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P Serwer

Publications and source records attributed to P Serwer.

At least 73 records · Page 4Linked to original sources
PubMed1988

Concatemerization and packaging of bacteriophage T7 DNA in vitro: determination of the concatemers' length and appearance kinetics by use of rotating gel electrophoresis.

During its morphogenesis both in intact infected cells (in vivo) and in lysates of infected cells (in vitro), bacteriophage T7 forms end-to-end concatemers of its mature DNA, a linear, nonpermuted, terminally repetitious DNA. During morphogenesis, in vivo T7 concatemers are packaged in preformed capsids and cut to mature size. In the present study the lengths and appearance kinetics of concatemers formed in vitro from mature T7 DNA have been determined. The following procedures are used here for the first time: (a) 20-35% efficient in vitro concatemerization and packaging of T7 DNA; the mixture used for packaging contained two lysates that together had all T7 gene products, and (b) fractionation of concatemers by rotating gel electrophorsis (RGE), which improves the resolution by length of concatemer-length DNA. Concatemerization at 30 degrees was so fast that some other process must be rate limiting for packaging. The concatemers formed were linear and joined left-end to right-end by complementary base pairing, not by blunt-end ligation. Concatemers formed at 30 degrees were reconverted to mature DNA by packaging in vitro. Reducing the temperature to 0 degrees both slowed concatemerization to the time scale (minutes) needed for control of the extent of concatemerization and reduced packaging to insignificant levels, thereby also uncoupling packaging from concatemerization. At both 30 degrees and 0 degrees bands of discrete-length concatemers were observed by RGE. The lengths were n times the length of mature T7 DNA; n was found to be any integer from 2 to 15. The bands were stronger at 0 degrees than they were at 30 degrees in comparison to a background of heterogeneous DNA. No evidence for the favoring of any value of n was found. In addition, it was found by two-dimensional agarose gel electrophoresis that a comparatively small amount of circular DNA was produced in vitro.

Cold Temperature↗
PubMed1988

The mechanism of DNA's fractionation during pulsed-field agarose gel electrophoresis: a hypothesis.

The resolution by length of linear double-stranded DNA longer than 10-20 kb is increased by periodically changing direction of the electrical field during agarose gel electrophoresis (pulsed field gel, or PFG, electrophoresis). Previously proposed mechanisms for this effect include viscoelastic relaxation of the DNA and DNA length-dependent path lengths through the gel. Several data are not explained by these mechanisms. To better explain the data, here is proposed: (a) the existence of flexible projections from the more rigid fibers that form the network of agarose gels; these projections slow DNA's motion by threading loops of DNA; (b) the bending of these projections in the direction of DNA's motion during electrophoresis; (c) hysteresis in the re-orientation of these projections when the direction of the electrical field is changed, and (d) increase in resistance to DNA's motion in a direction opposite to that of the projections' bending. By use of (a)-(d), the facts known about DNA fractionation during PFG electrophoresis are qualitatively explained.

Chemical Fractionation↗
PubMed1987

The mechanism for producing two symmetries at the head-tail junction of bacteriophages: a hypothesis.

Some double-stranded DNA bacteriophages consist of DNA packaged in a proteinaceous capsid. The capsid has a DNA-enclosing outer shell (head) attached to an external projection (tail). At the head-tail junction is a ring of subunits (connector) that has either six or twelve-fold rotational symmetry, and is joined to the head at an axis of the head's five-fold rotational symmetry. The head is made of subunits in either an icosahedral array or an array consisting of two icosahedral hemispheres separated by a cylinder of subunits. During infection of a host, the head with connector is assembled as a procapsid that subsequently packages DNA and joins a tail. The mechanism for producing two symmetries at the head-tail junction has in the past been an unsolved problem. The observation that the connector of bacteriophage T7 does not nucleate assembly of the outer shell of T7's icosahedral procapsid (P. Serwer and R. H. Watson [1982] J. Virol. 42, 595-601) places a constraint on a solution for the above problem. To solve the above problem for icosahedral procapsids, it is proposed here that: (a) assembly of the outer shell of procapsids is nucleated by a six-membered ring of hexameric aggregates of the major outer shell protein, (b) the connector is assembled in the center of this ring, (c) one of the hexamers dissociates from the ring, creating a five-membered ring and forcing the connector to the inside of the outer shell. A related mechanism is proposed for nucleation of the elongated procapsid of bacteriophage T4.

Bacteriophages↗
PubMed1987

Agarose gel electrophoresis of bacteriophages and related particles.

