expression of prokaryotic genes inserted into cole1 and pvh51 of the dna fragments obtained from ecori digests of guaa-transducing lambda phage dna contains the intact bacterial guaa gene at its one end and the lambda phage r gene at the other end. this dna fragment, named coslambda-guaa, does not contain promoter-operator regions of the gua operon and of the lambda phage r gene, coslambda-guaa dna fragments were inserted in two different orientations into respective dnas of ecori-cleaved cole1 and pvh151 (= mini cole1). mitomycin c stimulated the guaa ...19806444526
purification and characterization of the integration protein specified by bacteriophage lambda. 19806444632
[adsorption of lambda phage dna onto escherichia coli cells treated with ca2+ ions and onto frozen--thawed bacteria].the study of the biologically active tritium-labeled phage lambda dna adosrption on ca2+-treated and frozen--thawed e. coli cells showed the absence of a correlation between the adsorption level and transfection efficiency. thus the infectious phage lambda dna adsorption level does not change, while frozing--thawing of e. coli cells but it increases ten-fold when treating the cells with ca2+ in ice, the transfection efficiency level with this dna being equal for both types of recipients.19806446330
induction of prophage lambda without amplification of reca protein.the requirement for amplified synthesis of reca protein in the uv-promoted induction of coliphage lambda was studied. we confirmed that a low concentration of rifampicin inhibited specifically the increased synthesis of reca protein after an inducing treatment (satta and pardee, 1978). under these conditions, using an optimal dose of uv, e. coli lysogens were induced, producing active phage. the drug delayed the onset of induction and with increasing concentrations affected the yield of phage, b ...19806446647
pseudovirulent mutants of lambda b221poricasna resulting from mutations in or near oric, the e. coli origin of dna replication.mutants of the specialized transducing phage lambda b221poricasna have been isolated which form plaques on lambda lysogens. genetic and physical evidence is presented to show that the mutations responsible for the pseudovirulent phenotype map in or near oric, the origin of chromosomal replication in escherichia coli.19806446650
the phage promoter responsible for the expression of the inserted beta-galactosidase gene in bacteriophage lambda plac5.the lac transducing phage, lambda plac5, carries a segment of the e. coli lac operon on the left side of the b2 region of the lambda phage. in the absence of additional cyclic amp, beta-galactosidase can only be expressed from the phage promoter, and the expression of the inserted lac promoter is suppressed. this phage promoter responsible for beta-galactosidase synthesis is shown to be under the control of the ci and n gene products; however, the repressive action of the cro gene product at hig ...19806446653
transcription antitermination by bacteriophage lambda n gene product. 19806447798
promoter for the establishment of repressor synthesis in bacteriophage lambda.transcription of the lambda repressor gene (ci) is positively regulated by the phage-encoded proteins cii and ciii. we have isolated and characterized the 5'-terminal region of this rna and shown that it originates at a promoter (pe) located between genes cro and cii. the dna sequence of this promoter shows little homology to other known promoters. initiation of transcription from pe is abolished by the cis-dominant mutations cy; these mutations alter the "-10" and "-35" regions of the promoter. ...19806447872
[genetic properties of phage lambda recombinant molecules. the effect of an inserted fragment on the hybrid yield and recombination with respect to the h- and vir-markers].the yield of mature phages in lambda gt-lambda dm34, lambda gt-lambda dm225 and lambda gt-lambda dm234 is 2, 8 and 8 times lower (respectively) as compared to the wild type. the output of hvir-recombinants in lambda gt-lambda dm225 is more than an order lower as compared to the other phages. as distinct from two other hybrids, the decrease in lambda gt-lamba dm225 yield cannot be explained by red-genotype and a shortening of the phage genome as well as the decrease in the output hvir-recombinant ...19806449098
in vitro comparison of initiation properties of bacteriophage lambda wild-type pr and x3 mutant promoters.the in vitro initiation properties of the pr promoter of bacteriophage lambda and of a pr mutant, x3, were compared. using the abortive initiation reaction, we measured the lags in the approach to a final steady-state rate when dinucleotide synthesis was initiated with rna polymerase. these lags corresponded to the average times required for the formation of transcriptionally active open complexes. by measuring the lags at different rna polymerase concentrations, we could separate open complex f ...19806450417
[effect of coumermycin a1 and nalidixic acid on lambda phage integration and transducing lambdoid phages containing rna-polymerase genes].a specific inhibitor of dna gyrase, coumermycin a1, inhibits in vivo the interaction of bacteriophage lambda dna and dna of rifd transducing phages (lambda rifd47 and lambda att80 rifd35) carrying genes rpob and rpoc of rna polymerase. besides, coumermycin a1 inhibits reca-dependent recombination between the bacterial region of rifd phages and a homologous region of escherichia coli chromosome. the integration of transdusing phage dna into the chromosome of reca host is significantly more sensit ...19806450709
thermal (52 degrees c) inactivation and mutagenesis of bacteriophage lambda. 19806450895
[low transfecting efficiency of phage lambda ring chromosomes and their fragments formed by membrane nucleases].transfection efficiency of a number of lambda dna samples differing in ring to linear molecules ratio was determined. graphic extrapolation to the zero content of linear molecules showed that efficiency of ring molecules did not exceed 5% of that of linear molecules. probably, this difference is caused by more fast penetration of linear molecules into the cell and, therefore, by lower probability of their degradation by cell wall nucleases. fragments of both ring and linear molecules formed by c ...19816453042
evidence that ribosomal protein s10 participates in control of transcription termination.we report the isolation of an escherichia coli k-12 strain with a mutation, nuse71, that results in a change in ribosomal protein s10. phage lambda fails to grow in hosts carrying the nuse71 mutation because the lambda n gene product is not active. the n product regulates phage gene expression by altering transcription complexes so that they can overcome termination barriers. this suggests that a ribosomal protein is involved in antitermination of transcription.19816453343
