similar to those found in the wild type . Five of the linker insertion mutants were susceptible to cleavage by endogenous proteolytic activity: a second FhuA-related band that migrated at approximately 72 kilodaltons could be detected on Coomassie blue-stained gels and on Western blots (immunoblots) by using a carboxy terminus-specific anti-peptide antibody . Receptor functions were measured with the mutated genes present in a single copy on the chromosome . Some of the receptors conferred wild-type phenotypes: they demonstrated growth promotion by ferrichrome and the same efficiency of plating as that of wild-type FhuA; killing by colicin M was also unaffected . Several mutants were altered in their sensitivities to the lethal agents . TAB linker insertions after amino acids 69 and 128 abolished all receptor functions . Phage T5 id not bind to these mutant FhuA proteins in detergent extracts . The deletion mutant was also defective in all FhuA functions . Sensitivity to the lethal agents of cellsl that expressed mutant FhuAs with insertions after amino acids 59 and 135 was reduced by several orders of magnitude . Insertion at other selected sites decreased some or all receptor functions only slightly . An insertion after amino acid 321 selectively eliminated ferrichrome growth promotion . Finally, a strain carrying a mutant fhuA gene on the chromosome in which the linker insertion occurred after amino acid 82 showed a tonB phenotype . These subtle perturbations that were introduced into the FhuA protein resulted in changes in its stability and in the binding and uptake of its cognate ligands. Cell Differ Dev, 1990 Apr, 30(1), 77 - 85 Plasmid and bacteriophage lambda-DNA show differential replication characteristics following injection into fertilized eggs of Xenopus laevis: dependence on period and site of injection; Hofmann A et al.; The fate of DNA injected into in vitro fertilized eggs of Xenopus laevis during subsequent early embryogenesis was investigated by changing the time period and the area of injection . Form I/II plasmid DNA was found to be preferentially replicated in embryos which had been injected 60-65 min after fertilization into the animal half of the fertilized egg, irrespective of the presence of a eukaryotic origin of replication sequence element in the DNA probe used for injection . In the experiments where plasmid DNA and lambda-DNA were coinjected, only the latter was actively replicated, which suggests an inhibitory activity of this DNA on replication of coinjected plasmid DNA. J Clin Microbiol, 1990 Apr, 28(4), 787 - 8 Leakage of virus through used vinyl and latex examination gloves; Korniewicz DM et al.; A total of 480 examination gloves (240 vinyl and 240 latex) were stressed by using manipulations designed to mimic patient care . At the highest use level, 38 (63%) of 60 vinyl gloves leaked bacteriophage phi X174 compared with 4 (7%) of 60 latex gloves . At lower use levels, there was no statistically significant difference in leakage. Virology, 1990 Apr, 175(2), 586 - 90 Expression and regulation of genes coding for three bacteriophage T4 tail tube-associated proteins; Ishimoto LK et al.; The assembly and length regulation of the tail tube of bacteriophage T4 requires the function of three proteins: gp29, 48, and 54 . Six copies of each protein are found in the completed tail, and the genes for these proteins are adjacent on the T4 genome . Evidence is presented here that gp54 is also a tail tube-associated protein that remains bound to the tail tube after the baseplate is removed by guanidine hydrochloride, suggesting that all three proteins interact structurally . There is a strong polar effect of translation termination mutants in gene 48 upon the expression of the adjacent gene 54, in cis-trans tests . Gene dosage experiments that assay the in vivo expression of these genes show that only gene 48 is expressed at slightly higher than stoichiometric levels during T4 infection . Genes 48 and 54 were placed under the control of a T7 promoter and the corresponding proteins identified . When a frameshift mutation was introduced into gene 48, neither gp48 nor gp54 was made . Transcriptional termination was not the explanation of this result because genes distal to 48 and 54 in the plasmid were expressed . These data suggest that expression of genes 48 and 54 is translationally coupled. Virology, 1990 Apr, 175(2), 365 - 71 Characterization of infectious transcripts from a potato virus X cDNA clone; Hemenway C et al.; A full-length cDNA clone of potato virus X (PVX) has been constructed and fused to the bacteriophage T7 promoter in an in vitro transcription vector . Transcripts derived from this template (pMON 8660) were infectious when inoculated onto the local lesion host, Chenopodium amaranticolor . The infectivity of these transcripts was approximately 0.2% that of authentic PVX RNA . Lesions sampled from plants inoculated with these transcripts contained virus particles and virus aggregates typically observed in lesions from plants inoculated with authentic PVX RNA, as evidenced by electron microscopy . In addition, progeny virus isolated from these lesions was as infectious as progeny virus from an authentic PVX RNA infection when inoculated onto new local lesions plants . Infectious transcripts derived from PVX cDNA clones will facilitate analysis of the molecular aspects of PVX infection. Proc Natl Acad Sci U S A, 1990 Apr, 87(7), 2790 - 4 Evidence for the double-strand break repair model of bacteriophage lambda recombination; Takahashi N et al.; We have obtained evidence for the repair of double-strand gaps promoted by the Red function of bacteriophage lambda . A double-strand gap was made in one of the two regions of homology in an inverted orientation on a plasmid DNA molecule . The gapped plasmid was introduced into Escherichia coli cells expressing the red alpha (exo) and red beta (bet) genes of lambda . The gap was repaired by DNA synthesis copying an intact duplex . This gap repair was sometimes accompanied by reciprocal recombination (crossing over) . The gap stimulated recombination about 100-fold . Our results are compatible with previous proposals that lambda homologous recombination involves the following early steps: (i) generation of double-stranded ends by the packaging machinery or by the replication machinery; (ii) production of a single-stranded tail with a 3'-hydroxyl end by 5'----3' degradation by lambda exonuclease (red alpha gene product); (iii) pairing of the single-stranded tail with a complementary strand from a homologous duplex with the help of beta protein (red beta gene product); (iv) priming of DNA synthesis at this 3'-hydroxyl end to copy the second DNA molecule. Int J Biol Macromol, 1990 Apr, 12(2), 125 - 38 Model-building studies of Inovirus: genetic variations on a geometric theme; Marvin DA; Inovirus (filamentous bacteriophage) is a simple system for studying the rules by which protein primary structure (amino acid sequence) controls secondary and higher order structure, and thereby function . The virus occurs naturally as a number of different strains with similar secondary and higher order structure, but the protein subunit that assembles to form the virion coat has quite different primary structures in different virus strains . Despite these differences in primary structure, the subunits of all strains have much the same size, about 50 residues, which are distributed by type in much the same way into three domains of primary structure: a collection of acidic residues in the N-terminal region, a hydrophobic domain of about 19 residues near the middle, and a collection of basic residues near the C-terminus . Each subunit can be closely approximated by an alpha-helix with its long axis roughly parallel to the fibre axis, sloping from large to small radius in the virion and interleaving between subunits in the next turn or level . The acidic residues near the N-terminus of the subunit face outwards on the virion surface, and explain the low isoelectric point of the virion; the basic residues near the C-terminus face inwards, where they neutralize the charge on the DNA at the core of the virion; and the hydrophobic central domain is involved in interactions which bind neighbouring subunits . Detailed X-ray fibre diffraction analysis of one strain gives the subunit structure . Comparative model-building studies of different strains illustrate the common structural principles. Virus Genes, 1990 Apr, 3(4), 309 - 22 Marek's disease virus gene clones encoding virus-specific phosphorylated polypeptides and serological characterization of fusion proteins; Cui ZZ et al.; Marek's disease virus (MDV) gene clones, RA2 and GA8, constructed in E . coli bacteriophage lambda-gt11 (gt11) were identified by a monoclonal antibody (MAb), H19.47, against a putative transformation-related viral antigen consisting of a complex of three phosphorylated polypeptides, pp41, pp38, and pp24 . Both recombinants have a MDV-DNA insert of about 0.5 kb and are mapped to the region of BamHI-H or EcoRI-X fragments of the MDV genome by Southern blot hybridization . Immunoblot and immunoprecipitation with H19.47 identified a recombinant