t41 mutation in lac repressor is tyr282----asp.a monomeric mutant of the lac repressor protein, designated t41, produced originally by the in vivo mutt mutagenesis exhibited behavior similar to mutants identified as tyr282----ser or tyr282----gln [schmitz et al., j. biol. chem. 251 (1976) 3359-3366]. the t41 gene, encoded within a phage lambda prophage in the escherichia coli genome, was amplified by polymerase chain reaction and sequenced. the only mutation found in the nucleotide sequence corresponded to position 282 in the protein, and th ...19921547952
cloning and nucleotide sequence of the escherichia coli cytidine deaminase (ccd) gene.the structural gene that encodes cytidine deaminase (cdd) in escherichia coli was cloned from kohara phage lambda 365 (7f1), and its nucleotide sequence was determined. plasmids harboring the gene complemented chromosomal cdd mutations, enhanced cytidine deaminase activity in cell extracts, and directed the synthesis of a protein identical in mass and n-terminal amino acid sequence with cytidine deaminase purified from wild-type bacteria. metal analysis of the purified, plasmid-encoded deaminase ...19921567863
cyclic amp inhibits and putrescine represses expression of the spea gene encoding biosynthetic arginine decarboxylase in escherichia coli.the spea gene of escherichia coli encodes biosynthetic arginine decarboxylase (adc), the first of two enzymes in a putrescine biosynthetic pathway. the activity of adc is negatively regulated by mechanisms requiring cyclic amp (camp) and camp receptor protein (crp) or putrescine. a 2.1-kb bamhi fragment containing the spea-metk intergenic region, spea promoter, and 1,389 bp of the 5' end of the spea coding sequence was used to construct transcriptional and translational spea-lacz fusion plasmids ...19911646785
a combination of rnase h (rnh) and recbcd or sbcb mutations in escherichia coli k12 adversely affects growth.colony forming ability of escherichia coli strains carrying the rnh-339::cat mutant allele is strongly dependent on the recbcd and sbcb genes. a mutation inactivating either the recbcd nuclease or exonuclease i (sbcb) is sufficient to restrict severely the efficiency of plating of strains carrying the rnh-339::cat mutation. combining a non-lethal temperature-sensitive mutation in the recbcd nuclease, recb270 (ts) or recc271 (ts), with rnh-339::cat renders strains temperature sensitive for growth ...19911650908
effect of rfah (sfrb) and temperature on expression of rfa genes of escherichia coli order to study the regulation of a large block of contiguous genes at the rfa locus of escherichia coli k-12 which are involved in synthesis and modification of the lipopolysaccharide core, the transposon tnlacz was used to generate in-frame lacz fusions to the coding regions of five genes (rfaq, -g, -p, -b and -j) within this block. the beta-galactosidase activity of strains in which these fusions had been crossed into the chromosomal rfa locus was significantly decreased when the rfah11 (sf ...19911655711
immunoanalysis of ultraviolet radiation induced dna damage and repair within specific gene segments of plasmid dna.the region-specific heterogeneity of repairing dna damage has been established in several biological systems. a flexible and sensitive approach, based upon dna damage specific antibodies, is described to monitor the repair of specific lesions within discrete genomic segments. membrane transblotted dna restriction fragments are immunoanalyzed for the initial formation and repair of 254 nm radiation induced pyrimidine dimers. sensitivity of dimer immunodetection increases proportional to fragment ...19911657185
participation of rec genes of escherichia coli k 12 in w-reactivation of uv-irradiated phage lambda.the effect of the recombinational deficiency on w-reactivation of uv-damaged phage lambda was explored. in this paper we show that w-reactivation is reduced by the recb21 and recf143 mutations after bleomycin (bm) and uv treatment. combination of these mutations in the recb21recf143 double mutant blocks w-reactivation completely after bm induction, but leaves residual w-reactivation ability after uv-irradiation, which is abolished by the introduction of uvrb deficiency (delta(uvrb-chla]. w-react ...19901689458
renaturation of cobra venom phospholipase a2 expressed from a synthetic gene in escherichia coli.cobra venom (naja naja naja) phospholipase a2 (pla2) contains 14 cysteines in the form of 7 disulfide bonds amongst its 119 amino acids. a gene encoding the pla2 was synthesized and inserted into a bacterial expression vector containing the phage lambda pl promoter. in order to obtain protein without the initiating methionine at the n-terminus, a factor xa site was engineered upstream from the pla2 gene. upon heat-induction of the cells transformed with the expression plasmid, the protein is pro ...19921730025
saccharomyces cerevisiae elongation factor 2. genetic cloning, characterization of expression, and g-domain modeling.the elongation factor 2 (ef-2) genes of the yeast saccharomyces cerevisiae have been cloned and characterized with the ultimate goal of gaining a better understanding of the mechanism and control of protein synthesis. two genes (eft1 and eft2) were isolated by screening a bacteriophage lambda yeast genomic dna library with an oligonucleotide probe complementary to the domain of ef-2 that contains diphthamide, the unique posttranslationally modified histidine that is specifically adp-ribosylated ...19921730643
hu and integration host factor function as auxiliary proteins in cleavage of phage lambda cohesive ends by and integration host factor (ihf) are small, basic heterodimeric dna-binding proteins which participate in transcription initiation, dna replication, and recombination. we constructed isogenic escherichia coli strains in which hu, ihf, or both proteins were absent. bacteriophage lambda did not grow in hosts lacking both hu and ihf. phage dna replication and late gene transcription were normal in the double mutants, but packaging of lambda dna was defective. mature phage dna molecules were abs ...19911825651
isolation and preliminary characterization of escherichia coli mutants resistant to lethal action of the bacteriophage lambda p gene.both spontaneous and ntg-induced mutants of escherichia coli 594 insensitive to the lethal action of lambda p gene were isolated and called rpl (resistant to p lethality). these mutants were of two types, showing different phenotypes. on type i rpl mutants, lambda cl- and lambda v1v3 did not plate, while lambda vir, lambda cl- c17, lambda imm434, and lambda imm21 did; plasmid pmr45 carrying the lambda p gene could not complement lambda imm21p- phage in type i mutants. on the other hand, the type ...19911827225
identification of protein binding sites in genomic dna by two-dimensional gel electrophoresis.we describe a simple two-dimensional electrophoresis procedure to identify the recognition sites of dna-binding proteins within large dna molecules. using this approach, we have mapped e. coli ihf (integration host factor) binding sites within phage lambda (48 kb) and phage mu (39 kb) dna. we are also able to visualize ihf binding sites in e. coli chromosomal dna (4,700 kb). we present an extension of this technique using direct amplification by pcr of the isolated restriction fragments, which s ...19911827523
