Single-particle visualization of assembly: I. Dimerization in a planar zone.

Summary Single-particle fluorescence microscopy of association/dissociation is required for analysis of biological assembly reactions. Toward achieving this goal, Wang et al. (J. Microsc., 2004, 213, 101-109) used molten agarose to concentrate thermally diffusing particles in a thin zone of solution next to the surface of a coverglass (plane of concentration). The present study details the first real-time, single-particle analysis of the association/dissociation of thermally diffusing particles in the plane of concentration. The test particles were procapsids of bacteriophage lambda (radius = 31 nm). Quantification of thermal motion was developed and used to determine whether co-diffusing particles were bound to each other. The data are explained by (1) the presence of a molten agarose-generated barrier that is 93-155 nm from the coverglass surface, and (2) non-random orientation of procapsid dimers in the plane of concentration.

Fluorescence microscopy of colour-tagged nanoparticles that are undergoing thermal motion.

To bypass limitations of conventional biochemical analysis, single-particle biochemical analysis is used. To improve single-particle biochemical analysis, procedures are needed to keep nanometre-sized particles in focus while the particles are undergoing thermal motion. A simple, inexpensive procedure is developed here for keeping particles in focus during the continuous observing/discriminating/recording of two different particles, both of which are undergoing thermal motion. This procedure concentrates the particles in a plane of solution that is in focus when the cover glass surface is in focus. An essential component of the procedure is the addition of molten, low-melt agarose to the specimen. Motionless binding to glass is inhibited by inclusion of anti-stick additives in the specimen. Both carrier protein (gelatin) and non-ionic detergent (Triton X-100) are anti-stick additives successfully used here. Intact bacteriophages T3 and T7 are used as model particles, in anticipation of the use of the procedures developed here for the analysis of the assembly of bacteriophages. Observing/discriminating/recording of colour-tagged bacteriophages T3 and T7 is achieved at video frame rate with image splitting to discriminate colours.

Terminal protein-induced stretching of bacteriophage phi29 DNA.

Stretching of DNA molecules helps to resolve detail during the fluorescence microscopy of both single DNA molecules and single DNA-protein complexes. To make stretching occur, intricate procedures of specimen preparation and manipulation have been developed in previous studies. By contrast, the present study demonstrates that conventional procedures of specimen preparation cause DNA stretching to occur, if the specimen is the double-stranded DNA genome of bacteriophage phi29. Necessary for this stretching is a protein covalently bound at both 5' termini of phi29 DNA molecules. Some DNA molecules are attached to a cover glass only at the two ends. Others are attached at one end only with the other end free in solution. The extent of stretching varies from approximately 50% overstretched to approximately 50% understretched. The understretched DNA molecules are internally mobile to a variable extent. In addition to stretching, some phi29 DNA molecules also undergo assembly to form both linear and branched concatemers observed by single-molecule fluorescence microscopy. The assembly also requires the terminal protein. The stretched DNA molecules are potentially useful for observing DNA biochemistry at the single molecule level.

Fluorescence microscopy of single viral capsids.

To build a foundation for the single-molecule fluorescence microscopy of protein complexes, the present study achieved fluorescence microscopy of single, nucleic acid-free protein capsids of bacteriophage T7. The capsids were stained with Alexa 488 (green emission). Manipulation of the capsids' thermal motion was achieved in three dimensions. The procedure for manipulation included embedding the capsids in an agarose gel. The data indicate that the thermal motion of capsids is reduced by the sieving of the gel. The thermal motion can be reduced to any desired level. A semilogarithmic plot of an effective diffusion constant as a function of gel concentration is linear. Single, diffusing T7 capsids were also visualized in the presence of single DNA molecules that had been both stretched and immobilized by gel-embedding. The DNA molecules were stained with ethidium (orange emission). This study shows that single-molecule (protein and DNA) analysis is possible for both packaging of DNA in a bacteriophage capsid and other events of DNA metabolism. The major problem is the maintenance of biochemical activity.

Partially condensed DNA conformations observed by single molecule fluorescence microscopy.

