Bacteriophages

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 minor stop points exist at Q = 16, 18 and 20 . The existence of these well-defined classes of DNA that correlate with capsid structure suggest that a structural relationship between the scaffold protein and the capsid protein determines head length and thus DNA length.

J Bacteriol, 1990 Feb, 172(2), 1129 - 32
Determination of the packaging capacity of bacteriophage VWB; Anne J et al.; VWB is a temperate bacteriophage whose chromosome has cohesive ends . VWB can stably package modified chromosomes that contain insertions of up to about 4 kilobases of foreign DNA . Phage particles containing extra DNA differ from the wild type in their increased sensitivity to chelating agents . Because of these properties, VWB is a promising cloning vector for Streptomyces venezuelae.

Proc Natl Acad Sci U S A, 1990 Feb, 87(4), 1501 - 5
Cloning and expression of a protein-tyrosine-phosphatase; Guan KL et al.; A rat brain cDNA library was screened by using a mixture of oligonucleotides whose sequences were deduced from the amino acid sequence of a human placental protein-tyrosine-phosphatase (PTPase; EC 3.1.3.48) reported by Charbonneau et al . {Charbonneau, H., Tonks, N . K., Walsh, K . A . & Fischer, E . H . (1988) Proc . Natl . Acad . Sci . USA 85, 7182-7186} . The isolated clones encode a PTPase of 432 amino acids having a mass of 49,679 daltons and showing 97% sequence identity to the corresponding 321 residues of the placental enzyme . The coding sequence of the PTPase was placed behind a bacteriophage T7 promoter and the protein was expressed in Escherichia coli . The recombinant protein had a molecular weight of 50,000 by SDS/PAGE analysis and showed an absolute specificity for phosphotyrosine-containing substrates . Northern analysis documented that there were two sizes of RNA, 4.3 and 2.0 kilobases, which encode the PTPase . Both transcripts were present in a number of tissues, and the smaller RNA appears to arise by the use of an alternative polyadenylylation signal . The PTPase was also localized by in situ hybridization in the rat central nervous system . A diffuse pattern of hybridization signal is seen in a number of brain areas, with the hippocampus showing the highest levels of mRNA . Sequences located at the C terminus of the rat brain PTPase contain possible sites for phosphorylation as well as signals which could serve for membrane attachment.

Biochimie, 1990 Feb-Mar, 72(2-3), 191 - 200
Phi X174 protein E-mediated lysis of Escherichia coli; Witte A et al.; Bacteriophage PhiX174 encodes a single lysis gene, E, the function of which is necessary and sufficient to induce lysis of Escherichia coli . Here we present a novel model for E-lysis: physiological, genetic and biochemical data are presented which suggest that a transmembrane tunnel penetrating the inner and outer membrane is formed during the lytic action of protein E . Moreover, using high magnification scanning and transmission electron microscopy in this study, it was possible to visualize the transmembrane lysis structure directly.

Genet Anal Tech Appl, 1990 Feb, 7(1), 2 - 4
Purification of bacteriophage DNA by gel filtration chromatography; Gonzalez A et al.; Two fast and effective methods for high-scale purification of linear phage lambda DNA and circular double-stranded M13 replicative form are presented . A substantial reduction of time is attained by avoiding the long-term CsCl gradient centrifugations and dialysis common to standard procedures . Biologically active DNA preparations, free of chromosomal DNA and RNA, are obtained by including a simple gel filtration chromatography as the last step of purification . Yields are comparable to those from previously described methods.

Genet Anal Tech Appl, 1990 Feb, 7(1), 18 - 23
Direct cloning of cDNA inserts from lambda gt11 phage DNA into a plasmid vector by a novel and simple method; Chiu IM et al.; Bacteriophage lambda gt11 has been used quite extensively for producing cDNA libraries . The cDNA inserts are usually subcloned into a plasmid vector for large scale production and analysis . However, isolating the recombinant DNA of interest from the phage clones can be a tedious task . Since the E . coli strain Y1088 used for lambda gt11 phage infection carries a pBR322-derived plasmid endogenously, we reasoned that this endogenous plasmid could be used directly for cloning the cDNA phage insert . In this report, we describe a method in which cDNA inserts from lambda gt11 phage were cloned directly into the pBR322 plasmid vector, bypassing the time-consuming procedures of preparing plasmid DNA as a subcloning vector . This method is likely to be extended to the cloning of DNA inserts derived from other phage lambda vectors when bacteria containing endogenous pBR322 are used as host cells.

J Bacteriol, 1990 Feb, 172(2), 912 - 21
Oligomerization of the bacteriophage lambda S protein in the inner membrane of Escherichia coli; Zagotta MT et al.; Western blot (immunoblot) analysis of cell extracts from induced bacteriophage lambda lysogens probed with S-protein-specific antibody (raised against an S--beta-galactosidase fusion protein) demonstrated that the bacteriophage lambda S protein begins to appear 10 min after phage induction and is localized to the inner membrane at all times during the lytic cycle . Between 100 and 1,000 molecules of S protein per cell were present at the time of phage-induced lysis . Western blots of chemically cross-linked membranes from induced lysogens showed a ladder of bands at 18, 24, 32, and 42 kilodaltons (the S-protein monomer ran at 8 kilodaltons) that reacted with anti-S-protein antibody . Thus, the S protein appears to reside in the inner membrane as a multimer, and the molecular weights of the cross-linked species are consistent with those of S-protein homopolymers . Sodium dodecyl sulfate-resistant dimers were also detected when S protein was purified by immunoprecipitation.

J Bacteriol, 1990 Feb, 172(2), 1030 - 4
Transcription of a bacteriophage lambda DNA site blocks growth of Escherichia coli; Guzman P et al.; The rap mutation in Escherichia coli prevents the growth of bacteriophage lambda . Phage mutations that overcome rap inhibition (bar) have been mapped to loci in the pL operon . We cloned and sequenced three mutations in two of these loci: barIa to the left arm of the lambda attachment site (attP) and barII in the ssb (ea10) gene . The mutations represent single base-pair changes within nearly identical 16-base-pair DNA segments . Each mutation disrupts a sequence of dyad symmetry within the segment . Plasmids carrying a bar+ sequence downstream to an active promoter are lethal to rap, but not rap+, bacteria . The bar sequences isolated from the lambda bar mutants are not lethal . We synthesized a minimal lambda barIa+ sequence, 5'-TATATTGATATTTATATCATT, and cloned it downstream to an inducible promoter . When transcribed, this sequence is sufficient to kill a rap strain.

Semin Cell Biol, 1990 Feb, 1(1), 19 - 25
The Escherichia coli groE chaperonins; Georgopoulos C et al.; The E.coli groES and groEL genes have been shown to form an operon, to be essential for E . coli viability, and to belong to the so-called heat-shock class of genes whose expression is regulated by the intracellular levels of sigma factor sigma 32 . Both groE chaperonin proteins possess a seven-fold axis of symmetry, groES being composed of seven identical subunits of 97 amino acids each, and groEL of fourteen identical subunits of 548 amino acids each . The two groE chaperonins interact intimately as judged by both genetic and biochemical criteria . This interaction has been shown to be required for both bacteriophage morphogenesis and bacterial growth . The groEL chaperonin has been shown to bind to a number of incomplete or unfolded polypeptides in vitro . Such binding may prevent misfolding and promote rapid intra- or intermolecular folding of polypeptides in vivo . The proposed role of the groES chaperonin is to displace the polypeptides bound to groEL, thus effectively promoting the recycling of groEL.

Forensic Sci Int, 1990 Feb, 44(2-3), 209 - 24
Isolation of the DNA minisatellite probe MZ 1.3 and its application to DNA 'fingerprinting' analysis; Schacker U et al.; A minisatellite probe, MZ 1.3, detecting hypervariable fragment patterns was isolated from a human genomic library . A repetitive sequence of 27 bp length was identified which is contained in the probe approx . 40 times . The MZ 1.3 repeat shows variable homology of 53-73% to the repetitive sequence of the protein III gene of the bacteriophage M13 genome . Polymorphic restriction fragment patterns were found with MZ 1.3 using the enzymes Hinf I, BstN I, Hae III, Mbo I, PstI/Pvu II, and Rsa I . An average of 18 polymorphic fragments was observed using Hinf I as enzyme . The band sharing frequency after Hinf I digestion among unrelated individuals was determined to be 23.8 +/- 7.2% . An example for the application of MZ 1.3 to paternity testing in an incest case is given . The probe can be used with radioactive or non-radioactive detection systems . An approach is presented to compare polymorphic fragment patterns from individuals obtained by independent gel runs on the basis of relative band positions (RBP) and calculated in a computerized analysis.

Hum Genet, 1990 Feb, 84(3), 219 - 22
Isolation of anonymous, polymorphic DNA fragments from human chromosome 22q12-qter; Dumanski JP et al.; A series of 195 random chromosome 22-specific probes, equivalent to approximately 1% of the size of this chromosome, have been isolated from a chromosome 22-specific bacteriophage lambda genomic library . These probes were mapped to four different regions of chromosome 22 on a panel of five somatic cell hybrids . Restriction fragment length polymorphisms were detected by 28 of the probes mapping to 22q12-qter . Evolutionarily conserved sequences in human, mouse, and Chinese hamster DNA were detected by 12% of the isolated probes.

Proc Natl Acad Sci U S A, 1990 Feb, 87(3), 1023 - 7
Functions of replication factor C and proliferating-cell nuclear antigen: functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4; Tsurimoto T et al.; The proliferating-cell nuclear antigen (PCNA) and the replication factors A and C (RF-A and RF-C) are cellular proteins essential for complete elongation of DNA during synthesis from the simian virus 40 origin of DNA replication in vitro . All three cooperate to stimulate processive DNA synthesis by DNA polymerase delta on a primed single-stranded M13 template DNA and as such can be categorized as DNA polymerase accessory proteins . Biochemical analyses with highly purified RF-C and PCNA have demonstrated functions that are completely analogous to the functions of bacteriophage T4 DNA polymerase accessory proteins . A primer-template-specific DNA binding activity and a DNA-dependent ATPase activity copurified with the multisubunit protein RF-C and are similar to the functions of the phage T4 gene 44/62 protein complex . Furthermore, PCNA stimulated the RF-C ATPase activity and is, therefore, analogous to the phage T4 gene 45 protein, which stimulates the ATPase function of the gene 44/62 protein complex . Indeed, some primary sequence similarities between human PCNA and the phage T4 gene 45 protein could be detected . These results demonstrate a striking conservation of the DNA replication apparatus in human cells and bacteriophage T4.

New Biol, 1990 Feb, 2(2), 151 - 62
Generation of a 50,000-member human DNA library with an average DNA insert size of 75-100 kbp in a bacteriophage P1 cloning vector; Sternberg N et al.; A bacteriophage P1 cloning system that permits the isolation and amplification of cloned DNA fragments as large as 100 kbp was described previously . We have now utilized a similar system to generate a 50,000-member human DNA library with DNA inserts ranging in size from 75 to 100 kbp . Two major obstacles were overcome in constructing the library . The first concerned the mcrAB restriction system of Escherichia coli, which degrades DNA containing MeC and interferes with the recovery of cloned human DNA inserts . In the P1 cloning system, the effect of the Mcr restriction activity is to decrease recovery of cloned inserts by about 35-fold when the activity is in the host cell line and by about 3-fold when the activity is in the cells used to prepare the packaging extract . To circumvent this problem we inactivated, by mutation, the McrAB proteins in both components of the cloning system . The second obstacle concerned the preferential cloning of small DNA fragments from a population of fragments ranging in size from 20 to 100 kbp . To deal with this problem we first modified the P1 lysogen used to prepare the in vitro head-tail packaging extract so that it would produce 12 times as many large P1 heads (head capacity about 110 kbp) as small P1 heads (head capacity about 45 kbp) . We then restructured the P1 cloning vector so that it could be used to produce vector "arm" fragments that could be ligated to insert DNA at only one end . This prevented the formation of long concatamers consisting of alternating units of vector and insert DNA and prohibited the packaging of small inserts in large phage heads . Using the insert-biased large head extract, the arms vector, and size-selected human DNA fragments, we showed that as much as 90% of recovered transformants contained inserts in the desired high molecular weight range.

Anticancer Drug Des, 1990 Feb, 5(1), 21 - 9
New insight into drug-DNA interactions at individual drug binding sites probed by RNA polymerase during active transcription of the DNA; Phillips DR et al.; An in vitro transcription assay has been used to probe drug-DNA interactions during active transcription of the DNA . The method relies upon the formation of a stable, synchronized population of initiated transcripts comprising a short length of RNA, achieved by the absence of one nucleotide in the initiation mixture . Subsequent equilibration of the transcription complex with drug, followed by elongation of the initiated transcripts, yields lengths of RNA determined by transcription up to each drug-occupied site . A variety of site-specific phenomena have been observed, including delayed termination of transcription 5-10 bp downstream of actinomycin binding sites; 10-20% probability of termination at most echinomycin sites; drug residence-time-dependent termination of bacteriophage polymerases; enhanced residence time compared to physicochemical measurement of drug-DNA dissociation rates . The use of two counter-directed promoters separated by 100 bp results in a sensitive bidirectional transcription footprinting procedure able to resolve adjacent drug sites separated by only 1 bp . The significance of the method of in vitro transcription is the ability to quantitate a range of parameters describing individual drug sites in situations where multiple drug-DNA interactions exist.

Proc Natl Acad Sci U S A, 1990 Feb, 87(3), 1057 - 60
Nucleotide and amino acid sequence of lymphocyte-derived corticotropin: endotoxin induction of a truncated peptide; Smith EM et al.; To determine the degree of similarity between pituitary and lymphocyte proopiomelanocortin, the lymphocyte mRNA was reverse transcribed, cloned, and sequenced . Murine lymphocyte mRNA was first purified by oligo(dT)-cellulose affinity chromatography and was reverse transcribed by using a selective 3' antisense oligonucleotide primer directed at the boundary between the translated/nontranslated region on the 3' end of exon 3 . This cDNA was then amplified in a polymerase chain reaction with selective primers containing Sal I and Kpn I restriction endonuclease sites . Amplified cDNA was then directionally ligated into M13mp18 and M13mp19 bacteriophage and was sequenced . The nucleotide sequence encoding this peptide was identical to that of mouse pituitary corticotropin (ACTH) . Elevated levels of lymphocyte immunoreactive ACTH were then induced with bacterial lipopolysaccharide and the peptide(s) was purified by antibody affinity chromatography and reverse-phase high-performance liquid chromatography . The predominant immunoreactive ACTH species was approximately 3 kDa and its sequence was identical to pituitary ACTH(1-25) . These results conclusively demonstrate that lymphocytes produce authentic ACTH and harbor its mRNA.

Biochim Biophys Acta, 1990 Jan 30, 1048(1), 43 - 9
Properties of the bacteriophage alpha 3 mutants with deletion and/or insertion in the complementary strand origin; Nakano K et al.; Bacteriophage alpha 3 origin of complementary strand DNA synthesis (-ori) contains two potential secondary loop structures (I and II), which have been implicated in direct recognition sites for host Escherichia coli dnaG protein . We have introduced nucleotide deletion or insertion within the -ori region, by nuclease digestion and polymerase treatment of alpha 3 replicative form DNA . Deletion mutants (delo) showing the following in vivo properties were isolated: minute plaque size, longer latent period, and reduction both in phage yield and viral DNA synthesis as compared with the wild-type phage . In addition, several strains among the delo mutants did not grow on host E . coli dnaB cells at 42 degrees C . These results suggest that certain type of deletion in the -ori region converts simple priming of the complementary strand synthesis to a more complicated phi X174-type initiation that depends on the primosome, rather than on dnaG protein alone.

FEBS Lett, 1990 Jan 29, 260(2), 273 - 6
Correlation between the DNA supercoiling and the initiation of transcription by Escherichia coli RNA polymerase in vitro: role of the sequences upstream of the promoter region; Mishra RK et al.; Binding of Escherichia coli RNA polymerase and the abortive initiation of transcription at the A2 promoter of bacteriophage T7, separately cloned in pBR322, was found to be strongly dependent on the degree of supercoiling of the plasmid . Supercoiling does not seem to play any role in the initiation of transcription at the T7A1 promoter under identical conditions . Plasmid containing T7A2 promoter was found to be less amenable to S1 nuclease in comparison to that having T7A1 . Sequence comparison reveals a high G/C content upstream to the -35 region of T7A2 which by extra duplex stability probably renders the initiation of transcription more dependent on the state of supercoiling of the template.

Eur J Biochem, 1990 Jan 26, 187(2), 361 - 71
Selection of the 5'-proximal translation initiation site is influenced by mRNA and eIF-2 concentrations; Dasso MC et al.; A cDNA clone of the influenza virus NS (non-structural protein) gene in a vector carrying a bacteriophage T7 RNA polymerase promoter was manipulated so as to reiterate the initiation site to give two in-frame AUG codons 57 nucleotide residues apart . Each initiation site was in either a preferred context (...AUAAUGG...) or a less favourable context (...UUUAUGG...) and the four possible permutations were constructed . When capped mRNA transcripts of these clones were translated in the rabbit reticulocyte lysate system, products from initiation at both AUG codons were observed . At low RNA concentrations the frequency of initiation at the 5'-proximal AUG codon rather than the second was higher when the first AUG codon was in the preferred context, in qualitative agreement with the scanning ribosome model . However, a completely unexpected finding was that the ratio of initiation at the first AUG codon to initiation at the second decreased with increasing mRNA concentration, irrespective of the particular context involved . Several lines of evidence indicated that the increased frequency of initiation at the second AUG codon was not due solely to the lower density of ribosome loading per mRNA at high RNA concentrations, and may therefore be the result of high RNA concentrations out-titring the capacity of endogenous reticulocyte factors responsible for preferential initiation at the 5'-proximal AUG codon . The effect of supplementing the system with purified initiation factors was examined . Only eIF-2 was capable of decreasing the frequency of initiation at the second AUG codon and promoting use of the first AUG at high mRNA concentrations; eIF-3, 4A, 4B, 4C + 4D, 4F and 5 were inactive.

J Biol Chem, 1990 Jan 25, 265(3), 1623 - 7
Immunoelectron microscopic analysis of the A, B, and HU protein content of bacteriophage Mu transpososomes; Lavoie BD et al.; Stable protein-DNA complexes or transpososomes mediate the Mu DNA strand transfer reaction in vitro (Surette, M . G., Buch, S . J., and Chaconas, G . (1987) Cell 49, 253-262; Craigie, R., and Mizuuchi, K . (1987) Cell 51, 493-501) . Formation of the Type 1 complex, an intermediate in the strand transfer reaction, requires the Mu A and Escherichia coli HU proteins . Generation of the Type 2 complex, in which the Mu ends have been covalently linked to the target DNA, requires the Mu B protein, ATP, and target DNA in addition to A and HU . The protein content of these higher order synaptic complexes has been studied by immunoelectron microscopy using protein A-colloidal gold conjugates to visualize antibody-bound complexes . Under our in vitro transposition conditions, Type 1 complexes were found to contain A and HU; in addition, Type 2 complexes contained Mu B . However, both the HU and the Mu B protein were found to be loosely associated and could be quantitatively removed from the nucleoprotein core of both complexes by incubation in 0.5 M NaCl . Depletion of HU from the Type 1 complex did not affect the ability of this complex to be converted into the strand-transferred product . Hence, the indispensable role of the HU protein in the Mu DNA strand transfer reaction is limited to the formation of the Type 1 transpososome.

Nature, 1990 Jan 25, 343(6256), 381 - 3
Immunological activity of covalently linked T-cell epitopes; Ria F et al.; Immune responses to proteins necessarily involve the recognition by T lymphocytes of a peptide or peptides derived from a protein complexed with a major histocompatibility antigen . The T-cell response of BALB/c mice to the bacteriophage lambda cI repressor protein (residues 1-102) is directed predominantly towards the epitope contained within a single peptide encompassing residues 12-26 . Similar phenomena of immunodominance of a particular peptide have also been observed in other protein systems . The mechanisms that have been suggested to account for the focusing of the T-cell response are partial deletion in the T-cell repertoire, biased antigen processing, and competition for binding to the presenting molecule, the major histocompatibility complex encoded class II transplantation antigen . In a model system with a polypeptide containing two synthetically linked immunologically active epitopes, we now demonstrate the existence of a hierarchy between these epitopes, so that the immune response elicited is directed mainly towards the more immunogenic epitope, whereas the less immunogenic epitope elicits little or no T-cell reactivity . In addition, the same hierarchy of dominance is also apparent when the polypeptide is used to induce tolerance in the periphery in adult mice . The chimaeric peptide can induce tolerance only towards the more immunogenic epitope . These experiments indicate that the rules governing antigen processing and presentation that result in T-cell activation are apparently the same as the rules that govern the processes resulting in the induction of tolerance.

J Mol Biol, 1990 Jan 20, 211(2), 447 - 63
Secondary structure of the central region of bacteriophage MS2 RNA . Conservation and biological significance; Skripkin EA et al.; The RNA of the Escherichia coli RNA phages is highly structured with 75% of the nucleotides estimated to take part in base-pairing . We have used enzymatic and chemical sensitivity of nucleotides, phylogenetic sequence comparison and the phenotypes of constructed mutants to develop a secondary structure model for the central region (900 nucleotides) of the group I phage MS2 . The RNA folds into a number of, mostly irregular, helices and is further condensed by several long-distance interactions . There is substantial conservation of helices between the related groups I and II, attesting to the relevance of discrete RNA folding . In general, the secondary structure is thought to be needed to prevent annealing of plus and minus strand and to confer protection against RNase . Superimposed, however, are features required to regulate translation and replication . The MS2 RNA section studied here contains three translational start sites, as well as the binding sites for the coat protein and the replicase enzyme . Considering the density of helices along the RNA, it is not unexpected to find that all these sites lie in helical regions . This fact, however, does not mean that these sites are recognized as secondary structure elements by their interaction partners . This holds true only for the coat protein binding site . The other four sites function in the unfolded state and the stability of the helix in which they are contained serves to negatively control their accessibility . Mutations that stabilize helices containing ribosomal binding sites reduce their efficiency and vice versa . Comparison of homologous helices in different phage RNAs indicates that base substitutions have occurred in such a way that the thermodynamic stability of the helix is maintained . The evolution of individual helices shows several distinct size-reduction patterns . We have observed codon deletions from loop areas and shortening of hairpins by base-pair deletions from either the bottom, the middle or the top of stem structures . Evidence for the coaxial stacking of some helical segments is discussed.

J Mol Biol, 1990 Jan 20, 211(2), 297 - 9
Crystallization and preliminary X-ray characterization of maltoporin from Escherichia coli; Stauffer KA et al.; Crystals of maltoporin (the bacteriophage lambda receptor of Escherichia coli) that diffract X-rays to 3 A resolution can be grown reproducibly . Maltoporin is an integral membrane protein, which forms a channel in the E . coli outer membrane that specifically facilitates the diffusion of maltose and maltodextrins . The crystals have a rhombic prismatic habit and belong to the orthorhombic space group C222(1) with unit cell dimensions a = 130 A, b = 213 A and c = 216 A . X-ray structure determination is underway.

J Mol Biol, 1990 Jan 20, 211(2), 493 - 501
Analysis of the structure-function relationship of tumour necrosis factor . Human/mouse chimeric TNF proteins: general properties and epitope analysis; Tavernier J et al.; To analyse the structure-function relationship of tumour necrosis factor (TNF), a set of in-frame chimeric genes was constructed by coupling appropriate segments of the human and mouse TNF coding regions . Under control of the bacteriophage lambda inducible PL promoter high level expression of these chimeric genes was obtained in Escherichia coli . Although both human and mouse TNF were produced in E . coli as soluble proteins, a reduction of solubility was observed in some of the chimeric proteins . The specific activity was variable, but in some constructs comparable to human TNF, indicating that the structural conformation of these chimeric proteins resembled the human TNF structure . Neutralization analysis using two monoclonal antibodies directed against human TNF, indicated that the regions involved in the binding of these antibodies are distributed over multiple segments of the polypeptide . Further analysis by site-directed mutagenesis of one subregion allowed the identification of the Arg131 residue as involved in the binding of both neutralizing monoclonal antibodies; an Arg131----Gln replacement abolished antibody binding but did not affect the specific activity of TNF.

