Showing papers on "Lambda phage published in 2003"

A Highly Efficient Recombineering-Based Method for Generating Conditional Knockout Mutations

Abstract: Phage-based Escherichia coli homologous recombination systems have recently been developed that now make it possible to subclone or modify DNA cloned into plasmids, BACs, or PACs without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombineering, has many different uses for functional genomic studies. Here we describe a new recombineering-based method for generating conditional mouse knockout (cko) mutations. This method uses homologous recombination mediated by the lambda phage Red proteins, to subclone DNA from BACs into high-copy plasmids by gap repair, and together with Cre or Flpe recombinases, to introduce loxP or FRT sites into the subcloned DNA. Unlike other methods that use short 45-55-bp regions of homology for recombineering, our method uses much longer regions of homology. We also make use of several new E. coli strains, in which the proteins required for recombination are expressed from a defective temperature-sensitive lambda prophage, and the Cre or Flpe recombinases from an arabinose-inducible promoter. We also describe two new Neo selection cassettes that work well in both E. coli and mouse ES cells. Our method is fast, efficient, and reliable and makes it possible to generate cko-targeting vectors in less than 2 wk. This method should also facilitate the generation of knock-in mutations and transgene constructs, as well as expedite the analysis of regulatory elements and functional domains in or near genes.

Immunity profiles of wild-type and recombinant shiga-like toxin-encoding bacteriophages and characterization of novel double lysogens

Abstract: The pathogenicity of Shiga-like toxin (stx)-producing Escherichia coli (STEC), notably serotype O157, the causative agent of hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura, is based partly on the presence of genes (stx1 and/or stx2) that are known to be carried on temperate lambdoid bacteriophages. Stx phages were isolated from different STEC strains and found to have genome sizes in the range of 48 to 62 kb and to carry either stx1 or stx2 genes. Restriction fragment length polymorphism patterns and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles were relatively uninformative, but the phages could be differentiated according to their immunity profiles. Furthermore, these were sufficiently sensitive to enable the identification and differentiation of two different phages, both carrying the genes for Stx2 and originating from the same STEC host strain. The immunity profiles of the different Stx phages did not conform to the model established for bacteriophage lambda, in that the pattern of individual Stx phage infection of various lysogens was neither expected nor predicted. Unexpected differences were also observed among Stx phages in their relative lytic productivity within a single host. Two antibiotic resistance markers were used to tag a recombinant phage in which the stx genes were inactivated, enabling the first reported observation of the simultaneous infection of a single host with two genetically identical Stx phages. The data demonstrate that, although Stx phages are members of the lambdoid family, their replication and infection control strategies are not necessarily identical to the archetypical bacteriophage λ, and this could be responsible for the widespread occurrence of stx genes across a diverse range of E. coli serotypes.

Bacteriophage SPP1 Chu Is an Alkaline Exonuclease in the SynExo Family of Viral Two-Component Recombinases

Abstract: Many DNA viruses concatemerize their genomes as a prerequisite to packaging into capsids. Concatemerization arises from either replication or homologous recombination. Replication is already the target of many antiviral drugs, and viral recombinases are an attractive target for drug design, particularly for combination therapy with replication inhibitors, due to their important supporting role in viral growth. To dissect the molecular mechanisms of viral recombination, we and others previously identified a family of viral nucleases that comprise one component of a conserved, two-component viral recombination system. The nuclease component is related to the exonuclease of phage lambda and is common to viruses with linear double-stranded DNA genomes. To test the idea that these viruses have a common strategy for recombination and genome concatemerization, we isolated the previously uncharacterized 34.1 gene from Bacillus subtilis phage SPP1, expressed it in Escherichia coli, purified the protein, and determined its enzymatic properties. Like lambda exonuclease, Chu (the product of 34.1) forms an oligomer, is a processive alkaline exonuclease that digests linear double-stranded DNA in a Mg(2+)-dependent reaction, and shows a preference for 5'-phosphorylated DNA ends. A model for viral recombination, based on the phage lambda Red recombination system, is proposed.