purification of bacteriophage lambda o protein that specifically binds to the origin of means of a nitrocellulose filter binding assay, dna binding activities among proteins fractionated from extracts of escherichia coli carrying lambda dv have been surveyed. an activity was found that binds specifically to a fragment of 164 base pairs that specifies the lambda replication origin (lambda ori). this activity was not detected in an extract of cells not carrying the lambda dv plasmid. the activity was detected in extracts of cells carrying a hybrid plasmid in which the entire lambd ...19816454055
arrangement of bacteriophage lambda receptor protein (lamb) in the cell surface of escherichia coli: a reconstitution study.the lamb protein purified in a solution of sodium dodecyl sulfate was assembled into an ordered hexagonal lattice structure with a lattice constant of about 7.8 nm in the presence of lipopolysaccharide. the lamb alone formed aggregates with some lattice structure. however, the regularity of the lattice was only maintained within a very small area. an ordered hexagonal lattice was also formed when the wild-type lipopolysaccharide was replaced by heptoseless lipopolysaccharide, lipid a, and even f ...19816455415
fusion of the lac genes to the promotor for the cytidine deaminase gene of escherichia coli k-12.phage mu has been inserted into the structural gene for cytidine deaminase (cdd). by the use of phage lambda (lac, mu) the promoter for the cdd gene has been fused to lacz. in these strains lacz expression is regulated by the cytr repressor protein and is therefore induced by cytidine. the fusion strains were used for the isolation of cddo mutants. plaque forming lambda phages carrying the different cdd-lacz fusions were isolated. studies of the cdd-mu strains showed that the cdd gene is transcr ...19816455590
structure and function of the phage lambda att site: size, int-binding sites, and location of the crossover point. 19816457725
effects of recb21, recf143, and uvrd152 on recombination in lambda bacteriophage-prophage and hfr by f- crosses.the effects of the mutation pairs recb21 recf143 and recb21 uvrd152 on the frequency of genetic recombination were investigated in lambda phage-prophage crosses under homoimmune conditions. to prevent recombinants from being formed by the phage red system, these experiments were performed with phages and prophages carrying red and gam mutations. both spontaneous and damage-induced recombination was measured, the phages being either undamaged or treated with trimethylpsoralen and 360-nm light to ...19816457825
preferential cleavage of phage lambda repressor monomers by reca protease. 19816457991
general method for fine mapping of the escherichia coli k-12 lamb gene: localization of missense mutations affecting bacteriophage lambda adsorption.lamb is the structural gene for the bacteriophage lambda receptor, a multifunctional protein located in the outer membrane of escherichia coli k-12. we present a method for deletion mapping of any lamb mutations with a recognizable pheno-type. this method involves a transducing phage constructed by in vitro recombination which can also be used for complementation, deoxyribonucleic acid sequence, and in vitro protein synthesis studies with the mutated lamb gene. using this method, we mapped 18 la ...19816458595
direction of bacteriophage lambda dna replication in a thymine requiring escherichia coli k-12 strain. effect of thymidine concentration.the direction of replication was established for the first round of bacteriophage lambda dna replication in thymine requiring e. coli k-12 cells exposed to different concentrations of thymidine. it was found that a dramatic decrease in the proportion of bidirectionally replicating molecules followed a decrease in the concentration of thymidine. moreover, the rightward mode of replication appears to be exclusively favored in unidirectionally replicating molecules found at low concentrations of th ...19816460985
studies on the association of e. coli phage lambda dna and the host chromosome: lack of a role of membranes. 19826461964
a system for genetic analysis in gene lamb: first results with lambda-resistant tight mutants.we describe a system for genetic analysis in gene lamb. it consists of a phage which allows mapping, complementation and sequencing studies of lamb mutations and of the sequence of gene lamb. we present results obtained with this system for a set of mutations conferring tight resistant to phage lambda. this leads to a first identification of three residues in the lamb protein which are important for adsorption of phage lambda h+. residues 151 and 382 are important for reversible adsorption while ...19826462090
expression of pyruvate formate-lyase of escherichia coli from the cloned structural is shown here that a plasmid (p29) derived from the transducing phage lambda aspc2 (christiansen and pedersen 1981) codes for pyruvate formate-lyase. the identity of the 80 kilodaltons (kd) gene product of plasmid p29 with the pyruvate formate-lyase polypeptide was proven (i) by co-migration of the gene product expressed in the maxicell system with purified enzyme on o'farrell gels, and (ii) by comparison of the peptide maps obtained from limited proteolysis. in vivo the 80 kd form of the enz ...19826758723
sos induction and autoregulation of the hima gene for site-specific recombination in escherichia coli.the hima gene of escherichia coli controls the lysogenization of bacteriophage lambda at the level of catalysis of site-specific recombination and expression of the lambda int and ci genes required for lysogenic development. we have analyzed the regulation of hima by two methods: (i) beta-galactosidase synthesis from a lacz gene inserted into the hima gene and (ii) detection of radioactive hima protein after fractionation by two-dimensional gel electrophoresis. we find that hima- mutations produ ...19816796964
open reading frame cloning: identification, cloning, and expression of open reading frame dna.a plasmid was constructed that facilitates the cloning and expression of open reading frame dna. a dna fragment containing a bacterial promoter and the amino terminus of the ci gene of bacteriophage lambda was fused to an amino-terminally deleted version of the lacz gene. an appropriate cloning site was inserted between these two fragments such that a frameshift mutation was introduced upstream of the lacz-encoding dna. this cloning vehicle produces a relatively low level of beta-galactosidase a ...19826815653
regulation of phospholipid biosynthesis in escherichia coli. cloning of the structural gene for the biosynthetic sn-glycerol-3-phosphate dehydrogenase.the structural gene for the escherichia coli biosynthetic sn-glycerol-3-phosphate (glycerol-p) dehydrogenase gpsa, was transferred from a defective transducing phage (lambda dcyse, gpsa) into the eco ri site of plasmid pmb9 by recombinant dna techniques. the recombinant plasmids suppressed the glycerol-p requirement of gpsa- mutants and strains bearing one such plasmid, pdc2, overproduced the glycerol-p dehydrogenase about 60-fold. the glycerol-p dehydrogenase from a strain bearing the pdc2 was ...19806985897
