Showing papers on "Lambda phage published in 2004"

Rapid genetic engineering of human cytomegalovirus by using a lambda phage linear recombination system: demonstration that pp28 (UL99) is essential for production of infectious virus.

Abstract: A highly efficient lambda phage recombination system previously utilized for studies of bacterial artificial chromosome (BAC)-maintained mouse chromosomal DNA was adapted for the study of the role of human cytomegalovirus (HCMV)-encoded pp28 (UL99) in virus replication. Incorporating a two-step mutagenesis strategy with blue/white selection in Escherichia coli containing a HCMV AD169 BAC, we have shown that we can rapidly introduce point mutations into the HCMV BAC using linear PCR fragments. All manipulations were carried out in bacteria, which greatly accelerated the introduction and analysis of mutations in the viral genome. Our results indicated that HCMV pp28 was essential for the production of infectious virus and that introduction of a single base change that resulted in loss of the myristylation site on pp28 was also associated with the lack of production of infectious virus. Although the block in the viral morphogenesis cannot be determined from these studies, the latter finding suggested that authentic intracellular localization of pp28, not only the expression of the protein, is required for virus assembly.

Switching the polarity of a bacteriophage integration system.

Abstract: During lysogenic growth many temperate bacteriophage genomes are integrated into the host's chromosome and efficient integration and excision are therefore an essential part of the phage life cycle. The Streptomyces phage phiC31 encodes an integrase related to the resolvase/invertases and is evolutionarily and mechanistically distinct from the integrase of phage lambda. We show that during phiC31 integration the polarity of the recombination sites, attB and attP, is dependent on the sequences of the two base pairs (bp) where crossover occurs. A loss or switch in polarity of the recombination sites can occur by mutation of this dinucleotide, leading to incorrectly joined products. The properties of the mutant sites implies that phiC31 integrase interacts symmetrically with the substrates, which during synapsis can align apparently freely in either of two alternative forms that lead to correct or incorrect joining of products. Analysis of the topologies of the reaction products provided evidence that integrase can synapse and activate strand exchange even when recombinant products cannot form due to mismatches at the crossover site. The topologies of the recombination products are complex and indicative of multiple pathways to product formation. The efficiency of integration of a phiC31 derivative, KC859, into an attB site with switched polarity was assayed in vivo and shown to be no different from integration into a wild-type attB. Thus neither the host nor KC859 express a factor that influences the alignment of the recombination sites at synapsis.

Detection of extremely rare alleles by bidirectional pyrophosphorolysis-activated polymerization allele-specific amplification (Bi-PAP-A): measurement of mutation load in mammalian tissues

Abstract: Pyrophosphorolysis-activated polymerization (PAP) was developed to detect extremely rare mutations in complex genomes. In theory, PAP can detect a copy of a single base mutation present in 3 x 10(11) copies of the wild-type allele. In practice, the selectivity of detection is limited by a bypass reaction involving a polymerase extension error from the unblocked oligonucleotide annealed to the opposing strand. Bidirectional PAP allele-specific amplification (Bi-PAP-A) is a novel method that uses two opposing 3'-terminal blocked pyrophosphorolysis-activatable oligonucleotides (P*s) with one nucleotide overlap at their 3' termini. This eliminates the problematic bypass reaction. The selectivity of Bi-PAP-A was examined using lambda phage DNA as a model system. Bi-PAP-A selectively detected two copies of a rare mutated allele in the presence of at least 2 x 10(9) copies of the wild-type lambda phage DNA. Bi-PAP-A was then applied to direct detection of spontaneous somatic mutations in the mouse genome at a frequency as low as 3 x 10(-9). A 370-fold variation in the frequency of a specific somatic mutation among different mouse samples was found, suggesting clonal expansion of mutation occurring during early development and a hyper-Poisson variance. Bi-PAP-A is a rapid, general, and automatable method for the detection of rare mutations.

First-Time Isolation and Characterization of a Bacteriophage Encoding the Shiga Toxin 2c Variant, Which Is Globally Spread in Strains of Escherichia coli O157

Abstract: A bacteriophage encoding the Shiga toxin 2c variant (Stx2c) was isolated from the human Escherichia coli O157 strain CB2851 and shown to form lysogens on the E. coli K-12 laboratory strains C600 and MG1655. Production of Stx2c was found in the wild-type E. coli O157 strain and the K-12 lysogens and was inducible by growing bacteria in the presence of ciprofloxacin. Phage 2851 is the first reported viable bacteriophage which carries an stx2c gene. Electron micrographs of phage 2851 showed particles with elongated hexagonal heads and long flexible tails resembling phage lambda. Sequence analysis of an 8.4-kb region flanking the stx2c gene and other genetic elements revealed a mosaic gene structure, as found in other Stx phages. Phage 2851 showed lysis of E. coli K-12 strains lysogenic for Stx phages encoding Stx1 (H19), Stx2 (933W), Stx (7888), and Stx1c (6220) but showed superinfection immunity with phage lambda, presumably originating from the similarity of the cI repressor proteins of both phages. Apparently, phage 2851 integrates at a different chromosomal locus than Stx2 phage 933W and Stx1 phage H19 in E. coli, explaining why Stx2c is often found in combination with Stx1 or Stx2 in E. coli O157 strains. Diagnostic PCR was performed to determine gene sequences specific for phage 2851 in wild-type E. coli O157 strains producing Stx2c. The phage 2851 q and o genes were frequently detected in Stx2c-producing E. coli O157 strains, indicating that phages related to 2851 are associated with Stx2c production in strains of E. coli O157 that were isolated in different locations and time periods.

