Biomedical subjects

S J Hayes

Publications and source records attributed to S J Hayes.

At least 37 records · Page 2Linked to original sources

PubMed1997

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.

Bacteriophage T3↗

PubMed1997

Late failure of root canal therapy: a diagnostic and treatment planning challenge. Case report.

This report describes a case in which failure of conventional root canal therapy presented 17 years following initial treatment. The apical pathology was curetted, the tooth root resected and a root-end filling placed. The possible reasons for failure of the initial orthograde root canal therapy are discussed with the conclusion that the cause may not have been addressed fully by surgical intervention. The case highlights the challenge facing clinicians when assessing failed root fillings, not only in determining the exact cause of failure but deciding the appropriate treatment for the individual tooth and patient.

Adult↗

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

Ploidy analysis on Wilms' tumour touch imprints using ethidium bromide and automated image analysis integrated confocal laser scanning microscopy.

Although image analysis (IA) is increasingly being used to quantitate nuclear DNA, comparative data between fluorescence methods of IA and flow cytometry (FCM) is limited. In this study fluorescence IA was compared with FCM data in a series of Wilms' tumour touch preparations. Airdried touch imprints that had previously been Giemsa stained were restained with ethidium bromide. Confocal fluorescence images were obtained with a confocal laser scanning microscope and assessed by a fully automated IA package. Data was collected from 400 nuclei per imprint. The resulting DNA histograms were analysed and ploidy status and DNA indices determined using standard criteria. Results were compared with those derived from FCM analysis of nuclear suspensions. Ten of twelve tumours were concordant by both techniques. However in two cases assessed as diploid by FCM, IA identified aneuploidy. Excellent correlation between DNA indices as assessed by both techniques was observed (r = 0.987). In the three cases for which both unstained and Giemsa stained touch imprints were available for IA, the histogram configurations did not differ significantly. Fluorescence IA is an accurate and sensitive technique for DNA quantitation, which appears at least comparable to FCM assessment and which has a number of important advantages.

Child, Preschool↗

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↗

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↗

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

Conformation of DNA packaged in bacteriophage T7. Analysis by use of ultraviolet light-induced DNA-capsid cross-linking.

The conformation of the linear, double-stranded, 39,936 kilobase-pair DNA packaged in the protein capsid of bacteriophage T7 is investigated here by use of short wavelength ultraviolet light-induced DNA-capsid cross-linking. To detect both DNA-capsid and DNA-DNA cross-links, DNA is expelled from the T7 capsid and the products of expulsion are analyzed by use of Nycodenz buoyant density centrifugation, followed by either pulsed field gel electrophoresis or invariant field gel electrophoresis. Short wavelength ultraviolet light is found to progressively induce both DNA-DNA and DNA-protein cross-links in intact bacteriophage T7, but not in T7 from which DNA had been expelled before exposure to ultraviolet light. Protein-protein cross-links are not induced. When DNA expelled from previously cross-linked T7 is cleaved with restriction endonuclease (1 to 3 sites cleaved), analysis of the resulting fragments reveals no regions on T7 DNA that are excluded from cross-linking to the capsid. However, the efficiency of cross-linking decreases as the distance from the left end (last end packaged) of the packaged DNA increases. Electron microscopy of negatively stained capsid-DNA complexes reveals no DNA-retaining structure other than the outer shell of the capsid. Together with previously reported data that indicate lack of protein-based specificity for ultraviolet light-induced cross-linking, these observations are interpreted by the assumptions that, within the limits of resolution of these experiments: (1) no region of packaged T7 DNA is excluded from contact with the outer shell of the T7 capsid; (2) the probability of contacting the outer shell decreases as the distance from the left end of packaged T7 DNA increases. Thus, T7 DNA packaging concentrates the last end packaged near the inner surface of the outer shell of the T7 capsid.

Capsid↗

PubMed1990

Cardiovascular effect and stimulus-dependent inhibition of superoxide generation from human neutrophils by tibenelast, 5,6-diethoxybenzo(b)thiophene-2-carboxylic acid, sodium salt (LY186655).

Tibenelast (LY186655), 5,6,-diethoxybenzo(b)thiophene-2-carboxylic acid, sodium salt, is an orally active anti-anaphylactic compound in guinea pigs, and has been shown to prevent bronchospasm in moderately severe asthmatic patients. Pharmacological studies with tibenelast demonstrated that it is a selective phosphodiesterase (PDE) inhibitor in that it is moderately active against the lung and stomach enzyme while being a very weak inhibitor of the heart enzyme. The compound was without cardiovascular effects at anti-anaphylactic doses. In contrast to theophylline, tibenelast did not have a direct inotropic effect in the cat papillary muscle system. The concentration that inhibited 50% of the enzymatic activity (IC50) for tibenelast was 20- to 30-fold lower for neutrophil PDE than for PDE of other tissues. It was 100 times more potent than aminophylline in inhibiting superoxide generation from platelet-activating factor (PAF)-primed polymorphonuclear leukocytes (PMNL) challenged with chemotactic factor, N-formyl-methionyl-leucyl-phenylalanine. However, tibenelast was less effective in the tumor necrosis factor-primed system, and did not inhibit superoxide generation during phagocytosis or when other soluble stimuli, such as phorbo-12-myristate-13-acetate or the calcium ionophore A23187, were used. Furthermore, tibenelast did not inhibit enzymes involved in arachidonic acid metabolism. These results suggest that tibenelast probably inhibits superoxide release from PMNL via a selective inhibition on PDE.

