Showing papers on "Lambda phage published in 1992"
TL;DR: The X-ray crystal structure of Fis has been determined and insights into its mode of DNA binding and mechanisms of action in these disparate systems are being made.
Abstract: Higher-order nucleoprotein complexes are associated with many biological processes. In bacteria the formation of these macromolecular structures for DNA recombination, replication, and transcription often requires not only the participation of specific enzymes and co-factors, but also a class of DNA-binding proteins collectively known as 'nucleoid-associated' or 'histone-like' proteins. Examples of this class of proteins are HU, Integration Host Factor, H-NS, and Fis. Fis was originally identified as the factor for inversion stimulation of the homologous Hin and Gin site-specific DNA recombinases of Salmonella and phage Mu, respectively. This small, basic, DNA-bending protein has recently been shown to function in many other reactions including phage lambda site-specific recombination, transcriptional activation of rRNA and tRNA operons, repression of its own synthesis, and oriC-directed DNA replication. Cellular concentrations of Fis vary tremendously under different growth conditions which may have important regulatory implications for the physiological role of Fis in these different reactions. The X-ray crystal structure of Fis has been determined and insights into its mode of DNA binding and mechanisms of action in these disparate systems are being made.
312 citations
TL;DR: Results of this genetic analysis indicate that in some isolates, part of the binding site is encoded by flanking vector sequences, which may prove generally useful for identifying protein sequences able to recognize specific surfaces.
Abstract: Escherichia coli able to specifically adhere to iron oxide and not adhere to other metal oxides were constructed by genetic engineering. Concatamers of random oligonucleotides were introduced into a portion of a plasmid-borne lamB gene encoding an external domain of the phage lambda receptor. Bacteria able to adhere to iron oxide were selected by serial enrichment from the population of plasmid transformants. The concatameric nature of the inserted DNA allows a genetic analysis analogous to exons shuffling. Results of this genetic analysis indicate that in some isolates, part of the binding site is encoded by flanking vector sequences. This strategy may prove generally useful for identifying protein sequences able to recognize specific surfaces.
231 citations
TL;DR: The 21 kd NusG protein is essential for E. coli viability and inactivated the phage HK022 Nun termination factor and the factor-independent lambda tl terminator was fully active in Nus G-depleted cells and could be suppressed by phage lambda N function.
176 citations
TL;DR: It is suggested that RexB proteins form ion channels that, in response to lytic growth of bacteriophages, depolarize the cytoplasmic membrane and are likely to be a polytopic transmembrane protein.
Abstract: The rexA and rexB genes of bacteriophage lambda encode a two-component system that aborts lytic growth of bacterial viruses. Rex exclusion is characterized by termination of macromolecular synthesis, loss of active transport, the hydrolysis of ATP, and cell death. By analogy to colicins E1 and K, these results can be explained by depolarization of the cytoplasmic membrane. We have fractionated cells to determine the intracellular location of the RexB protein and made RexB-alkaline phosphatase fusions to analyze its membrane topology. The RexB protein appears to be a polytopic transmembrane protein. We suggest that RexB proteins form ion channels that, in response to lytic growth of bacteriophages, depolarize the cytoplasmic membrane. The Rex system requires a mechanism to prevent lambda itself from being excluded during lytic growth. We have determined that overexpression of RexB in lambda lysogens prevents the exclusion of both T4 rII mutants and lambda ren mutants. We suspect that overexpression of RexB is the basis for preventing self-exclusion following the induction of a lambda lysogen and that RexB overexpression is accomplished through transcriptional regulation.
141 citations
TL;DR: The common laboratory strain of bacteriophage lambda--lambda wild type or lambda PaPa--carries a frameshift mutation relative to Ur-lambda, the original isolate, which has expanded receptor specificity and adsorbs to Escherichia coli cells more rapidly.
Abstract: The common laboratory strain of bacteriophage lambda--lambda wild type or lambda PaPa--carries a frameshift mutation relative to Ur-lambda, the original isolate. The Ur-lambda virions have thin, jointed tail fibers that are absent from lambda wild type. Two novel proteins of Ur-lambda constitute the fibers: the product of stf, the gene that is disrupted in lambda wild type by the frameshift mutation, and the product of gene tfa, a protein that is implicated in facilitating tail fiber assembly. Relative to lambda wild type, Ur-lambda has expanded receptor specificity and adsorbs to Escherichia coli cells more rapidly.
133 citations
TL;DR: Reconstituted biologically relevant transcriptional antitermination in vitro by the phage lambda N protein, a newly identified Escherichia coli transcription elongation factor, and used RNA polymerase affinity chromatography to show that NusG binds to the core component of E. coliRNA polymerase.