a dnab analog function specified by bacteriophage p7 and its comparison to the similar function specified by bacteriophage p1.evidence is presented that bacteriophage p7 specifies an analog of the e. coli dna replication protein, dnab. as in the related bacteriophage p1 (d'ari et al., 1975; ogawa, 1975), in lysogens of p7, the production of the analog protein is repressed and constitutive mutants could be isolated. such constitutive mutants could suppress efficiently the thermosensitivity of several dnab(ts) mutations and also rescue a strain carrying a dnab amber mutation. while neither p7 nor the mutant p1bacban (def ...19806993853
replication control and switch-off function as observed with a mini-f factor is a fragment of the f plasmid, consisting of 9,000 base pairs, which carries all of the genes and sites required for replicon maintenance and control. its copy number is one to two per chromosome. this plasmid is joined to cole1, whose copy number is 16 to 20. under normal circumstances the composite plasmid replication exhibited cole1 characteristics, maintaining a high copy number. however, when cole1 replication was inhibited by deoxyribonucleic acid polymerase i inactivation, its rep ...19817021532
inducibility of a gene product required for uv and chemical mutagenesis in escherichia coli.the product of the umuc gene is required for uv and chemical mutagenesis in escherichia coli. by the use of the mud(ap, lac) bacteriophage, we have obtained an operon fusion of the lac structural genes to the promoter/regulatory region of the umuc gene. the strain containing the umuc::mud(ap, lac) fusion was identified on the basis of its uv nonmutability. strains containing this putative null allele of umuc were (i) nonmutable by uv and other agents, (ii) slightly uv sensitive, and (iii) defici ...19817029544
tif-1 mutation alters polynucleotide recognition by the reca protein of escherichia coli.the requirements for polynucleotide-dependent hydrolysis of atp and for proteolytic cleavage of phage lambda repressor have been examined for both the wild-type (reca+ protein) and the tif-1 mutant form [tif(reca) protein] of the reca gene product. the reca+ and tif(reca) proteins catalyze both reactions in the presence of long single-stranded dnas or certain deoxyhomopolymers. however, short oligonucleotides [(dt)12, (da)14] stimulate neither the protease nor the atpase activities of the reca+ ...19817031642
extensive sequence homology in the dna coding for elongation factor tu from escherichia coli and the chlamydomonas reinhardtii chloroplast.considerable dna sequence homology can be detected between the escherichia coli genes coding for translational components and chlamydomonas reinhardtii chloroplast dna. labeled chloroplast dna was found to hybridize to restriction fragments of the transducing phage lambda fus3 that code for elongation factor tu. the chloroplast probe also reacts with fragments coding for ribosomal proteins carried by this phage. the region homologous to the elongation factor genes was located on the physical map ...19827048316
klebsiella and enterobacter strains derived from hospital infections. ii. occurrence and characterization of r-, lac- and col- plasmids and their clinical-epidemiological significance.a total of 269 hospital klebsiella strains and 103 hospital enterobacter strains showed 34 and 10 different antibiotic resistance patterns, respectively. among multiple resistant klebsiella and enterobacter strains the ap sm cm tc resistance pattern was the most frequent (k. aerogenes). antibiotic resistant strains carried r-plasmids in 27.5%. the presence of r-plasmids was demonstrable in 2.9% of single antibiotic resistant, in 12.8% of double antibiotic resistant, and in 71.4% of multiple anti ...19817257874
coliphage hk243: biological and physicochemical characteristics.coliphage hk243 can form plaques on escherichia coli c and k-12, but not b. the plaques are 1-2 mm in diameter and are opaque areas which clear upon exposure to chloroform vapor. during one-step growth, the eclipse and the latent periods are 20 and 30 min, respectively. phage-infected cells continue to produce cell-free plaque-forming units for as long as 80 min after the end of the latent period, although at high multiplicities of infection (moi) most cells lyse. no lysogenic bacteria have been ...19807412594
glms of thermus thermophilus hb8: an essential gene for cell-wall synthesis identified immediately upstream of the s-layer gene.a 30 kbp chromosomal region containing the s-layer gene (slpa) from thermus thermophilus hb8 was cloned from a lambda phage gene library. dna sequence analysis of the region upstream to the slpa gene revealed the presence of an open reading frame (orf) which coded for a 604-amino-acid protein highly homologous to the glucosamine-6-p synthases (ec of both prokaryotic and eukaryotic origin. the identification of this orf as the glucosamine-6-p synthase gene from t. thermophilus (glmsth) ...19957476196
control of transcription processivity in phage lambda: nus factors strengthen the termination-resistant state of rna polymerase induced by n antiterminator.during transcription of phage lambda early operons, the n gene product alters host rna polymerase (rnap) so that transcription proceeds through multiple stop signals. here, we reproduce the essence of n activity with purified components in synthetic transcription units that contain lambda pl promoter and the n-recognition site, nutl, followed by a variety of intrinsic terminators. we show that three host factors (nusa, nuse, and nusg) are essential for n to allow appreciable transcription throug ...19947521531
differential replication of plasmids during stringent and relaxed response of escherichia coli.stringent control of dna replication has been demonstrated for a few replicons like oric, pbr322, and plasmids derived from coliphage lambda. in this study we investigated the replication of other plasmids harboring a well defined origin (orip15a, oripsc101, and orirk2 = oriv) in amino-acid-starved stringent and relaxed strains of escherichia coli. we found differential replication of plasmids during stringent and relaxed response. inhibition of dna synthesis or amplification of plasmid dna in a ...19947527562
a role for a small stable rna in modulating the activity of dna-binding proteins.the 10sa rna, encoded by the e. coli ssra gene, appears to modulate action of some dna-binding proteins. when ssra is inactivated, lacz expression from the lac operon, as well as galk from a gal operon fused to a phage lambda promoter, is reduced from that observed in bacteria wild-type for ssra. these differences are not observed if the relevant repressor is inactive, suggesting that in the absence of 10sa rna binding of laci and lambda ci repressors is enhanced. gel mobility shifts show that 1 ...19957585940