J Immunol, 1990 Jan 15, 144(2), 740 - 4
Structure of the gene for human complement protein decay accelerating factor; Post TW et al.; Decay accelerating factor (DAF) is a glycophospholipid-anchored membrane protein that is part of the regulators of complement activation (RCA) gene family located on human chromosome 1, band q32 . These proteins, beginning at their amino terminus, consist largely of multiple copies of an approximately 60 amino acid short consensus repeat (SCR) . A DAF cDNA clone was used to identify overlapping bacteriophage genomic clones . The human DAF gene spans approximately 40 kb and consists of 11 exons . The length of these exons and introns varies considerably, with the exons ranging from 21 to 956 bp and the introns ranging from approximately 0.5 to 19.8 kb . SCR I, II, and IV are all encoded by single exons; however, SCR III is encoded by two separate exons, with the splice junction occurring after the second nucleotide of the codon for the glycine residue at position 34 of the consensus sequence . This feature has also been found in CR1, CR2, membrane cofactor protein, and murine factor H . Following the SCR in DAF is a 76 amino acid serine/threonine-rich domain encoded on three separate exons . Exon 10 encodes the Alu family sequence that has been found as an insert in a minor class of DAF cDNA, thus indicating that this mRNA arises by standard alternative splicing . The last DAF exon, which comes after the largest intron of 19.8 kb, encodes the hydrophobic carboxy terminus and the 3'UT region . The nature of the signal that directs posttranslational attachment of a glycophospholipid anchor to DAF is not known, but that signal is apparently spread over three exons and greater than 20 kb . An analysis of the DAF gene provides additional evidence for the common evolutionary heritage of the RCA gene family . The exon/intron structure of this gene will facilitate experiments aimed at understanding the functions of the various domains of DAF.

J Mol Biol, 1990 Jan 5, 211(1), 91 - 101
Replication enhancer-independent mutation increases the co-operativity with which an initiator protein binds its origin; Greenstein D et al.; The plus-strand replication origin of bacteriophage fl has a bipartite structure consisting of a required core origin region and an adjacent A + T-rich enhancer sequence that potentiates replication approximately 100-fold . The core origin binds the initiator protein (gpII) and the enhancer binds the Escherichia coli integration host factor (IHF) . gpII binds the core origin in two steps, forming a binding intermediate (complex I) and a functional complex for nicking (complex II) . We have used a double-label gel binding assay to determine the stoichiometry of the gpII-origin interaction . The results indicate that complex I contains two gpII molecules per origin, and complex II contains four gpII molecules per origin . Enhancer-independent mutations in gpII allow wild-type levels of replication in the absence of either the enhancer or IHF . We have examined the binding of an enhancer-independent gpII mutant (mp1) protein to the replication origin . The mp1 mutation in gpII (Met40----Ile) increases the co-operativity with which the protein binds to form complex II . In addition, the mutant gpII forms both complexes with a DNA fragment containing only two (beta-gamma) of the three repeats from the core origin sequence, while the wild-type protein forms only complex I with this fragment . We discuss how a mutation that increases the co-operativity of binding of an initiator protein might stimulate DNA replication.

J Biol Chem, 1990 Jan 5, 265(1), 47 - 51
Cloning, sequence analysis, and expression of the bacteriophage T4 cd gene; Maley GF et al.; The cd gene of bacteriophage T4, which encodes the enzyme deoxycytidylate deaminase, was isolated as a 1.9-kilobase DNA fragment and completely sequenced . The deduced amino acid sequence was found to be 193 residues long compared with 188 for the corresponding enzyme from bacteriophage T2 . There were nine amino acid differences between the two enzymes in addition to a 5-residue insert near the carboxyl terminus of the T4 deaminase which was not present in the T2 deaminase . The cd-containing fragment also contained all of gene 31 (Nivinskas, R., and Black, L . W . (1988) Gene (Amst.) 73, 251-257) and thus precisely locates the two genes relative to one another within the T4 phage genomic map . Attempts to place the cd gene within a high expression vector have not been successful so far due to possible toxic effects of the gene product . However, placement of the gene within pUC18 resulted in a degree of expression which is about 10-20 times that found in T4-infected Escherichia coli . The enzyme was purified to homogeneity and found to possess properties similar to T2 phage deoxycytidylate deaminase.

Proc Natl Acad Sci U S A, 1990 Jan, 87(1), 103 - 7
Bacteriophage P1 cloning system for the isolation, amplification, and recovery of DNA fragments as large as 100 kilobase pairs; Sternberg N; The development of a bacteriophage P1 cloning system capable of accepting DNA fragments as large as 100 kilobase pairs (kbp) is described . The vectors used in this system contain a P1 packaging site (pac) to package vector and cloned DNA into phage particles, two P1 loxP recombination sites to cyclize the packaged DNA once it has been injected into a strain of Escherichia coli containing the P1 Cre recombinase, a kanr gene to select bacterial clones containing the cyclized DNA, a P1 plasmid replicon to stably maintain that DNA in E . coli at one copy per cell chromosome, and a lac promoter-regulated P1 lytic replicon to amplify the DNA before it is reisolated . An essential feature of the cloning system is a two-stage in vitro packaging reaction that packages vector DNA containing cloned inserts into phage particles that can deliver their DNA to E . coli with near unit efficiency . The packaging reaction can generate 10(5) clones with high molecular weight DNA inserts per microgram of vector DNA . Using NotI fragments from E . coli DNA, it was shown that the system can clone 95- and 100-kbp fragments but not a 106-kbp fragment . Presumably, the combined size of the latter fragment and the vector DNA (13 kbp) exceeds the headful capacity of P1.

Exp Parasitol, 1990 Jan, 70(1), 72 - 84
Schistosoma mansoni: cloning of antigen gene sequences in Escherichia coli; el-Sherbeini M et al.; Fischer rat protective antiserum (F-2x) prepared from Schistosoma mansoni-infected rats was used to screen an adult worm cDNA library constructed in a lambda gt11 bacteriophage expression vector . This led to the isolation of several clones yielding proteins reactive with antibodies in the infection serum . Counter-screening of these clones with Wistar-Furth rat nonprotective antiserum (W-2x) enabled identification of clones either uniquely or preferentially reacting with F-2x, in addition to clones of nearly equal reactivity with both antisera . Six clones were further characterized . Five expressed beta-galactosidase/S . mansoni fusion proteins which migrated more slowly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than beta-galactosidase and all were reactive in a Western immunoblot assay . The cDNA insert sizes in the clones ranged from 150 to 900 base pairs . Rabbit antibodies prepared against fusion proteins from three of the clones recognized biosynthetically radio-labeled 4-week worm proteins of sizes 20, 38, and 70 kDa, respectively . The 20- and 38-kDa proteins were among the protein antigens uniquely recognized by the F-2x protective antiserum . These proteins are therefore candidates for protective vaccine antigens and the recombinant lambda clones are now serving as useful reagents for obtaining the corresponding nucleotide gene sequences.

Mol Cell Biol, 1990 Jan, 10(1), 353 - 60
Signal-mediated import of bacteriophage T7 RNA polymerase into the Saccharomyces cerevisiae nucleus and specific transcription of target genes; Benton BM et al.; Bacteriophage T7 RNA polymerase and derivatives that contain the nuclear localization signal (NLS) from simian virus 40 T antigen (J . J . Dunn, B . Krippl, K . Bernstein, H . Westphal, and F . W . Studier, Gene 68:259-266, 1988) were expressed in Saccharomyces cerevisiae under the control of the inducible GAL1 promoter . As determined by indirect immunofluorescence, T7 RNA polymerase lacking the NLS remained mostly in the cytoplasm, whereas the protein containing the NLS localized to the nucleus . T7 RNA polymerase containing a mutated NLS remained mostly cytoplasmic . Hybrid proteins containing the NLS near the amino terminus were enzymatically active in the yeast cell, initiating transcription selectively at a T7 promoter placed in yeast chromosomal or plasmid DNA and stopping at a specific T7 terminator . At limiting enzyme concentrations, 5 to 10 times as much target RNA was produced when the polymerase contained the NLS, presumably because more enzyme reached the nucleus . Although substantial amounts of intact mRNA accumulated, no translation of target mRNAs in yeast cells was detected.

J Virol, 1990 Jan, 64(1), 24 - 36
Nonessential region of bacteriophage P4: DNA sequence, transcription, gene products, and functions; Ghisotti D et al.; We sequenced the leftmost 2,640 base pairs of bacteriophage P4 DNA, thus completing the sequence of the 11,627-base-pair P4 genome . The newly sequenced region encodes three nonessential genes, which are called gop, beta, and cII (in order, from left to right) . The gop gene product kills Escherichia coli when the beta protein is absent; the gop and beta genes are transcribed rightward from the same promoter . The cII gene is transcribed leftward to a rho-independent terminator . Mutation of this terminator creates a temperature-sensitive phenotype, presumably owing to a defect in expression of the beta gene.

J Virol, 1990 Jan, 64(1), 143 - 54
Structure of the bacteriophage T4 baseplate as determined by chemical cross-linking; Watts NR et al.; We have carried out a series of reversible chemical cross-linking experiments using the reagent ethylene glycol-bis(succinimidylsuccinate) with the goal of determining the three-dimensional structure of the bacteriophage T4 baseplate . In a previous report, we investigated the near-neighbor contacts in baseplate precursors and substructures (N.R.M . Watts and D.H . Coombs, J . Virol . 63:2427-2436, 1989) . Here we report completion of the analysis by examining finished baseplates and tails . Most of the previous contacts were confirmed, and we report several new contacts, including those within the central hub (gp5-gptd2, gp26-gptd), between the hub and the outer wedges (gp6-gp27(2}, between baseplate and sheath (gp54-gp18), and between sheath and core (gp19-gp18) . On the basis of this and other available information, a partial three-dimensional model of the baseplate is proposed.

Toxicon, 1990, 28(5), 575 - 85
Cloning and nucleotide sequences of crotamine genes; Smith LA et al.; A cDNA library containing snake toxin genes was constructed in bacteriophage lambda by using mRNA isolated from the glands of the South American rattlesnake, Crotalus durissus terrificus . The first high-density screening of 400,000 plaques for crotamine-containing genes yielded over 800 positives when a labeled cDNA probe with sequence homology to crotamine was used . Four of these clones with insert sizes from 270 to 400 base pairs were chosen and their inserts subcloned into pGEM-3Z and sequenced . Nucleotide sequence analysis of the cloned cDNAs predicted the existence of multiple variants of the crotamine toxin . The different forms, identified from the DNA sequences, displayed discrepancies in amino acid sequence for crotamine when compared with previously published reports . Direct amino acid sequencing of commercially purified crotamine and CNBr fragments thereof confirmed the structures predicted by the nucleic acid sequences.

Eur Biophys J, 1990, 18(5), 285 - 93
Analysis of time-resolved fluorescence anisotropy in lipid-protein systems . II . Application to tryptophan fluorescence of bacteriophage M13 coat protein incorporated in phospholipid bilayers; Peng K et al.; The subnanosecond fluorescence and motional dynamics of the tryptophan residue in the bacteriophage M13 coat protein incorporated within pure dioleoylphosphatidylcholine (DOPC) as well as dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) and dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) bilayers (80/20 w/w) with various L/P ratio have been investigated . The fluorescence decay is decomposed into four components with lifetimes of about 0.5, 2.0, 4.5 and 10.0 ns, respectively . In pure DOPC and DOPC/DOPG lipid bilayers, above the phase transition temperature, the rotational diffusion of the protein molecules contributes to the depolarization and the anisotropy of tryptophan is fitted to a dual exponential function . The longer correlation time, describing the rotational diffusion of the whole protein, shortens with increasing temperature and decreasing protein aggregation number . In DMPC/DMPG lipid bilayers, below the phase transition, the rotational diffusion of the protein is slowed down such that the subnanosecond anisotropy decay of tryptophan in this system reflects only the segmental motion of the tryptophan residue . Because of a heterogeneous microenvironment, the anisotropy decay must be described by three exponentials with a constant term, containing a negative coefficient and a negative decay time constant . From such a decay, the tryptophan residue within the aggregate undergoes a more restricted motion than the one exposed to the lipids . At 20 degrees C, the order parameter of the transition moment of the isolated tryptophan is about 0.9 and that for the exposed one is about 0.5.

Eur Biophys J, 1990, 18(5), 277 - 83
Analysis of time-resolved fluorescence anisotropy in lipid-protein systems . I . Application to the lipid probe octadecyl rhodamine B in interaction with bacteriophage M13 coat protein incorporated in phospholipid bilayers; Peng K et al.; Fluorescent probes located in heterogeneous environments give rise to anomalous time-resolved fluorescence anisotropy . A simple analytical expression of anisotropy has been derived for the case of a small difference in local fluorescence lifetimes . The expression has the diagnostic advantage that the time dependence of the fluorescence anisotropy can be predicted from the differences in fluorescence lifetimes and residual anisotropies of the probes located in different sites . Using this model, the local fluorescence anisotropy parameters and the relative contributions of the lipid probe octadecyl rhodamine B in a lipid environment and in the vicinity of bacteriophage M13 coat protein reconstituted in phospholipid bilayers, composed of 80% 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 20% 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol have been determined experimentally . At 40 degrees C, the correlation times for bound and free probes are 2.3 and 3.0 ns, respectively, while the corresponding order parameters are 0.85 and 0.62, respectively.

Mol Gen Genet, 1990 Jan, 220(2), 240 - 4
Novel replication mutant of microvirid phage alpha 3 deleted in the complementary strand origin; Kodaira K et al.; The bacteriophage alpha 3 origin of complementary strand DNA synthesis (-ori) contains two potential secondary loop structures (I and II), which have been implicated as direct recognition sites for host Escherichia coli DnaG protein . To elucidate to what extent such structures are essential, we introduced a nucleotide deletion within the -ori region, by nuclease digestion of alpha 3 replicative form DNA . A mutant, delB, thus constructed had a 121 nucleotide deletion within the -ori region and was completely lacking in the two putative hairpin loops, I and II . The delB mutant formed smaller plaques on the host E . coli C and had a longer latent period, but the mean burst size at 37 degrees C was almost the same (400 phages) as that of the wild type . In contrast to the parental phage, growth of the mutant depends on host dnaB and dnaC functions . These results indicate that the prototype secondary structures in the alpha 3 origin of complementary strand synthesis are dispensable for delB and that the alpha 3 mutant has an additional replication origin whose function is dependent on DnaB and DnaC proteins, rather than on DnaG protein alone.

Mutat Res, 1990 Jan, 243(1), 21 - 8
Specificity of spontaneous mutation in the lacI gene cloned into bacteriophage M13; Yatagai F et al.; We have studied the specificity of spontaneous mutation in the lacI gene of Escherichia coli cloned into bacteriophage M13 . The comparison of the spectrum of 85 spontaneous mutations with that of the lacI gene carried on an E . coli F' episone revealed the following characteristics: (i) base substitution was predominant, accounting for 80% of spontaneous events compared with only 11% on the F' episome; (ii) among the base substitutions, the majority were G:C----A:T transitions (86%); (iii) not one mutation recovered on M13 corresponded to a mutation at the spontaneous hotspots seen in the F' spectrum (i.e., neither the addition or deletion of the tetramer 5'-CTGG-3' at position 620-631 nor the A:T----G:C transition at position +6 of lacO were recovered) . The enhanced rate of cytosine deamination in single-stranded DNA, the unique replication mechanism and the refractory nature of single-stranded DNA to excision-repair processes present likely explanations for the observed mutational spectrum.

Lab Invest, 1990 Jan, 62(1), 96 - 103
Differential localization of type I and type III procollagen messenger ribonucleic acids in inflamed periodontal and periapical connective tissues by in situ hybridization; Larjava H et al.; Inflammatory lesions of periodontal and periapical connective tissue were studied by in situ hybridization to detect cells responsible for type I and type III collagen production . Formalin-fixed and paraffin-embedded tissue specimens from patients with oral lesions of various stages of inflammation were hybridized with cDNA probes specific for human pro alpha 1(I) and pro alpha 1(III) collagen mRNAs, and with bacteriophage lambda DNA as a control probe . This technique permitted us to localize fibroblasts active in type I collagen synthesis in the vicinity of inflammatory infiltrates in all the samples studied . Cells containing high levels of type III collagen mRNA were seen in early abscess formation and they were particularly abundant in pyogenic granuloma and irritation fibroma . Type I collagen mRNA was prominent in gingival fibrosis . In the infrabony lesions with active inflammatory infiltrations the production of collagen was confined mostly to the periphery of the lesions . These findings give indirect evidence that cytokines liberated during the early stages of the inflammatory process stimulate expression of the type III collagen gene by fibroblasts . In chronic lesions a gradual switch from type III to type I collagen gene expression occurs . The change in collagen types appears to underlie the observed isolation of the inflammation by a collagenous capsule . In all the samples studied fibroblasts exhibited marked variation in their levels of procollagen mRNAs, supporting previous views about their heterogeneity in connective tissues . The approach presented here offers new possibilities to study cellular interactions and metabolic activities in inflammatory lesions.

Virology, 1990 Jan, 174(1), 26 - 34
In vitro cleavage of the concatemer joint of bacteriophage T3 DNA; Fujisawa H et al.; Mature DNA from phage T3 or T7 is a linear duplex DNA with direct repeats at its ends known as "terminally redundant sequences." The DNA of these phages is synthesized as concatemers in which unit length molecules are joined together in a head-to-tail fashion through the terminally redundant sequences and processed to form mature DNA with coupling to DNA packaging . When linearized plasmid DNA carrying a concatemer joint, a terminally redundant sequence and its flanking sequences from the concatemer, was incubated in a defined in vitro system for packaging T3 DNA, composed of purified proheads and packaging proteins (gp 18 and gp 19), DNA was cleaved at the left end of the terminally redundant sequence . The cleavage reaction required all factors necessary for DNA packaging . The DNA fragment with the left end was preferentially protected from DNase I digestion, indicating that the cleavage reaction occurs at the left end of the terminally redundant sequence in the concatemer when DNA is packaged leftward, corresponding to the direction from the right to the left end of the T3 genome . The cleavage reaction was stimulated by high concentrations of NaCl and ATP, a condition in which DNA translocation into the head is slowed down . The cleavage reaction was not specific between T3 and T7 . The right end of the concatemer joint was not required for cleavage at the left end . In the absence of ATP, DNA was extensively degraded by gp 19 . gp 19 by itself had nonspecific endonuclease activity, making double-stranded breaks . The activity was inhibited by either ATP or gp 18.

J Bacteriol, 1990 Jan, 172(1), 204 - 11
Conservation of a dual-start motif in P22 lysis gene regulation; Nam K et al.; Gene 13 of bacteriophage P22 is functionally equivalent to lambda lysis gene S . Gene S codes for two products, the polypeptides S105 and S107, produced from translational initiation events at the third and first codon, respectively . We have shown that the two polypeptides have opposing functions in lysis: S105 is the lethal lysis effector, and S107 acts as an inhibitor of lysis (U . Blasi, K . Nam, D . Hartz, L . Gold, and R . Young, EMBO J . 11:3501-3510, 1989) . Gene 13 has a 108-codon reading frame and its product begins with a similar motif: Met-1-Lys-2-Lys-3-Met-4 . Here, we present in vivo and in vitro evidence for the expression of a 13(108) and a 13(105) product and show that the lambda lysis control mechanisms is evolutionarily conserved in phage P22 . In this case 13(108), like S107 in lambda, functions as the inhibitor of the lysis effector 13(105) . Although the DNA sequences upstream of the S and 13 gene starts showed less homology, the same structural characteristics, i.e., stem-loop structures immediately upstream and about 10 codons downstream of the start region, were present in both reading frames . Using in vitro mutagenesis and toeprinting, we show that the upstream stem-loop structures of genes 13 and S, containing the Shine-Dalgarno sequence for initiations at Met-1, are interchangeable . Moreover, our data indicate that the stability of the secondary structures present in the translational initiation regions of genes S and 13 is set to create a particular ratio of initiation events at Met-1 and Met-3 or Met-4 . The ratio of effector to inhibitor was much higher in P22 than in lambda . We propose that this reflects less transcriptional readthrough at the late terminator t(R) and suggests that the dual-start motif in genes 13 and S may be important for establishment of maintenance of the lysogenic state.

Mol Carcinog, 1990, 3(6), 330 - 4
Detection of genomic alterations in carcinogen-induced mouse liver tumors by DNA fingerprint analysis; Muller O et al.; DNA fingerprint analysis was used to study structural abnormalities in the genome of mouse liver tumor cells . Liver tumors were induced in three strains of mice, namely C57BL/6J, C3H/He and B6C3F1, by a single injection of 20 micrograms/g body wt . diethylnitrosamine on day 15 after birth . DNA from liver tumors was digested with Hinfl restriction enzyme and hybridized on Southern blots with wild-type bacteriophage M13 DNA as probe . The resulting fingerprints of tumor DNA were compared with those of DNA from normal liver tissue . Genomic aberrations were detected in two out of 68 tumors analyzed, one stemming from a C57BL/6J and the other from a C3H/He mouse.

Biorheology, 1990, 27(3-4), 485 - 9
Modification of mucin gene expression in the airways of rats exposed to sulfur dioxide; Basbaum C et al.; The molecular mechanisms mediating mucous cell metaplasia and hypersecretion in the respiratory tract are unknown . Previous work suggests that mucous metaplasia requires the induction of mucin gene expression . We are investigating this possibility by monitoring steady state levels of mucin mRNA in a model of mucous cell metaplasia induced by SO2 exposure . Male Sprague Dawley rats were exposed to 400 ppm SO2 gas in air for 3 h per day, 5 days per week for 0,1,2, or 3 weeks . Sham controls were exposed to air under similar conditions . After 3 weeks, morphological changes were apparent in the epithelium of SO2 exposed rats at all levels from the trachea to the distal airways . The epithelial thickness increased, as well as the number and size of glands in the trachea . Epithelial mucous (goblet) cells increased from 0 to 4.5 per mm in the trachea, 0.2 to 6.2 per mm in the main stem bronchi, and 0.2 to 22.7 per mm in the distal airways (mean values obtained for 3-6 tissue blocks per airway level per condition) . In parallel experiments, we used SMUC41, a 950 bp human intestinal cDNA to isolate a human airway cDNA, HAM-1 from a cDNA library constructed in bacteriophage from human bronchial poly A+RNA . HAM-1 is a 90 bp cDNA encoding a threonine- and proline-rich peptide with 96% homology to the human intestinal cDNA SMUC-41 . Next we probed total and poly A+ airway RNA from rats in each exposure condition with SMUC-41 or HAM-1 . Blots were then stripped and reprobed with cDNA encoding beta actin . Densitometry values normalized for the amount of RNA loaded per lane (as determined by actin hybridization intensity) showed that mucin mRNA increased 8-9 fold as a function of SO2 exposure . This is consistent with the possibility that mucin gene transcription is induced by SO2 exposure, and may represent a primary event in the development of mucous metaplasia and hypersecretion.

Yi Chuan Xue Bao, 1990, 17(4), 327 - 34
{The construction of shuttle plasmid vector of Streptomyces and E . coli}; Tan H et al.; A shuttle plasmid vector, pSE-3 was constructed using the Streptomyces plasmid pIJ486 and E . coli plasmid pGEM-3 digested with BgIII and BamHI respectively . The shuttle plasmid pSE-3 could replicate and express its neomycin resistance in both Streptomyces and E . coli well . The pSE-3 has opposed promoters containing bacteriophage ST6 and T7, the restriction enzyme digestion showed that the pSE-3 has a single site with HindIII and EcoRI respectively, it is very useful for the cloning and expression of the valuable gene inserts . The pSE-3 is stable in both of Streptomyces and E . coli after re-transformed pSE-3 is re-transformed from Streptomyces into E . coli and some 50 generations are propagated.

Biochem Cell Biol, 1990 Jan, 68(1), 318 - 29
Structure and dynamics of detergent-solubilized M13 coat protein (an integral membrane protein) determined by 13C and 15N nuclear magnetic resonance spectroscopy; Henry GD et al.; The major coat protein of the filamentous bacteriophage M13 is inserted as an integral protein in the inner membrane of the Escherichia coli host upon infection . M13 coat protein is an ideal model membrane protein and has been the target of many biophysical studies . An overview is presented here of the application of nuclear magnetic resonance spectroscopy to the study of the structure and dynamics of M13 coat protein in several lipid-mimetic environments . The coat protein may be biosynthetically enriched with 13C- and 15N-labelled amino acids, allowing the resolution and assignment of individual nuclei . Structural fluctuations at selected sites have been monitored using 13C relaxation and isotope-detected amide hydrogen exchange kinetics . A model is proposed for the structure of a coat protein dimer in detergent micelles.

