Biomedical subjects

Dwight L Anderson

Publications and source records attributed to Dwight L Anderson.

7 recordsLinked to original sources

PubMed2006

Structural changes of bacteriophage phi29 upon DNA packaging and release.

Cryo-electron microscopy three-dimensional reconstructions have been made of mature and of emptied bacteriophage phi29 particles without making symmetry assumptions. Comparisons of these structures with each other and with the phi29 prohead indicate how conformational changes might initiate successive steps of assembly and infection. The 12 adsorption capable 'appendages' were found to have a structure homologous to the bacteriophage P22 tailspikes. Two of the appendages are extended radially outwards, away from the long axis of the virus, whereas the others are around and parallel to the phage axis. The appendage orientations are correlated with the symmetry-mismatched positions of the five-fold related head fibers, suggesting a mechanism for partial cell wall digestion upon rotation of the head about the tail when initiating infection. The narrow end of the head-tail connector is expanded in the mature virus. Gene product 3, bound to the 5' ends of the genome, appears to be positioned within the expanded connector, which may potentiate the release of DNA-packaging machine components, creating a binding site for attachment of the tail.

Bacillus Phages↗

PubMed2006

Determinants of bacteriophage phi29 head morphology.

Bacteriophage phi29 requires scaffolding protein to assemble the 450 x 540 A prolate prohead with T = 3 symmetry end caps. In infections with a temperature-sensitive mutant scaffolding protein, capsids assemble predominantly into 370 A diameter isometric particles with T = 3 symmetry that lack a head-tail connector. However, a few larger, 430 A diameter, particles are also assembled. Cryo-electron microscopy shows that these larger particles are icosahedral with T = 4 symmetry. The prolate prohead, as well as the two isometric capsids with T = 3 and T = 4 symmetry, are composed of similar pentamers and differently skewed hexamers. The skewing of the hexamers in the equatorial region of proheads and in the T = 4 isometric particles reflects their different environments. One of the functions of the scaffolding protein, present in the prohead, may be to stabilize skewed hexamers during assembly.

Bacillus subtilis↗

PubMed2005

Mechanism of force generation of a viral DNA packaging motor.

A large family of multimeric ATPases are involved in such diverse tasks as cell division, chromosome segregation, DNA recombination, strand separation, conjugation, and viral genome packaging. One such system is the Bacillus subtilis phage phi 29 DNA packaging motor, which generates large forces to compact its genome into a small protein capsid. Here we use optical tweezers to study, at the single-molecule level, the mechanism of force generation in this motor. We determine the kinetic parameters of the packaging motor and their dependence on external load to show that DNA translocation does not occur during ATP binding but is likely triggered by phosphate release. We also show that the motor subunits act in a coordinated, successive fashion with high processivity. Finally, we propose a minimal mechanochemical cycle of this DNA-translocating ATPase that rationalizes all of our findings.

Adenosine Diphosphate↗

PubMed2005

Conservation of the capsid structure in tailed dsDNA bacteriophages: the pseudoatomic structure of phi29.

Bacteriophage phi29 is one of the smallest and simplest known dsDNA phages, making it amenable to structural investigations. The three-dimensional structure of a fiberless, isometric variant has been determined to 7.9 A resolution by cryo-electron microscopy (cryo-EM), allowing the identification of alpha helices and beta sheets. Their arrangement indicates that the folds of the phi29 and bacteriophage HK97 capsid proteins are similar except for an additional immunoglobulin-like domain of the phi29 protein. An atomic model that incorporates these two domains fits well into the cryo-EM density of the T = 3, fiberless isometric phi29 particle, and cryo-EM structures of fibered isometric and fiberless prolate prohead phi29 particles at resolutions of 8.7 A and 12.7 A, respectively. Thus, phi29 joins the growing number of phages that utilize the HK97 capsid structure, suggesting that this protein fold may be as prevalent in capsids of dsDNA phages as the jelly roll fold is in eukaryotic viruses.

Bacillus Phages↗

PubMed2005

Crystallization and initial X-ray diffraction studies of scaffolding protein (gp7) of bacteriophage phi29.