130 citations
TL;DR: Structural motifs provide a basis for postulating the mechanism of site-specific recombination and may also be relevant to other pathways in which two distant chromosomal sites become associated.
Abstract: The excisive recombination reaction of bacteriophage lambda involves a specific and efficient juxtaposition of two distant higher order protein-DNA complexes on the chromosome of Escherichia coli. These complexes, which mediate synapsis and strand exchange, consist of two DNA sequences, attL and attR, the bivalent DNA binding protein Int, and the sequence-specific DNA bending proteins, IHF, Xis, and Fis. The protein-protein and protein-DNA interactions within, and between, these complexes were studied by various biochemical techniques and the patterns of synergism among pairs of mutants with marginally impaired recombination function were analyzed. The DNA bending proteins facilitated long-range tethering of high- and low-affinity DNA sites by the bivalent Int protein, and a specific map is proposed for the resulting Int bridges. These structural motifs provide a basis for postulating the mechanism of site-specific recombination and may also be relevant to other pathways in which two distant chromosomal sites become associated.
126 citations
TL;DR: It is shown that, in the presence of NusA, N inhibits pausing by RNA polymerase and Rho-dependent termination in vitro at three sites in the lambda terminator tR1 which are located less than 100 base pairs downstream from nutR.
92 citations
TL;DR: The function of an open reading frame (orf-45) located upstream of the sbcC gene of Escherichia coli was investigated and it was concluded that the products of orf-45 and sBCC are functionally related.
Abstract: The function of an open reading frame (orf-45) located upstream of the sbcC gene of Escherichia coli was investigated. Mutations that inactivate sbcC improve the ability to propagate lambda red gam phage that carry a palindromic sequence in their DNA. They also act with sbcB mutations as cosuppressors of the defects in recombination, DNA repair, and cell viability associated with recBC mutations. A 1,282-bp cassette encoding resistance to kanamycin was used to disrupt orf-45. The mutation, which has a polar effect on the expression of sbcC, allowed stable propagation of palindromic lambda phage even when the sbcC gene product was provided in trans. Additional nonpolar mutations in orf-45 were isolated on the basis of their ability to improve the growth of recBC sbcB strains. These mutations also confer resistance to mitomycin C, allow efficient recombination in Hfr crosses, and facilitate stable propagation of palindromic phage. It is concluded that the products of orf-45 and sbcC are functionally related. The orf-45 gene is therefore renamed sbcD.
89 citations
TL;DR: Fragments from eleven different regions of lambda genome, encoding specific peptides or antisense RNA sequences, rendered E. coli resistant to the phage, and revealed some previously unknown functions of phage lambda, including suppression of the cellular lambda receptor by an 'accessory' gene of the phages.
Abstract: Selective inhibition of specific genes can be accomplished using genetic suppressor elements (GSEs) that encode antisense RNA, dominant negative mutant proteins, or other regulatory products. GSEs may correspond to partial sequences of target genes, usually identified by trial and error. We have used bacteriophage lambda as a model system to test a concept that biologically active GSEs may be generated by random DNA fragmentation and identified by expression selection. Fragments from eleven different regions of lambda genome, encoding specific peptides or antisense RNA sequences, rendered E. coli resistant to the phage. Analysis of these GSEs revealed some previously unknown functions of phage lambda, including suppression of the cellular lambda receptor by an 'accessory' gene of the phage. The random fragment selection strategy provides a general approach to the generation of efficient GSEs and elucidation of novel gene functions.
75 citations
TL;DR: It is shown here, by the use of reconstituted mutant RNA polymerases, that the C-terminal portion of the α subunit of RNA polymerase plays an essential role in the stimulation of transcription by IHF.
TL;DR: It is anticipated that the availability of spectrally enhanced proteins will significantly enhance the utility of both fluorescence and phosphorescence spectroscopies to study protein structure and function in complex interacting systems.
Abstract: We have used a tryptophan-requiring Escherichia coli auxotroph to replace the three tryptophan residues of lambda cI repressor with 5-hydroxy-L-tryptophan (5-OHTrp) By using a nonleaky promoter, we have achieved > 95% replacement of tryptophan in the repressor We show that the absorbance and fluorescence properties of 5-OHTrp-lambda cI are clearly distinct from lambda cI repressor and that the fluorescence of 5-OHTrp-lambda cI repressor can be observed selectively in the presence of exogenous tryptophan We also show that the 5-OHTrp-lambda cI repressor functional properties, as assessed by measurement of binding constants for self-association and for association to operator DNA, and structural properties, as assessed by fluorescence, are indistinguishable from the native repressor Based on these results, we anticipate that the availability of spectrally enhanced proteins will significantly enhance the utility of both fluorescence and phosphorescence spectroscopies to study protein structure and function in complex interacting systems
TL;DR: A sensitive genetic assay is developed to analyze DNA sequences and regulatory elements required for immunoglobulin heavy chain isotype switch recombination in substrates containing mu and gamma 3 chain switch (S)-region sequences.
