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K W Adolph

Publications and source records attributed to K W Adolph.

At least 37 records · Page 2Linked to original sources
PubMed1983

ADP-ribosylation of nonhistone proteins during the HeLa cell cycle.

ADP-ribosylation of nonhistone proteins during the HeLa cell cycle was investigated. Proteins were radiolabeled by incubating interphase nuclei and mitotic cells with the specific precursor, [32P]NAD. Autoradiograms of two-dimensional gels of total nuclear nonhistone proteins showed a large number of modified species (more than 140). A complex pattern was also found for interphase chromatin. Nuclear scaffolds showed a simpler pattern of four major groups of modified species, which appeared to be the lamins and poly(ADP-ribose) polymerase. The labeling pattern for nonhistones of metaphase chromosomes was fundamentally different than with interphase nuclei. Autoradiograms were dominated by the incorporation of label into poly(ADP-ribose) polymerase.

Adenosine Diphosphate Ribose↗
PubMed1983

Phosphorylation of nonhistone proteins during the HeLa cell cycle. Relationship to DNA synthesis and mitotic chromosome condensation.

Cell cycle variations in the phosphorylation of chromatin-associated nonhistones were determined. Cells were radiolabeled with [32P]orthophosphate and chromatin was obtained by mild digestion of nuclei with micrococcal nuclease. The experiments were performed in the presence of a substrate inhibitor of alkaline phosphatase, beta-glycerophosphate. The results show that, while similar molecular weight species of phosphorylated nonhistones are associated with interphase chromatin through the HeLa cell cycle, the incorporation (32P cpm/micrograms of protein) profiles of selected major phosphononhistones show substantial changes. The most prominent peaks of specific radioactivity occur in the DNA synthesis phase (S phase). The phosphorylation states of the proteins of isolated metaphase chromosomes were also determined. Nonhistone proteins of isolated metaphase chromosomes are strikingly dephosphorylated, especially in comparison to histone H1. The phosphorylation of the major phosphononhistone of chromatin, which has a molecular weight of 55,000, was further characterized by techniques that included one-dimensional peptide mapping in sodium dodecyl sulfate-polyacrylamide gels and nonequilibrium pH gradient slab gel electrophoresis. Phosphoproteins are also components of the nuclear scaffold, and cell cycle variations in these proteins were investigated. The primary phosphorylated species has a molecular weight of 119,000. As with chromatin-associated nonhistones, this nuclear scaffold protein shows substantial incorporation of 32P in S phase, and a high level of incorporation also occurs close to mitosis.

Cell Cycle↗
PubMed1983

Surface structure of isolated metaphase chromosomes.

The relationship between the surface protuberances of mitotic chromosomes isolated from HeLa cells and the underlying fiber organization was investigated by scanning electron microscopy (SEM). Chromosomes were isolated in the presence of 5.0 mM Mg2+ by a method which avoids the use of organic solvents and extremes of pH. Chromosomes in 5.0 mM Mg2+ are highly condensed with a relatively smooth surface structure. In 1.5 mM Mg2+, a knobby surface substructure became apparent, with the protuberances having a mean diameter of 691 +/- 96 A. The diameter was 647 +/- 76 A at a magnesium concentration of 0.5 mM, but was only 349 +/- 52 A at a concentration of 0.15 mM. In 0.05 mM Mg2+, the mean diameter had decreased to 299 +/- 47 A and the chromosomes had expanded such that the underlying fibers had become a predominant feature of the micrographs. The knobby appearance of the chromosomes most likely reflects a radial arrangement of the fibers, which loop back at the peripheries of the chromosomes.

Chromosomes, Human↗
PubMed1983

Occurrence of diphthamide in archaebacteria.

We examined the nature of the diphtheria toxin fragment A recognition site in the protein synthesis translocating factor present in cell-free preparations from the archaebacteria Thermoplasma acidophilum and Halobacterium halobium. In agreement with earlier work (M. Kessel and F. Klink, Nature (London) 287:250-251, 1980), we found that extracts from these organisms contain a protein factor which is a substrate for the ADP-ribosylation reaction catalyzed by diphtheria toxin fragment A. However, the rate of the reaction was approximately 1,000 times slower than that typically observed with eucaryotic elongation factor 2. We also demonstrated the presence of diphthine (the deamidated form of diphthamide, i.e., 2-[3-carboxyamide-3-(trimethylammonio)propyl]histidine) in acid hydrolysates of H. halobium protein in amounts comparable to those found in hydrolysates of similar preparations from eucaryotic cells (Saccharomyces cerevisiae and HeLa). Diphthine could not be detected in hydrolysates of protein from the eubacterium Escherichia coli. Whereas both archaebacterial and eucaryotic elongation factors contain diphthamide, they differ importantly in other respects.

Adenosine Diphosphate Ribose↗
PubMed1982

Role of non-histones in chromosome structure. Cell cycle variations in protein synthesis.