The Bacillus subtilis bacteriophage phi29 scaffolding protein (gp7) has been crystallized by the hanging-drop vapour-diffusion method at 293 K. Two new distinct crystal forms that both differed from a previously crystallized and solved scaffolding protein were grown under the same conditions. Form I belongs to the primitive tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 77.13, c = 37.12 A. Form II crystals exhibit an orthorhombic crystal form, with space group C222 and unit-cell parameters a = 107.50, b = 107. 80, c = 37.34 A. Complete data sets have been collected to 1.78 and 1.80 A for forms I and II, respectively, at 100 K using Cu Kalpha X-rays from a rotating-anode generator. Calculation of a VM value of 2.46 A3 Da(-1) for form I suggests the presence of one molecule in the asymmetric unit, corresponding to a solvent content of 50.90%, whereas form II has a VM of 4.80 A3 Da(-1) with a solvent content of 48.76% and two molecules in the asymmetric unit. The structures of both crystal forms are being determined by the molecular-replacement method using the coordinates of the published crystal structure of gp7.

Bacillus Phages↗

PubMed2003

Calcium hypochlorite as a disinfecting additive for dental stone.

STATEMENT OF PROBLEM: Dental casts come into direct contact with impression materials and other items that are contaminated by saliva and blood from a patient's mouth, leaving the casts susceptible to cross-contamination. Topical methods of disinfecting casts are difficult to control, while immersion methods are potentially destructive. Thus, an additional method to control cross-contamination between patients and laboratory personnel is needed. PURPOSE: This study was undertaken in an attempt to develop a dental stone with disinfecting properties and adequate compressive and tensile strengths. MATERIAL AND METHODS: Calcium hypochlorite [Ca(OCl)(2)] in aqueous solution in concentrations from 0 to 1.5% was tested as a disinfecting additive to type V dental stone. The compressive and tensile strength properties of the modified stone were measured (MPa) using a universal testing machine at a consistency similar to unmodified stone. Strength data were analyzed by 1-way ANOVA and post hoc Tukey-Kramer procedure (alpha < or =.05). To measure the disinfecting ability, the effect on Bacillis subtilis bacteriophage phi29 was tested in triplicate to find the minimum concentration at which no phage was detected. Additionally, 3 impressions were disinfected with CaviCide, and 3 impressions rinsed in water served as controls. RESULTS: In general, the effect of adding the disinfectant to the stone was a decrease in strength. Exceptions were the dry compressive strength, for which there was a significant increase in strength (P=.048) at 0.5%, and the wet compressive and wet tensile strength, which showed no significant difference between the 1.5% and the control. When Ca(OCl)(2) was added at the concentration 0.5% (2765 ppm available chlorine), the gypsum had acceptable mechanical properties; dry compressive strength was 78.86 +/- 4.12 MPa, and dry tensile strength was 10.64 +/- 1.27 MPa, compared to control values of 67.85 +/- 6.28 and 13.41 +/- 1.24 MPa, respectively. At concentrations of 0.3% and higher (36 1650 ppm of available chlorine), calcium hypochlorite was able to completely inactivate phi29. CONCLUSION: It is possible to prepare a type V dental stone that contains a disinfectant, has adequate mechanical properties, and will reduce numbers of residual microorganisms. For example, stone mixed with water containing 0.5% Ca(OCl)(2) meets these criteria.

Analysis of Variance↗

PubMed2003

Bacteriophage phi29 scaffolding protein gp7 before and after prohead assembly.

Three-dimensional structures of the double-stranded DNA bacteriophage phi29 scaffolding protein (gp7) before and after prohead assembly have been determined at resolutions of 2.2 and 2.8 A, respectively. Both structures are dimers that resemble arrows, with a four-helix bundle composing the arrowhead and a coiled coil forming the tail. The structural resemblance of gp7 to the yeast transcription factor GCN4 suggests a DNA-binding function that was confirmed by native gel electrophoresis. DNA binding to gp7 may have a role in mediating the structural transition from prohead to mature virus and scaffold release. A cryo-EM analysis indicates that gp7 is arranged inside the capsid as a series of concentric shells. The position of the higher density features in these shells correlates with the positions of hexamers in the equatorial region of the capsid, suggesting that gp7 may regulate formation of the prolate head through interactions with these hexamers.

Bacillus Phages↗