long-range effects in a supercoiled dna domain generated by transcription in vitro.the translocation of a transcription complex can transiently introduce positive and negative superhelical windings into the template dna. to gain further insight into this dynamic dna supercoiling mechanism and its possible involvement in biological processes, we employed an in vitro system in which site-specific recombination by gammadelta resolvase is topologically coupled to transcription-induced negative supercoiling. our kinetic experiments suggest that recombination is closely linked to th ...19979281422
oriented dna binding by one-armed lambda repressor heterodimers and contacts between repressor and rna polymerase at p(rm).bacteriophage lambda repressor activates transcription from p(rm) by contacting the sigma subunit of e. coli rna polymerase. although mutations in repressors that are defective in activation affect exposed residues in the repressor-operator co-crystal, the subunit in repressor dimers that is responsible for activation has not been determined experimentally. here, we describe an oriented heterodimer approach using one-armed repressor-leucine zipper fusion proteins to resolve this question. protec ...19979282743
roles for lambda orf and escherichia coli reco, recr and recf in lambda recombination.bacteriophage lambda lacking its red recombination functions requires either its own gene product, orf, or the product of escherichia coli's reco, recr and recf genes (recorf) for efficient recombination in recbc sbcb sbcc mutant cells (the recf pathway). phage crosses under conditions of a partial block to dna replication have revealed the following: (1) in the presence of orf, recf pathway recombination is similar to lambda red recombination; (2) orf is necessary for focusing recombination tow ...19979335578
chi-star, a chi-related 11-mer sequence partially active in an e. coli recc1004 strain.chi sequence (5'gctggtgg) of escherichia coli was first identified as a site that increased the plaque size of bacteriophage lambda. subsequent studies showed that this site is responsible for both the attenuation ofrecbcd exonuclease activity and the promotion of reca, recbcd-mediated recombination. it is known that bacteriophage lambda containing the chi site makes very small plaques on a recc* (recc1004) mutant because chi is not recognized by the recbc*d mutant enzyme.19979348042
regulation of the elongation-termination decision at intrinsic terminators by antitermination protein n of phage lambda.the mechanisms that control n-protein-dependent antitermination in the phage lambda life cycle have counterparts in the regulatory systems of other organisms. here we examine n-dependent antitermination at the intrinsic tr' terminator of lambda to elucidate the regulatory principles involved. the tr' terminator consists of a sequence of six base-pairs along the template at which the transcription complex is sufficiently destabilized to make rna release possible. within this "zone of opportunity" ...19979367773
the xcpr protein of pseudomonas aeruginosa dimerizes via its n-terminus.extracellular protein secretion by the main terminal branch of the general secretory pathway in pseudomonas aeruginosa requires a secretion machinery comprising the products of at least 12 genes. one of the components of this machinery, the xcpr protein, belongs to a large family of related proteins distinguished by the presence of a highly conserved nucleotide binding domain (walker box a). the xcpr protein is essential for the process of extracellular secretion and amino acid substitutions wit ...19979426126
mutation of d. radiodurans in a gene homologous to ruvb of e. coli.following the digestion of chromosomal dna of deinococcus radiodurans with a restriction enzyme a partial genomic library was constructed using lambda phage as a vector. a phage clone whose dna can complement the deficiency in a radiation-sensitive mutant of d. radiodurans was isolated. following the subcloning using phasmid vector, a hybrid plasmid containing 1.2 kb inserted dna was obtained. after the determination of nucleotide sequence, the deduced amino acid sequence showed close homology t ...19979447236
involvement of boxa nucleotides in the formation of a stable ribonucleoprotein complex containing the bacteriophage lambda n protein.the association of the transcriptional antitermination protein n of bacteriophage lambda with escherichia coli rna polymerase depends on nut site rna (boxa + boxb) in the nascent transcript and the host protein, nusa. this ribonucleoprotein complex can transcribe through rho-dependent and intrinsic termination sites located up to several hundred base pairs downstream of nut. for antitermination to occur farther downstream, this core antitermination complex must be stabilized by the host proteins ...19989461609
escherichia coli nusa is required for efficient rna binding by phage hk022 nun protein.the nun protein of phage hk022 is an rna binding protein of the arginine-rich motif family. nun binds the phage lambda boxb rna sequence (boxb) on nascent lambda transcripts and arrests transcription elongation. binding to boxb is inhibited by zn2+ and stimulated by the escherichia coli nusa protein. deletion of the nun c-terminal region enhances boxb binding and makes it independent of zn2+ and nusa. the c terminus of nun thus appears to interfere with the n-terminal rna binding motif. nusa rel ...19989465052
changes in the periplasmic linker and in the expression level affect the activity of toxr and lambda-toxr fusion proteins in escherichia order to assess the potentiality of vibrio cholerae toxr protein and of bacteriophage lambda repressor as indicators of the dimerization of periplasmic proteins in escherichia coli, we have constructed a series of plasmids encoding transmembrane fusion proteins. the amino-terminal part, containing the dna binding domain of either toxr or lambda repressor, is located in the cytoplasm and acts as reporter for dimerization. as models of periplasmic proteins we have used alkaline phosphatase (a d ...19989515742
design and implementation of a qualitative simulation model of lambda phage infection.motivation: molecular biology databases hold a large number of empirical facts about many different aspects of biological entities. that data is static in the sense that one cannot ask a database 'what effect has protein a on gene b?' or 'do gene a and gene b interact, and if so, how?'. those questions require an explicit model of the target organism. traditionally, biochemical systems are modelled using kinetics and differential equations in a quantitative simulator. for many biological process ...19989520505
crystallization and preliminary x-ray analysis of the dsdna bacteriophage hk97 mature empty capsid.hk97 is a temperate dsdna bacteriophage of escherichia coli that is structurally similar to phage lambda, with an icosahedral head of triangulation (7) number 7. although the capsids of several large dsdna phages have been studied extensively using a variety of biophysical approaches, no high-resolution structure is available. we have grown crystals of mature but empty bacteriophage hk97 capsids that diffract to at least 3.5 a using synchrotron radiation. the hk97 head ii crystals are the first ...19989527920
