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PubMed2007

Propagation of fluorescent viruses in growing plaques.

To study virus propagation, we have developed a method by which the propagation of the Lambda bacteriophage can be observed and quantified. This is done by creating a fusion protein of the capsid protein gpD and the enhanced yellow fluorescent protein (EYFP). We show that this fusion allows capsid formation and that the modified viruses propagate on a surface covered with host bacteria thus forming fluorescent plaques. The intensity of fluorescence in a growing plaque determines the distribution of phages. This provides a new tool to study the propagation of infection at the microscopic level.

Bacteriophage lambda↗

PubMed2004

An analytical system based on a compact flow cytometer for DNA fragment sizing and single-molecule detection.

BACKGROUND: Previous reports have demonstrated accurate DNA fragment sizing of linear DNA fragments, from 564 to approximately 4 x 10(5) bp, in a flow system. B-phycoerythrin (B-PE), commonly used in conventional cytometric applications that require high-sensitivity, was the first fluorophore detected in flow at the single-molecule level. METHODS: Dilute solutions of stained DNA fragments or B-PE were analyzed in a simplified, compact flow system, with enhanced performance and lower cost, utilizing a solid-state laser and a single-photon sensing avalanche photodiode detector (SSAPD). Extensive data processing and display software, developed specifically for the photon-counting data stream, extracts correlated height, width, and area features from bursts of photons due to discrete molecules passing through the sensing region in the flow channel. RESULTS: DNA fragment sizing in flow has now been demonstrated for SYTOX-orange-stained fragments ranging in size over 3.4 orders of magnitude, from 125 to 5 x 10(5) bp. For Lambda bacteriophage DNA (lambda DNA; 48.5 kbp) a CV of 1.2 % has been achieved. Analysis of a femtomolar B-PE solution demonstrates that the bursts of photons from individual molecules can be baseline-resolved with 0.5 mW of laser power at a signal to noise ratio (SNR) of approximately 30, with approximately 100 photons detected from each molecule. CONCLUSIONS: A compact, low-power, high-sensitivity system detects DNA fragments as small as 125 bp or individual B-PE molecules in a flowing liquid stream. Demonstrated linearity, sensitivity, and resolution indicate that <1.0 mW of laser power is optimal, permitting further miniaturization of the system and additional cost reduction. Comprehensive analytical software exploits the standard cytometric paradigm of multiple 2D graphs and gating to extract features from classes of individually analyzed biomolecules. This complete system is thus poised to engage high-sensitivity applications not amenable to conventional flow cytometric instrumentation.

DNA↗

PubMed1981

Characterization of cloned rat ribosomal DNA fragments.

Two Charon 4A lambda bacteriophage clones were characterized which contain all and part o the 18S ribosomal DNA of the rat. One clone contained two Eco RI fragments which include the whole 18S ribosomal RNA region and part of 28S ribosomal RNA region. The other clone contained an Eco RI fragment which covers part of 18S ribosomal RNA region. There were differences between the two clones in the non-transcribed spacer regions suggesting that there is heterogeneity in the non-transcribed spacer regions of rat ribosomal genes. The restriction maps of the two clones were compared to the restriction map of the cloned mouse ribosomal DNA. Eco RI, Hind III, Pst I, and Bam HI sites in 18S ribosomal RNA regions were in the same places in mouse and rat DNA but the restriction sites in the 5'-spacer regions were different.

Animals↗

PubMed2001

Identification of four families of peptidoglycan lytic transglycosylases.

The lytic transglycosylases are a class of autolysins which cleave the bacterial cell wall heteropolymer peptidoglycan (murein) to facilitate its biosynthesis and turnover. A search of the National Center for Biotechnology Information (NCBI) databases using the primary sequences of the six characterized lytic transglycosylases of Escherichia coli, a membrane-bound form of the enzyme from Pseudomonas aeruginosa, and the endolysins of lambda bacteriophage permitted the identification of a total of 127 known and hypothetical enzymes from a wide variety of bacteria and bacteriophage. These amino acid sequences have been arranged into four families based on alignments, and consensus motifs have been identified. Family 1 represents a superfamily comprising 86 sequences which are subdivided into five (1A--1E) subfamilies.

Amino Acid Motifs↗

PubMed1992

UV resistance of E. coli K-12 deficient in cAMP/CRP regulation.

Deletion of genes for adenylate cyclase (delta cya) or cAMP receptor protein (delta crp) in E. coli K-12 confers a phenotype that includes resistance to UV radiation (254 nm). Such mutations lead to UV resistance of uvr+, uvrA, lexA and recA strains which could partly be abolished by the addition of cAMP to delta cya but not to delta crp strain culture medium. This effect was not related to either inducibility of major DNA repair genes or growth rate of the bacteria. Enhanced survival was also observed for UV-irradiated lambda bacteriophage indicating that a repair mechanism of UV lesions was involved in this phenomenon.