Basic Life Sci, 1990, 52, 269 - 75
Genetic analyses of cellular functions required for UV mutagenesis in Escherichia coli; Battista JR et al.; In Escherichia coli, most UV and chemical mutagenesis is not a passive process and requires the participation of the umuD and umuC gene products . However, the molecular mechanism of UV mutagenesis is not yet understood and the roles of the UmuD and UmuC proteins have not been elucidated . The umuDC operon is induced by UV irradiation and regulated as part of the SOS response . Genetic evidence now indicates that RecA-mediated cleavage activates UmuD for its role in mutagenesis . The COOH-terminal fragment of UmuD is both necessary and sufficient for this role . The RecA protein appears to have a third role in UV mutagenesis besides mediating the cleavage of LexA and UmuD at the time of SOS induction . In addition, we have obtained evidence which indicates that the GroEL and GroES proteins also play a role in UV mutagenesis . Similarities of the amino acid sequence of UmuD to the sequence of gene 45 protein of bacteriophage T4 and of the sequence of UmuC to those of the gene 44 and gene 62 proteins suggest possible roles for UmuD and UmuC in mutagenesis that are supported by preliminary evidence.

Mol Microbiol, 1990 Jan, 4(1), 3 - 11
Integration host factor is necessary for lysogenization of Escherichia coli by bacteriophage P2; Saha S et al.; Whether infection by bacteriophage P2 results in lysogenization of the host or vegetative growth of the phage depends upon a race between transcription from the repressor promoter Pc and the early promoter Pe; transcription from these promoters is mutually exclusive, since the Pc repressor Cox is formed from the Pe transcript and the Pe repressor C from the Pc transcript . The involvement of integration host factor (IHF) in the lysogenization of Escherichia coli K12 by P2 was tested by comparing wild-type and IHF-deficient (himA and himD) mutants . No lysogenic clones were formed following infection of the mutant bacteria . A switch plasmid that contains Pc-C-cat and Pe-cox-kan was used to test the choice for expression of Pc versus Pe . In the wild-type K12 bacteria, 20% of the clones expressed Pe transcription and 80% Pc transcription, whereas all transformed IHF-defective clones expressed transcription from Pe only . The effects of IHF on the in vivo expression of the Pe and Pc promoters were only marginal . The IHF protein was found to bind upstream of the Pe promoter, where a potential ihf sequence is located.

Arch Microbiol, 1990, 154(1), 67 - 72
The cis-acting DNA sequences required in vivo for bacteriophage Mu helper-mediated transposition and packaging; Harel J et al.; The 37,000 bp double-stranded DNA genome of bacteriophage Mu behaves as a plaque-forming transposable element of Escherichia coli . We have defined the cis-acting DNA sequences required in vivo for transposition and packaging of the viral genome by monitoring the transposition and maturation of Mu DNA-containing pSC101 and pBR322 plasmids with an induced helper Mu prophage to provide the trans-acting functions . We found that nucleotides 1 to 54 of the Mu left end define an essential domain for transposition, and that sequences between nucleotides 126 and 203, and between 203 and 1,699, define two auxiliary domains that stimulate transposition in vivo . At the right extremity, the essential sequences for transposition require not more than the first 62 base pairs (bp), although the presence of sequences between 63 and 117 bp from the right end increases the transposition frequency about 15-fold in our system . Finally, we have delineated the pac recognition site for DNA maturation to nucleotides 32 to 54 of the Mu left end which reside inside of the first transposase binding site (L1) located between nucleotides 1-30 . Thus, the transposase binding site and packaging domains of bacteriophage Mu DNA can be separated into two well-defined regions which do not appear to overlap.

Avian Dis, 1990 Jan-Mar, 34(1), 99 - 105
Preparation of a detailed restriction map of the avian leukosis virus MAV-2(O); Aurigemma RE et al.; Unintegrated MAV-2(O) DNA was isolated from infected chicken embryo fibroblasts and inserted into the lambda bacteriophage vector lambda gtWES lambda B . Three x 10(6) bacteriophage plaques were screened, yielding a total of seven clones, six of which contained DNA representing the complete MAV-2(O) genome . Viral DNA was isolated from four of the clones and was used to transfect chicken embryo fibroblasts . All four clones produced virus as monitored by reverse transcriptase assay . When the four cloned viruses were inoculated into 10-day-old embryos, all hatched chickens developed osteopetrosis . One clone, lambda 9, induced osteopetrosis at a rate of onset and severity identical to that induced by the MAV-2(O) parental stock . This clone was selected for further study . To facilitate restriction mapping, the viral DNA from lambda 9 was subcloned into plasmid vector pUC 12 to construct a plasmid called p9 . Cleavage of p9 DNA with single and multiple restriction endonucleases and hybridization with gene-specific probes identified the restriction fragments obtained . A comprehensive restriction map of cloned MAV-2(O) was generated and is compared with published maps and sequences of other avian retroviruses.

Mutat Res, 1990 Jan, 240(1), 35 - 45
Hydrogen peroxide-induced DNA damage in bovine lens epithelial cells; Kleiman NJ et al.; The present investigation was undertaken to determine the types and extent of DNA damage resulting from incubation of primary cultures of bovine lens epithelial cells with hydrogen peroxide . Significant numbers of DNA single-strand breaks were detected by alkaline elution after exposure to as little as 25 microM H2O2 for 5 min at 37 degrees C . The extent of single-strand breakage was concentration dependent and linear from 25 to 200 microM H2O2 . The observed single-strand breaks appear primarily due to the action of the hydroxyl radical via a Fenton reaction as both an iron chelator, 1,10-phenanthroline and OH . scavengers, including DMSO, KI and glycerol, significantly inhibited the DNA-damaging effect of H2O2 . Diethyldithiocarbamate, an inhibitor of superoxide dismutase, further potentiated the DNA-damaging effects of H2O2, presumably by increasing the steady-state concentration of Fe2+ . DNA-protein cross-linking was not observed . In addition, significant levels of 5,6-saturated thymine residues or pyrimidine dimers were not detected after modification of the alkaline elution methodology to allow the use of either E . coli endonuclease III or bacteriophage T4 endonuclease V, respectively . No double-strand breaks were detected after incubation of epithelial cell cultures with H2O2 concentrations of up to 400 microM for 10 min and subsequent neutral filter elution . Since, in vivo, the lens epithelium contains populations of both quiescent and dividing cells, the degree of susceptibility to oxidative damage was also studied in actively growing and plateau-phase cultures . Reduced levels of single-strand breakage were observed when plateau-phase cultures were compared to actively growing cells . In contrast, essentially no differences in repair rates were noted at equitoxic doses of H2O2 . The above results suggest that lens epithelial cells may be particularly sensitive to oxidative damage and thus are a good model system in which to study the effects of oxidative stress.

Adv Biophys, 1990, 26, 1 - 18
Mechanism of length determination in bacteriophage lambda tails; Katsura I; The mechanism of length determination in bacteriophage lambda tails is discussed as a model for regulation in protein assembly systems . The lambda tail is a long flexible tube ending in a conical part and a single tail fiber . Its length is exactly determined in the sense that the number of major tail protein (gpV) molecules, which comprise more than 80% of the mass of the tail, is exactly the same in all tails . Assembly of gpV is regulated by the initiator complex, which contains the tail fiber and the conical part, and by the terminator protein gpU . There are two key points in the assembly of gpV with respect to length determination . (1) Assembly of gpV on the initiator pauses at the correct tail length . Binding of gpU to the tail only fixes the pause firmly . (2) When the tail length is too short, binding of gpU to tails is inhibited . Deletions and a duplication (both in frame) in gene H, which codes for one of the proteins in the initiator, result in production of phage particles with altered tail length . Moreover, the tail length is roughly proportional to the length of the mutated versions of gene H . This shows that the tail length is measured by the length of gene H protein (gpH), which seems to be approximately as long as the tail tube, if extended like a thread, according to secondary structure prediction (alpha-helices connected by other structures) . Various pieces of evidence show that about six molecules of gpH are attached to the remaining portion of the initiator by the C-terminal part and folded into a somewhat compact form, while they are elongated as they are enclosed in the tail tube during assembly of gpV . Unlike interaction between the length-measuring genome RNA and the coat protein of tobacco mosaic virus, the major tail protein gpV does not bind specifically to the ruler protein gpH . Rather, gpH determines the tail length by inhibiting the binding of gpU to short tails and by signalling the pause when the correct tail length is attained.

Microbios, 1990, 64(259), 73 - 83
Production of bacteriocins and siderophore-like activity by Azospirillum brasilense; Tapia-Hernandez A et al.; Sixty Azospirillum strains were tested for their bacteriocin production ability; twenty-seven (45%) were able to produce bacteriocins and inhibited the growth of one or more indicator strains in solid medium . Mitomycin C treatment enhanced the proportion to 80% . Sometimes large growth inhibition zones were formed, but not when FeCl3 was added in the medium . These inhibition zones probably result from the activity of siderophores . Partially purified bacteriocins produced by four strains were inactivated at pH 4, but were very stable between pH 5 to 10; bacteriocins produced by three strains lost their activity between 55 and 80 degrees C . Loss or decrease in the bacteriocin activity was observed with pronase E treatment; trypsin, lysozyme and alpha-amylase did not have an effect on bacteriocin activity . These findings show that the antagonism among azospirilla was due principally to the bacteriocins and sometimes probably due to siderophores, but not to bacteriophages or other substances.

Microbiol Immunol, 1990, 34(6), 485 - 96
Characterization of an Escherichia coli mutant pleiotropically altered in membrane-bound oxidoreductase activities; Cox JC et al.; An Escherichia coli mutant pleiotropically altered in membrane-bound oxidoreductase activities was isolated following nitrosoguanidine treatment . Mutant R23 was able to grow on glucose, but was unable to grow on succinate or other oxidizable substrates as a sole energy source . Isolated membranes prepared from R23 failed to oxidize succinate and formate; while NADH was oxidized at a reduced rate by membranes . The mutant also exhibited markedly reduced cytochrome content, but normal DL-lactate PMS reductase and H(+)-translocating ATPase activities relative to the parent strain . Bacteriophage Plkc was used to transduce R23 to growth on glycerol, DL-lactate or succinate; regardless of the selection procedure, each of the 179 transductants had gained the ability to grow on all three substrates . The suc- mutation in R23 appeared to be responsible for the loss of growth on oxidizable substrates, altered membrane-bound oxidoreductase activities, resistance to neomycin, and reduced levels of cytochrome components . The suc- mutation was localized in the 6 to 6.5 min region of the E . coli chromosome map utilizing episomal transfers.

FEMS Microbiol Lett, 1990 Jan 1, 54(1-3), 135 - 40
Mu gem3 as a tool to investigate the influence of chromosome supercoiling on gene expression in Escherichia coli K12; Bianchi E et al.; We have developed a rapid method to investigate the influence of chromosome supercoiling on gene expression in Escherichia coli K12 . This method exploits the ability of the gem3 mutant of the bacteriophage Mu, even in the prophagic state in immune cells, to induce relaxation of the host chromosome . The experiments can thus be performed under physiological conditions, and without the use of the drugs . In theory, this method can be applied to any bacterial gene . Here, we report the results obtained with four DNA replication and three cell division genes.

Mutat Res, 1990 Jan, 228(1), 81 - 7
Mutations in the bacteriophage lambda pL/oL region that spontaneously occur in plasmid pRPZ126; Chen JH et al.; In a previous study (Chen and Porter, 1988), we isolated spontaneous mutations in a test plasmid that had occurred under non-selective conditions and assigned them to 1 of 6 different categories or groups . The test plasmid, pRPZ126, is a pBR322 derivative containing the bacteriophage lambda immunity region with the cI857 allele so that plasmid-containing cells shifted to 42 degrees C survive only if the expression of the lambda kil gene is prevented by mutation . 75% of the total spontaneous mutations obtained fall into two of these groups where there is no readily detectable change in plasmid size . The two groups differ in that the plasmids from the group 4 mutations are missing a specific HincII site while the plasmids from the group 5 mutations had no detectable plasmid change whatsoever . In this study, we randomly selected ten group 4 mutants and ten group 5 mutants and sequenced the lambda pL/oL region of the mutant plasmid . Of the ten group 4 mutants (HincII site missing), five involved a 24- or 44-basepair deletion in the pL/oL region of the plasmid . The other five group 4 mutants and four of the ten group 5 mutants were A-T to G-C transitions in the pL/oL region . The remaining six group 5 mutants did not demonstrate any sequence change in the pL/oL region of the plasmids . 8 out of 9 of these transition mutations occurred next to the 3' end of 3 different 5'-PyGGNPuNTG-3' sequences in the lambda operator region, and this same sequence is found adjacent to the A-T to G-C transition hotspot in the lac operator region (Schaaper et al., 1986) . The 9th mutation, where the A-T to G-C transition occurred one basepair away from the lambda operator, was adjacent to a very similar sequence.

Virology, 1990 Jan, 174(1), 329 - 34
Structure of the bacteriophage lambda cohesive end site: bent DNA on both sides of the site, cosN, at which terminase introduces nicks during chromosome maturation; Yeo A et al.; Packaging of lambda DNA is mediated by the phage-encoded enzyme, terminase, which acts at a site termed cos . cos consists of cosB, the site where terminase binds lambda DNA, and cosN, the site where nicks are introduced to generate the cohesive ends of virion DNA . cos contains multiple binding sites for gpNu1, the small subunit of terminase, and integration host factor (IHF), an Escherichia coli DNA binding protein . Polyacrylamide gel electrophoresis of circularly permuted segments of cos DNA has been used to locate major bend loci in cos . Two major bends have been located; one bend is ca 146 bp to the left of cosN while the second major bend is located ca 92 bp to the right of cosN . The major bend at 92 coincides roughly with I1, the strongest IHF binding site in cos . The possible roles of static bending in DNA packaging are discussed.

Virology, 1990 Jan, 174(1), 192 - 203
Kinetics and regulation of transcription of bacteriophage Mu; Marrs CF et al.; Mu transcription was analyzed by hybridization of {3H}uridine pulse-labeled RNA from heat-induced Mu lysogens to Mu DNA restriction fragments on nitrocellulose blots . Based on their time of appearance and dependence on Mu functions, we have defined three classes of transcripts: early, middle, and late . Replication-defective prophages containing A or B amber mutations or a deletion of the beta (right) end produced only early RNA derived from the left-most 8 to 10 kb of the Mu genome . A replication-proficient C amber mutant exhibited similar early transcription but at later times also produced middle transcripts from a region including C, which encodes the activator of late transcription . The C mutant did not produce late transcripts from the right-most 26 kb of the Mu genome encoding genes involved in phage morphogenesis and release . These results indicate that Mu DNA replication is required for efficient expression of middle RNA, which is itself required for expression of late transcripts . Amber mutations in essential genes other than A, B, and C had no significant effect on transcription except for polarity of one E mutation . Uninduced Mu c+ and Mu cts prophages produced very low levels of Mu-specific RNA derived from several regions including the c (immunity) gene and the region between genes B and C.

J Bacteriol, 1990 Jan, 172(1), 361 - 71
Characterization of the C operon transcript of bacteriophage Mu; Stoddard SF et al.; Mu transcription occurs in three phases: early, middle, and late . Middle transcription occurs in the region of the C gene, which encodes the transactivator for late transcription . A middle promoter, Pm, was previously localized between 0.28 and 1.2 kilobase pairs upstream of C . We used S1 nuclease mapping with both unlabeled and radiolabeled capped RNAs from induced lysogens to characterize C transcription and identify its promoter . The C transcription initiation site was localized to a 4-base-pair region, approximately 740 base pairs upstream of C within the region containing Pm . Transcription of C was activated between 4 and 8 min after induction of cts and Cam lysogens and increased throughout the lytic cycle . Significant C transcription did not occur in replication-defective Aam lysogens . These kinetic and regulatory characteristics identify the C transcript as a middle RNA species and demonstrate that Pm is the C promoter . DNA sequence analysis of the Pm region showed a good -10, but poor -35, site homology to the Escherichia coli RNA polymerase consensus sequence . In addition, the sequence demonstrated that C is the distal gene in a middle operon containing several open reading frames . S1 mapping also showed an upstream transcript with a 3' end in the Pm region at a sequence strongly resembling a Rho-independent terminator . The regulatory characteristics of this RNA are consistent with this terminator, t9.2, being the early operon terminator.

Infect Immun, 1990 Jan, 58(1), 17 - 20
Isolation and characterization of recombinant lambda gt11 bacteriophages expressing four different Mycobacterium intracellulare antigens; Morris SL et al.; Four bacteriophages expressing different immunoreactive recombinant Mycobacterium intracellulare antigens were isolated from a lambda gt11 library with monoclonal antibodies to M . intracellulare . These four antibodies reacted with native M . intracellulare proteins of 54, 43, 40/38, and 22 kilodaltons . Southern blot hybridizations with DNA probes prepared from insert fragments of these bacteriophages confirmed the M . intracellulare derivation of the inserts . The physical maps of the immunoreactive phages were deduced by restriction enzyme digestions . The molecular weights of the expressed recombinant antigens were determined by Western (immuno-) blotting.

Acta Cient Venez, 1990, 41(1), 21 - 5
Isolation and characterization of mutants affected in the expression of the nar operon Escherichia coli; Orozco de Silva A et al.; In the nitrate reductase system of Escherichia coli, the maximal expression of the nar operon is obtained under anaerobiosis in the presence of nitrate . Mudl (Ap,lac) insertion mutants, which only grew on lactose anaerobically if supplemented with nitrate were mapped at the chlC locus at min 27 of the map . In these fusion strains which lack benzyl viologen dependent nitrate reductase (NR) activity as well as the formiate-linked NR activity, the synthesis of beta-galactosidase reflects the regulation of the wild type nar operon at the transcriptional level . From these strains, two classes of spontaneous regulatory mutants were isolated: class I mutants which synthesized beta-galactosidase in anaerobiosis in the absence of nitrate and class II mutants in which the synthesis of that enzyme was partially independent of nitrate and it was no longer repressed by oxygen . The class I regulatory mutation was tightly linked to the nar operon as shown by bacteriophage P1 transductions . It probably affects either a closely linked cis-active element or a gene coding for a negative regulatory protein.

Arch Insect Biochem Physiol, 1990, 14(1), 31 - 6
Cloning of the mitochondrial genome of Anopheles quadrimaculatus; Cockburn AF et al.; The entire 15 kilobase (kb) Anopheles quadrimaculatus mitochondrial DNA (mtDNA) was cloned as three EcoRI fragments in a bacteriophage vector and then subcloned into plasmid vectors . The cloned DNA was physically mapped with restriction endonucleases, and the maps were compared to the restriction patterns of native A . quadrimaculatus mtDNA . Several genes were mapped by sequencing the ends of A . quadrimaculatus mtDNA subclones and by hybridization with the previously characterized Aedes albopictus mtDNA clones . These portions of the genetic map were identical in gene order to those of Drosophila yakuba . The predicted amino acid sequence of the protein coding regions that were sequenced were between 72% and 98% homologous to D . yakuba . The cloned mtDNA will be useful as a probe for population genetic analysis of mosquitoes.

Biomed Sci, 1990 Jan, 1(1), 55 - 62
Cascade of overlapping late genes in bacteriophage T4; Selivanov NA et al.; The DNA sequences of genes 9, 10, 11, 12, and wac, which encode the structural proteins in the bacteriophage T4 base plate, were determined . These genes form a single operon which is transcribed in a clockwise direction from a single late promoter in the TATAAATA region located upstream of gene 9 at position -10 . A feature of the operon is an overlap between the termination codon of each upstream gene and the initiation codon of its downstream gene . With the exception of gene 10, the open reading frames encode proteins which have a calculated molecular mass close to that obtained experimentally . The reading frame of gene 10 encodes a polypeptide with a calculated molecular mass of 66.2 kDa, which is at least 22 kDa less than that in the phage particle . Thus the mature protein encoded by gene 10 is possibly a product of the fusion of two adjacent phage genes . The hybrid protein may be formed by a frameshift during the translation of messenger RNA at the end of gene 9 or gene 10.

J Immunoassay, 1990, 11(1), 89 - 95
A heterogeneous double antibody enzyme-linked immunoassay to measure beta-galactosidase fusion protein; Hayashibe K et al.; A rapid and sensitive enzyme-linked immunoassay (ELISA) to quantitate recombinant fusion proteins encoded by cloned cDNA in the bacteriophage lambda gt11 is described . Since the fusion protein is expressed in an equimolar ratio to beta-galactosidase, the assay derives the concentration of the recombinant protein in total bacterial lysates or pure preparations from the measurement of beta-galactosidase with an enzyme-linked immunoassay . This assay is a useful technique to measure the recombinant proteins for subsequent immunological and biochemical characterization.

J Virol, 1990 Jan, 64(1), 37 - 50
Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the beta-galactosidase gene; Vialard J et al.; An improved baculovirus expression vector was developed to expedite screening and facilitate oligonucleotide-directed mutagenesis . This vector contained twin promoters derived from the P10 and polyhedrin genes of Autographica californica nuclear polyhedrosis virus . The P10 promoter directed the synthesis of beta-galactosidase, whereas the polyhedrin promoter controlled the synthesis of foreign gene products . These two genes recombined with wild-type virus genome to yield recombinants which were polyhedrin negative, produced the foreign gene product, and formed blue plaques when beta-galactosidase indicator was present in the agarose overlay . An origin of replication derived from M13 or f1 bacteriophage was also included in the plasmid to permit the synthesis of single-stranded DNA . This template DNA was used to introduce or delete sequences through the process of site-specific mutagenesis . The measles virus virion possesses a membrane envelope which contains two glycoproteins: the hemagglutinin (H) and membrane fusion (F) proteins . The H polypeptide has receptor-binding and hemagglutinating activity, whereas the F protein mediates virus penetration of the host cell, formation of syncytia, and hemolysis of erythrocytes . Genes for these two glycoproteins were inserted into the NheI cloning site of the modified expression vector described above . The vector and purified wild-type viral DNA were introduced into Sf9 insect cells by calcium phosphate precipitation . A mixture of wild-type and recombinant virus was generated and used to infect Sf9 cells, which were subsequently overlaid with agarose . After 3 days, 0.1 to 1% of the plaques became blue in the presence of beta-galactosidase indicator . At least 70% of these blue viral colonies contained the foreign gene of interest as determined by dot blot analysis . Recombinant virus was separated from contaminating wild-type virus through several rounds of plaque purification . Insect cells were then infected with the purified recombinants, and synthesis of H and F proteins were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot detection and Coomassie blue staining . Glycosylation of the proteins appeared to be impaired somewhat, and the precursor to the F protein was not completely cleaved by the proteases present in insect host cells . On the other hand, both proteins appeared to be active in hemagglutination, hemolysis, and cell fusion assays . Levels of synthesis were in the order of 50 to 150 mg of protein per 10(8) cells.

Chin J Biotechnol, 1990, 6(2), 87 - 93
Cloning of the bacteriophage T7 lysozyme gene; Cui DS et al.; Plasmid pBR322 and its derivative containing strong promoter phi 10 of bacteriophage T7 RNA polymerase were used as vectors . A fragment of bacteriophage T7 DNA which was digested with two restriction endonucleases (AvaII and HaeIII) was cloned in the BamHI site of plasmid pBR322 and its derivative pAR951, respectively . The inserted DNA is a segment of 632 base pairs containing the complete coding sequence of both T7 gene 3.5 and weak promoter phi 3.8 for bacteriophage T7 RNA polymerase . The function of T7 gene 3.5 is known to code for bacteriophage T7 lysozyme . Transformants that carry the recombinant plasmid were tested for intracellular lysozyme by adding CHCl3 . Both cloned strains produce active T7 lysozyme . The gene product, T7 3.5 protein, was analyzed by 10 -20% gradient polyacrylamide- SDS electrophoresis . The result showed that the expression of inserted T7 gene 3.5 in pBR322 derivative is stronger than that in pBR322.