heat-inducible reactivation of uv-damaged bacteriophage lambda.induction of the sos response in uv-irradiated bacteria leads to an increase in the survival of an infecting irradiated bacteriophage lambda (weigle 1953). we report that a similar reactivation of irradiated phage lambda was induced by shifting the culture of recipient bacteria from 30 degrees to 47 degrees c. however, this repair process was nonmutagenic. the amplitude of the phenomenon was increased with the quantity of uv lesions in the phage dna. it was present despite mutations affecting th ...19911827875
dna recognition by the helix-turn-helix motif: investigation by laser raman spectroscopy of the phage lambda repressor and its interaction with operator sites ol1 and or3.the lambda repressor provides a model system for biophysical studies of dna recognition by the helix-turn-helix motif. we describe laser raman studies of the lambda operator sites ol1 and or3 and their interaction with the dna-binding domain of lambda repressor (residues 1-102). raman spectra of the two dna sites exhibit significant differences attributable to interstrand purine-purine steps that differ in the two oligonucleotides. remarkably, the conformation of each operator is significantly a ...19911828373
heteroduplex chain polarity in recombination of phage lambda by the red, recbcd, recbc(d-) and recf pathways.we have examined the chain polarity of heteroduplex dna in unreplicated, bacteriophage lambda splice recombinants when recombination was by the recbcd, recbc(d-), or recf pathway of escherichia coli or the red pathway of lambda. for each of these pathways, recombination is activated by the cutting of cos that accompanies chromosome packaging, and is effected by recombination enzymes acting at the right end created by that cutting. for exchanges occurring near cos, one parent makes a lesser physi ...19911829428
efficient excision of phage lambda from the escherichia coli chromosome requires the fis protein.the escherichia coli protein fis has been shown to bind a single site in the recombination region of phage lambda and to stimulate excisive recombination in vitro (j. f. thompson, l. moitoso de vargas, c. koch, r. kahmann, and a. landy, cell 50:901-908, 1987). we demonstrate that mutant strains deficient in fis expression show dramatically reduced rates of lambda excision in vivo. phage yields after induction of a stable lysogen are reduced more than 200-fold in fis cells. the defect observed in ...19911829453
the last duplex base-pair of the phage lambda chromosome. involvement in packaging, ejection and routing of lambda dna.cosn is the site at which the bacteriophage lambda dna packaging enzyme, terminase, introduces staggered nicks to generate the cohesive ends of mature lambda chromosomes. genetic and molecular studies show that cosn is recognized specifically by terminase and that effects of cosn mutations on lambda dna packaging and cosn cleavage are well correlated. mutations affecting a particular base-pair of cosn are unusual in being lethal in spite of causing only a moderate defect in cosn cleavage and dna ...19911830344
isolation and characterization of mutations in the bacteriophage lambda terminase genes.the terminase enzyme of bacteriophage lambda is a hetero-oligomeric protein which catalyzes the site-specific endonucleolytic cleavage of lambda dna and its packaging into phage proheads; it is composed of the products of the lambda nul and a genes. we have developed a simple method to select mutations in the terminase genes carried on a high-copy-number plasmid, based on the ability of wild-type terminase to kill reca strains of escherichia coli. sixty-three different spontaneous mutations and ...19911830578
molecular cloning and nucleotide sequence of the rhizobium phaseoli reca gene.a recombinant lambda phage carrying the reca gene of rhizobium phaseoli was isolated from a r. phaseoli genomic library by complementation of the fec- phenotype of the recombinant phage in escherichia coli. when expressed in e. coli, the cloned reca gene was shown to restore resistance to both uv irradiation and the dna alkylating agent methyl methanesulphonate (mms). the r. phaseoli reca gene also promoted homologous recombination in e. coli. the cloned reca gene was only weakly inducible in e. ...19911832737
dna sequence changes in mutations induced by ultraviolet light in the gpt gene on the chromosome of escherichia coli uvr+ and urva cells.sequence changes in mutations induced by ultraviolet light are reported for the chromosomal escherichia coli gpt gene in almost isogenic e. coli uvr+ and excision-deficient uvra cells. differences between the mutagenic spectra are ascribed to preferential removal of photoproducts in the transcribed strand by excision repair in uvr+ cells. this conclusion is confirmed by analysis of published results for genes in both uvr+ and uvr- cells, showing a similar selective removal of mutagenic products ...19911836051
[synthesis, secretion, and proteolytic degradation of diphtheria toxin in escherichia coli].the recombinant plasmids have been constructed encoding the synthesis of a full-sized diphtheria toxin from its own or pr, pl-promoters of bacteriophage lambda in escherichia coli cells. the high level constitutive synthesis of toxin results in slow cell growth and plasmid elimination. the toxin was mainly detected in the periplasm, partially in the membrane and to a less extent in the cytoplasm and culturing medium. the dimeric form of toxin was found in the cytoplasm. participation of toxin b- ...19911836836
the kila operon of promiscuous plasmid rk2: the use of a transducing phage (lambda pklaa-1) to determine the effects of the lethal klaa gene on escherichia coli cells.the kil-kor regulon of promiscuous plasmid rk2 includes the replication initiator gene trfa and several potentially host-lethal kil loci (kila, kilb, kilc, kile), whose functions may be involved in plasmid maintenance or broad host range. the kila locus consists of a single operon of three genes (klaa, klab, klac), each of which is lethal when expressed from the klaa promoter in the absence of repressors encoded by kora and korb. in this study, we examined the effects of the unregulated klaa gen ...19911838127
recombinagenic processing of uv-light photoproducts in nonreplicating phage dna by the escherichia coli methyl-directed mismatch repair system.nonreplicating lambda phage dna in homoimmune escherichia coli lysogens provides a useful model system for study of processes that activate dna for homologous recombination. we measured recombination by extracting phage dna from infected cells, using it to transfect reca recipient cells, and scoring the frequency of recombinant infective centers. with unirradiated phage, recombinant frequencies were less than 0.1%. however, recombination could be increased over 300-fold by prior uv irradiation o ...19911838344
selection of lacz operon fusions in genes of gluconate metabolism in e. coli. characterization of a gntt::lacz fusion.the initial steps involved in the utilization of gluconate by e. coli, its incorporation into the cell and subsequent phosphorylation to gluconate 6-phosphate, conform two systems that duplicate activities. these systems, gnti and gntii, are specified by two sets of genes distinctly regulated and located respectively at the mala-asd (75 min) and fdp-vals (96 min) regions of the bacterial chromosome. the presence of duplicate activities in the metabolism of gluconate of e. coli, has made difficul ...19911843569