Identification and Mutational Analysis of Bacteriophage PRD1 Holin Protein P35

Abstract: Holin proteins are phage-induced integral membrane proteins which regulate the access of lytic enzymes to host cell peptidoglycan at the time of release of progeny viruses by host cell lysis. We describe the identification of the membrane-containing phage PRD1 holin gene (gene XXXV). The PRD1 holin protein (P35, 12.8 kDa) acts similarly to its functional counterpart from phage lambda (gene S), and the defect in PRD1 gene XXXV can be corrected by the presence of gene S of lambda. Several nonsense, missense, and insertion mutations in PRD1 gene XXXV were analyzed. These studies support the overall conclusion that the charged amino acids at the protein C terminus are involved in the timing of host cell lysis.

Real-time PCR provides improved detection and titer determination of bacteriophage

Abstract: The plaque assay is the traditional method for the quantification of bacteriophage, particularly for lambda cloning vectors Unfortunately, this technique is fraught with procedural difficulties, and the quality of the data obtained from this "gold standard" assay may be inaccurate due to the subjective interpretation of the results The application of quantitative real-time PCR (QPCR) technology can address these issues and be a more accurate platform to evaluate phage growth conditions and quantify viral titers in phage preparations QPCR, with an improved primer set specific for lambda phage and coupled with fluorescent dye detection of PCR products, was used to detect and quantify phages in lysates with no prior DNA purification Phages were detected below one plaque-forming unit, and at least 89 viral copies were detected from a purified DNA sample When unknown concentrations of various phage preparations were assessed using QPCR, they were attained more efficiently, with greater sensitivity and precision, and the method produced more accurate quantitative data spanning a wider linear range than those obtained by the plaque assay (six logs vs one log, respectively) Finally, QPCR for the detection of phage has multiple applications, including conventional cloning and in alternative fields of study such as environmental sciences

Activation of site‐specific DNA integration in human cells by a single chain integration host factor

Abstract: The heterodimeric integration host factor (IHF) is a site-specific DNA-binding and DNA-bending protein from Escherichia coli. It plays essential roles in a variety of DNA transactions including recombination, transcription and DNA replication. IHF's ability for concerted binding and bending of DNA is key to its biological function. Here we report the design, characterization and application of a single polypeptide chain IHF, termed scIHF2. In a novel approach for protein engineering, we inserted almost the entire alpha-subunit of IHF into the beta-subunit. DNA binding and DNA bending assays revealed that purified wild-type IHF and scIHF2 behave very similarly. Further, scIHF2 is required for site-specific integrative recombination by phage lambda integrase and for pSC101 replication in a DeltaIHF E.coli host. It also triggers site-specific integrative and excisive recombination in vitro to the same extent as the wild-type protein. We also demonstrate that scIHF2 is stably expressed in HeLa cells, that it is localized primarily in the cell nucleus and that it triggers integrative recombination in mammalian cells by wild-type integrase. Hence, scIHF2 may be used as a novel regulatory cofactor for recombination or other DNA transactions in mammalian cells that require or benefit from sequence-specific high precision DNA bending.

New restriction enzymes discovered from Escherichia coli clinical strains using a plasmid transformation method.

Abstract: The presence of restriction enzymes in bacterial cells has been predicted by either classical phage restriction-modification (R-M) tests, direct in vitro enzyme assays or more recently from bacterial genome sequence analysis. We have applied phage R-M test principles to the transformation of plasmid DNA and established a plasmid R-M test. To validate this test, six plasmids that contain BamHI fragments of phage lambda DNA were constructed and transformed into Escherichia coli strains containing known R-M systems including: type I (EcoBI, EcoAI, Eco124I), type II (HindIII) and type III (EcoP1I). Plasmid DNA with a single recognition site showed a reduction of relative efficiency of transformation (EOT = 10(-1)-10(-2)). When multiple recognition sites were present, greater reductions in EOT values were observed. Once established in the cell, the plasmids were subjected to modification (EOT = 1.0). We applied this test to screen E.coli clinical strains and detected the presence of restriction enzymes in 93% (14/15) of cells. Using additional subclones and the computer program, RM Search, we identified four new restriction enzymes, Eco377I, Eco585I, Eco646I and Eco777I, along with their recognition sequences, GGA(8N)ATGC, GCC(6N)TGCG, CCA(7N)CTTC, and GGA(6N)TATC, respectively. Eco1158I, an isoschizomer of EcoBI, was also found in this study.

