Biomedical subjects

P Serwer

Publications and source records attributed to P Serwer.

At least 37 records · Page 2Linked to original sources

PubMed1996

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.

Borrelia↗

PubMed1995

Specific single-stranded breaks in mature bacteriophage T7 DNA.

Both rate zonal centrifugation and gel electrophoresis have revealed that the mature double-stranded DNA of bacteriophage T5 has single-stranded breaks (nicks) at specific sites. Neither of these procedures has previously revealed site-specific nicks in the double-stranded DNA of other bacteriophages, including T7. In the present study, denaturing gel electrophoresis, followed by specific DNA detection, reveals that a small fraction of mature T7 DNA molecules, like most T5 DNA molecules, has site-specific nicks. The procedure of specific detection is to probe with an oligonucleotide specific for one of the ends of T7 DNA. If position 0.0 is the left genetic end and position 100.0 is the right genetic end of T7 DNA, the nicks on the 5' left-oriented strand are at 11.3, 12.4, 65.7, 79.2, and 86.0; the nicks on the 5' right-oriented strand are at 23.3 and 26.5 (+/- 0.5). The positions of the three rightmost nicks are indistinguishable from those of double-stranded breaks that produce previously demonstrated shorter than mature length DNAs packaged in vivo. We propose that the T7 nicks are produced by premature activity of the T7 terminase during DNA packaging.

Bacteriophage T7↗

PubMed1995

Specific, nonproductive cleavage of packaged bacteriophage T7 DNA in vivo.

The morphogenesis of bacteriophage T7 includes assembly of a procapsid that subsequently both packages DNA and changes in structure. The DNA packaged by T7 is concatemeric and is cleaved to mature length during packaging. In the present study, packaged DNA obtained from T7-infected cells was analyzed after release from DNase-treated capsids. After fractionation by agarose gel electrophoresis, in-gel probing with oligonucleotides reveals that some of this DNA is shorter than mature T7 DNA; most of this short DNA has the T7 right end, but not the left end. Some of this short, packaged DNA is the product of left-to-right injection of DNA at the beginning of a T7 infection. However, subsequently produced short, packaged DNA has characteristics of a DNA that was produced during DNA packaging (incompletely packaged DNA or ipDNA). In contrast to results previously obtained in vitro, the profile of right-end-containing ipDNA is sometimes dominated by discrete bands. Some of the band-forming right-end-containing ipDNA appears with the kinetics of an abortive end product of packaging; cleavage in vivo appears to have arrested DNA packaging in this case. Other band-forming right-end-containing ipDNA appears with kinetics that have some characteristics expected of a precursor to the mature DNA; cleavage appears to have occurred after arrest of packaging in this case. The findings here of both left-to-right injection and right-to-left packaging is the most direct demonstration of polarity for these events in vivo.

Bacteriophage T7↗

PubMed1995

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. The procedures developed will be used for screening viral mutants; they also can potentially be used for diagnostic virology. Sensitivity of detection, the major current limitation, is being improved by use of both improved stains and scanning fluorimetry. The gels used for fractionation sometimes approximate random straight fiber gels, but become increasingly biphasic as the gel concentration is decreased.

Animals↗

PubMed1995

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.

Bacteriophages↗

PubMed1995

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.

DNA, Viral↗

PubMed1995

Gel electrophoretic analysis of bacteriophage assembly intermediates in bacteriophage plaques.

To increase the efficiency with which the phenotype of bacteriophage mutants is determined by gel electrophoresis, procedures are developed here for the preparation of the contents of bacteriophage plaques for gel electrophoresis. During the formation of plaques, the plaque-supporting upper layer gel is changed from the traditional agar gel to a gel made of a mixture of low-melt agaroses; the lower layer gel is eliminated. To extract particles from plaques, the plaque-supporting gel is disintegrated by both shaking and raising the temperature to 39-43 degrees C. During shaking, the gel is broken to domains that are 5-30 microns in diameter. After extraction, the contents of plaques are subjected to two electrophoretic analyses: (1) Nondenaturing agarose gel electrophoresis is performed after treatment with DNase. This procedure reveals both mature bacteriophage and immature capsids. (2) Nondenaturing agarose gel electrophoresis is performed after release of DNA from DNase-treated capsids. This latter procedure reveals both completely packaged (mature length) DNA and incompletely packaged (shorter than mature length) DNA. The amount of mature length DNA released per 2-3 mm plaque is 10-60 ng. In agreement with results previously obtained in liquid culture, most incompletely packaged DNA has the right, but not the left, mature T7 DNA end.

Bacteriophage T3↗

PubMed1994

Quantification of the effect of excluded volume on double-stranded DNA.