guanosine tetraphosphate (ppgpp)-mediated inhibition of the activity of the bacteriophage lambda pr promoter in escherichia was previously demonstrated that the activity of bacteriophage lambda promoter pr is decreased in wild-type escherichia coli cells starved for amino acids (during the stringent response). since pr activity is necessary for the transcriptional activation of ori lambda, this leads to inhibition of the replication of plasmids derived from phage lambda. these results led to the proposal that the pr promoter susceptible to control by the stringent response. however, subsequent studies demonstrated ...19989529531
dnaa-stimulated transcriptional activation of orilambda: escherichia coli rna polymerase beta subunit as a transcriptional activator contact site.we present evidence that escherichia coli rna polymerase beta subunit may be a transcriptional activator contact site. stimulation of the activity of the pr promoter by dnaa protein is necessary for replication of plasmids derived from bacteriophage lambda. we found that dnaa activates the pr promoter in vitro. particular mutations in the rpob gene were able to suppress negative effects that certain dnaa mutations had on the replication of lambda plasmids; this suppression was allele-specific. w ...19989539721
specific binding of escherichia coli ribosomal protein s1 to boxa transcriptional antiterminator rna.we show that ribosomal protein s1 specifically binds the boxa transcriptional antiterminator rnas of bacteriophage lambda and the escherichia coli ribosomal rna operons. although s1 competes with the nusb-s10 antitermination complex for binding to boxa, it does not affect antitermination by the lambda n protein in vitro, and its role, if any, in rrna synthesis is still unknown.19989555913
the helicobacter felis ftsh gene encoding an atp-dependent metalloprotease can replace the escherichia coli homologue for growth and phage lambda lysogenization.cloning and sequencing of an approximately 6.0-kb chromosomal dna fragment from helicobacter felis revealed five complete open reading frames. the deduced amino acid sequence of one orf exhibited sequence similarity to the ftsh protein, an atp-dependent metalloprotease, from various bacterial species. the encoded protein consists of 638 amino acid residues with a molecular mass of 70.2 kda. the hydropathy profile of the ftsh protein predicted two n-terminal transmembrane regions that were confir ...19989560419
mutations affecting cooperative dna binding of phage hk022 ci repressor.cooperative protein-dna interactions play critical roles in gene regulation in all organisms. among the best-studied cooperative interactions is that of phage lambda repressor, which binds cooperatively to two adjacent operators. similar cooperative interactions are also shown by several other lambdoid phage repressors, including hk022 ci repressor, which we study here. this protein has a much higher degree of cooperativity than seen with lambda repressor, and previous evidence has suggested tha ...19989636698
inactivation of bacteriophage lambda, escherichia coli, and candida albicans by ozone.the effects of ozone (o3) on three types of microbes were studied. test suspensions were exposed to 600 ppm o3 at room temperature. control experiments were performed under identical conditions using oxygen gas. bacteriophage lambda was completely inactivated at 10 min while escherichia coli and candida albicans were only inactivated by factors of 10(5) and 10(4) respectively at 40 min. exposure of a mixed microbial suspension to o3 for 5 min resulted in 100% killing of bacteriophages while the ...19989684309
oligohistidine tag mutagenesis of the lambda holin gene.holins are a diverse group of small integral membrane proteins elaborated by bacteriophages to lyse bacterial hosts and effect release of progeny phages in a precisely timed manner. recently, the holin s gene of phage lambda was overexpressed and the holin protein was purified to homogeneity by means of an oligohistidine tag procedure and immobilized metal affinity chromatography (d. l. smith, d. k. struck, j. m. scholtz, and r. young, j. bacteriol. 180:2531-2540, 1998). numerous locations withi ...19989696770
biochemical and genetic analysis of lambdaw, the newly isolated lambdoid phage.otherwise isogenic escherichia coli cp78 (rela+) and cp79 (rela-) strains are commonly used in studies on the stringent control, the bacterial response to amino acid starvation. we found that these strains are lysogenic for a phage which is spontaneously induced with a low frequency, producing virions able to infect other e. coli strains. genetic studies, restriction analysis of the phage dna genome, and electron microscopy revealed that this phage is very similar to, but not identical with, bac ...19989701518
the escherichia coli rna polymerase alpha subunit and transcriptional activation by bacteriophage lambda cii protein.bacteriophage lambda is not able to lysogenise the escherichia coli rpoa341 mutant. this mutation causes a single amino acid substitution lys271glu in the c-terminal domain of the rna polymerase alpha subunit (alphactd). our previous studies indicated that the impaired lysogenisation of the rpoa341 host is due to a defect in transcriptional activation by the phage cii protein and suggested a role for alphactd in this process. here we used a series of truncation and point mutants in the rpoa gene ...19989701520
rapid degradation of polyadenylated oop rna.the oop rna is a short (77 nucleotides (nt)) transcript encoded by bacteriophage lambda which acts as an antisense rna for lambda cii gene expression. recently we demonstrated that oop rna is specifically polyadenylated at its 3' end by poly(a) polymerase i (pap i), the pcnb gene product. here we demonstrate that the half life of oop rna is 3 times longer in the pcnb mutant relative to the pcnb+ host, indicating that polyadenylation of this transcript causes its accelerated degradation. although ...19989710253
the ihf mrna levels decline as neisseria gonorrhoeae enters the stationary growth phase.integration host factor (ihf) is a small heterodimeric dna binding protein found in all gram-negative bacteria and is implicated as a transcription cofactor of pile in neisseria gonorrhoeae (hill, s.a., samuels, d.s., carlson, j.h., wilson, j., hogan, d., lubke, l., belland, r.j., 1997. integration host factor is a transcriptional cofactor of pile in neisseria gonorrhoeae. mol. microbiol. 23, 649-656). the ihf genes (ihfa and ihfb) were cloned from n. gonorrhoeae through functional complementati ...19989714829