Abstract: We have developed a sensitive genetic assay to analyze DNA sequences and regulatory elements required for immunoglobulin heavy chain isotype switch recombination. Recombination substrates containing mu and gamma 3 chain switch (S)-region sequences, S mu and S gamma 3, are transiently introduced into primary murine B cells cultured with lipopolysaccharide to induce isotype switching. Recombination involving S-region sequences deletes a conditionally lethal marker, the leftward promoter of phage lambda (lambda PL), enabling recovered plasmids to transform Escherichia coli. In substrates carrying S mu-lambda PL-S gamma 3, about 2% of replicated molecules undergo deletion of lambda PL during transfection; insertion of either the immunoglobulin heavy chain promoter and enhancer sequences or cytomegalovirus IE1 promoter region upstream of S mu increases recombination 10-fold or more to 25% of replicated molecules. Guanosine-rich S-region sequences are essential for efficient recombination of these substrates.
TL;DR: It is shown that Xis has at least two functional domains and also demonstrates the importance of the cooperativity in DNA binding of FIS, Xis and Int in lambda excision.
Abstract: We have performed a mutational analysis of the xis gene of bacteriophage lambda. The Xis protein is 72 amino acids in length and required for excisive recombination. Twenty-six mutants of Xis were isolated that were impaired or deficient in lambda excision. Mutant proteins that contained amino acid substitutions in the N-terminal 49 amino acids of Xis were defective in excisive recombination and were unable to bind DNA. In contrast, one mutant protein containing a leucine to proline substitution at position 60 and two truncated proteins containing either the N-terminal 53 or 64 amino acids continued to bind lambda DNA, interact cooperatively with FIS and promote excision. However, these three mutants were unable to bind DNA cooperatively with Int. Cooperativity between wild-type Xis and Int required the presence of FIS, but not the Int core-type binding sites. This study shows that Xis has at least two functional domains and also demonstrates the importance of the cooperativity in DNA binding of FIS, Xis and Int in lambda excision.
TL;DR: The function of the Hsp70 complex in sequestering and releasing P protein provides for the critical step in the disassembly pathway, and an ATP-dependent ternary interaction between P, DnaK, and DnaJ is discovered.
Abstract: Initiation of DNA replication by phage lambda requires the ordered assembly and disassembly of a specialized nucleoprotein structure at the origin of replication. In the disassembly pathway, a set of Escherichia coli heat shock proteins termed the Hsp70 complex--DnaK, DnaJ, and GrpE--act with ATP to release lambda P protein from the nucleo-protein complex, freeing the DnaB helicase for its DNA-unwinding reaction. To investigate the mechanism of the release reaction, we have examined the interaction between P and the three heat shock proteins by glycerol gradient sedimentation and gel electrophoresis. We have discovered an ATP-dependent ternary interaction between P, DnaK, and DnaJ; this P.DnaK.DnaJ complex is dissociated by GrpE. We have concluded that the function of the Hsp70 complex in sequestering and releasing P protein provides for the critical step in the disassembly pathway. Based on our data and other work on protein folding, the formation of the P.DnaK.DnaJ complex might involve a conformational shift to a folding intermediate of P.
TL;DR: The integration host factor of Escherichia coli is a small, basic protein that is required for lambda site‐specific recombination and a variety of cellular processes and a model of the IHF‐DNA complex proposed by Yang and Nash is discussed.
Abstract: The integration host factor (IHF) of Escherichia coli is a small, basic protein that is required for lambda site-specific recombination and a variety of cellular processes. It is composed of two subunits, alpha and beta, that are encoded by the himA and hip (himD) genes, respectively. IHF is a sequence-specific DNA-binding protein and bends the DNA when it binds. We have used the bacteriophage P22-based challenge phage selection to isolate suppressor mutants with altered, expanded DNA binding specificities. The suppressors were isolated by selecting mutants that recognize variants of the phage lambda H'IHF recognition site. Two of the mutants recognize both the wild-type and a single variant site and contain amino acid substitutions at positions 64 (Pro to Leu) or 65 (Lys to Ser) of the alpha subunit. These substitutions are in a region of the protein that is predicted to contain a flexible arm that interacts with DNA. Three other mutants, which recognize the wild-type and a different variant site, contain amino acid substitutions at position 44 (Glu to Lys, Val or Gly) of the beta subunit. These substitutions are in the middle of a predicted beta-strand of the subunit. We discuss the possible mechanisms of suppression by the mutants in terms of a model of the IHF-DNA complex proposed by Yang and Nash [Cell, 57, 869-880 (1989)].