By steric exclusion of volume, neutral polymers both condense and increase the effective concentration of DNA random coils. Neutral polymers also stimulate the in vitro packaging of DNA in the capsids of some double-stranded DNA bacteriophages. In the present study, the physical effects of neutral polymers on DNA random coils have been quantified by assaying the DNA products at equilibrium of the following two reactions of the 12 nucleotide single-stranded complementary (cohesive) ends of mature bacteriophage lambda DNA: bimolecular joining of half-molecules of mature lambda DNA, and cyclization of intact lambda DNA; cyclization is used as a probe for unimolecular DNA condensation. The smaller neutral molecules, including polyethylene glycol of molecular mass 200 Da (PEG200), shift both reactions towards dissociation; this shift is partially correlated with reduced water activity. The larger PEGs (molecular mass of 1540 or more) shift both reactions towards association. Water activity-corrected equilibrium constants for the larger PEGs are found to increase as a function of PEG concentration. Below 2% to 3% (w/v) PEG, these equilibrium constants are independent of PEG molecular mass; at higher PEG concentrations, these equilibrium constants increase as the molecular mass of the PEG increases. The following conclusions are drawn. (1) Volume exclusion among PEG molecules is the primary cause of the PEG molecular mass-dependence of excluded volume. (2) At the lower PEG concentrations, the PEG radius obtained by quantification of excluded volume is usually equal to the hydrodynamic PEG radius. (3) At any given PEG concentration, the PEG-DNA excluded volume is approximately the same for bimolecular DNA joining as it is for unimolecular DNA cyclization. (4) Polymer-induced stimulation of in vitro bacteriophage DNA packaging is derived primarily from alteration of water activity, not alteration of excluded volume.

Bacteriophage T7↗

PubMed1994

Quantitative analysis of macromolecular conformational changes using agarose gel electrophoresis: application to chromatin folding.

Quantitative analysis of chromatin electrophoretic mobility (mu) in agarose gels provides a measure of three structural parameters: average surface electrical charge density, which is proportional to the gel-free mu (mu 0), effective radius (Re), and particle deformability [Fletcher, T. M., Krishnan, U., Serwer, P., & Hansen, J. C. (1994) Biochemistry 33, 2226-2233]. To determine whether the intramolecular conformational changes associated with salt-dependent chromatin folding influence these electrophoretic parameters, defined oligonucleosomes were reconstituted from monodisperse tandemly repeated 5S DNA and varying amounts of histone octamers. These oligonucleosomes were subjected to both quantitative agarose gel electrophoresis and analytical velocity ultracentrifugation in buffers containing 0-2 mM MgCl2. Ionic conditions that caused a 40% increase in the oligonucleosome sedimentation coefficient (s20,w) also caused both a 30% decrease in Re and a 60% decrease in the magnitude of the mu 0. Furthermore, the Mg(2+)-dependent changes in s20,w, Re, and mu 0 each exhibited the same nonlinear dependence on the degree of nucleosome saturation of the DNA. These data demonstrate that quantitative agarose gel electrophoresis can be used to detect and characterize the process of chromatin folding. In addition, they suggest that this approach can be used for characterization of the conformational dynamics of many other types of macromolecular assemblies, including those systems that are not yet amenable for study by more traditional quantitative biophysical techniques.

Animals↗

PubMed1994

Quantitative agarose gel electrophoresis of chromatin: nucleosome-dependent changes in charge, sharp, and deformability at low ionic strength.

The surface electrical charge density and the deformability of nucleosomal arrays have been characterized by quantitative agarose gel electrophoresis. Monodisperse linear DNA (2.5-3.3 kbp) was reconstituted with histone octamers into either saturated (approximately 1 nucleosome/200-bp DNA) or subsaturated (< 1 nucleosome/200-bp DNA) nucleosomal arrays. The electrophoretic mobility (mu) of both nucleosome-free DNA and nucleosomal arrays was determined at low ionic strength in 0.2-3.0% agarose gels. A semilogarithmic plot of mu vs gel concentration was linear for DNA and convex for saturated nucleosomal arrays. By extrapolating the mu to 0% agarose, the magnitude of the gel-free mu of saturated nucleosomal arrays was found to be approximately 20% lower than that of nucleosome-free DNA molecules. This difference is explained by the net neutralization of approximately 85 DNA negative charges by each histone octamer. By using an internal standard to measure the effective pore size (Pe) of the agarose gel, the effective radius (R) of DNA and nucleosomal arrays was determined at each agarose concentration. In the more dilute gels (Pe > or = 400 nm), the differences between the effective R values of DNA, subsaturated nucleosomal arrays, and saturated nucleosomal arrays are consistent with the differences in their hydrodynamic shapes as measured by analytical velocity centrifugation. However, as Pe decreases, the effective R of both nucleosome-free DNA and subsaturated nucleosomal arrays decreases significantly. This is in contrast to the effective R of saturated nucleosomal arrays, which remains constant at all Pe.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Pulsed field agarose gel electrophoresis in the study of morphogenesis: packaging of double-stranded DNA in the capsids of bacteriophages.