analysis of the dnak molecular chaperone system of francisella tularensis.we have cloned the francisella tularensis (ft) grpe-dnak-dnaj heat-shock genes which are organized in that order. these genes allow heterologous genetic complementation of each respective mutant strain of escherichia coli (ec) for bacteriophage lambda growth. the nucleotide sequences of the ft grpe-dnak-dnaj genes and the deduced amino-acid sequences share significant homologies with their respective ec counterparts. the ft dnak and dnaj proteins cross-react with polyclonal antibodies raised aga ...19957590305
the recombination hot spot chi activates recbcd recombination by converting escherichia coli to a recd mutant phenocopy.the products of the recb and recc genes are necessary for conjugal recombination and for repair of chromosomal double-chain breaks in escherichia coli. the recd gene product combines with the recb and recc proteins to comprise recbcd enzyme but is required for neither recombination nor repair. on the contrary, recbcd enzyme is an exonuclease that inhibits recombination by destroying linear dna. the recd ejection model proposes that recbcd enzyme enters a dna duplex at a double-chain end and trav ...19957603978
rate of translocation of bacteriophage t7 dna across the membranes of escherichia coli.translocation of bacteriophage t7 dna from the capsid into the cell has been assayed by measuring the time after infection that each gatc site on the phage genome is methylated by cells containing high levels of dna adenine methylase. methylation at gatc sites on t7 dna renders both the infecting genome and any newly synthesized molecules sensitive to the restriction enzyme dpni. in a normal infection at 30 degrees c, translocation of the t7 genome into the cell takes between 9 and 12 min. in co ...19957608081
temperature induction of bacteriophage lambda mutants in escherichia coli.the paper presents temperature induction in escherichia coli cells with phage lambda on the target-protein production and cell growth. replicated lambda-dna particles in the q- and s- mutants remain naked for a longer time by preventing dna packaging and cell lysis, and therefore the expression of the foreign genes is high. however, the parasitic infection of phage-lambda causes on significant losses of host cell viability in the induction phase. the temperature effects on cell growth and target ...19957612243
gene cloning, sequence analysis, purification, and secretion by escherichia coli of an extracellular lipase from serratia marcescens.the gene encoding extracellular lipase of serratia marcescens has been identified from a phage lambda genomic library. formation of orange-red fluorescent plaques on rhodamine b-triolein plates was used to identify phages carrying the lipase gene. a 2.8-kb sali fragment was subcloned into a plasmid, and lipase was expressed in escherichia coli. extracellular lipase was detected in the presence of the secretion plasmid pgsd6 carrying the genes prtd, -e, and -f, which guide the secretion of protea ...19957618881
expression, purification and characterisation of the product from the bacillus subtilis hemd gene, uroporphyrinogen iii synthase.uroporphyrinogen iii synthase, the product of the hemd gene, is the enzyme responsible for the cyclisation of the linear tetrapyrrole, hydroxymethylbilane. the hemd gene isolated from bacillus subtilis was manipulated by pcr to enable direct cloning behind a synthetic ribosome-binding site downstream of tandem bacteriophage lambda pr and pl promoters in a pce30-derived vector. following thermal induction of transcription, the resulting plasmid (pps21) directed the synthesis of uroporphyrinogen i ...19957628476
recognition of boxa antiterminator rna by the e. coli antitermination factors nusb and ribosomal protein s10.the boxa sequences of the e. coli ribosomal rna (rrn) operons are sufficient to cause rna polymerase to read through rho-dependent transcriptional terminators. we show that a complex of the transcription antitermination factors nusb and ribosomal protein s10 interacts specifically with boxa rna. neither nusb nor s10 binds boxa rna on its own, and neither nusa nor nusg affects the interaction of the nusb-s10 complex with boxa rna. mutations in boxa that impair its antitermination activity comprom ...19937678781
in vivo regulatory responses of four escherichia coli operons which encode leucyl-trnas.four escherichia coli operons, the leuv operon which encodes trna(1leu), the leux operon which encodes trna(6leu), the mett operon which encodes trna(3leu), and the argt operon which encodes trna(1leu), were examined for the stringent response induced by serine hydroxamate and for growth rate-dependent regulation. in nuclease protection assays, the leuv operon displayed the stringent response in response to leucine starvation, analog inhibition, and growth of a temperature-sensitive leucyl-trna ...19937680341
the rhizobium meliloti groelc locus is required for regulation of early nod genes by the transcription activator nodd.the molecular chaperones related to groel (hsp60, cpn60) interact with partially folded proteins and appear to assist them to attain active and correctly folded conformation. they are required for cell viability but are probably more important for some processes than for others. through a random genetic search to find loci that are required for expression of the rhizobium meliloti nod (nodulation) genes, we isolated a mutant (b4) defective in luteolin-dependent activation of nod gene expression, ...19957729688
the requirement for molecular chaperones in lambda dna replication is reduced by the mutation pi in lambda p gene, which weakens the interaction between lambda p protein and dnab helicase.during the initiation of lambda dna replication, the host dnab helicase is complexed with phage lambda p protein in order to be properly positioned near the ori lambda-lambda o initiation complex. however, the lambda p-dnab interaction inhibits the activities of dnab. thus, the concerted action of bacterial heat shock proteins, dnak, dnaj, and grpe, is required to activate the helicase. wild-type phage lambda cannot grow on the e. coli dnab, dnak, dnaj, and grpe mutants. however, lambda phage wi ...19957730358
cloning and expression of cdna encoding a protein that binds a palindromic promoter element essential for induction of fungal cutinase by plant cutin.previous studies showed that a palindromic sequence located at -159 base pairs is essential for induction of cutinase gene in fusarium solani f. sp. pisi (nectria haematococca mating type vi) by the hydroxy fatty acids from plant cutin and that a 50-kda nuclear protein binds to a promoter that contains this element. screening of a phage lambda gt11 expression library with the concatenated palindromic sequence as the probe identified a cdna encoding a palindrome-binding protein (pbp). nucleotide ...19957744822