bacteriophage lambda promoters pl and pr: sequence determinants of in vivo activity and of sensitivity to the dna gyrase inhibitor, coumermycin.sequence encompassing the region between bp -43 and +8 of the pl and pr promoters of bacteriophage lambda, as well as sequence variants of these promoters, were compared with respect to their ability to drive a promoterless cat gene in vivo. for both pl- and pr-based promoters, variants with one nonconsensus bp rather than the consensus promoter were found to be maximally active. determination of promoter function in csh26 and c600 revealed a marked strain dependence in the activity of some prom ...19911847348
rna polymerases from pseudomonas aeruginosa and pseudomonas syringae respond to escherichia coli activator proteins.the activities of rna polymerases (rnaps) from pseudomonas aeruginosa and pseudomonas syringae were compared with that of escherichia coli rnap. all three enzymes are able to initiate transcription at the trpba promoter of p. aeruginosa and at the coliphage lambda promoters, prm and pre, in response to heterospecific activators (trpi protein, repressor, and cii protein, respectively). however, both pseudomonas polymerases have less stringent requirements for promoter recognition in the absence o ...19911898924
cloning, sequencing and overexpression of the gene for prokaryotic factor ef-p involved in peptide bond synthesis.a soluble protein ef-p (elongation factor p) from escherichia coli has been purified and shown to stimulate efficient translation and peptide-bond synthesis on native or reconstituted 70s ribosomes in vitro. based on the partial amino acid sequence of ef-p, 18- and 24-nucleotide dna probes were synthesized and used to screen lambda phage clones from the kohara gene bank. the entire ef-p gene was detected on lambda clone #650 which contains sequences from the 94 minute region of the e.coli genome ...19911956781
a cdna encoding 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase from solanum tuberosum l.a cdna encoding potato (solanum tuberosum l.) 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway, was cloned into phage lambda gt11. the clone represents the first cdna for this enzyme from any eukaryotic source. the nucleotide sequence of the cdna was determined, and its identity was confirmed through partial amino acid sequence analysis of the encoded enzyme. the cdna contains a 1527-base pair open reading frame that encodes a polypeptide with a cal ...19901967256
characterization of the pilin gene of moraxella bovis dalton 2d and expression of pili from m. bovis in pseudomonas aeruginosa.the pilin gene of moraxella bovis dalton 2d was isolated by cloning in pseudomonas aeruginosa. the nucleotide sequence of this gene encodes a prepilin of 156 amino acid residues. when high levels of pilin were expressed from the gene in p. aeruginosa, by using the pl promoter of bacteriophage lambda inserted upstream of the coding sequence, pili which were indistinguishable from pili of m. bovis were produced.19901971258
structural and functional comparison between the stability systems pard of plasmid r1 and ccd of plasmid f.the stability determined by the systems pard of plasmid r1 and ccd of plasmid f is due to the concerted action of two proteins, a cytotoxin and an antagonist of this function. in this paper we report that ccda and kis proteins, the antagonists of the ccd and pard systems respectively, share significant sequence homologies at both ends. in kis, these regions seem to correspond to two different domains. despite the structural similarities, kis and ccda are not interchangeable. in addition we have ...19912017133
construction and characterization of a single-chain catalytic antibody.the antigen-binding (fab) fragment of the catalytic monoclonal antibody npn43c9 has recently been cloned by using bacteriophage lambda. by inserting the variable regions of this fab coding sequence into a (nh2)-vl-linker-vh-(cooh) construct (where vl and vh represent the heavy and light chain variable regions), we have assembled a recombinant gene encoding a catalytic single-chain antigen-binding protein. this protein has been expressed in escherichia coli and exhibits the same catalytic paramet ...19912023948
celb, a gene coding for a bifunctional cellulase from the extreme thermophile "caldocellum saccharolyticum"."caldocellum saccharolyticum" is an obligatory anaerobic thermophilic bacterium. a gene from this organism, designated celb, has been cloned in escherichia coli as part of a bacteriophage lambda gene library. this gene produces a thermostable cellulase that shows both endoglucanase and exoglucanase activities on test substrates and is able to degrade crystalline cellulose to glucose. the sequence of celb has homology with both exo- and endoglucanases described by others. it appears to have a cen ...19902126700
integration of bacteriophage lambda into the cryptic lambdoid prophages of escherichia coli.bacteriophage lambda missing its chromosomal attachment site will integrate into reca+ escherichia coli k-12 and c at the sites of cryptic prophages. the specific regions in which these recombination events occur were identified in both lambda and the bacterial chromosomes. a noti restriction site on the prophage allowed its physical mapping. this allowed us to identify the locations of rac, qin, and qsr' cryptic prophages on the noti map of e. coli k-12 and, by analogy, to identify the cryptic ...19902139644
a minor arginine trna mutant limits translation preferentially of a protein dependent on the cognate codon.the escherichia coli argu gene encodes a rare arginine trna (anticodon ucu) that translates the similarly rare aga codon. the argu10(ts) mutation is a transition that changes the first nucleotide of the mature trna from g to a, presumably destabilizing the acceptor stem. this mutation, when present in haploid condition in the chromosome, reduces the growth rate at 30 degrees c and results in cessation of growth after 60 to 90 min at 43 degrees c. the mutation also preferentially limits (compared ...19902139647
stimulation of mutations suppressing the loss of replication control by small alcohols.transient exposure of lysogenic escherichia coli cells to small alcohols stimulated the frequency of mutations suppressing the lethal loss of replication control from a prophage fragment of bacteriophage lambda. the stimulation in mutation frequency paralleled the effect of mutagenic agents, and in this sense the alcohols behaved as mutagens. 10-min treatments above distinct threshold concentrations at 23%, 18%, 10% and 4% (v/v) were required in order for methanol, ethanol, isopropanol and propa ...19902143557
lysis protein s of phage lambda functions in saccharomyces cerevisiae.the lambda s lysis gene was cloned into a saccharomyces cerevisiae expression vector under gal1 control. induction with galactose in s. cerevisiae terminated cell growth and prevented colony formation. several membrane proteins immunoreactive with anti-s antibody accumulated in the membranes, indicating that sodium dodecyl sulfate-resistant oligomers of s are formed, similar to those observed in the membranes of escherichia coli cells killed by expression of the s gene. these observations sugges ...19902147680
a short dna sequence from lambda phage inhibits protein synthesis in escherichia coli rap.the escherichia coli rap mutant inhibits vegetative growth of bacteriophage lambda. phage mutations termed bar, which overcome the rap defect, have been mapped to three genetic loci in the pl operon. plasmids with a lambda wild-type bar dna segment cloned downstream from an active promoter cannot be maintained in rap mutant bacteria. the viability of a rap mutant strain decreases rapidly after induction of transcription through bar regions present on plasmids. under these (restrictive) condition ...19902147720