Viruses and related particles have been fractionated by electrophoresis through gels. For agarose gels, the radius at the exclusion limit for spheres varies from 1500 nm in a 0.04% gel to 3.6 nm in a 4.0% gel. Thus, the size of the gel's pores can be adjusted to sieve all known viruses. By measurement of electrophoretic mobility (mu) as a function of agarose concentration, the mu in the absence of a solid support (mu 0) can be determined for any particle. From the shape of a semilogarithmic plot of mu as a function of agarose percentage, a rod-shaped particle can be discriminated from a spherical particle. The sphere's radius can be determined from this plot with an accuracy of +/- 8%. Accuracy of +/- 1% has been more recently achieved using two-dimensional agarose gel electrophoresis. Though bacteriophages have been the primary object of study, the above techniques of agarose gel electrophoresis have also been applied to plant viruses and should be applicable to animal viruses. The mu 0 values measured for bacteriophages with and without their tail fibers suggest a mechanism of controlling attachment to a host. A related mechanism is proposed for the control of the virulence of animal viruses. Measurement of outer radius for different forms of the capsid of bacteriophage P22 reveals variability in outer radius too small to be detected by electron microscopy.

Bacteriophages↗
PubMed1987

Multidimensional analysis of intracellular bacteriophage T7 DNA: effects of amber mutations in genes 3 and 19.

By use of rate-zonal centrifugation, followed by either one- or two-dimensional agarose gel electrophoresis, the forms of intracellular bacteriophage T7 DNA produced by replication, recombination, and packaging have been analyzed. Previous studies had shown that at least some intracellular DNA with sedimentation coefficients between 32S (the S value of mature T7 DNA) and 100S is concatemeric, i.e., linear and longer than mature T7 DNA. The analysis presented here confirmed that most of this DNA is linear, but also revealed a significant amount of circular DNA. The data suggest that these circles are produced during DNA packaging. It is proposed that circles are produced after a capsid has bound two sequential genomes in a concatemer. The size distribution of the linear, concatemeric DNA had peaks at the positions of dimeric and trimeric concatemers. Restriction endonuclease analysis revealed that most of the mature T7 DNA subunits of concatemers were joined left end to right end. However, these data also suggest that a comparatively small amount of left-end to left-end joining occurs, possibly by blunt-end ligation. A replicating form of T7 DNA that had an S value greater than 100 (100S+ DNA) was also found to contain concatemers. However, some of the 100S+ DNA, probably the most branched component, remained associated with the origin after agarose gel electrophoresis. It has been found that T7 protein 19, known to be required for DNA packaging, was also required to prevent loss, probably by nucleolytic degradation, of the right end of all forms of intracellular T7 DNA. T7 gene 3 endonuclease, whose activity is required for both recombination of T7 DNA and degradation of host DNA, was required for the formation of the 32S to 100S molecules that behaved as concatemers during gel electrophoresis. In the absence of gene 3 endonuclease, the primary accumulation product was origin-associated 100S+ DNA with properties that suggest the accumulation of branches, primarily at the left end of mature DNA subunits within the 100S+ DNA.

Centrifugation, Zonal↗
PubMed1986

Arrangement of double-stranded DNA packaged in bacteriophage capsids. An alternative model.

Toroidal winding of double-stranded DNA in the protein capsids of bacteriophages has been proposed previously. An alternative model for the packaging and arrangement of DNA in bacteriophage capsids is presented here. By introducing sharp folds, the alternative model avoids toroidal winding and its accompanying difficulties. This alternative model is in agreement with the current data obtained with several different bacteriophages.

Bacteriophages↗
PubMed1986

Determination of a particle's radius by two-dimensional agarose gel electrophoresis.

Electrophoresis in an agarose gel dilute enough to be almost nonretarding, followed by electrophoresis in an orthogonal direction into a more concentrated agarose gel, has been developed as a procedure to determine the radius of spherical particles. Unlike procedures of unidirectional electrophoresis in a single gel, the above procedure can be used to compare the radii of particles that differ in solid-support-free electrophoretic mobility. Accuracy of 0.3 nm has been achieved with particles 30 nm in radius. It was found that the apparent radius of the spherical capsid of bacteriophage P22 decreased by 3% during elevated temperature-induced ejection of DNA from the capsid. Though originally designed for use with multimolecular particles, the procedure described here should also be useful with monomolecular particles.

Bacteriophages↗
PubMed1986

Exclusion of spheres by agarose gels during agarose gel electrophoresis: dependence on the sphere's radius and the gel's concentration.