production of a biologically active recombinant teleostean growth hormone in e. coli cells.we have isolated and characterized several recombinant lambda phage clones carrying growth hormone (gh) cdna of striped bass (morone saxatilis). nucleotide sequence and the predicted amino acid sequence of sbgh was determined from a recombinant clone carrying the longest cdna insert. the sbgh cdna encodes a pre-hormone of 204 amino acid residues. comparison of the predicted amino acid sequence of sbgh with those of other vertebrates revealed different degrees of sequence identity: approximately ...19957758842
characterization of gene expression in recombinant escherichia coli cells infected with phage lambda.phage lambda infection was investigated for possible production of toxic foreign proteins in escherichia coli. the target gene transcription was regulated by a t7 promoter, which was initiated under the action of t7 rna polymerase delivered by infecting phage. two types of phage infection were investigated. in both cases, deletion of the int gene prevents lysogenic integration. one phage, lambda ce6, contains the sam7 lysis mutation, so that infectious phage particles remain intracellular. the o ...19937763591
phage t4 dna [n6-adenine]methyltransferase. overexpression, purification, and characterization.the bacteriophage t4 dam gene, encoding the dam dna [n6-adenine]methyltransferase (mtase), has been subcloned into the plasmid expression vector, pjw2. in this construct, designated pint4dam, transcription is from the regulatable phage lambda pr and pl promoters, arranged in tandem. a two-step purification scheme using deae-cellulose and phosphocellulose columns in series, followed by hydroxyapatite chromatography, was developed to purify the enzyme to near homogeneity. the yield of purified pro ...19957782299
cloning of the na(+)-translocating nadh-quinone reductase gene from the marine bacterium vibrio alginolyticus and the expression of the beta-subunit in escherichia coli.the na(+)-translocating nadh-quinone reductase purified from the marine bacterium vibrio alginolyticus is composed of three subunits, alpha, beta and gamma. from the n-terminal amino acid sequences of each subunit and its polypeptide fragment obtained by partial digestion with v8 protease, oligonucleotides corresponding to forward and reverse primers for each gene (nqr a, b and c) encoding the alpha, beta and gamma subunit, respectively, were synthesized. using these primers, a part of each gene ...19947805866
very short patch repair of t:g mismatches in vivo: importance of context and accessory escherichia coli, t:g mismatches in specific contexts are corrected by a very short patch (vsp) repair system. previous studies have shown that the product of gene vsr mediates correction of t:g to c:g in the 5'ctagg/3'ggtcc context and in some related contexts. amber mutations that arose in cag sequences in gene ci of bacteriophage lambda were used to determine the effect of flanking bases on the repair of t:g mispairs arising during phage recombination. the experimental findings were combin ...19957836300
repair of phage lambda dna damaged by near ultraviolet light plus 8-methoxypsoralen.treatment of phage lambda with 8-methoxypsoralen plus near ultraviolet light (puva) and its subsequent infection and growth on various mutant and non-mutant hosts were investigated. a number of escherichia coli dna repair-deficient mutants, particularly those deficient in genes producing proteins known to participate in interstrand crosslink repair, were used as hosts to assess the roles of these gene products in the activation of phage affected by puva. results show that puva, uvra, uvrd, reca, ...19957897361
the gene fimu affects expression of salmonella typhimurium type 1 fimbriae and is related to the escherichia coli trna gene argu.the gene fimu, located on a recombinant plasmid carrying the salmonella typhimurium type 1 fimbrial gene cluster is closely related to the escherichia coli trna gene argu. the fimu gene complements an e. coli argu mutant that is a p2 lysogen, thereby allowing the phage p4 to grow in this strain but preventing the growth of phage lambda. in addition, fimu was shown to be involved in fimbrial expression since transformants of the e. coli argu mutant could produce fimbriae only in the presence of f ...19947914347
a novel escherichia coli expression-export vector containing alkaline phosphatase as an insertional inactivation screening escherichia coli expression-export vector was constructed (pcgv1, 6.3 kb) containing the alkaline phosphatase structural gene (phoa) located downstream from the phage lambda pr and pl promoters positioned in tandem and the cits857 gene encoding lambda thermosensitive repressor. the phoa gene is fused to dna encoding a hybrid signal sequence that contains the n-terminal portion of the beta-lactamase (bla) signal sequence and the c-terminal region of the phoa signal sequence. within the dna enc ...19947926833
cloning and expression of the gene for group b streptococcal hyaluronate b streptococci (gbs) are a major cause of serious human perinatal infections. most clinical isolates of gbs secrete hyaluronate lyase, and production of high levels of the enzyme has been associated with strain virulence. degenerate oligonucleotide primers, designed on the basis of the amino acid sequences of tryptic peptides prepared from the purified enzyme, permitted the polymerase chain reaction amplification from gbs chromosomal dna of a 363-base pair internal dna fragment of the gbs ...19947982914
topology of the product binding site in rna polymerase revealed by transcript slippage at the phage lambda pl the presence of transcription substrates atp, ctp, and utp, a stable ternary complex containing tetranucleotide auca is formed on the phage lambda pl promoter (starting sequence c-3a-2c-1a+1u+2c+3a+4g+5). we show that in the absence of gtp or at undersaturating gtp concentrations the auca transcript synthesized at the +1 to +4 segment slips back by 3 nucleotides and is stabilized in the ternary complex in such a way that only its 2 3'-proximal bases remain paired to the -1/+1 positions of the ...19947989343
high-level expression of staphylococcal nuclease a in escherichia coli.the staphylococcal nuclease a gene has been successfully cloned and overexpressed in e. coli under the transcriptional control of the bacteriophage lambda prpl promoters regulated by the temperature sensitive repressors. the sds-page analysis demonstrates that the nuclease a is produced to the extent of as much as 60% of the total cellular protein. the n-terminal analysis of the nuclease a shows that the amino terminal formyl methionine residue of the enzyme is precisely processed. the recombina ...19947993969
the vibrio cholerae toxin-coregulated-pilus gene tcpi encodes a homolog of methyl-accepting chemotaxis proteins.virulence gene activation in vibrio cholerae is under the control of the toxr-toxt regulatory cascade. the toxr regulon consists of genes required for toxin-coregulated-pilus (tcp) biogenesis, accessory colonization factor genes, cholera toxin genes, and toxr-activated genes (tag) of unknown function. the tagb gene was isolated by using a tagb::tnphoa fusion junction to probe a v. cholerae )395 bacteriophage lambda library. nucleotide sequence analysis revealed that tagb is identical to tcpi, a ...19948005659