transcription-dependent competition for a host factor: the function and optimal sequence of the phage lambda boxa transcription antitermination signal.ordered development of lambdoid phages relies on systems of transcription termination and antitermination. the phage-encoded n early regulatory proteins, acting with the nus proteins of escherichia coli, modify rna polymerase to a form that overrides many transcription termination signals. these modifications require cis-acting sites, nut, located downstream of the early phage promoters. the nut sites in phages lambda, 21, and p22, which share similarities but are not identical, contain two sign ...19902148536
the use of transgenic mice for short-term, in vivo mutagenicity order to develop a short-term, in vivo assay to study the mutagenic effects of chemical exposure, transgenic mice were generated using a lambda shuttle vector containing a lacz target gene. following exposure to mutagens, this target can be rescued efficiently from genomic dna prepared from tissues of the treated mice using restriction minus, in vitro lambda phage packaging extract and restriction minus escherichia coli plating cultures. mutations in the target gene appear as colorless plaque ...19902151115
site-directed insertion mutagenesis with cloned fragments in escherichia coli by p1 phage transduction.a cloned gene with an insertion, which was made by introducing cat, was ligated to the cloning site of the phage lambda gt11. p1 phage grown on cells lysogenized with the recombinant lambda phage could transduce the mutant gene into the original site on the escherichia coli chromosome.19902157955
vectors for constructing kan gene fusions: direct selection of mutations affecting is10 gene expression.we describe several vectors for constructing translational fusions to the kan gene of tn5. fusions are constructed in vitro using multi-copy vectors containing unique cloning sites situated between upstream transcriptional terminators and a downstream kan gene lacking transcriptional and translational start signals. multi-copy fusions can be converted to single-copy chromosomal fusions by in vivo recombination with specific phage lambda vectors and vice versa. we find that kan fusions are often ...19902165970
effects of medium quality on the expression of human interleukin-2 at high cell density in fermentor cultures of escherichia coli k-12.we examined the ability of transformed escherichia coli cells in fermentor cultures to accumulate interleukin-2 (il-2) intracellularly under temperature-regulated control of the phage lambda pl promoter. induction of expression was undertaken at different culture optical densities, and specific il-2 accumulation was found to decrease with increasing cell density at induction. induction at higher culture optical densities was also accompanied by decreased growth during induction and increased ace ...19902180368
detection of protein-dna complex formation by time-resolved fluorescence depolarization of bound ethidium bromide.we introduce the use of time-resolved fluorescence spectroscopy to probe the interaction between gene regulatory proteins and dna. changes in the decay kinetics of fluorescence polarization anisotropy of ethidium bromide bound to dna segments report changes in hydrodynamic volume and shape which occurs upon complex formation between protein and dna. we have used the decay of fluorescence polarization anisotropy as a spectroscopic handle on the interaction between several site-specific dna-bindin ...19902291477
e. coli nusa protein binds in vitro to an rna sequence immediately upstream of the boxa signal of bacteriophage lambda.the nusa protein of escherichia coli is a factor which mediates termination of transcription. in this paper, we demonstrate that the nusa protein can bind in vitro to a specific site on the mrna of bacteriophage lambda. several rnas were synthesized by in vitro transcription of truncated lambda dna templates, and the activity of nusa binding to these rnas was examined by a millipore filter-binding assay. rnas containing the sequence immediately upstream of the boxa site were trapped on the filte ...19852416564
the rna component of the bacillus subtilis rnase p. sequence, activity, and partial secondary structure.the gene defining the catalytic rna component of rnase p in bacillus subtilis 168 was cloned into bacteriophage lambda and plasmid vectors. the nucleotide sequence of the gene and its surroundings was determined from the cloned dna and by directly sequencing or reverse transcribing the rnase p rna. the b. subtilis rnase p rna sequence (400-401 nucleotides) is remarkably different from that of escherichia coli (377 nucleotides) (reed, r. e., baer, m. f., guerrier-takada, c., donis-keller, h., and ...19862423526
a rapid and improved method for generating cdna libraries in plasmid and phage lambda vectors.we have developed a fast and efficient procedure for generating cdna libraries in plasmid or phage lambda vectors. we used mo-mulv reverse transcriptase to synthesize the first strand and directly added escherichia coli dna polymerase i with rnase h to synthesize the second strand. a special advantage of our procedure is the use of oligodeoxynucleotide adapters to insert the cdna into the vector, avoiding the use of methylating enzymes and subsequent digestion with massive amounts of restriction ...19872445632
overproduction of an antisense rna containing the oop rna sequence of bacteriophage lambda induces clear plaque formation.we have constructed an iptg-inducible plasmid which overexpresses oop rna sequences in escherichia coli. infection of these transformed e. coli cells (sb221/poop5) with lambda+ phage produced clear plaques, whereas lambda+ infection of cells transformed with the plasmid vector (sb221/pjdc406) or the plasmid expressing the oop rna transcript in the other orientation (sb221/poop9) gave rise to turbid plaques characteristic of lambda+. calculations of the percentage of infected cells forming lysoge ...19872448589
mutagenesis of bleomycin-damaged lambda phage in sos-deficient and repair endonuclease-deficient escherichia coli.previous dna sequence analysis of bleomycin-induced forward mutations in repackaged lambda phage has suggested sos-dependent replicative bypass of oxidized apyrimidinic sites as a possible mechanism of mutagenesis. in order to evaluate this hypothesis further, frequencies of mutation to a clear-plaque phenotype were compared for bleomycin-damaged phage grown in various repair-deficient strains of escherichia coli. survival of bleomycin-damaged phage was virtually identical in all host strains. s ...19882453358
monoclonal antibodies to a proenkephalin a fusion peptide synthesized in escherichia coli recognize novel proenkephalin a precursor forms.monoclonal antibodies have been generated to a chimeric peptide comprised of escherichia coli beta-galactosidase fused to the amino acid sequence 69-207 of human preproenkephalin a. two monoclonal antibodies, pe-1 and pe-2, were identified by their ability to recognize the same segment of proenkephalin a fused to the cii gene product of the e. coli bacteriophage lambda. the binding domains of pe-1 and pe-2 have been broadly located, with respect to the primary translation product, within the ami ...19882461943