Bacteriophage lambda↗

PubMed1981

Characterization of coliphage lambda hybrids carrying DNA fragments from Herpes simplex virus type 1 defective interfering particles.

We describe the characterization of 34 hybrid lambda bacteriophages carrying EcoRI fragments obtained from DNA of defective interfering particles of the Patton strain of Herpes simplex virus type 1 (HSV-1). All cloned fragments contained S region terminal repeat sequences (TRs) fused to unique HSV-1 DNA. Several fragments contained deletions and rearrangements not described previously for DNA of HSV-1 defective interfering particles. A model describing the generation of defective interfering DNA based on recombination events involving the terminal "a" sequence as presented.

Bacteriophage lambda↗

PubMed1989

A PstI polymorphism for the human erythrocyte surface protein band 3 (EPB3) demonstrates close linkage of EPB3 to the nerve growth factor receptor.

Erythrocyte surface protein band 3 (EPB3) plays an important role in CO2 transport in the blood. We have isolated a recombinant lambda bacteriophage that contains coding sequence for the human gene. Sequence analysis demonstrated that the human insert contains a portion of exon 13. A 1.1-kb BamHI fragment revealed a two-allele polymorphism with PstI. Alleles of 1.4 and 0.9/0.5 kb were present in Caucasoids at frequencies of 0.74 and 0.26, respectively. EPB3 has previously been mapped to 17q21-qter by in situ hybridization, and linkage analysis showed that EPB3 is tightly linked to the gene for the nerve growth factor receptor (NGFR). The maximum likelihood estimate of recombination (theta) is 0.00, with a lod score of 11.40 and confidence interval of 0.00 to 0.04.

Alleles↗

PubMed2005

Production in Saccharomycescerevisiae of MS2 virus-like particles packaging functional heterologous mRNAs.

Recently, DNA bacteriophages (M13, lambda) have been genetically engineered to transfer genes into mammalian cells. Although efficiencies observed are still relatively low, this opens the possibility of using these viruses as a new class of transfection agents not only for fundamental research purposes but also in gene therapy protocols or in other applications like vaccination. In this respect, it has been shown that a lambda bacteriophage engineered to express the hepatitis B surface antigen in mammalian cells could elicit an immune response against this antigen in mice and rabbits without any specific targeting of the bacteriophage. These impressive results would be even more encouraging if they could be obtained with an RNA bacteriophage, as RNA vaccines are preferred over DNA vaccines for safety reasons. Up to now, RNA bacteriophages have never been engineered for gene delivery. In this paper, we have sought to determine whether such a vector could be obtained by engineering the RNA bacteriophage MS2. We show that MS2 can be produced as virus-like particles (VLPs) in Saccharomyces cerevisiae and is able to package functional heterologous mRNAs, provided that these mRNAs contain the MS2 packaging sequence. For instance, linking the MS2 packaging sequence to the human growth hormone (hGH) mRNA enabled the packaging of this particular mRNA in MS2 VLPs. Functionality in eukaryotic systems of packaged mRNAs was confirmed by showing that mRNAs purified from VLPs can be efficiently translated in vitro and in cell cultures. The high stability of MS2 could, therefore, make MS2 VLPs a very powerful carrier for RNA vaccines.

Genetic Engineering↗

PubMed1998

Induction of a SOS repair system in lysogenic bacteria by zearalenone and its prevention by vitamin E.

Zearalenone (Zen) is an oestrogenic mycotoxin produced by several Fusarium species in cereals. It induces modifications of haematological parameters in rats with cytotoxicity and inhibition of macromolecular synthesis (nucleic acids and protein). Zen and its metabolites have oestrogenic and anabolic activities and interact with human oestrogen receptors. Zen and its metabolites showed a positive DNA damaging effect in recombination tests with Bacillus subtilis. It induces sister chromatid exchange and chromosomal aberration in CHO cells. Zen was found to be capable of inducing DNA-adduct formation in mouse liver. The genotoxicity of Zen was questionable until the last decade when increasing data tended to show this toxin to be genotoxic in vivo. However the mechanism of its genotoxicity and mutagenicity has not been completely clarified. The present investigations were designed to show whether Zen induces an SOS-DNA repair response in lysogenic bacteria which have an integrated lambda-bacteriophage in their genome. Zen was found to be genotoxic in the bacterial systems from a concentration of 1.50 mM and it was also bactericidal (IC50 = 1.45 mM). In addition vitamin E (6.0-12.0 mM) added 1 h prior to the toxin proved to prevent both the genotoxic and bactericidal effects of Zen. This vitamin could be active both as an antioxidant and as a radical scavenger. The specificity of this prevention is probably due to the similarity of structure between vitamin E and Zen.

