Relationship of DNA tertiary and quaternary structure to carcinogenic processes.
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Chromatin↗
Biomedical subjects
R E Stephens
Publications and source records attributed to R E Stephens.
Specific localization of scallop gill epithelial calmodulin in cilia.
Calmodulin has been isolated and characterized from the gill of the bay scallop aequipecten irradians. Quantitative electrophoretic analysis of epithelial cell fractions show most of the calmodulin to be localized in the cilia, specifically in the detergent- solubilized membrane-matrix fraction. Calmodulin represents 2.2 +/- 0.3 percent of the membrane-matrix protein or 0.41 +/- 0.5 percent of the total ciliary protein. Its concentration is at least 10(-4) M if distributed uniformly within the matrix. Extraction in the presence of calcium suggests that the calmodulin is not bound to the axoneme proper. The ciliary protein is identified as a calmodulin on the basis of its calcium- dependent binding to a fluphenazine-sepharose affinity column and its comigration with bovine brain calmodulin on alkaline-urea and SDS polyacrylamide gels in both the presence and absence of calcium. Scallop ciliary calmodulin activates bovine brain phosphodiesterase to the same extent as bovine brain and chicken gizzard calmodulins. Containing trimethyllysine and lacking cysteine and tryptophan, the amino acid composition of gill calmodulin is typical of known calmodulins, except that it is relatively high in serine and low in methionine. Its composition is less acidic than other calmodulins, in agreement with an observed isoelectric point approximately 0.2 units higher than that of bovine brain. Comparative tryptic peptide mapping of scallop gill ciliary and bovine brain calmodulins indicates coincidence of over 75 percent of the major peptides, but at least two major peptides in each show no near-equivalency. Preliminary results using ATP-reactivated gill cell models show no effect of calcium at micromolar levels on ciliary beat or directionality of the lateral cilia, the cilia which constitute the vast majority of those isolated. However, ciliary arrest will occur at calcium levels more than 150 muM. Because calmodulin usually functions in the micromolar range, its role in this system is unclear. Scallop gill ciliary calmodulin may be involved in the direct regulation of dyneintubule sliding, or it may serve some coupled calcium transport function. At the concentration in which it is found, it must also at least act as a calcium buffer.
Amino Acids↗
Equimolar heterodimers in microtubules.
Two equimolar beta chains can be resolved from sea urchin sperm flagellar and scallop gill ciliary tubulins, and from certain brain tubulins as well, using the Triton X-100-acid-urea polyacrylamide gel system commonly used for histone analysis. The beta chains are identified as such from their mobility on urea-free SDS PAGE, from amino acid composition, and from tryptic peptide distribution. Scallop beta chains have almost identical amino acid profiles but they differ by one tryptic peptide. Optimal conditions for beta chain resolution are very species-dependent, with some closely related species showing either maximal or no beta chain separation. In addition, beef brain tubulin on Triton X-100-acid-urea electrophoresis and scallop gill ciliary tubulin upon isoelectric focusing in the presence of SDS show two approximately equimolar alpha chains. These data, indicating equimolar amounts of two potentially different tubulin heterodimers from a variety of microtubule types, support a model for microtubule structure wherein protofilaments consist of alternating heterodimers of two kinds, generating a 16-nm (2-dimer) axial repeat.
Animals↗
Chemical differences distinguish ciliary membrane and axonemal tubulins.
Tubulin was prepared by exhaustive dialysis solubilization from axonemal A and B subfibers and by detergent solubilization of the membrane of cilia from the scallop aequipecten irradians. The respective alpha and beta chains were isolated by preparative sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis and then compared by amino acid analysis and high-resolution two-dimensional tryptic peptide mapping. Minor amino acid and peptide differences distinguished A- and B-subfiber-derived tubulin subunits from each other, but far more significant amino acid differences distinguished the membrane-derive subunits from those of the axoneme. Peptide mapping revealed that each membrane tubulin subunit contained two major peptides not coincident with those of the axoneme and vice versa, but many corresponding peptides differed markedly in relative intensity. The alpha and beta subunits from these sources showed virtually identical isoelectric points. Certain NaDodSO4-polyacrylamide gel systems, sensitive to differential detergent binding, allowed the membrane-derived subunits to be distinguished from those of the axoneme. Under nondenaturing conditions, the membrane-derived tubulin bound both anionic and cationic detergents more strongly than axonemal tubulin. These data indicate that ciliary membrane tubulin is a distinct molecule whose subunits have the same molecular weight and isoelectric point as those of axonemal tubulin but differs chiefly in terms of nonpolar, conservative substitutions. These chemical differences argue against the artifactual origin of the protein from breakdown of the axoneme.