To understand how comparatively simple macromolecular components become biological systems, studies are made of the morphogenesis of bacteriophages. Pulsed field agarose gel electrophoresis (PFGE) has contributed to these studies by: (i) improving the length resolution of both mature, linear, double-stranded bacteriophage DNAs and the concatemers formed both in vivo and in vitro by the end-to-end joining of these mature bacteriophage DNAs, (ii) improving the resolution of circular conformers of bacteriophage DNAs, (iii) improving the resolution of linear single-stranded bacteriophage DNAs, (iv) providing a comparatively simple technique for analyzing protein-DNA complexes, and (v) providing a solid-phase quantitative assay for all forms of bacteriophage DNA; solid-phase assays are both less complex and more efficient than liquid-phase assays such as rate zonal centrifugation. Conversely, studies of bacteriophages have contributed to PFGE the DNA standards used for determining the length of nonbacteriophage DNAs. Among the solid-phase assays based on PFGE is an assay for excluded volume effects.

Bacteriophages↗

PubMed1993

Progress in developing improved programs for pulsed field agarose gel electrophoresis of DNA.

Details are described here for using a rotating gel to perform pulsed field agarose gel electrophoresis (PFGE) with programmable control of the following variables: magnitude of the electrical field, polarity of the electrical field, temperature of the gel and position of the rotating disk upon which the agarose gel rests. By use of this procedure for programmable control, modes of PFGE have been explored that have the following characteristics: (i) resolution by DNA length is completely lost for DNA shorter than a critical length that increases as the pulse times increase, and (ii) resolution by DNA length is enhanced for longer DNAs that are shorter than a second critical length. This window of resolution can be moved to the position of the 2-6 Mb chromosomes of Schizosaccharomyces pombe.

DNA↗

PubMed1993

The direction and rate of bacteriophage T7 DNA packaging in vitro.

To determine the direction of the entry of DNA during in vitro bacteriophage T7 DNA packaging, incompletely packaged DNA (ipDNA) was fractionated by agarose gel electrophoresis after degradation of DNA outside of capsids and then release of packaged DNA from capsids. After fractionation, quantitative in-gel probing with a right end-specific oligonucleotide detects heterogeneous ipDNA (called right-end ipDNA). Most of the right-end ipDNA appears with kinetics expected of a precursor to the mature T7 DNA. In-gel probing with a left-end-specific oligonucleotide detects ipDNA (left-end ipDNA); the molar amount of left end ipDNA is always at least 50x less than the molar amount of right-end ipDNA. Left-end ipDNA appears with the kinetics of an abortive end product of T7 DNA packaging. Thus, productive T7 DNA packaging occurs in a right-to-left direction. Quantitation of the conversion of right-end ipDNA to mature-length DNA yields an estimate of the mean rate of right-to-left in vitro T7 DNA packaging: 28 +/- 6 kbp/min for the last 20-50% of the DNA packaged.

Bacteriophage T7↗

PubMed1993

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. As the agarose concentration decreases below 1.5%, the semilogarithmic F versus Pc plot becomes progressively less linear. Two straight lines represent the data; the plot is steeper at the lower Pc values. As the percentage of agarose increases above 1.5%, the semilogarithmic F versus Pc plot becomes steeper at the higher Pc values. This change in the shape of semilogarithmic F versus Pc plots is possibly explained by the existence in agarose gels of two zones, one whose Pc distribution is more sensitive to the average agarose concentration than the other. To compare the structure of agarose gels to their sieving during electrophoresis, the root mean square value of Pc (Pc) is compared to the sieving-based radius of the effective pore (PE; Griess et. al. (16)) for both underivatized agarose and a derivatized agarose that has a smaller PE at any given agarose percentage. For 0.8-2.0% gels of either underivatized or derivatized agarose, PE/Pc is a constant within experimental error. Deviations from this constant are observed at lower gel percentages. This relationship of PE to Pc constrains theoretical descriptions of the motion of spheres in fibrous networks.