Animals↗

PubMed2001

Identification of a mutation causing increased expression of the tas gene in Escherichia coli FX-11.

Studies of N-ethyl-N-nitrosourea (ENU)-induced mutagenesis with a tyrosine auxotroph of Escherichia coli revealed a new type of revertant. This mutant strain was interesting because: (i) it was not a true revertant of the nonsense (ochre) defect nor a tRNA suppressor mutation; and (ii) it was induced by ENU to greater extent in a UmuC-defective host. Genetic mapping located the probable mutation to a region of the E. coli chromosome containing a newly described gene called tas. To investigate this mutation, the upstream region of the tas gene from both wild-type and mutant cells was cloned into a promoterless lacZ expression vector and recombined onto a lambda bacteriophage. Recombinant bacteriophage were inserted into the bacterial chromosome and beta-galactosidase (betaGal) assays were performed. These assays revealed an almost three-fold greater expression of betaGal from the mutant DNA than from the wild-type DNA. Sequence analysis of the region directly upstream of the tas gene revealed a G:C to A:T transition at base number 2263 (numbering based on GenBank Accession #AE000367), located within a potential promoter site. Further sequencing indicated no other mutations within the 1454bp region analyzed; however, there were several nucleotide differences seen in our B/r strain of E. coli, when compared with the published E. coli K-12 sequence. A total of 10 base differences were discovered; one in mutH, six within a potential open reading frame (ORF-o237) and three in non-coding regions. Yet, none of the changes altered the predicted amino acid sequences. These results provide evidence of a mechanism for increased expression of the novel gene tas and support the neutral drift hypothesis for the evolution of DNA sequences.

Amino Acid Sequence↗

PubMedDate not supplied

Imaging stretched single DNA molecules by pulsed-force-mode atomic force microscopy.

The effect of a surface water layer on DNA strands deposited on a substrate was studied by atomic force microscopy (AFM). DNA molecules were deposited and stretched on chemically modified glass coverslips by a molecular combing method. Lambda bacteriophage DNA molecules were aligned on the organosilane-modified substrate surfaces by chemical and physical adsorption during the molecular combing. The combed DNA molecules were observed in humidity-controlled air and in aqueous solutions by pulsed-force-mode AFM (PFM-AFM). Chemical modification of cantilevers with an Au-coated tip by organothiol compounds was also applied to DNA observation. Mapping adhesive forces in aqueous media was useful to discriminate chemically the DNA strands from the substrate surface. The results suggest that PFM-AFM can be used widely to image the stretched DNA molecules on the silane-modified substrates.

Adsorption↗

PubMed1993

Rapid purification of double-stranded DNA by triple-helix-mediated affinity capture.

A simple and rapid method for the preparation of highly pure plasmid DNA has been developed. The DNA is directly captured from bacterial cell lysates by formation of a triple-helical structure between the plasmid dsDNA and a 20-base biotinylated oligonucleotide attached to streptavidin-coated magnetic beads and then eluted from the beads in pH 9 buffer solution. No phenol extraction, ethanol precipitation, RNase digestion, or CsCl gradient centrifugation is required. A general purpose cloning vector, pHJ19, was constructed for this application from pUC19 DNA by insertion of a 40-base sequence suitable for triple-helix formation. The approach was also found suitable for the purification of lambda bacteriophage DNA.

Bacterial Proteins↗

PubMed1985

Nucleotide sequence of the gene for the gamma chain of human fibrinogen.

A human genomic DNA library was screened for the gene coding for the gamma chain of fibrinogen by using a human cDNA for the gamma chain as a hybridization probe. The gene was identified in three overlapping recombinant lambda bacteriophage, and its sequence, including the immediate 5' and 3' flanking regions, was determined. The DNA sequence analysis revealed the presence of 10 exons coding for 411 amino acids present in the mature protein and a signal sequence of 26 amino acids. Two 30 base pair (bp) direct repeats of 93% identity were found 468 bp upstream from the transcription initiation site. The DNA sequence of the gene for the gamma chain of human fibrinogen showed considerable sequence homology with a partial sequence reported for the gene for the gamma chain of rat fibrinogen.

Amino Acid Sequence↗

PubMed2006

Generic technique to generate large branched DNA complexes.

The inherent self-recognition properties of DNA have led to its use as a scaffold for various nanotechnology self-assembly applications, with macromolecular complexes, metallic and semiconducting nanoparticles, proteins, inter alia, being assembled onto a designed DNA scaffold. Such structures may typically comprise a number of DNA molecules organized into macromolecules. Many studies have used synthetic methods to produce the constituent DNA molecules, but this typically constrains the molecules to be no longer than around 100 base pairs (30 nm). However, applications that require larger self-assembling DNA complexes, several tens of nanometers or more, need to be generated by other techniques. Here, we present a generic technique to generate large linear, branched, and/or circular DNA macromolecular complexes. The effectiveness of this technique is demonstrated here by the use of Lambda Bacteriophage DNA as a template to generate single- and double-branched DNA structures approximately 120 nm in size.

