Showing papers on "Lambda phage published in 2005"

Isolation of mouse x-chromosome specific DNA from an x-enriched lambda phage library derived from flow sorted chromosomes.

Abstract: A lambda phage library enriched in X(7) chromosomal material has been constructed from flow sorted chromosomes isolated from mice carrying the Cattanach translocation T(X;7)1Ct. The flow sorted fraction that was cloned contained 40% X(7) chromosomes, so that the resulting lambda phage library should be more than 10-fold enriched for X chromosomal DNA. Approximately 100,000 lambda phage clones were obtained; of these, at least 80% were recombinant. Three quarters of recombinants were positive for mouse repetitive DNA as detected either by phage plaque filter hybridization or by Southern blotting. Recombinant DNA inserts were prepared from some of the remaining nonrepetitive phage fraction. The X-chromosome specificity of cloned DNA inserts was tested by hybridization to DNA from mouse-hamster somatic cell hybrids that had retained all or most of the mouse X as the only mouse chromosome and by comparison of the extent of hybridization to DNA from male and female mice. Out of nine cloned unique sequence segments successfully examined thus far, two were presumably derived from the X. Possession of a phage library highly enriched for mouse X DNA should facilitate molecular studies of the control of X chromosome gene expression.

A simple method for displaying recalcitrant proteins on the surface of bacteriophage lambda

Abstract: Bacteriophage lambda (lambda) permits the display of many foreign peptides and proteins on the gpD major coat protein. However, some recombinant derivatives of gpD are incompatible with the assembly of stable phage particles. This presents a limitation to current lambda display systems. Here we describe a novel, plasmid-based expression system in which gpD deficient lambda lysogens can be co-complemented with both wild-type and recombinant forms of gpD. This dual expression system permits the generation of mosaic phage particles that contain otherwise recalcitrant recombinant gpD fusion proteins. Overall, this improved gpD display system is expected to permit the expression of a wide variety of peptides and proteins on the surface of bacteriophage lambda and to facilitate the use of modified lambda phage vectors in mammalian gene transfer applications.

Functional analysis of the lysis genes of Staphylococcus aureus phage P68 in Escherichia coli

Abstract: Double-stranded DNA phages of both Gram-positive and Gram-negative bacteria typically use a holin-endolysin system to achieve lysis of their host. In this study, the lysis genes of Staphylococcus aureus phage P68 were characterized. P68 gene lys16 was shown to encode a cell-wall-degrading enzyme, which causes cell lysis when externally added to clinical isolates of S. aureus. Another gene, hol15, was identified embedded in the -1 reading frame at the 3' end of lys16. The deduced Hol15 protein has three putative transmembrane domains, and thus resembles class I holins. An additional candidate holin gene, hol12, was found downstream of the endolysin gene lys16 based on two predicted transmembrane domains of the encoded protein, which is a typical trait of class II holins. The synthesis of either Hol12 or Hol15 resulted in growth retardation of Escherichia coli, and both hol15 and hol12 were able to complement a phage lambda Sam mutation. The hol15 gene has a dual start motif beginning with the codons Met1-Lys2-Met3.... Evidence is presented that the hol15 gene encodes a lysis inhibitor (anti-holin) and a lysis effector (actual holin). As depolarization of the membrane converted the anti-holin to a functional holin, these studies suggested that hol15 functions as a typical dual start motif class I holin. The unusual arrangement of the P68 lysis genes is discussed.

An action spectrum of the riboflavin-photosensitized inactivation of Lambda phage.

Abstract: The Action Spectrum of riboflavin (RB) sensitized inactivation of lambda phage was determined between 266 and 575 nm. Below 304 nm, RB depresses the phage reduction by screening phage from radiation that it would otherwise absorb directly. Between 308 and 525 nm, RB sensitizes the inactivation of phage. Enhanced phage reduction is observed at 320 and 500 nm because of binding of RB to the phage and the shifting of the absorption curve of the phage-bound flavin relative to free flavin in phosphate-buffered saline. Enhanced inactivation at 320 and 500 nm and depressed phage inactivation between 360 and 410 nm is also influenced by the inner filter effect.

Functional similarities between phage lambda Orf and Escherichia coli RecFOR in initiation of genetic exchange.