cloning and characterization of the dnak heat shock operon of the marine bacterium vibrio harveyi.we cloned the dna region of the vibrio harveyi chromosome containing the heat shock genes dnak and dnaj and sequenced them. these genes are arranged in the chromosome in the order dnak-dnaj, as in other proteobacteria of the alpha and gamma subdivisions. the dnak gene is 1923 nucleotides in length and codes for a protein of 640 amino acid residues, with a predicted molecular mass of 69,076 da and 81.2% similarity to the dnak protein of escherichia coli. the v. harveyi dnaj gene has a coding sequ ...19989747709
escherichia coli dnaa gene function and bacteriophage lambda replication.allele specificity of the escherichia coli dnaa gene function in the replication of plasmids derived from bacteriophage lambda has been demonstrated previously. here, using a series of dnaa temperature-sensitive mutants, we investigated dnaa allele specificity of the replication of phages lambda p+ and lambda pts 1 pi a66. we found that phage lambda p+ produces its progeny efficiently at 43 degrees c irrespective of the dnaa allele, whereas lambda pts 1 pi a66, which is unable to develop lytical ...19989785448
identification and characterization of the fis operon in enteric bacteria.the small dna binding protein fis is involved in several different biological processes in escherichia coli. it has been shown to stimulate dna inversion reactions mediated by the hin family of recombinases, stimulate integration and excision of phage lambda genome, regulate the transcription of several different genes including those of stable rna operons, and regulate the initiation of dna replication at oric. fis has also been isolated from salmonella typhimurium, and the genomic sequence of ...19989811652
formation of the preprimosome protects lambda o from rna transcription-dependent proteolysis by clpp/clpx.using the bacteriophage lambda dna replication system, composed entirely of purified proteins, we have tested the accessibility of the short-lived lambda o protein to the clpp/clpx protease during the various stages of lambda dna replication. we find that binding of lambda o protein to its orilambda dna sequence, leading to the so-called "o-some" formation, largely inhibits its degradation. on the contrary, under conditions permissive for transcription, the lambda o protein bound to the orilambd ...19989860956
the geometry of a synaptic intermediate in a pathway of bacteriophage lambda site-specific recombination.bacteriophage lambda uses site-specific recombination to move its dna into and out of the escherichia coli genome. the recombination event is mediated by the phage-encoded integrase (int) at short dna sequences known as attachment ( att ) sites. int catalyzes recombination via at least four distinct pathways, distinguishable by their requirements for accessory proteins and by the sequence of their substrates. the simplest recombination reaction catalyzed by int does not require any accessory pro ...19999927749
malk forms a dimer independent of its assembly into the malfgk2 atp-binding cassette transporter of escherichia coli.the maltose transport complex (mtc) is a member of the atp-binding cassette superfamily of membrane transport proteins and is a model for understanding the folding and assembly of hetero-oligomeric membrane protein complexes. the mtc is made up of two integral membrane proteins, malf and malg, and a peripheral membrane protein, malk. these proteins associate with a stoichiometry of 1:1:2 to form the complex malfgk2. in our studies of the oligomerization of this complex, we have shown that the at ...199910037713
cloning and expression in escherichia coli of an azoreductase gene from clostridium perfringens and comparison with azoreductase genes from other bacteria.a genomic library of clostridium perfringens atcc 3626 was constructed in phage lambda gt11 and screened with an antibody against the c. perfringens azoreductase, which catalyzes the reduction of azo dyes to aromatic amines. a positive recombinant phage, containing a 3.8 kb dna fragment insert was selected and purified. lytic and lysogenic escherichia coli cultures infected with the recombinant phage had higher azoreductase activity than cultures infected only with the vector lambda gt11. the 3. ...199910071864
characterization of the recd gene of neisseria gonorrhoeae ms11 and the effect of recd inactivation on pilin variation and dna transformation.pilin antigenic variation in neisseria gonorrhoeae may result following intrachromosomal recombination between homologous pil genes. despite extensive study, reca is the only previously characterized gene known to be involved in this process. in this study, the gonococcal recd gene, encoding one subunit of the putative recbcd holoenzyme, was characterized and its role in pilin variation assessed. the complete recd gene of n. gonorrhoeae ms11 was cloned and its nucleotide sequence determined. the ...199910075421
ftsh--a single-chain charonin?the ftsh gene encodes an atp- and zn(2+)-dependent metalloprotease with a molecular mass of about 70 kda. it was first identified in escherichia coli where it is also designated hflb, tolz or mrsc, and seems to be present in most if not all bacteria. the ftsh protein is anchored to the cytoplasmic membrane via two transmembrane regions in such a way that the very short amino- and the long carboxy-termini are exposed into the cytoplasm. ftsh is member of the aaa family (atpases associated with a ...199910077851
crystallographic analysis of the dsdna bacteriophage hk97 mature empty capsid.hk97 is a member of the siphovirus family of dsdna bacteriophages. it is similar in architecture to bacteriophage lambda, the type member of this family, with an icosahedral capsid of triangulation number t = 7. no high-resolution structural information is available for the dsdna phages, and hk97 is the only dsdna bacteriophage capsid to produce crystals which diffract x-rays. at 650 a in diameter, the large size of the particle and resultant large unit cell create crystallographic challenges. t ...199910089306
cloning of mycoplasma synoviae genes encoding specific antigens and their use as species-specific dna probes.a genomic library of mycoplasma synoviae (ms) was generated by using bacteriophage lambda gt11 as a cloning and expression vector. identification of recombinant clones highly specific to ms was achieved by screening the library for expression of ms proteins with polyclonal antiserum that had been preadsorbed with 6 heterologous avian mycoplasma species antigens. expression of the recombinant clones in escherichia coli followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the t ...199910098689
replication of bacteriophage lambda in the escherichia coli dnaa deltarac hosts. 199910328680
evidence for a holin-like protein gene fully embedded out of frame in the endolysin gene of staphylococcus aureus bacteriophage 187.we have cloned, sequenced, and characterized the genes encoding the lytic system of the unique staphylococcus aureus phage 187. the endolysin gene ply187 encodes a large cell wall-lytic enzyme (71.6 kda). the catalytic site, responsible for the hydrolysis of staphylococcal peptidoglycan, was mapped to the n-terminal domain of the protein by the expression of defined ply187 domains. this enzymatically active n terminus showed convincing amino acid sequence homology to an n-acetylmuramoyl-l-alanin ...199910419939