Annu Rev Genet, 1990, 24, 465 - 90
Interactions between satellite bacteriophage P4 and its helpers; Christie GE et al.; The helper dependence of satellite phage P4 superimposes an additional set of regulatory interactions on those required for the independent maintenance of P4 or its helpers . These interactions allow P4 to exploit a helper phage under a variety of circumstances and can affect expression of the immunity functions and late genes of both phages . The phage P2 lysis/lysogeny decision involves two competing repressors regulating mutually exclusive promoters in the early control region . In the absence of a helper phage, the P4 immunity function plays a role in the choice between lysogeny or the multicopy plasmid state . No evidence exists for a P4-encoded immunity repressor; in P4-lysogenic cells, expression of the P4 DNA replication gene alpha appears to be prevented by premature termination of transcription . Immunity-independent expression of alpha in the multicopy plasmid state involves initiation of transcription at an alternative upstream promoter that is positively regulated by P4 delta protein; the same promoter is activated by P2 Cox protein during derepression of P4 by P2 . The mechanism of derepression of P2 by P4 remains to be determined, and the relationship between the P4 immunity and derepression functions and the mutations that allow P4 to grow with a P3 prophage helper is an intriguing area for further exploration . Expression of P2 and P4 late genes is regulated by phage-encoded, zinc-binding transcriptional activators that appear to interact directly with the alpha subunit of RNA polymerase of E . coli . Stimulation of P2 late transcription by P2 Ogr protein depends upon phage DNA replication, whereas activation of transcription from the same promoters by the related P4 delta gene product is replication-independent . Elucidation of the mechanisms underlying these interactions promises to provide new insights into strategies for control of gene expression.

Acta Biochim Pol, 1990, 37(1), 39 - 52
Structural proteins of adenovirus . Expression in Escherichia coli; Chroboczek J et al.; The fiber proteins of adenovirus serotype 2 Ad2 and serotype 3 Ad3 and structural protein IIIa of wild type Ad2 and Ad2 ts 112 mutant were cloned and expressed in E . coli . For the expression of both fiber proteins a gene expression system based on bacteriophage T7 RNA polymerase was used . The expressed proteins constituted 1-3% of total host cell protein . Both proteins were insoluble and inclusion bodies were observed . The proteins could be purified from cellular debris by extraction with 6 M urea followed by chromatography in the presence of diminishing concentration of urea . The folding of recombinant fiber proteins was assessed by sensitivity to proteases and gel filtration . Both proteins were synthetized as trimers . Ad2 recombinant fiber has a much less compact structure than native Ad2 fiber, since on gel filtration it is excluded before the native fiber . It is also much more sensitive to chymotrypsin digestion than the native protein . Contrary to that, Ad3 recombinant fiber is much less sensitive to proteolytic cleavage and on gel filtration has the same exclusion volume as the trimeric native fiber of Ad3.

J Basic Microbiol, 1990, 30(9), 679 - 83
Cloning of the resistant EcoRII recognition site of phage T7 into an EcoRII-sensitive plasmid makes the site susceptible to the restriction enzyme; Kruger DH et al.; The recognition sequence 5'-CC(A/T)GG for EcoRII in the bacteriophage T7 genome is refractory to this restriction endonuclease, despite not bearing the specific (protective) methylation . Following the integration of this site as part of a 219 bp fragment (in which the recognition sequence is flanked by about 100 bp of T7 origin) into the EcoRII-sensitive vector pUC18, the T7 site becomes susceptible to cleavage, too . The same is true of recombinant pBR322 plasmids containing the T7-derived recognition site . The results show that the flanking sequences are not immediately responsible for the refractory behaviour of EcoRII sites and are in agreement with data according to which EcoRII requires the coordinated presence of at least two recognition sites in its DNA substrate.

Electron Microsc Rev, 1990, 3(1), 43 - 72
Negative staining of proteins; Kiselev NA et al.; Negative staining, some closely related alternative preparation techniques and radiation stability are considered . An attempt is made to clarify the mechanism of action and ultimate resolution limit of negative staining . The results of electron diffraction investigation of thermitase microcrystals embedded in glucose and glucose + stains are presented . It is shown that at doses not exceeding 10 electrons/nm2 electron diffraction from thermitase crystals demonstrate diffraction fields up to 0.2 nm . When adding heavy-atom salts to glucose or using negative staining, the relative intensities of reflections change and electron diffraction patterns for every type of heavy-atom additive (or negative stain) have their specific features . Such characteristic changes of reflection intensities indicate specific interaction of these additives (or stains) with the object . In the case of electron diffraction from the crystals stained using the routine negative staining technique the ordering was preserved down to 0.4-0.5 nm . Increasing the dose up to the normal value results in fading of distant reflections . Thus, negative staining with radiation doses less than the critical one could yield resolution down to 0.4 nm . Yet, the structure may change due to interaction with the stain . Nevertheless, the possibility that such resolution could be obtained for a limited number of objects should not be excluded . Some examples of the application of negative staining for investigation of quaternary and domain structure of proteins (nitrogenase, glutamine synthetase, mitochondrial ATP-synthase, membrane monooxygenase enzymes), tubular and two-dimensional protein crystals (catalase, phosphorylase, HWV protein, hydrogenase), as well as ribosomes and bacteriophages are given in the review.

Acta Microbiol Pol, 1990, 39(1-2), 13 - 22
Construction and characterization of hybrids containing genes coding for proteins of the central part of bacteriophage T4 baseplate; Nieradko J et al.; The central part (hub or plug) of bacteriophage T4 baseplate consist of several proteins which are present in only few copies per phage particle . The presence of these minor baseplate components was inferred from the genetic data but only some of them were identified as distinct proteins species by biochemical analysis . We have constructed a number of plasmids containing segments of bacteriophage T4 genome coding for baseplate proteins . The following genes were cloned into expression vectors: 54, 48, 29, 28, 27, 51, 26 and 25 . The presence of a particular gene product was confirmed by in vivo complementation test . On the basis of these results we could more precisely localize the position of a particular gene on T4 phage genetic map . The hybrids contain sets of genes which make aggregation impossible, so bacteria harbouring these plasmids are convenient starting point for the purification of baseplate proteins.

Biochem Int, 1990, 20(6), 1033 - 40
T7 RNA polymerase: use of limited proteolysis for the study of enzyme's interaction with substrates and inhibitors; Tunitskaya VL et al.; The specific limited trypsinolysis of bacteriophage T7 RNA polymerase (T7RP) was performed in the presence of various components of the polymerase reaction and some GTP-analogs--irreversible inhibitors of the enzyme . The differences in the rate and sites of proteolysis were observed . Basing on the data obtained the role of the N-terminal domain of the T7RP in the interaction with promoter-containing template is proposed.

Infect Immun, 1990 Jan, 58(1), 245 - 51
Identification of a 35-kilodalton Mycobacterium tuberculosis protein containing B- and T-cell epitopes; Vismara D et al.; Screening of a Mycobacterium tuberculosis genomic DNA library in the lambda gt11 expression vector was carried out by using, as probes, sera from tuberculous patients and murine monoclonal antibody H61.3 recognizing a mycobacterial 35-kilodalton protein present only on the M . tuberculosis complex . The recombinant beta-galactosidase-fused protein present in the crude lysate induced the proliferation of T lymphocytes from patients with tuberculous pleuritis . As the recombinant insert contains an internal EcoRI restriction site, it was possible to identify two fragments, one proximal to the lacZ gene and 1.7 kilobases (kb) in size and the other distal to the lacZ gene and 2.2 kb in size . Southern blot analysis showed that both of them hybridized with the genomic DNA from M . tuberculosis and M . bovis but not with the DNA from other mycobacterial species . To perform extensive immunological studies, the amount of beta-galactosidase-fused protein being very low, we fused the 1.7-kb fragment to the N-terminal part of the gene coding for the DNA polymerase of bacteriophage MS2 in the expression vector pEx34 . The fusion protein was partially purified, and subsequent Western blotting (immunoblotting) and T-cell proliferation experiments confirmed the presence of B- and T-cell mycobacterial epitopes . Furthermore, to isolate the chromosomal region containing the 35-kilodalton gene, we constructed another mycobacterial genomic library in the lambda 2001 vector by cloning 15 to 20 kb of foreign DNA . Screening of this library was carried out by using 1.7- and 2.2-kb recombinant fragments as probes . Restriction maps of some clones isolated were determined.

Biochemistry, 1989 Dec 26, 28(26), 9995 - 10001
Spin-label ESR of bacteriophage M13 coat protein in mixed lipid bilayers . Characterization of molecular selectivity of charged phospholipids for the bacteriophage M13 coat protein in lipid bilayers; Wolfs JA et al.; Bacteriophage M13 major coat protein has been incorporated at different lipid/protein ratios in lipid bilayers consisting of various ratios of dimyristoylphosphatidylcholine (DMPC) to dimyristoylphosphatidylglycerol (DMPG) . Spin-label ESR experiments were performed with phospholipids labeled at the C-14 position of the sn-2 chain . For M13 coat protein recombinants with DMPC alone, the relative association constants were determined for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels and found to be 1.0, 1.0, and 2.1 relative to the background DMPC, respectively . The number of association sites for each phospholipid on the protein was found to be 4 per protein monomer . The intrinsic off-rates for lipid exchange at the intramembranous surface of the protein in DMPC alone at 30 degrees C were found to be 5 X 10(6), 6 X 10(6), and 2 X 10(6) s-1 for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels, respectively . Adding DMPG to the DMPC lipid system increased the exchange rates of the lipids on and off the protein . By gel filtration chromatography, it is found that protein aggregation is reduced after addition of DMPG to the lipid system . This is in agreement with measurements of tryptophan fluorescence, which show a decrease in quenching efficiency after introduction of DMPG in the lipid system . The results are interpreted in terms of a model relating the ESR data to the size of the protein-lipid aggregates.

Nucleic Acids Res, 1989 Dec 25, 17(24), 10353 - 66
Functional domains in the bacteriophage phi 29 terminal protein for interaction with the phi 29 DNA polymerase and with DNA; Zaballos A et al.; Deletion mutants at the amino- and carboxyl-ends of the phi 29 terminal protein, as well as internal deletion and substitution mutants, whose ability to prime the initiation of phi 29 DNA replication was affected to different extent, have been assayed for their capacity to interact with DNA or with the phi 29 DNA polymerase . One DNA binding domain at the amino end of the terminal protein has been mapped . Two regions involved in the binding to the DNA polymerase, an internal region near the amino-terminus and a carboxyl-terminal one, have been also identified . Interaction with both DNA and phi 29 DNA polymerase are required to led to the formation of terminal protein-dAMP initiation complex to start phi 29 DNA replication.

J Biol Chem, 1989 Dec 25, 264(36), 21788 - 92
Localization of DNA repair synthesis by human cell extracts to a short region at the site of a lesion; Hansson J et al.; Double-stranded bacteriophage M13 DNA molecules were constructed containing a single specifically placed 2-(acetylamino)fluorene adduct or a single 4'-hydroxymethyl-4,5',8-trimethylpsoralen monoadduct . These circular DNA molecules were used to analyze in vitro DNA repair synthesis by cell extracts from normal human lymphoid cell lines . Both types of lesions stimulate DNA repair synthesis at the site of the adduct . DNA repair synthesis induced by the 2-(acetyl-amino)fluorene adduct took place in the damaged strand and was confined to a region within a 31-base pair restriction fragment around the adduct.

Nucleic Acids Res, 1989 Dec 25, 17(24), 10203 - 12
Regulation of phage Mu repressor transcription by IHF depends on the level of the early transcription; van Rijn PA et al.; Integration Host Factor (IHF) of E . coli can stimulate both early and repressor transcription of bacteriophage Mu . We introduced several mutations in the early promoter (Pe) and studied the effect of these mutations on the stimulation of early and repressor transcription by IHF . All mutant promoters are still positive regulated by IHF, but the level of stimulation is dependent on the strength of the promoter . The strength of the early promoter has an even greater impact on the regulation of the repressor promoter by IHF: stimulation is observed in the presence of a relatively weak Pe, whereas with a strong Pe the repressor promoter Pc is inhibited by IHF . This inhibition is most probably due to an interference of the early transcription with the opposing repressor transcription . The implication of this type of regulation for the Mu life cycle is discussed.

Gene, 1989 Dec 21, 85(1), 53 - 8
The organization of the right-end early region of bacteriophage PRD1 genome; Pakula TM et al.; Bacteriophage PRD1 is the only protein-primed DNA replication system known to operate in Escherichia coli . The left-genome end of PRD1 contains the early genes for the terminal protein and the DNA polymerase . These genes have been sequenced and the proteins have been produced separately . In this investigation we completed the analysis of the PRD1 early DNA regions by cloning and sequencing the right end genome containing early genes XII and XIX . We compared the structure of the right- and left-terminal regions . The genome organization of both ends was found to be rather uniform . The inverted terminal repeats, the first promoters and the first translation start codons are located almost exactly at the same distance from the genome ends . The PRD1 early gene products, P12 and P19, do not share similarities with proteins found in other protein-primed replication systems.

Gene, 1989 Dec 21, 85(1), 45 - 51
Bacteriophage PRD1 terminal protein: expression of gene VIII in Escherichia coli and purification of the functional P8 product; Savilahti H et al.; The gene VIII coding for the bacteriophage PRD1 terminal protein P8 has been cloned under the control of the lambda pL promoter . The recombinant plasmid thus obtained (pUSH20) was able to complement a mutation in the phage terminal-protein gene VIII . High expression of the cloned gene from this plasmid could be obtained by raising the growth temperature from 28 to 42 degrees C . This heat induction resulted in an increased synthesis of a protein of 30 kDa, the size expected for the P8 protein . When complemented with an extract of cells carrying the PRD1 DNA polymerase gene, the extract from the cells harboring the plasmid pUSH20 was able to form the P8-dGMP replication initiation complex . The PRD1 replication initiation reaction was optimized and used to detect the biological activity of the expressed terminal protein . Subsequently, P8 protein was purified to almost homogeneity and shown to be biologically functional after the various purification steps.

Gene, 1989 Dec 21, 85(1), 25 - 33
The P2 phage old gene: sequence, transcription and translational control; Haggard-Ljungquist E et al.; The old (overcoming lysogenization defect) gene product of bacteriophage P2 kills Escherichia coli recB and recC mutants and interferes with phage lambda growth {Sironi et al., Virology 46 (1971) 387-396; Lindahl et al., Proc . Natl . Acad . Sci . USA 66 (1970) 587-594} . Specialized transducing lambda phages, which lack the recombination region, can be selected by plating lambda stocks on E . coli that carry the old gene on a prophage or plasmid {Finkel et al., Gene 46 (1986) 65-69} . Deletion and sequence analyses indicate that the old-encoded protein has an Mr of 65,373 and that its transcription is leftward . Primer extension analyses locate the transcription start point near the right end of the virion DNA . A bacterial mutant, named pin3 and able to suppress the effects of the old gene, has been isolated {Ghisotti et al., J . Virol . 48 (1983) 616-626} . In a pin3 mutant strain, carrying the old gene on a prophage or plasmid, the amount of old transcript is greatly reduced . The effect of the pin3 mutation is abolished by the wild-type allele of argU, an arginine tRNA that reads the rare Arg codons AGA and AGG, which are used for eight of the 14 Arg codons in the old gene . Thus the pin3 allele probably stalls translation of the old mRNA, causing this mRNA to be degraded . Isoelectric focusing and electrophoretic analysis identify the old gene product as a basic protein of approx . 65 kDa.

Gene, 1989 Dec 21, 85(1), 199 - 204
Direct sequencing of bacteriophage T4 DNA with a thermostable DNA polymerase; Kricker MC et al.; We present a simple and convenient protocol for the direct sequencing of bacteriophage T4 genomic DNA . The method utilizes the thermostable DNA polymerase from Thermus aquaticus (Taq) and 32P-end-labeled oligodeoxyribonucleotide primers to produce extension products that allow the analysis of at least 200 nucleotides (nt) on a single sequencing gel . Single-nt changes in the template were easily detectable following an overnight exposure of the autoradiograms . Comparison of sequences from fully modified T4 DNA containing glucosylated hydroxymethyldeoxycytosine or from templates containing cytosine showed little difference in sequence clarity . These techniques considerably simplify the molecular analysis of T-even bacteriophages and should be compatible with automated sequencing methods which employ 5'-end-labeled primers.

Gene, 1989 Dec 21, 85(1), 75 - 81
Saturation mutagenesis of the inside end of insertion sequence IS50; Dodson KW et al.; A 19-bp segment at the inside (I) end of IS50 (Tn5) is needed for efficient transposition . The importance of each position was assayed by making at least one base substitution at each position by either chemical-or oligodeoxyribonucleotide-directed mutagenesis . Mutant I ends were paired with a wild-type (wt) segment from the outside (O) end of IS50 and the transposase (tnp) gene was placed either between the ends or 1200 bp from the O end . The frequency of transposition of the resultant elements to bacteriophage lambda was measured . At least one substitution at each of the 19 I-end positions decreased transposition activity to less than 25% of wt, and most substitutions (25 of 28) decreased it to less than 5% of wt from one or both donor plasmids . These results show that each position in the I end is important during transposition.

Gene, 1989 Dec 21, 85(1), 15 - 23
Bending of DNA by gene-regulatory proteins: construction and use of a DNA bending vector; Kim J et al.; The binding of a protein to its specific sequence, borne on a DNA fragment, retards the mobility of the fragment in a characteristic way during gel electrophoresis . If the protein induces bending in the DNA, the contortion can also be monitored by gel electrophoresis, because the amount of retardation of the mobility of the DNA-protein complex is dependent upon the position and the degree of the bend induced in the DNA fragment {Wu and Crothers, Nature 308 (1984) 509-513} . We have constructed a plasmid, pBend2, which can generate a large number of DNA fragments of identical length in which the protein-binding nucleotide sequence is located in circular permutations . The vector contains two identical DNA segments containing 17 restriction sites in a direct repeat spanning a central region containing cloning sites . The protein-binding sequence is inserted at one of these cloning sites . To investigate the functional significance of bending, we have compared, using pBend2, the cAMP.cAMP-receptor protein (CPR)-induced bending of CRP-binding sites found in five different genes of Escherichia coli . We have also shown that the bacteriophage lambda 0R1 operator DNA is bent when complexed with the CI or Cro repressor of the phage.

J Mol Biol, 1989 Dec 20, 210(4), 687 - 701
Sequence of bacteriophage T3 DNA from gene 2.5 through gene 9; Beck PJ et al.; The nucleotide sequence of bacteriophage T3 DNA, from gene 2.5 through gene 9 has been determined . In addition to regulatory sites, the sequence predicts 19 close-packed genes plus two genes that overlap, in a different reading frame, another gene . The majority of these genes are highly homologous to those in the corresponding region of bacteriophage T7 . However, there are some genes that are present in one, but not the other, phage . These apparent deletions are almost exactly gene size and thus the close-packed organization of genes remains the same in T3 as in T7 . The varying levels of homology between T3 and T7 DNAs, first noted by Davis and Hyman in their study of DNA heteroduplexes, are also demonstrated here by a comparison of T3 and T7 nucleotide sequences . Many regions of extremely high homology immediately abut sequences that have no apparent homology . These data suggest that bacteriophages T3 and T7 have recombined, both with each other and with other members of a pool of T7-like phages, during their co-evolution.

Biochem Biophys Res Commun, 1989 Dec 15, 165(2), 817 - 25
Expression of rat hepatic glucokinase in Escherichia coli; Chien CT et al.; Rat liver glucokinase was expressed in Escherichia coli by using an expression system based on bacteriophage T7 RNA polymerase . The expressed protein starts with the predicted initiator methionine residue and ends at the appropriate carboxyl terminal residue . It was partially purified by ammonium sulfate precipitation and gel filtration and had kinetic and physical properties similar to the purified rat liver enzyme . The efficient expression of this low abundance hepatic protein in bacteria provides a system for in vitro analysis of mutations of the enzyme.

Gene, 1989 Dec 14, 84(2), 209 - 19
Regulation of coliphage T3 and T7 RNA polymerases by the lac repressor-operator system; Giordano TJ et al.; The single-polypeptide RNA polymerases that are encoded by bacteriophage T7 and its relatives form the basis of highly specific and efficient transcription systems . Here, we describe the regulation of transcription from phage promoters by the lac repressor-operator system of Escherichia coli . A synthetic oligodeoxyribonucleotide that contains the core sequence of the lac operator (lacO) was cloned at various distances downstream from the transcription start point (tsp) of the T3 and T7 promoters . The ability of lac repressor to prevent transcription from the phage promoters in vitro was dependent on the position of the operator . Efficient repression was observed when the center of the operator was placed between +14 and +27 (+1 being the tsp), whereas the repressor had little effect when bound to operators centered at +64 . For in vivo studies, the chloramphenicol acetyltransferase (CAT)-encoding reporter gene was placed under the control of various promoter-operator constructs, and introduced into bacterial cells containing the genes for the lac repressor and T3 or T7 RNA polymerase . As with in vitro studies, high levels of repression (greater than 4000-fold) of T3 and T7 RNA polymerase activity were achieved, and repression was reversed by the inducer isopropyl-beta-D-thiogalactopyranoside . When the T3 promoter-lacO constructs are used to regulate the expression of a target gene in combination with an inducible RNA polymerase gene under control of the lacUV5 promoter, the doubly regulated system provides extremely tight levels of repression, yet allows high levels of expression after induction . In such a system, we observed a greater than 10(5)-fold increase in CAT activity within 30 min after induction . This system should prove useful in cloning and expressing genes that are potentially toxic to the host cells.

Gene, 1989 Dec 14, 84(2), 247 - 55
Primary structure of bacteriophage M2 DNA polymerase: conserved segments within protein-priming DNA polymerases and DNA polymerase I of Escherichia coli; Matsumoto K et al.; Bacteriophage M2 encodes its own DNA polymerase which catalyses the formation of a primer protein-5'dAMP initiation complex for DNA replication . To understand the relation of structure to function of this 'protein-priming DNA polymerase', we have determined the nucleotide sequence of the M2 DNA polymerase-encoding gene (gene G) . The deduced 572-amino acid sequence of M2 DNA polymerase shows 82.3% overall homology to that of phi 29 DNA polymerase . A homology search with the mutation data matrix revealed that six segments (A-F, from the N terminus) of M2 and phi 29 DNA polymerases are homologous with the sequence of Escherichia coli DNA polymerase I (PolI) . Segments D and F coincide with the conserved segments of many other DNA polymerases . Therefore, M2 and phi 29 DNA polymerases have structural features, at least in the conserved segments, similar to those of PolI and other DNA polymerases . Based on the homology with PolI and the location of the mutations for aphidicolin resistance and nucleoside analog resistance of M2, phi 29 and herpes simplex virus type-1 DNA polymerases, we propose that segments A-D of the M2 and phi 29 DNA polymerases constitute a structure which forms the cleft for holding template DNA and that segment D is a region for interacting with dNTP.

Biochemistry, 1989 Dec 12, 28(25), 9826 - 33
Sequence-specific 1H NMR assignment and secondary structure of the Arc repressor of bacteriophage P22, as determined by two-dimensional 1H NMR spectroscopy; Breg JN et al.; The Arc repressor of bacteriophage P22 is a DNA binding protein that does not belong to any of the known classes of such proteins . We have undertaken a 1H NMR study of the protein with the aim of elucidating its three-dimensional structure in solution and its mode of binding of operator DNA . Here we present the 1H nuclear magnetic resonance (NMR) assignments of all backbone protons and most of the side-chain protons of Arc repressor . Elements of secondary structure have been identified on the basis of networks of characteristic sequential and medium-range nuclear Overhauser enhancements (NOEs) . Two alpha-helical regions have been found in the peptide regions 16-29 and 35-45 . The ends of the helices could not yet be firmly established and could extend to residue 31 for the first helix and to residue 49 for the second . Immediately before the first helix, between residues 8 and 14, a region is present with beta-sheet characteristics dominated by a close proximity of the alpha-protons of residues 9 and 13 . Because of the dimeric nature of the protein there are still two possible ways in which the NOEs in the beta-sheet region can be interpreted . If the NOEs are intramonomer, this requires a tight turn involving residues 10-12 . Alternatively, if the NOEs are intermonomer, then and antiparallel beta-sheet would be implicated comprising two strands of different Arc monomers . While the data presently do not allow an unambiguous choice between these two possibilities, some evidence is discussed that favors the latter (beta-sheet between monomers).(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleic Acids Res, 1989 Dec 11, 17(23), 9861 - 70
Internal deletion mutants of Xenopus transcription factor IIIA; Hanas JS et al.; Xenopus transcription factor IIIA (TFIIIA) or TFIIIA mutants with internal deletions were expressed in E . coli utilizing a bacteriophage T7 RNA polymerase system . TFIIIA or deletion mutant TFIIIAs, isolated from E.coli cell extracts, were identified by SDS PAGE and immunoblotting with rabbit antiserum against native TFIIIA purified from Xenopus immature oocytes . Specific DNA binding of intact or internally deleted TFIIIA was compared by analyzing their abilities to protect the internal control gene (ICR) of the Xenopus 5S RNA gene from DNase I digestion . Intact protein, synthesized from a full-length TFIIIA cDNA, bound specifically to the entire ICR (+96 to +43) and promoted 5S RNA gene transcription in vitro . One TFIIIA deletion mutant, expressed from cDNA lacking the coding sequence for the putative fourth zinc finger (designated from the N-terminus, amino acids 103-132) protected the ICR from DNase I digestion from nucleotide positions +96 to +78 . A second TFIIIA mutant resulting from fusion of putative zinc fingers 7 and 8 (deletion of amino acids 200-224) protected the 5S gene ICR from positions +96 to +63 . The DNase I protection patterns of these mutant proteins are consistent with the formation of strong ICR contacts by those regions of the protein on the N-terminal side of the mutation but not by those regions on the C-terminal side of the mutation . The regions of the protein comprising the N-terminal 3 fingers and N-terminal six fingers appear to be in contact with approximately 18 and 33 bp of DNA respectively on the 3' side of the 5S gene ICR . These internal deletion mutants promoted 5S RNA synthesis in vitro and DNA renaturation.

