Biomedical subjects
Publications and source records attributed to K W Adolph.
X-ray diffraction shows that complete virus particles and empty capsids crystallize isomorphously. The surface morphology of the protein coat, as revealed by electron microscopy, is the dominant structural feature determining the intensity of x-ray reflections to a resolution of approximately 30 angstroms. The structure and variability of the viral chromatin core can now be analyzed by comparison of electron density maps.
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We have recently shown that, after the histones and most of the nonhistone proteins are gently removed from HeLa metaphase chromosomes, the chromosomal DNA is still highly organized and relatively compact. The structure of these histone-depleted chromosomes is due to the presence of a number of nonhistone proteins that form a central scaffold that retains the approximate size and shape of intact chromosomes and to which the DNA is attached, predominantly forming loops. We now demonstrate that the protein scaffold may be isolated independently of the DNA by treating HeLa chromosomes with micrococcal nuclease before removing the histones.The chromosomal scaffolds may be isolated by sucrose density gradient centrifugation as a well-defined peak that is stable in 2 M sodium chloride, but is dissociated by treatment with proteases, 4 M urea, or 0.1% sodium dodecyl sulfate. Polyacrylamide gel electrophoresis reveals that the protein content of scaffold preparations is identical to that of histone-depleted chromosomes. Fluorescence microscopy of purified scaffolds in isolation buffer shows that the particles still possess the familiar chromosome morphology. When the scaffolds are examined in the electron microscope, a fibrous structure with the approximate size and shape of intact, paired chromatids is seen. Less than 0.1% of the chromosomal DNA and virtually no histones are associated with the purified scaffold structures.
The conditions previously reported as necessary for the reassembly of spherical viruses have been distinctly unphysiological and such reassembly cannot be related directly to the in vivo reaction. Mild conditions for the in vitro reassembly of cowpea chlorotic mottle virus (CCMV) from its isolated components have now been described (Adolph & Butler 1975) and the reassembled virus characterized. This reassembly involved the co-aggregation of the RNA and protein around neutrality and at ionic strength 0.2, giving yields of 70% encapsidation at pH 6.0. The reaction was independent of temperature over the range 5-25 degrees C and did not require the presence of Mg2+ ions. The reassembled virus shows a stability similar to that of native CCMV, with the same change in sedimentation coefficient around pH 6.5. The molecular mass and buoyant density in CsCl are also the same as those of native CCMV, while the electron microscope reveals a surface morphology on the reassembled particles like that on native CCMV. Analysis of the number-average, mass-average, and Z-average molecular masses of the purified protein at both pH 6.0 and pH 7.5 suggests that the active unit for reassembly is a dimer of the protein subunit.
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The binding of the dye acridine orange to cowpea chlorotic mottle virus (CCMV) and its purified RNA has been studied to obtain the number of dye-binding sites as a function of pH and, through further analysis, to estimate the degree of RNA secondary structure in situ. Acridine organe does not bind to CCMV protein and so the dye binding directly reflects the accessibility and structure of the RNA. The number of dye molecules per nucleotide which can be bound by native virus (pH 4.5, I = 0.1 buffer) is 0.13-0.18, the precise value depending upon the assumption of either heterogeneous binding sites or weak binding forces. The number of binding sites increases by a factor of about 2.7 to 0.34-0.48 when the pH is raised to pH 7.5 and "swelling" of the virus occurs. About 50% of the sites on the free RNA are available to bind dye in the swollen virus. The stacking coefficient, which is a measure of the degree of base pairing in a polynucleotide, has been calculated for the native and swollen virus and for the isolated RNA. The values of the stacking coefficient for the RNA in the virus and following extraction are comparable, which suggests that the structure of the RNA in both cases is similar, and the values are low in magnitude, which indicates the existence of extensive regions of double-helix.