regulation of copy number and stability of phage lambda derived ptc lambda 1 plasmid in the light of the dimer/multimer catastrophe hypothesis.the dimer catastrophe hypothesis has been proposed previously to explain instability of multicopy plasmids whose partitioning is random, contrary to low copy number plasmids which are stably maintained and actively partitioned. until now, this hypothesis has been investigated using multicopy cole1 plasmids. however, for more detailed testing of the dimer/multimer catastrophe hypothesis, one should use a plasmid which can be maintained at either low or high copy number and still possesses the sam ...199910427732
minicircle: an improved dna molecule for in vitro and in vivo gene transfer.minicircles are a new form of supercoiled dna molecule for nonviral gene transfer which have neither bacterial origin of replication nor antibiotic resistance marker. they are thus smaller and potentially safer than the standard plasmids currently used in gene therapy. they were obtained in e. coli by att site-specific recombination mediated by the phage lambda integrase, which was used to excise the expression cassette from the unwanted plasmid sequences. we produced two minicircles containing ...199910435105
background mutations and polymorphisms in lacz-plasmid transgenic mice.transgenic animal models harboring chromosomally integrated shuttle vectors with bacterial reporter genes are now widely used to measure in vivo mutant frequencies. the lacz-plasmid transgenic mouse model has a unique sensitivity to large rearrangements compared to systems using bacteriophage lambda vectors, which mainly detect point mutations and small deletions or insertions. in this study, the background mutant frequencies and spectra in the lacz-plasmid transgenic mouse model were investigat ...199910529734
addiction modules and programmed cell death and antideath in bacterial bacteria, programmed cell death is mediated through "addiction modules" consisting of two genes. the product of the second gene is a stable toxin, whereas the product of the first is a labile antitoxin. here we extensively review what is known about those modules that are borne by one of a number of escherichia coli extrachromosomal elements and are responsible for the postsegregational killing effect. we focus on a recently discovered chromosomally borne regulatable addiction module in e. co ...199910547685
functional importance of regions in escherichia coli elongation factor nusa that interact with rna polymerase, the bacteriophage lambda n protein and rna.the association of the essential escherichia coli protein nusa with rna polymerase increases pausing and the efficiency of termination at intrinsic terminators. nusa is also part of the phage lambda n protein-modified antitermination complex that functions to prevent transcriptional termination. we have investigated the structure of nusa using various deletion fragments of nusa in a variety of in vitro assays. sequence and structural alignments have suggested that nusa has both s1 and kh homolog ...199910564494
a genetic screen to identify sequences that mediate protein oligomerization in escherichia coli.many proteins assemble as oligomeric complexes and in several cases a distinct domain mediates the interaction between the subunits. the identification of new oligomerization modules is relevant to comprehend both the architecture and the evolution of protein sequences and also for protein engineering applications. using the bacteriophage lambda repressor dimerization assay, we searched escherichia coli genomic libraries for sequences able to mediate protein oligomerization in vivo. we identifie ...199910581196
a mutation correcting the dna interaction defects of a mutant phage lambda terminase, gpnu1 k35a terminase.terminase, the dna packaging enzyme of bacteriophage lambda, is a heteromultimer composed of gpnu1 (181 aa) and gpa (641 aa) subunits, encoded by the lambda nu1 and a genes, respectively. similarity between the deduced amino acid sequences of gpnu1 and gpa and the nucleotide binding site consensus sequence suggests that each terminase subunit has an atp reactive center. terminase has been shown to have two distinct atpase activities. the gpnu1 subunit has a low-affinity atpase stimulated by nons ...199910600592
genetic system for reversible integration of dna constructs and lacz gene fusions into the escherichia coli chromosome.a plasmid system for site-specific integration into and excision and recovery of gene constructs and lacz gene fusions from the escherichia coli chromosome was developed. plasmid suicide vectors utilizing the origin of replication of r6k plasmids and containing the attp sequence of bacteriophage lambda, multiple cloning site, and antibiotic resistance markers facilitate reversible integration into the e. coli chromosome by site-specific recombination. additional vectors permit construction of la ...200010610816
proteolysis of bacteriophage lambda cii by escherichia coli ftsh (hflb).ftsh (hflb) is a conserved, highly specific, atp-dependent protease for which a number of substrates are known. the enzyme participates in the phage lambda lysis-lysogeny decision by degrading the lambda cii transcriptional activator and by its response to inhibition by the lambda ciii gene product. in order to gain further insight into the mechanism of the enzymatic activity of ftsh (hflb), we identified the peptides generated following proteolysis of the phage lambda cii protein. it was found ...200010809689
the crystal structure of nusb from mycobacterium tuberculosis.both prokaryotes and eukaryotes regulate transcription through mechanisms that suppress termination signals. an antitermination mechanism was first characterized in bacteriophage lambda. bacteria have analogous machinery that regulates ribosomal rna transcription and employs host factors, called the n-utilizing (where n stands for the phage lambda n protein) substances (nus), nusa, nusb, nuse and nusg. here we report the crystal structure of nusb from mycobacterium tuberculosis, the bacterium th ...200010881194
escherichia coli dna polymerase iv mutator activity: genetic requirements and mutational specificity.the dinb gene of escherichia coli is known to be involved in the untargeted mutagenesis of lambda phage. recently, we have demonstrated that this damage-inducible and sos-controlled gene encodes a novel dna polymerase, dna pol iv, which is able to dramatically increase the untargeted mutagenesis of f' plasmid. at the amino acid level, dna pol iv shares sequence homologies with e. coli umuc (dna pol v), rev1p, and rad30p (dna polymerase eta) of saccharomyces cerevisiae and human rad30a (xpv) prot ...200010913093
