Showing papers on "Lambda phage published in 2001"
TL;DR: The role of high-frequency double-strand break repair/recombination in the life cycle of the lambdoid phages is discussed and the Red-mediated recombination system, called Red, is discussed.
Abstract: Recent studies have generated interest in the use of the homologous recombination system of bacteriophage λ for genetic engineering. The system, called Red, consists primarily of three proteins: λ exonuclease, which processively digests the 5′-ended strand of a dsDNA end; β protein, which binds to ssDNA and promotes strand annealing; and γ protein, which binds to the bacterial RecBCD enzyme and inhibits its activities. These proteins induce a ‘hyper-rec’ state in Escherichia coli and other bacteria, in which recombination events between DNA species with as little as 40 bp of shared sequence occur at high frequency. Red-mediated recombination in the hyper-rec bacterium proceeds via a number of different pathways, and with the involvement of different sets of bacterial proteins, depending in part on the nature of the recombining DNA species. The role of high-frequency double-strand break repair/recombination in the life cycle of the lambdoid phages is discussed.
127 citations
TL;DR: In this paper, a temperate lambdoid bacteriophage that adsorbs to the O-antigen of its host, Escherichia coli H, was identified.
104 citations
TL;DR: The lambda (lambda) family of bacteriophages continues to provide significant insights into the understanding of basic biological processes, as well as useful technological innovations in areas in which recent advances have occurred.
90 citations
TL;DR: It is shown that the lambda display system has a unique ability to display, at high density, proteins ranging in size from a few to at least 300 amino acid residues, which permits attenuation of the size bias in the selection procedure and offers a sensitive plaque assay that permits us to do away with the ligand background without unduly increasing the number of selection cycles.
70 citations
TL;DR: It is shown that the T holin accumulates in the membrane during LIN, forming SDS‐resistant oligomers, and by cross‐linking experiments that a T‐RI heterodimer is formed, demonstrating that RI belongs to the functional class of antiholins, such as the S107 protein of λ, whichheterodimerizes with its cognate holin, S105.
Abstract: Phage T4 effects lysis by its holin T and its endolysin E. Lysis is inhibited (LIN) if the infected cell is subjected to secondary infections by T4 phage particles. The T4 rI gene is required for LIN in all hosts tested. Here, we show that a cloned rI gene can impose a T-specific LIN on T-mediated lysis in the context of the phage lambda infective cycle, in the absence of other T4 genes and without secondary infection by T4. Moreover, it is shown that the T holin accumulates in the membrane during LIN, forming SDS-resistant oligomers. We show by cross-linking experiments that a T-RI heterodimer is formed during LIN, demonstrating that RI belongs to the functional class of antiholins, such as the S107 protein of lambda, which heterodimerizes with its cognate holin, S105. Finally, we show that the addition of Ni(2+) ions to the medium can block lysis by a T protein hexahistidine-tagged at its C-terminus, suggesting that liganding of the periplasmic domain is sufficient to impose lysis inhibition. The results are discussed in terms of a model in which the LIN-inducing signal of the secondary infecting phage influences a conformational equilibrium assumed by RI in the periplasm.
58 citations
TL;DR: In two-dimensional nuclear Overhauser enhancement spectroscopy experiments boxB RNA in complex with Nun-(20-44) exhibits the same pattern of resonances as it does incomplex with N peptides containing the ARM, and it is shown that Nun-( 20- 44) forms a bent alpha-helix upon binding to the boxBRNA.
32 citations
TL;DR: A simple system for cloning and expression of genes in single copy in the E. coli chromosome is described, using a non-antibiotic selection for transgene insertion and a vector containing homology to the chromosomal region flanking the attachment site for phage lambda.
25 citations
TL;DR: The molecular nature of mutations induced by AAT in the lambda cII gene, a phenotypically selectable marker in thelambda transgene, has 294bp, which makes it easier to sequence than the original target, the 3kb lacZ gene, is revealed.
Abstract: The o-aminoazotoluene (AAT) has been evaluated as a possible human carcinogen by the International Agency for Research on Cancer. In rodents, it is carcinogenic mainly in the liver, and also in lung following long term administration. We previously examined in lambda/lacZ transgenic mice for the induction of lacZ mutations in liver, lung, urinary bladder, colon, kidney, bone marrow, and testis. AAT induced gene mutations strongly in the liver and colon. In the present report, we reveal the molecular nature of mutations induced by AAT in the lambda cII gene (the cII gene, a phenotypically selectable marker in the lambda transgene, has 294 bp, which makes it easier to sequence than the original target, the 3 kb lacZ gene). The cII mutant frequency in liver and colon was five and nine times higher, respectively, in AAT-treated mice than in control mice. Sequence analysis revealed that AAT induced G:C to T:A transversions, whereas spontaneous mutations consisted primarily of G:C to A:T transitions at CpG sites.
