Gene, 1988, 62(1), 45 - 54 In vivo functional characterization of a yeast nucleotide sequence: construction of a mini-Mu derivative adapted to yeast; Daignan-Fornier B et al.; We have constructed a derivative of the bacteriophage Mu (called MudIIZZ1), which contains the lacZ gene coding for beta-galactosidase (beta Gal) and markers suited for yeast transformation (2 mu circle replication origin and LEU2) . This new transposon is an efficient tool for studying the expression of cloned yeast nucleotide sequences through beta Gal-protein fusions . It is also adapted for one-step disruption experiments so that a functional map of the same sequence can be drawn . We have used this MudIIZZ1 transposon to study a 5-kb DNA fragment which had been cloned by complementation of a cold-sensitive respiration-deficient phenotype . By testing the expression of the beta Gal fusions and the disruption phenotype, we have confirmed the presence of a gene required for mitochondrial functions, and revealed another two open reading frames in the same fragment; one of these also interferes with mitochondrial biogenesis . The method is fast and reliable, and has potential for more general purposes which are discussed. Gene, 1988, 62(1), 111 - 9 Identification of the bacteriophage D108 kil gene and of the second region of sequence nonhomology with bacteriophage Mu; Waggoner BT et al.; To identify the second region of sequence nonhomology between the genomes of the transposable bacteriophages Mu and D108 originally observed by electron-microscopic analysis of DNA heteroduplexes and to localize functions ascribed to the 'accessory' or 'semi-essential' early regions of the phages between genes B and C, a 0.9-kb fragment of each genome located immediately beyond the B gene was cloned and sequenced . Three open reading frames (ORFs) were identified in each . The region of nonhomology is located within the 3' portion of the third ORF . D108 is shown to possess a Kil function similar to that previously shown for Mu, and that function is encoded by the first ORF. Genetika, 1988 Jan, 24(1), 42 - 52 {Determination of the direction of the transcription of bacteriophage T4 genes by heat induction of the transcription from recombinant plasmid Pl-promoter: 2 directions in the region of genes 25-29}; Klausa VI et al.; The bacterial strains with recombinant plasmids constructed on the basis of vector plasmid pSCC31 and containing BglII fragment of bacteriophage T4 DNA with genes 25-29 have been used in this study . Restriction analysis and subcloning demonstrated that in the case of the recombinant plasmid pRL705, the phage DNA fragment had right orientation for phage late genes transcription, while the opposite one in the plasmid pRL707 . Heat induction of plasmids transcription under control of pL promoter led to the substantial change in phage burst size and the production of recombinants, as shown in complementation experiments, the changes in the burst size being dependent both on the host strain and the amber mutant used . On the basis of these data, the conclusion has been drawn that the genes 26 and 25, in contrast to genes 51, 27, 28 and 29, are being transcribed in the counter-clockwise direction on the genomic map, i.e . in the direction of transcription of T4 early genes. Genetika, 1988 Jan, 24(1), 34 - 41 {Cloning of the DNA BglII fragments of bacteriophage T4 in the vector plasmid pSCC31}; Nivinskas RG; An attempt has been made to clone six BglII fragments of T4 DNA in the range of 3.3-8.1 kb in the vector plasmid pSCC31 containing a single BglII site within the gene for endonuclease EcoRI and pL promoter of phage lambda . DNA fragments were extracted from the corresponding bands of agarose gel . The following BglII fragments were cloned: the 3.3 kb fragment No . 9 containing a portion of gene 20, the gene 21 and a portion of gene 22; the 4.2 kb fragment No . 8.1 with genes 17, 18, 19 and a portion of gene 20; the 5.2 kb fragment No . 7.1 with genes 25-29 and a portion of gene 48 . In the case of the fragment No . 7.1, the recombinant plasmids pRL705 and pRL707 with different orientation of phage DNA fragment were obtained . An attempt to clone the fragments No . 8.2 (4.2 kb), No . 7.2 (5.45 kb) and No . 6 (8.1 kb) was unsuccessful and this probably indicates the presence of the genes, whose products are deleterious to the growth of bacterial cell. Mol Gen Genet, 1988 Jan, 211(1), 63 - 71 Use of recombination techniques to examine the structure of the csg locus of Myxococcus xanthus; Shimkets LJ et al.; The myxobacteria are among the simplest organisms with a developmental cycle that is dependent on cell cooperation, and they provide an outstanding system with which to study genes involved in cell interactions . Myxococcus xanthus cells which acquire a csg mutation (formerly known as spoC) lose three different traits, the ability to sporulate, the ability to stimulate adjacent Csg cells to sporulate, and the ability to ripple . The boundaries of the csg locus were determined by transferring a recombinant DNA molecule containing all or part of the locus to Csg mutants and examining the sporulation and rippling phenotypes of the transductants . Three methods were used to integrate the csg locus into the chromosome . First, the entire molecule was integrated into the chromosome by a single homologous crossover . Second, a portion of the molecule was integrated into the chromosome by two flanking homologous crossovers . Third, the entire molecule was integrated into the chromosome by site-specific recombination at a bacteriophage attachment site . Together, these techniques suggested that all of the functions of the csg locus are carried on a DNA fragment of 1.9 kbp or less . The locus appears to contain two smaller units of function . Transposon insertions or deletions in the right end of the locus disrupted sporulation and intercellular complementation of Csg mutants for sporulation, but did not disrupt rippling . The intercellular complementation of Csg mutants may reflect a natural and necessary step in the sporulation of wild-type cells, since the ability to sporulate and the ability to stimulate Csg mutants to sporulate were inseparable by any of these methods. Proc Natl Acad Sci U S A, 1988 Jan, 85(2), 396 - 400 A 7-kDa region of the bacteriophage T7 gene 4 protein is required for primase but not for helicase activity; Bernstein JA et al.; Bacteriophage T7 gene 4 protein, purified from phage-infected cells, consists of a mixture of 56- and 63-kDa species that provides helicase and primase activities required for T7 DNA replication . The 56-kDa species has been purified independently of the colinear 63-kDa species . Like a mixture of the two proteins, the 56-kDa protein binds single-stranded DNA in the presence of dTTP, catalyzes DNA-dependent hydrolysis of dTTP, and has helicase activity . In contrast to the mixture, the 56-kDa protein cannot catalyze template-dependent RNA primer synthesis . In the absence of a DNA template, both the 56-kDa protein and the mixture of the two species synthesize low levels of diribonucleotide . A putative "zinc finger" present near the amino terminus of the 63-kDa protein but absent from the 56-kDa protein may play a major role in the recognition of primase sites in the template. Mutat Res, 1988 Jan, 197(1), 23 - 37 Characterization of the types of mutational events that spontaneously occur in a plasmid system; Chen JH et al.; The immunity region from a cI857 derivative of bacteriophage lambda has been cloned into the EcoRI site of pBR322 to produce a plasmid that can be used to analyze spontaneous mutagenesis . Cells containing this plasmid are temperature-sensitive for growth unless mutations have occurred that somehow prevent the expression of the kil gene in the lambda fragment at non-permissive temperature . 678 such temperature-resistant mutants from 10 independent subcultures each of 2 different recA- E . coli strains have been collected, and the nature of the plasmid mutations obtained has been analyzed . All of the subcultures contained mutants that allowed growth at the restrictive temperature without showing a detectable change in plasmid size . 75% of the total mutants fell in this class . More than half of these mutations involved the lambda leftward promoter, pL, and such mutants were found in all 20 subcultures . The remaining 25% of the mutations involved a change in plasmid size and mutations of this class were found in 18 of 20 subcultures . 12% of the total mutants (found in 16 of 20 subcultures) had an insertion of IS1 in the region between pL and the lambda kil gene . 6% of the total mutants had undergone an IS1-mediated deletion, while 1% were mixed colonies in which multiple IS1-mediated events had occurred . About 1% of the total mutants had undergone complex IS1-mediated DNA rearrangement(s) that have not yet been characterized . In total, 11 of 20 subcultures yielded isolates where IS1-mediated rearrangements had occurred . The remaining 4% of the mutations included insertions of IS5, IS30, and an IS1 family member that appears to be IS1T as well as IS1T-mediated deletions and deletions that do not appear to have been mediated by any insertion sequence . A mutant with both an IS1 insertion and an alteration involving pL has also been isolated. J Bacteriol, 1988 Jan, 170(1), 352 - 8 Sequence requirements of Escherichia coli attTn7, a specific site of transposon Tn7 insertion; McKown RL et al.; Transposon Tn7 transposes at high frequency to a specific site, attTn7, in the Escherichia coli chromosome . We devised a quantitative assay for Tn7 transposition in which Tn7-end derivatives containing the cis-acting transposition sequences of Tn7 transpose from a bacteriophage lambda vector upon infection into cells containing the Tn7-encoded transposition proteins . We used this assay to identify a 68-base-pair DNA segment containing the sequences essential for attTn7 target activity . This segment is positioned asymmetrically with respect to the specific point of Tn7 insertion in attTn7 and lacks obvious homology to the sequences at the ends of Tn7 which participate directly in transposition . We also show that some sequences essential for attTn7 target activity are contained within the protein-coding sequence of a bacterial gene. J Bacteriol, 1988 Jan, 170(1), 108 - 16 Double negative and positive control of tsx expression in Escherichia coli; Bremer E et al.; The Escherichia coli tsx gene encodes an outer membrane protein that is involved in nucleoside uptake and serves as the receptor protein for colicin K and several bacteriophages . Regulation of its expression was studied by using tsx-lacZ protein and operon fusion strains carrying mutations in deoR, cytR, and crp . The cytR-encoded repressor had a stronger influence on tsx transcription than the DeoR repressor did, and the level of tsx expression in a deoR cytR double mutant was approximately the sum of those found in the single deoR and cytR strains . This double negative control of Tsx synthesis was superceded by a positive control mechanism mediated by the cyclic AMP-catabolite activator protein (cAMP-CAP) complex . Our results suggest that tsx expression is controlled at two separate and differently regulated promoters: the weaker promoter (P1) is repressible by DeoR, while the stronger promoter (P2) is subject to negative and positive control by the CytR repressor and the cAMP-CAP complex, respectively . A mutant was isolated that showed unaltered tsx regulation by DeoR and the cAMP-CAP complex but strongly reduced repression by CytR . This tsx operator mutant was used to obtain a suppressor mutation located on a plasmid carrying the cloned cytR gene that restored CytR control of tsx expression . The direction of tsx transcription was determined and found to be counterclockwise on the E . coli chromosome. J Biol Chem, 1987 Dec 25, 262(36), 17651 - 8 Bacteriophage lambda N gene leader RNA . RNA processing and translational initiation signals; Steege DA et al.; The positions of RNA processing events mediated by RNase III and of two ribosome binding sites have been defined in an in vitro transcript of 321 nucleotides initiated at the major leftward promoter (PL) of bacteriophage lambda . Purified RNase III makes two specific endonucleolytic cleavages in the transcript at points 88 and 197 nucleotides from the PL start . The positions of these cuts suggest a secondary structure for the RNase III recognition site which is similar to other RNase III sites in which double-stranded cleavage occurs . Structure mapping experiments reveal a pattern of cleavages made in the PL transcript by nucleases specific for single- or double-stranded RNA that support the structure proposed for the RNase III stem and provide clear evidence for the stem-loop formed in the RNA at the position of the N recognition (nutL) site . Our finding that ribosomes bind efficiently in vitro to the region of the PL transcript which includes the AUG codon at position 223 supports other evidence that this triplet is the N protein start codon . The existence of an additional ribosome binding site in the N gene leader region just downstream from the nutL site identifies a second position possibly used for translational initiation or regulation . Its occurrence suggests potential roles for ribosome interaction and/or translation of the leader RNA in regulating phage development and N gene expression. J Biol Chem, 1987 Dec 25, 262(36), 17437 - 42 The grpE protein of Escherichia coli . Purification and properties; Zylicz M et al.; The grpE gene of Escherichia coli was first identified because a mutation in it, grpE280, prevented bacteriophage