Nucleic Acids Res, 1989 Dec 11, 17(23), 10047 - 68
Bacteriophage T4 regA protein binds to the Shine-Dalgarno region of gene 44 mRNA; Webster KR et al.; We have overproduced and purified wild type regA protein, a translational repressor encoded by bacteriophage T4 . The repressor activity of the cloned regA protein has been tested on four known regA target genes (T4 genes: 44, 45, rpbA and regA) using in vitro coupled transcription-translation reactions . We have demonstrated the sensitivity of two additional T4 genes coding for alpha- and beta-glucosyltransferases to regA protein in vitro . The regA target site on the gene 44 messenger RNA has been identified through deletion analysis and RNase protection assays, using plasmids containing gene 44-lacZ fusions . The effect of regA protein on expression of 44P-beta-galactosidase fusion proteins was assayed in vitro, in coupled transcription-translation reactions . Analysis of deletion mutants of gene 44-lacZ localized the regA recognition region to between nucleotides -11 and +9 of the mRNA . RNase protection assays of g44-lacZ transcripts further defined the site of regA protein interaction to between nucleotides -10 and +2 of the mRNA . This region overlaps the gene 44 Shine-Dalgarno region and the A and U of the initiation codon.

Science, 1989 Dec 8, 246(4935), 1275 - 81
Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda; Huse WD et al.; A novel bacteriophage lambda vector system was used to express in Escherichia coli a combinatorial library of Fab fragments of the mouse antibody repertoire . The system allows rapid and easy identification of monoclonal Fab fragments in a form suitable for genetic manipulation . It was possible to generate, in 2 weeks, large numbers of monoclonal Fab fragments against a transition state analog hapten . The methods described may supersede present-day hybridoma technology and facilitate the production of catalytic and other antibodies.

Gene, 1989 Dec 7, 84(1), 9 - 16
Mutational analysis of the primer RNA template region in the replication origin (oric) of bacteriophage G4: priming signal recognition by Escherichia coli primase; Hiasa H et al.; The primase-dependent phage G4 origin of complementary DNA strand synthesis (G4oric) contains three stable stem-loops (I, II, and III) upstream from the initiation point of primer RNA (pRNA) . Site-directed mutagenesis was used to introduce alterations into the nucleotide (nt) sequence of the G4oric pRNA template region . Mutations in stem-loop I, that changed the length of the stem and the sequence of the loop, slightly depressed, but did not abolish, G4oric activity . However, functional G4oric activity was destroyed when the sequence containing the starting position of pRNA synthesis was deleted, or when insertions were introduced between the pRNA starting position (5'-CTG-3') and stem-loop I . Reintroducing a CTG as part of a PstI linker close to stem-loop I, however, resulted in recovery of G4oric functional activity . These results suggest that the specific nt sequence, containing 5'-CTG-3', between nt 3994 and 4007, and also the distance between the starting position of pRNA synthesis and stem-loop I, are essential structural features for G4oric function.

J Mol Biol, 1989 Dec 5, 210(3), 453 - 60
Specificity and mechanism of antitermination by Q proteins of bacteriophages lambda and 82; Yang XJ et al.; Lambdoid phage late gene operons are positively regulated by genome-specific antiterminator proteins encoded by the Q gene of each phage . In this paper, we compare the activity of phage lambda and phage 82 Q proteins . Q82-mediated antitermination, like that of Q lambda, involves a transcription pause during which the regulator can modify RNA polymerase . We show that the activities of both Q82 and Q lambda are genome-specific in chasing RNA polymerase out of the early pause sites and in mediating antitermination . Finally, we show that the length of the RNA in the paused complex, or the exact position of the pause, affects the efficiency with which Q82 chases RNA polymerase out of the pause.

J Biol Chem, 1989 Dec 5, 264(34), 20331 - 8
Purification of a ubiquitin protein peptidase from yeast with efficient in vitro assays; Liu CC et al.; In eukaryotic cells ubiquitin is synthesized as a polyubiquitin protein or as a protein fused at the carboxyl terminus to other polypeptides . An enzyme activity, ubiquitin protein peptidase, has been proposed to process these precursors by cleaving the peptide bond between adjoining ubiquitin molecules or between ubiquitin and the fused peptides . Using the cleavage of a 35S-labeled yeast ubiquitin protein fused to a synthetic 38-residue peptide obtained by in vivo metabolic labeling in Escherichia coli in an expression system based on the interaction of bacteriophage T7 RNA polymerase and its promoter, it is possible to detect a processing activity in soluble yeast extract . The specificity of the cleavage suggests this activity could be the in vivo processing activity for various ubiquitin precursor proteins in yeast cells . A similarly labeled ubiquitin protein fused to one cysteine residue was also utilized to detect an activity capable of removing a single cysteine residue from ubiquitin in a soluble extract . Employing assays based on the cleavage of labeled ubiquitin protein fusions, a ubiquitin protein peptidase activity from Saccharomyces cerevisiae was purified about 15,000-fold to yield a protein mixture consisting of only a few protein species . The major protein band which comigrated with the activities in in vitro assays has an apparent molecular weight of 29,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Two other protein species, about 20,000 and 10,000 in molecular weight, also comigrated with the in vitro activities throughout the purification procedure . Though our most purified protein fraction was shown to cleave various artificial ubiquitin protein fusions under our experimental conditions, it cannot cleave a ubiquitin dimer protein, suggesting the existence of functionally distinct ubiquitin protein peptidases . Our experimental protocol for preparing various labeled ubiquitin protein precursors provides a means to explore various processing enzymes existing in cells . The same protocol may also be adapted to prepare substrates for the study of other specific protein processing enzymes.

J Bacteriol, 1989 Dec, 171(12), 6600 - 9
Nucleotide sequences of the tolA and tolB genes and localization of their products, components of a multistep translocation system in Escherichia coli; Levengood SK et al.; Various mutations in the tolQRAB gene cluster of Escherichia coli render the bacteria tolerant to high concentrations of the E, A, or K colicins as well as tolerant to infection by the single-stranded filamentous bacteriophage . The nucleotide sequence of a 2.8-kilobase fragment containing the tolA and tolB genes was determined . This sequence predicts TolA to be a 421-amino-acid protein of molecular mass 44,190 daltons . Studies using minicells show it to be associated with the inner membrane, presumably via a 21-amino-acid hydrophobic sequence between residues 13 and 35 . The remaining 387 residues on the carboxyl side of this region are located in the periplasm . Within this region of TolA is a 230-residue portion that is predicted to form a very long helical segment . This region is rich in alanine, lysine, and glutamic and aspartic acids . The TolB protein is predicted to contain 431 amino acids . Localization studies using minicells show two proteins encoded by this open reading frame . The larger protein of 47.5 kilodaltons appears to be associated with the membrane fractions . The smaller protein is 43 kilodaltons in size and is found with the periplasmic components of the cell.

J Bacteriol, 1989 Dec, 171(12), 6586 - 92
Alternative route for biosynthesis of amino sugars in Escherichia coli K-12 mutants by means of a catabolic isomerase; Vogler AP et al.; By inserting a lambda placMu bacteriophage into gene glmS encoding glucosamine 6-phosphate synthetase (GlmS), the key enzyme of amino sugar biosynthesis, a nonreverting mutant of Escherichia coli K-12 that was strictly dependent on exogenous N-acetyl-D-glucosamine or D-glucosamine was generated . Analysis of suppressor mutations rendering the mutant independent of amino sugar supply revealed that the catabolic enzyme D-glucosamine-6-phosphate isomerase (deaminase), encoded by gene nagB of the nag operon, was able to fulfill anabolic functions in amino sugar biosynthesis . The suppressor mutants invariably expressed the isomerase constitutively as a result of mutations in nagR, the locus for the repressor of the nag regulon . Suppression was also possible by transformation of glmS mutants with high-copy-number plasmids expressing the gene nagB . Efficient suppression of the glmS lesion, however, required mutations in a second locus, termed glmX, which has been localized to 26.8 min on the standard E . coli K-12 map . Its possible function in nitrogen or cell wall metabolism is discussed.

J Bacteriol, 1989 Dec, 171(12), 6526 - 33
Point mutations in a conserved region (TonB box) of Escherichia coli outer membrane protein BtuB affect vitamin B12 transport; Gudmundsdottir A et al.; Uptake of cobalamins and iron chelates in Escherichia coli K-12 is dependent on specific outer membrane transport proteins and the energy-coupling function provided by the TonB protein . The btuB product is the outer membrane receptor for cobalamins, bacteriophage BF23, and the E colicins . A short sequence near the amino terminus of mature BtuB, previously called the TonB box, is conserved in all tonB-dependent receptors and colicins and is the site of the btuB451 mutation (Leu-8----Pro), which prevents energy-coupled cobalamin uptake . This phenotype is partially suppressed by certain mutations in tonB . To examine the role of individual amino acids in the TonB box of BtuB, more than 30 amino acid substitutions in residues 6 to 13 were generated by doped oligonucleotide-directed mutagenesis . Many of the mutations affecting each amino acid did not impair transport activity, although some substitutions reduced cobalamin uptake and the Leu-8----Pro and Val-10----Gly alleles were completely inactive . To test whether the btuB451 mutation affects only cobalamin transport, a hybrid gene was constructed which encodes the signal sequence and first 39 residues of BtuB fused to the bulk of the ferrienterobactin receptor FepA (residues 26 to 723) . This hybrid protein conferred all FepA functions but no BtuB functions . The presence of the btuB451 mutation in this fusion gene eliminated all of its tonB-coupled reactions, showing that the TonB box of FepA could be replaced by that from BtuB . These results suggest that the TonB-box region of BtuB is involved in active transport in a manner dependent not on the identity of specific side chains but on the local secondary structure.

Mol Cell Biol, 1989 Dec, 9(12), 5480 - 3
Miniribozymes, small derivatives of the sunY intron, are catalytically active; Doudna JA et al.; The self-splicing sunY intron from bacteriophage T4 has the smallest conserved core secondary structure of any of the active group I introns . Here we show that several nonconserved regions can be deleted from this intron without complete loss of catalytic activity . The 3' stems P9, P9.1, and P9.2 can be deleted while retaining 5' cleaving activity . Two base-paired stems (P7.1 and P7.2) that are peculiar to the group IA introns can also be deleted; however, the activities of the resulting derivatives depend greatly on the choice of replacement sequences and their lengths . The smallest active derivative is less than 180 nucleotides long . These experiments help to define the minimum structural requirements for catalysis.

Forensic Sci Int, 1989 Dec, 43(3), 275 - 80
M13 Bioprints: non-isotopic detection of individual-specific human DNA fingerprints with biotinylated M13 bacteriophage; Medeiros AC et al.; M13 bacteriophage was labelled by primer extension with biotin-11-dUTP and was successfully used as probe to study human hypervariable minisatellites (DNA fingerprints) in Southern blots . The method, which we called M13 Bioprints, presents two advantages over conventional DNA fingerprinting . First, it is easily accessible, since it utilizes the widely available M13 phage as a probe . Second, it makes use of biotin, which is safer, simpler and more economical than isotopic labels.

Curr Genet, 1989 Dec, 16(5-6), 407 - 18
DNA sequence analysis of the apocytochrome b gene of Podospora anserina: a new family of intronic open reading frame; Cummings DJ et al.; The 5,969 bp (base pair) DNA sequence of the apocytochrome b mitochondrial (mt) gene of race A Podospora anserina was located in a 8.5 Kbp region . This gene contained a 2,499 bp subgroup IB and a 1,306 bp subgroup ID intron as well as a 990 bp subgroup IB intron which is present in race A but not race s . The large subgroup IB intron and the race A specific IB intron both contained potential alternate splice sites which brought their open reading frames into phase with their upstream exon sequences . All three introns were compared with regard to their secondary structures and open reading frames to the other 30 group I introns in Podospora anserina, as well as to other fungal introns . We detected a new family of intronic ORFs comprising seven P . anserina introns, several N . crassa introns, as well as the T4td bacteriophage intron . Sequence similarities to intron-encoded endonucleases were noteworthy . The DNA sequences reported here and in the accompanying paper complete the analysis of race s and race A mitochondrial DNA.

Mol Gen Genet, 1989 Dec, 220(1), 8 - 11
An inhibitory effect of RGD peptide on protein-priming reaction of bacteriophages phi 29 and M2; Kobayashi H et al.; The amino acid sequence, arginine-glycine-aspartic acid (RGD), found in some cell adhesive proteins, is a recognition signal for the receptor protein . It is interesting that we have found the RGD sequence in terminal protein (TP) of bacteriophages phi 29 and M2 near an amino acid, the serine residue at 232, covalently linked to the terminal nucleotide of their DNAs . At the initiation of protein-primed DNA replication, TP is essential for the recognition of replication machinery containing DNA polymerase and primer protein (PP; PP becomes TP upon linking the first nucleotide, and hence the primary structure of TP is the same as that of PP) . Synthetic peptide RGD specifically inhibited transfection of phi 29 and M2 . The target of the RGD peptide is shown to be TP by marker rescue experiments, suggesting that a receptor for the RGD sequence exists in TP . Furthermore, the peptide inhibited the in vitro protein-priming reaction of DNA replication . We propose that the RGD sequence of PP and a putative receptor on TP is utilized for the molecular recognition initiating DNA replication.

Mol Gen Genet, 1989 Dec, 220(1), 161 - 4
Organization of Methanobacterium thermoautotrophicum bacteriophage psi M1 DNA; Jordan M et al.; psi M1 is a virulent bacteriophage of Methanobacterium thermoautotrophicum strain Marburg . Restriction enzyme analysis of the linear, 30.4 kb phage DNA led to a circular map of the 27.1 kb psi M1 genome . psi M1 is thus circularly permuted and exhibits terminal redundancy of approximately 3 kb . Packaging of psi M1 DNA from a concatemeric precursor initiates at the pac site which was identified at coordinate 4.6 kb on the circular genome map . It proceeds clockwise for at least five packaging rounds . Headful packaging was also shown for psi M2, a phage variant with a 0.7 kb deletion at coordinate 23.25 on the map.

J Gen Virol, 1989 Dec, 70 ( Pt 12), 3381 - 90
Analysis of the complete nucleotide sequence of Chp1, a phage which infects avian Chlamydia psittaci; Storey CC et al.; We report the complete nucleotide sequence of bacteriophage Chp1 . The genome was found to be 4877 bases long and it potentially codes for 11 proteins . Open reading frames (ORFs) 6 and 7 lie within ORFs 2 and 1 respectively but are in a second reading frame . No significant DNA homology was found when Chp1 was compared to the EMBL database . The N-terminal amino acid sequences of the three structural proteins VP1, VP2 and VP3 were determined and it was found that they were encoded by ORFs 1, 2 and 3 respectively . Amino acid homology studies revealed that VP1 has homology with the major structural protein of bacteriophages phi X174 and S13, and that the protein inferred from ORF 4 shows homology to the A proteins of phi X174, S13 and G4 . The genome of Chp1 has an organization similar to that of phi X174 although it is 509 bases smaller . We propose that Chp1 is a member of the Microviridae but that it is sufficiently different to warrant its own subfamily which we have called the Chlamydiavirinae.

J Virol, 1989 Dec, 63(12), 5386 - 92
In vitro construction of poliovirus defective interfering particles; Hagino-Yamagishi K et al.; To construct poliovirus defective interfering (DI) particles in vitro, we synthesized an RNA from a cloned poliovirus cDNA, pSM1(T7)1, which carried a deletion in the genome region corresponding to nucleotide positions 1663 to 2478 encoding viral capsid proteins, by using bacteriophage T7 RNA polymerase . The RNA was designed to retain the correct reading frame in nucleotide sequence downstream of the deletion . HeLa S3 monolayer cells were transfected with the deletion RNA and then superinfected with standard virus as a helper . The DI RNA was observed in the infected cells after three passages at high multiplicity of infection . The sequence analysis of RNA extracted from the purified DI particle clearly showed that this DI RNA had the same deletion in size and location as that in the RNA used for the transfection . Thus, we succeeded in construction of a poliovirus DI particle in vitro . To gain insight into the mechanism for DI generation, we constructed poliovirus cDNAs pSM1(T7)1a and pSM1(T7)1b that, in addition to the same deletion as that in pSM1(T7)1, had insertion sequences of 4 bases and 12 bases, respectively, at the corresponding nucleotide position, 2978 . The RNA transcribed from pSM1(T7)1a was not a template for synthesis of poliovirus nonstructural proteins and therefore was inactive as an RNA replicon . On the other hand, the RNA from pSM1(T7)1b replicated properly in the transfected cells . Superinfection of the transfected cells with standard virus resulted in production of DI particles derived from pSM1(T7)1b and not from pSM1(T7)1a . These observations indicate that deletion RNAs that are inactive replicons have little or no possibility of being genomes of DI particles suggesting the existence of a nonstructural protein(s) that has an inclination to function as a cis-acting protein(s) . The method described here will provide a useful technique to investigate genetic information essential for poliovirus replication.

Virology, 1989 Dec, 173(2), 378 - 89
Identification of the bacteriophage Mu kil gene; Waggoner BT et al.; The kil gene encoded in bacteriophage Mu DNA was previously shown to reside between the end of the B gene at 4.3 kb and the EcoRI site at 5.1 kb from the left end . To precisely map the kil gene within this region, two series of BAL-31 deletion derivatives were created: one removed Mu DNA rightward from the Hpal site (4.2 kb) and the other removed Mu DNA leftward from the EcoRI site . The deleted Mu DNA was subcloned into the expression vector pUC19 under lac promoter control and tested for the expression of the killing function following IPTG induction . Using DNA sequencing analysis, the Mu DNA in Kil+ and Kil- clones was precisely determined, and the kil gene was mapped to the first open reading frame beyond the B gene . The expression of the kil gene was sufficient to induce dramatic morphological changes: cells became enlarged and predominantly spherical, reminiscent of the phenotype of certain cell mutants.

EMBO J, 1989 Dec 1, 8(12), 3843 - 51
Stereoselective arginine binding is a phylogenetically conserved property of group I self-splicing RNAs; Hicke BJ et al.; We have examined the reaction of GTP with RNA polymerase transcripts containing the self-splicing RNA precursors from the Neurospora crassa Cob1 intron, and from introns in the sunY, nrdB and td genes of bacteriophage T4 . In each case, we find a low Km for GTP (between 0.8 and 11 microM), accompanied by competitive inhibition of the GTP reaction by L-arginine, as was found for the previously examined Tetrahymena nuclear pre-rRNA intron . Trials with the 20 standard amino acids show that inhibition in all cases is specific to the arginine side-chain . L-arginine binds with similar affinity to all introns studied, the Ki's ranging from 4.3 to 21 mM . Strikingly, the relative binding preference of the RNAs for L- versus D-arginine is highly conserved: the ratio of L-arg Ki/D-arg Ki, the stereoselectivity, is always close to 2 . Because of the conservation of GTP and arginine binding constants and particularly because of the conserved stereoselectivity, we conclude that the evolution of an effective group I RNA transesterification catalyst necessarily produces a specific and stereoselective RNA binding site for a single amino acid . This suggests that selection for an ancient group I RNA could have fortuitously initiated the specific association of RNA sequences with amino acids, a first step toward the genetic code.

J Mol Graph, 1989 Dec, 7(4), 186 - 95
Modeling loop structures in proteins and nucleic acids: an RNA stem-loop; Haneef I et al.; We have used a novel modeling technique, based on combining information from several preexisting structures, to generate a three-dimensional (3D) model for the RNA stem-loop responsible for translational repression of the MS2 RNA bacteriophage replicase . Specific features of the model have been tested experimentally by chemical and enzymatic structural probes; results from these experiments have been used to "improve" the model by fixing initial assumptions . The new model and chemical modification data are in part consistent, and further predictions are being tested . The modeling algorithm has a wide range of potential applications, particularly to loop regions in proteins and nucleic acids.

Gene, 1989 Nov 30, 83(2), 367 - 70
Site-directed transcription initiation with a mobile promoter; Loewy ZG et al.; A method is described for the high-level transcription of any DNA segment using bacteriophage RNA polymerases (RNAPs) . A synthetic mobile promoter with a template-complementary 3' extension is ligated to the target sequence of interest . Transcription with an appropriate RNAP results in an amplification of approx . 70-fold . In the presence of heterologous DNA, bacteriophage RNAPs are shown to be specific for their cognate mobile promoters.

Biochem Biophys Res Commun, 1989 Nov 30, 165(1), 512 - 8
Minute amounts of RNA are synthesized from several regions of the bacteriophage Mu DNA during the lysogenic state; Barron C et al.; The transcription of phage Mu DNA during the lysogenic state has been quantitatively analysed . For this purpose pulse-labelled RNA from two lysogens and from their nonlysogenic parental strains were hybridized to non-overlapping Mu DNA restriction fragments covering the whole phage genome . The data revealed that all regions of the prophage are transcribed at low rates and that phage promoters are involved in this transcription . For this study an improved assay for quantitative filter hybridization was employed . The high sensitivity and reproducibility that can be obtained with the assay make it suitable for the quantitative analysis of minute amounts of mRNA.

Biochemistry, 1989 Nov 28, 28(24), 9521 - 7
The missing nucleoside experiment: a new technique to study recognition of DNA by protein; Hayes JJ et al.; We report a new technique for quickly determining which nucleosides in a DNA molecule are contacted by a sequence-specific DNA-binding protein . Our method is related to the recently reported "missing contact" experiment {Brunelle, A., & Schleif, R . F . (1987) Proc . Natl . Acad . Sci . U.S.A . 84, 6673-6679} . We treat the DNA molecule with the hydroxyl radical to randomly remove nucleosides . The ability of protein to bind to gapped DNA is assayed by gel mobility shift . Nucleosides important to protein binding are identified by sequencing gel electrophoresis . The missing nucleoside experiment can be used to scan a DNA molecule at single-nucleotide resolution in one experiment . The bacteriophage lambda repressor-OR1 and cro-OR1 complexes were analyzed to evaluate the method . For both proteins, the most important contacts are located in the protein monomer that binds to the consensus half of the operator . These contacts correspond well to those found by mutational studies, and in the cocrystal structure of the lambda repressor-operator . The missing nucleoside data show that the amino-terminal arms of lambda repressor make energetically important contacts with positions 7 and 8 and the central dyad base pair of the operator . The amino-terminal arm that makes the most extensive contacts to DNA appears to be the one that emanates from the repressor monomer that binds to the consensus half of the operator, in agreement with the cocrystal structure . The lambda cro protein does not have an amino-terminal arm, and the missing nucleoside experiment clearly shows a lack of contacts to DNA in the central region of the operator in this complex.