Agarose gel electrophoresis of spheres (radius = R) has been used to determine the effective radius (PE) of the pores of an agarose gel (percentage of agarose in a gel = A). The value of PE at a given A was taken to be the R of the largest sphere that enters the gel. When log PE is plotted as a function of log A, the results can be represented by: PE = 118A-0.74 for 0.2 less than or equal to A less than or equal to 4.0 (PE in nm). However, the data suggest significant nonlinearity in this plot, the magnitude of the exponent of the PE vs A relationship increasing by about 20% as A increases from 0.2 to 4.0. From these data, PE's as big as 1500 nm and as small as 36 nm can be achieved with agarose gels formed with unmodified, unadulterated agarose and usable for electrophoresis.

Electrophoresis↗
PubMed1985

Alterations of the bacteriophage T7 and T3 DNA packaging pathway in Escherichia coli mutant TSN B.

Data previously obtained indicate that, during assembly of the related bacteriophages T7 and T3, a DNA-free procapsid (capsid I) is produced and that subsequently capsid I: (1) binds to a longer than mature (concatemeric) DNA and then becomes structurally altered to a particle isolated as a capsid (capsid II) physically resembling the mature bacteriophage capsid more than the procapsid (initiation phase of packaging), (2) draws DNA to its interior (entry phase of packaging), (3) participates in cutting the concatemeric DNA to mature size. It was found that, after infection of Escherichia coli mutant tsnB (selected for a deficiency in plating T7; M. Chamberlin [1974], J. Virol. 14, 509-516), T7 and T3 capsid I is assembled at a rate not significantly different from its rate of assembly in the wild-type host. However, the conversion of capsid I to capsid II was slowed in E. coli tsnB, suggesting that the tsnB mutation interferes with the initiation of DNA packaging. Although some T3 and T7 DNA enters capsids and is cut to mature size in the tsnB mutant, the data further suggest that the entry rate of DNA into capsid II is lower in the tsnB mutant than it is in an unaltered host. T7 capsid II-concatemeric DNA complexes accumulate during infection of the tsnB mutant. These observations suggest that use of the tsnB mutant as a host will simplify studies of bacteriophage T7 and T3 DNA packaging.

Capsid↗
PubMed1985

Two-dimensional agarose gel electrophoresis without gel manipulation.

The apparatus and procedure to perform two-dimensional agarose gel electrophoresis without manipulating the gel used for the first electrophoresis (first-dimension gel) have been developed. The procedure is less complex, less damaging to first-dimension gels, and more precise than procedures that require manipulation of the first-dimension gel. When combined with gel-embedding techniques, the procedure presented can be used to perform the second electrophoresis in a gel different from the first-dimension gel. A first-dimension gel too dilute to be manipulated and a more concentrated gel for the second electrophoresis have been used to separate DNA open circles from a mixture of variable-length linear DNAs.

DNA↗
PubMed1985

Heterogeneity of the procapsid of bacteriophage T3.

Like other double-stranded DNA bacteriophages, bacteriophage T3 assembles a DNA-free procapsid that subsequently packages the bacteriophage DNA. By agarose gel electrophoresis, it has been found that the T3 procapsid has a negative electrophoretic mobility (mu) that progressively increases in magnitude by as much as 3% after assembly of the procapsid. This increase is (i) caused by an increase in the solid support-free mu (muo) of the procapsid, not a decrease in its radius, and (ii) not prevented by either genetically or chemically (use of proflavine) blocking DNA packaging. However, inhibition of the formation of high-energy compounds with a mixture of cyanide and fluoride ions does block the time-dependent increase in the magnitude of muo. This increase appears to be accompanied by addition of an unidentified T3 protein to the procapsid.

Capsid↗
PubMed1984

Conformation of double-stranded DNA during agarose gel electrophoresis: fractionation of linear and circular molecules with molecular weights between 3 X 10(6) and 26 X 10(6).