To detect partially condensed conformations of a double-stranded DNA molecule, single molecule fluorescence microscopy is performed here. The single DNA molecules are ethidium stained, 670 kilobase pair bacteriophage G genomes that are observed both during and after expulsion from capsids. Expulsion occurs in an agarose gel. Just after expulsion, the entire G DNA molecule typically has a partially condensed conformation not previously described (called a balloon). A balloon subsequently extrudes a filamentous segment of DNA. The filamentous segment becomes gently elongated via diffusion into the network that forms the agarose gel. The elongated DNA molecule usually has bright spots that undergo both appearance/disappearance and apparent motion. These spots are called dynamic spots. A dynamic spot is assumed to be the image of a zone of partially condensed DNA segments (globule). The positions of globules along an elongated DNA molecule 1) are restricted primarily to time-stable regions with comparatively high thermal motion-induced, micrometer-scale bending of the DNA molecule and 2) move within a given region on a time scale smaller than the time scale of recording. Less mobile globules are observed when either magnesium cation or ethanol is added before gel-embedding DNA molecules. These observations are explained by globules induced at equilibrium by a bending-dependent, inter-DNA segment force. Theory has previously predicted that globules are induced by electrostatic forces along an electrically charged polymer at equilibrium. The hypothesis is proposed that intracellular DNA globules assist action-at-a-distance during DNA metabolism.

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. Chim. Acta 1998, 372, 299-306). The electrical oscillations are the following electrical field pulses: high field --> low field --> high field, etc. The field is inverted after each pulse; the time-integral of the field can be zero. Response to the ratchet is caused by steric trapping in the high field-direction, but not in the low field-direction. Trapping and, therefore, response to the ratchet decrease as R decreases. The smaller spheres do not respond to the ratchet. In the present study, spheres with R values smaller than 95 nm are made, for the first time, to respond to a similar gel electrophoretic ratchet. To achieve this objective, the heterogeneity of pore size is increased for the gel used. The heterogeneity of pore size is increased by (i) forming the gel with degraded hydroxyethyl agarose, and (ii) gelling at comparatively high temperature. If a particle still does not respond to the ratchet (because the particle is too small), this particle has a net migration in the high field-direction, when the above-described pulsed field is biased in the high field-direction. If a particle does respond to the improved ratchet, the particle has a net migration in the low field-direction. Here, the R of ratchet-responding spheres is reduced to 30-50 nm. These ratchet-responding spheres include both intact bacteriophage particles (R = 30 nm) and latex spheres. The smaller ratchet-responding spheres have an electrophoretic mobility that decreases in magnitude as the electrical field increases in magnitude. A ratchet-based procedure is developed here to achieve continuous preparative gel electrophoresis.

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. This analysis-stage reveals the length distribution of the shortest DNA molecules not previously analyzed. The second, enhancement-stage of each cycle is zero-integrated field electrophoresis (ZIFE). The enhancement-stage improves the DNA length-fractionation for the next DNA molecules to be analyzed. A slight reverse migration occurs in the enhancement-stage. Increase in both peak separation and peak sharpness contribute to improvement in the length-fractionation of DNA molecules.

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. Subsequently, gel electrophoresis is performed in the reverse direction by pulsing the electrical field (PFGE). A newly developed type of pulsing is used, which causes inversion of a double-stranded DNA ladder: the distance migrated increases as the length of the DNA molecule increases. Thus, the resolution of DNA bands continues to increase during the PFGE. These two stages of electrophoresis are serially repeated. Eventually, both the short and the long DNA ends of the ladder migrate out of the gel while a selected region of the ladder undergoes progressive increase in resolution during back-and-forth migration. Improved resolution of DNA bands is achieved, without a known limit.

Single-event analysis of the packaging of bacteriophage T7 DNA concatemers in vitro.

Bacteriophage T7 packages its double-stranded DNA genome in a preformed protein capsid (procapsid). The DNA substrate for packaging is a head-to-tail multimer (concatemer) of the mature 40-kilobase pair genome. Mature genomes are cleaved from the concatemer during packaging. In the present study, fluorescence microscopy is used to observe T7 concatemeric DNA packaging at the level of a single (microscopic) event. Metabolism-dependent cleavage to form several fragments is observed when T7 concatemers are incubated in an extract of T7-infected Escherichia coli (in vitro). The following observations indicate that the fragment-producing metabolic event is DNA packaging: 1) most fragments have the hydrodynamic radius (R(H)) of bacteriophage particles (+/-3%) when R(H) is determined by analysis of Brownian motion; 2) the fragments also have the fluorescence intensity (I) of bacteriophage particles (+/-6%); 3) as a fragment forms, a progressive decrease occurs in both R(H) and I. The decrease in I follows a pattern expected for intracapsid steric restriction of 4',6-diamidino-2-phenylindole (DAPI) binding to packaged DNA. The observed in vitro packaging of a concatemer's genomes always occurs in a synchronized cluster. Therefore, the following hypothesis is proposed: the observed packaging of concatemer-associated T7 genomes is cooperative.