beta-galactosidase-MDV 140-kD fusion protein for RA2 and a 127-kD fusion protein for GA8 . Immunoprecipitation of 35S-methionine-labeled, MDV-infected chicken embryo fibroblasts (CEF) with antisera against RA2 and GA8 fusion proteins recognized five polypeptides, of which three (p41, p38, and p24) are specified by H19.47 and the remaining two, p135 and p20, have not been previously identified . Immunoprecipitation of 32P-phosphate-labeled or 3H-glucosamine-labeled, GA-MDV-infected CEF with the antiserum against RA2 fusion protein identified a phosphorylated polypeptide of 38 kD and two glycoproteins of 60 and 49 kD, respectively . The antisera against recombinant fusion proteins thus revealed the existence of epitopes common to the phosphorylated polypeptides and other MDV-specific polypeptides . Sera from chickens or mice hyperimmunized with the purified fusion proteins reacted with serotype 1, MDV-infected CEF in the fluorescent antibody (FA) test to significant titers . These immune sera did not react with either serotype II or III, indicating the serotype specificity of the phosphorylated polypeptides. Gene, 1990 Mar 30, 88(1), 25 - 36 Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning; Palazzolo MJ et al.; We describe the construction and use of two classes of cDNA cloning vectors . The first class comprises the lambda EXLX(+) and lambda EXLX(-) vectors that can be used for the expression in Escherichia coli of proteins encoded by cDNA inserts . This is achieved by the fusion of cDNA open reading frames to the T7 gene 10 promoter and protein-coding sequences . The second class, the lambda SHLX vectors, allows the generation of large amounts of single-stranded DNA or synthetic cRNA that can be used in subtractive hybridization procedures . Both classes of vectors are designed to allow directional cDNA cloning with non-enzymatic protection of internal restriction sites . In addition, they are designed to facilitate conversion from phage lambda to plasmid clones using a genetic method based on the bacteriophage P1 site-specific recombination system; we refer to this as automatic Cre-loxP plasmid subcloning . The phage lambda arms, lambda LOX, used in the construction of these vectors have unique restriction sites positioned between the two loxP sites . Insertion of a specialized plasmid between these sites will convert it into a phage lambda cDNA cloning vector with automatic plasmid subcloning capability. Gene, 1990 Mar 30, 88(1), 7 - 14 The bacteriophage T4 gene mrh whose product inhibits late T4 gene expression in an Escherichia coli rpoH (sigma 32) mutant; Frazier MW et al.; In an Escherichia coli rpoH mutant (affecting sigma 32, heat-shock sigma factor) infected at high temperatures with wild-type T4 phage, late T4 transcription and consequently progeny production are dramatically impaired . This defect is due, in part, to insufficient activity of sigma 70 {Frazier and Mosig, J . Bacteriol . 170 (1988) 1384-1388}, which is necessary to initiate early T4 transcription . Unexpectedly, however, we found that, in this rpoH host, late T4 transcription is also impaired when the temperature is raised from 30 to 42 degrees C late after infection, when T4 transcription is directed by the T4-encoded sigma factor, sigma gp55 . Here, we show that a T4 gene that we call mrh (modulates rpoH), located at 14 kb on the T4 map, is responsible for the inhibition of late T4 transcription in the rpoH mutant host . T4 deletion mutants that lack the mrh gene can produce progeny in the rpoH host, but the Mrh protein, provided in trans from a plasmid-borne mrh gene, inhibits this growth . We have cloned and sequenced this T4 gene and synthesized the Mrh protein in a T7 RNA polymerase-dependent expression system . The Mr of the Mrh protein deduced from the nucleotide sequence is 13419 . Gene mrh is cotranscribed with several other, yet unidentified genes, both from an early promoter downstream from the late soc gene (encoding the small outer capsid protein) and from the late soc promoter further upstream.(ABSTRACT TRUNCATED AT 250 WORDS) Biochem Biophys Res Commun, 1990 Mar 30, 167(3), 1196 - 9 Bacteriophage T4 gene 32 protein shares antigen determinants with reconstituted HeLa hnRNP proteins in western blots; Schenkel J et al.; A polyclonal antiserum against purified bacteriophage T4 gene 32 protein was raised in rabbits . In Western blots it detected a number of SDS-PAGE separated nuclear and ribosomal proteins of HeLa cells . Using a renaturing blotting system, however, larger hnRNP proteins ranging between 66.000 and 82.000 Da preferentially reacted with this antiserum . In addition hnRNP group A core proteins were detected to a minor extent . Nucleic acid binding proteins like histones or ribosomal proteins were not stained by this antibody after renaturation. Biochemistry, 1990 Mar 27, 29(12), 3071 - 7 Folding kinetics of phage T4 thioredoxin; Borden KL et al.; The folding mechanism for bacteriophage T4 thioredoxin is best described by a four-state box mechanism, N----Uc----Ut----It----N, where N indicates native, Uc the unfolded form with the cis proline isomer, Ut unfolded with the trans proline isomer, and It a compact form with a trans proline isomer . Both manual mixing fluorescence and size-exclusion chromatography indicate that there is a cis-trans proline isomerization that is important to the folding pathway . Furthermore, the data suggest that the cis-trans isomerization can also occur in a compact nativelike state which is referred to as It . The slow phase seen in fluorescence seems to be monitoring the cis-trans isomerization in the compact form, not the isomerization which occurs in the denatured state. Carbohydr Res, 1990 Mar 25, 197, 171 - 80 The structure of Escherichia coli K31 antigen; Dutton GG et al.; The capsular polysaccharide of Escherichia coli K31 has been found by methylation analysis and n.m.r . spectroscopy to be based on the hexasaccharide shown . The sequence of the repeating unit was deduced from the combined results of beta-elimination, lithium-ethylenediamine degradation, and hydrogen-fluoride and selective hydrolyses . The nature of the anomeric linkages, established by chromic acid oxidation, was confirmed by 1H-coupled 13C-n.m.r . spectroscopy . Two dimensional n.m.r . studies on a low molecular weight polymer obtained by bacteriophage depolymerization are also reported . (formula; see text) J Biol Chem, 1990 Mar 25, 265(9), 5303 - 16 Transcriptional mapping of a DNA replication gene cluster in bacteriophage T4 . Sites for initiation, termination, and mRNA processing; Hsu T et al.; A phage T4 genetic cluster that encodes DNA polymerase, several other DNA replication proteins, transcriptional factors, and the translation repressor RegA has been shown to be controlled by overlapping modes of transcription which initiate at several promoters . The promoters were mapped by using a combination of assays including Northern blotting, S1-mapping, RNA sequencing, and analysis of products of radioactive labeling of 5' ends on T4-induced RNA in vitro via the reaction catalyzed by eukaryotic guanylyl transferase (RNA capping assay) . The most proximal in the cluster are two promoters that do not require any phage-induced factors for activation, i.e . they are T4 early promoters . Initiation at these promoters yields several RNA species having overlapping 5'-terminal sequences, the largest of which is estimated to be about 15,000 nucleotides long and to include all the cistrons of the cluster . A third early promoter maps inside the protein encoding segment of one of the cistrons (T4 gene 47), while at least five additional promoters map in intercistronic regions and are T4 middle promoters, i.e . they require the T4-induced DNA-binding transcription factor MotA . Transcriptional readthrough at a termination site within the T4 gene 45-44 intercistronic region is required for synthesis of gp44 and gp62, two essential T4 DNA-polymerase (gp43) accessory proteins . In contrast, transcription of T4 gene 43 is serviced by readthrough across a termination site in the regA-43 intercistronic region as well as by a MotA-dependent promoter that maps downstream of the termination site, and the region contains a site for processing by a T4-induced enzyme that also cleaves elsewhere in the polycistronic mRNA from the cluster (i.e . in the Shine-Dalgarno sequence of the gene 45.2 mRNA) . The termination events in the gene 45-44 and regA-43 intercistronic regions both occur downstream of RNA stem-loop structures containing the sequence 5'CUUCGG3' in the loop segments . Transcription termination in the 78-base-pair regA-43 intercistronic region occurs about 60 nucleotides away from the gp43 initiator AUG, transcription initiation occurs at 38-40 nucleotides upstream from the AUG, and T4-dependent RNA processing occurs at several sites (including a GGAG sequence) between the transcription termination and initiation sites . Thus, all gp43-encoding mRNAs contain the translational operator (residues -40 to -1 relative to the AUG) for autogenous repression by this DNA polymerase (Andrake et al., 1988).