Animals↗

PubMed1990

Quantized viral DNA packaging revealed by rotating gel electrophoresis.

Two classes of missense mutations in the bacteriophage T4 gene coding for the major head protein produce phage with different length heads. The pt (petite) mutations produce phage with normal, intermediate, and isometric heads, whereas ptg (petite and giant) mutations also produce greatly elongated (giant) heads. DNA from petite, normal, and giant particles was clearly resolved by discontinuous rotating gel electrophoresis, and several new species of headful length DNA were found. These results confirm the idea that the major stop points for head length regulation are at Q = 13, 17, and 21, and also show that minor stop points exist at Q = 16, 18 and 20. The existence of these well-defined classes of DNA that correlate with capsid structure suggest that a structural relationship between the scaffold protein and the capsid protein determines head length and thus DNA length.

DNA, Viral↗

PubMed1989

Optimization of the in vitro packaging efficiency of bacteriophage T7 DNA: effects of neutral polymers.

The in vitro DNA packaging of several DNA bacteriophages is stimulated by the presence of neutral polymers. To optimize bacteriophage T7 DNA packaging and to understand the basis for optimization, the efficiency of T7 DNA packaging has been determined at completion, as a function of the type, molecular mass, and concentration of the polymer added. When the polymer used was polyethylene glycol (PEG) of 0.2, 0.6 or 12.6 kDa, the efficiency of DNA packaging reached maximum at an intermediate concentration of polymer. The osmotic pressure (Pos) at maximum efficiency was either in, or close to, the range of colloid Pos measured for the intact host cell. The optimum Pos increased as the size of the polymer used decreased. PEG-100 (of 0.1 kDa) did not stimulate in vitro T7 DNA packaging. Dextran of 10 kDa also stimulated packaging and produced maximum efficiency at a physiological Pos. The degree of stimulation increases as DNA packaging extract concentration decreases; stimulation by as much as two to three orders of magnitude is observed. The presence of added polymer reduces fluctuations in DNA packaging efficiency caused by variability in the concentration of DNA packaging extracts. For reproducible and high efficiency packaging, the dextran was more reliable than the PEGs, possibly because the Pos of the dextran solutions is less sensitive to polymer concentration than is the Pos of PEG solutions. The optimum concentration of dextran at completion was also the optimum at all times before completion.

Capsid↗

PubMed1989

Atypical sieving of open circular DNA during pulsed field agarose gel electrophoresis.

Pulsed field agarose gel (PFG) electrophoresis, originally used to improve the resolution by length of linear DNA [Cantor et al. (1988) Annu. Rev. Biophys. Biophys. Chem. 17, 287-304], is found here to cause atypical sieving of 48.5-97.0-kb open circular DNA. Two procedures of PFG electrophoresis are used: rotating gel electrophoresis with rotation of 2 pi radians [2 pi RGE; Serwer, P., & Hayes, S.J. (1989) Appl. Theor. Electrophor. (in press)] and field inversion gel electrophoresis [FIGE; Carle, G.F., Frank, M., & Olson, M. V. (1986) Science 232, 65-68]. During 2 pi RGE at 6 V/cm, the electrophoretic mobility (mu) of 48.5-kb open circular DNA increases in magnitude as agarose percentage (A) increases from 0.4 to 1.5. The sieving revealed by this mu vs A relationship is highly atypical (possibly unique) for any particle. The extent of this atypical sieving increases as electrical potential gradient, DNA length, and pulse time increase. In some cases a maximum is observed in a plot of mu's magnitude vs A. The mu of open circular lambda DNA is smaller in magnitude than the mu of equally long linear lambda DNA. Atypical sieving has also been observed by use of FIGE. As pulse times used during FIGE decrease below those achievable by 2 pi RGE, the progressive loss of circular DNA's atypical sieving is accompanied by both a dramatic increase in mu's magnitude at the lower A values and a decrease in mu's magnitude at the higher A values. At the lower A values, open circular DNA sometimes migrates more rapidly than linear DNA of the same length.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage lambda↗

PubMed1989

A new mode of rotating gel electrophoresis for fractionating linear and circular duplex DNA: the effects of electrophoresis during the gel's rotation.

Linear and circular DNA have been fractionated by use of rotating gel electrophoresis (RGE; RGE is a form of pulsed-field gel [PFG] electrophoresis) in a mode not previously described. This new mode consists of electrophoresis while repeating the following two-step process: rotating the gel by 2 pi radians, followed by holding the gel motionless. With this new mode of RGE: (1) The fractionation by length of linear DNA is greater than that achieved without changing the direction of the field. (2) The simultaneous separation of open circular DNA (48.5-97 Kb) from linear DNA (1-180 Kb) and fractionation by length of open circular DNA is achieved. These latter separations have not previously been achieved by a one-dimensional electrophoresis. The mechanism of linear DNA's sieving in the new mode appears different from that of all previously described procedures of PGF electrophoresis.

Bacteriophage lambda↗

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↗

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

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↗