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 response of electrophoretic mobility to increasing the magnitude of the electrical field is used to detect rod-shaped particles. To increase the capacity of nondenaturing gel electrophoresis to characterize comparatively large particles, some current research is directed towards either determining the structure of gels used for electrophoresis or inducing steric trapping of particles in dead-end regions within the fibrous network that forms a gel. A trapping-dependent technique of pulsed-field gel electrophoresis is presented with which a DNA-protein complex can be made to electrophoretically migrate in a direction opposite to the direction of migration of protein-free DNA.

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. This result is confirmed by using the electrophoretic sieving of spheres to determine the radius (PE) of the effective pore of the gel. At a given concentration of gel less than 1%, the PE for a water-cast carboxymethyl agarose gel is 0.25-0.30x the PE for a water-cast underivatized agarose gel. The value of PE predicts the extent of the electrophoretic sieving that is observed when double-stranded DNA is subjected to electrophoresis through a water-cast carboxymethyl agarose gel; DNA bands formed in a water-cast carboxymethyl agarose gel are comparable in quality to DNA bands formed in a water-cast underivatized agarose gel of equal PE. The following observation supports the hypothesis that electrical charge-charge repulsion among carboxymethyl agarose molecules inhibits the formation of suprafibers in water-cast carboxymethyl agarose gels: Increased content of suprafibers in carboxymethyl agarose gels is observed when the ionic strength is raised by the presence of NaCl, MgCl2, or any of several buffers during gelation of carboxymethyl agarose.

Conformation and interactions of the packaged double-stranded DNA genome of bacteriophage T7.

The structure of the packaged double-stranded DNA genome of bacteriophage T7 was compared to that of unpackaged T7 DNA using digital difference Raman spectroscopy. Spectral data were obtained at 25 degrees C from native T7 virus (100 mg/mL), empty T7 capsids (50 mg/mL), and purified T7 DNA (40 mg/mL) in buffer containing 200 mM NaCl, 10 mM MgCl2, and 10 mM Tris at pH 7.5. At these conditions, the local conformation of T7 DNA was not affected by packaging. Specifically, the local B-form secondary structure of unpackaged T7 DNA, including furanose C2'-endo pucker, anti glycosyl torsion, Watson-Crick base pairing, and base stacking, were essentially fully (> 98%) retained when the genome was condensed within the viral capsid. However, the average electrostatic environment of T7 DNA phosphates was altered dramatically by packaging as revealed by large perturbations in the Raman bands associated with localized vibrations of the DNA phosphate groups. The change in the phosphate environment was attributed to Mg2+ ions that were packaged with the genomic DNA, and the observed Raman perturbations of genomic DNA were equivalent to those generated by a 50-100-fold increase in Mg2+ concentration in aqueous phosphodiester model compounds. The T7 data were qualitatively and quantitatively similar to those observed previously for packaged DNA of bacteriophage P22 and imply that genomic DNAs of T7 and P22 are both organized in a similar fashion within their respective capsids. The results show that the condensed genome does not contain kinks or folds that would disrupt the local B conformation by more than 2%. The present findings are discussed in relation to previously proposed models for condensation and organization of double-stranded and single-stranded viral DNA.

Polymorphism of bacteriophage T7.

For viruses made of nucleic acid and protein, the structure of the protein outer shell has, in the past, been found to be uniquely determined by the viral genome. However, here, non-denaturing agarose gel electrophoresis of bacteriophage T7 reveals two states of the mature T7 capsid; the conditions of growth are found to alter the population by T7 of these two electrophoretically defined states. Both states have been previously observed for a genetically altered T7 and they are observed here for wild-type T7. The average electrical surface charge density of a bacteriophage particle (delta) determines its state; the delta of particles in both states is negative. For a given condition of growth, the population of these two states is influenced by the extent to which the major T7 outer shell protein, p10A, is accompanied by its minor readthrough variant, p10B. Comparison of the two electrophoretic states reveals the following. (1) No difference in radius is present in the outer shell (+/-2%). (2) As the pH of electrophoresis is either increased or decreased from neutrality, the state becomes more highly populated for which delta is greater in magnitude (state 1). By changing the pH, some T7 particles are made to change state. (3) Particles in state 1 adsorb less quickly to host cells than do the particles in the alternative state (state 2). This latter observation suggests the hypothesis that state 1 evolved to reduce the probability of re-initiating an infection when conditions are not favorable for growth. This hypothesis is supported by the observation that, as conditions of growth become apparently more unfavorable, progeny increasingly populate state 1.