Abstract: Genetic recombination in bacteriophage λ relies on DNA end processing by Exo to expose 3′-tailed strands for annealing and exchange by β protein. Phage λ encodes an additional recombinase, Orf, which participates in the early stages of recombination by supplying a function equivalent to the Escherichia coli RecFOR complex. These host enzymes assist loading of the RecA strand exchange protein onto ssDNA coated with ssDNA-binding protein. In this study, we purified the Orf protein, analyzed its biochemical properties, and determined its crystal structure at 2.5 A. The homodimeric Orf protein is arranged as a toroid with a shallow U-shaped cleft, lined with basic residues, running perpendicular to the central cavity. Orf binds DNA, favoring single-stranded over duplex and with no obvious preference for gapped, 3′-tailed, or 5′-tailed substrates. An interaction between Orf and ssDNA-binding protein was indicated by far Western analysis. The functional similarities between Orf and RecFOR are discussed in relation to the early steps of recombinational exchange and the interplay between phage and bacterial recombinases.

Bacteriophage Evolution and the Role of Phages in Host Evolution

Abstract: Comparative analyses of phage genome sequences imply that a major component of phage evolution is large numbers of intrinsically very improbable events In addition, there are comparable numbers of prophage sequences that are found in the genomic sequences of bacteria and, sometimes, archaea There are stretches of sequence that match well, with abrupt transitions to regions with no detectable similarity or sometimes a different level of similarity These transition points are considered the products of nonhomologous recombination events in the ancestry of one of the phages being compared; in this sense, they are fossils of past events in the history of the genome It is believed that there are at least two factors that can restrict the horizontal flow of genes across the expanses of phage sequence space Many of the beneficial genes carried by prophages appear to be morons, ie, the genes that have entered the genome recently and are typically flanked by a transcription promoter and a terminator The understanding of how phages evolve began in the late 1960s with the hetero duplex mapping of the chromosomes of phage lambda and some close relatives, showing that these molecules are mosaic with respect to each other Genome sequences for such phages are just now becoming available, and the largest genome to date is 10 times as big as that of phage lambda, with a corresponding increase in gene number

Polarity within pM and pE promoted phage lambda cI-rexA-rexB transcription and its suppression.

Abstract: The cI-rexA-rexB operon of bacteriophage lambda confers 2 phenotypes, Imm and Rex, to lysogenic cells. Immunity to homoimmune infecting lambda phage depends upon the CI repressor. Rex exclusion of T4rII mutants requires RexA and RexB proteins. Both Imm and Rex share temperature-sensitive conditional phenotypes when expressed from cI[Ts]857 but not from cI+ lambda prophage. Plasmids were made in which cI-rexA-rexB was transcribed from a non-lambda promoter, pTet. The cI857-rexA-rexB plasmid exhibited Ts conditional Rex and CI phenotypes; the cI+-rexA-rexB plasmid did not. Polarity was observed within cI-rexA-rexB transcription at sites in cI and rexA when CI was nonfunctional. Renaturation of the Ts CI857 repressor, allowing it to regain functionality, suppressed the polar effect on downstream transcription from the site in cI. The second strong polar effect near the distal end of rexA was observed for transcription initiated from pE. The introduction of a rho Ts mutation into the host genome suppressed both polar effects, as measured by its suppression of the conditional Rex phenotype. Strong suppression of the conditional Rex[Ts] phenotype was imparted by ssrA and clpP (polar for clpX) null mutations, suggesting that RexA or RexB proteins made under conditions of polarity are subject to 10Sa RNA tagging and ClpXP degradation.

The FI‐Gene Product of Bacteriophage

Abstract: The FI gene product of bacteriophage lambda has been purified extensively using a biochemical assay that measures assembly of lambda phage particles in vitro. The molecular weight of the native protein was estimated to be 21700 with an s20,w of 2.1 S and a Stokes radius of 2.5 nm. The molecular weight in dodecylsulfate was estimated to be 19000. The protein is highly acidic with an isoelectric point less than 4.1.

RECOMBINANT PLASMID DNA p30NE2, ENCODING 181-AMINO ACID N-TERMINAL FRAGMENT OF GLYCOPROTEIN E2 OF CLASSICAL BOG CHOLERA VIRUS AND PROVIDING EXPRESSION THEREOF IN CELLS

Abstract: FIELD: genetic engineering ^ SUBSTANCE: recombinant plasmid DNA p30NE2 contains DNA fragment of pasmid pQE30, including promoter/operator of T5 phage; ribosome-binding site; phage lambda transcription terminator, and beta-lactomase gene as genetic marker, as well as BamHI/HindIII fragment, encoding 181-amino acid N-terminal fragment of glycoprotein E2 of classical bog cholera virus Plasmid provides expression of glycoprotein E2 of classical bog cholera virus in E/coli bacteria cells Invention is useful in diagnostic investigation and in vaccine production ^ EFFECT: genetically engineered plasmid for vaccine preparations ^ 3 dwg, 6 ex