Nucleic Acids Res, 1989 Nov 25, 17(22), 9101 - 12
The bacteriophage T2 and T4 DNA-{N6-adenine} methyltransferase (Dam) sequence specificities are not identical; Schlagman SL et al.; Bacteriophages T2 and T4 encode DNA-{N6-adenine} methyltransferases (Dam) which differ from each other by only three amino acids . The canonical recognition sequence for these enzymes in both cytosine and 5-hydroxymethylcytosine-containing DNA is GATC; at a lower efficiency they also recognize some non-canonical sites in sequences derived from GAY (where Y is cytosine or thymine) . We found that T4 Dam fails to methylate certain GATA and GATT sequences which are methylated by T2 Dam . This indicates that T2 Dam and T4 Dam do not have identical sequence specificities . We analyzed DNA sequence data files obtained from GenBank, containing about 30% of the T4 genome, to estimate the overall frequency of occurrence of GATC, as well as non-canonical sites derived from GAY . The observed N6methyladenine (m6A) content of T4 DNA, methylated exclusively at GATC (by Escherichia coli Dam), was found to be in good agreement with this estimate . Although GATC is fully methylated in virion DNA, only a small percentage of the non-canonical sequences are methylated.

J Biol Chem, 1989 Nov 25, 264(33), 19514 - 27
Structure of the gene for human von Willebrand factor; Mancuso DJ et al.; von Willebrand factor is a large multimeric plasma protein composed of identical subunits which contain four types of repeated domains . von Willebrand factor is essential for normal hemostasis, and deficiency of von Willebrand factor is the most common inherited bleeding disorder of man . Four human genomic DNA cosmid libraries and one bacteriophage lambda library were screened with von Willebrand factor cDNA probes . Twenty positive overlapping clones were characterized that span the entire von Willebrand factor gene . A high-resolution restriction map was constructed for approximately 75% of the locus and a total of approximately 33.8 kilobases was sequenced on both strands including all intron-exon boundaries . The gene is approximately 178 kilobases in length and contains 52 exons . The exons vary from 40 to 1379 base pairs in length, and the introns vary from 97 base pairs to approximately 19.9 kilobases in length . The signal peptide and propeptide (von Willebrand antigen II) of von Willebrand factor are encoded by 17 exons in approximately 80 kilobases of DNA while the mature subunit of von Willebrand factor and 3' noncoding region are encoded by 35 exons in the remaining approximately 100 kilobases of the gene . A number of repetitive sequences were identified including 14 Alu repeats and a approximately 670-base pair TCTA simple repeat in intron 40 that is polymorphic . Regions of the gene that encode homologous domains have similar structures, supporting a model for their origin by gene segment duplication.

FEBS Lett, 1989 Nov 20, 258(1), 171 - 4
Lambda plac10 transducing bacteriophage: DNA primary structure of the region of the abnormal excision; Shpakovski GV et al.; In studying molecular mechanisms of the formation of transducing bacteriophages, we have elucidated the primary structure of the phage-bacterial DNA junction which resulted from the abnormal excision of the lambda plac10 phage . The process is structurally similar to the excision of the lambda plac5 phage and involves, in both cases, highly homological DNA stretches approximately 20 bp long, one of them being a part of the Z-Y spacer of the lac operon and possessing a developed secondary structure . The conception of regioselective recombination as a type of illegitimate recombinational process with a certain degree of site-specificity is suggested.

J Mol Biol, 1989 Nov 20, 210(2), 265 - 80
Alternative mRNA structures of the cIII gene of bacteriophage lambda determine the rate of its translation initiation; Altuvia S et al.; The bacteriophage lambda cIII gene product has a regulatory function in the lysis-lysogeny decision following infection . The availability of a set of cIII expression mutants allowed us to establish the structure-function relationship of the cIII mRNA . We demonstrate, using defined in vitro systems, that the cIII mRNA is present in two conformations at equilibrium . Mutations that have been shown to lead to cIII overexpression were found to freeze the RNA in one conformation (structure B), and permit efficient binding to the 30 S ribosomal subunit . Mutations that have been shown to prevent cIII translation cause the mRNA to assume the alternative conformation (structure A) . In this structure, the translation initiation region is occluded, thereby preventing 30 S ribosomal subunit binding . By varying the temperature or Mg2+ concentration it was possible to alter the relative proportion of the alternative structures in wild-type mRNA . We suggest that the regulation of the equilibrium between the two mRNA conformations provides a mechanism for the control of cIII gene expression.

FEBS Lett, 1989 Nov 20, 258(1), 177 - 9
Inhibition of the first phosphodiester bond formation catalyzed by Escherichia coli RNA polymerase in the presence of bovine seminal plasmin: promoter dependency; Gopal V et al.; Inhibition of the abortive initiation of transcription catalyzed by E . coli RNA polymerase has been studied here in the presence of bovine seminal plasmin . Seminal plasmin, which is known to be a stronger inhibitor than rifampicin binds at the same site as rifampicin to RNA polymerase . However, unlike rifampicin, seminal plasmin showed the inhibition of the formation of both the first and second phosphodiester bonds . We observed, in vitro, that the degree of inhibition of transcription was different at different promoters . Thus, the percent of inhibition of transcription initiation by seminal plasmin was much less at r-RNA promoters in comparison to that at the early promoters of bacteriophage T7.

Anal Biochem, 1989 Nov 15, 183(1), 89 - 93
A simple purification procedure for lambda gt bacteriophage DNA with hybridization size screening for isolation of longest length cDNA clones; Elliott RM et al.; An improved procedure for isolating lambda DNA and screening lambda gt10 or lambda gt11 libraries is described . Recombinant lambda gt11 bacteriophage particles (150,000) were amplified on three agarose plates (50,000 per plate) with Escherichia coli Y1090 as plating bacteria . After confluent lysis, recombinant bacteriophage was extracted with SM buffer . Bacterial debris was removed by centrifugation . A small aliquot of amplified lambda gt11 bacteriophage was kept to rescreen the bacteriophage, should a large or full-length clone be found to be present, after analysis of the size of the cDNA inserts . The major portion of the bacteriophage particles was purified by treatment with equilibrated DEAE-cellulose, pH 7.5 . Purified phage particles were precipitated with polyethylene glycol from the DEAE supernatant and extracted with phenol, phenol-chloroform, and chloroform . Such lambda gt11 DNA was readily digested with EcoRI . Liberated insert cDNA was separated on 1.2% agarose gels, transferred onto a nylon membrane, and hybridized with an alkaline phosphatase cDNA probe in an iterative procedure that allows isolation of the largest cDNA clones present in the library . We have used this procedure to isolate a full-length alkaline phosphatase cDNA . The method is quick, reliable, and less costly than conventional procedures for the isolation of full-length cDNAs.

Biochemistry, 1989 Nov 14, 28(23), 9158 - 65
Aggregation-related conformational change of the membrane-associated coat protein of bacteriophage M13; Spruijt RB et al.; The state of the coat protein of bacteriophage M13, reconstituted into amphiphilic media, has been investigated . The in situ conformation of the coat protein has been determined by using circular dichroism . Minimum numbers for the protein aggregation in the system have been determined after disruption of the lipid-protein system and subsequent uptake of the protein in cholate micelles . The aggregational state and conformation of the protein were affected by (1) the method of coat protein isolation (phenol extraction vs cholate isolation), (2) the nature of amphiphiles used (variation in phospholipid headgroups and acyl chains), and (3) the ratio of amphiphiles and protein . Under all conditions, phenol-extracted coat protein was in a predominantly beta-structure and in a highly aggregated polymeric form . Cholate-isolated coat protein was initially oligomeric and contained a substantial amount of alpha-helix . Below an aggregation number of 20, this protein showed a reversible aggregation with no change in conformation . Upon further aggregation, a conformational change was observed, and aggregation was irreversible, resulting in predominantly beta-structured coat protein polymers . This effect was observed upon uptake in phospholipids at low lipid to protein molar ratios (L/P ratios) and with phosphatidylcholines (PC) and phosphatidic acids (PA) containing saturated acyl chains . After reconstitution in phospholipids with unsaturated acyl chains and with phosphatidylglycerols (PG) at high L/P ratios, the original alpha-helix-containing state of the coat protein was maintained . Cross-linking experiments demonstrated that the beta-polymers are able to form reversible superaggregates within the vesicle system . An aggregation-related conformational change mechanism for the coat protein in phospholipid systems is proposed.

J Mol Biol, 1989 Nov 5, 210(1), 181 - 93
Contributions of left-handed helical residues to the structure and stability of bacteriophage T4 lysozyme; Nicholson H et al.; Non-glycine residues in proteins are rarely observed to have "left-handed helical" conformations . For glycine, however, this conformation is common . To determine the contributions of left-handed helical residues to the stability of a protein, two such residues in phage T4 lysozyme, Asn55 and Lys124, were replaced with glycine . The mutant proteins fold normally and are fully active, showing that left-handed non-glycine residues, although rare, do not have an indispensable role in the folding of the protein or in its activity . The thermodynamic stability of the Lys124 to Gly variant is essentially identical with that of wild-type lysozyme . The Asn55 to Gly mutant protein is marginally less stable (0.5 kcal/mol) . These results indicate that the conformational energy of a glycine and a non-glycine residue in the left-handed helical conformation are very similar . This is consistent with some theoretical energy distributions, but is inconsistent with others, which suggest that replacements of the sort described here might increase the stability of the protein by up to 5 kcal/mol . Crystallographic analysis of the mutant proteins shows that the backbone conformation of the Lys124 to Gly variant is essentially identical with that of the wild-type structure . In the case of the Asn55 to Gly replacement, however, the (phi, psi) values of residue 55 change by about 20 degrees . This suggests that the energy minimum for left-handed glycine residues is not the same as that for non-glycine residues . This is strongly indicated also by a survey of accurately determined protein crystal structures, which suggests that the energy minimum for left-handed glycine residues is near (phi = 90 degrees, psi = 0 degrees), whereas that for non-glycine residues is close to (phi = 60 degrees, psi = 30 degrees) . This apparent energy minimum for glycine is not clearly predicted by any of the theoretical (phi, psi) energy contour maps.

Virology, 1989 Nov, 173(1), 214 - 22
Molecular cloning of a type D retrovirus from human cells (PMFV) and its homology to simian acquired immunodeficiency type D retroviruses; Krause H et al.; Unintegrated circular proviral DNA of a type D retrovirus (PMFV) isolated from a permanent human cell line was molecularly cloned in the bacteriophage vector L47.1 and subcloned in the plasmid vector pGEM-2 . A restriction endonuclease map of PMFV DNA was established using 10 different enzymes for single and multiple digestions of closed circular and cloned DNA molecules . By restriction endonuclease analysis cloned PMFV DNA represented full-length viral DNA with one long terminal repeat (LTR) . The comparison of the physical map of cloned PMFV to those of other cloned type D retroviruses revealed closest homology to the map of retrovirus D/New England (pD398) and SAIDS retrovirus type 1 (SRV-1) . The relatedness of PMFV to further type D retroviruses (Mason-Pfizer monkey virus, MPMV; SAIDS retrovirus type 2, SRV-2) was also demonstrated by cross-hybridization of cloned DNAs under different stringencies (i) using full-length genomic probes of PMFV, MPMV, and SRV-2 and (ii) by DNA sequence analysis of regions of the group specific antigen (gag) protease (prt), polymerase (pol), and envelope (env) genes.

J Bacteriol, 1989 Nov, 171(11), 5763 - 7
Unique organization of Leptospira interrogans rRNA genes; Fukunaga M et al.; We cloned Sau3AI fragments containing the rRNA genes for Leptospira interrogans serovar canicola strain Moulton in the BamHI site of lambda EMBL3 bacteriophage DNA . Physical maps of the fragments were constructed, and the locations of the rRNA genes were determined by Southern blot hybridization and S1 protection . Each fragment of the 23S or the 16S rRNA gene contained at least one copy of the 23S or the 16S sequence . Genomic hybridization showed that there were two genes for the 23S rRNA and the 16S rRNA but only one gene for the 5S rRNA on the chromosome of L . interrogans . The results revealed the important fact that each rRNA gene is located far from the other rRNA genes . Our findings, accordingly, also suggest that these rRNA genes are expressed independently in this organism.

Proc Natl Acad Sci U S A, 1989 Nov, 86(21), 8237 - 41
Hydrophobic packing in T4 lysozyme probed by cavity-filling mutants; Karpusas M et al.; To probe the nature of the hydrophobic cores of proteins and to test potential ways of increasing protein thermostability, an attempt was made to improve the packing within T4 bacteriophage lysozyme by engineered amino acid replacements . Two mutations, Leu-133----Phe and Ala-129----Val, which were designed to fill the largest cavities that exist in the folded structure of the native protein, were constructed . The mutant proteins have normal activities and their thermal stabilities are marginally lower than that of wild-type lysozyme . Crystal structure analysis of the mutant proteins shows that the introduced amino acids are accommodated with very little perturbation of the three-dimensional structure . Incorporation of the more bulky hydrophobic residues within the core of the protein is expected to provide an increase in hydrophobic stabilization, but this is seen to be offset by the introduction of strain . Inspection of the mutant structures shows that in each case the introduced amino acid side chain is forced to adopt a non-optimal dihedral angle X1 . Strain is also observed in the form of bond angle distortion and in unfavorable van der Waals contacts . The results illustrate how the observed core structures of proteins represent a compromise between the hydrophobic effect, which will tend to maximize the core packing density, and the strain energy that would be incurred in eliminating all packing defects . The results also suggest that mutations designed to increase protein stability by filling existing cavities may be effective in some cases but are unlikely to provide a general method for increasing protein stability.

J Virol, 1989 Nov, 63(11), 4762 - 6
Locations of amino acid substitutions in bacteriophage T4 tsL56 DNA polymerase predict an N-terminal exonuclease domain; Reha-Krantz LJ; The amino acid substitutions responsible for the temperature-sensitive (ts) and mutator phenotypes of the classical bacteriophage T4 DNA polymerase mutant tsL56 were determined . tsL56 DNA polymerase has two mutations in the 5' end of the DNA polymerase gene (g43) that produce two amino acid substitutions: codon 89, alanine to threonine, and codon 363, aspartate to asparagine . Both mutations are required for the strong ts and mutator phenotypes . The increased error rate of the tsL56 DNA polymerase is due to a reduction in 3'----5' exonuclease activity relative to polymerase activity (N . Muzyczka, R . L . Poland, and M . J . Bessman, J . Biol . Chem . 247:7116-7122, 1972) . Thus, the locations of the tsL56 mutations suggest that the 3'----5' exonuclease domain resides in the N-terminal region . Several other ts DNA polymerase mutant strains isolated with tsL56 also have mutator or antimutator phenotypes . The nucleotide changes in these important mutant strains were also determined . This mutant collection, combined with collections of g43 amber mutants and mutants selected on the basis of a strong mutator phenotype (L . J . Reha-Krantz, J . Mol . Biol . 202:711-724, 1988), contains nearly 70 different DNA polymerase mutations . The numerous T4 DNA polymerase mutations are valuable for DNA polymerase structure-function and fidelity studies.

J Virol, 1989 Nov, 63(11), 4736 - 43
Analysis of T4 bacteriophage deletion mutants that lack td and frd genes; Wang Y et al.; The roles of bacteriophage T4-encoded thymidylate synthase and dihydrofolate reductase as virion structural components have been further investigated . Two mutants, del(63-32)7 and del(63-32)9, bearing deletions in the gene 63 to 32 region of the T4 genome, were characterized by Southern blotting analysis, as well as by enzyme and immunological assays . Our results have confirmed the original report of Homyk and Weil (Virology 61:505-523, 1974) that del7 and del9 each carries a deletion of about 4.0 kilobases, which totally eliminates the frd gene, encoding dihydrofolate reductase, and the td gene, encoding thymidylate synthase . With the well-characterized deletion mutants, along with newly prepared antisera against T4-encoded thymidylate synthase and dihydrofolate reductase, we have reevaluated the experimental results supporting the idea that T4-induced dihydrofolate reductase and thymidylate synthase are essential T4 baseplate components and antigenic determinants of phage particles . These deletion mutant phages are not targets for neutralization by antisera against either dihydrofolate reductase or thymidylate synthase purified from cloned genes . Furthermore, these newly prepared antisera also cannot neutralize the infectivity of T4D . Those results suggest that the phage-neutralizing components in the old antisera used in the earlier studies were not antibodies against either dihydrofolate reductase or thymidylate synthase but were antibodies against minor components of the purified enzyme preparations . Study of the biological properties of the deletion mutants indicates that T4-induced thymidylate synthase and dihydrofolate reductase play significant roles in growth of the phage beyond their known roles in nucleotide biosynthesis, even though they are apparently not essential for phage viability . The deletion mutants should be useful in defining these roles.

Gene Anal Tech, 1989 Nov-Dec, 6(6), 111 - 9
Prediction of operator-binding protein by discriminant analysis; Nakata K et al.; A number of operator-binding proteins contain similar sequence features to Cro and cI repressors of bacteriophage and CAP protein of Escherichia coli, such as conserved amino acids at constant positions . However, these sequence patterns also occur in proteins that are not operator-binding . We use sequence analogy information in conjunction with a pattern recognition algorithm . The functional and structural properties, e.g., distributions of hydrophobicity, hydrophilicity, charged amino acids, electrostatic free energy, and helical structures of protein are also considered . Within the framework of discriminant analysis, we calculate the above variables and search for a better combination of variables . To assess the discriminatory power of these variables, we allocated additional sequences and predict DNA-binding regions of regulatory proteins not included in the training set.

Genetics, 1989 Nov, 123(3), 431 - 40
Genetic analysis of bacteriophage P22 lysozyme structure; Rennell D et al.; The suppression patterns of 11 phage P22 mutants bearing different amber mutations in the gene encoding lysozyme (19) were determined on six different amber suppressor strains . Of the 60 resulting single amino acid substitutions, 18 resulted in defects in lysozyme activity at 30 degrees; an additional seven were defective at 40 degrees . Revertants were isolated on the "missuppressing" hosts following UV mutagenesis; they were screened to distinguish primary- from second-site revertants . It was found that second-site revertants were recovered with greater efficiency if the UV-irradiated phage stocks were passaged through an intermediate host in liquid culture rather than plated directly on the nonpermissive host . Eleven second-site revertants (isolated as suppressors of five deleterious substitutions) were sequenced: four were intragenic, five extragenic; three of the extragenic revertants were found to have alterations near and upstream from gene 19, in gene 13 . Lysozyme genes from the intragenic revertant phages were introduced into unmutagenized P22, and found to confer the revertant plating phenotype.

Biopolymers, 1989 Nov, 28(11), 1861 - 73
A CD determination of the alpha-helix contents of the coat proteins of four filamentous bacteriophages: fd, IKe, Pf1, and Pf3; Clack BA et al.; The CD spectra of four filamentous bacteriophages--fd, IKe, Pf1, and Pf3--were analyzed to determine the alpha-helix contents of their major coat proteins . Measured spectra included the 192-nm band so that analyses could be carried out over the full wavelength range of the reference spectra for protein secondary structures available (a) from globular proteins {J.T . Yang, C.S.C . Wu, and H.M . Martinez (1986) Methods in Enzymology 130, 208-269} and (b) from poly(L-lysine) {N . Greenfield and G.D . Fasman (1960) Biochemistry 8, 4108-4116} . Extended analyses were also performed with the addition of the spectrum of a model beta-turn to the Greenfield and Fasman reference set, with the spectrum of a short alpha-helix in the Yang et al . reference set, and with an estimate of the spectrum of Trp added to both reference sets . The reference set based on the simple poly(L-lysine) polypeptide, plus a spectrum of a model beta-turn or of Trp, gave reasonably good fits to the measured spectra for all four phages and yielded the largest percentages of alpha-helix . The class I phages--fd and IKe--had large percentages of alpha-helix of 98 +/- 2 and 97 +/- 5%, respectively, while the two class II phages--Pf1 and Pf3--had similar but smaller alpha-helix contents of 83 +/- 6 and 84 +/- 2, respectively . While these alpha-helix contents were within the ranges previously reported from CD spectra of these phages in solution, they were more precise, and they indicated that the coat proteins of the intact phages have CD spectra that are probably modeled better by the reference spectra of polypeptides than by those of globular proteins.

Somat Cell Mol Genet, 1989 Nov, 15(6), 545 - 53
Molecular cloning of human A1S9 locus: an X-linked gene essential for progression through S phase of the cell cycle; Zacksenhaus E et al.; The temperature-sensitive (ts) A1S9 mouse L-cell mutant is defective in an X-linked gene essential for the progression of cells through the S phase of the cell duplication cycle . We recently reported the complementation of the ts A1S9 cell defect with total human DNA and the isolation of independent temperature-resistant transformants that retained a common set of human specific Alu-containing fragments . Here we describe the molecular cloning of these human DNA sequences from one of the secondary transformants . ST-1-0 . A genomic library prepared from ST-1-0 was screened with a total human DNA probe, and two recombinant bacteriophages carrying overlapping segments were isolated . The cloned region was extended in both directions using a human X-chromosome specific library . In total, a human region spanning 42 kb in length, and containing all the Alu-specific DNA sequences found in ST-1-0, was isolated in five overlapping recombinant phages . The A1S9 gene appeared to be larger than the DNA recovered in individual phage isolates, as was assessed by transfection experiments . A single-copy probe derived from the phage DNA was shown to be conserved in independent primary, secondary, and tertiary transformants of ts A1S9 cells and mapped to the X chromosome by molecular hybridization . Northern blot hybridization of this probe with poly(A)+ mRNA derived from ST-1-0 cells identified a transcript of about 3.6 kb.

Genomics, 1989 Nov, 5(4), 857 - 65
Genomic structure of the human cytosolic aldehyde dehydrogenase gene; Hsu LC et al.; We have isolated and characterized several overlapping clones from two human genomic libraries constructed in cosmid and bacteriophage vectors . They span about 80 kbp and include the entire human cytosolic aldehyde dehydrogenase (ALDH1) gene . Restriction endonuclease mapping, Southern blotting with cDNA and specific oligonucleotide probes, and DNA sequencing were performed to analyze the cloned genomic DNA . The ALDH1 gene is about 53 kbp long and is divided into 13 exons which encode 501 amino acid residues . Primer extension results defined the transcription initiation site to 53 bp upstream from the A of the initiation codon ATG . The promoter region of the gene contains an ATA box and a CCAAT box, which are located 32 and 74 bp upstream, respectively, from the transcription initiation site . The possible functional domains of the protein encoded by exons are discussed . A similar intron-exon organization between the genes of cytosolic ALDH1 and its mitochondrial ALDH2 isozyme in which both enzymes are encoded by 13 exons and 9 of the 12 introns interrupt the coding sequence at homologous positions was observed . This is consistent with the model that the two isozyme genes evolved after the duplication of a common ancestor gene.

Clin Chem, 1989 Nov, 35(11), 2196 - 201
Automated DNA sequencing methods involving polymerase chain reaction; McBride LJ et al.; Polymerase chain reaction (PCR) as a method for preparing DNA templates has been used for several DNA sequencing applications . An in situ procedure for directly sequencing PCR products by the dideoxy-termination method has been developed by using fluorophore-labeled sequencing primers . Completed sequence reactions were combined and loaded into a single electrophoretic lane of a fluorescence-based DNA sequence analyzer . DNA targets devoid of a universal primer sequence could be sequenced with labeled universal primers by incorporating a universal primer sequence into the PCR product . With this method, the sequence of a 351-bp region in the bacteriophage lambda genome was fully analyzed in a single lane with automatic base identification accuracy of greater than 99% . An unknown sequence, 1.7 kb long, also was sequenced by this procedure, in combination with a "PCR gene walking" strategy . Comparison of the 1110 bases in overlapping sequence data from both strands yielded only two single-base ambiguities . Automated DNA sequence analysis of the highly polymorphic HLA-DQA-1 (alpha) region in the human genome can be performed with this simple methodology . Use of this PCR-sequencing method to analyze DNA extracted from a one-month-old blood sample from an individual who is heterozygous at this locus allowed unambiguous assignment of genotype.

EMBO J, 1989 Nov, 8(11), 3517 - 21
Effects of mutations in heat-shock genes groES and groEL on protein export in Escherichia coli; Kusukawa N et al.; Escherichia coli heat-shock proteins GroES and GroEL are essential cytoplasmic proteins, which have been termed 'chaperonins' because of their ability to assist protein assembly of bacteriophage capsids and multimeric enzymes of foreign origin . In this report we show that temperature-sensitive mutations in groES and groEL genes cause defective export of the plasmid-encoded beta-lactamase (Bla) in vivo . Since efficient translocation of proteins across biological membranes is thought to be supported by cytoplasmic factors that protect presecretory molecules from being misfolded, these results suggest that both GroES and GroEL proteins possess a chaperone function by which they facilitate export of Bla . The translocation of other secretory proteins, however, appears to depend minimally on GroE, suggesting that GroE interacts only with a specific class of secreted proteins.