1,3-butadiene: cancer, mutations, and adducts. part ii: roles of two metabolites of 1,3-butadiene in mediating its in vivo genotoxicity.1,3-butadiene (bd) is carcinogenic in mice and rats, with mice being more susceptible than rats to its carcinogenic effects. 1,3-butadiene is mutagenic in the bone marrow and spleen cells of b6c3f1 laci transgenic mice. the goal of this research was to assess the roles of two bd metabolites, 1,2-epoxy-3-butene (bdo) and 1,2,3,4-diepoxybutane (bdo2), in the mutagenicity and mutational spectrum of the parent compound bd by determining the mutagenicity and mutational spectra of bdo and bdo2 in huma ...200010925839
detection of beta-amyloid peptide aggregation using dna electrophoresis.dna could readily associate with the aggregated forms of the beta-amyloid peptides beta(1-40) and beta(25-35), giving rise to a shift in the electrophoretic mobility of dna. as a result, dna was retained at the top of a 1% agarose gel. in contrast, the electrophoretic mobility of dna was little influenced by the monomeric forms of beta(1-40) and beta(25-30). dna from different sources such as lambda phage, escherichia coli plasmid, and human gene showed similar results. however, the electrophore ...200010964426
replication of orij-based plasmid dna during the stringent and relaxed responses of escherichia coli.the orij-based plasmids contain the origin of dna replication from the cryptic rac prophage, present in the chromosomes of most escherichia coli k-12 strains. the organization of the orij replication region resembles that of the bacteriophage lambda, although sequence similarity is small. here we investigated the regulation of replication of the orij-based plasmid in e. coli rela(+) and rela(-) hosts during amino acid starvation and limitation, i.e., during the stringent and relaxed responses. w ...200010964622
survey and summary: holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories.holliday junction resolvases (hjrs) are key enzymes of dna recombination. a detailed computer analysis of the structural and evolutionary relationships of hjrs and related nucleases suggests that the hjr function has evolved independently from at least four distinct structural folds, namely rnase h, endonuclease, endonuclease vii-colicin e and rusa. the endonuclease fold, whose structural prototypes are the phage lambda exonuclease, the very short patch repair nuclease (vsr) and type ii restrict ...200010982859
endonuclease and helicase activities of bacteriophage lambda terminase: changing nearby residue 515 restores activity to the gpa k497d mutant enzyme.terminase, the dna packaging enzyme of bacteriophage lambda, is a heteromultimer of gpnu1 and gpa subunits. in an earlier investigation, a lethal mutation changing gpa residue 497 from lysine to aspartic acid (k497d) was found to cause a mild change in the high-affinity atpase that resides in gpa and a severe defect in the endonuclease activity of terminase. the k497d terminase efficiently sponsored packaging of mature lambda dna into proheads. in the present work, k497d terminase was found to h ...200011062051
mechanism for a transcriptional activator that works at the isomerization step.transcriptional activators in prokaryotes have been shown to stimulate different steps in the initiation process including the initial binding of rna polymerase (rnap) to the promoter and a postbinding step known as the isomerization step. evidence suggests that activators that affect initial binding can work by a cooperative binding mechanism by making energetically favorable contacts with rnap, but the mechanism by which activators affect the isomerization step is unclear. a well-studied examp ...200011087868
increased bar minigene mrna stability during cell growth inhibition.bacteriophage lambda is unable to grow vegetatively on escherichia coli mutants defective in peptidyl-trna hydrolase (pth) activity. mutations which allow phage growth on the defective host have been located at regions named bar in the lambda genome. expression of wild-type bar regions from plasmid constructs results in inhibition of protein synthesis and lethality to pth-defective cells. two of these wild-type bar regions, bari+ and barii+, contain minigenes with similar aug-aua-stop codon sequ ...200111136457
a new expression cloning strategy for isolation of substrate-specific kinases by using phosphorylation site-specific antibody.signal transduction from cell surface receptors to the nucleus is regulated in most part by protein phosphorylation. for the purpose of identification of kinases which play an important role at a particular phosphorylation step in a series of signal transduction pathways, we have developed a new expression-screening method using a phosphorylation site specific antibody and a vector encoding substrate polypeptide. we have applied this method for screening kinases which phosphorylate stat3 at seri ...200111150545
genetic footprinting in vivo genetic footprinting was developed in the yeast saccharomyces cerevisiae to simultaneously assess the importance of thousands of genes for the fitness of the cell under any growth condition. we have developed in vivo genetic footprinting for escherichia coli, a model bacterium and pathogen. we further demonstrate the utility of this technology for rapidly discovering genes that affect the fitness of e. coli under a variety of growth conditions. the definitive features of this system incl ...200111160101
bacteriophage lambda: alive and well and still doing its thing.the lambda (lambda) family of bacteriophages continues to provide significant insights into the understanding of basic biological processes, as well as useful technological innovations. areas in which recent advances have occurred include transcription elongation, repressor interactions, genomics and post-transcriptional regulation. the homologous lambda recombination functions have been exploited as an efficient in vivo recombinant engineering system for functional genomic studies. the virulenc ...200111282477
specific and non-specific interactions of integration host factor with dna: thermodynamic evidence for disruption of multiple ihf surface salt-bridges coupled to dna dna binding of architectural protein integration host factor (ihf) is involved in formation of functional multiprotein-dna assemblies in escherichia coli, while non-specific binding of ihf and other histone-like proteins serves to structure the nucleoid. here, we report an isothermal titration calorimetry study of the thermodynamics of binding ihf to a 34 bp fragment composed entirely of the specific h' site from lambda-phage dna. at low to moderate [k(+)] (60-100 mm), strong compe ...200111428896
what makes the bacteriophage lambda red system useful for genetic engineering: molecular mechanism and biological function.recent studies have generated interest in the use of the homologous recombination system of bacteriophage lambda for genetic engineering. the system, called red, consists primarily of three proteins: lambda exonuclease, which processively digests the 5'-ended strand of a dsdna end; beta protein, which binds to ssdna and promotes strand annealing; and gamma protein, which binds to the bacterial recbcd enzyme and inhibits its activities. these proteins induce a 'hyper-rec' state in escherichia col ...200111445160
