Showing papers on "Lambda phage published in 1998"
TL;DR: The deltarecBCD::Plac-red kan replacement allele can be P1 transduced to other E. coli strains, making the hyper-Rec phenotype easily transferable.
Abstract: Replacement of Escherichia coli's RecBCD function with phage lambda's Red function generates a strain whose chromosome recombines with short linear DNA fragments at a greatly elevated rate. The rate is at least 70-fold higher than that exhibited by a recBC sbcBC or recD strain. The value of the system is highlighted by gene replacement with a PCR-generated DNA fragment. The deltarecBCD::Plac-red kan replacement allele can be P1 transduced to other E. coli strains, making the hyper-Rec phenotype easily transferable.
627 citations
TL;DR: Observations suggest that stx‐I expression can be enhanced by transcription from pR′ as a model for toxin release through cell lysis mediated by action of phage‐encoded lysis functions.
Abstract: Analysis of the DNA sequence of a 17 kb region of the coli lambdoid phage H-19B genome located the genes encoding shiga-like toxin I (Stx-I) downstream of the gene encoding the analogue of the phage lambda Q transcription activator with its site of action, qut at the associated pR' late promoter, and upstream of the analogues of lambda genes encoding lysis functions. Functional studies, including measurement of the effect of H-19B Q action on levels of Stx expressed from an H-19B prophage, show that the H-19B Q acts as a transcription activator with its associated pR'(qut) by promoting readthrough of transcription terminators. Another toxin-producing phage, 933W, has the identical Q gene and pR'(qut) upstream of the stx-II genes. The H-19B Q also activates Stx-II expression from a 933W prophage. An ORF in H-19B corresponding to the holin lysis genes of other lambdoid phages differs by having only one instead of the usual two closely spaced translation initiation signals that are thought to contribute to the time of lysis. These observations suggest that stx-I expression can be enhanced by transcription from pR' as well as a model for toxin release through cell lysis mediated by action of phage-encoded lysis functions. Functional studies show that open reading frames (ORFs) and sites in H-19B that resemble components of the N transcription antitermination systems controlling early operons of other lambdoid phages similarly promote antitermination. However, this N-like system differs significantly from those of other lambdoid phages.
234 citations
TL;DR: Investigation of the binding of beta protein to DNA has revealed a new mechanism of renaturation, and it is observed that beta protein bound more strongly to a presumed intermediate in the renaturation reaction that beta itself catalyzed, and beta thereby protected all of a renatured duplex 83-mer oligonucleotide from nuclease digestion.
98 citations
TL;DR: Overexpression of Xis in wild-type cells from a multicopy plasmid inhibited integration of lambda and promoted curing of established lysogens, confirming that accumulation of XIS interferes with the ability of Int to establish and maintain an integrated prophage.
Abstract: Lambda Xis, which is required for site-specific excision of phage lambda from the bacterial chromosome, has a much shorter functional half-life than Int, which is required for both integration and excision (R. A. Weisberg and M. E. Gottesman, p. 489–500, in A. D. Hershey, ed., The Bacteriophage Lambda, 1971). We found that Xis is degraded in vivo by two ATP-dependent proteases, Lon and FtsH (HflB). Xis was stabilized two- to threefold more than in the wild type in a lon mutant and as much as sixfold more in a lon ftsH double mutant at the nonpermissive temperature for the ftsH mutation. Integration of lambda into the bacterial chromosome was delayed in the lon ftsH background, suggesting that accumulation of Xis in vivo interferes with integration. Overexpression of Xis in wild-type cells from a multicopy plasmid inhibited integration of lambda and promoted curing of established lysogens, confirming that accumulation of Xis interferes with the ability of Int to establish and maintain an integrated prophage.
68 citations
TL;DR: The central 65 to 169 domain of lambda Int was cloned and purified and found to bind to core-type DNA autonomously and specifically, and Int C65, which was shown to be as competent as Int, in binding to, and cleaving, core- type sites.
66 citations
TL;DR: These mutations identify a locus of sigma70 that is important for the formation and stability of open promoter complex, likely because it mediates protein interactions with RNAP core.