As part of a study of the role of non-histone proteins in chromosome structure, the synthesis of non-histones associated with interphase chromatin was investigated. Synchronized suspension cultures of HeLa cells were pulse-labeled with [35S]methionine, and chromatin was prepared by mild micrococcal nuclease digestion. Two-dimensional polyacrylamide gel electrophoresis, in addition to one-dimensional electrophoresis, was used to resolve the patterns of incorporation of radioactive label. Significant variations in non-histone synthesis were seen during the cell cycle. A strong correlation was not found between DNA synthesis in mid-S phase and variations in non-histone synthesis. The non-histone proteins of purified metaphase chromosomes were also characterized by two-dimensional gel electrophoresis and compared to the proteins of interphase chromatin. The pattern of non-histones is not identical with that of interphase chromatin, although a number of major species may be shared by interphase chromatin and metaphase chromosomes. The HeLa nuclear scaffold, the framework that maintains the overall morphology of the interphase nucleus, shows relatively few proteins on two-dimensional gels. The synthesis of nuclear scaffold proteins was quantitated by excising each of 19 proteins from two-dimensional gels and determining the incorporated radioactivity by scintillation counting. Substantial variations in protein synthesis were found, with several species showing changes of about 2-fold in the percentage of incorporation.

Cell Cycle↗
PubMed1981

A serial sectioning study of the structure of human mitotic chromosomes.

The organization of chromosomes in mitotic HeLa cells was investigated by serial sectioning and electron microscopy. Mitotic HeLa cells were resuspended in a hypotonic buffer containing a magnesium ion concentration of 1.0 to 1.5 mM prior to fixation and sectioning. By treating the cells in this way, most of the material adjacent to the chromosomes were removed, and the chromosomes were slightly expanded, thereby separating the chromatin fibers and allowing their arrangement to be observed. Gazing sections that cut across the chromatids longitudinally and transversely are especially informative. The typical pattern of radially oriented fibers which is found for sections through the body of the chromatids is replaced by a dot pattern. The array of dots, which is almost exclusively found as the sections approach the chromosome boundary, must result from the sectioning knife cutting across chromatin strands. Longitudinal and transverse sections both show the transition from radial fibers to dots and demonstrate that chromosome organization is the same around the chromatid arms. As was previously found with random sections [1], consecutive, transverse sections through the body of the chromatid arms show the primary mode of organization to be a radial distribution of fibers. These consecutive sections firmly establish that this characteristic feature of chromosome organization extends through the particles and is not merely a feature of occasional, untypical sections. Serial sections that intersect the body of the particles approximately parallel to the chromatid arms further demonstrate that the fundamental orientation of the fibers is radial and extends uniformly through and along the chromatids.

Chromatids↗
PubMed1980

Isolation and structural organization of human mitotic chromosomes.

New methods are presented for the bulk isolation of metaphase chromosomes from HeLa cells, and an electron microscopic study of thin sections of these chromosomes is presented. The techniques for chromosome isolation were developed to utilize solution conditions that are as mild as possible, so that further biochemical and structural studies can be directly related to the in situ state of chromosomes. - Electron micrographs of thin sections of isolated HeLa metaphase chromosomes reveal the general organization of the nucleosome-containing fibers. Chromosomes in isolation buffer show a dense, relatively uniform distribution of material across the chromatids. Swollen chromosomes reveal the primary mode of organization of the fibers to be a radial distribution from the central axes of the chromatids. A significant proportion of the fibers could also be oriented longitudinally.

Chromosomes, Human↗
PubMed1980

Organization of chromosomes in HeLa cells: isolation of histone-depleted nuclei and nuclear scaffolds.

Histone-depleted nuclei were prepared from isolate HeLa nuclei by extracting the histones and other proteins with polyanions (dextran sulphate and heparin) or with high salt concentrations as used previously. The particles were characterized by sucrose density gradient sedimentation, thin sectioning and electron microscopy, and by polyacrylamide gel electrophoresis. The general result of the experiments is that the DNA in the histone-depleted nuclei is highly organized, and that this residual, higher-order structure is maintained by a reproducible subset of nuclear proteins, and perhaps by RNA. Furthermore, the residual proteins remain associated, in some conditions, as rapidly sedimenting structures even when the DNA is digested with nucleases. These nuclear scaffolds can resemble extracted nuclei. Histone-depleted HeLa nuclei sediment in sucrose density gradients as well defined peaks with sedimentation coefficients of around 12 000 S, when 2M NaCl is used to extract the histones, or 6 000 S, when dextran sulphate is used. The rate of sedimentation is drastically decreased by treating the particles with trypsin, and reduced to a lesser extent with RNase A. Thin sectioning and electron microscopy show that histone-depleted nuclei possess the nuclear periphery and that internal material is also present. These general features are also seen in thin sections of nuclear scaffolds, which are prepared by treating the nuclei with micrococcal nuclease of DNase I in addition to extracting the histones. Two groups of major proteins are associated with histone-depleted HeLa nuclei and the nuclear scaffolds: One group has molecular weights of 50 000-55 000 Daltons. The major species of this latter group of proteins have mobilities that are similar to the proteins of the metaphase chromosomal scaffold.

Cell Nucleus↗
PubMed1979

Polyoma virion and capsid crystal structures.

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.

Crystallization↗
PubMed1977

Isolation of a protein scaffold from mitotic HeLa cell chromosomes.

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.

Centrifugation, Density Gradient↗
PubMed1976

Assembly of a spherical plant virus.

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.

Capsid↗
PubMed1975

The conformation of the RNA in cowpea chlorotic mottle virus: dye-binding studies.

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.

Acridines↗