(ABSTRACT TRUNCATED AT 400 WORDS) J Mol Biol, 1990 Mar 20, 212(2), 345 - 50 Preliminary investigation of the phage phi X174 crystal structure; Willingmann P et al.; Crystals of the single-stranded DNA bacteriophage phi X174 have been grown . They have a monoclinic unit cell with space group P2(1), unit cell dimensions of a = 306.0 (+/- 0.2) A, b = 361.1 (+/- 0.2) A, c = 299.7 (+/- 0.2 degrees) A, beta = 92.91 degrees (+/- 0.02 degrees) and diffract to at least 2.7 A resolution . There are two virus particles per unit cell . Packing considerations show that the mean diameter of the virus particles is 280 A . The virus separates into two bands in a sucrose gradient . The ratio between the absorbance at 260 nm and 280 nm is 1.45 to 1.65 for the faster and 1.15 to 1.35 for the slower bands, but both bands contain intact particles . Crystals derived from these bands are isomorphous and there is no detectable difference in their structure amplitudes. J Biol Chem, 1990 Mar 15, 265(8), 4411 - 9 The gene 1.2 protein of bacteriophage T7 interacts with the Escherichia coli dGTP triphosphohydrolase to form a GTP-binding protein; Nakai H et al.; Escherichia coli encodes a dGTP triphosphohydrolase (dGTPase) that cleaves dGTP to deoxyguanosine and tripolyphosphate . dGTP is hydrolyzed with a Michaelis constant (Km) of 5 microM and a maximal velocity (Vmax) of 1.8 mumols/min/mg . The ribonucleotide GTP is a poor substrate with a much lower affinity . It is hydrolyzed with a Km of 150 microM and Vmax of 0.07 mumols/min/mg . Bacteriophage T7 encodes a specific inhibitor of dGTPase, the gene 1.2 protein, that forms a tight complex with the enzyme . The enzyme-inhibitor complex binds dGTP with a dissociation constant (KD) of 1.5 microM, but the bound dGTP is not hydrolyzed . It remains stably bound to the complex with a half-life of approximately 5 min . In contrast, dGTP is unable to bind to gene 1.2 protein alone, and dGTP bound to dGTPase alone is quickly hydrolyzed and released . Surprisingly, the dGTPase-gene 1.2 protein complex has a higher affinity for GTP than for dGTP . GTP is stably bound to the dGTPase-gene 1.2 protein complex with a half-life greater than 30 min and KD of 0.8 microM; GTP is not stably bound to either dGTPase or gene 1.2 protein alone . Both GTP and dGTP bind to and stabilize the dGTPase-gene 1.2 protein complex, inhibiting its dissociation . Although the presence of dGTP induces conformation changes in dGTPase so that it is unable to associate with the gene 1.2 protein, saturating concentrations of GTP have no such effect . The enzyme efficiently associates with its inhibitor in the presence of GTP . These results indicate that E . coli dGTPase and gene 1.2 protein interact to form a high affinity GTP-binding site . dGTP is most effective in preventing the association of the enzyme with the inhibitor whereas GTP is most effective in preventing the dissociation of the enzyme-inhibitor complex. Biochemistry, 1990 Mar 13, 29(10), 2532 - 7 A sensitive genetic assay for the detection of cytosine deamination: determination of rate constants and the activation energy; Frederico LA et al.; Previously it has not been possible to determine the rate of deamination of cytosine in DNA at 37 degrees C because this reaction occurs so slowly . We describe here a sensitive genetic assay to measure the rate of cytosine deamination in DNA at a single cytosine residue . The assay is based on reversion of a mutant in the lacZ alpha gene coding sequence of bacteriophage M13mp2 and employs ung- bacterial strains lacking the enzyme uracil glycosylase . The assay is sufficiently sensitive to allow us to detect, at a given site, a single deamination event occurring with a background frequency as low as 1 in 200,000 . With this assay, we determined cytosine deamination rate constants in single-stranded DNA at temperatures ranging from 30 to 90 degrees C and then calculated that the activation energy for cytosine deamination in single-stranded DNA is 28 +/- 1 kcal/mol . At 80 degrees C, deamination rate constants at six sites varied by less than a factor of 3 . At 37 degrees C, the cytosine deamination rate constants for single- and double-stranded DNA at pH 7.4 are 1 x 10(-10) and about 7 x 10(-13) per second, respectively . (In other words, the measured half-life for cytosine in single-stranded DNA at 37 degrees C is ca . 200 years, while in double-stranded DNA it is on the order of 30,000 years.) Thus, cytosine is deaminated approximately 140-fold more slowly when present in the double helix . These and other data indicate that the rate of deamination is strongly dependent upon DNA structure and the degree of protonation of the cytosine . The data suggest that agents which perturb DNA structure or facilitate direct protonation of cytosine may induce deamination at biologically significant rates . The assay provides a means to directly test the hypothesis. FEBS Lett, 1990 Mar 12, 262(1), 145 - 8 A new superfamily of putative NTP-binding domains encoded by genomes of small DNA and RNA viruses; Gorbalenya AE et al.; Statistically significant similarity was revealed between amino acid sequences of NTP-binding pattern-containing domains which are among the most conserved protein segments in dissimilar groups of ss and dsDNA viruses (papova-, parvo-, geminiviruses and P4 bacteriophage), and RNA viruses (picorna-, como- and nepoviruses) with small genomes . Within the aligned domains of 100-120 amino acid residues, three highly conserved sequence segments have been identified, i.e . 'A' and 'B' motifs of the NTP-binding pattern, and a third, C-terminal motif 'C', not described previously . The sequence of the 'B' motif in the proteins of the new superfamily is unusually variable, with substitutions, in some of the members, of the Asp residue conserved in other NTP-binding proteins . The 'C' motif is characterized by an invariant Asn residue preceded by a stretch of hydrophobic residues . As the new superfamily included a well studied DNA and RNA helicase, T antigen of SV40, helicase function could be tentatively assigned also to the other related viral putative NTP-binding proteins . On the other hand, the possibility of different and/or multiple functions for some of these proteins is discussed. J Biol Chem, 1990 Mar 5, 265(7), 3823 - 30 Transcription termination by bacteriophage T7 RNA polymerase at rho-independent terminators; Jeng ST et al.; We have investigated the mechanism of transcription termination by T7 RNA polymerase using templates encoding variants of the transcription-termination structure (attenuator) of the regulatory region of the threonine (thr) operon of Escherichia coli . The thr attenuator comprises the following two distinct structural elements: a G + C-rich inverted repeat, which encodes an RNA hairpin structure, and A + T-rich regions, one of which contains a continuous sequence of template deoxyadenosine residues within which the transcription terminates . Fourteen attenuator variants were analyzed and we find that not only the hairpin structure itself but also its sequence influences termination . Furthermore, the formation of a hairpin in the RNA encoded by the A + T-rich regions of the attenuator is not mandatory for termination . A series of seven deletion variants that successively shorten the deoxyadenosine tract in the attenuator template were also analyzed . Results from these experiments indicate that complete readthrough occurs when there are four or fewer deoxyadenosine residues . With 5 template deoxyadenosine residues there is 5% termination increasing to 32% with 8 deoxyadenosines, the value produced by the wild-type attenuator . In addition, a comparison with E . coli RNA polymerase shows that T7 RNA polymerase requires a more perfect region of dyad symmetry and a longer deoxyadenosine tract than does the bacterial enzyme to terminate with maximum efficiency. J Mol Biol, 1990 Mar 5, 212(1), 53 - 66 Structural and functional properties of the segments of lambda cro mRNA that interact with transcription termination factor Rho; Faus I et al.; Termination of transcription at tR1, the Rho-dependent terminator between genes cro and cII of bacteriophage lambda, is dependent upon the structure of segments near the 3' end of the nascent cro gene transcript and on contacts between Rho protein and a 3' proximal segment called rut . The characteristics of the structure of cro RNA in the region from residue 220 to residue 355 in free, isolated RNA and in the presence of Rho or NusA proteins were analyzed by measuring relative rates of reactivity of individual nucleotides with chemicals and enzymes of defined specificities . The results indicate that the rut segments are single-stranded and become blocked to the action of the various probes in the presence of Rho factor . They also show that this region contains two stem-loop structures; one involves the boxB sequence of nutR, the other precedes the tR1 subsite II end points . The results provide direct evidence for a primary binding contact between Rho protein and the rut segment of cro RNA and demonstrate that this binding contact remains stable when the cro RNA is serving as a cofactor for ATP hydrolysis, an observation that is consistent with a mechanism in which