16 citations
TL;DR: lambda vectors have been designed to be readily converted into plasmids or phagemids, and there are a variety of promoters and fusions that can be used to drive expression of foreign genes.
Abstract: Bacteriophage lambda has been in use as a cloning vector for over 25 years, and has been used extensively as an expression vector. The efficiency of packaging and infection, and the simplicity of plaque screening are advantages of lambda as a cloning vector. A number of ingenious modifications help overcome the disadvantages associated with its mode of growth and its size. Some lambda vectors have been designed to be readily converted into plasmids or phagemids, and there are a variety of promoters and fusions that can be used to drive expression of foreign genes. Screening lambda libraries with antibodies or ligands is a powerful way of identifying novel genes.
14 citations
TL;DR: An excellent system for quantifying mutations at the cII locus in Muta™Mouse is reported, written in the context of lambda recombinant genetics, which explores the question further how does cII selection work in the MutA™Mouse.
Abstract: The lambda CII protein is an essential component in the lytic vs. lysogeny decision a bacteriophage makes upon infection of a host at low temperatures. The protein interacts with numerous phage promoters modulating the expression of the CI repressor, thus providing the mechanism for lysogenization soon after infection. The Big Blue and Muta Mouse are two widely used in vivo mutational model systems. The assays rely on retrievable lambda-based transgenes housing mutational targets (lacI or lacZ, respectively). The transgenes provide an elegant vehicle for the quantification of mutations sustained in virtually any tissue of the rodent. The use of the bacteriophage cII locus as an alternative, or additional mutational target for use with the Big Blue rodent system was first reported by Jakubczak et al. ([1996]: Proc Natl Acad Sci USA 93:9073-9078). More recently, this selection assay has been applied successfully to the Muta Mouse (Swiger et al. [1999]: Environ Mol Mutagen 33:201-207). The use of an Hfl bacterial strain and low temperature allows the determination of mutations sustained at the cII locus in either system, with high fidelity. The cII selection assay in the Big Blue relies on the presence of the lambda repressor protein CI. In contrast, the recombinant construct used to make the Muta Mouse transgene lacks functional CI protein. Nevertheless, we report an excellent system for quantifying mutations at the cII locus in Muta Mouse. Just how does cII selection work in the Muta Mouse? Written in the context of lambda recombinant genetics, this paper explores the question further.
12 citations
TL;DR: The data suggest that T2 Dam P126S can modify 28 recognition sequences, and when bacteriophage lambda DNA was used as a substrate, maximal protection from restriction nuclease cleavage in vitro was achieved on the sequences GATC, GATN and GACY, while protection of GACR sequences was less efficient.
Abstract: Properties of a mutant bacteriophage T2 DNA [N:(6)-adenine] methyltransferase (T2 Dam MTase) have been investigated for its potential utilization in RecA-assisted restriction endonuclease (RARE) cleavage. Steady-state kinetic analyses with oligonucleotide duplexes revealed that, compared to wild-type T4 Dam, both wild-type T2 Dam and mutant T2 Dam P126S had a 1.5-fold higher k(cat) in methylating canonical GATC sites. Additionally, T2 Dam P126S showed increased efficiencies in methylation of non-canonical GAY sites relative to the wild-type enzymes. In agreement with these steady-state kinetic data, when bacteriophage lambda DNA was used as a substrate, maximal protection from restriction nuclease cleavage in vitro was achieved on the sequences GATC, GATN and GACY, while protection of GACR sequences was less efficient. Collectively, our data suggest that T2 Dam P126S can modify 28 recognition sequences. The feasibility of using the mutant enzyme in RARE cleavage with BCL:I and ECO:RV endonucleases has been shown on phage lambda DNA and with BCL:I and DPN:II endonucleases on yeast chromosomal DNA embedded in agarose.
TL;DR: It is demonstrated that activities of clpP and clpX genes influence the lysis-versus-lysogenization decision of bacteriophage λ under certain growth conditions of the host cells.
Abstract: The initiator of bacteriophage λ DNA replication, the O protein, is rapidly degraded in Escherichia coli by the ClpP/ClpX protease encoded by the host. Although the biochemical mechanism of this degradation has been investigated intensively, a physiological role for this process remained unknown since little effect of dysfunction of clpP and clpX genes on the lytic development of the phage was observed. Here we demonstrate that activities of clpP and clpX genes influence the lysis-versus-lysogenization decision of bacteriophage λ under certain growth conditions of the host cells. This decision is influenced specifically by ClpP/ClpX-mediated O degradation and resultant inhibition of early λ DNA replication because mutations in clpP and clpX genes have little effect on stability of other λ proteins involved in the regulation of the phage developmental switch.
TL;DR: The usefulness of the genetic system for the regulatable production of a protein encoded by a gene inserted into pTCλ3 plasmid is demonstrated by dependence of β-galactosidase activity on the lacZ gene dosage.