lambda DNA replication in vivo . Subsequent work resulted in the identification of the grpE protein in two-dimensional gels and its classification as a heat shock protein . Here we report the purification of the grpE protein . We show that overproduction of grpE occurs in dnaK 103 bacteria which do not produce a functional Mr 72,000 dnaK protein . The grpE protein was purified from this strain primarily by its specific retention on a dnaK affinity column . The interaction between these two proteins, which is stable in the presence of 2 M KCl, allowed other proteins to be washed from this column . grpE was then eluted by ATP, which disrupts the interaction . During purification, grpE activity was monitored by its ability to complement an in vitro lambda dv DNA replication system dependent on the lambda O and lambda P proteins . The effect of ATP on the dnaK-grpE complex was also observed during sedimentation of the two proteins in glycerol gradients . Purified grpE protein has a Mr of approximately 23,000 under both denaturing and native conditions, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sedimentation, respectively . However, in the presence of dnaK under native conditions, grpE cosediments with dnaK . When ATP is added to the gradient, the complex is disrupted, and the two proteins sediment independently as monomers. J Mol Biol, 1987 Dec 20, 198(4), 579 - 87 Purification and DNA-binding properties of FIS and Cin, two proteins required for the bacteriophage P1 site-specific recombination system, cin; Haffter P et al.; An Escherichia coli chromosomally coded factor termed FIS (Factor for Inversion Stimulation) stimulates the Cin protein-mediated, site-specific DNA inversion system of bacteriophage P1 more than 500-fold . We have purified FIS and the recombinase Cin, and studied the inversion reaction in vitro . DNA footprinting studies with DNase I showed that Cin specifically binds to the recombination site, called cix . FIS does not bind to cix sites but does bind to a recombinational enhancer sequence that is required in cis for efficient recombination . FIS also binds specifically to sequences outside the enhancer, as well as to sequences unrelated to Cin inversion . On the basis of these data, we discuss the possibility of additional functions for FIS in E . coli. J Biol Chem, 1987 Dec 15, 262(35), 16858 - 64 Structural analysis of the temperature-sensitive mutant of bacteriophage T4 lysozyme, glycine 156----aspartic acid; Gray TM et al.; The structure of the mutant of bacteriophage T4 lysozyme in which Gly-156 is replaced by aspartic acid is described . The lysozyme was isolated by screening for temperature-sensitive mutants and has a melting temperature at pH 6.5 that is 6.1 degrees C lower than wild type . The mutant structure is destabilized, in part, because Gly-156 has conformational angles (phi, psi) that are not optimal for a residue with a beta-carbon . High resolution crystallographic refinement of the mutant structure (R = 17.7% at 1.7 A resolution) shows that the Gly----Asp substitution does not significantly alter the configurational angles (phi, psi) but forces the backbone to move, as a whole, approximately 0.6 A away from its position in wild-type lysozyme . This induced strain weakens a hydrogen bond network that exists in the wild-type structure and also contributes to the reduced stability of the mutant lysozyme . The introduction of an acidic side chain reduces the overall charge on the molecule and thereby tends to increase the stability of the mutant structure relative to wild type . However, at neutral pH this generalized electrostatic stabilization is offset by specific electrostatic repulsion between Asp-156 and Asp-92 . The activity of the mutant lysozyme is approximately 50% that of wild-type lysozyme . This reduction in activity might be due to introduction of a negative charge and/or perturbation of the surface of the molecule in the region that is assumed to interact with peptidoglycan substrates. Carbohydr Res, 1987 Dec 15, 170(2), 193 - 206 The structure of the neuraminic acid-containing capsular polysaccharide of Escherichia coli serotype K9; Dutton GG et al.; The acidic capsular polysaccharide isolated from Escherichia coli O9:K9:H12 was investigated by using n.m.r . spectroscopy, methylation analysis, periodate oxidation, and bacteriophage-borne enzyme degradation . The polysaccharide, the structure of which is shown below, is the third E . coli capsular polysaccharide reported to contain neuraminic acid, the others being the K1 and K92 polysaccharides, and it is the first in the E . coli series shown to contain a 4-linked neuraminic acid unit . (formula; see text). J Immunol, 1987 Dec 15, 139(12), 3973 - 80 T lymphocyte response to bacteriophage lambda repressor cI protein . Recognition of the same peptide presented by Ia molecules of different haplotypes; Lai MZ et al.; The murine T cell response to bacteriophage lambda cI repressor protein has been investigated . Isolation and characterization of class II-restricted T cell hybridomas from BALB/c and A/J mice undergoing a primary response has revealed that a single region of the protein, residues 12-26, is the immunodominant site . Fine specificity analysis using truncated peptides (P12-24 and P15-26) reveals a great deal of heterogeneity at the clonal level of I-Ad-restricted T cells . I-Ek-restricted T cells are less heterogeneous in their reactivity toward P12-24 and P15-26, but show diversity in their responses to peptide analogues with substitution at Tyr22 . The specificity difference between T cell hybridomas of I-Ad-restriction and I-Ek-restriction and the inhibition effect of different inactive peptides suggest that the same peptide is presented in different configurations by different Ia molecules . Further, no cross-reactivity can be detected between T cells of these two haplotypes, Ia molecules and Ia bound-peptides. Philos Trans R Soc Lond B Biol Sci, 1987 Dec 15, 317(1187), 421 - 8 Mammalian DNA polymerase alpha: a replication-competent holoenzyme form from calf thymus; Hubscher U et al.; Calf thymus DNA polymerase alpha, like the replication-specific DNA polymerase III holoenzyme of Escherichia coli, can be isolated as a distinct complex . A specific multiprotein form of the polymerase alpha, a form designated replication-competent (RC) holoenzyme, consists of a complex of a polymerase-primase core and at least six other polypeptides . The RC holoenzyme can efficiently replicate several naturally occurring templates, including the genomic DNA of the porcine circovirus (PCV) . The DNA of this virion consists of a single-stranded circle with a defined replication origin, and its replication requires the cellular DNA replication machinery . It might therefore provide an invaluable opportunity to investigate chromosomal replication mechanisms, analogous to the way that studies on E . coli bacteriophage DNA replication elucidated host DNA replication mechanisms . Calf RC holoenzyme alpha selectively initiates PCV DNA replication in vitro at a site that possibly represents a consensus sequence of cellular DNA replication origins . The cell-free PCV replication system will be exploited for the in vitro dissection and reconstitution of the RC holoenzyme and the functional analysis of its component polypeptides. J Biol Chem, 1987 Dec 5, 262(34), 16575 - 9 The ban operon of bacteriophage P1 . Localization of the promoter controlled by P1 repressor; Lurz R et al.; Repression of a strong promoter localized 5' to the P1 ban gene is a prerequisite for cloning the ban operon in the multicopy plasmid pBR325 . Repression is brought about by the binding of P1 repressor to the operator of the ban operon (Heisig, A., Severin, I., Seefluth, A . K., and Schuster, H . (1987) Mol . Gen . Genet . 206, 368-376) . Binding of RNA polymerase in vitro overlaps with the operator and is inhibited by P1 repressor as shown by electron microscopy . The mutant P1 bac, which renders ban expression constitutive, contains a single base pair exchange within the operator . As a consequence, more repressor is required (i) for the inhibition of binding of RNA polymerase, and (ii) for the electrophoretic retardation of a P1 bac DNA fragment when compared to the corresponding bac+ fragment . A P1 ban recombinant plasmid containing a 4-base pair deletion close to the operator still allows binding of repressor but not of RNA polymerase . By that means, a repressible promoter is located at the P1 map position 72 in a distance of about 2.5 kilobase pairs to the beginning of the ban gene. J Biol Chem, 1987 Dec 5, 262(34), 16558 - 65 Accessory proteins bind a primed template and mediate rapid cycling of DNA polymerase III holoenzyme from Escherichia coli; O'Donnell ME; DNA polymerase III holoenzyme was assembled from pure proteins onto a primer template scaffold . The assembly process could be divided into two stages . In the time-consuming first stage, beta subunit and gamma.delta subunit complex were required in forming a tightly bound ATP-activated "preinitiation complex" with a single-stranded DNA bacteriophage circle uniquely primed with a synthetic pentadecadeoxyribonucleotide . This finding substantiates an earlier study using crude protein preparations in a homopolymer system lacking Escherichia coli single-stranded DNA binding protein (Wickner, S . (1976) Proc . Natl . Acad . Sci . U . S . A . 73, 3511-3515) . In the second stage, the polymerase III core and the tau subunit rapidly seek out and bind the preinitiation complex to form DNA polymerase III holoenzyme capable of rapid and entirely processive replication of the circular DNA . ATP is not required beyond formation of the preinitiation complex . It is remarkable that the fully assembled DNA polymerase III holoenzyme is so stably bound to the primed DNA circle (4-min half-time of dissociation), yet upon completing a round of synthesis the polymerase cycles within 10 s to a new preinitiation complex on a challenge primed DNA circle . Efficient polymerase cycling only occurred when challenge primed DNA was endowed with a preinitiation complex implying that cycling is mediated by a polymerase subassembly which dissociates from its accessory proteins and associates with a new preinitiation complex . These subunit dynamics suggest mechanisms for polymerase cycling on the lagging strand of replication forks in a growing chromosome. J Mol Biol, 1987 Dec 5, 198(3), 393 - 404 Structure of cryptic lambda prophages; Redfield RJ et al.; When Escherichia coli cells lysogenic for bacteriophage lambda are induced with ultraviolet light, cells carrying cryptic lambda prophages are occasionally found among the apparently cured survivors . The lambda variant crypticogen (lambda crg) carries an insertion of the transposable element IS2, which increases the frequency of cryptic lysogens to about 50% of cured cells: 43 of these cryptic prophages have been characterized . They all contain substitutions that replace the early segment of the prophage genome (from the IS2 to near the cos site) with a duplicate copy of a large segment of the host chromosome . The right end of the substitution always results from recombination between the nin-QSR-cos region of the prophage and the homologous incomplete lambdoid prophage Qsr' at 12.5 minutes in the E . coli chromosome . The left end of the substitution is usually a crossover that recombines the IS2 element in the prophage with an E . coli IS2 at 8.5 minutes, near the lac gene, or with a second IS2 located counterclockwise from leu at 2 minutes, generating duplications of at least 200,000 bases . Five cryptic lysogens derived from cells lysogenic for a reference strain of lambda (which lacks the IS2 present in lambda crg) have been characterized . They contain substitutions whose right termini are generated by a crossover with the Qsr' prophage . The left termini of these substitutions are formed either by a crossover between the lambda exo gene and a short exo-homologous segment of Qsr' (2/5), or by a crossover between sequences to the left of attL and an unmapped distant region of the host chromosome (3/5) . The large duplications carried by these cryptic lysogens are stable, unlike tandem duplications, and so may significantly influence the cell's evolutionary potential. Science, 1987 Dec 4, 238(4832), 1413 - 5 Bacteriophage M13 procoat protein inserts into the plasma membrane as a loop structure; Kuhn A; The major coat protein of bacteriophage M13 is synthesized as a precursor, the procoat, with a typical leader (signal) sequence of 23 residues at its NH2-terminus . A fusion protein that contains the NH2-terminal 141 residues of cytoplasmic ribulokinase and all but the first ten residues of M13 procoat was made . The fusion protein inserts into the plasma membrane of Escherichia coli and is processed by leader peptidase to give rise to a leader peptide of 155 residues and the mature coat protein of 50 residues . The NH2-terminus of the leader peptide remains in the cytoplasm and is protected from protease added to the medium outside of the cell . This indicates that M13 procoat inserts into the membrane as a loop structure and that the NH2-terminus of a leader peptide remains within the cytoplasm during membrane insertion. Proc Natl Acad Sci U S A, 1987 Dec, 84(23), 8248 - 52 Cloning genes for the biosynthesis of a macrolide antibiotic; Fishman SE et al.; Macrocin-O-methyltransferase (MacOMeTase) catalyzes the final enzymatic step in the biosynthesis of tylosin in Streptomyces fradiae . A 44-base mixed oligonucleotide probe containing only guanosine and cytidine in the third position of degenerate codons was synthesized based on the