Rho maintains contact with the rut region while it makes additional interactions with RNA that are coupled to ATP hydrolysis. J Biol Chem, 1990 Mar 5, 265(7), 3757 - 62 The N-terminal domain of the insertion sequence 30 transposase interacts specifically with the terminal inverted repeats of the element; Stalder R et al.; The gene for the insertion sequence (IS) 30 transposase is placed under the control of the tac promoter, and large quantities of transposase are expressed upon induction . The resulting protein precipitates inside the Escherichia coli cells in the form of inclusion bodies which, upon cell lysis, cannot be dissolved under nondenaturing conditions . In contrast, the N-terminal third of the transposase, a 17-kDa protein produced by a truncated gene, can be purified and is able to interact site specifically with the ends of the IS30 element . In DNase I footprint experiments, regions of 26 nucleotides on one DNA strand and 19 nucleotides on the other strand at either end of the element are protected from nuclease digestion . It is concluded that a functional DNA-binding domain can be formed by expression of only one-third of the complete IS30 transposase . Sequence comparison shows a homology of the IS30 ends to the ends of IS4351 and to the L1 end of bacteriophage Mu. J Bacteriol, 1990 Mar, 172(3), 1587 - 94 Identification, cloning, and characterization of the Escherichia coli sohA gene, a suppressor of the htrA (degP) null phenotype; Baird L et al.; Two extragenic suppressors which allow temperature-sensitive htrA mutant Escherichia coli bacteria to grow at 42 degrees C and simultaneously acquire a cold-sensitive phenotype at 30 degrees C were isolated . The cold-sensitive phenotype exhibited by one of the mutants was used to clone the corresponding wild-type copy of the suppressor gene . This was done through complementation with a mini-mu plasmid E . coli DNA library, by selection for colonies which were no longer cold sensitive, at 30 degrees C . The cloned suppressor gene was shown to complement the cold-sensitive phenotype of both suppressor mutations . It was mapped to 68 min on the E . coli chromosome through hybridization to the Kohara library of overlapping lambda transducing bacteriophages, which covers the entire E . coli chromosome . The complementing gene was further subcloned on an 830-base-pair (bp) DNA fragment . DNA sequencing revealed the presence of an open reading frame (ORF) of 333 bp which could encode a protein of 12,359 Mr . Subcloning of various DNA fragments from within this 830-bp DNA fragment suggests that this ORF is most likely responsible for suppression of the cold-sensitive phenotype of the htrA suppressor bacteria . By using a T7 polymerase system to overproduce plasmid-encoded proteins, a protein of approximately 12,000 Mr was produced by this cloned DNA fragment . This ORF defines a previously undiscovered gene in E . coli, called sohA (suppressor of htrA). FASEB J, 1990 Mar, 4(5), 1488 - 93 Membrane morphogenesis from cloned fragments of bacteriophage PM2 DNA that contain the sp6.6 gene; Armour GA et al.; The formation of new membrane vesicles normally occurs during eukaryotic organellogenesis and maturation of bacteriophage PM2 . This virus was studied as a simple model for membrane morphogenesis . Previous biochemical and genetic studies suggest that a major structural protein of PM2, sp6.6, is an integral membrane protein involved in viral membrane morphogenesis . To establish the necessity of sp6.6 in membrane formation, restriction fragments of PM2 that contained the sp6.6 coding sequence were cloned into several plasmid vectors for expression in Escherichia coli . A construction in pBR322 containing two HindIII fragments of PM2 DNA caused production of intracellular membrane vesicles of the same size as those produced in the course of natural infection of Alteromonas espejiana . Similar results were obtained with a smaller construct of HindIII fragments in the plasmid vector pPL-lambda . Expression of sp6.6 was detected via incorporation of 35S-labeled methionine after SDS-polyacrylamide gel electrophoresis and with a specific rabbit antiserum on immunoblots . Other constructs did not produce recognizable vesicles or sp6.6 . These results are the first to suggest that a hydrophobic membrane protein can cause development of new membrane structure. J Gen Microbiol, 1990 Mar, 136 ( Pt 3), 573 - 9 Saccharopolyspora hirsuta strain 367 releases JHJ-1, a bacteriophage capable of propagation on old mycelium; Haket J Jr et al.; Bacteriophage JHJ-1 was isolated from Saccharopolyspora hirsuta strain 367 NRRL 12045 as an endogenous but virulent phage . The plaque size was not self-limiting, since a few p.f.u . could completely lyse a lawn . Electron microscopy showed that this phage belonged to group B of Bradley's morphological classification . The JHJ-1 genome is a linear DNA molecule of 41.1 kbp with cohesive ends and a G + C content of 68.8-70.0 mol% . The DNA cleavage map was established for 12 restriction endonucleases . The host range is apparently very narrow, being limited to two strains of S . hirsuta (NRRL 12045 and NRRL B-5792) . However, JHJ-1 did not lytically infect S . hirsuta strain 367 UC 8106 . Phage JHJ-1 was shown, by Southern blot analysis, to lysogenize both S . hirsuta NRRL 12045 and UC 8106 . It thus appears to behave as a virulent mutant of a temperate phage on one, but not on the other, JHJ-1 lysogen. Biofizika, 1990 Mar-Apr, 35(2), 263 - 8 {Energy of cooperative transitions in bacteriophage SD}; Mrevlishvili GM et al.; Heat denaturation of native phages SD suspensions, phage "shadows", and isolated phage DNA solutions were studied by scanning microcalorimetry and viscosimetry . Energetic parameters of cooperative transitions of protein fraction and DNA were measured . DNA melting was shown to be preceded by the destruction of capsid and protein denaturation . The melting curve of isolated DNA and DNA in the presence of protein component is characterized by a fine structure which is completely restored at repeated denaturation only in the presence of the protein component . "Creeping" of DNA out of the capsid in heated suspensions at 50-52 degrees C was shown to proceed with "zero" enthalpy without significant endo- and exo-thermal effects . No change of specific heat capacity of the suspension was also observed . It is emphasized that the mechanism of DNA going out of the capsid can be understood by studying DNA hydration inside the phage and its change in the course of liberation of the phage genome from the protein capsid. Mol Biol (Mosk), 1990 Mar-Apr, 24(2), 541 - 7 {Changes in the secondary structure bacteriophage T4 envelope protein after its polymerization}; Serysheva II et al.; Circular dichroism (CD) spectra of the structural protein of the bacteriophage T4 sheath (gp 18) in a monomeric native state, helices, polysheaths and contracted sheaths were measured in the range 184-310 nm . The secondary structure of the protein studied was calculated from the spectra in the range 190-240 nm according to Provencher and Glockner . It has been shown that the polymerization is proceeded without change of the alpha-helical content in the secondary structure of gp 18: estimated alpha-helix in monomeric gp 18, helices and polysheaths was 39% . The beta-form content in monomeric gp 18, helices and polysheaths was 33, 32 and 37%, respectively . Tail sheath contraction is attended by a 14% decrease in gp 18 alpha-helicity and a 5% increase in its beta-form content. Mol Biol (Mosk), 1990 Mar-Apr, 24(2), 379 - 90 {Gene 26 of bacteriophage T4 basal plate . I . Two expression products of the cloned gene}; Vaishkunaite RI et al.; We have cloned the 1.9 kb EcoRV-BglII DNA fragment with T4 genes 51, 26, and 25 into the expression plasmid pT7-5 carrying a T7 promoter . The resulting recombinant plasmid, pRR5-3, contained T4 genes 26 and 25 in the correct orientation for expression . We expressed these genes using the T7 RNA polymerase/promoter system and the synthesis of three polypeptides with the molecular masses of approximately 24, 15, and 8-9 kDa was observed . Expression of genes from the subcloned DNA fragments and from the fragments carrying deletions was studied as well and the 15 kDa protein appeared to be the product of gene 25, while 24 kDa and 8-9 kDa proteins were identified as products of gene 26 . The 8-9 kDa protein was shown to be expressed from the end region of gene 26 . Having analysed the proteins expressed from the fragments carrying fusion of genes 26 and 25 we supposed two products of gene 26 to be encoded by the same open reading frame. Biol Chem Hoppe Seyler, 1990 Mar, 371(3), 239 - 48 Expression of the E . coli nadB gene and characterization of the gene product L-aspartate oxidase; Seifert J et al.; Quinolinic acid is synthesized in E . coli by the enzymes L-aspartate oxidase and quinolinate synthase A, the genes of which are named nadB and nadA . In our previous work we cloned and characterized the two genes (Flachmann, R., Kunz, N., Seifert, J., Gutlich, M., Wientjes, F.J., Laufer, A . & Gassen, H.G . (1988) Eur . J . Biochem . 