Modulation of DNA Repair and Recombination by the Bacteriophage λ Orf Function in Escherichia coli K-12

Abstract: The orf gene of bacteriophage λ, fused to a promoter, was placed in the galK locus of Escherichia coli K-12. Orf was found to suppress the recombination deficiency and sensitivity to UV radiation of mutants, in a Δ( recC ptr recB recD ):: P tac gam bet exo pae cI Δ recG background, lacking recF , recO , recR , ruvAB , and ruvC functions. It also suppressed defects of these mutants in establishing replication of a pSC101-related plasmid. Compared to orf , the recA803 allele had only small effects on recF , recO , and recR mutant phenotypes and no effect on a ruvAB mutant. In a fully wild-type background with respect to known recombination and repair functions, orf partially suppressed the UV sensitivity of ruvAB and ruvC mutants.

Initial cos cleavage of bacteriophage λ concatemers requires proheads and gpFI in vivo.

Abstract: The development of bacteriophage lambda and double-stranded DNA viruses in general involves the convergence of two separate pathways: DNA replication and head assembly. Clearly, packaging will proceed only if an empty capsid shell, the prohead, is present to receive the DNA, but genetic evidence suggests that proheads play another role in the packaging process. For example, lambda phages with an amber mutation in any head gene or in FI, the gene encoding the accessory packaging protein gpFI, are able to produce normal amounts of DNA concatemers but they are not cut, or matured, into unit length chromosomes for packaging. Similar observations have been made for herpes simplex 1 virus. In the case of lambda, a negative model proposes that in the amber phages, unassembled capsid components are inhibitory to maturation, and a positive model suggests that assembled proheads are required for cutting. We tested the negative model by using a deletion mutant devoid of all prohead genes and FI in an in vivo cos cleavage assay; in this deleted phage, the cohesive ends were not cut. When lambda proheads and gpFI were provided in vivo via a second prophage, cutting was restored, and gpFI was required, results that support the positive model. Phage 21 is a sister phage of lambda, and although its capsid proteins share approximately 60% residue identity with lambda's, phage 21 proheads did not restore cutting, even when provided with the accessory protein gpFI. Models for the role of proheads and gpFI in cos cutting are discussed.

Book review: A Genetic Switch–Third Edition Phage Lambda Revisited

Abstract: In undergoing this life, many people always try to do and get the best. New knowledge, experience, lesson, and everything that can improve the life will be done. However, many people sometimes feel confused to get those things. Feeling the limited of experience and sources to be better is one of the lacks to own. However, there is a very simple thing that can be done. This is what your teacher always manoeuvres you to do this one. Yeah, reading is the answer. Reading a book as this a genetic switch third edition phage lambda revisited and other references can enrich your life quality. How can it be?

The recognition and modification sites for the bacterial type I restriction systems KpnAI, StySEAI, StySENI and StySGI

Abstract: Using an in vivo plasmid transformation method, we have determined the DNA sequences recognized by the KpnAI, StySEAI, StySENI and StySGI R-M systems from Klebsiella oxytoca strain M5a1, Salmonella eastbourne, Salmonella enteritidis and Salmonella gelsenkirchen, respectively. These type I restriction-modification systems were originally identified using traditional phage assay, and described here is the plasmid transformation test and computer program used to determine their DNA recognition sequences. For this test, we constructed two sets of plasmids, pL and pE, that contain phage lambda and Escherichia coli K-12 chromosomal DNA fragments, respectively. Further, using the methylation sensitivities of various known type II restriction enzymes, we identified the target adenines for methylation (listed in bold italics below as A or T in case of the complementary strand). The recognition sequence and methylation sites are GAA(6N)TGCC (KpnAI), ACA(6N)TYCA (StySEAI), CGA(6N)TACC (StySENI) and TAAC(7N)RTCG (StySGI). These DNA recognition sequences all have a typical type I bipartite pattern and represent three novel specificities and one isoschizomer (StySENI). For confirmation, oligonucleotides containing each of the predicted sequences were synthesized, cloned into plasmid pMECA and transformed into each strain, resulting in a large reduction in efficiency of transformation (EOT).

Bacterial sensitivity to bacteriophage in the aquatic environment.