Aluminum Silicates↗

PubMed2006

Individually resolved DNA molecules stretched and embedded in electrospun polymer nanofibers.

We have used the flow characteristics of an electrospinning jet to elongate and fix DNA molecules. We embedded and observed fluorescently labeled lambda bacteriophage DNA molecules in polyethylene oxide nanofibers. The embedded DNA molecules were imaged using fluorescence microscopy and found to be stretched to lengths approaching the full dyed contour length.

Bacteriophage lambda↗

PubMed1997

Optimization of PCR/lambda exonuclease-mediated synthesis of sense and antisense DNA probes for in situ hybridization.

In situ hybridization experiments are stringently dependent on the quality of the probes, which should be single-stranded when efficient comparison of signals obtained with antisense and control sense probes are needed. In this report, we describe an optimized synthesis of radioactive single-stranded DNA probes, without vector cloning and requiring a unique polymerization step. The sequence region selected as probe is amplified by polymerase chain reaction in the presence of radiolabelled nucleotides. The sense and antisense probes are then yielded by the action of the lambda bacteriophage exonuclease, which can specifically eliminate one out of the two strands of the amplified fragments. In this way, sense and antisense probes with identical length and specific activity can be generated by selecting the primer to be phosphorylated. We have verified the efficiency of our probes for in situ hybridization of the clusterin transcripts within the peripheral olfactory system, after surgical lesion of its synaptic target.

Animals↗

PubMed2002

Genetic instability favoring transversions associated with ErbB2-induced mammary tumorigenesis.

It has been argued that genetic instability is required to generate the myriad mutations that fuel tumor initiation and progression and, in fact, patients with heritable cancer susceptibility syndromes harbor defects in specific genes that normally maintain DNA integrity. However, the vast majority of human cancers arise sporadically, in the absence of deficiencies in known "mutator" genes. We used a cII-based mutation detection assay to show that the mean frequency of forward mutations in primary mammary adenocarcinomas arising in mouse mammary tumor virus-c-erbB2 transgenic mice harboring multiple copies of the lambda bacteriophage genome was significantly higher than in aged-matched, wild-type mammary tissue. Analysis of the cII mutational spectrum within the mammary tumor genomic DNA demonstrated a >6-fold elevation in transversion mutation frequency, resulting in a highly unusual inversion of the transition/transversion ratio characteristic of normal epithelium; frameshift mutation frequencies were unaltered. Arising oncogenic point mutations within the c-erbB2 transgene of such tumors were predominantly transversions as well. Data from this model system support the notion that elaboration of a mutator phenotype is a consequential event in breast cancer and suggest that a novel DNA replication/repair gene is a relatively early mutational target in c-erbB2-induced mammary tumorigenesis.

Adenocarcinoma↗

PubMed1996

Function of the htrB high temperature requirement gene of Escherichia coli in the acylation of lipid A: HtrB catalyzed incorporation of laurate.

By assaying lysates of Escherichia coli generated with the hybrid lambda bacteriophages of an ordered library (Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50, 495-508), we identified two clones (lambda232 and lambda233) capable of overexpressing the lauroyl transferase that functions after 3-deoxy-D-manno-octulosonic acid (Kdo) addition in lipid A biosynthesis (Brozek, K. A., and Raetz, C. R. H. (1990) J. Biol. Chem. 265, 15410-15417). The E. coli DNA inserts in lambda232 and lambda233 suggested that a known gene (htrB) required for rapid growth above 33 degrees C might encode the lauroyl transferase. Using the intermediate (Kdo)2-lipid IVA as the laurate acceptor, extracts of strains with transposon insertions in htrB were found to contain no lauroyl transferase activity. Cells harboring hybrid htrB+ plasmids overproduced transferase activity 100-200-fold. The overproduced transferase was solubilized with a non-ionic detergent and purified further by DEAE-Sepharose chromatography. With lauroyl acyl carrier protein as the donor, the purified enzyme rapidly incorporated one laurate residue into (Kdo)2-lipid IVA. The rate of laurate incorporation was reduced by several orders of magnitude when either one or both Kdos were absent in the acceptor. With a matched set of acyl-acyl carrier proteins, the enzyme incorporated laurate 3-8 times faster than decanoate or myristate, respectively. Transfer of palmitate, palmitoleate, or R-3-hydroxymyristate was very slow. Taken together with previous studies, our findings indicate that htrB encodes a key, late functioning acyltransferase of lipid A biosynthesis.

Acylation↗