175, 221-228) . Here we report on the expression of the nadB gene under control of the inducible left promoter of the bacteriophage lambda . The yield of the active gene product L-aspartate oxidase was enhanced up to 20% of the soluble cell protein . The enzyme was purified to homogeneity in a three-step procedure and the reading frame of the L-aspartate oxidase gene was confirmed by Edman degradation of five cyanogen bromide peptides . L-Aspartate oxidase shows no classical Michaelis-Menten behaviour but is subject to a substrate inactivation . The apparent Km values were different for substrate concentrations below and above 1mM and were determined to 0.5 mM and 4.1mM, respectively . The active form of the enzyme is a monomer of 60,284 Da and contains one molecule of FAD and nine cysteine residues, four of which built up two disulfide bonds . The isoelectric point of the protein was determined to be at pH 5.6 . Chemical modifications of the enzyme showed that at least one tyrosine and one histidine residue are essential for enzyme activity . The coenzyme-binding domain is located in the amino-terminal part of the polypeptide chain as revealed by a sequence comparison to other dinucleotide binding enzymes . Furthermore, there is evidence for a relationship to fumarate reductase and succinate dehydrogenase of E . coli. Gene, 1990 Mar 1, 87(1), 1 - 5 Specificity of Escherichia coli mutD and mutL mutator strains; Wu TH et al.; The products of the mutD and mutL genes of Escherichia coli are involved in proofreading by DNA polymerase III and DNA adenine MTase (Dam)-dependent mismatch repair, respectively . We have used the plasmid-borne bacteriophage P22 mnt gene as a target to determine the types of mutations produced in mutL25 and mutD5 strains . Of 60 mutations identified from mutL25 cells, 52 were transition mutations and of these the AT----GC subset predominated (40 out of 52) . The majority of AT----GC mutations were found at the same three sites (hotspots) . In contrast, transversion mutations (47 out of 76) were found about twice as frequently as transitions (28 out of 76) from mutD5 bacteria . Two hotspots were identified but at different sites than those in the mutL25 cells . These results suggest that the proofreading function of DNA polymerase III primarily repairs potential transversion mutations while Dam-dependent mismatch repair rectifies potential transition mutations. DNA Cell Biol, 1990 Mar, 9(2), 139 - 47 Retrovirus-mediated insertional mutagenesis: phagemid rescue of flanking DNA by selecting plasmid ori; Rodgers JR et al.; A method for screening recombinant lambda libraries was devised to select phage containing genomic regions containing provirus insertions of retroviruses that carry the kanamycin and G418 resistance factor neo and the origin of replication derived from pBR322 (oripBR) . Such recombinants are phagemids, able to replicate as bacteriophages or as plasmids under lambda repressor control . lambda repressor was cloned into a plasmid derived from pSC101 that is compatible with pBR322-derived phagemids . A strain carrying this plasmid may be used to select phagemids derived from a single proviral insertion with 100% efficiency from complex recombinant libraries . Homologous recombination between proviral long terminal repeats was observed at a rate of 10(-4)/plaque-forming unit in recABC+ strains . Despite this frequency, intact phagemids are easily recovered as phage after temperature shift to 42 degrees C . Since oripBR itself is a selectable marker in this system, the method could be applied to recover any sequence carrying the ori sequence from pBR322. Anal Biochem, 1990 Mar, 185(2), 230 - 4 A bacteriophage lambda DNA purification procedure suitable for the analysis of DNA from either large or multiple small lysates; Lockett TJ; A method for the efficient preparation of high quality bacteriophage lambda DNA from cleared lysates is described . Advantages of the method include high DNA yields (typically around 0.8 micrograms of DNA/1 ml of cleared lysate), speed of processing (approximately 2 h from lysate to DNA), economy, and the absence of any requirement for phenol or chloroform extractions . The technique involves the concentration of phage particles by standard polyethylene glycol precipitation followed by enzymatic treatment to remove contaminating RNA and DNA . Phage particles are then lysed with sodium dodecyl sulfate (SDS) at elevated pH and temperature . Contaminating protein/SDS complexes are rendered insoluble by the addition of potassium acetate and removed by centrifugation . The quality of the resultant DNA is comparable to that prepared by cesium chloride banding for all standard molecular biological purposes providing that spermidine is included in all restriction endonucleases digestions. Mol Gen Genet, 1990 Mar, 221(1), 27 - 32 Mutational alteration of the breakage/resealing subunit of bacteriophage T4 DNA topoisomerase confers resistance to antitumor agent m-AMSA; Huff AC et al.; Bacteriophage T4 provides a simple model system in which to examine the mechanism of action of antitumor agents that have been proposed to attack type II DNA topoisomerases . Prior results demonstrated that T4 type II DNA topoisomerase is the target of antitumor agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in phage-infected Escherichia coli: a point mutation in topoisomerase structural gene 39 was shown to confer both m-AMSA-resistant phage growth and m-AMSA-insensitive topoisomerase activity . We report here that a point mutation in T4 topoisomerase structural gene 52 can also independently render both phage growth and topoisomerase activity resistant to m-AMSA . The DNA relaxation and DNA cleavage activities of this newly isolated mutant topoisomerase were significantly insensitive to m-AMSA . The drug-resistance mutation in gene 52, as well as that in gene 39, alters the DNA cleavage site specificity of wild-type T4 topoisomerase . This finding is consistent with a mechanism of drug action in which both topoisomerase and DNA participate in formation of the drug-binding site. Biochem J, 1990 Mar 1, 266(2), 379 - 84 Effects of DNA-binding drugs on T4 DNA ligase; Montecucco A et al.; A number of DNA intercalating and externally binding drugs have been found to inhibit nick sealing, cohesive and blunt end ligation, AMP-dependent DNA topoisomerization and EDTA-induced DNA nicking mediated by bacteriophage T4 DNA ligase . The inhibition seems to arise from drug-substrate interaction so that formation of active DNA-Mg2(+)-AMP-enzyme complex is impaired while assembled and active complexes are not disturbed by drug binding to the substrate. J Bacteriol, 1990 Mar, 172(3), 1529 - 38 Genetic analysis of bacteriophage lambda integrase interactions with arm-type attachment site sequences; Lee EC et al.; The bacteriophage P22-based challenge phage system was used to study lambda integrase (Int) protein binding to its arm-type recognition sequences in the bacteriophage lambda attachment site . Challenge phages were constructed that carried inserts containing either the contiguous P'123 arm-type sites or the single P'1 site within the P22 phage promoter, Pant, which is required for expression of antirepressor . If Int protein binds to these sequences in vivo, it represses transcription from Pant . We found that Int repressed Pant in phages carrying the P'123 sites more efficiently than those carrying only the P'1 site, suggesting that the protein binds cooperatively at the three adjacent sites . The Int protein from a related lambdoid phage, HK022, also repressed transcription by binding to the same arm-type sites . Mutations in the P'123 or P'1 sites that impair Int binding were isolated by selecting mutant phages that express antirepressor in the presence of Int . DNA sequence analyses showed that most of the mutants in the challenge phages carrying the P'123 sites contained multiple changes and that two mutants contained only single-base-pair changes at positions that are completely conserved among all arm-type sites . Thirty-five mutants were isolated and analyzed from phages containing only the P'1 site . Most mutants contained single-nucleotide changes, and mutations were isolated at 8 of the 10 positions of the site, suggesting that most if not all base pairs in the conserved recognition sequence are involved in Int binding. J Bacteriol, 1990 Mar, 172(3), 1436 - 40 Properties of new Escherichia coli Hfr strains constructed by integration of pSC101-derived conjugative plasmids; Francois V et al.; Conjugative temperature-sensitive plasmids were derived from pSC101 . These plasmids are useful in genetic analysis for two reasons: (i) they render possible the construction of new Hfr lines by plasmid integration at predetermined chromosomal loci via Tn10 inverse transposition, and (ii) the Hfr characters are transducible via bacteriophage P1 . We also showed that replication from pSC101 origin is deleterious for the plasmid-chromosome fusion. Gene, 1990 Mar 1, 87(1), 123 - 6 Modified bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli; Elvin CM et al.; A new series of expression vectors that direct high-level overproduction of gene products in Escherichia coli is described . All contain strong bacteriophage lambda promoters, PR and PL, arranged in tandem so that both promote transcription into genes inserted into or between unique restriction sites . The vectors also direct expression of the lambda cI857 gene (from its natural promoter, PM), which enables their use in any E . coli host strain to effect controlled expression by shifting the temperature of cultures from 30 to 42 degrees C . The vectors pCE30, pND201, pPT150 and pMA200U are derivatives of the high-copy-number plasmid pUC9 . Vector pCE33 is an analogous derivative of the heat-inducible runaway-replication plasmid, pMOB45, and directs overproduction of proteins by virtue of increase in both gene dosage and transcription following treatment at 42 degrees C . The vectors pND201 and pPT150 bear a ribosome-binding site (RBS) perfectly complementary to the 3' end of E . coli 16-S rRNA a few bp upstream from a unique HpaI site . Ways in which they may be used to improve the efficiency of translation of mRNA by substitution of a natural RBS with selection for optimal spacing from an ATG (or GTG) start codon are described . The phagemid vector pMA200U is a direct analog of pCE30 designed to facilitate preparation of single-stranded DNA templates for use in oligodeoxyribonucleotide-directed mutagenesis of overexpressed genes. J Bacteriol, 1990 Mar, 172(3), 1424 - 9 Activation of the bacteriophage Mu lys promoter by Mu C protein requires the sigma 70 subunit of Escherichia coli RNA polymerase; Margolin W et al.; Bacteriophage Mu C protein, a product of the middle operon, is required for activation of the four Mu late promoters . To address its mechanism of action, we overproduced the approximately 16.5-kilodalton C protein from a plasmid containing the C gene under the control of a phage T7 promoter and ribosome-binding site . A protein fraction highly enriched for Escherichia coli RNA polymerase (E sigma 70) and made from the overproducing strain was able to activate transcription in vitro from both the tac promoter (Ptac) and a Mu late promoter, Plys . The behavior of Plys was similar in vivo and in vitro; under both conditions, transcription was C dependent and the RNA 5' ends were identical . When anti-sigma 70 antibody was added to C-dependent transcription reactions containing both Ptac and Plys templates, transcription from both promoters was inhibited; transcription was restored by the addition of excess E sigma 70 . This result suggests that C-dependent activation of Plys requires sigma 70 . Further supporting evidence was provided by a reconstitution experiment in which an E sigma 70-depleted fraction containing C was unable to activate transcription from Plys unless both purified sigma 70 and core polymerase were added . These results strongly suggest that C is not a new sigma factor but acts as an activator for E sigma 70-dependent transcription. Antibiot Khimioter, 1990 Mar, 35(3), 25 - 7 {Double-stranded RNA from phage F6: its interferon-inducing and antiviral properties}; Nosik NN et al.; Double stranded RNA was isolated from bacteriophage phi 6 parasitizing on phytobacteria . Its interferon-inducing and antiviral activities were shown in vitro and in vivo . In the culture of L-929 cells, interferon resistant to heat and acids was synthesized . The interferon could be practically completely neutralized by specific anti-interferon serum . The phage phi 6 preparation dsRNA in the lyophilized form was studied . The preparation retained its biological activity . It was shown that a preparation containing 30 per cent of dsRNA was less toxic than a preparation containing 100 per cent of dsRNA, the difference in the interferon-inducing activity being insignificant. J Bacteriol, 1990 Mar, 172(3), 1660 - 2 Isolation of an Lc-specific Escherichia coli bacteriophage; Fralick JA et al.; We isolated an OmpF-specific bacteriophage whose host range mutant, SQ108h2, requires the presence of the Lc porin for its attachment and which can be used to screen or select for Lc-defective mutants among Escherichia coli K-12 strains lysogenic for the PA-2 converting phage. J Bacteriol, 1990 Mar, 172(3), 1174 - 9 Structure and function of conjugative pili: monoclonal antibodies as probes for structural variants of F pili; Grossman TH et al.; The lac-tra operon fusion plasmid pTG801 contains the known F plasmid DNA transfer (tra) genes required by Escherichia coli to elaborate functional F pili (T . Grossman and P . M . Silverman, J . Bacteriol . 171:650-656, 1989) . Here, we show that these pili are actually structural variants of normal F pili and that the F plasmid must contain additional genes that affect pilus structure and function . We confirmed a previous report that two monoclonal antibodies that recognize epitopes at and near the amino terminus of F pilin do not decorate the sides of normal F pili, as determined by immunogold electron microscopy . However, both antibodies laterally decorated pTG801 pili . The epitope for one of the antibodies has been shown to include the amino-terminal acetyl group of F pilin, which must therefore also be present on pTG801 pilin . Normal antibody staining was restored to pTG801 pili when cells contained, in addition to pTG801, the compatible plasmid pRS31, which must therefore include at least one gene affecting F-pilus structure . One candidate, traD, was excluded as the sole such gene, since traD+ derivatives of a pTG801 strain still elaborated pili that could be laterally decorated with antibody . Moreover, although traD alone restored RNA bacteriophage R17 infectivity to pTG801 cells, as expected, it did not mimic pRS31 in restoring to pTG801 pili other characteristics of normal F pili . We conclude that pRS31 contains as yet uncharacterized genes required for elaboration of structurally normal F pili . Finally, we identified vesicular material, especially abundant in cultures of pTG801 transformants, that stained heavily with the anti-F-pilin monoclonal antibodies . This material may reflect the inner membrane pool of F pilin. Nucleic Acids Res, 1990 Feb 25, 18(4), 865 - 70 Readthrough transcription occurs at the rho dependent signal F1 TIV in suppressor cells; La Farina M et al.; Suppressor cells infected with bacteriophage f1 yield phage encoded gene IV transcripts longer than those present in the supo host and identical to those found in a rho- host . However, such longer transcripts do not appear in the suppressor-infected cell when, by changing the translation frame of gene IV, the ribosome is not allowed to proceed to the end of the gene IV message and thus to reach the rho dependent transcription terminator f1 TIV . This suggests that ribosome movement beyond the natural gene IV stop codon disturbs the activity of that termination signal . In contrast to the rho- behaviour, the suppressor does not accumulate high levels of gene IV messages indicating that the accumulation occurring in the rho- mutant may not be a primary effect of the readthrough per se. J Biol Chem, 1990 Feb 25, 265(6), 3022 - 9 Physical interactions between bacteriophage and Escherichia coli proteins required for initiation of lambda DNA replication; Liberek K et al.; The process of initiation of lambda DNA replication requires the assembly of the proper nucleoprotein complex at the origin of replication, ori lambda . The complex is composed of both phage and host-coded proteins . The lambda O initiator protein binds specifically to ori lambda . The lambda P initiator protein binds to both lambda O and the host-coded dnaB helicase, giving rise to an ori lambda DNA.lambda O.lambda P.dnaB structure . The dnaK and dnaJ heat shock proteins have been shown capable of dissociating this complex . The thus freed dnaB helicase unwinds the duplex DNA template at the replication fork . In this report, through cross-linking, size chromatography, and protein affinity chromatography, we document some of the protein-protein interactions occurring at ori lambda . Our results show that the dnaK protein specifically interacts with both lambda O and lambda P, and that the dnaJ protein specifically interacts with the dnaB helicase. Nature, 1990 Feb 22, 343(6260), 719 - 26 In vitro replication through nucleosomes without histone displacement; Bonne-Andrea C et al.; A well-characterized set of proteins encoded by bacteriophage T4 replicates DNA in vitro and generates replication forks that can pass nucleosomes . The histone octamers remain associated with newly replicated DNA even in the presence of excess DNA competitor, and intact nucleosomes re-form on the two daughter DNA helices . It is concluded that nucleosomes are designed to open up transiently to allow the passage of a replication fork without histone displacement. Biochemistry, 1990 Feb 20, 29(7), 1791 - 8 Effects of the bacteriophage T4 gene 41 and gene 32 proteins on RNA primer synthesis: coupling of leading- and lagging-strand DNA synthesis at a replication fork; Cha TA et al.; We have demonstrated previously that the template sequences 5'-GTT-3' and 5'-GCT-3' serve as necessary and sufficient signals for the initiation of new DNA chains that start with pentaribonucleotide primers of sequence pppApCpNpNpN or pppGpCpNpNpN, respectively . Normally, the complete T4 primosome, consisting of the T4 gene 41 (DNA helicase) and gene 61 (primase) proteins, is required to produce RNA primers . However, a high concentration of the 61 protein alone can prime DNA chain starts from the GCT sites {Cha, T.