Abstract: We have developed a genetic system allowing for precise regulation of plasmid copy number in Escherichia coli cells. A cloning vector based on this system is described in this article. The pTC lambda 3 plasmid is a lambda replicon, but transcription controlling initiation of plasmid DNA replication starts from the PtetA promoter instead of phage lambda PR promoter. Additionally, activity of PtetA promoter is negatively controlled by the TetR repressor whose gene is located on the same plasmid vector and is induced by an analog of tetracycline, autoclaved chlortetracycline (aCT). Using different concentrations of the inducer it is possible to strictly regulate the copy number of pTC lambda 3 and thus the copy number of a cloned gene. The usefulness of the system for the regulatable production of a protein encoded by a gene inserted into pTC lambda 3 plasmid is demonstrated by dependence of beta-galactosidase activity on the lacZ gene dosage.
TL;DR: It is found that colony direct sequencing and plaque direct sequencing are estimated to be very useful for rapid and high-throughout screening of genomic and cDNA libraries.
Abstract: Direct sequencing using lambda phage DNA and E. coli colonies with plasmid DNA is a very powerful technique. Almost all of the reported direct sequencing methods involve either radioactive sequencing or fluorescent dye-primer sequencing. We present a direct colony sequencing strategy that uses a dye terminator (BigDye terminator kit) together with dye primer sequencing. We found that single-colony sequencing with the terminator yielded about 500 base pairs of sequence information. Signal strength was not improved when the number of cycles increased to 40. The colony used for the sequencing was estimated to contain about 5.6 x 10(7) cells. In addition, although a single plaque consisted of 2 x 10(6) cells, the pfu was not high enough to read with single-cycle sequencing, and only about 300 base pairs of sequence information were obtained from a single plaque using two cycle-sequencing reactions (re-cycle sequencing). The optimal amounts of the template were 500 ng of purified lambda DNA and 1 x 10(7) pfu of the lambda phage suspension, but with BigDye terminator it was possible to detect as little as 50 ng of purified lambda DNA and 2 x 10(6) pfu for lambda phage suspensions. Thus, colony direct sequencing and plaque direct sequencing are estimated to be very useful for rapid and high-throughout screening of genomic and cDNA libraries.
TL;DR: The genome of the unicellular cyanobacterium Synechococcus sp.
Abstract: The genome of the unicellular cyanobacterium Synechococcus sp. strain PCC6301 (Anacystis nidulans 6301) is 2.7 Mb in size. To understand the genome structure and function we constructed a set of ordered lambda clones that covered the entire region of the Synechococcus PCC6301 genome. The insert DNA of lambda phage clones was amplified by LA PCR and sheared to short DNA fragments. The resultant DNA fragments were subcloned into a SmaI site of pUC18, and then shotgun sequenced. We present here the gene organization of Synechococcus PCC6301 genome.
Journal Article•
TL;DR: It was shown that formation of the DNA-binding sites for the three structurally different protein domains involves polar residues selected from the largest polar cluster on the surface of the protein molecule, and that the same polar residues bind with different points of DNA sites.
Abstract: IPolar interactions between protein and the major groove of double-stranded DNA have been analyzed on the basis of a structural study of the complexes formed by two transcription factors: phage lambda repressor and murine immunoglobulin transcription factor NF kappa B-p50. Two identical molecules of these two factors form two binding sites within two different parts of a single DNA molecule. This allows one to study formation of the recognition module by comparing the binding pattern of two different parts of the DNA operator. We have shown that formation of the DNA-binding sites for the three structurally different protein domains (one in repressor and two in the immunoglobulin factor) involves polar residues selected from the largest polar cluster on the surface of the protein molecule. It was also shown that the same polar residues bind with different points of DNA sites. This result provides understanding of the recognition module adaptation to varying nucleotide sequence of the operator sites and shows the way of binding site formation.
TL;DR: Construction of a broad-host-range plasmid expressing the lamB gene, which made Vibrio harveyi, one of the most frequently investigated species of marine bacteria, susceptible to bacteriophage λ adsorption and λ DNA injection.
Abstract: The remarkable success of Escherichia coli as a model organism in molecular genetics was dependent, among other things, on its susceptibility to genetic manipulation. Many versatile and sophisticated genetic tools for molecular biology studies are derived from bacteriophage lambda. However, this bacteriophage is specific for E. coli, and thus lambda-based techniques have been restricted to this bacterium. Plasmids expressing the E. coli gene coding for bacteriophage lambda receptor were reported previously, and introduction of such plasmids into cells of some other bacteria made them sensitive to phage lambda infection. However, we found that these systems were not efficient for Vibrio harveyi, one of the most frequently investigated species of marine bacteria. Here we describe construction of a broad-host-range plasmid expressing the lamB gene. Introduction of this plasmid to V. harveyi cells and expression of lamB made this strain susceptible to bacteriophage lambda adsorption and lambda DNA injection. Foreign genetic material could be introduced into cells of this strain using a cosmid vector.