single-strand interruptions in replicating chromosomes cause double-strand breaks.replication-dependent chromosomal breakage suggests that replication forks occasionally run into nicks in template dna and collapse, generating double-strand ends. to model replication fork collapse in vivo, i constructed phage lambda chromosomes carrying the nicking site of m13 bacteriophage and infected with these substrates escherichia coli cells, producing m13 nicking enzyme. i detected double-strand breaks at the nicking sites in lambda dna purified from these cells. the double-strand break ...200111459959
cell toxicity caused by products of the p(l) operon of bacteriophage lambda.induction of a lambda prophage causes the death of the host cell even in the absence of phage replication and lytic functions due to expression of functions from the lambda p(l) operon. we genetically modified the lambda prophage to determine which lambda p(l) operon functions were involved in cell killing. viability assays and flow cytometry were used to monitor cell death and filamentation. the kil gene was shown to cause cell death and filamentation as described previously. another killing ac ...200111470529
purification and crystallization of cii: an unstable transcription activator from phage lambda.the cii protein of the temperate bacteriophage lambda is a transcriptional activator involved in the lysis-lysogeny switch of the phage. it is an unstable protein of 97 amino acids and is known to exist as a tetramer in the native state. the cii gene has been cloned and expressed in escherichia coli using a t7 promoter based over-expression system. the recombinant cii protein has been purified to homogeneity by ammonium sulfate fractionation followed by two steps of ion-exchange chromatography. ...200111689008
arm-site binding by lambda -integrase: solution structure and functional characterization of its amino-terminal domain.the integrase protein (int) from bacteriophage lambda catalyzes the insertion and excision of the viral genome into and out of escherichia coli. it is a member of the lambda-int family of site-specific recombinases that catalyze a diverse array of dna rearrangements in archaebacteria, eubacteria, and yeast and belongs to the subset of this family that possesses two autonomous dna-binding domains. the heterobivalent properties of int can be decomposed into a carboxyl-terminal domain that executes ...200211904406
rapid construction of adenoviral vectors by lambda phage genetics.continued improvements of adenoviral vectors require the investigation of novel genome configurations. since adenovirus can be generated directly by transfecting packaging cell lines with viral genomes isolated from plasmid dna, it is possible to separate genome construction from virus production. in this way failure to generate a virus is not associated with an inability to generate the desired genome. we have developed a novel lambda-based system that allows rapid modification of the viral gen ...200211907206
the cell surface protein ag43 facilitates phage infection of escherichia coli in the presence of bile salts and was found that infection of escherichia coli by bacteriophage lambda is inhibited in the presence of certain bile salts and carbohydrates when cells are in the "off" state for production of the phase-variable cell surface protein antigen 43 (ag43). the inhibition of phage growth was found to be due to a significant impairment in the process of phage adsorption. expression of the gene encoding ag43 (agn43) from a plasmid or inactivation of the oxyr gene (encoding an activator of genes importan ...200211988528
alterations of the portal protein, gpb, of bacteriophage lambda suppress mutations in cosq, the site required for termination of dna packaging.the cosq site of bacteriophage lambda is required for dna packaging termination. previous studies have shown that cosq mutations can be suppressed in three ways: by a local suppressor within cosq, an increase in the length of the lambda chromosome, and missense mutations affecting the prohead's portal protein, gpb. in the present work, revertants of a set of lethal cosq mutants were screened for suppressors. seven new cosq suppressors affected gene b, which encodes the portal protein of the proh ...200212019220
requirement for nusg for transcription antitermination in vivo by the lambda n protein.transcription antitermination by the bacteriophage lambda n protein is stimulated in vitro by the escherichia coli nusg protein. earlier work suggested that nusg was not required for n activity in vivo. here we present evidence that nusg also stimulates n-mediated transcription antitermination in intact cells.200212029062
excretion of human beta-endorphin into culture medium by using outer membrane protein f as a fusion partner in recombinant escherichia coli.escherichia coli bl21 strains were found to excrete a large amount of outer membrane protein f (ompf) into culture medium during high-cell-density cultivation. from this interesting phenomenon, a novel and efficient ompf fusion system was developed for the excretion of recombinant proteins by e. coli. the ompf gene of e. coli bl21(de3) was first knocked out by using the red operon of bacteriophage lambda to construct e. coli mbel-bl101. for the excretion of human beta-endorphin as a model protei ...200212324347
protein-protein and protein-dna interactions of sigma70 region 4 involved in transcription activation by lambdaci.the ci protein of bacteriophage lambda (lambdaci) activates transcription from promoter p(rm) through an acidic patch on the surface of its dna-binding domain. genetic evidence suggests that this acidic patch stimulates transcription from p(rm) through contact with the c-terminal domain (region 4) of the sigma(70) subunit of escherichia coli rna polymerase. here, we identify two basic residues in region 4 of sigma(70) that are critical for lambdaci-mediated activation of transcription from p(rm) ...200212421556
scanning mutagenesis identifies amino acid residues essential for the in vivo activity of the escherichia coli dnaj (hsp40) j-domain.the dnaj (hsp40) cochaperone regulates the dnak (hsp70) chaperone by accelerating atp hydrolysis in a cycle closely linked to substrate binding and release. the j-domain, the signature motif of the hsp40 family, orchestrates interaction with the dnak atpase domain. we studied the j-domain by creating 42 mutant e. coli dnaj variants and examining their phenotypes in various separate in vivo assays, namely, bacterial growth at low and high temperatures, motility, and propagation of bacteriophage l ...200212454054
generation and pcr screening of bacteriophage lambda sublibraries enriched for rare clones (the "sublibrary method"). 200212494671