-A., & Alberts, B . M . (1986) J . Biol . Chem . 261, 7001-7010} . We show here that the 61 protein can catalyze a single-stranded DNA template-dependent reaction in which the dimers pppApC and pppGpC are the major products and much longer oligomers of various lengths are minor ones . Further addition of the 41 protein is needed to form a primosome that catalyzes efficient synthesis of the physiologically relevant pentaribonucleotides that are responsible for the de novo DNA chain starts on the lagging strand of a replication fork . The helicase activity of the 41 protein is necessary and sufficient to ensure a high rate and processivity of DNA synthesis on the leading strand {Cha, T.-A., & Alberts, B . M . (1989) J . Biol . Chem . 264, 12220-12225} . Coupling an RNA primase to this helicase in the primosome therefore coordinates the leading- and lagging-strand DNA syntheses at a DNA replication fork . Our experiments reveal that the addition of the T4 helix-destabilizing protein (the gene 32 protein) is required to confine the synthesis of RNA primers to those sites where they are used to start an Okazaki fragment, causing many potential priming sites to be passed by the primosome without triggering primer synthesis. Biochemistry, 1990 Feb 20, 29(7), 1737 - 43 Response of phage T4 polynucleotide kinase toward dinucleotides containing apurinic sites: design of a 32P-postlabeling assay for apurinic sites in DNA; Weinfeld M et al.; We have examined the capacity of bacteriophage T4 polynucleotide kinase (EC 2.7.1.78) to phosphorylate the partially depurinated products of d-ApA, namely, d-SpA and d-ApS (where S represents an apurinic deoxyribose group) . It was observed that the enzyme acted only on the latter isomer . Since molecules of this type (d-NpS) are the sole apurinic site containing products resulting from the combined digestion of lightly depurinated DNA by snake venom phosphodiesterase and calf alkaline phosphatase {Weinfeld, M., Liuzzi, M., & Paterson, M . C . (1989) Nucleic Acids Res . 17, 3735-3745}, we were able to devise a postlabeling assay for these biologically important DNA lesions . The method offers several advantages, including (a) elimination of the need for prelabeled DNA, (b) high (femtomole range) sensitivity, and (c) nearest-neighbor analysis of bases 5' to apurinic/apyrimidinic sites . Using this assay, we obtained a value for the rate of depurination of form I pRSVneo plasmid DNA, incubated at pH 5.2 at 70 degrees C, of approximately 3.3 apurinic sites per plasmid molecule per hour . This value compares favorably with previously published data of others, acquired by alternative approaches . The rate of depurination of poly(dA), treated in a similar fashion, was found to be approximately 1 base per 10(3) nucleotides per hour. J Biol Chem, 1990 Feb 15, 265(5), 2596 - 602 A new sialic acid analogue, 9-O-acetyl-deaminated neuraminic acid, and alpha -2,8-linked O-acetylated poly(N-glycolylneuraminyl) chains in a novel polysialoglycoprotein from salmon eggs; Iwasaki M et al.; A new polysialoglycoprotein, designated PSGP(On), was isolated from the unfertilized eggs of the kokanee salmon, Oncorhynchus nerka adonis . 400-MHz 1H NMR analyses showed the O . nerka adonis PSGP contained alpha -2,8-linked oligo- and polysialic acid (polySia) chains that were made up of 4-O-Ac-, 7-O-Ac-, and 9-O-Ac esters of N-glycolylneuraminic acid (Neu5Gc) residues . The presence of a new sialic acid derivative, identified by 1H NMR as 9-O-acetyl-2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (trivial name, 9-O-acetyldeaminated neuraminic acid; 9-O-Ac-KDN), was also shown to be present as a minor component . The O-acetylated KDN residues appear to cap the nonreducing termini of the O-acetylated poly(Neu5Gc) chains . The O-acetylated polySia chains were resistant to depolymerization by bacterial exosialidases and a bacteriophage-derived endo-N-acylneuraminidase that is specific for catalyzing the hydrolysis of alpha -2,8-linkages in polySia containing either N-acetylneuraminic acid or Neu5Gc residues . After de-O-acetylation by mild alkali, the polySia chains were sensitive to digestion by endo-N-acylneuraminidase, yet partially resistant to exosialidase . These data confirm the alpha -2,8-ketosidic linkage in these chains and the nonreducing terminal location of the KDN residues . These results extend further the range of structural diversity in polySia-containing glycoconjugates, and in the family of naturally occurring sialic acids . They also suggest that the O-acetylated Neu5Gc and 9-O-Ac-KDN residues may have an important role during oogenesis. Anal Biochem, 1990 Feb 15, 185(1), 187 - 93 Generation of deletion subclones for sequencing by partial digestion with restriction endonucleases; Lamperti ED et al.; A method for creating a group of deletion subclones for DNA sequencing by partial digestion of M13 bacteriophage constructions is outlined . The M13 construct is linearized at a unique site and then subjected to partial digestion with a frequent-cutting restriction endonuclease . The insert is truncated at different locations . The vector DNA is also partially digested . The products of a single partial digestion are repaired, recircularized by ligation, and used for bacterial transfection to generate subclones with a spectrum of deletions in the insert; most deletions in the vector DNA will disrupt vital viral genes and will thus disappear in the transfection . The subclones are sorted by size by gel electrophoresis of single-stranded viral DNA . This method is simpler and thus may be more reliable than established subcloning schemes. Gene, 1990 Feb 14, 86(2), 275 - 8 Deduced amino acid sequence of mouse blood-coagulation factor IX; Wu SM et al.; A mouse fetal liver cDNA library was screened with a cDNA clone encoding human blood coagulation factor IX protein (hBCFIX) . A bacteriophage lambda clone was isolated and the nucleotide sequence of a 2710-bp insert was determined . An open reading frame of 459 amino acids (aa) was identified within the sequence that has an 80% sequence similarity with hBCFIX . The cDNA contains a long 3'-untranslated sequence similar to that of BcfIX gene from human and canine sources . However, instead of a sequence that might form two hair-pins such as those found in hBcfIX, a (GA)16 repeat that has been reported to form H-DNA {Htun and Dahlberg, Science 241 (1988) 1791-1796; Johnston, Science 241 (1988) 1800-1804} was found in the 3'-untranslated region . The predicted aa sequence of mouse BCFIX serves as a comparative sequence for identifying key residues within hBCFIX where epitopes recognized by monoclonal antibodies produced from an immunized mouse are compared with respect to the human and mouse primary BCFIX sequence. FEBS Lett, 1990 Feb 12, 261(1), 1 - 4 Structure of the DNA binding wing of the gene-V encoded single- stranded DNA binding protein of the filamentous bacteriophage M13; van Duynhoven JP et al.; The structure in solution of a beta-loop in mutant Y41H of the single-stranded DNA binding protein encoded by gene-V of the filamentous phage M13 has been elucidated using 2-dimensional 1H-nuclear magnetic resonance techniques . Furthermore, these studies enabled us to demonstrate that an identical structural element is present in wild-type gene-V-protein and that this element intimately is involved in the binding of gene-V-protein to single-stranded DNA . It is shown that the structure of the DNA binding wing deviates from that proposed for the same amino acid sequence on the basis of X-ray diffraction data . The structure is, however, identical to that of the DNA binding wing present in the single-stranded DNA binding protein encoded by the genome of the evolutionary distantly related filamentous phage IKe . The latter observations support our current view that in the binding of these proteins to single-stranded DNA a common structural motif is involved. J Biol Chem, 1990 Feb 5, 265(4), 1903 - 12 Sequence analysis of the translational elongation factor 3 from Saccharomyces cerevisiae; Qin SL et al.; The gene YEF-3 encoding the elongation factor for protein synthesis in Saccharomyces cerevisiae is an essential gene as shown by one-step gene disruption and is located on chromosome XII as determined by orthogonal field alternation gel electrophoresis . The nucleotide sequence of the gene was determined from a sequential series of subclones generated from the YEF-3 gene cloned into bacteriophage M13 . The HOMOL1 sequence and the RPG box, which are considered to be enhancer elements involved in coordinate regulation of transcription of the genes coding for yeast ribosomal proteins and protein synthesis factors, are found in the 5'-flanking region of the gene . A dyad symmetry that enables hairpin loop formation in the DNA molecule is found in the 3'-terminal at the termination site of transcription . An open reading frame of 3132 nucleotides codes