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. The negatively stained packaged DNA segments of this hybrid bacteriophage are found to be wrapped around the axis of the internal cylinder. To obtain additional information about the conformation of packaged T7 DNA, electron microscopy is performed of negatively stained capsids that are incompletely filled with DNA (ipDNA-capsids); a procedure is described for improved isolation of ipDNA-capsids from lysates of hybrid bacteriophage T7-infected cells. The packaged DNA segments of ipDNA-capsids are found not to be wrapped around any axis. Images of ipDNA-capsids are explained by the hypothesis that DNA does not achieve its wrapped condition until the capsid is more than 40% full of DNA. Wrapping via folding is, therefore, proposed to explain the images of DNA packaged in bacteriophage T7.

Formation and cleavage of a DNA network during in vitro bacteriophage T7 DNA packaging: light microscopy of DNA metabolism.

To understand in vivo DNA metabolism, in vitro systems are developed that perform DNA metabolism, while maintaining in vivo (physiological) character. To determine the state of DNA during in vitro physiological metabolism, the present study develops procedures of fluorescence light microscopy for observation of stained DNA molecules during in vitro physiological metabolism in a crude extract of bacteriophage T7-infected cells. The extract inhibits illumination-induced breakage of DNA. The following DNA metabolism remains active for 2-3 min during microscopy: exonuclease-dependent end-to-end joining (concatemerization) of T7 DNA and subsequent cleavage of concatemers. When the T7 gene 3-encoded DNA debranching endonuclease is absent during in vitro T7 DNA concatemerization, DNA progressively partitions to form a continuous, mostly immobile (i.e., no detected Brownian motion) fibrous network that encloses the DNA-depleted solution; presumably because of reduced branching, a less extensive network forms when the gene 3-encoded debranching endonuclease is present. Most strands of the network consist of multiple DNA segments. After a time interval of 5-10 min, the DNA network undergoes cleavage that depends on the presence of both ATP, capsids, and the DNA packaging accessory proteins encoded by genes 18 and 19; multiple cleavages eventually disrupt the continuity of the DNA network. The dependence of the observed cleavage on these factors is explained by the hypothesis that this cleavage is the first of two cleavages known to occur during the packaging of T7 DNA concatemers both in vivo and in vitro. The first cleavage is also known to initiate entry of DNA into a T7 capsid. The cleavage observed here is usually preceded by an approximately 10 s burst of oscillatory motion of the DNA network near the point of eventual cleavage. If the in vivo presence of a similar concatemer-containing DNA network is assumed, requirement for DNA packaging-associated release of DNA from this network is a possible explanation for the evolution of a T7 DNA packaging pathway that is initiated by cleavage of a concatemer.

The conformation of DNA packaged in bacteriophage G.

When packaged in a bacteriophage capsid, double-stranded DNA occupies a cavity whose volume is roughly twice the volume of the DNA double helix. The data thus far have not revealed whether the compactness of packaged bacteriophage DNA is achieved by folding of the DNA, undirectional winding of the DNA, or a combination of both folding and winding. To assist in discriminating among these possibilities, the present study uses electron microscopy, together with ultraviolet light-induced DNA-DNA cross-linking, to obtain the following information about the conformation of DNA packaged in the comparatively large bacteriophage, G: 1) At the periphery of some negatively stained particles of bacteriophage G, electron microscopy reveals standards of DNA that are both parallel to each other and parallel to the polyhedral bacteriophage G capsid. However, these strands are not visible toward the center of the zone of packaged DNA. 2) Within some positively stained particles, electron microscopy reveals DNA-associated stain in relatively high concentration at corners of the polyhedral bacteriophage G capsid. 3) When cross-linked DNA is expelled from its capsid during preparation for electron microscopy, some DNA molecules consist primarily of a compacted central region, surrounded by DNA strands that appear to be unravelling at multiple positions uniformly distributed around the compacted DNA region. The above results are explained by a previously presented model in which DNA is compacted by folding to form 12 icosahedrally arranged pear-shaped rings.