Biochem Int, 1989 Nov, 19(5), 959 - 67
Yeast genomic clones encoding polypeptides immunologically related to an 18 kDa subunit of mitochondrial ATP synthase; Grasso DG et al.; A yeast genomic library in the bacteriophage expression vector lambda gt11 was screened with a polyclonal anti-holo-ATPase antiserum resulting in the isolation of 54 immunoreactive clones . Four of these phage clones express in bacteria a polypeptide antigenically related to an 18 kDa subunit (P18) of the yeast mitochondrial ATPase complex . Molecular analysis of the yeast DNA inserts in these phage clones revealed two classes of yeast DNA that share little homology at the nucleotide sequence level and therefore may represent distinct separate genes . The polypeptides potentially encoded by these yeast DNA segments do show scattered short blocks of strong amino acid sequence homology, which may underlie the observed immunochemical relatedness between the proteins expressed in bacteria.

Genetics, 1989 Nov, 123(3), 465 - 76
Enhancement of Escherichia coli plasmid and chromosomal recombination by the Ref function of bacteriophage P1; Laufer CS et al.; The Ref activity of phage P1 enhances recombination between two defective lacZ genes in the Escherichia coli chromosome (lac- x lac- recombination) . Plasmid recombination, both lac- x lac- and tet- x tet-, was measured by transformation of recA strains, and was also assayed by measurement of beta-galactosidase . The intracellular presence of recombinant plasmids was verified directly by Southern blotting . Ref stimulated recombination of plasmids in rec+ and rec(BCD) cells by 3-6-fold, and also the low level plasmid recombination in recF cells . RecA-independent plasmid recombination, either very low level (recA cells) or high level (recB recC sbcA recA cells), was not stimulated . Ref stimulated both intramolecular and intermolecular plasmid recombination . Both normal and Ref-stimulated lac- x lac- chromosomal recombination, expected to be mostly RecBC-dependent in wild-type bacteria, were affected very little by a recF mutation . We have previously reported Ref stimulation of lac- x lac- recombination in recBC sbcB bacteria, a process known to be RecF-dependent . Chromosomal recombination processes thought to involve activated recombination substrates, e.g., Hfr conjugation, P1 transduction, were not elevated by Ref activity . We hypothesize that Ref acts by unknown mechanisms to activate plasmid and chromosomal DNA for RecA-mediated recombination, and that the structures formed are substrates for both RecF-dependent (plasmid, chromosomal) and Rec(BCD)-dependent (chromosomal) recombination pathways.

J Gen Virol, 1989 Nov, 70 ( Pt 11), 3085 - 9
Identification of 21 genes of infectious laryngotracheitis virus using random sequencing of genomic DNA; Griffin AM; DNA from infectious laryngotracheitis virus (ILTV) was randomly sheared and cloned into the M13 bacteriophage . Clones containing ILTV DNA were sequenced and the predicted amino acid sequences were compared to the known sequences of other herpesviruses using computer analysis . Twenty-one ILTV genes were identified, 20 by comparison to varicella-zoster virus and 19 by comparison to herpes simplex virus type 1; only 12 genes, giving consistently lower homology scores, were found by comparison with the gammaherpesvirus Epstein-Barr virus, indicating that ILTV sequences bear greater similarity to other alpha- than to gammaherpesvirus sequences.

EMBO J, 1989 Nov, 8(11), 3523 - 33
Heteroduplex substrates for bacteriophage lambda site-specific recombination: cleavage and strand transfer products; Nash HA et al.; Lambda's Int protein acts as a specific topoisomerase at attachment sites, the DNA segments that are required for site-specific recombination . Int cleaves each strand of an attachment site at a unique place and creates strand exchanges by joining broken ends from two different parents . To study the action of Int topoisomerase in more detail, heteroduplex attachment sites were made by annealing strands that are complementary except for a few base pairs that lie in the region between the points of top and bottom strand exchange in the attachment site core . These heteroduplexes appear to interact normally with Int and its accessory proteins IHF and Xis . Although the heteroduplex sites are specifically cleaved by Int topoisomerase, rejoining of the broken DNA is hindered by the lack of Watson--Crick complementarity adjacent to the break . Because of this, heteroduplexes accumulate broken intermediates which are then processed in novel ways . We have used this feature to provide new information about functional differences between attachment sites, to investigate the way Xis protein controls directionality of site-specific recombination, and to demonstrate that Int protein can join strands indiscriminately and can therefore generate recombinants with either of two genetic polarities.

J Bacteriol, 1989 Nov, 171(11), 6387 - 90
The structurally related exbB and tolQ genes are interchangeable in conferring tonB-dependent colicin, bacteriophage, and albomycin sensitivity; Braun V; Double exbB tolQ mutants of Escherichia coli were completely resistant to bacteriophages T1 and phi 80, in contrast to strains with exbB or tolQ mutations, which were sensitive . Cells carrying mutations in exbB were partially tolerant to colicins B, D, and M and became fully tolerant by the introduction of tolQ mutations . This suggested involvement of both exbB and tolQ in tonB-dependent uptake.

J Bacteriol, 1989 Nov, 171(11), 6345 - 8
Analysis of Bordetella pertussis virulence gene regulation by use of transcriptional fusions in Escherichia coli; Miller JF et al.; The virulence regulon of Bordetella pertussis includes a trans-acting regulatory locus, bvg, that is required for expression of several virulence factors . The virulence control system also responds to environmental signals . We have reconstructed a bvg-dependent regulatory system in Escherichia coli by using bacteriophage lambda vectors carrying transcriptional fusions to lacZYA . Single-copy lacZYA fusions to the B . pertussis fhaB locus, which encodes the attachment factor filamentous hemagglutinin, were activated nearly 400-fold by pBR322 replicons carrying sequences that included bvg . In contrast, bvg had no effect on the pertussis toxin operon (ptxA-E) promoter in E . coli as measured by ptxA-lacZ expression . Environmental signals that modulate expression of virulence genes in B . pertussis had a pronounced effect on bvg-mediated activation of fhaB-lacZ . MgSO4, nicotinic acid, and low temperature resulted in decreases in beta-galactosidase activities of 175-, 115-, and 45-fold respectively . Sensory transduction and transcriptional activation were tightly coupled, and both required an intact bvg locus as determined by 5' and 3' deletions that eliminated both activities.

J Bacteriol, 1989 Nov, 171(11), 6197 - 205
Characterization of the cryptic lambdoid prophage DLP12 of Escherichia coli and overlap of the DLP12 integrase gene with the tRNA gene argU; Lindsey DF et al.; The argU (dnaY) gene of Escherichia coli is located, in clockwise orientation, at 577.5 kilobases (kb) on the chromosome physical map . There was a cryptic prophage spanning the 2 kb immediately downstream of argU that consisted of sequences similar to the phage P22 int gene, a portion of the P22 xis gene, and portions of the exo, P, and ren genes of bacteriophage lambda . This cryptic prophage was designated DLP12, for defective lambdoid prophage at 12 min . Immediately clockwise of DLP12 was the IS3 alpha 4 beta 4 insertion element . The argU and DLP12 int genes overlapped at their 3' ends, and argU contained sequence homologous to a portion of the phage P22 attP site . Additional homologies to lambdoid phages were found in the 25 kb clockwise of argU . These included the cryptic prophage qsr' (P . J . Highton, Y . Chang, W . R . Marcotte, Jr., and C . A . Schnaitman, J . Bacteriol . 162:256-262, 1985), a sequence homologous to a portion of lambda orf-194, and an attR homolog . Inasmuch as the DLP12 att int xis exo P/ren region, the qsr' region, and homologs of orf-194 and attR were arranged in the same order and orientation as the lambdoid prophage counterparts, we propose that the designation DLP12 be applied to all these sequences . This organization of the DLP12 sequences and the presence of the argU/DLP12 int pair in several E . coli strains and closely related species suggest that DLP12 might be an ancestral lambdoid prophage . Moreover, the presence of similar sequences at the junctions of DLP12 segments and their phage counterparts suggests that a common mechanism could have transferred these DLP12 segments to more recent phages.

Proc Natl Acad Sci U S A, 1989 Nov, 86(21), 8343 - 7
Extension of mismatched 3' termini of DNA is a major determinant of the infidelity of human immunodeficiency virus type 1 reverse transcriptase; Perrino FW et al.; The unusually high error rate of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) suggests that polymerization errors by this enzyme contribute to the genetic variability of the AIDS virus . We have analyzed the mechanism for HIV-1 RT infidelity by studying two distinct steps that might lead to base substitution mutations: nucleotide misinsertions and elongation from 3'-terminal DNA mispairs . Our results indicate that the capacity of HIV-1 RT to polymerize nucleotides onto mispaired termini is a major factor in the production of mutations by this enzyme . When a noncomplementary dAMP was inserted opposite a template adenine by HIV-1 RT, the nascent 3'-terminal A.A mispair was readily extended by subsequent incorporation of the next complementary nucleotide . The frequencies of nucleotide addition onto 3'-terminal A-A, A-C, and A-G mispairs were determined by quantitating the amount of extended primers with a gel electrophoresis assay and by measuring mutagenesis after hybridization of mismatched primers opposite an amber mutation in bacteriophage phi X174 DNA . The mispair extension frequencies are approximately 50-fold higher by HIV-1 RT than by the mammalian replicative enzyme DNA polymerase alpha.

Biochemistry, 1989 Oct 31, 28(22), 8699 - 705
Site-directed mutagenesis of the T4 endonuclease V gene: the role of arginine-3 in the target search; Dowd DR et al.; Endonuclease V, a pyrimidine dimer specific endonuclease in T4 bacteriophage, is able to scan DNA, recognize pyrimidine dimer photoproducts produced by exposure to ultraviolet light, and effectively incise DNA through a two-step mechanism at the damaged bases . The interaction of endonuclease V with nontarget DNA is thought to occur via electrostatic interactions between basic amino acids and the acidic phosphate DNA backbone . Arginine-3 was chosen as a potential candidate for involvement in this protein-nontarget DNA interaction and was extensively mutated to assess its role . The mutations include changes to Asp, Glu, Leu, and Lys and deleting it from the enzyme . Deletion of Arg-3 resulted in an enzyme that retained marginal levels of AP specificity, but no other detectable activity . Charge reversal to Glu-3 and Asp-3 results in proteins that exhibit AP-specific nicking and low levels of dimer-specific nicking . These enzymes are incapable of affecting cellular survival of repair-deficient Escherichia coli after irradiation . Mutations of Arg-3 to Lys-3 or Leu-3 also are unable to complement repair-deficient E . coli . However, these two proteins do exhibit a substantial level of in vitro dimer- and AP-specific nicking . The mechanism by which the Leu-3 and Lys-3 mutant enzymes locate pyrimidine dimers within a population of heavily irradiated plasmid DNA molecules appears to be significantly different from that for the wild-type enzyme . The wild-type endonuclease V processively incises all dimers on an individual plasmid prior to dissociation from that plasmid and subsequent reassociation with other plasmids, yet neither of these mutants exhibits any of the characteristics of this processive nicking activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1989 Oct 31, 28(22), 8828 - 32
1H NMR studies of T4 gene 32 protein: effects of zinc removal and reconstitution; Pan T et al.; Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol bound in a tetrahedral ligand field . 113Cd NMR studies of Cd-substituted wild-type and mutant (Cys166----Ser166) g32Ps show Cys77, Cys87, and Cys90 to provide three sulfur donor atoms as ligands to the metal ion {Giedroc, D . P., Johnson, B . A., Armitage, I . M., & Coleman, J . E . (1989) Biochemistry 28, 2410} . Proton NMR signals from the His and Trp side chains of the protein have been followed as a function of pH and metal ion removal by biosynthesizing the protein with amino acids carrying protons at specific positions in a background of perdeuteriated aromatic amino acids . Only one of the two pairs of His resonances (from His64 and His81) titrates over the pH range 8.0-5.9 . The nontitrating His side chain is most likely ligated to the metal ion . Upon Zn(II) removal, 1H NMR spectra of the fully protonated g32P-(A + B) exhibit substantial signal broadening in several regions of the spectrum, while the His 2,4-1H resonances are broadened beyond detection . The 1H NMR spectral characteristics of the original protein are restored by reconstitution with stoichiometric Zn(II) . The broadening of the 1H NMR signals is not due to oligomerization of the protein, since small-angle X-ray scattering experiments show that the average radius of gyration of the apo-g32P-(A + B) is 25.0 A and that of the reconstituted Zn(II)-g32P-(A + B) is 31.2 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1989 Oct 30, 82(2), 321 - 5
Optimization of the in vitro packaging efficiency of bacteriophage T7 DNA: effects of neutral polymers; Son M et al.; The in vitro DNA packaging of several DNA bacteriophages is stimulated by the presence of neutral polymers . To optimize bacteriophage T7 DNA packaging and to understand the basis for optimization, the efficiency of T7 DNA packaging has been determined at completion, as a function of the type, molecular mass, and concentration of the polymer added . When the polymer used was polyethylene glycol (PEG) of 0.2, 0.6 or 12.6 kDa, the efficiency of DNA packaging reached maximum at an intermediate concentration of polymer . The osmotic pressure (Pos) at maximum efficiency was either in, or close to, the range of colloid Pos measured for the intact host cell . The optimum Pos increased as the size of the polymer used decreased . PEG-100 (of 0.1 kDa) did not stimulate in vitro T7 DNA packaging . Dextran of 10 kDa also stimulated packaging and produced maximum efficiency at a physiological Pos . The degree of stimulation increases as DNA packaging extract concentration decreases; stimulation by as much as two to three orders of magnitude is observed . The presence of added polymer reduces fluctuations in DNA packaging efficiency caused by variability in the concentration of DNA packaging extracts . For reproducible and high efficiency packaging, the dextran was more reliable than the PEGs, possibly because the Pos of the dextran solutions is less sensitive to polymer concentration than is the Pos of PEG solutions . The optimum concentration of dextran at completion was also the optimum at all times before completion.

J Biol Chem, 1989 Oct 25, 264(30), 17924 - 30
Molecular cloning and DNA sequence analysis of Escherichia coli topB, the gene encoding topoisomerase III; DiGate RJ et al.; Escherichia coli contains two type 1 topoisomerases, topoisomerase I and III . Although topoisomerase III can be purified as a potent decatenase, its role in DNA metabolism is unclear . In order to address this issue, the gene encoding topoisomerase III from E . coli has been molecularly cloned and its DNA sequence determined . The cloned fragment of DNA contains an open reading frame that can encode a polypeptide of 73.2 kDa . The first 20 amino acids of this open reading frame are identical to those of topoisomerase III as determined by amino-terminal gas-phase microsequencing . Expression of the polypeptide encoded by this open reading frame, using a bacteriophage T7 transient expression system, results in the accumulation of a 74-kDa polypeptide . Soluble extracts prepared from cells overexpressing this gene product show a dramatic increase in topoisomerase activity when compared with control extracts . We propose that this gene be designated topB.

FEBS Lett, 1989 Oct 23, 257(1), 55 - 8
Bacterial expression of human cysteine proteinase inhibitor stefin A; Fong D et al.; Stefin A, a cysteine proteinase inhibitor of the cystatin superfamily, has been found to be most abundant in epidermal cells . In order to determine its cellular function, we have expressed human stefin A in Escherichia coli using plasmid expression vectors under the control of bacteriophage T7 RNA polymerase . The heat-stable, antibody-positive bacterial product was isolated using a papain-Sepharose affinity column and was shown to inhibit two cysteine proteinases, papain and human cathepsin B . Recombinant stefin A may have commercial and therapeutic potential in situations requiring inhibition of cysteine proteinase activities, and in cosmetics, as an ingredient in skin creams.

J Mol Biol, 1989 Oct 20, 209(4), 525 - 38
Organization of the immunity region immI of bacteriophage P1 and synthesis of the P1 antirepressor; Heisig A et al.; The immI region of bacteriophage P1 includes the ant/reb gene, which encodes the antirepressor protein, and the c4 gene, which encodes a repressor molecule that negatively regulates antirepressor synthesis . The antirepressor interferes with the activity of the P1 repressor of lytic function, the product of the c1 gene . We have determined the DNA sequences of the immI region of P1 wild-type and the mutants virs, ant16, ant17, and reb22 . Using suitable P1 immI DNA subfragments cloned into a vector of the T7 bacteriophage RNA polymerase expression system the antirepressor protein(s) was overproduced . On the basis of positions of immI mutations and the sizes of ant gene products, the following organizational feature of the P1 immI region is suggested: (1) the genes c4 and ant are cotranscribed in that order from the same promoter in the clockwise direction of the P1 genetic map; (2) an open reading frame for an unknown gene is located in between c4 and ant; (3) the site at which the c4 repressor acts is located within the c4 structural gene; (4) two antirepressor proteins of molecular weights 42,000 and 32,000 are encoded by a single open reading frame, with the smaller protein initiating at an in-frame start codon; (5) transcription of immI is regulated via a c1-controlled operator, Op51, indicating a communication between the immunity systems immC and immI.

FEMS Microbiol Lett, 1989 Oct 15, 52(3), 335 - 9
Cysteine-22 and cysteine-38 are not essential for the functions of maltoporin (LamB protein); Ferenci T et al.; Maltoporin in the outer membrane of Escherichia coli contains two cysteine residues, at positions 22 and 38 in the primary sequence . The role of these residues in determining structural stability, and their contributions to the maltoporin binding sites for maltodextrins and bacteriophage lambda, was investigated . Site-directed mutagenesis was used to alter each of these residues to a serine . A double mutant lacking both cysteines was also isolated . None of the substitutions affected maltodextrin binding or the binding of phage lambda, suggesting the variant proteins retain a native binding-site conformation . The mutants were assembled at wild-type levels into the outer membrane as maltoporin trimers but the temperature-stability of the trimer greater than monomer dissociation was slightly reduced in the presence of the Cys 38 substitution . However, it is unlikely that the stability of trimers was due to disulfide linkages between subunits since the native trimers are stable under highly reducing conditions in the presence of SDS; more likely the Cys greater than Ser substitutions slightly perturb intra- or inter-subunit hydrophobic interactions in regions predicted to span across the outer membrane.

Gene, 1989 Oct 15, 82(1), 77 - 82
The catalytic core of the sunY intron of bacteriophage T4; Xu MQ et al.; The self-splicing sunY intron of bacteriophage T4 shares a common secondary structure with other group I introns . A long open reading frame within the intron is entirely 3' of the structural elements conserved in all group I introns . This catalytic core is the smallest yet described for a self-splicing intron . An internal deletion of 728 nucleotides (nt), leaving 196 nt at the 5' end and 109 nt at the 3' end, allows normal self-splicing . Transcripts terminating 196 nt 3' of the 5' splice site retain catalytic activity.

J Biol Chem, 1989 Oct 15, 264(29), 16973 - 6
A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli; Olins PO et al.; We recently reported that a ribosome binding site (RBS) derived from gene 10 of bacteriophage T7 (g10-L) causes a pronounced stimulation of expression when placed upstream of a variety of genes, and that this effect is probably due to a stimulation of translation efficiency in Escherichia coli (Olins, P . O., Devine, C . S., Rangwala, S . H., Kavka, K . S . (1988) Gene (Amst.) 73, 227-235) . Here we present a model for the mechanism of action of the g10-L: the RBS contains a 9-base sequence which has the potential for forming a novel base-paired interaction with bases 458-466 of the 16 S rRNA of E . coli . Although such sequence homologies are rare in E . coli RBS regions, a number of similar sequences were found in the RBS regions of other bacteriophage structural genes . When an isolated homology sequence was placed upstream of a synthetic RBS, there was a 110-fold increase in the translation efficiency of the lacZ gene . Surprisingly, the homology sequence also stimulated translation when placed downstream of the initiator codon, indicating that this sequence is acting as a translational "enhancer."

Nucleic Acids Res, 1989 Oct 11, 17(19), 7681 - 92
The c1 repressor of bacteriophage P1 operator-repressor interaction of wild-type and mutant repressor proteins; Heinrich J et al.; The c1 repressor gene of bacteriophage P1 and the temperature-sensitive mutants P1c1.100 and P1c1.162 was cloned into an expression vector and the repressor proteins were overproduced . A rapid purification procedure was required for the isolation of the thermolabile repressor proteins . Identification of the highly purified protein of an apparent molecular weight of 33,000 as the product of the c1 gene was verified by (i) the coincidence of partial amino acid sequences determined experimentally to that deduced from the c1 DNA sequence, and (ii) the temperature-sensitive binding to the operator DNA of the thermolabile repressor proteins . Analysis of the products of c1-c1.100 recombinant DNAs relates the thermolability to an unknown alteration in the C-terminal half of the c1.100 repressor . Binding to the operator DNA of c1 repressor is sensitive to N-ethylmaleimide . Since the only three cysteine residues are located in the C-terminal half of the repressor it is suggested that this part of the molecule is important for the binding to the operator DNA . This assumption is supported by the findings that a 14-kDa C-terminal repressor fragment obtained by cyanogen bromide cleavage retains DNA binding properties.

Cell, 1989 Oct 6, 59(1), 207 - 18
Sequence-specific recognition of RNA hairpins by bacteriophage antiterminators requires a conserved arginine-rich motif; Lazinski D et al.; We have dissected the protein and nucleic acid determinants that direct a group of transcriptional antiterminators to their specific target operons . These antiterminators, the N gene products of phages lambda, 21, and P22, function solely with their respective recognition sites, nut, to modify RNA polymerase to a termination-resistant form . We demonstrate that a unique hairpin sequence within each nut site, called boxB, confers genome specificity by interacting with a small amino-terminal domain of the cognate N protein . This interaction is dependent upon an arginine-rich subdomain, which is conserved not only among the N proteins but also in many RNA binding proteins from ribosomes and RNA virus capsids . Notably, this motif constitutes an essential domain of the HIV protein Tat whose function as a trans-activator requires a specific hairpin sequence.

J Biol Chem, 1989 Oct 5, 264(28), 16880 - 6
Sequence-specific pausing during in vitro DNA replication on double-stranded DNA templates; Bedinger P et al.; Sequence-specific pausing occurs during DNA synthesis catalyzed by the bacteriophage T4 DNA polymerase holoenzyme in the presence of the T4 helix destabilizing protein (gene 32 protein) . Two of the six strongest pause sites on a double-stranded bacteriophage fd DNA template are in regions where hairpin helices are predicted to form when the DNA is single stranded . However, the other pause sites are in regions that are not obviously involved in secondary structure . The positions of the DNA chain ends produced at one pause site of each type were determined to within +/- 2 nucleotides . At this resolution, a clustering of sites is observed, suggesting that the polymerase holoenzyme may become destabilized when moving along selected regions of the DNA and then pause at one or more of several closely spaced positions . The addition of the T4 gene 41 protein (a DNA helicase that forms part of the T4 primosome) to the above replication system greatly increases the rate of fork movement and eliminates detectable pausing . In contrast, the addition of the T4 dda protein (a second DNA helicase that increases the rate of fork movement to a similar extent) has no affect on replication fork pausing . This difference could either be due to specific protein-protein interactions formed between the polymerase holoenzyme and the 41 protein or to the highly processive movement of the 41 protein along the displaced DNA strand.

J Mol Biol, 1989 Oct 5, 209(3), 433 - 45
Orientation of the bases of single-stranded DNA and polynucleotides in complexes formed with the gene 32 protein of bacteriophage T4 . A linear dichroism study; van Amerongen H et al.; Linear dichroism measurements were performed in the wavelength region 250 to 350 nm on complexes between the single-stranded DNA binding protein of bacteriophage T4 (gp32) and single-stranded DNA and a variety of homopolynucleotides in compressed polyacrylamide gels . The complexes appeared to orient well, giving rise to linear dichroism spectra that showed contributions from both the protein aromatic residues and the bases of the polynucleotides . In most cases the protein contribution appeared to be very similar, and the linear dichroism of the bases could be explained by similar orientations of the bases for most of the complexes . Assuming a similar, regular structure for most of the polynucleotides in complex, only a limited set of combinations of tilt and twist angles can explain the linear dichroism spectra . These values of tilt and twist are close to (-40 degrees, 30 degrees), (-40 degrees, 150 degrees), (40 degrees, -30 degrees) or (40 degrees, -150 degrees), with an uncertainty in both angles of about 15 degrees . Although the linear dichroism results do not allow a choice between these possible orientations, the latter two combinations are not in agreement with earlier circular dichroism calculations . For the complexes formed with poly(rC) and poly(rA), the linear dichroism spectra could not be explained by the same base orientations . In these two cases also the protein contribution to the linear dichroism appeared to be different, indicating that for some aromatic residues the orientations are not the same as those in the other complexes . The different structures of these complexes are possibly related to the relatively low binding affinity of gp32 to poly(rC), and to a lesser extent to poly(rA).