Abstract: There are several unusual features about phage when you first encounter them as a biologist. They are small, but conform to one of a few morphological types. Next their genomes can be composed of DNA or RNA and be single or double stranded. Finally they are numerically more abundant than prokaryotes and a significant proportion of them form an association in their microbial host populations termed lysogeny. The latter findings indicate that they are numerically significant in microbial populations. Since bacterial and phage abundance or lack of it is related in environments, this implies that the phage populations 'titrate' their hosts, and more probably the host's physiological status. Microbial populations wax and wane with nutritional inputs and there is a dynamic relationship between phage population sizes and host numbers and physiology. Overlay this with the different phage life cycle strategies, exemplified at the extremes by phage lambda (temperate) and phage T4 (virulent), then it becomes apparent that phage are a component in nutrient cycling in ecology. But their contribution does not stop there. Many are capable of transduction, moving DNA from one cell into another. So they can also aid the evolutionary progress of microbial populations by allowing them to share genes, just as gene exchange via plasmids and transformation does. Our perception of bacteria has been derived from pure culture studies and we are just being able to appreciate how subtle their ecological interactions are. This is no less true of the studies on bacteriophage, which are almost all based on laboratory experimentation, where the hosts are physiologically stressed by growing in 'high nutritional and optimum conditions'. The natural environment is naturally discontinuous and life evolved in this. Thus our perceptions of bacteriophage and their life cycle patterns derived from laboratory experimentation may be a little off the mark when we come to understand how they and their hosts interact in the niches available to them. It is worth just considering this as you read the article, as I suspect phage behaviours are more intimately involved in, and moderated by the physiological stresses in the life cycle of bacteria than we currently believe.

Morphing molecular specificities between Arm‐peptide and NUT‐RNA in the antitermination complexes of bacteriophages λ and P22

Abstract: Bacteriophage lambda's N-protein includes a 17-amino-acid segment, Arm, rich in arginine and having specific affinity for a 15-nucleotide RNA stem-loop called BOX-B. Parallel but different Arm/BOX-B sequences in lambda's cousin, phage P22, account for some of the type specificity that distinguishes lambda from P22: the N of each works only with its cognate BOX-B in vivo. We find that the specificity of N(lambda) can be shifted gradually to that of N(22) by substituting sets of particular amino acids from Arm(22) into Arm of N(lambda). The determinative amino acids are generally those shown by nuclear magnetic resonance to contact BOX-B RNA; gain or loss of these contact amino acids is reasonably expected to contribute to the affinity of each amino acid sequence. Intermediate sequences may show no function with either BOX-B, or weak function with both BOX-B(lambda) and BOX-B(22), the latter suggesting possible evolutionary paths for specificity shifts.

Over-expression of rexA nullifies T4rII exclusion in Escherichia coli K(λ) lysogens

Abstract: Dosage and relative cellular levels of RexA and RexB proteins encoded by the rexA–rexB genes of a λ prophage are important for the Rex+ phenotype, which was nullified when greater RexA or RexB was provided than was necessary for the complementation of a rexA– or a rexB– prophage.Key words: bacteriophage lambda (λ), T4rII exclusion (Rex) phenotype, lambda pM–cI–rexA–rexB–timm operon.

Evidence of bar minigene expression and tRNA2Ile sequestration as peptidyl-tRNA2Ile during lambda bacteriophage development.

Abstract: Lambda bacteriophage development is impaired in Escherichia coli cells defective for peptidyl (pep)-tRNA hydrolase (Pth). Single-base-pair mutations (bar−) that affect translatable two-codon open reading frames named bar minigenes (barI or barII) in the lambda phage genome promote the development of this phage in Pth-defective cells (rap cells). When the barI minigene is cloned and overexpressed from a plasmid, it inhibits protein synthesis and cell growth in rap cells by sequestering as . Either or Pth may reverse these effects. In this paper we present evidence that both barI and barII minigenes are translatable elements that sequester as . In addition, overexpression of the barI minigene impairs the development even of bar− phages in rap cells. Interestingly, tRNA or Pth may reestablish lambda phage development. These results suggest that lambda bar minigenes are expressed and is sequestered as during lambda phage development.

Screening method for ubiquitin and ubiquitin-like protein specific proteases using ci gene of lamda phage, in which the enzymes are useful for development of drugs for treating brain neuronal diseases

Abstract: PURPOSE: A screening method for ubiquitin and ubiquitin-like protein specific proteases using cI gene of lambda phage is provided, thereby easily screening ubiquitin and ubiquitin-like protein specific proteases and simultaneously measuring activity of enzymes, so that the enzymes are useful for development of drugs for treating brain neuronal diseases. CONSTITUTION: The screening method for ubiquitin and ubiquitin-like protein specific proteases using cI gene of lambda phage comprises the steps of: (1) cloning Ub, UbI and cI genes, preparing a mutant of cI gene, preparing an expression plasmid of Ub-cI* or UbI-cI* fusion protein, and preparing transformed E. coli using the expression plasmid; (2) introducing cDNA library into the transformed E. coli using recombinant lambda-phage; (3) selecting E. coli expressing desired protein by heat shock at 42 deg. C for 2 hours; and analyzing enzyme activity of the expressed protein, wherein the Ub-cI* or UbI-cI* fusion protein is produced by fusing ubiquitin-like protein Nedd8 or SUMO-1 with cI protein mutant(cI*); and the fusion protein expression plasmid is pACYC184.