Abstract: The σ initiation factor σ70 of Escherichia coli acts not only in promoter recognition and DNA strand opening, but also to mediate the transformation of RNA polymerase (RNAP) to an antiterminating form by the phage λ gene Q protein. Q is able to bind and modify RNAP when α70, still present in the initially elongating enzyme, recognizes a repeat of the −10 promoter element and induces a transcription pause. We have isolated mutations in the rpoD gene for σ70 that impair Q function because they reduce the ability of σ70 to recognize the downstream pause site. These mutations identify a locus of σ70 that is important for the formation and stability of open promoter complex, likely because it mediates protein interactions with RNAP core.
47 citations
TL;DR: In this article, a bacteriophage lambda has been used for the study of genetic recombination, showing that it is possible to correlate inheritance of DNA with the inheritance of information encoded by DNA through density-label equilibrium centrifugation.
41 citations
TL;DR: The direct application of lambda phage in targeted mutagenesis is described and it is demonstrated that in vivo phage-plasmid recombination can be used to introduce neo and lacZ - neo mutations into precise positions within a lambda2TK subclone via double crossover recombination.
Abstract: Targeted mutagenesis is an extremely useful experimental approach in molecular medicine, allowing the generation of specialized animals that are mutant for any gene of interest. Currently the rate determining step in any gene targeting experiment is construction of the targeting vector (TV). In order to streamline gene targeting methods and avoid problems encountered with plasmid TVs, we describe the direct application of lambda phage in targeted mutagenesis. The recombination-proficient phage vector lambda2TK permits generation of TVs by conventional restriction-ligation or recombination-mediated methods. The resulting lambdaTV DNA can then be cleaved with restriction endonucleases to release the bacteriophage arms and can subsequently be electroporated directly into ES cells to yield gene targets. We demonstrate that in vivo phage-plasmid recombination can be used to introduce neo and lacZ - neo mutations into precise positions within a lambda2TK subclone via double crossover recombination. We describe two methods for eliminating single crossover recombinants, spi selection and size restriction, both of which result in phage TVs bearing double crossover insertions. Thus TVs can be easily and quickly generated in bacteriophage without plasmid subcloning and with little genomic sequence or restriction site information.
25 citations
TL;DR: It is suggested that in rich media phage lambda produces some early proteins in excess of that needed for its effective propagation and indicate that replication proteins may be limiting factors for phage lytic growth in poor media.
Abstract: Bacteriophage lambda is unable to lysogenize Escherichia coli hosts harbouring the rpoA341 mutation due to a drastic reduction in transcription from CII-activated lysogenic promoters (pE, pI and paQ). In addition, the level of early transcripts involved in the lytic pathway of lambda development is also decreased in this genetic background due to impaired N-dependent antitermination. Here, it is demonstrated that despite the reduced level of early lytic pL- and pR-derived transcripts, lytic growth of bacteriophage lambda is not affected in rich media. The level of the late lytic, pR-derived transcripts also remains unaffected by the rpoA341 mutation under these conditions. However, it was found that whilst there is no significant difference in the phage burst size in rpoA+ and rpoA341 hosts growing in rich media, phage lambda is not able to produce progeny in the rpoA341 mutant growing in minimal medium, in contrast to otherwise isogenic rpoA+ bacteria. Provision of an excess of the phage replication proteins O and P in trans or overproduction of the antitermination protein N restore the ability of phage lambda to produce progeny in the rpoA341 mutant under the latter conditions. These results suggest that in rich media phage lambda produces some early proteins in excess of that needed for its effective propagation and indicate that replication proteins may be limiting factors for phage lytic growth in poor media.
23 citations
TL;DR: To increase the expression in the lambda system, a Q- mutant lambda vector is constructed that can be used in long-term operations such as a two-stage continuous operation and it is expected that the replicated Q- lambda DNA containing a cloned gene would not be coated by a phage head and would remain naked for ample expression of theCloned gene and host cells would not lyse easily and consequently would produce larger amounts of cloned-gene products.