Amino Acids↗
7-Chloro-4-nitrobenzeno-2-oxa-1,3-diazole actin as a probe for actin polymerization.
Lysine 372 of N-ethylmaleimide actin was specifically (60%) labeled by 7-chloro-4-nitrobenzeno-2-oxa-1,3-diazole chloride (NBD-Cl), which also reacted with lysines on cyanogen bromide fragment 17 (20%) and other undetermined residues (20%). Isolation of N-ethylmaleimide peptides and two-dimensional peptide mapping demonstrated that 90% of bound N-ethylmaleimide was attached to an adjacent residue, cysteine 373, independent of the polymerization state of actin during the labeling reaction. Formation of NBD cysteine severely inhibited lysine modification. After N-ethylmaleimide blockage of cysteine 373, lysine labeling with NBD was greatly accelerated. The kinetics of formation of fluorescent compounds were biphasic, with fluorescence decreasing upon prolonged incubation of actin in NBD-Cl. Lysine 372 of purified NBD actin reproducibly responded to polymerization by a 2.2- to 2.3-fold enhancement of fluorescence. By contrast, interaction of NBD actin with several actin-binding proteins caused only very small or undetectable changes in fluorescence intensity: 10% enhancement on myosin subfragment 1 binding, about 6% quenching by DNase I, and no change at all by tropomyosin-troponin. Despite its sensitivity to polymerization the probe did not affect it. Native and modified actin polymerized randomly indicating that the rate constants for polymerization remained the same. Labeling actin with NBD did not diminish its cofactor activity for myosin ATPase activity. Contrary to previous reports we observed that myosin subfragment 1 (single myosin heads) caused actin polymerization in the absence of salt.
4-Chloro-7-nitrobenzofurazan↗
Growth characteristics of human glioma-derived and fetal neural cells in culture.
Growth characteristics of human fetal neural cells (CH) and human glioblastoma multiforme-derived cells (12-18) in culture were compared. Cells were grown to confluent densities of 38,000 to 42,500 cells/cm2 for CH and 85,800 to 87,100 for 12-18. Population doubling times were 40.0 +/- 5.1 hr and 66.5 +/- 9.8 hr for CH and 12-18 cells, respectively. The mean DNA content per cell of the glioma-derived cells was twice that of the fetal brain cells at sparse, log, and confluent cell densities. High concentrations (40%) of serum in growth medium increased DNA contents in confluent CH, but not 12-18, cells. The amount of protein per cell also was consistently higher in glioma cells than CH cells, but, as cell densities increased, protein contents decreased for both: 1200 to 700 pg/cell in glioma cells, and 840 to 560 pg/cell in CH cells. In each cell line, initial rates of [3H]ThdR incorporation into TCA precipitable material decreased as cell density increased, but confluent glioma-derived cells incorporated 10 times more [3H]ThdR than confluent fetal cells. Almost all CH cells had a normal diploid chromosome number of 46. A histogram showing the relative frequencies of chromosome numbers of glioma-derived cells had peaks of 52, 79, and 105 chromosomes per metaphase, indicating a haploid number of 26 for most cells. Lengths of cell cycle phases, determined using autoradiographic techniques, indicate that glioma-derived cells had a longer generation time and S period than fetal neural cells. These data demonstrate several biological differences between glioblastoma-derived cells and non-neoplastic fetal neural cells, indicating that this system is of potential value for comparative studies on growth control and contact inhibition.
Cell Count↗
Microtubule-membrane interactions in cilia. II. Photochemical cross-linking of bridge structures and the identification of a membrane-associated dynein-like ATPase.