PCR-directed formation of viral hybrids in vitro.

When constructing viruses that have desired hybrid phenotypes, anticipated difficulties include the nonviability of many, possibly most, of the hybrid genomes that can be constructed by incorporation of DNA fragments. Therefore, many different hybrid genomes may have to be constructed in order to find one that is viable. To perform this combinatorial work in a single experiment, we have used bacteriophage T7-infected cell extracts to transfer DNA in vitro. In an extract, we have incubated T7 DNA, together with DNA obtained by polymerase chain reaction (PCR) amplification of the gene (gene 17) for the tail fiber of the T7-related bacteriophage, T3. After in vitro packaging of DNA in the extract, hybrid progeny bacteriophage were detected by probing with a T3-specific oligonucleotide; hybrids are found at a frequency of 0.1%. By determination of the nucleotide sequence of the entire gene 17 of 14 independently isolated hybrids, both right and left ends of the PCR fragment are found to be truncated in all hybrids. For all 14 hybrids, the right end is in the same location; the left end is found at 3 different locations. The nonrandom location of the ends is explained by selection among different inserts for viability; that is, most of the hybrid genomes are nonviable. Some hybrids acquire from T3 the desirable phenotype of nonadherence to agarose gels during agarose gel electrophoresis.

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. Griess et al., J. Struct. Biol. 1993, III, 39-47), pore size heterogeneity has been increased for agarose gels by a combination of both derivatization and molecular weight reduction of the polysaccharide chains of agarose. In the present study, pore size heterogeneity is increased by a mechanism that appears to have an origin different from the origin of this previously observed increase in heterogeneity: Pore size heterogeneity is increased by addition of a polyethylene glycol (PEG) of high molecular weight (18,500) to molten agarose before gelation. In contrast, the use of a lower molecular weight PEG (either 4,000 or 7,500) causes the formation of micron-sized precipitates within a gelled network of agarose fibers. Thus far, the PEG-induced heterogeneity of pore size occurs primarily in 100-1,000 microns scale zones separated from each other by interzone regions of decreased agarose fiber density. More uniform gels are needed for the study of sieving.

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. hermsii, B. turicatae, and B. anserina with pulsed-field gel electrophoresis. All four species contained a linear chromosome approximately 1 Mb in size and multiple linear plasmids in the 16- to 200-kb size range. Plasmids 180 and 170 kb in size, present in the relapsing fever agents B. hermsii and B. turicatae but not in the other two species, behaved as linear duplex DNA molecules under different electrophoretic conditions. A variant of strain HSI of B. hermsii had a 180-kb circular instead of linear plasmid. There were no detectable differences in the growth rates or in the expression of cellular proteins between cells bearing linear forms and those bearing circular forms of the plasmid. The conversion to a circular conformation of monomeric length was demonstrated by the introduction of strand breaks with irradiation, restriction endonuclease analysis, and direct observation of the DNA molecules by fluorescent microscopy. Consideration of different models for the replication of linear DNA suggests that circular intermediates may be involved in the replication of linear replicons in Borrelia spp.

Video light microscopy of 670-kb DNA in a hanging drop: shape of the envelope of DNA.

Although its conformation has not been observed directly, double-stranded DNA in solution is usually assumed to be randomly coiled at the level of the DNA double helix. By video light microscopy of ethidium-stained DNA at equilibrium in a nonturbulent hanging drop, in the present study, the 670 kb linear bacteriophage G DNA is found to form a flexible filament that has on average 17 double helical segments across its width. This flexible filament 1) has both asymmetry and dimensions expected of a random coil and 2) has ends that move according to the statistics expected of a random walk. After unraveling the flexible filament-associated DNA double helix near the surface of a hanging drop, recompaction occurs without perceptible rotation of the DNA. Both conformational change and intermolecular tangling of the DNA are observed when G DNA undergoes nondiffusive motion in a hanging drop. The characteristics of the G DNA flexible filament are explained by the assumption that the flexible filament is a random coil of double helical segments that are unperturbed by motion of the suspending medium.