for a deduced protein of 115,860 Da . A striking feature of the elongation factor 3 deduced polypeptide is the internal repeat of a region with approximately 200 amino acids which includes an ATP-binding site and shares similarity with some transport and drug-resistant proteins . Another characteristic is the presence of a highly charged C-terminal region composed of three basic polylysine blocks, suggesting interaction with RNA . The sequence supports the hypothesis that YEF-3 encodes a protein synthesis factor and suggests that its main role may be to transduce nucleoside triphosphate energy into mechanical energy for translocation during translation. J Mol Biol, 1990 Feb 5, 211(3), 537 - 49 Deletion-tolerance and trans-splicing of the bacteriophage T4 td intron . Analysis of the P6-L6a region; Galloway Salvo JL et al.; Non-directed mutagenesis and phylogenetic comparison suggest that certain elements of the bacteriophage T4 td group Ia intron are dispensable to self-splicing . The L6-P6a-L6a region was identified as a potential non-essential element, and was removed by sequential deletions extending from the L6a loop toward the P6 pairing . Assays for splicing indicate that as long as the P6 pairing is maintained, the 1016 nucleotide td intron can be reduced to less than 250 nucleotides while maintaining function in vivo and in vitro . The P6 pairing appears to be essential for splicing while P6a is not . In addition, a spontaneous pseudorevertant of a splicing-defective deletion was isolated and shown to result from a single nucleotide change in the predicted L6a loop . This genetic suppressor mimics the ability of Mg2+ to reverse the phenotype of the deletion, suggesting that function is restored by structural stabilization of P6 . The tolerance of this region to deletion prompted us to split the ribozyme core in L6a, to generate precursors that might function in trans . Indeed, the two half-molecules do associate to form a bimolecular complex that yields accurately ligated exons both in vitro and in vivo . The biological implications of these results, as well as the usefulness of trans-splicing for generating unprocessed precursors in vitro are discussed. Biotechniques, 1990 Feb, 8(2), 184 - 9 Optimization of asymmetric polymerase chain reaction for rapid fluorescent DNA sequencing; Wilson RK et al.; A high-throughput method for the preparation of single-stranded template DNA, which is suitable for sequence analysis using fluorescent labeling chemistry, is described here . In this procedure, the asymmetric polymerase chain reaction is employed to amplify recombinant plasmid or bacteriophage DNA directly from colonies or plaques . The use of amplification primers located at least 200 base pairs 5' to the site of sequencing primer annealing removes the need for extensive purification of the asymmetric polymerase chain reaction product . Instead, the single-stranded product DNA is purified by a simple isopropanol precipitation step and then directly sequenced using fluorescent dye-labeled oligonucleotides . This method significantly reduces the time and labor required for template preparation and improves fluorescent DNA sequencing strategies by providing a much more uniform yield of single-stranded DNA. J Biomol Struct Dyn, 1990 Feb, 7(4), 943 - 57 A refined calculation of the solution dimensions of the complex between gene 32 protein and single stranded DNA based on estimates of the bending persistence length; Kuil ME et al.; The rotation diffusion coefficient of a complex of GP32, the single stranded DNA binding protein of the bacteriophage T4, with a single stranded DNA fragment with about 270 bases was determined to obtain further information on the flexibility of this particle . The rotation diffusion of these molecules is used as a sensitive measure of the flexibility of different DNA protein complexes . Using the theory of Hagerman and Zimm (Biopolymers 20, 1481 (1981)) and assuming a bending persistence length of about 35 nanometer it can be shown that the axial increment for GP32 complexes with single stranded DNA is close to 0.5 nm per base . The value for the bending persistence length is in agreement with values found for much larger DNA protein complexes using light scattering experiments . This value for the persistence length also implies that the complex is thin . The radius is estimated to be around 1.7 nm, which shows a moderate degree of hydration . With this set of parameters we can describe all the hydrodynamic experiments on GP32 complexes from 76 to more than 7000 bases obtained using electric birefringence, quasi-elastic light scattering and sedimentation experiments performed in our group over the last few years. J Biomol Struct Dyn, 1990 Feb, 7(4), 773 - 94 Solution structure of bacteriophage T4D and icosahedral capsid geometry visualized in freeze-fractured, deep-etched replicas; Aksiyote-Benbasat J et al.; The prolate icosahedral capsid geometry of wild type bacteriophage T4D has been determined by direct visualization of the triangular faces in stereoimages of transmission electron micrographs of phage particles . Bacteriophage T4 was prepared for transmission electron microscopy (TEM) following a protocol of freeze-fracturing, deep-etching (FDET) and replication by vertical deposition (80 degrees angle) of a thin platinum-carbon (Pt-C) metal layer of 1.01 nm . From direct statistical measurements of the ratio of the head length to width and of stereometric angles on T4 heads, we have estimated a Q number of 21 . This confirms previous indirect studies on T4 and agrees with determinations on bacteriophage T2 . Many of the structural features of T4 observed in FDET preparations differ significantly from those observed by classical negative staining methods for TEM imaging . Most important among the differences are the conformation of the baseplate (a closed rosebud) and the positioning of the tail fibers (retracted) . The retracted position of the tail fibers in the FDET preparations has been confirmed by negatively staining phage previously fixed suspended in solution with 2% glutaraldehyde . The FDET protocols appear to reveal important structural features not seen in negative stained preparations . These have implications for bacteriophage T4 conformation in solution, viral assembly and phage conformation states prior to tail contraction and DNA ejection. Genetics, 1990 Feb, 124(2), 213 - 20 Genetic evidence for two protein domains and a potential new activity in bacteriophage T4 DNA polymerase; Reha-Krantz LJ; Intragenic complementation was detected within the bacteriophage T4 DNA polymerase gene . Complementation was observed between specific amino (N)-terminal, temperature-sensitive (ts) mutator mutants and more carboxy (C)-terminal mutants lacking DNA polymerase polymerizing functions . Protein sequences surrounding N-terminal mutation sites are similar to sequences found in Escherichia coli ribonuclease H (RNase H) and in the 5'----3' exonuclease domain of E . coli DNA polymerase I . These observations suggest that T4 DNA polymerase, like E . coli DNA polymerase I, contains a discrete N-terminal domain. Virology, 1990 Feb, 174(2), 585 - 92 In vivo analysis of the initiation of bacteriophage T7 DNA replication; Rabkin SD et al.; We have examined the initiation of bacteriophage T7 DNA replication in vivo using a pulse-labeling technique . The pulse-labeling technique permits the rapid identification of initiation sites on the T7 chromosome and a determination of the rate of movement of the replication fork . This technique has been used to analyze a number of phage mutants having alterations in the nucleotide sequence of the primary origin . The experiments confirm the results obtained by electron microscope analysis on the mapping of the primary origin region and demonstrate the requirement for a T7 promoter in the primary origin . The secondary origins were found to be located near the center and at the right end of the genome . Analysis of T7 phage harboring mutations in the essential replication genes of T7 shows that they fell into three classes . The first, including those mutated in genes 4 and 5, do not initiate DNA synthesis . The second, in genes 3, 6, and 1.2, initiate and elongate as wild-type phage, albeit some with lower rates of synthesis, during the first round of replication and then cease DNA synthesis . Mutations in gene 2 have no apparent effect on initiation or elongation. Virology, 1990 Feb, 174(2), 472 - 8 Quantized viral DNA packaging revealed by rotating gel electrophoresis; Lane T et al.; Two classes of missense mutations in the bacteriophage T4 gene coding for the major head protein produce phage with different length heads . The pt (petite) mutations produce phage with normal, intermediate, and isometric heads, whereas ptg (petite and giant) mutations also produce greatly elongated (giant) heads . DNA from petite, normal, and giant particles was clearly resolved by discontinuous rotating gel electrophoresis, and several new species of headful length DNA were found . These results confirm the idea that the major stop points for head length regulation are at Q = 13, 17, and 21, and also show that