amino acid sequence of the amino terminus of MacOMeTase . Plaque blot hybridization to a bacteriophage lambda library and colony blot hybridization to a cosmid library of S . fradiae DNA identified recombinants that contained overlapping fragments of chromosomal DNA . The nucleotide sequence of the cloned DNA verified that the DNA contained the coding sequence for MacOMeTase . Recombinant plasmids transformed mutants blocked in tylosin biosynthesis and complemented tylF (the structural gene for MacOMeTase) and tyl mutations of eight other classes. Mol Gen Mikrobiol Virusol, 1987 Dec, (12), 29 - 34 {Properties of bacteriophage T4 modified with cetyltrimethylammonium bromide}; Berezhneva GI et al.; Treating of the bacteriophage T4B with cetyltrimethylammonium bromide results in extension of long fibers and contraction of tail sheaths . The unreorganized hexagonal baseplate attachment to the distal end of the tail core remains intact . Such aberrantly contracted phages are shown to retain the ability to absorb on the bacterial surface . The absorption is inhibited by sucrose and does not require the presence of 1-tryptophan . The aberrantly contracted phages lose the infection ability. EMBO J, 1987 Dec 1, 6(12), 3779 - 85 An efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus IBV; Brierley I et al.; The polymerase-encoding region of the genomic RNA of the coronavirus infectious bronchitis virus (IBV) contains two very large, briefly overlapping open reading frames (ORF), F1 and F2, and it has been suggested on the basis of sequence analysis that expression of the downstream ORF, F2, might be mediated through ribosomal frame-shifting . To examine this possibility a cDNA fragment containing the F1/F2 overlap region was cloned within a marker gene and placed under the control of the bacteriophage SP6 promoter in a recombinant plasmid . Messenger RNA transcribed from this plasmid, when translated in cell-free systems, specified the synthesis of polypeptides whose size was entirely consistent with the products predicted by an efficient ribosomal frame-shifting event within the overlap region . The nature of the products was confirmed by their reactivity with antisera raised against defined portions of the flanking marker gene . This is the first non-retroviral example of ribosomal frame-shifting in higher eukaryotes. Biochemistry, 1987 Dec 1, 26(24), 7870 - 5 The DNA sequence of the human beta-globin region is strongly biased in favor of long strings of contiguous purine or pyrimidine residues; Behe MJ; The DNA sequence of the human beta-globin region, comprising over 67 kilobase pairs, has been analyzed for the occurrence of strings of contiguous purine or pyrimidine residues . Tracts of 10 or more contiguous residues are found 4 times more frequently than would be expected with a random distribution of bases, so that a long string occurs at an average of every 250 base pairs . A survey of six other human gene sequences, totaling 86 kilobase pairs, shows a remarkably similar result . No such overrepresentation of contiguous purine or pyrimidine residues is found in the bacteriophages lambda or T7. J Gen Microbiol, 1987 Dec, 133 ( Pt 12), 3505 - 11 Highly efficient uptake of a rifamycin derivative via the FhuA-TonB-dependent uptake route in Escherichia coli; Pugsley AP et al.; Rifamycin CGP 4832 is a semisynthetic rifamycin derivative . It is at least 200 times more active than rifampicin against Escherichia coli and related bacteria . This increased activity is shown here to be due to the efficient uptake of CGP 4832 across the E . coli outer membrane via the ferrichrome transport system comprising the outer membrane FhuA (TonA) protein, the ferrichrome receptor, and the inner membrane TonB protein . CGP 4832 competed with ferrichrome and other iron siderophore complexes, and with bacteriophage T5 and colicin M for binding sites on the FhuA protein . Mutations in fhuA or tonB genes reduce CGP 4832 sensitivity to a level comparable to that to rifampicin . There is no evidence that CGP 4832 or rifampicin utilize the inner membrane ferrichrome transport system to gain entry into the cytoplasm. J Biochem (Tokyo), 1987 Dec, 102(6), 1539 - 46 Affinity chromatography on immobilized anhydrotrypsin: general utility for selective isolation of C-terminal peptides from protease digests of proteins; Kumazaki T et al.; Recently we have succeeded in the efficient isolation of the C-terminal peptides from tryptic digests of the tail sheath protein (with C-terminal Gly) and the tube protein (with C-terminal Glu) of bacteriophage T4, by taking advantage of a unique property of immobilized anhydrotrypsin, that is, a strong specific affinity for peptides containing Arg or Lys residues at their C-termini . In this study, the utility of affinity chromatography on immobilized anhydrotrypsin was further demonstrated in the cases of Streptomyces subtilisin inhibitor (as a reduced and S-carboxymethylated form, with C-terminal Phe) and alpha 1-antitrypsin (with C-terminal Lys) . By subjecting a tryptic digest of the former protein and a chymotryptic digest of the latter protein to the affinity chromatography, the C-terminal peptides were specifically recovered in the breakthrough fraction and in the adsorbed fraction, respectively . It was further shown that immobilized anhydrotrypsin can also adsorb peptides with C-terminal S-aminoethyl-Cys residues and exerts adsorptive ability even toward the peptides in solution containing urea at a high concentration if appropriate precautions are taken . These findings suggest the general utility of this simple method for C-terminal peptide isolation, which is extremely helpful for studies to confirm amino acid sequences deduced from nucleotide sequences of the cDNA (or genomic DNA) of proteins. Virology, 1987 Dec, 161(2), 561 - 9 Pathogen-derived resistance to viral infection using a negative regulatory molecule; Grumet R et al.; The principle of pathogen-derived resistance (the use of pathogen-derived genes to interfere with the pathogenic process and thereby confer disease resistance to the host) has been put forward as a broadly applicable conceptual tool for use in the genetic engineering of resistance to pathogens and parasites . It was previously predicted that four mechanisms of pathogen-derived resistance could be established using the bacteriophage QB and its host, Escherichia coli, as a model system . This paper demonstrates and helps ellucidate the first of these mechanisms by using a viral regulatory protein, the QB coat protein, to block viral replication . The QB coat protein gene was transferred to susceptible E . coli . Expression of this gene had no obvious detrimental effect on the host . Low-level, constitutive expression of the coat protein conditions very high levels of resistance to QB infection . The resulting resistance is not associated with RNA interference or loss of pili as attachment sites, and does not appear to be associated with premature encapsidation . This low-level expression of the QB coat protein also produces an intermediate level of resistance to the closely related phage SP, but fails to protect against the unrelated phage f2 . Thus the resistance does not result from a generalized antiviral host response induced by the presence of the coat protein . We conclude that the QB coat protein blocks viral infection, as was predicted, due to its action as a negative regulatory molecule . The use of negative regulatory molecules may provide an effective mechanism for use in the genetic engineering of pathogen-derived resistance. J Bacteriol, 1987 Dec, 169(12), 5385 - 92 Increased expression in Escherichia coli of a synthetic gene encoding human somatomedin C after gene duplication and fusion; Schulz MF et al.; A synthetic gene coding for human somatomedin C (SMC) was inserted into an Escherichia coli plasmid vector that contains the bacteriophage lambda pL promoter . Intracellular accumulation of the gene product after induction of the promoter was found to be low . A 200-fold greater yield was obtained with a similar plasmid containing two translationally fused copies of the SMC gene . A series of such tandem genes truncated at their 3' ends were generated with nuclease Bal 31 . These gave intermediate expression levels that correlated with the expected sizes of their gene products . Comparison of RNAs extracted from cells containing either the monomer or tandem SMC gene constructions showed that there was no significant difference in expression at the transcriptional level . Pulse-chase experiments demonstrated that the tandem SMC protein was far more stable than the monomer SMC product. Infect Immun, 1987 Dec, 55(12), 3117 - 25 Role of a 60-megadalton plasmid and Shiga-like toxins in the pathogenesis of infection caused by enterohemorrhagic Escherichia coli O157:H7 in gnotobiotic piglets; Tzipori S et al.; Enterohemorrhagic Escherichia coli (EHEC) of serotype O157:H7 has two putative virulence factors: (i) a fimbrial adhesin, specified by a 60-megadalton (MDa) plasmid, and (ii) bacteriophage-specified cytotoxin(s), known as Shiga-like toxin (SLT) or verotoxin . The contribution of these factors to the pathogenesis of EHEC-induced disease in gnotobiotic piglets was examined . The bacterial strains included the following: two EHEC strains and their corresponding plasmid-cured derivatives; another EHEC isolate and its derivative which had spontaneously lost the ability to produce SLT; one E . coli K-12 transconjugatant containing a 60-MDa plasmid from an EHEC strain; two K-12 strains into which an SLT-producing phage had been transduced (one of these strains also carried a 60-MDa EHEC-derived plasmid); and the parent K-12 strain . Each strain was fed to four piglets, which were observed for diarrhea and examined for development of characteristic mucosal lesions 3 or 5 days after inoculation . All 24 piglets inoculated with the three EHEC strains and their respective derivatives (two plasmid cured and one SLT negative) showed the typical mucosal lesions of bacterial attachment: effacement of microvillous border and cell membrane dissolution culminating in destruction of surface and glandular epithelium in the cecum and colon . No such lesions were observed in 12 piglets inoculated with three strains of E . coli K-12, including the strain which carried both the 60-MDa plasmid and a phage which specified production of SLT . Moderate to severe diarrhea was observed in 16 piglets inoculated with two EHEC strains and their derivatives (one plasmid cured and one SLT negative) . The third EHEC strain and its plasmid-cured derivative produced fewer typical mucosal lesions and no diarrhea . The reason for the reduced virulence of this strain was not clear . These results demonstrate that neither the 60-MDa plasmid nor the capacity to produce SLT is essential for expression of virulence by E . coli O157:H7 in gnotobiotic piglets. J Immunogenet, 1987 Dec, 14(6), 273 - 83 DNA sequence analysis of two bovine immunoglobulin CH gamma pseudogenes; Symons DB et al.; A bovine calf liver DNA library in lambda 2001 bacteriophage has been screened with a human Ig gamma 4 heavy-chain constant-region gene probe . Four hybridizing clones have been identified, and the DNA sequences in two of these, which have high homology with CH gamma genes, are reported here . Within the bovine sequences, four separate exons can be identified, corresponding to the three CH domains and the hinge of gamma heavy-chain genes . Both of these genes contain atypical sequences around one or more of their exon/intron boundaries with consequent loss of splice sites, indicating that these are probably gamma pseudogenes . One sequence codes for a C-terminal peptide which matches the 18-mer C-terminal heavy-chain peptide of bovine serum IgG2, the other encodes a C-terminal peptide unknown in the bovine . These results suggest that evolutionary duplication of CH gamma genes has occurred in the bovine. Infect Immun, 1987 Dec, 55(12), 3023 - 9 Cloning and sequence analysis of cytadhesin P1 gene from Mycoplasma pneumoniae; Su CJ et al.; Mycoplasma pneumoniae cytadhesin P1 was purified by monoclonal antibody affinity chromatography followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The N-terminal 18-amino-acid sequence of P1 was determined and used to design two synthetic oligonucleotides, a 14-mer corresponding to amino acids 1 to 5 and an 18-mer corresponding to amino acids 7 to 12 . These oligonucleotides served as hybridization probes for the identification of the P1 gene by Southern blot analysis of M . pneumoniae DNA . The P1 gene was cloned into plasmid pUC19 and mapped by using appropriate restriction endonucleases . The DNA sequence of the entire P1 gene was determined by subcloning appropriate DNA fragments into bacteriophage M13 and sequencing the DNA by the dideoxy-chain-termination method . The P1 gene contains an open reading frame of 4,881 nucleotides coding for a protein of 1,627 amino acids with a calculated molecular weight of 176,288 . Properties of the amino-terminal sequence suggest that protein P1 may be synthesized as a precursor with subsequent processing to a mature protein of a calculated molecular weight of 169,758 . Potential antigenic sites were determined by hydrophilicity plots . A computer search revealed that part of the predicted P1 sequence is homologous to cytoskeletal keratin of mammalian species and human fibrinogen alpha chain precursor . These results demonstrate the uniqueness of P1 as a cytadhesin and virulence determinant. Genitourin Med, 1987 Dec, 63(6), 355 - 60 Molecular cloning of Treponema pallidum outer envelope fibronectin binding proteins, P1 