Abstract: We previously demonstrated that the lambda system integrated into the host chromosome can overcome the instability encountered in continuous operations of unstable plasmid-based expression vectors. High stability of a cloned gene in a lysogenic state and a high copy number in a lytic state provide cloned-gene stability and overexpression in a two-stage continuous operation. But the expression by the commonly used S- mutant lambda was only twice as high as that of the single copy. To increase the expression in the lambda system, we constructed a Q- mutant lambda vector that can be used in long-term operations such as a two-stage continuous operation. The Q- mutant phage lambda is deficient in the synthesis of proteins involved in cell lysis and lambda DNA packaging, while the S- mutant is deficient in the synthesis of one of two phage proteins required for lysis of the host cell and liberation of the progeny phage. Therefore, it is expected that the replicated Q- lambda DNA containing a cloned gene would not be coated by a phage head and would remain naked for ample expression of the cloned gene and host cells would not lyse easily and consequently would produce larger amounts of cloned-gene products. The beta-galactosidase expression per unit cell by the Q- mutant in a lytic state was about 30 times higher than that in a lysogenic state, while the expression by the commonly used S- mutant in a lytic state was twice as high as that in a lysogenic state. The optimal switching time of the Q- mutant from the lysogenic state to the lytic state for the maximum production of beta-galactosidase was 5.3 h, which corresponds to an early log phase in the batch operation.
20 citations
TL;DR: A physical map of the Zymomonas mobilis ZM4 genome has been constructed from the results of reciprocal Southern hybridization with PmeI, PacI, and NotI-digested genomic DNA fragments and linking cosmid clones.
TL;DR: First in vitro studies on the interactions between J and LamB are described, showing that J could be expressed at high levels (up to 28% of whole cell proteins), in an insoluble form, and solubilized J was able to interact with LamB trimers in vitro.
TL;DR: It is demonstrated that the burst size of phage λcIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37° C, while no phage progeny are observed at 20°‹C.
Abstract: It was previously demonstrated that while lysogenic development of bacteriophage lambda in Escherichia coli proceeds normally at low temperature (20-25 degrees C), lytic development is blocked under these conditions owing to the increased stability of the phage CII protein. This effect was proposed to be responsible for the increased stimulation of the pE promoter, which interferes with expression of the replication genes, leading to inhibition of phage DNA synthesis. Here we demonstrate that the burst size of phage lambda cIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37 degrees C, while no phage progeny are observed at 20 degrees C. Contrary to previous reports, it is possible to demonstrate that pE promoter activation by CII may be more efficient at lower temperature. Using density-shift experiments, we found that phage DNA replication is completely blocked at 20 degrees C. Phage growth was also inhibited in cells overexpressing cII, which confirms that CII is responsible for inhibition of phage DNA replication. Unexpectedly, we found that replication of plasmids derived from bacteriophage lambda is neither inhibited at 20 degrees C nor in cells overexpressing cII. We propose a model to explanation the differences in replication observed between lambda phage and lambda plasmid DNA at low temperature.
TL;DR: Differences in the efficiency of pR activation observed in dnaA mutants at 30 and 43°C can explain the mechanisms of allele specificity of DnaA gene function in the replication of bacteriophage λ and plasmids derived from this phage.
Abstract: Allele specificity of the Escherichia coli dnaA gene function in the replication of plasmids derived from bacteriophage λ has been demonstrated previously. Here, using a series of dnaA temperature-sensitive mutants, we investigated dnaA allele specificity of the replication of phages λP+ and λPts1πA66. We found that phage λP+ produces its progeny efficiently at 43°C irrespective of the dnaA allele, whereas λPts1πA66, which is unable to develop lytically in the dnaA+ host at this temperature, can replicate with different efficiency in certain dnaA mutants. Since the main role of DnaA in λ development seems to be stimulation of transcription from the pR promoter, we measured the activity of this promoter (using a pR-lacZ fusion) and the abundance of pR-derived transcripts (by Northern blotting analysis) in dnaA+ host and dnaA(ts) mutants at 30 and 43°C. We found significant differences in the activity of pR in various dnaA(ts) mutants at 30°C, which indicate different levels of stimulation of this promoter by products of particular dnaA alleles at permissive temperature. Differential levels of DnaA-mediated stimulation of pR in various dnaA(ts) mutants were also found at 43°C. Stimulation of the pR promoter by DnaA is necessary for both efficient production of the λ replication proteins, O and P, and effective transcriptional activation of oriλ. The differences in the efficiency of pR activation observed in dnaA mutants at 30 and 43°C can explain the mechanisms of allele specificity of dnaA gene function in the replication of bacteriophage λ and plasmids derived from this phage.