inactivation and repair of bacteriophage lambda by furfural.furfural is a dietary mutagen and is present in various frequently consumed food products. in earlier work we have shown that it induces single strand breaks in duplex dna which occur preferentially in at base pairs. experiments on the conservation of ecori cleavage site in mutant plasmids further established that repair of furfural damaged plasmid dna occurs on propagation in the host cells. in this paper it is shown that the strand scissions induced by furfural in dna account for its biologica ...19948019442
functions involved in bacteriophage p2-induced host cell lysis and identification of a new tail gene.successful completion of the bacteriophage p2 lytic cycle requires phage-induced lysis of its escherichia coli host, a process that is poorly understood. genetic analysis of lysis-deficient mutants defined a single locus, gene k, which lies within the largest late transcription unit of p2 and maps between head gene l and tail gene r. we determined and analyzed the dna sequence of a ca. 2.1-kb ecorv fragment that spans the entire region from l to r, thus completing the sequence of this operon. th ...19948051010
[bacteriophage lambda:lux: design and expression of bioluminescence in e. coli cells].the bacteriophages lambda:lux and lambda:luxab have been constructed by ligation of phage arms generated by bamhi or salgi restriction endonucleases digestion of embl4 to bamhi digested plasmid pf1 lux+ or to salgi digested plasmid pf2 lambda:luxa+b+. cells of escherichia coli prototrophic strain cs were infected with lambda:lux or lambda:luxab and intensity of bioluminiscence of the samples registered at different time intervals determined. the signal of bioluminiscence was first detected 15 mi ...19948065385
synthesis and assembly of the f0 proton channel from f0 genes cloned into bacteriophage lambda and integrated into the escherichia coli chromosome.the promoter region and the first four genes of the escherichia coli proton-translocating atpase (unc) operon, uncibef, were cloned into bacteriophage lambda, enabling this region to be recombined into an unc-deleted e. coli chromosome at the lambda att site. the resultant e. coli strain, carrying single-copy f0 genes, was tested for synthesis and assembly of functional f0 proton channels. membranes isolated from this strain contained all three f0 subunits and were capable of binding purified f1 ...19948125942
generation of bacteriophage lambda lysogens by electroporation. 19948179876
generating bacteriophage lambda sublibraries enriched for rare clones. 19948185914
bacterially expressed fabs of monoclonal antibodies neutralizing tumour necrosis factor alpha in vitro retain full binding and biological activity.antibody fragments specific for the human tumour necrosis factor alpha (tnf alpha) have been cloned from lambda combinatorial expression libraries using total rna obtained from three different hybridoma cell lines of therapeutic interest. the previously described bacteriophage lambda vectors, lambda hc2 and lambda lc1, were modified to create unique antibody cloning sites in the combinatorial construct and a novel tag peptide was inserted at the c-terminal end of the expressed fd chain. sequence ...19938232337
expression and biochemical properties of a protein serine/threonine phosphatase encoded by bacteriophage lambda.the predicted amino acid sequence encoded by the open reading frame 221 (orf221) of bacteriophage lambda exhibited a high degree of similarity to the catalytic subunits of a variety of protein serine/threonine phosphatases belonging to pp1, pp2a, and pp2b groups. cloning and expression of the orf221 gene in escherichia coli provided direct evidence that the gene codes for a protein serine/threonine phosphatase. the single-subunit recombinant enzyme was purified in soluble form and shown to posse ...19938248155
from the double-helix to novel approaches to the sequencing of large genomes.elucidation of the structure of dna by watson and crick [nature 171 (1953) 737-738] has led to many crucial molecular experiments, including studies on dna replication, transcription, physical mapping, and most recently to serious attempts directed toward the sequencing of large genomes [watson, science 248 (1990) 44-49]. i am totally convinced of the great importance of the human genome project, and toward achieving this goal i strongly favor 'top-down' approaches consisting of the physical map ...19938276270
an analogue of the dnaj molecular chaperone in escherichia coli.escherichia coli dnaj functions as a typical molecular chaperone in coordination with other heat shock proteins such as dnak and grpe in a variety of cellular processes. in this study, it was found that e. coli possesses an analogue of dnaj, as judged from not only its primary structure but also its possible function. this protein, named cbpa (for curved dna-binding protein), was first identified as a dna-binding protein that preferentially recognizes a curved dna sequence. cloning and nucleotid ...19948302830
targeted mutagenesis of dna using triple helix-forming oligonucleotides linked to psoralen.oligonucleotides can bind as third strands of dna in a sequence-specific manner in the major groove in homopurine/homopyrimidine stretches in duplex dna. here we use a 10-base triplex-forming oligonucleotide linked to a psoralen derivative at its 5' end to achieve site-specific, targeted mutagenesis in an intact, double-stranded lambda phage genome. site-specific triplex formation delivers the psoralen to the targeted site in the lambda dna, and photoactivation of the psoralen produces adducts a ...19938356097
localization and nucleotide sequences of genes mediating site-specific recombination of the slp1 element in streptomyces lividans.slp1 is a 17.2-kbp genetic element indigenous to the streptomyces coelicolor chromosome. during conjugation, slp1 can undergo excision and subsequent site-specific integration into the chromosomes of recipient cells. we report here the localization, nucleotide sequences, and initial characterization of the genes mediating these recombination events. a region of slp1 adjacent to the previously identified site of integration, attp, was found to be sufficient to promote site-specific integration of ...19938387993
identification of algf in the alginate biosynthetic gene cluster of pseudomonas aeruginosa which is required for alginate acetylation.mucoid strains of pseudomonas aeruginosa produce a high-molecular-weight exopolysaccharide called alginate that is modified by the addition of o-acetyl groups. to better understand the acetylation process, a gene involved in alginate acetylation called algf was identified in this study. we hypothesized that a gene involved in alginate acetylation would be located within the alginate biosynthetic gene cluster at 34 min on the p. aeruginosa chromosome. to isolate algf mutants, a procedure for loca ...19938394313