and P2; Peterson K et al.; Phages directing the synthesis of Treponema pallidum fibronectin binding adhesin proteins, P1 and P2, were isolated from an EMBL-3 bacteriophage lambda library of T pallidum deoxyribonucleic acid (DNA) . The recombinant phages were identified using antisera generated to treponemal proteins purified in fibronectin-Sepharose . Recombinant P1 and P2 proteins possessed the same relative molecular weights as the native surface polypeptides of spirochaetes . The structural genes for these proteins were subcloned into the plasmid vector pUC19, and transformed Escherichia coli expressed and translocated recombinant P1 and P2 to their outer membranes . Finally, the recombinant adhesin proteins, P1 and P2, were purified from detergent solubilised E coli outer membrane preparations using fibronectin-Sepharose affinity chromatography, which confirmed that the fibronectin binding properties of the cloned proteins were retained. Ann Rheum Dis, 1987 Dec, 46(12), 889 - 97 Antibody producing capacity to the bacteriophage phi X174 in rheumatoid arthritis; Bucknall R et al.; A study of antibody production in response to a primary immunogen, the bacteriophage phi X174, was performed in 27 patients with rheumatoid arthritis and 15 controls . All patients produced a primary (IgM) response to initial immunisation . The frequency distribution of peak antibody titres after secondary immunisation showed a marked difference between the patients and controls, with 10 patients having peak titres below 5000 . The IgG component of the antibody response expressed as a percentage of total phage antibody on the 10th day after secondary immunisation was less in the patients than in the control group . There was no correlation between antibody titres and indices of disease activity, rheumatoid factor titres, or the presence of DRw4, DRw3, and DRw2 . After secondary immunisation the patients with rheumatoid arthritis were treated with D-penicillamine, azathioprine, levamisole, or maintained on a non-steroidal anti-inflammatory drug . Assessment of response to tertiary immunisation again showed an impairment of antibody production in the rheumatoid group receiving non-steroidal anti-inflammatory drugs compared with the controls . None of the drugs, D-penicillamine, azathioprine, or levamisole, produced further suppression or augmentation of antibody production in response to the immunogen. Child Dev, 1987 Dec, 58(6), 1420 - 30 Psychological factors capable of preventing the inhibition of antibody responses in separated infant monkeys; Coe CL et al.; The capacity of infant squirrel monkeys to mount an antibody response to viral challenge was evaluated after removal from their mothers in several social and physical environments . Control and separated infants were injected with a benign virus, the bacteriophage X174, and levels of neutralizing antibody were assessed for 3 weeks . Infants separated alone in an unfamiliar environment showed a significant reduction in antibody levels as compared to control infants . Allowing infants to remain in the home environment, either alone or with peers, prevented this inhibition of antibody responses from occurring . Similarly, providing familiar peers in the novel environment facilitated the normal expression of antibody responses . These results indicate that the trauma of maternal separation is significantly reduced when infants are familiar with the separation environment or familiar social companions are available . The reduced antibody response was associated with the highest level of adrenal activation induced by the unfamiliar separation condition, but antibody titers and plasma cortisol levels could not be specifically correlated in individual infants. Virology, 1987 Dec, 161(2), 305 - 14 A novel in vitro DNA packaging system demonstrating a direct role for the bacteriophage lambda FI gene product; Davidson A et al.; A new in vitro bacteriophage lambda DNA packaging system is described in which all the proteins necessary for head morphogenesis are supplied by extracts of plasmid-transformed cells . This assay is used to demonstrate that the lambda FI gene product (gpFI) is necessary for maximal packaging efficiency when proheads and terminase are present in limiting amounts . A 100- to 200-fold decrease in packaging is seen when gpFI is omitted . gpFI is shown to act at and/or after the stage in packaging where proheads bind to the DNA:terminase complex. J Virol, 1987 Dec, 61(12), 3790 - 4 Wild-type bacteriophage T4 is restricted by the lambda rex genes; Shinedling S et al.; The bacteriophage T4 rII genes and the lambda rex (r exclusion) genes interact; rII mutants are unable to productively infect rex+ lambda lysogens . The relationship between rex and rII has been found to be quantitative, and plasmid clones of rex have excluded not only rII mutants but T4 wild type and most other bacteriophages as well . Mutations in the T4 motA gene substantially reversed exclusion of T4 by rex. J Bacteriol, 1987 Dec, 169(12), 5884 - 6 An open reading frame in the Escherichia coli bacteriophage lambda genome encodes a protein that functions in assembly of the long tail fibers of bacteriophage T4; Montag D et al.; Assembly of the long tail fibers of the Escherichia coli bacteriophage T4 requires the catalytic action of two auxiliary proteins . It was found that a gene of the entirely unrelated phage lambda codes for a protein which can substitute for one of these T4 polypeptides, protein 38 . The lambda gene was designated tfa (tail fiber assembly) . Protein 38 consists of 183 residues, and the Tfa protein consists of 194 residues; the two polypeptides are about 40% homologous . Although the tfa gene is dispensable for the growth of phage lambda, these results indicate that it may have a function in lambda morphogenesis. J Bacteriol, 1987 Dec, 169(12), 5504 - 9 Involvement of heat shock proteins in bacteriophage Mu development; Pato M et al.; Growth of bacteriophage Mu was severely inhibited at elevated temperature in mutants defective in the heat shock genes dnaK, groEL, and groES and in the rpoH (htpR) regulatory mutant, but not in mutants defective in the heat shock genes dnaJ or grpE; growth of a mutant of Mu deficient in functions encoded in the accessory region of the Mu genome was inhibited in the latter two host mutants . Phage production in the dnaJ mutant was restored by growth in low-salt medium . The stage in Mu development primarily affected in all except the groE mutants was phage late transcription . In contrast, the groE mutants did not support growth of Mu at any temperature; neither Mu DNA replication nor transcription was inhibited in these strains, suggesting that groE is required for phage morphogenesis as observed with several other coliphages. Appl Environ Microbiol, 1987 Dec, 53(12), 2708 - 13 Isolation and characterization of a temperate bacteriophage from Streptomyces galilaeus; Kuhn SP et al.; A new temperate actinophage from Streptomyces galilaeus ATCC 31133 was purified after that strain was crossed with S . peucetius ATCC 29050 . Sensitive hosts became lysogenized and yielded turbid plaques of 2 to 3 mm in diameter . Host-range analysis indicated that 16 of 27 Streptomyces strains tested were sensitive to infection on solid medium . S . lividans and S . coelicolor A3(2) were among those not infected by this new actinophage . The new actinophage, designated phi SPK1, belongs to the Bradley group B morphological type, the pH optimum for infection is 6.75 to 7.0, it is not efficiently induced by mitomycin C or UV irradiation, it has a circular chromosome of 35.8 +/- 0.5 kilobase pairs in length containing overlapping (cohesive) ends, and the G+C content of its DNA was calculated from the buoyant density of 1.7240 to be 69 mol% . The DNA of phage phi SPK1 was cleaved by the restriction endonucleases ApaI, AluII, EcoRI, PvuII, and SalI, but, in all cases except that with EcoRI, treatment yielded greater than 20 restriction fragments . No sites were detected for BamHI, BclI, BglII, ClaI, HindIII, MluI, PstI, SmaI, SphI, SstI, XbaI, or XhoI. Biochemistry, 1987 Dec 1, 26(24), 7571 - 4 Spin-label electron spin resonance study of bacteriophage M13 coat protein incorporation into mixed lipid bilayers; Datema KP et al.; The major coat protein of bacteriophage M13 was incorporated in mixed dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (80/20 w/w) vesicles probed with different spin-labeled phospholipids, labeled on the C-14 atom of the sn-2 chain . The specificity for a series of phospholipids was determined from a motionally restricted component seen in the electron spin resonance (ESR) spectra of vesicles with the coat protein incorporated . At 30 degrees C and pH 8, the fraction of motionally restricted phosphatidic acid spin-label is 0.36, 0.52, and 0.72 for lipid/protein ratios of 18, 14, and 9 mol/mol, respectively . The ESR spectra, analyzed by digital subtraction, resulted in a phospholipid preference following the pattern cardiolipin = phosphatidic acid greater than stearic acid = phosphatidylserine = phosphatidylglycerol greater than phosphatidylcholine = phosphatidylethanolamine . The specificities found are related to the composition of the target Escherichia coli cytoplasmic membrane. J Virol, 1987 Dec, 61(12), 3694 - 700 Mapping the B and D gene promoters of bacteriophage S13 by footprinting and exonuclease III analysis; Arquint M et al.; The region of the bacteriophage S13 genome which contains the B, K, and C genes and most of the A, D, and E genes (map positions 627 to 2198) was analyzed for Escherichia coli RNA polymerase-binding sites by combined DNase I footprinting, exonuclease III analysis, and DNA sequencing . Two high-affinity binding sites that correspond to the B and D gene promoters were mapped at positions 936 to 995 and 1779 to 1848, respectively . Positions 936 to 995 are in gene A, preceding the B gene, and positions 1779 to 1848 are in gene C, just preceding the D gene . In addition, two lower-affinity binding sites were identified at positions 1527 to 1538 and 1705 to 1756 preceding the C and D genes, respectively . The footprinting studies described here, in combination with previous studies on transcription, provide definitive evidence on the position of the B and D gene promoters in S13 and the closely related phage phi X174. J Bacteriol, 1987 Dec, 169(12), 5700 - 7 In vivo mutagenesis of bacteriophage Mu transposase; Toussaint A et al.; We devised a method for isolating mutations in the bacteriophage Mu A gene which encodes the phage transposase . Nine new conditional defective A mutations were isolated . These, as well as eight previously isolated mutations, were mapped with a set of defined deletions which divided the gene into 13 100- to 200-base-pair segments . Phages carrying these mutations were analyzed for their ability to lysogenize and to transpose in nonpermissive hosts . One Aam mutation, Aam7110, known to retain the capacity to support lysogenization of a sup0 host (M . M . Howe, K . J . O'Day, and D . W . Shultz, Virology 93:303-319, 1979) and to map 91 base pairs from the 3' end of the gene (R . M . Harshey and S . D . Cuneo, J . Genet . 65:159-174, 1987) was shown to be able to complement other A mutations for lysogenization, although it was incapable of catalyzing either the replication of Mu DNA or the massive conservative integration required for phage growth . Four Ats mutations which map at different positions in the gene were able to catalyze lysogenization but not phage growth at the nonpermissive temperature . Phages carrying mutations located at different positions in the Mu B gene (which encodes a product necessary for efficient integration and lytic replication) were all able to lysogenize at the same frequency . These results suggest that the ability of Mu to lysogenize is not strictly correlated with its ability to perform massive conservative and replicative transposition. J Bacteriol, 1987 Dec, 169(12), 5556 - 62 Transposition of Tn1000: in vivo properties; Tsai MM et al.; Transposition mediated by the Tn1000 transposase was investigated by using transposon variants carrying synthetic or wild-type termini but no intact Tn1000 genes . Transposon Tn1001, whose only homologies to Tn1000 are in its 38-base-pair terminal inverted repeats, transposed at the same rate as Tn1005, an artificial construct carrying wild-type Tn1000 termini and approximately 1 kilobase of flanking Tn1000 DNA at each end, when transposase was supplied in trans . The majority of the transpositions into pOX38 gave rise to cointegrates, but approximately 10% of the products expressed phenotypes of direct transpositions . The expression and temperature dependence of the tnpA gene product were examined by studying transposition of Tn1001 to bacteriophage lambda . The temperature optimum for transposition was 37 degrees C, and the transposase was stable for up to 2 h at this temperature. J Bacteriol, 1987 Dec, 169(12), 5523 - 9 Processing of Escherichia coli 16S rRNA with bacteriophage lambda leader sequences; Krych M et al.; To test whether any specific 5' precursor sequences are required for the processing of pre-16S rRNA, constructs were studied in which large parts of the 5' leader sequence were replaced by the coliphage lambda pL promoter and adjacent sequences . Unexpectedly, few full-length transcripts of the rRNA were detected after the pL promoter was induced, implying that either transcription was poor or most of the rRNA chains with lambda leader sequences were unstable . Nevertheless, sufficient transcription occurred to permit the detection of processing by S1 nuclease analysis . RNA transcripts in