a spring-loaded state of nusg in its functional cycle is suggested by x-ray crystallography and supported by site-directed mutants.transcription factor nusg is present in all prokaryotes, and orthologous proteins have also been identified in yeast and humans. nusg contains a 27-residue kow motif, found in ribosomal protein l24 where it interacts with rrna. nusg in escherichia coli (ecnusg) is an essential protein and functions as a regulator of rho-dependent transcription termination, phage lambda n and rrna transcription antitermination, and phage hk022 nun termination. relative to ecnusg, aquifex aeolicus nusg (aanusg) an ...200312600194
growth-rate dependent rna polyadenylation in escherichia coli.rna polyadenylation occurs not only in eukaryotes but also in bacteria. in prokaryotes, polyadenylated rna molecules are usually degraded more efficiently than non-modified transcripts. here we demonstrate that two transcripts, which were shown previously to be substrates for poly(a) polymerase i (pap i), escherichia coli lpp messenger rna and bacteriophage lambda oop rna, are polyadenylated more efficiently in slowly growing bacteria than in rapidly growing bacteria. intracellular levels of pap ...200312612607
seqa-mediated stimulation of a promoter activity by facilitating functions of a transcription was demonstrated recently that the seqa protein, a main negative regulator of escherichia coli chromosome replication initiation, is also a specific transcription factor. seqa specifically activates the bacteriophage lambda pr promoter while revealing no significant effect on the activity of another lambda promoter, pl. here, we demonstrate that lysogenization by bacteriophage lambda is impaired in e. coli seqa mutants. genetic analysis demonstrated that cii-mediated activation of the phage p ...200312622820
the mechanism of regulation of bacteriophage lambda pr promoter activity by escherichia coli dnaa protein.apart from its function as an initiator of dna replication, the escherichia coli dnaa protein is also a specific transcription factor. it activates and represses a number of promoters. however, mechanisms of transcription stimulation by dnaa remained unknown. bacteriophage lambda pr promoter is one of the promoters activated by dnaa. it was reported previously that dnaa binds downstream of the pr promoter and perhaps interacts with the rna polymerase beta subunit. here we demonstrate that dnaa p ...200312654908
on the origin of unusual mutations recovered from the laci transgenic assay.amongst approximately 25,000 mutants recovered from tissues of the laci mouse and rat transgenic mutation assay, we identified seven mutants that carry changes that are unlike the majority of mutations that are normally recovered in these systems. the recovered mutants feature replacements and insertions of sequences that originate in the animal's genome, in the bacteriophage lambda construct that harbors the laci gene, and in the genome of the e. coli plating host. these mutants demonstrate tha ...200312694740
stationary phase-like properties of the bacteriophage lambda rex exclusion phenotype.the rex genes of bacteriophage lambda were found to protect lysogenic escherichia colik host cells against killing by phage t4 rii, when compared in parallel to isogenic rex(-) lysogens and nonlysogens. this protective effect was abrogated upon mutation of the host stationary-phase sigma factor rpos. rex(+) lysogens infected by t4 rii contracted, formed aggregates and shed flagella, thus resembling cells entering stationary phase. these phenotypes were accentuated in nonlysogenic cells carrying ...200312715152
constitutive versus thermoinducible expression of heterologous proteins in escherichia coli based on strong pr,pl promoters from phage lambda.constitutive and thermoinducible expression plasmids based on strong p(r),p(l) promoters from phage lambda were compared for production of tnf-alpha and its analogs under various conditions. much higher accumulation of tnf was obtained in a constitutive system, so the wider applicability of such systems was studied. in constitutive systems, proteolytically susceptible proteins can be produced easily at low cultivation temperatures and the addition of expensive or toxic chemical inducers is not r ...200312768624
bacteriophage lambda repressor allelic modulation of the rex exclusion phenotype.the sensitivity of delta red-gam delta ren mutants of bacteriophage lambda to rex exclusion by lambda rexa+ rexb+ lysogens is modulated by the prophage ci repressor allele. we show the following: (i) lambda spi156 delta nin5 forms plaques on a ci+-rexa+-rexb+ lysogen with 10(5)-fold higher efficiency than on ci[ts]-rexa+-rexb+ derivatives. (ii) the ci[ts]857 allele augmentation of rex exclusion is recessive to ci+. (iii) the ci857-mediated increase in rex exclusion activity involves the particip ...200312795410
identification and mutational analysis of bacteriophage prd1 holin protein p35.holin proteins are phage-induced integral membrane proteins which regulate the access of lytic enzymes to host cell peptidoglycan at the time of release of progeny viruses by host cell lysis. we describe the identification of the membrane-containing phage prd1 holin gene (gene xxxv). the prd1 holin protein (p35, 12.8 kda) acts similarly to its functional counterpart from phage lambda (gene s), and the defect in prd1 gene xxxv can be corrected by the presence of gene s of lambda. several nonsense ...200312813073
the optimal eukaryotic signal for translation initiation from non-aug codons, present upstream of bacteriophage lambda p cistron, is inactive in escherichia coli.expression of the replication genes of bacteriophage lambda, o and p, is believed to be translationally coupled. however, it was previously noted that, under conditions of amino acid starvation, when o is not synthesized, p continues to be expressed at a relatively high level. the results presented in this report, contrary to the previously presented hypothesis, suggest that an agacuggau sequence (an optimal context for translation initiation from non-aug codons in eukaryotes, and present upstre ...200312813564
role of the cgta gene function in dna replication of extrachromosomal elements in escherichia coli.the cgta gene codes for a common gtp-binding protein whose homologues were found in all prokaryotic and eukaryotic organisms investigated so far. although cgta is an essential gene in most bacterial species, its precise functions in the regulation of cellular processes are largely unknown. in escherichia coli, dysfunction or overexpression of the cgta gene causes problems in various chromosomal functions, like synchronization of dna replication initiation and partitioning of daughter chromosomes ...200312826057
osmotic pressure inhibition of dna ejection from phage.bacterial viral capsids in aqueous solution can be opened in vitro by addition of their specific receptor proteins, with consequent full ejection of their genomes. we demonstrate that it is possible to control the extent of this ejection by varying the external osmotic pressure. in the particular case of bacteriophage lambda, the ejection is 50% inhibited by osmotic pressures (of polyethylene glycol) comparable to those operative in the cytoplasm of host bacteria; it is completely suppressed by ...200312881484