phage genetic sites involved in lambda growth inhibition by the escherichia coli rap mutant.the rap mutation of escherichia coli prevents the growth of bacteriophage lambda. we have isolated phage mutants that compensate for the host deficiency. the mutations, named bar, were genetically located to three different loci of the lambda genome: bari in the attp site, barii in the ciii ea10 region, and bariii within or very near the imm434 region. the level of lambda leftward transcription correlates with rap exclusion. phage lambda mutants partially defective in the pl promoter or in pl-tr ...19892523838
effects of all single base substitutions in the loop of boxb on antitermination of transcription by bacteriophage lambda's n protein.the 'n' antitermination proteins of lambdoid bacteriophages are essential for overcoming multiple transcription terminators located within the major early operons of these phages (1). in order for n proteins to function, a genome sequence specifying n utilization, nut, must be located within an operon, between the promoter and the terminators (2). two components have been identified within nut: 8-base boxa, conserved among different phages and implicated in the recognition of host nusa protein, ...19892527353
high level expression of genes cloned in phage lambda gt11.plasmid cloning vectors have been constructed which allow genes originally cloned in lambda gt11 to be expressed at a high level in escherichia coli. they are based on the pembl and puc vectors, with the genes transcribed from the lac promoter. the ecori site in the vector has been altered to be in the same reading frame as the site used for cloning in lambda gt11. cloned proteins are expressed fused to a 2-kda leader sequence containing a run of six aparagine residues which considerably improve ...19892527780
translesion synthesis is the main component of sos repair in bacteriophage lambda dna.agents that interfere with dna replication in escherichia coli induce physiological adaptations that increase the probability of survival after dna damage and the frequency of mutants among the survivors (the sos response). such agents also increase the survival rate and mutation frequency of irradiated bacteriophage after infection of treated bacteria, a phenomenon known as weigle reactivation. in uv-irradiated single-stranded dna phage, weigle reactivation is thought to occur via induced, erro ...19892527845
phasing of protein-induced dna bends in a recombination complex.many of the structures responsible for replication, transcription initiation and recombination arise from complex sets of protein-protein interactions and the folding of dna in three dimensions, with protein-induced bending of dna often playing an integral role. the magnitude and orientation of dna bending induced by various single proteins has been estimated by gel mobility shift methods and by modelling of crystallographic data. the site-specific recombination by which bacteriophage lambda (ph ...19892528698
transcriptional activation of bacteriophage lambda dna replication in vitro: regulatory role of histone-like protein hu of escherichia coli.initiation of bacteriophage lambda dna replication in vivo and in crude in vitro systems is strongly dependent on transcription at or near the lambda replication origin (ori lambda). through its capacity to prevent rna polymerase-mediated 'transcriptional activation' of lambda dna replication, the lambda ci repressor is capable of negatively regulating initiation of lambda dna replication, even when all required replication proteins are present. surprisingly, the strict requirement for transcrip ...19892529119
generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda.a novel bacteriophage lambda vector system was used to express in escherichia coli a combinatorial library of fab fragments of the mouse antibody repertoire. the system allows rapid and easy identification of monoclonal fab fragments in a form suitable for genetic manipulation. it was possible to generate, in 2 weeks, large numbers of monoclonal fab fragments against a transition state analog hapten. the methods described may supersede present-day hybridoma technology and facilitate the producti ...19892531466
the terminus of the escherichia coli chromosome is flanked by several polar replication pause sites.replication of two small 'constrained' regions of the escherichia coli chromosome, one bordered by replication terminator t1 and the other by t2, displays normal velocity in the normal direction whereas it is much slower in the opposite direction (de massy et al., 1987). the presence of multiple polar terminators has been investigated, using a bacteriophage lambda derivative which provides a replication origin movable to predetermined loci and inducible on demand. the amount of dna made from thi ...19892532703
bending of dna by gene-regulatory proteins: construction and use of a dna bending vector.the binding of a protein to its specific sequence, borne on a dna fragment, retards the mobility of the fragment in a characteristic way during gel electrophoresis. if the protein induces bending in the dna, the contortion can also be monitored by gel electrophoresis, because the amount of retardation of the mobility of the dna-protein complex is dependent upon the position and the degree of the bend induced in the dna fragment [wu and crothers, nature 308 (1984) 509-513]. we have constructed a ...19892533576
homologous recombination in prokaryotes: enzymes and controlling sites.a common step in prokaryotic recombination appears to be the synapsis of the 3'-end of single-stranded dna with duplex dna to form a d-loop. the enzymatic mechanisms by which 3'-ends are produced and by which d-loops are converted into recombinant molecules are illustrated by proposed mechanisms of recombination by the escherichia coli recbcd pathway and the phage lambda red pathway. the enzymes promoting recombination and the special dna sites at which they act are emphasized. recombination by ...19892534386
cloning and dna sequence of plasmid determinant iss, coding for increased serum survival and surface exclusion, which has homology with lambda dna.escherichia coli k12 cells carrying a cloned 1.4 kb hindiii fragment from plasmid colv2-k94, showed increased survival in guinea pig serum. the recombinant plasmid also conferred group ii surface exclusion, i.e. the cells were reduced in recipient ability towards the incoming plasmid r538drd in conjugation experiments. southern blotting suggested homology with bacteriophage lambda dna and to the insertion element is2. determination of the dna sequence of the fragment demonstrated the presence of ...19892546040
the escherichia coli protein, fis: specific binding to the ends of phage mu dna and modulation of phage growth.we show, using gel retardation, that crude escherichia coli cell extracts contain a protein which binds specifically to dna fragments carrying either end of the phage mu genome. we have identified this protein as fis, a factor involved in several site-specific recombinational switches. furthermore, we show that induction of a mucts62 prophage in a fis lysogen occurs at a lower temperature than that of a wild-type strain, and that spontaneous induction of mucts62 is increased in the fis mutant. d ...19892548061