which 2/3 of the normal rRNA leader was deleted (from the promoter up to the normal RNase III cleavage site) were processed to form the normal 5' terminus . Thus, most of the double-stranded stem that forms from sequences bracketing wild-type 16S pre-rRNA is apparently not required for proper processing; the expression of such modified transcripts, however, must be increased before the efficiency of processing of the 16S rRNA formed can be assessed. J Biol Chem, 1987 Nov 25, 262(33), 16224 - 32 Escherichia coli thioredoxin stabilizes complexes of bacteriophage T7 DNA polymerase and primed templates; Huber HE et al.; The DNA polymerase activity induced after bacteriophage T7 infection of Escherichia coli is found in a complex of two proteins, the T7 gene 5 protein and a host protein, thioredoxin . Gene 5 protein is a DNA polymerase and a 3' to 5' exonuclease . Thioredoxin binds tightly to the gene 5 protein and increases the processivity of polymerization some 1000-fold . Gene 5 protein forms a short-lived complex with the primer-template, poly(dA).oligo(dT), in the absence of Mg2+ and nucleotides . Thioredoxin increases the half-life of the preformed primer-template-polymerase complex from less than a second to approximately 5 min . The dissociation is accelerated by excess single-stranded DNA in an apparent second order reaction, indicating direct transfer of polymerase between DNA fragments . Thioredoxin also reduces the equilibrium dissociation constant, Kd, of the gene 5 protein -poly(dA).oligo(dT) complex 20- to 80-fold . The salt dependence of Kd indicates that thioredoxin stabilizes the primer-template-polymerase complex mainly through additional charge-charge interactions, increasing the estimated number of interactions from 2 to 7 . The affinity of gene 5 protein for single-stranded DNA is at least 1000-fold higher than for double-stranded DNA and is little affected by thioredoxin . Under conditions of steady state synthesis the effect of thioredoxin on the polymerization rate is determined by two competing factors, an increase in processivity and a decrease of the dissociation rate of polymerase and replicated template. J Biol Chem, 1987 Nov 25, 262(33), 16212 - 23 Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7; Tabor S et al.; Bacteriophage T7 gene 5 protein has been purified to apparent homogeneity from cells overexpressing its gene several hundred-fold . Gene 5 protein is a DNA polymerase with low processivity; it dissociates from the primer-template after catalyzing the incorporation of 1-50 nucleotides, depending on the salt concentration . Escherichia coli thioredoxin, a host protein that is tightly associated with the gene 5 protein in phage-infected cells, is not required for this activity . Thioredoxin acts as an accessory protein to bestow processivity on the polymerizing reaction; DNA synthesis catalyzed by the gene 5 protein-thioredoxin complex on a single-stranded DNA template can polymerize thousands of nucleotides without dissociation . Conditions that increase the stability of secondary structures in the template (i.e., low temperature or high ionic strength) decrease the processivity . E . coli single-stranded DNA-binding protein stimulates both the rate of elongation and the processivity of the gene 5 protein-thioredoxin complex. J Biol Chem, 1987 Nov 25, 262(33), 15869 - 74 Purification and characterization of the repressor for the sn-glycerol 3-phosphate regulon of Escherichia coli K12; Larson TJ et al.; The glpR gene encoding the repressor for the sn-glycerol 3-phosphate regulon of Escherichia coli was cloned downstream from the strong pL promoter of bacteriophage lambda . This allowed overproduction of the repressor upon thermal induction of a cryptic lambda lysogen harboring the cI857 gene . The repressor was purified 40-fold to homogeneity from an induced strain . The purification scheme utilized polyethyleneimine and ammonium sulfate fractionation, followed by phosphocellulose and DEAE-Sephadex chromatography . Purification was monitored by measuring the binding of radiolabeled inducer (sn-glycerol 3-phosphate) to the repressor . The purified repressor migrated as a single band exhibiting a subunit molecular weight of 30,000 assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The molecular weight of the repressor under nondenaturing conditions was 100,000-130,000 suggesting the repressor is a tetramer under native conditions . Interaction of the repressor with sn-glycerol 3-phosphate was studied using flow dialysis . Scatchard analysis of the data indicated four binding sites/repressor tetramer and a dissociation constant of 31 microM . Interaction of the repressor with DNA was studied using band-shift electrophoresis . The repressor specifically bound DNA fragments containing the control regions for the glpD, glpK, and glpT-A genes . Binding of DNA by the repressor was diminished in the presence of sn-glycerol 3-phosphate. J Mol Biol, 1987 Nov 20, 198(2), 281 - 93 Characterization of the binding sites of c1 repressor of bacteriophage P1 . Evidence for multiple asymmetric sites; Eliason JL et al.; The repressor of bacteriophage P1, encoded by the c1 gene, is responsible for maintaining a P1 prophage in the lysogenic state . In this paper we present: (1) the sequence of the rightmost 943 base-pairs of the P1 genetic map that includes the 5'-terminal 224 base-pairs of the c1 gene plus its upstream region; (2) the construction of a plasmid that directs the production of approximately 5% of the cell's protein as P1 repressor; (3) a deletion analysis that establishes the startpoint of P1 repressor translation; (4) filter binding experiments that demonstrate that P1 repressor binds to several regions upstream from the c1 gene; (5) DNase I footprint experiments that directly identify two of the P1 repressor binding sites . Sequences very similar to the identified binding sites occur in at least 11 sites in P1, in most cases near functions known, or likely, to be controlled by repressor . From these sites we have derived the consensus binding site sequence ATTGCTCTAATAAATTT . We suggest that, unlike other phage operators, the P1 repressor binding sites lack rotational symmetry. Cell, 1987 Nov 20, 51(4), 631 - 41 An elongation control particle containing the N gene transcriptional antitermination protein of bacteriophage lambda; Horwitz RJ et al.; The N gene transcriptional antitermination protein of bacteriophage lambda is incorporated in vitro into transcriptional elongation complexes containing the E . coli proteins NusA and NusB . The binding of NusA to elongating RNA polymerase is sequence-independent and follows the release of sigma 70 . Incorporation of N into the elongation complex requires an N utilization site (nut site) on the DNA template . Incorporation of NusB into the complex requires NusA, ribosomal protein S10, and the boxA component of the nut site . T1 RNAase releases N, but not NusB, from the elongation complex . We therefore propose that an N-modified termination-resistant elongation complex includes an elongation control particle (ECP) containing at least NusA, NusB, S10, N, and an RNA transcript of the nut site. Carbohydr Res, 1987 Nov 15, 169, 213 - 20 Structure of Escherichia coli capsular antigen K34; Dutton GG et al.; The structure of the heat-stable K antigen of Escherichia coli serotype K34 has been determined by n.m.r . spectroscopy, methylation analysis, and Smith degradation to be (formula; see text) The assignment of the single alpha-linkage to the D-glucosyl residue was confirmed by the positive reaction of an alpha-D-glucosidase on the oligosaccharide obtained by bacteriophage depolymerization . It is noteworthy that the same enzyme was without action on the native polysaccharide. J Biol Chem, 1987 Nov 15, 262(32), 15796 - 802 Functional activity and regulation of human beta 2-adrenergic receptors expressed in Xenopus oocytes; Kobilka BK et al.; The recently cloned human beta-adrenergic cDNA and several mutated forms have been expressed in Xenopus laevis oocytes by injection of RNA made from the cDNA under the control of the bacteriophage SP6 promoter . The cDNA and gene of the beta 2-adrenergic receptor possess the unusual feature of having a second upstream ATG (-101 base pairs) and a 19-codon open reading frame 5' to the initiator methionine codon of the receptor (Kobilka, B . K., Dixon, R . A . F., Frielle, T., Dohlman, H . G., Bolanowski, M., Sigal, I . S., Yang-Feng, T . L., Francke, U., Caron, M . G., and Lefkowitz, R . J . (1987) Proc . Natl . Acad . Sci . U.S.A . 84, 46-50) . RNA lacking this upstream AUG and open reading frame was translated approximately 10-fold more efficiently both in an in vitro rabbit reticulocyte system and in oocytes . Injected oocytes but not water injected controls expressed typical beta 2-adrenergic receptors as assessed by ligand binding (450 fmol/mg membrane protein) and catecholamine-stimulated adenylate cyclase (approximately 20 fold) . Moreover, these receptors displayed typical agonist-induced homologous desensitization when oocytes were incubated with isoproterenol at room temperature for 3-24 h . Among a series of mutations, truncations of the membrane-anchored core of the receptor eliminated receptor binding and cyclase stimulating activity . In contrast, disruption of one of the cAMP-dependent protein kinase phosphorylation sites or removal of the serine/threonine-rich carboxyl terminus had little or no effect on these functions or on the extent of agonist-induced desensitization relative to that observed with native receptor . These studies validate the beta 2-adrenergic nature of the cloned human beta-adrenergic cDNA, document the utility of the Xenopus oocyte system for studying functional and regulatory properties of receptors coupled to adenylate cyclase, and suggest the possibility that elements in the 5' untranslated region of the beta 2-adrenergic receptor RNA may regulate its translation in vivo. J Biol Chem, 1987 Nov 15, 262(32), 15330 - 3 Selective oxidation of the exonuclease domain of bacteriophage T7 DNA polymerase; Tabor S et al.; Bacteriophage T7 DNA polymerase, the product of gene 5 of the phage, has both polymerase and single-and double-stranded DNA 3'-to 5'-exonuclease activities . The exonuclease activities can be inactivated selectively by an oxidation reaction that requires molecular oxygen, a reducing agent, and iron at a concentration less than or equimolar to that of the gene 5 protein . Both exonuclease activities can be diminished by several thousandfold, with only a small decline in the polymerase activity . Escherichia coli thioredoxin, an accessory protein that binds tightly to the gene 5 protein and increases the processivity of the polymerization reaction, has no effect on the rate of oxidation . We propose that iron binds specifically to the exonuclease domain and, in the presence of molecular oxygen and a reducing agent, generates reactive oxygen species that selectively modify amino acid residues essential for the exonuclease activities. Nucleic Acids Res, 1987 Nov 11, 15(21), 8999 - 9009 Primary structure of the DNA terminal protein of bacteriophage PRD1; Hsieh JC et al.; The genome of a lipid-containing phage, PRD1, is replicated by a protein-priming mechanism . We have determined the nucleotide sequence of the PRD1 gene 8 which specifies the terminal protein, the protein primer for DNA synthesis . The coding region is 780 base pairs long and encodes for 259 amino acids (29,326 daltons) . The predicted amino acid sequence of the PRD1 terminal protein reveals no substantial homology with that of any known terminal protein . However, hydropathy profiles of the PRD1, phi 29, and Nf terminal proteins are remarkably similar, suggesting a common evolutionary origin . A particular tyrosine residue is predicted to be covalently linked to the 5' end of the PRD1 DNA . The initiation codon ATG of gene 8 is preceded by the identifiable ribosome binding site, and putative promoter sequences . There are unique palindromic sequences between the ribosome binding site and "-10" region. Nucleic Acids Res, 1987 Nov 11, 15(21), 8831 - 44 Interactions of the transposase with the ends of Mu: formation of specific nucleoprotein structures and non-cooperative binding of the transposase to its binding sites; Groenen MA et al.; Transposition of the E . coli bacteriophage Mu requires the phage encoded A and B proteins, the host protein HU and the host replication proteins . The ends of the genome of the phage, on which some of these proteins act, both contain three transposase (A) binding sites . The organization of these binding sites on each end, however, is different . Here we show, using DNase footprinting experiments with purified A protein, that mutant A binding sites, which affect transposition, have decreased affinity for the transposase . Furthermore the transposase binds non-cooperatively to all A binding sites both in the left and right end of Mu . Electron microscopic studies show that the A protein forms specific nucleoprotein structures upon binding to the ends of Mu . The A and B proteins interact with the ends of Mu to generate larger structures than with the A protein alone. J Mol Biol, 1987 Nov 5, 198(1), 13 - 20 Molecular cloning and sequence of the gene for p9Ka . A cultured myoepithelial cell protein with strong homology to S-100, a calcium-binding protein; Barraclough R et al.; The gene for p9Ka, a protein of molecular weight 9000 that is expressed in cultured rat mammary myoepithelial cells, has been isolated from a normal rat genomic library in bacteriophage lambda, by its ability to hybridize to a cloned complementary DNA corresponding