TL;DR: The individual mutations with the highest compensatory information for W138P repair are the substitutions A146F and A146M, selected after targeted randomization of three residues in the neighborhood of Trp138 by combinatorial mutagenesis.
Abstract: Model-free approaches (random mutagenesis, DNA shuffling) in combination with more "rational," three-dimensional information-guided randomization have been used for directed evolution of lysozyme activity in a defective T4 lysozyme mutant. A specialized lysozyme cloning vector phage, derived from phage lambda, depends upon T4 lysozyme function for its ability to form plaques. The substitution W138P in T4 lysozyme totally abolishes its plaque-forming ability. Compensating mutations in W138P T4 lysozyme after sequential random mutagenesis of the whole gene as well as after targeted randomization of residues in the vicinity of Trp138 were selected. In a second stage, these mutations were randomly recombined by the recombinatorial PCR method of DNA shuffling. Shuffled and selected W138P T4 lysozyme variants provide the hybrid lambda phage with sufficient lysozyme activity to produce normal-size plaques, even at elevated temperature (42 degrees C). The individual mutations with the highest compensatory information for W138P repair are the substitutions A146F and A146M, selected after targeted randomization of three residues in the neighborhood of Trp138 by combinatorial mutagenesis. The best evolved W138P T4 lysozymes, however, accumulated mutations originating from both randomly mutagenized as well as target-randomized variants.
TL;DR: In this article, the authors examined several experimental parameters of the lambda cI/cII transgenic mutation assay and found that the mean mutant frequency of packaged phage stored 28 days at 4 degrees C was consistently higher than phage plated the same day as packaging.
Abstract: We examined several experimental parameters of the lambda cI/cII transgenic mutation assay. In the assay, clear plaque lambda phage mutants are identified in a positive selection scheme following rescue of the lambda/LIZ shuttle vector from frozen tissues of Big Blue" transgenic mice. Mutant frequency and titer of phage from various tissues of control and ENU-treated animals was essentially the same on LB or TB1 plating medium, and storage of isolated DNA at 4 degrees C for up to 4 months did not affect either mutant frequency or titer. Storage of packaged phage for 28 days at 4 degrees C did not affect titer. The mean mutant frequency of packaged phage stored 28 days at 4 degrees C was consistently higher than phage plated the same day as packaging (day 0), though the difference was statistically significant in only two of the four samples tested. Reconstruction experiments in which numerically defined titers of known cII mutants were plated on both G1217 and G1225 E. coli strains and incubated at 37 degrees C or 24 degrees C showed highest titers on G1217 at 37 degrees C. The fraction of the G1217, 37 degrees C titer seen in the other strains and conditions varied widely with the cII mutation.
TL;DR: The present work was undertaken to analyze the possibility that mechanisms other than DNA methylation could account for the viability of several clones exhibiting a r+m‐ phenotype, and found no evidence for an inhibitory agent or endonuclease compartmentation.
Abstract: Bacterial restriction and modification systems must be regulated to avoid self-restriction. It is generally accepted that cognate DNA methyltransferases normally protects both, the host's chromosome and extrachromosomal elements from the activity of their endonuclease counterparts. When the bstVIRM genes from Bacillus stearothermophilus V were subcloned into Escherichia coli, several clones exhibiting a r+m- phenotype were originated. The present work was undertaken to analyze the possibility that mechanisms other than DNA methylation could account for the viability of these cells. No evidence was found for an inhibitory agent or endonuclease compartmentation. In vivo experiments showed that lambda phage multiplication was poorly restricted by the heterologous enzyme. The restricting activity against the incoming phase increased however when phage adsortion was performed at higher temperatures. Analogous experiments in which a DNA-repair deficient strain was used as a host for the thermophilic R-M system suggested, to some extent, the participation of the repair machinery in the viability of r+m- clones.