Biophysical Phenomena↗

PubMed1993

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 0.2% agarose gel at 22 degrees C: (a) When a comparatively high field (3.0 V cm-1) is used, inelastic collisions result in field-induced trapping of spheres; no elastic collisions are observed. (b) Reduction of the field from 3.0 to 0.0 V cm-1 results in reverse migration of previously trapped spheres. (c) In the absence of trapping, the electrical field does not cause an alteration in the tortuosity of motion (i.e. motion in a field-perpendicular direction). (d) When results are obtained for a constant time between images (0.2 s), gel-dependent deviations from a true random walk are not observed in the absence of trapping. (e) When results are obtained as a function of time between images, significant gel-dependent deviation from a random walk is observed. In the absence of trapping, the data presented here indicate that retardation is derived primarily from dissipative processes that are concentrated near gel fibers. However, steric effects have not yet been distinguished from hydrodynamic effects.

Buffers↗

PubMed1992

A small (58-nm) attached sphere perturbs the sieving of 40-80-kilobase DNA in 0.2-2.5% agarose gels: analysis of bacteriophage T7 capsid-DNA complexes by use of pulsed field electrophoresis.

Although the icosahedral bacteriophage T7 capsid has a diameter (58 nm) that is 234-fold smaller than the length of the linear, double-stranded T7 DNA, binding of a T7 capsid to T7 DNA is found here to have dramatic effects on the migration of the DNA during both pulsed field agarose gel electrophoresis (PFGE; the field inversion mode is used) and constant field agarose gel electrophoresis (CFGE). For these studies, capsid-DNA complexes were obtained by expelling DNA from mature bacteriophage T7; this procedure yields DNA with capsids bound at a variable position on the DNA. When subjected to CFGE at 2-6 V/cm in 0.20-2.5% agarose gels, capsid-DNA complexes arrest at the electrophoretic origin. Progressively lowering the electrical potential gradient to 0.5 V/cm results in migration; most complexes form a single band. The elevated electrical potential gradient (3 V/cm) induced arrest of capsid-DNA complexes is reversed when PFGE is used instead of CFGE. For some conditions of PFGE, the mobility of capsid-DNA complexes is a function of the position of the capsid on the DNA. During either CFGE (0.5 V/cm) or PFGE, capsid-DNA complexes increasingly separate from capsid-free DNA as the percentage of agarose increases. During these studies, capsid-DNA complexes are identified by electron microscopy of enzymatically-digested pieces of agarose gel; this is apparently the first successful electron microscopy of DNA from an agarose gel.(ABSTRACT TRUNCATED AT 250 WORDS)

Capsid↗

PubMed1992

Formation of the right before the left mature DNA end during packaging-cleavage of bacteriophage T7 DNA concatemers.

During bacteriophage T7 morphogenesis in a T7-infected cell, mature length T7 DNA molecules join end-to-end to form concatemers that are subsequently both packaged in the T7 capsid and cut to mature size. In the present study, the kinetics of the appearance in vivo of the mature right and left T7 DNA ends have been analyzed. To perform this analysis, the intercalating dye proflavine is used to interrupt DNA packaging. When used at 0.5 to 8.0 micrograms/ml, proflavine progressively inhibits events in the T7 DNA packaging pathway, without either altering protein synthesis or degrading intracellular T7 DNA. Restriction endonuclease kinetic analysis reveals that proflavine (8 micrograms/ml) completely blocks formation of the mature T7 DNA left end, but only partially blocks formation of the mature T7 DNA right end. Both these and other observations are explained by the hypothesis that, in the T7 DNA packaging pathway, events occur in the following sequence: (1) formation of a mature right end; (2) packaging of at least some of the genome; (3) formation of the mature left end.

Capsid↗

PubMed1992

Bacteriophage P22 portal protein is part of the gauge that regulates packing density of intravirion DNA.

The complex double-stranded DNA bacteriophages assemble DNA-free protein shells (procapsids) that subsequently package DNA. In the case of several double-stranded DNA bacteriophages, including P22, packaging is associated with cutting of DNA from the concatemeric molecule that results from replication. The mature intravirion P22 DNA has both non-unique (circularly permuted) ends and a length that is determined by the procapsid. In all known cases, procapsids consist of an outer coat protein, an interior scaffolding protein that assists in the assembly of the coat protein shell, and a ring of 12 identical portal protein subunits through which the DNA is presumed to enter the procapsid. To investigate the role of the portal protein in cutting permuted DNA from concatemers, we have characterized P22 portal protein mutants. The effects of several single amino acid changes in the P22 portal protein on the length of the DNA packaged, the density to which DNA is condensed within the virion, and the outer radius of the capsid have been determined. The results obtained with one mutant (NT5/1a) indicate no change (+/- 0.5%) in the radius of the capsid, but mature DNA that is 4.7% longer and a packing density that is commensurately higher than those of wild-type P22. Thus, the portal protein is part of the gauge that regulates the length and packaging density of DNA in bacteriophage P22. We argue that these findings make models for DNA packaging less likely in which the packing density is a property solely of the coat protein shell or of the DNA itself.

Base Sequence↗