TL;DR: The results suggest that NusG may act as a component of the N antitermination complex and imply a role for Nus g in Rho-dependent termination, and also suggest that nusA binds both N and RNA polymerase and may also interact with other Nus factors.
Abstract: The Escherichia coli Nus factors act in conjunction with the bacteriophage lambda N protein to suppress transcription termination on the lambda chromosome. NusA binds both N and RNA polymerase and may also interact with other Nus factors. To search for additional components of the N antitermination system, we isolated host revertants that restored N activity in nusA1 mutants. One revertant, nusG4, was mapped to the rif region of the E. coli chromosome and shown to represent a point mutation near the 3' end of the nusG gene. The nusG4 mutation also suppressed nusE71 but not nusASal, nusB5, nusC60 (rpoB60), or nusD026 (rho026). However, nusG+ expressed from a multicopy plasmid suppressed nusD026 and related rho mutants for both lambda and phage T4 growth. These results suggest that NusG may act as a component of the N antitermination complex. In addition, the data imply a role for NusG in Rho-dependent termination.
TL;DR: The lambdoid phages are a group of related temperate bacteriophages that lysogenize by site-specific recombination with the bacterial chromosome that suggest that, at an earlier time, phages may have arrived at their present locations by a process of chromosomal walking.
Abstract: The lambdoid phages are a group of related temperate bacteriophages that lysogenize by site-specific recombination with the bacterial chromosome. Various members of the group have different specific chromosomal insertion sites, despite the fact that the enzymes catalyzing the insertion (integrases) appear to be all descended from a common ancestor. Insertion sites are not located randomly on the E. coli chromosome but are restricted to one segment of the map; also, most prophages are oriented in the same direction along the chromosome. Lambdoid phage 21 inserts within the isocitrate dehydrogenase gene and introduces an alternative 165 bp 3' end for that gene. A defective element (e14) inserts at the same position. We suggest that this mode of insertion arose from insertion of an ancestral phage to the right of icd which then picked up part of the icd gene by abnormal excision and speculate that, at an earlier time, phages may have arrived at their present locations by a process of chromosomal walking.
TL;DR: This work describes the characterization of mutations within the A gene which lead to the loss of terminase endonuclease activity without affecting the ability of the enzyme to package monomeric mature (cut) lambda DNA.
TL;DR: By measuring beta-galactosidase levels in Escherichia coli strains lysogenized with lambda phage carrying a metA-lacZ gene fusion, the MetR protein was shown to activate the metA gene.
Abstract: The DNA sequence of the Salmonella typhimurium metA control region is presented. S1 nuclease mapping was used to determine the transcription initiation site. By measuring beta-galactosidase levels in Escherichia coli strains lysogenized with lambda phage carrying a metA-lacZ gene fusion, the MetR protein was shown to activate the metA gene. Homocysteine, an intermediate in methionine biosynthesis, plays a negative role in the MetR-mediated activation mechanism. Gel mobility shift assays and DNase I protection experiments showed that the MetR protein binds to a DNA fragment carrying the metA control region and protects a 26-bp region beginning 9 bp upstream of the -35 promoter sequence. Images
TL;DR: The sensitizer without light and 658 nm photolysis of lambda phage in the absence of Sensitizer do not lead to a measurable decrease in viral inactivity.
Abstract: Exposure of lambda phage to 658 nm light in the presence of 5,10,15,20-tetrakis-(1-methyl-4-pyridyl)-21H,23H-porphine, tetra-p-tosylate leads to complete (greater than 7 logs) inactivation as measured by the plaque assay. The sensitizer without light and 658 nm photolysis of lambda phage in the absence of sensitizer do not lead to a measurable decrease in viral inactivity. Viral inactivation is not dependent upon the presence of oxygen.
TL;DR: Besides the availability of a wide range of lambda vectors, many related techniques such as rapid isolation of lambda DNA, a high efficiency of commercially available in vitro packaging extracts, and in vitro amplification of DNA via the polymerase chain reaction have collectively contributed to lambda's becoming one of the most powerful and popular tools for molecular cloning.
TL;DR: It is demonstrated that the rpoA341 allele prevents lysogenization of the host strain by bacteriophage lambda, a process dependent upon the action of two phage-encoded activators, and proposed that the inhibition of lysagenization arises from a defective interaction between the phage lambda transcriptional activator CII and the mutant RNA polymerase at the phages promoters pI and pE.