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TL;DR: Computer analysis revealed palindromes and repeats within 450 bp of N15, including the cohesive termini including the cos region, which are notably homologous only within the 50 bp region from the left and right ends.
Abstract: The cohesive termini including the cos region (altogether 414 bp) of the DNA of the temperate coliphage N15 are sequenced. The termini are complementary 12-nucleotide single-stranded 5'-extended DNAs. The sequence of the left terminus is 5'-GGGCGGCGTCCG-3', that of the right 5'CGGACGCCGCCC-3'. Ten nucleotides of the N15 termini are identical to those of phage lambda. The N15 and lambda sequences are notably homologous only within the 50 bp region from the left and right ends. Phage N15 has a region with the nucleotide sequence identical to the R4 site of phage lambda, presumably reacting with terminase. This region is situated in the same site with regard to the cohesive sequence as in phage lambda. The cos region of N15 has no sequences similar to R1, R2, and R3 of lambda. N15 has a sequence similar to IHF of phage lambda, but in N15 this sequence is located near the right but not left (as in phage lambda) terminus. Computer analysis revealed palindromes and repeats within 450 bp of N15, including the cohesive termini.
TL;DR: It is established that lambda poriCc infection of a lambda lysogen is a legitimate assay for oriC function and it is demonstrated using this assay that the UV induced inhibition of initiation of DNA replication from oriC is not a SOS function nor a heat shock function.
Abstract: The oriC transducing phage lambda poriCc is a pseudovirulent phage capable of forming plaques on a lambda lysogen. This phenotype is dependent upon the presence of the oriC insert. The ability of lambda poriCc to form plaques on a lambda lysogen represents a potential phage assay system for studying aspects of oriC function. In the present study we establish that lambda poriCc infection of a lambda lysogen is a legitimate assay for oriC function. We use this assay to confirm the previously reported observation that initiation of DNA replication from oriC is transiently inhibited in a ultra violet (UV) irradiated cell at doses greater than 60 J/m2. We further demonstrate using this assay that the UV induced inhibition of initiation of DNA replication from oriC is not a SOS function nor a heat shock function. In the course of these studies, we found that lambda poriCc infection of a non-lysogenic cell is extremely sensitive to pre-irradiation of the Escherichia coli host. We postulate that the sensitivity of lambda poriCc replication to host cell pre-irradiation reflects in some way the transient inhibition of initiation of DNA replication from oriC following UV irradiation.
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TL;DR: Induction of the mutagenic SOS system by UV light caused a 1.5-fold increase in the frequency of c mutations induced by 8-3H-purines in fPG+ cells over that in fpg-cells, which suggests that apurinic AP sites produced after the excision of 8-oxo-G by the Fpg protein are substrates for mutagenics SOS repair.
Abstract: The lethal and mutagenic effects of the decay of 3H incorporated in phage lambda DNA as 8-3H-adenosine and 8-3H-guanosine were studied, using the DNA of 8-3H-adenine as a labeled DNA precursor. A transmutation component of 3H decay is involved in formation of 8-oxoguanine (8-oxo-G) and 8-oxoadenine (8-oxo-A) residues in phage DNA. The efficiency of phage inactivation (the number of lethal lesions per one tritium decay in the phage genome) for 3H decay in position 8 of purines was the same as that measured in positions 5 and 6 of pyrimidines (alpha = 0.14 +/- 0.01) and virtually did not depend on the fpg-1::kan mutation in the host gene encoding the Fpg protein (formamidepyrimidine-DNA-glycosylase). The efficiency of the mutagenic effect of 3H-purines Em (frequency of c mutations per one 3H decay in the phage genome) was (2.9 +/- 0.3) x 10(-5) in the fpg+ host and (4.6 +/- 0.4) x 10(-5) in the fpg-host. This means that the Fpg protein excised approximately 40% of premutational DNA lesions (probably, 8-oxo-G residues). Induction of the mutagenic SOS system by UV light caused a 1.5-fold increase in the frequency of c mutations induced by 8-3H-purines in fpg+ cells over that in fpg-cells. This suggests that apurinic AP sites produced after the excision of 8-oxo-G by the Fpg protein are substrates for mutagenic SOS repair.
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