The Escherichia coli Fis Protein Stimulates Bacteriophage λ Integrative Recombination In Vitro

Abstract: The Escherichia coli nucleoid-associated protein Fis was previously shown to be involved in bacteriophage lambda site-specific recombination in vivo, enhancing the levels of both integrative recombination and excisive recombination. While purified Fis protein was shown to stimulate in vitro excision, Fis appeared to have no effect on in vitro integration reactions even though a 15-fold drop in lysogenization frequency had previously been observed in fis mutants. We demonstrate here that E. coli Fis protein does stimulate integrative lambda recombination in vitro but only under specific conditions which likely mimic natural in vivo recombination more closely than the standard conditions used in vitro. In the presence of suboptimal concentrations of Int protein, Fis stimulates the rate of integrative recombination significantly. In addition, Fis enhances the recombination of substrates with nonstandard topologies which may be more relevant to the process of in vivo phage lambda recombination. These data support the hypothesis that Fis may play an essential role in lambda recombination in the host cell.

Genetic Screen for Monitoring Hepatitis C Virus NS3 Serine Protease Activity

Abstract: We have developed a genetic system to monitor the activity of the hepatitis C virus (HCV) NS3 serine protease. This genetic system is based on the bacteriophage lambda regulatory circuit where the viral repressor c I is specifically cleaved to initiate the switch from lysogeny to lytic infection. An HCV protease-specific target, NS5A-5B, was inserted into the lambda phage c I repressor. The target specificity of the HCV NS5A-5B repressor was evaluated by coexpression of this repressor with a β-galactosidase (βgal)-HCV NS3 2-181 /4 21-34 protease construct. Upon infection of Escherichia coli cells containing the two plasmids encoding the cI.HCV5AB-cro and the βgal-HCV NS3 2-181 /4 21-34 protease constructs, lambda phage replicated up to 8,000-fold more efficiently than in cells that did not express the HCV NS3 2-181 /4 21-34 protease. This simple, rapid, and highly specific assay can be used to monitor the activity of the HCV NS3 serine protease, and it has the potential to be used for screening specific inhibitors.

Novel lambda phage display system and the process

Abstract: The present invention relates to a process of obtaining recombinant lambdoid bacteriophage with high density display of functional peptides and proteins on surface of said phage comprising of: constructing a donor plasmid having a nucleotide sequence that defines the elements for replication of the vector in bacteria, a selectable marker, a nucleotide sequence flanked by two non-compatible recombination sequences, and an inducible cistron for expression of a capsid protein and a fusion protein; constructing a recipient phage having a nucleotide sequence that defines the lambdoid elements for replication and packaging of the vector into an assembled bacteriophage and encodes an inducible cistron for expression of a selectable marker flanked by two non-compatible recombination sequences; transferring the said donor plasmid to said recipient plasmid to obtain cointegrates; growing said cointegrates in selective liquid medium; harvesting phages displaying protein encoded by the foreign DNA encapsulated in said harvested phage particle.

Methods, compositions, and kits for enhancing oligonucleotide-mediated nucleic acid sequence alteration using compositions comprising a histone deacetylase inhibitor, lambda phage beta protein, or hydroxyurea

Abstract: Improved methods, compositions, and kits for oligonucleotide-mediated nucleic acid sequence alteration are presented.

Phage having nuclear location signal

Abstract: A lambda phage with a nuclear localization signal has been obtained by constructing a vector capable of expressing a fused protein between a gpD protein constituting the head of a lambda phage and a nuclear localization signal sequence, transforming Escherichia coli with this vector, and propagating a mutant lambda phage which cannot express the gpD protein in E. coli in this transformant. It has been confirmed that the resulting lambda phage is capable of packaging lambda phage DNAs of 80% and 100% genome sizes. After further confining that the nuclear localization signal exposed on the outside of the head of this phage, this phage has been microinjected into cells to analyze its nuclear localization activity. Thus, it has been clarified that this phage has a nuclear localization activity.

Bacteriophage lambda repressor allelic modulation of the Rex exclusion phenotype.