new derivatives of transposon tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in gram-negative bacteria.three types of new variants of the broad-host-range transposon tn5 are described. (i) tn5-mob derivatives with the new selective resistance (r) markers gmr, spr and tcr facilitate the efficient mobilization of replicons within a wide range of gram-negative bacteria. (ii) promoter probe transposons carry the promoterless reporter genes lacz, nptii, or luc, and nmr, gmr or tcr as selective markers. these transposons can be used to generate transcriptional fusions upon insertion, thus facilitating ...19892551782
characterization of the cryptic lambdoid prophage dlp12 of escherichia coli and overlap of the dlp12 integrase gene with the trna gene argu.the argu (dnay) gene of escherichia coli is located, in clockwise orientation, at 577.5 kilobases (kb) on the chromosome physical map. there was a cryptic prophage spanning the 2 kb immediately downstream of argu that consisted of sequences similar to the phage p22 int gene, a portion of the p22 xis gene, and portions of the exo, p, and ren genes of bacteriophage lambda. this cryptic prophage was designated dlp12, for defective lambdoid prophage at 12 min. immediately clockwise of dlp12 was the ...19892553674
mapping of insertion elements is1, is2 and is3 on the escherichia coli k-12 chromosome. role of the insertion elements in formation of hfrs and f' factors and in rearrangement of bacterial chromosomes.the chromosome of an escherichia coli k-12 strain w3110 contains seven copies of insertion element is1, 12 copies of is2 and six copies of is3. we determined the approximate locations of six copies of is1 (named is1a to is1f), ten copies of is2 (named is2a to is2j), and five copies of is3 (named is3a to is3e) on the w3110 chromosome by plaque hybridization using the "mini-set" of the lambda phage library that includes 476 clones carrying chromosomal segments that cover the w3110 chromosome almos ...19892553981
location and molecular cloning of the structural gene for the deoxyguanosine triphosphate triphosphohydrolase of escherichia coli.the structural gene for deoxyguanosine triphosphate triphosphohydrolase (dgtpase) (ec and its regulator, opta, have been located on a lambda phage carrying a 17.5kb escherichia coli dna insert. the dna fragment has been excised and ligated into pbr325 and also transferred to another lambda vector. from the results of transduction and transformation experiments, we find that the structural gene for dgtpase is very closely linked to opta and dapd, which locates it at approximately 3.6 min ...19892559296
sumo15a: a lambda phasmid that permits easy selection for and against cloned inserts.we report the construction of a phasmid vector, sumo15a, designed for recombination-based screening of recombinant dna libraries [seed, nucleic acids res. 11 (1983) 2427-2445]. this vector permits rapid selection in escherichia coli for homology-mediated integration and excision between homologous dna inserts cloned in a supf-carrying plasmid and in sumo15a. the region available for recombination spans the homologous sequence shared by the plasmid and the phasmid. supf is the selection tool that ...19892559877
overexpression of n antitermination proteins of bacteriophages lambda, 21, and p22: loss of n protein specificity.the n protein of bacteriophage lambda (n lambda) modifies escherichia coli rna polymerase in such a way that it transcribes through termination signals, a process called antitermination. n antitermination normally occurs only if the template contains a specific utilization or nut site upstream of the terminators and only in the presence of host-encoded nus proteins. the lambda-related phages 21 and p22 produce n analogs, n21 and n22, but these require different nut sites and show a different pat ...19892651405
characterization and vaccine potential of a novel recombinant coccidial antigen.a cdna clone derived from sporulated oocysts of eimeria tenella and encoding the expression product gx3262 was identified using a monoclonal antibody raised against eimeria acervulina sporozoites. the cdna fragment containing the coccidial antigen gene was cloned in bacteriophage lambda gt11, transferred to a plasmid, and introduced into escherichia coli for analysis of the gene products. the strain carrying the plasmid produced gx3262 as part of a fusion protein consisting of the first 1,006 am ...19892659532
genetic analysis of the lytic replicon of bacteriophage p1. ii. organization of replicon elements.the region of bacteriophage p1 dna containing a lytic (vegetative) replicon has been identified by cloning p1 fragments into a phage lambda vector. we present the sequence of that replicon. using a novel fusion vector containing two p1 loxp recombination sites, we have developed a transformation assay for replicon function and have used that assay to identify some of the components of the p1 lytic replicon. among those components is a transcription promoter, p53, whose activity is essential for ...19892661830
cysteine-22 and cysteine-38 are not essential for the functions of maltoporin (lamb protein).maltoporin in the outer membrane of escherichia coli contains two cysteine residues, at positions 22 and 38 in the primary sequence. the role of these residues in determining structural stability, and their contributions to the maltoporin binding sites for maltodextrins and bacteriophage lambda, was investigated. site-directed mutagenesis was used to alter each of these residues to a serine. a double mutant lacking both cysteines was also isolated. none of the substitutions affected maltodextrin ...19892693195
bacteriophage t4 early promoter regions. consensus sequences of promoters and ribosome-binding sites.twenty-nine early promoters from bacteriophage t4 and 14 early promoters from bacteriophage t6 were isolated using vector m13hdl17, a promoterless derivative of m13mp8 carrying a linker sequence, the bacteriophage lambda-terminator tr1, and the lacz' gene including part of its ribosome-binding site. the consensus sequence for the t4 promoters is: (sequence; see text). ribosome-binding sites of t4 share the sequence: 5'...g.ggaga..aa.atgaa.a...3' the consensus sequence of the t4 early promoter re ...19892810355
a system for in vivo selection of genomic rearrangements with predetermined endpoints in escherichia coli using modified tn10 transposons.using recombinant dna techniques, the tn10-specific teta gene (coding for tetracycline resistance) has been mutagenized by insertion of a streptomycin-resistance or a kanamycin-resistance gene. the insertions occurred at loci separated by 920 bp. the mutated teta fragments, respectively designated as tes (for tetracycline-streptomycin) and tek (for tetracycline-kanamycin), were subsequently cloned into a phage lambda ciii+cits857cii+ in replacement of the att lambda region. the two recombinant p ...19872824289
transposition of tn1000: in vivo properties.transposition mediated by the tn1000 transposase was investigated by using transposon variants carrying synthetic or wild-type termini but no intact tn1000 genes. transposon tn1001, whose only homologies to tn1000 are in its 38-base-pair terminal inverted repeats, transposed at the same rate as tn1005, an artificial construct carrying wild-type tn1000 termini and approximately 1 kilobase of flanking tn1000 dna at each end, when transposase was supplied in trans. the majority of the transposition ...19872824438
characterization of the types of mutational events that spontaneously occur in a plasmid system.the immunity region from a ci857 derivative of bacteriophage lambda has been cloned into the ecori site of pbr322 to produce a plasmid that can be used to analyze spontaneous mutagenesis. cells containing this plasmid are temperature-sensitive for growth unless mutations have occurred that somehow prevent the expression of the kil gene in the lambda fragment at non-permissive temperature. 678 such temperature-resistant mutants from 10 independent subcultures each of 2 different reca- e. coli str ...19882827020