TL;DR: It is demonstrated further that restriction enzymes could substitute for the intron-encoded endonuclease, indicating that the end onuclease does not have an essential role in recombination and provides insight into the recombination pathway.
Abstract: Intron mobility requires cleavage of an intronless allele by an intron-encoded endonuclease, followed by transfer of the intron into the cleaved recipient. The mobile phage introns provide an opportunity to identify accessory functions involved in the intron inheritance process. To test for trans and cis requirements of mobility in Escherichia coli, we have exploited the td intron of phage T4 in both phage T4 and lambda genetic backgrounds. Mobility depends on host or phage recombinase functions, RecA or UvsX, respectively. The process also requires a phage-encoded 5'----3' exonuclease activity and associated annealing function that can be provided by phage lambda. Finally, host-encoded 3'----5' exonuclease activities are also implicated in intron inheritance. We demonstrated further that restriction enzymes could substitute for the intron-encoded endonuclease, indicating that the endonuclease does not have an essential role in recombination. Neither the precise position nor the nature of the double-strand break was critical to intron transfer. These features provide insight into the recombination pathway and are factors impacting on the spread of introns throughout natural populations.
TL;DR: A set of plasmid vectors which allow single-step cloning and expression of PCR-amplified DNA coding sequences has been constructed and a significant increase in expression of 14-3-3 protein was observed when synthetic TIRs were used in the place of the cII TIR.
Abstract: A set of plasmid vectors which allow single-step cloning and expression of PCR-amplified DNA coding sequences has been constructed. The vectors contain the phage lambda PL promoter, a synthetic translation initiation region (TIR), and convenient cloning sites. The cloning sites provide all or part of an AUG translation initiation codon and facilitate the precise fusion of target DNA sequences to vector transcriptional and translational signals. The vectors were constructed with synthetic TIRs because there is evidence which suggests that the efficiency of the phage lambda cII gene TIR present in the parental vector depends strongly on information contained within the cII N-terminal coding sequence. Bovine brain 14-3-3 eta chain cDNA was PCR-amplified and used to demonstrate the expression capacity of the newly constructed vectors. A significant increase in expression of 14-3-3 protein was observed when synthetic TIRs were used in the place of the cII TIR. Expression levels vary from 15% to 48% of total cell protein. The effects of a reported translational enhancer from phage T7 on expression of the 14-3-3 protein are also discussed. The vectors should be generally useful for high level heterologous protein expression in Escherichia coli.
TL;DR: The results suggest that T acts as a pore that is specific for this type of enzyme, which is required to allow the phage's lysozyme to reach the murein layer of the cell envelope and cause lysis.
Abstract: Lysis protein T of phage T4 is required to allow the phage's lysozyme to reach the murein layer of the cell envelope and cause lysis. Using fusions of the cloned gene t with that of the Escherichia coli alkaline phosphatase or a fragment of the gene for the outer membrane protein OmpA, it was possible to identify T as an integral protein of the plasma membrane. The protein was present in the membrane as a homooligomer and was active at very low cellular concentrations. Expression of the cloned gene t was lethal without causing gross leakiness of the membrane. The functional equivalent of T in phage λ is protein S. An amber mutant of gene S can be complemented by gene t, although neither protein R of λ (the functional equivalent of T4 lysozyme) nor S possess any sequence similarity with their T4 counterparts. The murein-degrading enzymes (including that of phage P22) have in common a relatively small size (molecular masses of ca. 18 000) and a rather basic nature not exhibited by other E. coli cystosolic proteins. The results suggest that T acts as a pore that is specific for this type of enzyme.
TL;DR: The IHF-dependent phenotype of a lambda mutant carrying a deletion of the R1 sequence indicates that IHF obviates the need for terminase binding to the R 1 site, and a lambda phage with mutations in both R2 and R3 fails to form plaques on either IHF+ or I HF- cells, indicating terminasebinding of R2 is involved in suppression of R mutants by IHF.
TL;DR: This in vitro genetic approach identified a mutation which significantly increased binding to gp120 which should be generally applicable to other high affinity protein/ligand interactions that are compatible with expression in Escherichia coli.
TL;DR: In contrast to lambda cII, the C1 protein exhibited little cooperativity with Escherichia coli RNA polymerase for DNA binding, but because of its stronger inherent binding ability, achieved an overall promoter affinity similar to that observed for cII at its cognate promoter signals.
TL;DR: A combination of gel exclusion chromatography and velocity sedimentation gradient data indicates that gpNu3 possesses an unusually elongated shape, which indicates its classification as a scaffolding protein.