To answer several questions concerning the mechanisms of DNA fractionation during agarose gel electrophoresis, the electrophoretic mobility (mu) of double-stranded DNA has been measured as a function of (1) DNA topological conformation (linear, open circular, closed circular) and molecular weight (Mr) (molecular weights were between 2.9 X 10(6) and 26.4 X 10(6)), (2) gel concentration (A) and temperature, and (3) voltage gradient. It was found that mu extrapolated to an A of 0 (mu 0') was independent of DNA conformation. The effect of temperature was to raise values of mu 0' in inverse proportion to buffer viscosity. Semilogarithmic mu vs. A plots for linear DNAs had curvature that was opposite to the curvature for spherical particles (plots for linear DNA were concave). As A approached 0, the plots became increasingly linear. For the larger DNAs, the negative slope (KR) in the region of linearity was decreased as voltage gradient increased. These and other data indicate deformation of linear DNA random coils during agarose gel electrophoresis. The data suggest both an asymmetric and a symmetric collapse of linear DNA random coils during agarose gel electrophoresis. However, end-first migration of linear DNA, previously suggested by others, does not explain the data. The semilogarithmic mu vs. A plots were more linear for closed and open circular DNAs than they were for linear DNAs. Closed circular DNAs had KR's lower than KR's of either open circular or linear DNAs of the same molecular weight. At the lower voltage gradients, open circular DNA had the same KR as linear DNA of the same molecular weight. However, as voltage gradient and molecular weight increased, the KR of open circular DNA became smaller than the KR of linear DNA (of the same molecular weight). This and the concave curvature of semilogarithmic mu vs. A plots for linear DNA resulted in a previously unreported reversal of the relative migration of linear and open circular DNAs as A increased.

DNA, Circular↗
PubMed1984

Bacteriophage P22 capsids with a subgenome length of packaged DNA.

Bacteriophage P22 assembles a DNA-free procapsid that subsequently packages P22 DNA. To study the packaging of bacteriophage P22 DNA, attempts were made to isolate P22 capsids with a subgenome length of packaged DNA. With the use of cesium chloride buoyant density sedimentation and agarose gel electrophoresis, the following capsids with a subgenome length of packaged DNA were isolated and characterized: (i) a capsid with the solid-support-free electrophoretic mobility and radius of the DNA-free P22 procapsid; (ii) a capsid with the solid-support-free electrophoretic mobility and radius of the mature P22 bacteriophage; and (iii) a capsid with a solid-support-free electrophoretic mobility and possibly a radius intermediate to those of the procapsid and bacteriophage.

Bacteriophages↗
PubMed1983

Comparison of the physical properties and assembly pathways of the related bacteriophages T7, T3 and phi II.

To understand constraints on the evolution of bacteriophage assembly, the structures, electrophoretic mobilities (mu) and assembly pathways of the related double-stranded DNA bacteriophages T7, T3 and phi II, have been compared. The characteristics of the following T7, T3 and phi II capsids in these assembly pathways have also been compared: (1) a DNA-free procapsid (capsid I) that packages DNA during assembly; (b) a DNA packaging-associated conversion product of capsid I (capsid II). The molecular weights of the T3 and phi II genomes were 25.2 X 10(6) and 25.9 (+/- 0.2) X 10(6) (26.44 X 10(6) for T7, as previously determined), as determined by agarose gel electrophoresis of intact genomes. The radii of T7, T3 and phi II bacteriophages were indistinguishable by sieving during agarose gel electrophoresis (+/- 4%) and measurement of the bacteriophage hydration (+/- 2%) (30.1 nm for T7, as previously determined). Assuming a T = 7 icosahedral lattice for the arrangement of the major capsid subunits (p10A) of T7, T3 and phi II best explains these data and data previously obtained for T7. At pH 7.4 and an ionic strength of 1.2, the solid-support-free mu values (mu 0 values) of T7, T3 and phi II bacteriophages, obtained by extrapolation of mu during agarose gel electrophoresis to an agarose concentration of 0 and correction for electro-osmosis, were -0.71, -0.91 and -1.17(X 10(-4) cm2V-1 s-1. The mu 0 values of T7, T3 and phi II capsids I were -1.51, -1.58 and -2.07(X 10(-4] cm2V-1 s-1. For the capsids II, these mu 0 values were -0.82, -1.07 and -1.37(X 10(-4] cm2V-1 s-1. The tails of all three bacteriophages were positively charged and the capsid envelopes (heads) were negatively charged. In all cases the procapsid had a negative mu 0 value larger in magnitude than the negative mu 0 value for bacteriophage or capsid II. A trypsin-sensitive region in capsid I-associated, but not capsid II-associated, T3 p10A was observed (previously observed for T7). The largest fragment of trypsinized capsid I-associated p10A had the same molecular weight in T7 and T3, although the T3 p10A is 18% more massive than the T7 p10A. It is suggested that the trypsin-resistant region of capsid I-associated p10A determines the radius of the bacteriophage capsid.

Capsid↗