to p9Ka mRNA . The cloned rat genomic DNA fragment hybridized to translatable p9Ka mRNA . A nucleotide sequence of 2340 base-pairs of genomic DNA surrounding the p9Ka cDNA sequence has been obtained; the gene contains one intervening sequence of 675 nucleotides . The 3' end of the p9Ka mRNA has been identified on the gene sequence to be 13 nucleotides downstream from a poly(A) addition signal AATAAA . The gene contains an open reading frame of 101 amino acid residues, which is the only open reading frame in the entire gene long enough to encode a protein of molecular weight at least 9000 . This hypothetical protein sequence shows greater than 40% homology to rat or bovine S-100 protein and over 30% homology to bovine intestinal calcium-binding protein . The results suggest that p9Ka may be related to a family of low molecular weight calcium-binding proteins. J Mol Biol, 1987 Nov 5, 198(1), 43 - 9 Tau factor from Escherichia coli mediates accurate and efficient termination of transcription at the bacteriophage T3 early termination site in vitro; Briat JF et al.; The termination signal that limits transcription through the early region of bacteriophage T3 (T3Te) has been cloned and sequenced . The nucleotide sequence of T3Te is identical with that of T7Te, with the exception of a single G to U substitution in the 3' tail of the terminated transcript, and addition of an AC to the loop in the terminator stem-loop, enlarging the loop to six residues . Previous studies of the properties of T3Te have shown that this site is rho independent and is highly efficient for termination in vivo, but is used poorly in vitro during transcription with purified Escherichia coli RNA polymerase . In contrast, the equivalent site in bacteriophage T7 (T7Te) is an efficient termination signal both in vivo and in vitro . However, T3Te becomes an efficient termination site in vitro in the presence of preparations of tau factor . This factor also alters the sites of RNA chain termination found in vitro at T3Te . Transcripts formed in the presence of tau are several nucleotides shorter than those produced with RNA polymerase alone, and have 3' termini that are almost identical with transcripts found in vivo . These latter results are similar to our earlier findings with T7Te, and suggest that other rho independent terminators may act with transcription termination factors in vivo. J Immunol Methods, 1987 Nov 5, 103(2), 211 - 20 The production of recombinant HLA-DR beta and invariant chain polypeptides by cDNA expression in E . coli; Koch S et al.; In this report we describe the production of recombinant fusion proteins of the HLA-DRw6 beta chain and the murine Ia-associated invariant chain . cDNAs encoding the human HLA-DRw6 beta chain and the murine Ia-associated invariant chain were introduced into bacterial expression plasmids . These plasmids direct the synthesis of the respective molecules as fusion proteins of the bacteriophage MS-2 polymerase by E . coli . Fusion proteins purified from crude E . coli lysates were used to raise antisera in rabbits . These antisera were able to immunoprecipitate biosynthetically labelled class II and invariant chain antigens . Additionally, two anti-DR antisera were raised against single domains of the HLA-DR beta chain thus generating reagents with a defined fine specificity . The anti-murine invariant chain serum was shown to cross-react with the human invariant chain and therefore may be useful for studying invariant chain and Ia antigen expression in different species . The method described here permitted us to produce large quantities of immunologically relevant proteins, for use in the production of polyclonal and monoclonal antibodies . Soluble fragments of the fusion proteins representing certain DR domains may also be useful in functional immunological studies. J Mol Biol, 1987 Nov 5, 198(1), 63 - 71 Studies on the arrangement of DNA inside viruses using a breakable bis-psoralen crosslinker; Welsh J et al.; We have developed a new DNA-DNA crosslinking strategy based on a cleavable bispsoralen reagent and used this strategy to study the structures of bacteriophage lambda and the animal virus SV40 . Our results show that in both lambda and SV40, all restriction fragments examined can be crosslinked to all other restriction fragments . In bacteriophage lambda, the crosslinking data are consistent with a random packaging model, while in SV40 there is some deviation from the random model . These results imply that the structures of DNA inside these viruses are either highly disordered or very complex. J Mol Biol, 1987 Nov 5, 198(1), 51 - 61 Mutational analysis of the bacteriophage phi X174 replication origin; Baas PD; Bacteriophage phi X174 mutants within the 30 base-pair replication origin were constructed using oligodeoxynucleotide-directed mutagenesis . A total of 18 viable base substitution mutants at 13 different positions within the origin region were obtained . The majority of these ori mutants have a plaque morphology and burst size comparable to that of wild-type phi X174 . Two phi X174 ori mutants with a reduced growth ability spontaneously acquired additional mutations that enhanced the growth rate . The additional mutation was located at the same site as the original mutation or was located in the N-terminal part of the gene A protein . This latter secondary mutation is responsible for a better binding and/or recognition of the gene A protein to the mutated origin . In a Darwinian experiment wild-type phi X174 outgrows all phi X174 ori mutants, indicating the superiority of the wild-type ori sequence for the reproduction of bacteriophage phi 174 . Insertions and deletions were constructed at different positions within the phi X174 replication origin cloned in a plasmid . Small insertions and deletions in the A + T-rich spacer region do not inhibit phi X174 gene A protein cleavage in vitro, but severely impair packaging of single-stranded plasmid DNA in viral coats. Eur J Biochem, 1987 Nov 2, 168(3), 621 - 7 Effect of bacteriophage M13 infection on phosphorylation of dnaK protein and other Escherichia coli proteins; Rieul C et al.; 1 . The effects of infection with the filamentous phage M13 on the phosphorylation of Escherichia coli proteins were studied . Phosphorylated proteins were labeled with {32P}orthophosphate and analyzed by the O'Farrell two-dimensional gel technique and autoradiography . 2 . Phage infection was shown to induce significant changes in the pattern of protein phosphorylation . At least eight different proteins were found to be phosphorylated to a larger extent while seven others were, by contrast, much less labeled than in uninfected bacteria . 3 . Labeling experiments with {35S}methionine demonstrated that these quantitative changes in protein phosphorylation were not connected, in any case, with changes in the amount of protein synthesized . They rather seemed to result from a variation of the phosphorylating capacity of the relevant protein kinase(s) . 4 . The individual proteins, whose phosphorylation was affected by phage infection, were characterized by both their molecular mass and isoelectric point . One of them, whose phosphorylation was increased by a factor of 7, was identified as the dnaK protein which is necessary for both cellular and phage DNA replication . 5 . The chemical analysis of the phosphorylated moiety of dnaK protein showed that it was modified exclusively at serine residues during normal growth of cells, and mostly at threonine residues after phage infection . These results were discussed in terms of stimulation of the protein activity by phosphorylation. Virology, 1987 Nov, 161(1), 228 - 33 On the molecular mechanism of DNA translocation during in vitro packaging of bacteriophage T3 DNA; Fujisawa H et al.; The process of packaging of bacteriophage T3 DNA in a defined in vitro system can be separated into two stages: formation of a precursor complex (50 S complex) in the presence of adenosine-5'-O-(3'-thiotriphosphate) (ATP-gamma-S) and subsequent translocation of DNA into the head by the addition of ATP . Packaged DNA exits when DNA translocation is interrupted by the addition of ATP-gamma-S (M . Shibata, H . Fujisawa, and T . Minagawa, 1987, Virology, in press; M . Shibata, H . Fujisawa, and T . Minagawa, 1987, J . Mol . Biol., in press) . The in vitro system packaged nicked and cross-linked DNAs but did not package single-stranded DNA . DNA packaging was inhibited by intercalating reagents such as ethidium bromide, acridine orange, and 4',6-diamino-2-phenylindole dihydrochloride . The inhibitory effect was proportional to the ability of intercalating agents to unwind DNA . Ethidium bromide did not inhibit the formation of 50 S complex but blocked translocation of DNA into and out of the capsid . DNA packaging was inhibited by actinomycin D and distamycin A which bind to the minor groove of the DNA helix . From these results, we conclude that DNA packaging mechanism utilizes the exterior structure of duplex DNA for translocating the DNA into the capsid. J Clin Periodontol, 1987 Nov, 14(10), 605 - 9 The presence of phage-infected Actinobacillus actinomycetemcomitans in localized juvenile periodontitis patients; Preus HR et al.; Electron microscopy revealed 2 different types of bacteriophages isolated from Actinobacillus actinomycetemcomitans colonizing exclusively diseased sites in 4 patients with localized juvenile periodontitis (LJP) . All sites infected with phage were undergoing periodontal destruction, as judged from consecutive routine radiographs . The phages isolated had a wide host range as assessed from their ability to infect a series of reference strains of A . actinomycetemcomitans . A 5th patient harboured non-infected A . actinomycetemcomitans in a surgically treated site which had undergone no bone destruction during the last 12 months . The present findings suggested that the pathogenic potential of A . actinomycetemcomitans in LJP may increase due to phage infection. Proc Natl Acad Sci U S A, 1987 Nov, 84(21), 7547 - 51 Liver fatty acid binding protein is the mitosis-associated polypeptide target of a carcinogen in rat hepatocytes; Bassuk JA et al.; Hepatocytes in normal rat liver were found previously to contain a cytoplasmic 14,000-dalton polypeptide (p14) that is associated with mitosis and is the principal early covalent target of activated metabolites of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene) . The level of immunohistochemically detected p14 was low when growth activity of hepatocytes was low, was markedly elevated during mitosis in normal and regenerating livers, but was very high throughout interphase during proliferation of hyperplastic and malignant hepatocytes induced in rat liver by a carcinogen (N-2-fluorenylacetamide or 3'-methyl-4-dimethylaminoazobenzene) . We report here that p14 is the liver fatty acid binding protein . The nucleotide sequence of p14 cDNA clones, isolated by screening a rat liver cDNA library in bacteriophage lambda gt11 using p14 antiserum, was completely identical to part of the sequence reported for liver fatty acid binding protein . Furthermore, the two proteins shared the following properties: size of mRNA, amino acid composition, molecular size according to NaDodSO4 gel electrophoresis, and electrophoretic mobilities in a Triton X-100/acetic acid/urea gel . Their pI values overlapped in 2-dimensional isoelectric focusing/NaDodSO4 gel electrophoresis and showed the same response to delipidation . Either polypeptide reacted with and blocked the antiserum raised against the other polypeptide . The two polypeptides bound oleic acid similarly . Finally, identical elevations of cytoplasmic immunostain were detected specifically in mitotic hepatocytes with either antiserum . The collected findings are suggestive that liver fatty acid binding protein may carry ligands that promote hepatocyte division and may transport certain activated chemical carcinogens. J Gen Microbiol, 1987 Nov, 133 ( Pt 11), 3065 - 70 Characterization of Bdellovibrio bacteriovorus bacteriophage MAC-1; Roberts RC et al.; The bacteriophage MAC-1, which specifically infects Bdellovibrio bacteriovorus, was plaque purified and raised to high titre . The phage was purified by NaCl/polyethylene glycol precipitation, followed by two cycles of isopycnic density gradient centrifugation in CsCl . The purified phage exhibited a density of 1.363 g cm-3 and a sedimentation coefficient of 94S . Nucleic acid isolated from purified phage was resistant to hydrolysis under alkaline conditions and to digestion with RNAase, but it was hydrolysed by DNAase, providing evidence that the phage genome is made up of DNA . The lack of hyperchromic effect upon denaturation, hydrolysis of phage DNA by S1 nuclease, characteristic fluorescent staining with acridine orange, and resistance to digestion with a variety of restriction endonucleases are consistent with the DNA being single-stranded . A buoyant density of 1.722 g cm-3 and a sedimentation coefficient of 17.9S were obtained for the phage DNA . The molecular mass of phage DNA was determined as 1.58 MDa by agarose gel electrophoresis with single-stranded DNA as standards . Electron microscopy of the DNA showed that the genome is circular in nature . In addition, using Southern blots, the two replicative forms, RF1 (supercoiled) and RF2 (circular) have been identified and isolated from infected cell extracts. Mol Cell Biol, 1987 Nov, 7(11), 4065 - 74 Human acidic ribosomal phosphoproteins P0, P1, and P2: analysis of cDNA clones, in vitro synthesis, and assembly; Rich BE et al.; cDNA clones encoding three antigenically related human ribosomal phosphoproteins (P-proteins) P0, P1, and P2 were isolated and sequenced . P1 and P2 are analogous to Escherichia coli ribosomal protein L7/L12, and P0 is likely to be an analog of L10 . The three proteins have a nearly identical carboxy-terminal 17-amino-acid sequence (KEESEESD(D/E)DMGFGLFD-COOH) that is the basis of their immunological cross-reactivity . The identities of the P1 and P2 cDNAs were confirmed by the strong similarities of their encoded amino acid sequences to published primary structures of the homologous rat, brine shrimp, and Saccharomyces cerevisiae proteins . The P0 cDNA was initially identified by translation of hybrid-selected mRNA and immunoprecipitation of the products . To demonstrate that the coding sequences are full length, the P0, P1, and P2 cDNAs were transcribed in vitro by bacteriophage T7 RNA polymerase and the resulting mRNAs were translated in vitro . The synthetic P0, P1, and P2 proteins were serologically and electrophoretically identical to P-proteins extracted from HeLa cells . These synthetic P-proteins were incorporated into 60S but not 40S ribosomes and also assembled into a complex similar to that described for E . coli L7/L12 and L10. Proc Natl Acad Sci U S A, 1987 Nov, 84(22), 7822 - 6 Bacteriophage T4 regA protein binds to mRNAs and prevents translation initiation; Winter RB et al.; The bacteriophage T4 regA protein is a translational repressor of a subset of phage mRNAs . We show here that purified regA protein binds specifically to target mRNAs near the initiating AUG and occludes binding of ribosomes . Translational repression by regA protein diminishes expression of many genes whose mRNA sequences around the initiating AUG codons are different . A comparison of nucleotide sequences from several regA-repressed mRNAs suggests that the initiating AUG is an important, but not sufficient, sequence for regA binding. Bioorg Khim, 1987 Nov, 13(11), 1561 - 9 {Construction and properties of the expression vector based on the temperature-regulated P'R promoter in phage lambda DNA}; Petrenko LA et al.; Plasmid expression vector using the temperature-regulated promoter P'R of bacteriophage lambda is described . The vector carries a combination of two regions of lambda cI857indgenome, that contain: 1) gene cI and promoter PR, and 2) gene Q and promoter P'R . Transcription or gene Q is initiated at promoter PR, which is controlled by cI857 repressor . The Q gene product acts as a positive regulator of RNA synthesis from P'R . At 37 degrees C, sufficient amounts of protein Q are synthesised to initiate the expression of the cloned gene from P'R . Inactivation of Q gene (by elimination of a single NcoI site) results in the loss of P'R expression activity in the vector . E . coli beta-galactosidase gene (lacZ) and human leukocyte interferon alpha 2 gene (ifn alpha 2) were cloned into a single EcoRI cleavage site under the control of P'R . These constructs express high levels of beta-galactosidase and interferon alpha 2 in E . coli at 37 degrees C. J Biochem (Tokyo), 1987 Nov, 102(5), 1075 - 82 Nucleotide sequence of a human liver cytochrome P-450 related to the rat male specific form; Yasumori T et al.; P-450 human-2 is a human cytochrome P-450 that is immunochemically related to a constitutive male-specific cytochrome P-450 (P-450-male) and the phenobarbital-inducible P-450b/e in rat liver . By screening a human liver cDNA library in bacteriophage lambda gt11, we isolated a clone with an insert length of 1,847 bases (pHY13) . The clone was sequenced and shown to code for a protein of 487 amino acids . The N-terminal 11-amino-acid sequence was in agreement with the protein sequence of P-450 human-2 . The nucleotide sequence of pHY13 showed less than 50% similarity with those of human cytochrome P-450s, pHP-450(1), HLp, P-450NF, P1-450 4, and P3(450), but the nucleotide sequence of pHY13 is 80% similar to the reported sequence of rat cytochrome P-450, P-450(M-1) . In addition, the coding sequence of pHY13 showed close similarity to that of MP-8, which was recently reported as the sequence corresponding to human cytochrome P-450MP, although no apparent similarity was observed in their 3' non-coding sequences except for the first 75 bases and the expected length of the complete sequences . These results, together with the immunochemical data, indicate that P-450 human-2 is closely related, but not identical, to P-450MP, and may belong to the category of developmentally regulated constitutive cytochrome P-450s. Genes Dev, 1987 Nov, 1(9), 1005 - 13 OOP RNA, produced from multicopy plasmids, inhibits lambda cII gene expression through an RNase III-dependent mechanism; Krinke L et al.; OOP RNA is a major short (77 bases) transcript that is made from bacteriophage lambda DNA both in vivo and in vitro . OOP RNA is synthesized in the opposite direction to mRNA for the lambda cII gene, and the final 55 bp of the OOP region overlaps the 3' end of the cII gene . We find that a multicopy plasmid containing an OOP DNA fragment inhibits cII expression from a derepressed prophage by approximately 100-fold, using an in vivo assay in which cII protein activates galactokinase synthesis from a cII-dependent promoter on a multicopy plasmid . A large inhibitory effect is also observed when the po promoter for OOP RNA is replaced by the strong lambda pL promoter, but not when po is deleted . Plasmids that provide a large excess of "anti-OOP" RNA (RNA that is complementary to OOP RNA) make OOP RNA a less effective inhibitor of cII expression . Inhibition by the OOP DNA plasmid is not observed in an Escherichia coli strain deficient in RNase III . We propose that the 3' end of cII mRNA and OOP RNA form a double-stranded complex that is a substrate for the host enzyme RNase III, resulting in degradation of cII mRNA . Deletion studies on the OOP DNA plasmid indicate that no specific sequence between the promoter and terminator stem structure is required for the inhibitory effect . Lambda cII expression from an induced prophage is increased twofold in the presence of a large excess of anti-OOP RNA . This experiment, in which the prophage is the sole source of OOP RNA, suggests a physiological role for OOP RNA in regulating cII-gene expression. Genetics, 1987 Nov, 117(3), 381 - 90 Some features of base pair mismatch and heterology repair in Escherichia coli; Raposa S et al.; We have used artificially constructed heteroallelic heteroduplex molecules of bacteriophage lambda DNA to transfect Escherichia coli, and E . coli mutants deficient in various functions involved in the adenine methylation-directed mismatch repair system, MutL, MutS, MutH, and UvrD (MutU) . Analysis of the allele content of single infective centers shows that this repair system often acts on several mismatches, separated by as many as 2000 bp, on one of the strands of a heteroduplex molecule . When the methyl-directed mismatch repair system is disabled by mutH or uvrD mutations, localized mismatch repair becomes prominent . This prominent localized repair that can result in separation of very closely linked markers requires the functions MutL and MutS, is independent of adenine methylation, and appears to reflect another mechanism of mismatch repair . Heterology-containing heteroduplex molecules with a deletion in one strand often escape processing . However, when the heterology includes the stem and loop structure of a transposon, Tn10, the transposon is lost. J Bacteriol, 1987 Nov, 169(11), 5289 - 97 Isolation of Escherichia coli rpoB mutants resistant to killing by lambda cII protein and altered in pyrE gene attenuation; Hammer K et al.; Escherichia coli mutants simultaneously resistant to rifampin and to the lethal effects of bacteriophage lambda cII protein were isolated . The sck mutant strains carry alterations in rpoB that allow them to survive cII killing (thus the name sck), but that do not impair either the expression of cII or the activation by cII of the lambda promoters pE and pI . The sck-1, sck-2, and sck-3 mutations modify transcription termination . The growth of lambda, but not of the N-independent lambda variant, lambda nin-5, is hindered by these mutations, which act either alone or in concert with the bacterial nusA1 mutation . In contrast to their effect on lambda growth, the three mutations reduce transcription termination in bacterial operons . The E . coli pyrE gene, which is normally regulated by attenuation, is expressed constitutively in the mutant strains . The sck mutations appear to prevent pyrE attenuation by slowing the rate of transcriptional elongation of the pyrE leader sequence . The sck-6 mutation, unlike the other sck mutations, neither increases pyrE expression nor inhibits the ability of lambda to suppress transcription termination . Instead, the sck-6 mutation blocks the growth of the lambda variants lambda nin-5 and lambda red-3. J Bacteriol, 1987 Nov, 169(11), 5241 - 6 Bacterial genes mutL, mutS, and dcm participate in repair of mismatches at 5-methylcytosine sites; Lieb M; Certain amber mutations in the cI gene of bacteriophage lambda appear to recombine very frequently with nearby mutations . The aberrant mutations included C-to-T transitions at the second cytosine in 5'CC(A/T)GG sequences (which are subject to methylation by bacterial cytosine methylase) and in 5'CCAG and 5'CAGG sequences . Excess cI+ recombinants arising in crosses that utilize these mutations are attributable to the correction of mismatches by a bacterial very-short-patch (VSP) mismatch repair system . In the present study I found that two genes required for methyladenine-directed (long-patch) mismatch repair, mutL and mutS, also functioned in VSP mismatch repair; mutH and mutU (uvrD) were dispensable . VSP mismatch repair was greatly reduced in a dcm Escherichia coli mutant, in which 5-methylcytosine was not methylated . However, mismatches in heteroduplexes prepared from lambda DNA lacking 5-methylcytosine were repaired in dcm+ bacteria . These results indicate that the product of gene dcm has a repair function in addition to its methylase activity. Mol Gen Genet, 1987 Nov, 210(1), 77 - 85 Phage Mu transposase: deletion of the carboxy-terminal end does not abolish DNA-binding activity; Betermier M et al.; We demonstrate that a specific site on the transposase protein, pA, of bacteriophage Mu is highly susceptible to proteolytic cleavage . Cleavage is observed in a minicell system on solubilisation with the non-ionic detergent Triton X-100 or following addition of a solubilised minicell preparation to pA synthesised in a cell-free coupled transcription/translation system . Cleavage occurs at the carboxy-terminal end of the protein and generates a truncated polypeptide of 64 kDa, pA*, which retains some of the DNA-binding properties of pA . These results suggest that pA may be divided into functional domains for DNA binding and for interaction with the proteins involved in phage replication. J Virol, 1987 Nov, 61(11), 3621 - 4 Fusions of bacteriophage P22 late genes to the Escherichia coli lacZ gene; Riggs PD et al.; The late genes of bacteriophage P22 were fused to lacZ to study their differential expression from the late operon transcript . No instances of posttranscriptional regulation were uncovered, thus supporting the model that the late genes are expressed, by and large, in fixed ratios based on their translational efficiency and message stability. J Virol, 1987 Nov, 61(11), 3550 - 4 Mapping and nucleotide sequence of the vaccinia virus gene that encodes a 14-kilodalton fusion protein; Rodriguez JF et al.; A library of rabbit poxvirus DNA fragments contained in the expression cloning vector lambda gt11 was screened with monoclonal antibodies that react specifically against a 14-kilodalton envelope protein of vaccinia virus and rabbit poxvirus . The 14-kilodalton protein appears to play an important role in virus penetration at the level of cell fusion; it also elicits neutralizing antibodies, and it forms covalently linked trimers on the surface of virions and in infected cells (Rodriguez et al., J . Virol . 56:482-488, 1985; Rodriguez et al., J . Virol . 