expression of the escherichia coli ftsz gene: trials and tribulations of gene fusion studies.the ftsz gene of escherichia coli, which codes for an essential cell division protein, is subjected to multiple regulation, as shown in part with studies using an ftsz::lacz operon fusion located on phage lambda jfl100. using this same fusion, we sought to isolate regulatory mutants overexpressing ftsz by selecting mutants able to grow on lactose. one lac+ mutant was obtained which overexpressed the ftsz::lacz fusion 70-fold. the mutation responsible for the overexpression lies in a new gene, co ...19938468005
biosynthetic incorporation of 7-azatryptophan into the phage lambda lysozyme: estimation of tryptophan accessibility, effect on enzymatic activity and protein stability.the phage lambda lysozyme (lambda l) contains four tryptophans. these have been efficiently replaced by 7-azatryptophan (7aw) through biosynthetic incorporation into the overexpressed protein. comparative analysis of the effect of temperature or ph on the fluorescence of the wild-type lambda l and 7aws-containing protein (a lambda l) shows that the stability of the protein is only mildly reduced by 7aw incorporation above ph 5 but that it is strongly decreased below ph 4 on protonation of inacce ...19958532666
functional analysis of isolated cpn10 domains and conserved amino acid residues in spinach chloroplast co-chaperonin by site-directed mutagenesis.the possibilities of independent function of the two chaperonin 10 (cpn10) domains of the cpn10 homologue from spinach chloroplasts and the role of five conserved amino acid residues in the n-terminal cpn10 unit were investigated. recombinant single domain proteins and complete chloroplast cpn10 proteins carrying amino acid exchanges of conserved residues in their n-terminal cpn10 domain were expressed in escherichia coli and partially purified. the function of the recombinant proteins was teste ...19958555447
transformation of naturally competent streptococcus mutans with replicative and non-replicative tn916-containing plasmids: implications for a mechanism of transposition.based on the observations reported here and what is known concerning transformation of naturally competent strains of s. mutans and other streptococcal species such as s. gordonii, we propose the model shown in figure 2. the tn916-intermediate transforms s. mutans as originally proposed for b. subtilis by scott and coworkers [8]. it is not clear in either system (b. subtilis or s. mutans) whether the tn916 intermediate enters the cell as ds-dna or ss-dna. because it is likely that transformation ...19958586174
role of escherichia coli rna polymerase alpha subunit in modulation of pausing, termination and anti-termination by the transcription elongation factor nusa.the alpha subunit (alpha) of rna polymerase (rnap) is critical for assembly of polymerase and positive control of transcription initiation in escherichia coli. here, we report that alpha also plays a role in transcription elongation, and this involves a direct interaction between alpha and nusa factor. during in vitro transcription without nusa, alpha interacts with the nascent rna, as revealed by photocrosslinking. when nusa is present, rna crosslinks to nusa rather than to alpha. we show that ...19968598198
long-circulating bacteriophage as antibacterial agents.the increased prevalence of multidrug-resistant bacterial pathogens motivated us to attempt to enhance the therapeutic efficacy of bacteriophages. the therapeutic application of phages as antibacterial agents was impeded by several factors: (i) the failure to recognize the relatively narrow host range of phages; (ii) the presence of toxins in crude phage lysates; and (iii) a lack of appreciation for the capacity of mammalian host defense systems, particularly the organs of the reticuloendothelia ...19968622911
escherichia coli thymidylate kinase: molecular cloning, nucleotide sequence, and genetic organization of the corresponding tmk locus.thymidylate kinase (dtmp kinase; ec catalyzes the phosphorylation of dtmp to form dtdp in both de novo and salvage pathways of dttp synthesis. the nucleotide sequence of the tmk gene encoding this essential escherichia coli enzyme is the last one among all the e. coli nucleoside and nucleotide kinase genes which has not yet been reported. by subcloning the 24.0-min region where the tmk gene has been previously mapped from the lambda phage 236 (e9g1) of the kohara e. coli genomic library ...19968631667
identification of functional regions of the nun transcription termination protein of phage hk022 and the n antitermination protein of phage lambda using hybrid nun-n genes.phages lambda and hk022 express proteins n and nun, respectively, each of which acts with a number of escherichia coli host nus factors at lambda nut rna sites, to influence transcription elongation. the lambda nut sites, nearly identical sequences located downstream of the early promoters, pl and pr, were first identified as cis-acting signals required for the action of n in forming termination-resistant transcription complexes. surprisingly, the nun protein, resembling n and expressed by anoth ...19968632463
the rna chain elongation rate of the lambda late mrna is unaffected by high levels of ppgpp in the absence of amino acid this study, the effects of high levels of guanosine tetraphosphate (ppgpp) on the decay and rna chain elongation kinetics of the bacteriophage lambda late transcript in escherichia coli were examined in the absence of amino acid starvation. the accumulation, mrna decay kinetics, and rna chain elongation rate of the lambda late mrna were determined after heat induction of lambdaci857 lysogens in the presence of high levels of ppgpp induced from a relaalpha fragment-overproducing plasmid. the a ...19968663373
rho-dependent termination of transcription is governed primarily by the upstream rho utilization (rut) sequences of a terminator.a rho-dependent transcription terminator in escherichia coli dna consists of an upstream part for rho utilization (rut) and the transcription stop point (tsp) region. to test the role of the tsp region variants of the coliphage lambda cro gene terminator, tr1, containing inserts of non-terminator sequences between its rut and tsp regions were tested for termination function. the results showed that termination occurred with high efficiency at multiple sites in each of the new sequences with the ...19968702947
transcription antitermination: the lambda paradigm updated.coliphage lambda employs systems of transcription termination and antitermination to regulate gene expression. early gene expression is regulated by the phage-encoded n protein working with a series of escherichia coli proteins, nus, at rna sites, nut, to modify rna polymerase to a termination-resistant form. expression of lambda late genes is regulated by the phage-encoded q antitermination protein. q, which appears to use only one host factor, acts at a dna site, qut, to modify rna polymerase ...19958709839
