In situ fluorescence microscopy of bacteriophage aggregates.

Virus aggregation is analyzed because of its potential use for both classifying viruses and understanding virus ecology and evolution. Virus aggregation is, however, problematic because aggregation causes loss of virions during processing for microscopy of any type. Thus, here we detect virus aggregation by fluorescence microscopy of wet-mounted dissections of dilute gel-supported plaques (in situ fluorescence microscopy) of a test virus, the long-tail aggregating Bacillus thuringiensis bacteriophage, 0305phi8-36.

Routine fluorescence microscopy of single untethered protein molecules confined to a planar zone.

To bypass limitations of ensemble averaging biochemical analysis, microscopy-based detection and tracking are needed for single protein molecules that are diffusing in aqueous solution. Confining the molecules to a planar zone dramatically assists tracking. Procedures of microscopy should be routine enough so that effort is focused on the biochemistry.

Rapid determination of genomic DNA length for new bacteriophages.

dsDNA viruses with long genomes (>200 kb) are expected to be a major source of novel genes. To rapidly characterize the genomes of newly isolated dsDNA bacteriophages, we develop here a procedure for the PFGE of intact long DNA genomes from bacteriophage particles in unfractionated, infected cell lysates of either liquid or gelled cultures. The DNA used for PFGE is suitable for sequencing after extraction with phenol.

Temperature-dependence of preconditioning for lengthened capillary DNA sequencing.

A previous study shows that electrophoretic preconditioning of a commercial polymer solution increases the spacing and resolution of DNA fragments fractionated in this solution by CE at 50 degrees C (Griess, G. A. et al.

Matrix conditioning for lengthened capillary DNA sequencing.

Capillary electrophoresis (CE) is currently the preferred format for both DNA sequencing and small DNA fragment analysis. The present study provides a simple revision of the procedure used for CE of DNA with a commercial DNA sequencing apparatus from Applied Biosystems. The revision is electrophoretic conditioning of the sieving matrix (typically POP-6) before sample injection.

Cyclic capillary electrophoresis.

A strategy is described here for increasing both the resolution and the flexibility of capillary electrophoresis performed in a sieving medium of ungelled polymer. This strategy is based on analysis and, sometimes, re-analysis that is done in several stages of constant-field electrophoresis. Enhancement-stages are between the analysis-stages.

Improving the length-fractionation of DNA during capillary electrophoresis.

The present study develops a path-lengthening strategy for capillary electrophoresis of short double-stranded DNA molecules, in an aqueous solution of neutral polymer (hydroxypropylmethylcellulose). Tests of the dependence of fractionations on pulse times reveal the operation of at least one mechanism in addition to increase in effective path length. Electrophoresis is performed in the following two-stage cycles (cyclic electrophoresis): The first analysis-stage of each cycle is a constant field (forward) capillary electrophoresis.

Application of the concept of an electrophoretic ratchet.

Fractionation via a gel electrophoretic ratchet has previously succeeded for comparatively large (radius R > or = 95 nm) spheres (Serwer, P, Griess, G.A., Anal.

Unlimited increase in the resolution of DNA ladders.

Fractionation of DNA ladders by gel electrophoresis is limited by the progressive compressing of the long DNA end of a ladder. Improvement in the resolution of this DNA is achieved by use of the following two-step electrophoresis. Initially, the DNA ladder is fractionated by conventional constant field agarose gel electrophoresis.

Advances in the separation of bacteriophages and related particles.

Nondenaturing gel electrophoresis is used to both characterize multimolecular particles and determine the assembly pathways of these particles. Characterization of bacteriophage-related particles has yielded strategies for characterizing multimolecular particles in general. Previous studies have revealed means for using nondenaturing gel electrophoresis to determine both the effective radius and the average electrical surface charge density of any particle.

The formation of small-pore gels by an electrically charged agarose derivative.

Previous studies have shown that, during the formation of an underivatized agarose gel, agarose molecules laterally aggregate to form thicker fibers called suprafibers; the suprafibers branch to form a gelled network. In the present study, electron microscopy of thin sections is used to investigate both the thickness and the spacing of the fibers of gels formed by agarose chemically derivatized with carboxymethyl (negatively charged) groups. For carboxymethyl agarose, electron microscopy reveals that gels cast in water consist of both fibers narrower and pores smaller than those observed for water-cast underivatized agarose gels at the same concentration.

Intracellular Ca2+ thresholds that determine survival or death of energy-deprived cells.

Increase of intracellular ionized or free Ca2+ is thought to play a central role in cell death due to ATP depletion. However, concurrently operative mechanisms of injury that do not require intracellular Ca2+ increases have made it difficult to test this hypothesis or to determine the concentrations at which intracellular Ca2+ becomes lethal. The predominant Ca2+-independent mechanism of injury during ATP depletion involves the loss of cellular glycine.

The conformation of packaged bacteriophage T7 DNA: informative images of negatively stained T7.

Within the icosahedral protein outer shell of bacteriophage T7, a 40-kbp DNA genome occupies a cavity also occupied by a protein cylinder that projects into the DNA from the outer shell. However, neither the internal cylinder nor separately resolved DNA segments are revealed in the conventional negatively stained specimens of intact bacteriophage T7. In the present study, a procedure of negative staining is used that reveals both internal proteins and separately resolved segments of packaged DNA during electron microscopy of intact particles of a hybrid T7 bacteriophage; the hybrid is genetically T7, except for a tail fiber gene that has a segment from the T7-related bacteriophage, T3.