61:395-404, 1987) . Two recombinant bacteriophages expressing beta-galactosidase fusion proteins were isolated . Restriction enzyme analysis and hybridization studies mapped the 14-kilodalton encoding sequences in the middle of vaccinia virus HindIII A DNA fragment . Nucleotide sequence analysis revealed an open reading frame (ATG) preceded by a characteristic TAA sequence of late genes . The sequence spans 330 nucleotides and codes for a protein with a molecular weight of 12,500 and an isoelectric point of 6.3 . There are two small hydrophobic regions, one at the C terminus (11 amino acids) and the other at the N terminus (5 amino acids) . The protein contains two cysteines for oligomer formation and one glycosylation site . Inspection of the deduced amino acid sequence of the 14-kilodalton protein revealed consensus sites with the hemagglutinin precursor of influenza A virus and with adenylate kinase and cytochrome c of various species. J Virol, 1987 Nov, 61(11), 3499 - 509 Multidimensional analysis of intracellular bacteriophage T7 DNA: effects of amber mutations in genes 3 and 19; Serwer P et al.; By use of rate-zonal centrifugation, followed by either one- or two-dimensional agarose gel electrophoresis, the forms of intracellular bacteriophage T7 DNA produced by replication, recombination, and packaging have been analyzed . Previous studies had shown that at least some intracellular DNA with sedimentation coefficients between 32S (the S value of mature T7 DNA) and 100S is concatemeric, i.e., linear and longer than mature T7 DNA . The analysis presented here confirmed that most of this DNA is linear, but also revealed a significant amount of circular DNA . The data suggest that these circles are produced during DNA packaging . It is proposed that circles are produced after a capsid has bound two sequential genomes in a concatemer . The size distribution of the linear, concatemeric DNA had peaks at the positions of dimeric and trimeric concatemers . Restriction endonuclease analysis revealed that most of the mature T7 DNA subunits of concatemers were joined left end to right end . However, these data also suggest that a comparatively small amount of left-end to left-end joining occurs, possibly by blunt-end ligation . A replicating form of T7 DNA that had an S value greater than 100 (100S+ DNA) was also found to contain concatemers . However, some of the 100S+ DNA, probably the most branched component, remained associated with the origin after agarose gel electrophoresis . It has been found that T7 protein 19, known to be required for DNA packaging, was also required to prevent loss, probably by nucleolytic degradation, of the right end of all forms of intracellular T7 DNA . T7 gene 3 endonuclease, whose activity is required for both recombination of T7 DNA and degradation of host DNA, was required for the formation of the 32S to 100S molecules that behaved as concatemers during gel electrophoresis . In the absence of gene 3 endonuclease, the primary accumulation product was origin-associated 100S+ DNA with properties that suggest the accumulation of branches, primarily at the left end of mature DNA subunits within the 100S+ DNA. J Bacteriol, 1987 Nov, 169(11), 5188 - 92 Genetic and physical location of the Escherichia coli rap locus, which is essential for growth of bacteriophage lambda; Guarneros G et al.; The Escherichia coli rap mutant does not support the growth of bacteriophage lambda (D . Henderson and J . Weil, Virology 71:546-559, 1976) . We located the rap site at 26 min in the E . coli genetic map and determined the gene order fadR-rap-supF-trp from our transduction experiments . Plasmid pHO1 harbors a 5.6-kilobase-pair segment of the E . coli chromosome which contains the pth gene (B . Hove-Jensen, Mol . Gen . Genet . 201:269-276, 1985) . This plasmid complemented rap bacteria, suggesting that it carries the dominant allele rap+ . Subcloning experiments reduced the rap-complementing segment to 1.5 kilobase pairs . This segment still contained pth; thus, both loci are tightly linked . The lit mutations that inhibit phage T4 growth in E . coli are located nearby at 25 min (W . Cooley, K . Sirotkin, R . Green, and L . Snyder, J . Bacteriol . 140:83-91, 1979) . We showed that rap and lit mutations are phenotypically and genetically different. Infect Immun, 1987 Nov, 55(11), 2774 - 6 Transposon Tn5 mutagenesis of Brucella abortus; Smith LD et al.; We demonstrate that transposon-mediated mutagenesis can be used to construct mutations in the pathogen Brucella abortus . We have used both a plasmid and a bacteriophage to introduce either Tn5 or Tn5 lac into the Brucella chromosome . B . abortus is naturally sensitive to kanamycin . We have selected 22 independent kanamycin-resistant colonies in strain US-19 and 19 colonies in strain 2308 . We have demonstrated by Southern hybridization that Tn5 was inserted into the chromosome. Infect Immun, 1987 Nov, 55(11), 2675 - 80 Cloning of genes for production of Escherichia coli Shiga-like toxin type II; Newland JW et al.; Genes controlling production of Shiga-like toxin type II (SLT-II) in Escherichia coli were cloned from the SLT-II-converting bacteriophage 933W and compared with the Shiga-like toxin type I (SLT-I) genes previously isolated and described from phage 933J . Subcloning analysis identified a region within the 4.9-kilobase EcoRI fragment of phage 933W that was associated with SLT-II production . Experiments with E . coli minicells containing these subclones demonstrated that the 4.9-kilobase EcoRI fragment encodes the structural genes for SLT-II . These experiments additionally showed the genetic organization of the SLT-II genes to be the same as that of the SLT-I genes, with the coding sequence for the large A subunit adjacent to that for the smaller B subunit . The mobilities of the SLT-II subunits in sodium dodecyl sulfate-polyacrylamide gels were slightly greater than those determined for the SLT-I subunits . Although apparent processing of the SLT-I subunits was observed with polymyxin B treatment of the labeled minicells, no processing of the SLT-II subunits was detected . Southern blot hybridization studies suggested that the DNA fragment carrying the SLT-II structural genes shares approximately 50 to 60% homology with the DNA of the SLT-I structural genes. Virus Genes, 1987 Nov, 1(1), 83 - 96 Translation products of cauliflower mosaic virus ORF V, the coding region corresponding to the retrovirus pol gene; Pietrzak M et al.; Open reading frame (ORF) V of cauliflower mosaic virus (CaMV), the candidate for the reverse transcriptase gene, has been expressed in E . coli under control of the PR promoter of bacteriophage lambda either as an N-terminal polypeptide fused to beta-galactosidase or as the total ORF V without fusion . Antibodies against these proteins were used to analyze extracts from CaMV-infected plants by immunoblotting . ORF V-specific polypeptides of 80, 62, 58, 22, and 18 kD apparent molecular weights were detected, with the largest species corresponding to the full length translation product . The 62 and 22 kD species could be assigned to the N-terminus and the remaining two species to the C-terminus of the ORF. Mol Gen Genet, 1987 Nov, 210(1), 184 - 6 Overproduction of an antisense RNA containing the oop RNA sequence of bacteriophage lambda induces clear plaque formation; Takayama KM et al.; We have constructed an IPTG-inducible plasmid which overexpresses oop RNA sequences in Escherichia coli . Infection of these transformed E . coli cells (SB221/pOOP5) with lambda+ phage produced clear plaques, whereas lambda+ infection of cells transformed with the plasmid vector (SB221/pJDC406) or the plasmid expressing the oop RNA transcript in the other orientation (SB221/pOOP9) gave rise to turbid plaques characteristic of lambda+ . Calculations of the percentage of infected cells forming lysogens show a 6-fold decrease in the absence of isopropyl beta-D-thiogalactoside (IPTG) and a 20-fold decrease in the presence of IPTG for SB221/pOOP5 as compared to both SB221/pJDC406 and SB221/pOOP9 . We have thus shown that the overexpression of oop RNA favors the lytic mode of lambda development. Nucleic Acids Res, 1987 Oct 26, 15(20), 8521 - 30 Competition between sigma factors for core RNA polymerase; Malik S et al.; The switch of RNA polymerase specificity from early to late promoters of bacteriophage T4 is achieved by substitution of host sigma factor, sigma 70, with the T4 induced factor, sigma gp55 . However, overproduction of sigma gp55 from an expression vector is not detrimental to Escherichia coli growth . Direct competition binding assays demonstrate that sigma 70 readily displaces sigma gp55 from RNA polymerase and thereby reverses the promoter specificity of the enzyme . The displacement also occurs with the core enzyme modified by bacteriophage T4 infection . We postulate that an antagonist of sigma 70 should be formed in T4-infected cells to aid sigma gp55 in the early/late switch. J Biol Chem, 1987 Oct 25, 262(30), 14826 - 36 The site-specific cleavage of synthetic Holliday junction analogs and related branched DNA structures by bacteriophage T7 endonuclease I; Dickie P et al.; Various branched DNA structures were created from synthetic, partly complementary oligonucleotides combined under annealing conditions . Appropriate mixtures of oligonucleotides generated three specific branched duplex DNA molecules: (i) a Holliday junction analog having a fixed (immobile) crossover bounded by four duplex DNA branches, (ii) a similar Holliday junction analog which is capable of limited branch migration and, (iii) a Y-junction, with three duplex branches and fixed branch point . Each of these novel structures was specifically cleaved by bacteriophage T7 gene 3 product, endonuclease I . The cleavage reaction "resolved" the two Holliday structure analogs into pairs of duplex DNA products half the size of the original molecules . The point of cleavage in the fixed-junction molecules was predominantly one nucleotide removed to the 5' side of the expected crossover position . Multiple cleavage positions were mapped on the Holliday junction with the mobile, or variable, branch point, to sites consistent with the unrestricted movement of the phosphodiester crossover within the region of limited dyad symmetry which characterizes this molecule . Based on the cleavage pattern observed with this latter substrate, the enzyme displayed a modest degree of sequence specificity, preferring a pyrimidine on the 3' side of the cleavage site . Branched molecules that were partial duplexes (lower order complexes which possessed single-stranded as well as duplex DNA branches) were also substrates for the enzyme . In these molecules, the cleaved phosphodiester bonds were in duplex regions only and predominantly one nucleotide to the 5' side of the branch point . The phosphodiester positions 5' of the branch point in single-stranded arms were not cleaved . Under identical reaction conditions, individually treated oligonucleotides were completely refractory . Thus, cleavage by T7 endonuclease I displays great structural specificity with an efficiency that can vary slightly according to the DNA sequence. J Mol Biol, 1987 Oct 20, 197(4), 617 - 26 Repair of thymine.guanine and uracil.guanine mismatched base-pairs in bacteriophage M13mp18 DNA heteroduplexes; Shenoy S et al.; Repair of thymine.guanine (T.G) and uracil.guanine (U.G) mismatched base-pairs in bacteriophage M13mp18 replicative form (RF) DNA was compared upon transfection into repair-proficient or repair-deficient Escherichia coli strains . Oligonucleotide-directed mutagenesis was used to prepare covalently closed circular heteroduplexes that contained the mismatched base-pair at a restriction recognition site . The heteroduplexes were unmethylated at dam (5'-GATC-3') sites to avoid methylation-directed biasing of repair . In an E . coli host containing uracil-DNA glycosylase (ung+), about 97% of the transfecting U.G-containing heteroduplexes had the U residue excised by the uracil-excision repair system . With the analogous T.G mispair, mismatch repair operated on almost all of the transfecting heteroduplexes and removed the T residue in about 75% of them when the mismatched T was on the minus strand of the RF DNA . Similar preferential excision of the minus-strand's mismatched base was observed whether the heteroduplex RF DNA molecules had only one or both strands unmethylated at dcm (5'-CC(A/T)GG-3') sites and whether the RF DNA was prepared by primer extension in vitro or by reannealing mutant and non-mutant DNA strands . Also, the extent and directionality of repair was the same at a U.G mispair in ung- host cells as at the analogous T.G mispair in ung- or ung+ cells . Only in a mismatch repair-deficient (mutH-) host was the plus strand of the transfecting M13mp18 heteroduplex DNA preferentially repaired . It is suggested that the plus strand nick made by the M13-encoded gene II protein might be employed by a mutH- host to initiate repair on that strand. J Biol Chem, 1987 Oct 15, 262(29), 14178 - 89 Promoter search by Escherichia coli RNA polymerase on a circular DNA template; Singer P et al.; Using the rapid-mixing/photocross-linking technique developed in our laboratory, we have investigated the kinetics of interaction between Escherichia coli RNA polymerase and pAR1319, a recombinant plasmid DNA containing the bacteriophage T7 A2 early promoter . By monitoring the time-dependent density of bound RNA polymerase along the relaxed circular DNA molecule using this technique, we have been able to demonstrate kinetic evidence for linear diffusion of RNA polymerase along DNA in a different system from that previously described (Park, C . S., Hillel, Z., and Wu, C.-W . (1982) J . Biol . Chem . 251, 6950-6956) . The nonspecific association rate constant kon was measured to be 7.7 x 10(4) M-1 s-1 at a DNA chain concentration of 22.4 nM . By taking advantage of the fact that rapid mixing displaces bound protein molecules from DNA, but leaves them within the domain of the DNA, the rate of intradomain binding of RNA polymerase to pAR1319 DNA was determined to be 8.2 s-1 . Since the plasmid is described by a radius of gyration of 0.22 microns, the intradomain concentration of base pairs could be calculated . Using this concentration (180 microM), the rate constant for intradomain nonspecific association of RNA polymerase to pAR1319 DNA was estimated to be 4.6 x 10(4) M-1 s-1 . In addition, a mathematical model has been used to fit the other two important rate constants to the experimental data: koff, which describes the dissociation of RNA polymerase from nonspecific binding sites, and D1, the one-dimensional diffusion coefficient of the enzyme along the DNA molecule . In this model, the circular DNA molecule is described as a ring of interconnected binding sites which together comprise a DNA "domain." RNA polymerase, which enters the domain via three-dimensional diffusion and binds to each site, is allowed to diffuse linearly between adjacent sites and three-dimensionally on and off the DNA molecule . The rate equations for the time-dependent occupancy of each site by RNA polymerase could be written, based on general principles . By solving th