structure of cryptic lambda prophages.when escherichia coli cells lysogenic for bacteriophage lambda are induced with ultraviolet light, cells carrying cryptic lambda prophages are occasionally found among the apparently cured survivors. the lambda variant crypticogen (lambda crg) carries an insertion of the transposable element is2, which increases the frequency of cryptic lysogens to about 50% of cured cells: 43 of these cryptic prophages have been characterized. they all contain substitutions that replace the early segment of the ...19872828640
[alleviation of type i restriction in the presence of plasmids of group inc i. general characteristics and molecular cloning of the ard gene].the capability of a number of plasmids of incn and inci groups to alleviate an action of type i ecok, ecob, ecod, and ecoa restriction endonucleases on the unmodified dna was revealed. the efficiency of ecok action on lambda 0 dna is alleviated about 10 divided by 100 fold in e. coli k12 ab 1157 bacteria containing the plasmid of incn group (pkm101, n3, pja4733) or inci group (r144, r648; r621a; colib-p9). we have cloned ard gene of colib-p9 plasmid (sali-c fragment) in pbr322 multicopying vecto ...19882836721
sequences in the 5' proximal segment of the paused transcript affect nusa-mediated enhancement of transcriptional pausing.nusa protein is a transcription elongation and termination factor that acts to enhance pausing of rna chain growth by rna polymerase at specific sites on dna templates. we demonstrate that this enhancement of pausing in tr1, the transcription termination site between genes cro and cii of phage lambda, is inhibited by dna oligonucleotides complementary to a segment of the nascent rna just preceding the sequence that is thought to be a part of the stem of an rna hairpin that is responsible for pau ...19882839506
structural and functional analysis of tn4430: identification of an integrase-like protein involved in the co-integrate-resolution process.the 4149-bp transposon tn4430 from bacillus thuringiensis is delineated by 38-bp inverted repeats and codes for a 113-kd protein that shares homology with the transposases (tnpa) of tn3, tn21 and tn501. through transpositional recombination, this protein generates the formation of co-integrates between both donor and target replicons, with duplication of tn4430 molecules. these features are characteristic of transposons of the tn3 family (class ii elements). the second step of the transposition ...19882842151
initiation of lambda dna replication reconstituted with purified lambda and escherichia coli replication proteins.using highly purified bacteriophage lambda and e. coli replication proteins, we were able to reconstitute an in vitro system capable of replication ori lambda-containing plasmid dna. the addition of a new e. coli factor, the grpe gene product, to this replication system reduced the level of dnak protein required for efficient dna synthesis by at least 10-fold, and also allowed the isolation of a stable dna replication intermediate. based on all available information, we propose a molecular mecha ...19882850011
jekyll, a family of phage-plasmid shuttle vectors.a series of shuttle vectors has been constructed, which consist of a plasmid carrying a polylinker sequence and an m13 origin integrated into a lambda vector. a short direct repeat flanking the plasmid allows plasmid excision by homologous recombination. sequences are cloned into unique restriction sites within the plasmid, and can be recovered either in phage or plasmid form, or can be packaged further as single-stranded dna phage. these vectors therefore combine the efficiency of phage lambda ...19882854093
characterization of the gene encoding glutamine synthetase i (glna) from bradyrhizobium japonicum.we have isolated the bradyrhizobium japonicum gene encoding glutamine synthetase i (glna) from a phage lambda library by using a fragment of the escherichia coli glna gene as a hybridization probe. the rhizobial glna gene has homology to the e. coli glna gene throughout the entire length of the gene and can complement an e. coli glna mutant when borne on an expression plasmid in the proper orientation to be transcribed from the e. coli lac promoter. high levels of glutamine synthetase activity c ...19852859270
the role of adenylyltransferase and uridylyltransferase in the regulation of glutamine synthetase in escherichia coli.the regulation of gs activity involves two nucleotidylation cycles, the uridylylation cycle of pii and the adenylylation cycle of gs, which are catalyzed by two converter enzymes, uridylyltransferase and adenylyltransferase, respectively. the converter enzymes sense the fluctuation in the availability of nitrogen and accordingly regulate the activity of gs. on the other hand, the posttranslational modification of gs is tightly coupled to the transcriptional regulation of the glna gene by unmodif ...19852868842
nucleotide sequence of the gdh gene coding for the nadp-specific glutamate dehydrogenase of saccharomyces cerevisiae.the isolation of the saccharomyces cerevisiae gene for nadp-dependent glutamate dehydrogenase (nadp-gdh) by cross hybridization to the neurospora crassa am gene, known to encode for nadp-gdh is described. two dna fragments selected from a yeast genomic library in phage lambda gt11 were shown by restriction analysis to share 2.5 kb of common sequence. a yeast shuttle vector (cv13) carrying either to the cloned fragments complements the gdh- strain of s. cerevisiae and directs substantial overprod ...19852932370
effect of an umuc mutation on phage lambda induction.a possible role of the umuc gene product in the induction of the sos responses was examined. we compared the expression of a genetic fusion, in which gene lacz, encoding beta-galactosidase in escherichia coli is under the direct control of the ci repressor from prophage lambda, in a umuc+ strain and in an otherwise isogenic umuc- mutant. we found that two times higher uv doses were required to obtain a similar induction in the umuc+ strain as in the umuc mutant. in addition we showed that, at th ...19852935710
activity of chi recombinational hotspots in salmonella typhimurium.chi sites have previously been shown to stimulate homologous recombination by the escherichia coli recbc pathway. to test the activity of chi in another organism, bacteriophage lambda crosses were carried out in salmonella typhimurium strains bearing the e. coli lambda receptor protein. chi is active in these crosses in s. typhimurium, but is less active than in the same crosses carried out in e. coli. the lower chi activity in s. typhimurium appears to be intrinsic to the s. typhimurium recbc e ...19862937685
relationship between cellular reca protein concentration and untargeted mutagenesis in escherichia coli.we measured the production of untargeted mutations in the ci and cii genes of untreated lambda phage undergoing a lytic cycle in uv-irradiated bacterial hosts. as previously shown, treatment with 4 micrograms/ml of rifampicin during post-irradiation incubation inhibited amplification of the reca protein in these cells. in addition, we observed a decreased mutation rate compared to the untreated, irradiated bacteria. treatment with 4 micrograms/ml or 8 micrograms/ml rifampicin did not prevent the ...19862938000