Use of excluded volume to increase the heterogeneity of pore size in agarose gels.

When testing theoretical models that quantitatively describe the sieving of macromolecules during gel electrophoresis, investigators have been limited by absence of control of the heterogeneity of the size of pores in the gel. In a recent study performed by electron microscopy of thin sections (G. A.

Conversion of a linear to a circular plasmid in the relapsing fever agent Borrelia hermsii.

Spirochetes of the genus Borrelia have genomes composed of both linear and circular replicons. We characterized the genomic organization of B. burgdorferi, B.

Non-denaturing gel electrophoresis of biological nanoparticles: viruses.

Although gel electrophoresis is usually used for the fractionation of monomolecular particles, it is also applicable to the fractionation of the multimolecular complexes produced during both cellular metabolism and assembly of viruses in virus-infected cells. Gel electrophoretic procedures have been developed for determining both the size of a spherical particle and some aspects of the shape of a non-spherical particle. Capsids bound to DNA outside of the capsid can also be both fractionated and characterized.

A desktop, low-cost video fluorometer for quantitation of macromolecules after gel electrophoresis.

After gel electrophoresis, quantification of in-gel fluorescence is sometimes used to assay the macromolecules fractionated. Both procedures and equipment that have the following improvements are presented here for direct video fluorometry of gels used for electrophoresis: comparatively low cost, high ease of use and low consumption of space. This equipment has a linear response to the amount of ethidium-stained DNA that forms a band in an agarose gel.

The relationship of agarose gel structure to the sieving of spheres during agarose gel electrophoresis.

To understand the organization of fibers in an agarose gel, digitized electron micrographs are used here to determine the frequency distribution of interfiber distance (2Pc) in thin sections of agarose gels. For a preparation of underivatized agarose, a 1.5% gel has a Pc distribution that is indistinguishable from the Pc distribution of a computer-generated, random-fiber gel; the log of the occurrence frequency (F) decreases linearly as a function of Pc.

The trajectories of spheres during agarose gel electrophoresis.

To develop a physical description of the gel-induced retardation of spheres during gel electrophoresis, the microscopic motion of single electrically charged latex spheres is statistically quantified here, by digital image analysis. To obtain adequate resolution in space, comparatively large spheres, 240 nm in radius, are used. The following observations are made during electrophoresis in a 0.

Assembly-associated structural changes of bacteriophage T7 capsids. Detection by use of a protein-specific probe.

To detect changes in capsid structure that occur when a preassembled bacteriophage T7 capsid both packages and cleaves to mature-size longer (concatameric) DNA, the kinetics and thermodynamics are determined here for the binding of the protein-specific probe, 1,1'-bi(4-anilino)naphthalene-5,5'-di-sulfonic acid (bis-ANS), to bacteriophage T7, a T7 DNA deletion (8.4%) mutant, and a DNA-free T7 capsid (metrizamide low density capsid II) known to be a DNA packaging intermediate that has a permeability barrier not present in a related capsid (metrizamide high density capsid II). Initially, some binding to either bacteriophage or metrizamide low density capsid II occurs too rapidly to quantify (phase 1, duration < 10 s).

Desktop digital imaging : application to detection of length heterogeneity after hyperresonant pulsed-field gel electmphoresis of mature bacteriophage p22 DNA.

The multisample slab gels used for pulsed-field gel electrophoresis (PFGE) can produce gel patterns of hundreds of samples per day (see, for example, ref. 1). Because of this efficiency in the production of data, the rate-limiting steps in obtaining information at times are analysis, organization, reproduction, storage, and retrieval of data.

Variation of the permeability of bacteriophage T4: analysis by use of a protein-specific probe for the T4 interior.

The permeability of bacteriophage T4 and the change in T4 permeability caused by mutation to osmotic shock resistance are investigated here by quantification of the kinetics with which both a DNA-specific probe (ethidium) and a protein-specific probe [1,1'-bi(4-anilino)naphthalene-5,5'-di-sulfonic acid, or bis-ANS)] bind to T4. In the case of an osmotic shock-resistant mutant, T40s41, both ethidium and bis-ANS bind with first order kinetics. The first-order rate constant (k*) for both bis-ANS and ethidium is a function of anion type and concentration.

Gel electrophoresis of micron-sized particles: a problem and a solution.

The gel electrophoresis of spherical particles with a radius above 0.2 micron has not been reported yet. In the present study, video phase-contrast light microscopy is used to observe the motion of individual latex spheres, 0.

The sieving of rod-shaped viruses during agarose gel electrophoresis. I. Comparison with the sieving of spheres.

The sieving of rod-shaped viruses during agarose gel electrophoresis is quantitatively analyzed here with a previously proposed model [G. A. Griess et al.

The sieving of spheres during agarose gel electrophoresis: quantitation and modeling.

By use of agarose gel electrophoresis, the sieving of spherical particles in agarose gels has been quantitated and modeled for spheres with a radius (R) between 13.3 and 149 nm. For quantitation, the electrophoretic mobility has been determined as a function of agarose percentage (A).