pathogenic leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily.proteins with bacterial immunoglobulin-like (big) domains, such as the yersinia pseudotuberculosis invasin and escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. here, we report the identification and characterization of a new family of big domain proteins, referred to as lig (leptospiral ig-like) proteins, in pathogenic leptospira. screening of l. interrogans and l. kirschneri expression libraries with sera from leptospirosis patien ...200312890019
the pair of arginine codons aga agg close to the initiation codon of the lambda int gene inhibits cell growth and protein synthesis by accumulating analyse the mechanism by which rare codons near the initiation codon inhibit cell growth and protein synthesis, we used the bacteriophage lambda int gene or early codon substitution derivatives. the lambda int gene has a high frequency of rare ata, aga and agg codons; two of them (aga agg) located at positions 3 and 4 of the int open reading frame (orf). escherichia coli pth (rap) cells, which are defective in peptidyl-trna hydrolase (pth) activity, are more susceptible to the inhibitory effe ...200312890027
the mobile element is1207 of brevibacterium lactofermentum atcc21086: isolation and use in the construction of tn5531, a versatile transposon for insertional mutagenesis of corynebacterium glutamicum.is1207 is the insertion most frequently found among the spontaneous mutations that abolish the activity of an escherichia coli phage lambda ci gene integrated in the corynebacterium brevibacterium lactofermentum atcc21086 genome. we examined the transposition of transposon-like structures composed of a selective kanamycin resistance gene (aph3), and one or two is1207 sequences. one of these, the tn5531 transposon, transposed efficiently in corynebacterium glutamicum. a replicative and a non-repl ...200312948647
[spontaneous induction of the development of bacteriophage lambda during genetic recombination in escherichia coli k12]. 195413200000
[study of the lysogenization process of e. coli. i. single-cycle development curve of the temperate lambda phage and its virulent mutant lambda v in e. coli k12 s]. 195413236148
interference phenomenon with lambda phage and cellular changes in induced e. coli, k12 (lambda) infected with t2 phage. 195613428420
recombination and phenotypic mixing during phage growth in strains of escherichia coli doubly lysogenic for coliphage lambda. 195813544106
effect of infection with phage lambda on the synthesis of protein, rna and dna in escherichia coli. 196414135546
bacteriophage hk022 nun protein: a specific transcription termination factor that excludes bacteriophage lambda. 200314712713
assignments of 1h and 15n resonances of the bacteriophage lambda capsid stabilizing protein gpd. 200414739644
a novel cell-free protein synthesis efficient cell-free protein synthesis system has been developed using a novel energy-regenerating source. using the new energy source, 3-phosphoglycerate (3-pga), protein synthesis continues beyond 2 h. in contrast, the reaction rate slowed down considerably within 30-45 min using a conventional energy source, phosphoenol pyruvate (pep) under identical reaction conditions. this improvement results in the production of twice the amount of protein obtained with pep as an energy source. we have ...200415163516
the recognition and modification sites for the bacterial type i restriction systems kpnai, styseai, styseni and stysgi.using an in vivo plasmid transformation method, we have determined the dna sequences recognized by the kpnai, styseai, styseni and stysgi r-m systems from klebsiella oxytoca strain m5a1, salmonella eastbourne, salmonella enteritidis and salmonella gelsenkirchen, respectively. these type i restriction-modification systems were originally identified using traditional phage assay, and described here is the plasmid transformation test and computer program used to determine their dna recognition sequ ...200415199175
translation repression by an rna polymerase elongation complex.bacteriophage lambda n and bacterial nus proteins together with a unique site nut in the leader of the early viral n gene transcript bind rna polymerase (rnap) and form a highly processive antitermination complex; n bound at nut also represses n translation. in this study, we investigate whether n and nut cause n translation repression as part of the antitermination complex by testing conditions that inhibit the formation of the n-modified transcription complex for their effect on n-mediated tra ...200415255895
nonspecific binding of the or repressors ci and cro of bacteriophage lambda.we estimate the gibbs free energy for nonspecific binding (deltagnsb) to the escherichia coli dna for two regulatory proteins of the lambda phage, ci and cro. by means of a statistical-mechanical approach, we calculate the ci and cro activities associated with the operator or of an introduced lambda phage genome (prophage). in this statistical model we apply in vitro-measured binding free energies to fit in vivo experimental data for ci and cro activities, respectively, where deltagnsb is introd ...200415488529
electrokinetic bioprocessor for concentrating cells and molecules.bioprocessors for concentrating bioparticles, such as cells and molecules, are commonly needed in bioanalysis systems. in this microfluidic processor, a global flow field generated by ac electroosmosis transports the embedded particles to the regions near the electrode surface. the processor then utilizes electrophoretic and dielectrophoretic forces, which are effective in short range, to trap the target cells and molecules on the electrode surface. by optimizing the operating parameters, we hav ...200415571340
role of c-terminal residues in oligomerization and stability of lambda cii: implications for lysis-lysogeny decision of the phage.a crucial element in the lysis-lysogeny decision of the temperate coliphage lambda is the phage protein cii, which has several interesting properties. it promotes lysogeny through activation of three phage promoters p(e), p(i) and p(aq), recognizing a direct repeat sequence ttgcn6ttgc at each. the three-dimensional structure of cii, a homo-tetramer of 97 residue subunits, is unknown. it is an unstable protein in vivo, being rapidly degraded by the host protease hflb (ftsh). this instability is e ...200515571724
restoration of gene function by homologous recombination: from pcr to gene expression in one step.we have developed a simple method for single-step cloning of any pcr product into a plasmid. a novel selection principle has been applied, in which activation of a drug selection marker is achieved following homologous recombination. in this method a dna fragment is amplified by pcr with standard oligonucleotides that contain flanking tails derived from the host plasmid and the complete lambdapr or rrna1 promoter regions. the resulting pcr product is then electroporated into an escherichia coli ...200415574912
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