Abstract: The sensitivity of delta red-gam delta ren mutants of bacteriophage lambda to Rex exclusion by lambda rexA+ rexB+ lysogens is modulated by the prophage cI repressor allele. We show the following: (i) lambda spi156 delta nin5 forms plaques on a cI+-rexA+-rexB+ lysogen with 10(5)-fold higher efficiency than on cI[Ts]-rexA+-rexB+ derivatives. (ii) The cI[Ts]857 allele augmentation of Rex exclusion is recessive to cI+. (iii) The cI857-mediated increase in Rex exclusion activity involves the participation of a genetic element mapping outside of cI-rexA-rexB.

Lambda phage display system and the process

Abstract: The present invention relates to a process of obtaining recombinant lambdoid bacteriophage with high density display of functional peptides and proteins on surface of said phage comprising of: constructing a donor plasmid having a nucleotide sequence that defines the elements for replication of the vector in bacteria, a selectable marker, a nucleotide sequence flanked by two non-compatible recombination sequences, and an inducible cistron for expression of a capsid protein and a fusion protein; constructing a recipient phage having a nucleotide sequence that defines the lambdoid elements for replication and packaging of the vector into an assembled bacteriophage and encodes an inducible cistron for expression of a selectable marker flanked by two non-compatible recombination sequences; transferring the said donor plasmid to said recipient plasmid to obtain cointegrates; growing said cointegrates in selective liquid medium; harvesting phages displaying protein encoded by the foreign DNA encapsulated in said harvested phage particle.

Enhanced oligonucleotide-mediated nucleic acid sequence alteration

Abstract: The invention is directed to oligonucleotide-mediated repair or alteration of genetic information, such as nucleic acid sequence alteration, and methods, compositions and kits for enhancing the efficiency of such alteration. Specifically, the invention incorporates the use of factors such as Histone Deacetylase Inhibitor (HDAC), Lambda phage beta protein, or hydroxyurea to achieve such enhanced efficiency.

Hybrid plasmid pzzsa coding the synthesis of angiogenis of angiogenin protein and escherichia coli bl21(des) pzzsa strain as the the superoducer of the recombinant chimeric protein of human angiogenin

Abstract: The invention relates to biotechnology and enhances the expression efficiency of the hybrid gene and stability of the angiogenin protein, as well as provides a possibility of affinity purification of chimeric protein on IgG sorbents The hybrid plasmid pZZSA with a molecular weight of 3814 megadalton (MDa) (6192 bp) which drives the synthesis of the chimeric protein of angiogenin is disclosed, comprising an XhoI/EcoRI fragment of the pGM280 plasmid DNA comprising a tandem of E coli tryptophan operon promoters, a lambda phage transcription terminator, a β-lactamase gene and an ori replication initiation site; an EcoRI/EcoNI fragment of the PfM plasmid comprising a synthetic translation enhancer of the bacteriophage T7 gene and gene Ap coding for the amino acid sequence corresponding to 3988-4845 bp; synthetic chimeric angiogenin gene (Ang) fused with Spa; unique recognition sites for restriction nucleases The strain Escherichia coli BL21 (DE3) pZZSA (the Collection of Microorganisms of the Interregional Center for Human Microcenosis Correction, Novosibirsk, Registration No MCKM B-127) that is the superproducer of recombinant chimeric protein of human angiogenin is described

Dendritic Cell-Targeted Phage Vectors for Breast Cancer Vaccine Development

Abstract: : We hypothesize that one can use specific protein or peptide sequences to direct bacteriophage vectors to dendritic cells. We further propose that one can then use such re-targeted phage vectors to deliver potentially important antigens to dendritic cells, and that this may allow one to derive vectors capable of eliciting potent immune responses to breast cancer antigens such as her2. These hypotheses are being experimentally tested. During the period covered by this progress report, we have used phage display technology to identify peptide sequences which bind to cellular receptors expressed on dendritic cells, and we have conducted proof-of-concept studies to show that we can selectively target adenovirus vectors to dendritic cells using these peptide sequences. We have also shown that one can enhance intracellular internalization of T7 bacteriophage vectors using cell-binding peptides. We are now examining whether can use these same peptide sequences to deliver T7 and lambda phage vectors encoding a mammalian expression cassette, to primary dendritic cells. These experiments are expected to provide direct support for our hypothesis, that one can use phage vectors to express foreign genes in dendritic cells (including antigenic molecules, such as her2).

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 β-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