spontaneous lambda or mutations suppress inhibition of bacteriophage growth by nonimmune exclusion phenotype of defective lambda prophage.survivor clones with defects in gene functions that participate in the replicative killing of thermally induced escherichia coli constructs with integrated lambda n through p or ciii through p gene fragments were selected at a frequency of about 10(-6). among the population of survivors, clones were identified that exhibited normal lambda immunity at 30 degrees c, as shown by their ability to prevent the plating of lambda wild type and to support the plating of a nearly identical heteroimmune ba ...19862939262
the homologous recombination system of phage lambda. pairing activities of beta protein.the red genes of phage lambda specify two proteins, exonuclease and beta protein, which are essential for its general genetic recombination in reca- cells. these proteins seem to occur in vivo as an equimolar complex. in addition, beta protein forms a complex with another polypeptide, probably of phage origin, of mr 70,000. the 70-kda protein appears to be neither a precursor nor an aggregated form of either exonuclease or beta protein, since antibodies directed against the latter two proteins f ...19862940241
plasmid expression vector using the lambda late promoter.a plasmid expression vector called pqte1 based on the late promoter, pr', and positive control gene q of bacteriophage lambda has been constructed. this vector has unique cloning sites for placing exogenous dna under control of pr'. induction of expression of genes cloned into the pqte1 plasmid leads to massive overproduction of the gene products. also, transcription from the pr' promoter on pqte1 appears to be insensitive to polarity effects.19862940611
promoter recognition by escherichia coli rna polymerase. effects of substitutions in the spacer dna separating the -10 and -35 regions.a family of variants of the prm promoter of lambda phage was constructed, bearing nine base pair substitutions in a stretch of the spacer dna separating the contacted -10 and -35 regions. the substituted sequences were chosen for their potential to adopt structures different from those of average b-form dna and thus to affect the interaction of rna polymerase with the two contacted regions. characterization of the promoters in vitro and in vivo provides additional support for the lack of specifi ...19862942546
selective inhibition of escherichia coli recbc activities by plasmid-encoded gams function of phage lambda.the gam locus of bacteriophage lambda encompasses two coding sequences with the same reading frame and translational stop, one corresponding to an mr 11646 polypeptide (gams gene), the other to an mr 16349 polypeptide (gaml gene). a dna segment encoding gams but not gaml was placed under lambda pr promoter control (regulated by the cits857-coded repressor) on a multicopy plasmid, and an insertion mutation (gams201) was constructed. expression of gams+, but not gams201, inhibited escherichia coli ...19862943636
an improved method for the isolation of high yields of bacteriophage lambda dna. 19862947045
wavelength dependence for the induction of bacteriophage lambda by antitumor agent gilvocarcin v. 19862949330
chi-stimulated patches are heteroduplex, with recombinant information on the phage lambda r chain.generalized recombination in escherichia coli is elevated near chi sites. in vitro, recbcd enzyme can nick chi a few nucleotides 3' of the terminal gg of the chi sequence (5'-gctggtgg). the simplest model in which this nick at chi participates in chi function predicts that in phage lambda, chi-stimulated recombinants not crossed-over for flanking markers (patches) should be heteroduplex, with recombinant information on the lambda i chain. i report here that patches are heteroduplex, but that rec ...19872949853
structures of mismatched base pairs in dna and their recognition by the escherichia coli mismatch repair system.the escherichia coli mismatch repair system does not recognize and/or repair all mismatched base pairs with equal efficiency: whereas transition mismatches (g x t and a x c) are well repaired, the repair of some transversion mismatches (e.g. a x g or c x t) appears to depend on their position in heteroduplex dna of phage lambda. undecamers were synthesized and annealed to form heteroduplexes with a single base-pair mismatch in the centre and with the five base pairs flanking each side correspond ...19862951250
large palindromes in the lambda phage genome are preserved in a rec+ host by inhibiting lambda dna replication.a large palindrome carried by phage lambda has been shown to prevent growth of the phage on a rec+ strain of escherichia coli. the phage do form plaques on recbc sbcb strains, but the palindrome is not stable--deletions that either destroy the palindrome or diminish its size overgrow the original engineered palindrome-containing phage. we have prepared stocks of lambda carrying a palindrome that is 2 x 4200 base pairs long. these phage stocks are produced by induction of a lysogen in which the t ...19872951734
mutations that improve the pre promoter of coliphage lambda.the dya5 mutation, a c----t change at position -43 of the lambda pre promoter, results in a twofold increase in pre activity in vivo. smaller increases in pre activity are found for the dya2 mutation, a t----c change at position -1 of pre, and the dya3 mutation, an a----g change at +5 of pre. the mutant pre promoters retain complete dependence on cii protein for activity. these observations argue, at least for pre-like promoters, that promoter activities are influenced by nucleotide sequences at ...19872953648
role of homology in site-specific recombination of bacteriophage lambda: evidence against joining of cohesive ends.bacteriophage lambda integration and excision take place at specific loci called attachment sites. earlier work has shown that efficient recombination requires the identical sequence to be present in both attachment sites throughout the seven-base-pair region between the points of strand exchange. a plausible model for the role of homology postulates that int, the site-specific recombinase, makes double-strand breaks at attachment sites such that each broken end has a short single-strand protrus ...19872954163
construction of a dna-polymerase i overproducing plasmid and isolation of the enzyme.the pola gene of escherichia coli coding for dna polymerase i was cloned under the control of bacteriophage lambda promoter pl and gene n in a high copy number plasmid vector. the chromosomally located lambda cits repressor gene kept the synthesis of the pola gene product at 28 degrees c at a low level. raising the temperature to 43 degrees c resulted in inactivation of the repressor and overproduction of dna polymerase i, which could easily be purified to homogeneity.19872955618
mismatch repair of deaminated 5-methyl-cytosine.deamination of 5-methyl-cytosine in double-stranded dna produces a g-t mismatch. heteroduplexes of bacteriophage lambda dna containing a g-t mismatch at the site of a g-5-mec base-pair in one of the parental phages were constructed and used to transfect escherichia coli cells. genetic analysis of the progeny phages derived from such heteroduplexes suggests that, in e. coli, mismatches resulting from the deamination of 5-methyl-cytosine are repaired by a system requiring the e. coli dcm methylase ...19872956428
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