function of e. coli rna polymerase sigma factor sigma 70 in promoter-proximal pausing.the sigma factor sigma 70 of e. coli rna polymerase acts not only in initiation, but also at an early stage of elongation to induce a transcription pause, and simultaneously to allow the phage lambda gene q transcription antiterminator to act. we identify the signal in dna that induces early pausing to be a version of the sigma 70 -10 promoter consensus, and we show that sigma 70 is both necessary for pausing and present in the paused transcription complex. regions 2 and 3 of sigma 70 suffice to ...19968756730
[organization of the gene for the beta-subunit of human photoreceptor cyclic gmp phosphodiesterase].two recombinant bacteriophage lambda clones encoding a 27.8-kb fragment of the human phosphodiesterase beta-subunit gene were isolated from a human genomic library. the nucleotide sequences of 19 exons (from the 4th to 22nd), 18 introns, and the 3'-flanking region were determined. the analysis of the nucleotide sequence of the phosphodiesterase beta-subunit gene revealed four alu repeats and four minisatellite regions.19968768262
device for the simultaneous screening of bacteriophage lambda clones. 19968770396
conjugative transposon tn916: evidence for excision with formation of 5'-protruding termini.conjugative transposons are genetic elements able to promote their own intracellular transposition and intercellular conjugal transfer. they move by an excision-integration system related to that of lambdoid phages, in which the first step is the excision of the transposon from the donor replicon to form a covalently closed circular intermediate which contains a heteroduplex joint. in this work, sequencing both strands of the circular intermediate heteroduplex joint, it was found that, as during ...19968824634
modulation of lambda integrase synthesis by rare arginine trna.lambda's int gene contains an anomalously high frequency of the rare arginine codons aga and agg when compared to genes of escherichia coli or to the rest of phage lambda. these are the least frequent codons in genes of e. coli and are recognized by the rarest trnas. the presence of these codons reduces the translation rate and, depending on the context, this can strongly modulate translational efficiency by a variety of mechanisms. in this study, we show that expression of the natural int gene ...19968843435
identification and characterization of the escherichia coli rbn gene encoding the trna processing enzyme rnase bn.the gene encoding rnase bn was localized to 88 min on the escherichia coli chromosome by a novel suppressor assay and conjugational and transductional analysis. assay of subclones derived from lambda phage 543 of the kohara library, which encompasses this region of the chromosome, for elevated rnase bn activity identified o290, a previously reported open reading frame, as the gene encoding rnase bn. interruption of this gene with a kan(r) cassette and introduction into the chromosome eliminated ...19968955422
[lux-regulon from vibrio fisheri reduces type 1 restriction in escherichia coli k12 cells].the ecok restriction of nonmodified phage lambda.0 controlled by escherichia coli k12 lon- cells is alleviated 10-20-fold in the presence of a hybrid plasmid containing the entire lux regulon of vibrio fischeri. the la protease participates in degradation of the regulatory luxr-luxi proteins; therefore, transcription of the right lux operon begins significantly earlier than in the lon+ bacteria and is characterized by high content of the n-(3-oxohexanoyl) homoserine lactone autoinducer in medium ...19968964486
a novel bacterial vector system for monitoring protein-protein interactions in the camp-dependent protein kinase complex.a bacterial expression vector is described for investigation of protein-protein interactions. important features of the vector include partition of the ci repressor of bacteriophage lambda into two functional domains separated by a multicloning site, and low level auto-regulated expression of human genes as c-terminal fusions to the dna-binding domain of ci. two different reporter systems have been employed; expression of either a suppressor trna or the alkaline phosphatase gene is dependent in ...19979034306
cloning and characterization of bacteriophage-like dna from haemophilus somnus homologous to phages p2 and an attempt to identify and characterize components of a heme uptake system of haemophilus somnus, an escherichia coli cosmid library of h. somnus genomic dna was screened for the ability to bind hemin (hmb+). the hmb+ phenotype was associated with a 7,814-bp hindiii fragment of h. somnus dna that was subcloned and sequenced. thirteen open reading frames (orfs) were identified, all transcribed in one direction, and transposon mutagenesis identified orf7 as the gene associated with the hmb+ phe ...19979068631
gene replacement with linear dna fragments in wild-type escherichia coli: enhancement by chi sites.during conjugation and transduction of escherichia coli even numbers of recombinational exchanges are required for replacement of a gene on the circular chromosome. we studied gene replacement using a related method of gene transfer (transformation with 6.5-kb linear dna fragments) as an experimental model for conjugation and transduction. two properly situated chi sites, 5' gctggtgg 3', stimulated gene replacement approximately 50-fold, more than the sum of the stimulation by the individual chi ...19979093843
immunological screening of lambda-phage cdna expression libraries. 19979116850
protein interaction cloning by far-western screening of lambda-phage cdna expression libraries. 19979116852
genetic and transcriptional analysis of flgb flagellar operon constituents in the oral spirochete treponema denticola and their heterologous expression in enteric bacteria.oral spirochetes possess many potential virulence factors, including the capacity for tissue invasion and persistence despite a vigorous host immune response. in an attempt to identify treponemal immunoreactive components, sera derived from individuals with advanced periodontal disease were used as a reagent to isolate recombinant bacteriophage lambda clones expressing antigens of the oral spirochete treponema denticola atcc 35405. nucleotide sequence analysis of a clone expressing three immunor ...19979169730
cloning, expression, and mutagenesis of trypsin inhibitor etib from erythrina variegata seeds.erythrina variegata trypsin inhibitors designated etia and etib belong to the kunitz family trypsin inhibitor, but etia is unique in its ability to inhibit tissue-type plasminogen activator, while etib is not. the cdna clone encoding etib was isolated from the seed cdna library constructed in the lambda phage lambda gt11. the etib cdna insert consists of 765 bp, including an open reading frame of 606 pb from atg to tga codons. the deduced amino acid sequence consists of 202 amino acids, having t ...19979214769
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