J Biol Chem, 1989 Oct 5, 264(28), 16451 - 7
The phage T4 uvs Y recombination protein stabilizes presynaptic filaments; Kodadek T et al.; The bacteriophage T4 uvsY protein is required for efficient recombination in T4-infected Escherichia coli cells . Previous in vitro work has shown that the purified uvsY protein is an accessory protein; it stimulates homologous pairing catalyzed by the phage uvsX protein (a RecA-like recombinase) under certain conditions . We show here that this effect can be traced, at least in part, to a UvsY-dependent stabilization of uvsX protein-single-stranded DNA complexes . These presynaptic filaments are one of the early obligatory intermediates in the strand exchange reaction between homologous single- and double-stranded DNAs . The mechanism of filament stabilization seems to involve a slower loss of UvsX subunits . A model that accounts for the data is presented in which both recombination proteins are incorporated into the presynaptic filament.

Biochemistry, 1989 Oct 3, 28(20), 8234 - 41
Antibodies against synthetic peptides and the topology of LamB, an outer membrane protein from Escherichia coli K12; Molla A et al.; LamB, an outer membrane protein from Escherichia coli K12, is involved in the transport of maltose and maltodextrins across the outer membrane and constitutes a receptor for a number of bacteriophages . A recent folding model proposes that LamB spans the outer membrane through a number of transmembranous segments separated by regions exposed either to the cell exterior or to the periplasm . This model is essentially based on predictions of structure and genetic arguments relying on the hypothesis that the mutations studied did not alter the folding of the protein . In order to obtain direct evidence with the unaltered protein, we elicited polyclonal antibodies against synthetic peptides corresponding to several LamB sequences . We chose four regions . Three of them {aa 147-161 (peptide 2), aa 371-385 (peptide 3), and aa 399-413 (peptide 4)} are predicted to face the outside of the cell, and the fourth (aa 19-33 (peptide 1)} is predicted to be periplasmic . By immunoblotting against extracts of various mutants, these antibodies were shown to be specific for LamB and targeted to the selected regions . In some cases, the recognition sites for antibodies were narrowed down to parts of a region . In vivo, on intact cells, anti-peptides 2, 3, and 4 reacted with LamB in an ELISA; this confirmed that regions of peptide 2 and 3 are located, at least in part, at the cell exterior and provided the first proof for a similar, situation of the region of peptide 4 . Under the same conditions, anti-peptide 1 did not react with LamB.(ABSTRACT TRUNCATED AT 250 WORDS)

J Virol, 1989 Oct, 63(10), 4409 - 16
Common cleavage pattern of polysialic acid by bacteriophage endosialidases of different properties and origins; Pelkonen S et al.; The cleavage specificities of seven bacteriophage endosialidases degrading the alpha 2-8-linked polysialic acid common to bacterial polysaccharides and to the cell adhesion molecule N-CAM were investigated . The bacteriophages studied represented five different phenotypic groups by protein and DNA fragment analysis and two different morphology groups by electron microscopy . Characterization of the fragments arising from the native or chemically modified substrates of different sizes showed that cleavage specificity was influenced by enzyme concentration . At the initial phase of degradation, at concentrations ranging from 20- to 100-fold, the minimum substrate size was an oligomer of eight (in one case, nine) sialic acid units that was preferably cleaved at the same position . Under exhaustive conditions, the oligomers were degraded further, and each enzyme type had its own specificity . The similar initial cleavage of polysialic acid by endosialidases associated with phages of different properties and morphology suggests a conserved mechanism of enzyme-substrate interaction . This mechanism may be conformationally determined and related to the specific properties of polysialic acid in other molecular interactions.

Sud Med Ekspert, 1989 Oct-Dec, 32(4), 39 - 42
{Genomic "dactyloscopy" with the use of bacteriophage M13 as a DNA probe (the expertise of material evidence and personal identification)}; Ivanov PL et al.; In this article the authors give a scientific evaluation of genetic dactyloscopy method in which the sites of human chromosomal DNA, possessing structural polymorphism, act as genetic markers . Technology of genome "dactyloscopy" including both the series of standard conventional methods and new methods is presented . The method is highly sensitive and requires small amounts of material for investigation . A practical case is described when genome "dactyloscopy" gave positive results which led to a conclusion on suspect's involvement in the crime.

Mol Gen Genet, 1989 Oct, 219(1-2), 256 - 62
A.T----C.G transversions and their prevention by the Escherichia coli mutT and mutHLS pathways; Schaaper RM et al.; Escherichia coli mutT strains are strong mutators yielding only A.T----C.G transversion mutations . These are thought to result from uncorrected (or unprevented) A.G mispairings during DNA replication . We have investigated the interaction of mutT-induced replication errors with the mutHLS-encoded postreplicative mismatch repair system . By measuring mutation frequencies in both forward and reversion systems, we have demonstrated that mutTmutL and mutTmutS double mutators produce no more mutants than expected from simple additivity of the frequencies in the single mutators . We conclude that mutT-induced A.G replication errors are not recognized by the MutHLS system . On the other hand, direct measurements of mismatch repair by transfection of bacteriophage M13mp2 heteroduplex DNA as well as mutational data from strains other than muT, indicate that the MutHLS system can repair at least certain A.G mispairs . We suggest that A.G mispairs may exist in several different conformations, some of which are recognized by the MutHLS system . However, the A.G mispairs normally prevented by the mutT function are refractory to mismatch repair, indicating that they may represent a structurally distinct class.

Proc Natl Acad Sci U S A, 1989 Oct, 86(20), 7756 - 60
Heterologous expression of a wheat high molecular weight glutenin gene in Escherichia coli; Galili G; An engineered DNA fragment containing a DNA sequence encoding a wheat high molecular weight glutenin subunit was cloned into a bacterial expression vector that is based on bacteriophage T7 RNA polymerase . The resulting plasmid directed the synthesis of large amounts of the mature form of the wheat high molecular weight glutenin subunit in Escherichia coli . This protein comigrated in SDS/PAGE with a native high molecular weight glutenin subunit extracted from wheat endosperm and cross-reacted with antibodies raised against purified wheat high molecular weight glutenins . The wheat subunit synthesized in E . coli also exhibited pI and solubility characteristics identical to those of native wheat subunits . Moreover, the wheat glutenin subunit produced in E . coli cells self-assembled into oligomers linked by intermolecular disulfide bonds, a process that plays an important role in the assembly of native glutenins during gluten formation . The large amounts of a purified wheat subunit obtained from E . coli will enable investigators to analyze the three-dimensional structure of that protein and to identify protein sequences that affect the bread-making quality of wheat dough.

Proc Natl Acad Sci U S A, 1989 Oct, 86(20), 7677 - 81
Genetic effects of thymine glycol: site-specific mutagenesis and molecular modeling studies; Basu AK et al.; The mutational specificity and genetic requirements for mutagenesis by 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), one of the principal DNA lesions induced by oxidation and ionizing radiation, has been investigated in Escherichia coli . Thymine glycol was positioned at a unique site in the single-stranded genome of a bacteriophage M13mp19 derivative . Replication of the genome in E . coli yielded targeted mutations at a frequency of 0.3%; the mutations were exclusively T----C . Mutagenesis was independent of SOS and nth (nth encodes endonuclease III, a thymine glycol repair enzyme) . The adduct was not detectably mutagenic in duplex DNA . A chemical rationalization for the mutation observed for thymine glycol was developed by applying molecular modeling and molecular mechanical calculations to the same DNA sequence studied in vivo . Modeling suggested that the 5R,6S isomer of cis-thymine glycol, when not base paired, was displaced laterally by approximately 0.5 A toward the major groove in comparison to the position that thymine would otherwise occupy . This perturbation of DNA structure should increase the likelihood of a guanine.thymine glycol wobble base pair during replication, which would explain the mutational specificity of the base observed in the genetic experiments.

Proc Natl Acad Sci U S A, 1989 Oct, 86(19), 7485 - 9
Use of yeast artificial chromosome clones for mapping and walking within human chromosome segment 18q21.3; Silverman GA et al.; Well-characterized large genomic clones obtained from yeast artificial chromosome (YAC) libraries provide the framework to localize genes and approach genetic disease . We developed universally applicable approaches to establish authenticity, localize and orient internal genes, map restriction sites, and rescue the distal ends of large human genomic DNA inserts . We selected human chromosome segment 18q21.3 as a model system . Molecular cloning of this segment was initiated by characterizing three plasminogen activator inhibitor type 2 (PAI-2) clones {290, 180, and 60 kilobases (kb)} isolated from a YAC library . Comparison of YAC and bacteriophage lambda genomic DNA clones confirmed the fidelity of the PAI-2 locus . Detailed rare cutting restriction maps were generated by ramped contour-clamped homogeneous electric field electrophoresis . The PAI-2 locus was located and oriented within the YACs, which span a distance 70 kb 5' to 220 kb 3' of PAI-2 . Moreover, both left and right ends of the YAC genomic DNA inserts were rescued by amplifying circularized cloning sites with an inverted form of the polymerase chain reaction . These unique terminal genomic DNA fragments were used to rescreen the YAC library and isolate overlapping clones that extend the map . These approaches will enable neighboring loci to be definitively linked and establish the feasibility of using YAC technology to clone and map chromosomal segments.

Mol Gen Genet, 1989 Oct, 219(1-2), 39 - 48
Transcription and messenger RNA processing upstream of bacteriophage T4 gene 32; Carpousis AJ et al.; Bacteriophage T4 gene 32 lies at the 3' end of a complex transcription unit which includes genes 33, 59, and several open reading frames . In the course of an infection, four major transcripts are synthesized from this unit: two overlapping polycistronic transcripts about 3800 and 2800 nucleotides in length, and two monocistronic gene 32 transcripts about 1150 and 1100 nucleotides in length . These transcripts are made at different times in infection and the polycistronic transcripts have segmental differences in stability . Messenger RNA processing yields a 1025 nucleotide monocistronic gene 32 transcript, and a 135 nucleotide transcript containing part of the gene 59 coding sequence . Processing depends on Escherichia coli encoded ribonuclease E . This pattern of transcription and processing leads to the synthesis of gene 32 mRNA throughout infection, whereas transcripts encoding the upstream genes are present only early in infection . The 3800 nucleotide polycistronic transcript initiates at a promoter that does not require T4 encoded factors for activity . However, full-length synthesis of this transcript depends on the T4 mot gene product . The region upstream of gene 32 also contains four E . coli-like promoters that are active on chimeric plasmids in uninfected cells, but inactive in bacteriophage T4 . The location of these cryptic T4 promoters is intriguing in that they lie near the 5' ends of open reading frame B, gene 59 and gene 32 . They could play a role in phage development under particular conditions of growth or in bacterial hosts other than those examined here.

Antonie Van Leeuwenhoek, 1989 Oct, 56(3), 263 - 71
Purification and characterization of bacteriophage receptor on Veillonella rodentium cells . Phage-receptor on Veillonella; Totsuka M et al.; Veillonellophage N2 adsorbed to polysaccharides (PSs) on Veillonella rodentium ATCC 17743 cell wall, and the bacteriophage receptor contained only glucosamine . D(+)-glucosamine hydrochloride (Sigma) also adsorbed the veillonellophage N2 . These results therefore indicate that the receptor to the veillonellophage N2 is cell wall PSs . The PSs of the host cells as receptor have been characterized . Glucosamine accounted for approximately 100% of the weight of the PSs . The PSs which were partially resolved by Sephadex G-75 chromatography comprised approximately four glucosamine units . Their primary structure was determined by 400 MHz n.m.r . spectroscopy . One- and two-dimensional 1H-nmr experiments showed the PS to be a branched polymer . Glucosamine linkage was detected in one of the branches.

Genomics, 1989 Oct, 5(3), 633 - 5
A PstI polymorphism for the human erythrocyte surface protein band 3 (EPB3) demonstrates close linkage of EPB3 to the nerve growth factor receptor; Stewart EA et al.; Erythrocyte surface protein band 3 (EPB3) plays an important role in CO2 transport in the blood . We have isolated a recombinant lambda bacteriophage that contains coding sequence for the human gene . Sequence analysis demonstrated that the human insert contains a portion of exon 13 . A 1.1-kb BamHI fragment revealed a two-allele polymorphism with PstI . Alleles of 1.4 and 0.9/0.5 kb were present in Caucasoids at frequencies of 0.74 and 0.26, respectively . EPB3 has previously been mapped to 17q21-qter by in situ hybridization, and linkage analysis showed that EPB3 is tightly linked to the gene for the nerve growth factor receptor (NGFR) . The maximum likelihood estimate of recombination (theta) is 0.00, with a lod score of 11.40 and confidence interval of 0.00 to 0.04.

Am J Med, 1989 Oct, 87(4), 421 - 4
Detection of X-linked lymphoproliferative disease using molecular and immunovirologic markers; Purtilo DT et al.; PURPOSE, PATIENTS, AND METHODS: Detection of males affected with the X-linked lymphoproliferative disease (XLP) was sought using immunovirologic and molecular genetic linkage techniques . The study population consisted of 20 males in six families with XLP . RESULTS: Concordance for detection of affected males was 100% when linkage analysis using DXS42 and DXS37 DNA probes and antibody responses to challenge with bacteriophage phi X174 were both determined . Most affected males showing IgG subclass immune deficiency could not produce antibodies to Epstein-Barr virus nuclear antigen and had deficient responses to challenge with bacteriophage phi X174 . CONCLUSION: Use of only one of the techniques described can fail to lead to the diagnosis of XLP, because problems can prevail with each individual determination.

Int J Radiat Biol, 1989 Oct, 56(4), 401 - 11
Mutations induced by 60Co gamma-irradiation in double-stranded M13 bacteriophage DNA in nitrous-oxide saturated solutions are characterized by a high specificity; Hoebee B et al.; Irradiation of double-stranded M13 mp10 DNA in a diluted aqueous solution under N2O leads to a very specific mutation spectrum . Fifteen of 28 mutations induced in a 144 base pair (bp) target are C/G to G/C transversions, the other five bp substitutions are C/G to A/T transversions . Six mutations were single bp deletions, one is a large deletion of 180 bp and one is a 10 bp duplication which is probably from spontaneous origin . The mutations are not randomly distributed throughout the 144 bp mutation target but concentrated around two sites . The differences and similarities with the radiation-induced mutation spectrum previously obtained under oxygen are discussed.

Infect Immun, 1989 Oct, 57(10), 3022 - 9
Type 1 pili are not necessary for colonization of the streptomycin-treated mouse large intestine by type 1-piliated Escherichia coli F-18 and E . coli K-12; McCormick BA et al.; Escherichia coli F-18, an excellent colonizer of the streptomycin-treated mouse large intestine, produces type 1 pili . E . coli F-18 FimA-, type 1 pilus negative, and E . coli F-18 FimH-, type 1 pilus positive but adhesin negative, were constructed by bacteriophage P1 transduction of defective fimA and fimH genes from the E . coli K-12 strains ORN151 and ORN133, respectively, into E . coli F-18 . Adhesion of E . coli F-18 to an immobilized mannose-bovine serum albumin glycoconjugate was about sixfold greater than that of either E . coli F-18 FimA- or E . coli F-18 FimH-, and adhesion of E . coli F-18 to immobilized cecal epithelial cell brush border membranes was between two- and threefold greater than that of E . coli F-18 FimA- or E . coli F-18 FimH- . When either E . coli F-18 FimA- or E . coli FimH- was fed to streptomycin-treated mice together with E . coli F-18, the pilus-negative and adhesin-negative strains colonized as well as their type 1-piliated parent . Essentially the same result was observed when the type 1-piliated E . coli K-12 strain ORN152 was fed to streptomycin-treated mice together with a nearly isogenic K-12 FimA- strain, ORN151 . Furthermore, when streptomycin-treated mice were fed E . coli F-18 FimA- or E . coli F-18 FimH- together with E . coli F-18 Col-, which also makes type 1 pili but is a poor colonizer relative to E . coli F-18 because it grows poorly in mucus in the presence of E . coli F-18, the F-18 FimA- and F-18 FimH- strains colonized well (10(6) to 10(7) CFU/g of feces), whereas the number of E . coli F-18 Col- in feces decreased rapidly to 10(2) CFU/g of feces . These data show that in streptomycin-treated mice, the inability to produce functional type 1 pili has no effect on the ability of E . coli F-18 and E . coli K-12 to colonize the large intestine.

Mol Gen Mikrobiol Virusol, 1989 Oct, (10), 16 - 9
{Cloning and analysis of the primary structure of an element, related to the murine mammary cancer virus, from the genome of the Djungarian hamster}; Vasetskii NS et al.; 11 recombinant bacteriophages from the genomic library of Djungarian hamster genome, that carry MMTV-related sequences (MRS-Ps), have been cloned with the murine mammary cancer virus (MMTV) as a hybridization probe . The sequences are repeated 50 times in the genome . MRS-Ps contain the tracts of homology with the long end repeat MMTV, the genes pol and, possibly, env, but not with the gag gene . Sequencing of the 0,87 kb restrict, common to all EcoRI-BamHI clones, and the analysis of the sequence have demonstrated the high level of homology with the pol gene of MMTV and its origination from the somewhat functioning gene.

Biochem J, 1989 Oct 1, 263(1), 19 - 23
Secondary structural features of the bacteriophage Mu-encoded A and B transposition proteins; Chaconas G et al.; The role of the bacteriophage Mu-encoded A and B proteins is to direct the transposition of Mu DNA . These are the first active DNA transposition proteins to have been purified and their mechanism of action at the biochemical level is under intensive study . Structural studies on these proteins, however, have lagged behind their biochemical characterization . We report here near- and far-u.v . c.d . spectra for these proteins and their secondary structural features derived from these data . The Mu A protein appears to be composed of primarily beta-sheet (40%) with 24% alpha-helix, 9% beta-turn and 27% random coil . In contrast, the Mu B protein contains 55% alpha-helix with only 13% beta-sheet and 3+ beta-turn and 29% random coil . The near-u.v . c.d . spectrum of the A protein was not unusual; however, the profile of the B protein suggested either buried or restricted chromophores within the protein or short-range interactions between aromatic residues.

Proc Natl Acad Sci U S A, 1989 Oct, 86(19), 7316 - 20
Expression of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and its kinase domain in Escherichia coli; Tauler A et al.; The rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (ATP:D-fructose-6-phosphate 2-phosphotransferase/D-fructose-2,6-bisphosphate 2-phosphohydrolase, EC 2.7.1.105/EC 3.1.3.46) and its separate kinase domain were expressed in Escherichia coli by using an expression system based on bacteriophage T7 RNA polymerase . The bifunctional enzyme (470 residues per subunit) was efficiently expressed as a protein that starts with the initiator methionine residue and ends at the carboxyl-terminal tyrosine residue . The expressed protein was purified to homogeneity by anion exchange and Blue Sepharose chromatography and had kinetic and physical properties similar to the purified rat liver enzyme, including its behavior as a dimer during gel filtration, activation of the kinase by phosphate and inhibition by alpha-glycerol phosphate, and mediation of the bisphosphatase reaction by a phosphoenzyme intermediate . The expressed 6-phosphofructo-2-kinase also started with the initiator methionine but ended at residue 257 . The partially purified kinase domain was catalytically active, had reduced affinities for ATP and fructose 6-phosphate compared with the kinase of the bifunctional enzyme, and had no fructose-2,6-bisphosphatase activity . The kinase domain also behaved as an oligomeric protein during gel filtration . The expression of an active kinase domain and the previous demonstration of an actively expressed bisphosphatase domain provide strong support for the hypothesis that the hepatic enzyme consists of two independent catalytic domains encoded by a fused gene.

Proc Natl Acad Sci U S A, 1989 Oct, 86(20), 7971 - 5
Efficient rescue of integrated shuttle vectors from transgenic mice: a model for studying mutations in vivo; Gossen JA et al.; To study gene mutations in different organs and tissues of an experimental animal, we produced transgenic mice harboring bacteriophage lambda shuttle vectors integrated in the genome in a head-to-tail arrangement . As a target for mutagenesis, the selectable bacterial lacZ gene was cloned in the vector . The integrated vectors were rescued from total genomic DNA with high efficiency by in vitro packaging and propagation of the phages in a LacZ- strain of Escherichia coli C . The background mutation frequencies in brain and liver DNA appeared to be low, as was indicated by the absence of colorless plaques among 138,816 and 168,160 phage isolated from brain and liver DNA, respectively . Treatment of adult female transgenic mice with N-ethyl-N-nitrosourea resulted in a dose-dependent increase of the frequency of mutated vectors isolated from brain DNA, up to 7.4 x 10(-5) at 250 mg of the alkylating agent per kilogram of body weight . At this dose, in liver DNA of the same mice, mutation frequencies were approximately 3 x 10(-5) . DNA sequence analysis of four mutant vectors isolated from brain DNA indicated predominantly G.C----A.T transitions . These results demonstrate the value of this transgenic mouse model in studying gene mutations in vivo . In addition to its use in fundamental research, the system could be used as a sensitive, organ-specific, short-term mutagenicity assay.

New Biol, 1989 Oct, 1(1), 54 - 65
Identification of a T4 gene required for bacteriophage mRNA processing; Ruckman J et al.; A ribonucleolytic activity that cleaves within the Shine/Dalgarno sequences of the bacteriophage T4 motA and ORF2 mRNAs was recently described . We have identified additional sites of processing within several other ribosome binding sites, including two sites in the polycistronic frd transcript . Deletion mutants (farP) that overproduce the product of frd are defective in this mRNA processing . The mutants were used to identify processing events dependent on the T4 activity including attack at nuclease-sensitive sites within the coding sequences of some genes and within the intercistronic region 5' of gene 43 . All known processing sites lie within similar sequences . Another mutant in mRNA processing carries a point mutation in one of the open reading frames (orf61.9) removed by the farP deletions . Introduction of a cloned copy of this open reading frame into a unique site in the chromosome of farP phage is sufficient to restore mRNA processing capability . The open reading frame probably encodes the T4 regB protein.

Mol Gen Mikrobiol Virusol, 1989 Oct, (10), 24 - 9
{Polymerization of DNA fragments coding epitopes of the surface antigen of hepatitis B in Escherichia coli cells}; Sergienko OV et al.; Synthetic oligonucleotides coding for the amino acid residues 135-151 (A, B) and 93-109 (C) of the hepatitis B surface antigen (HBsAg) have been polymerized . The obtained polymers (AC)n and (BCAC)n were inserted into the beginning of the lacZ gene under the control of the chloramphenicol promoter or the right promoter PR of bacteriophage lambda . Stability of the obtained plasmids was investigated during transformation, cell growth and gene expression.

Gene, 1989 Sep 30, 81(2), 267 - 74
Synthesis of human adenovirus type 2 fiber protein in Escherichia coli cells; Chatellard C et al.; We have cloned and expressed in Escherichia coli the gene encoding the trimeric fiber protein of human adenovirus type 2 . A gene expression system based on bacteriophage T7 RNA polymerase was used . Optimal gene expression was obtained with 1-h induction, at a temperature of 30 degrees C . The synthesized protein constituted about 1% of total host-cell protein . During induction, the growth of bacteria carrying the plasmid containing the fiber gene, was retarded compared with that of bacteria carrying the plasmid without the fiber gene . This toxic effect of fiber protein on bacterial hosts could be diminished by addition of glucose to the medium and by maintaining the pH above 7, thus improving the yield of recombinant fiber protein . The fiber protein produced in E . coli is stable during the course of induction . It is insoluble in buffers at physiological pH, in various salt solutions, and in the presence of nonionic detergents . It can be solubilized in 1% sodium dodecyl sulfate or in urea solutions above 2 M . There are indications that recombinant fiber trimerizes spontaneously, since after the removal of urea by dialysis at pH 8, recombinant fibers runs similarly to native trimeric fiber, on nondenaturing polyacrylamide gels . This trimer has, however, a less compact structure than native Ad2 fiber, since during gel filtration recombinant protein is excluded before native protein . It is also more sensitive to chymotrypsin digestion than native fiber.

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