Photochemical cross-linking of both Tetrahymena and Aequipecten ciliary membrane proteins with the lipophilic reagent 4,4'-dithiobisphenylazide links together a high molecular weight dynein-like ATPase, membrane tubulin, and at least two other proteins. Electron microscopy of detergent-extracted cilia reveals that the cross-linked complex remains attached to the outer-doublet microtubules by a microtubule-membrane bridge. Cleavage of the reagent's disulfide bond releases the bridge-membrane complex and the dynein-like membrane-associated ATPase. Electron microscopy was used to ensure that the dynein-like protein did not result from the solubilization of the dynein arms attached to the outer-doublet microtubules. The dynein-like protein has been isolated using sucrose gradients and is similar to axonemal dynein with respect to its sedimentation characteristics nucleotide specificity, and divalent cation requirements. Photochemical cross-linking of ciliary membrane porteins in vivo results initially in the modification of ciliary beat and, eventually, in the cessation of ciliary movement. These results suggest that a dynein-like ATPase comprises the bridge which links the ciliary membrane to the outer-doublet microtubules and that this bridge is involved in the modulation of normal ciliary movement.
Adenosine Triphosphatases↗
Brush-border alpha-actinin? Comparison of two proteins of the microvillus core with alpha-actinin by two-dimensional peptide mapping.
The bundle of filaments within the intestinal microvillus contains four major polypeptides in addition to actin calmodulin, a 70-kdalton subunit and two polypeptides with molecular masses similar to that of the Z-line component alpha-actinin (95 and 105 kdaltons). Two-dimensional mapping of tryptic peptides indicates that (a) alpha-actinins from chicken skeletal, cardiac, and smooth muscle are similar but not identical proteins and that skeletal alpha-actinin in more similar to the cardiac subunit than to the alpha-actinin from gizzard; (b) the brush-border 95- and 105-kdalton subunits are closely related to each other, but the smaller subunit is not a proteolytic fragment of the 105-kdalton subunit; and (c) although there is considerable peptide overlap between the brush-border subunits and the three alpha-actinins, the peptide maps of the 95- and 105-kdalton proteins are substantially distinct from the various alpha-actinin maps, suggesting that neither brush-border subunit is a bona fide alpha-actinin. Nevertheless, on the basis of peptide mapping criteria alone, one cannot exclude the possibility that the brush-border subunits are "alpha-actinin-like." However, there is no immunological cross-reactivity between the brush-border subunits and alpha-actinins, using antibodies prepared against gizzard alpha actinin.
Actinin↗
Motile statocyst cilia transmit rather than directly transduce mechanical stimuli.
We have investigated the role of motile cilia in mechanotransduction by statocysts of the nudibranch mollusk Hermissenda crassicornis. Movement of the cilia that experience the weight of statoconia causes increased variance of voltage noise and membrane depolarization of the statocyst hair cell. Two complementary approaches were used to immobilize the cilia. Vanadate anion was iontophoretically injected into hair cells. This reversible inhibitor of vibratile form and to assume a more classic, pliable beat pattern. Voltage noise decreased as the cilia slowed and bent more extremely, nearly disappearing as motility was lost. When the intracellular vanadate concentration approached 10(-5) M, the cilia were arrested in an effective stroke against the cell membrane. The cell no longer depolarized upon gravitational or local mechanical stimulation. Rapid reversal of ciliary inhibition by norepinephrine or slow reversal with time restored both the voltage noise and depolarization response. Cilia were rendered rigid and upright by covalent cross-linkage of their membrane "sleeve" to the 9 + 2 axoneme, using the photoactivated, lipophilic, bifunctional agent 4,4'-dithiobisphenyl azide. In the initial stages of cross-linkage, the cilia remained vibratile but slowed and moved through wider excursions. Voltage noise decreased in frequency but increased in amplitude. When the cilia were fully arrested, voltage noise was minimized while the resting potential and membrane resistance remained essentially constant. Mechanical stimulation of the rigid cilia, normal to the cell membrane, elicited a generator potential of the same amplitude but of greater duration than before treatment. Because cilia that are partially arrested by vanadate undergo increased bending, although the hair cell shows decreased noise, neither the axoneme nor the ciliary membrane proper would appear to be sites of direct transduction. In cells with beating but stiffened cilia, however, the voltage noise becomes amplified, implying an increased efficiency of transduction. We suggest that active but rigid flexure of the axoneme is involved in amplification and continuous signal detection. The basal insertion area is the most likely transduction site, being the terminal leverage point through which force is applied to the plasma membrane via the flexing ciliary shaft.
Animals↗
Primary structural differences among tubulin subunits from flagella, cilia, and the cytoplasm.
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Amino Acids↗
Fluorescent thin-layer peptide mapping for protein identification and comparison in the subnanomole range.
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Actins↗
Major membrane protein differences in cilia and flagella: evidence for a membrane-associated tubulin.
The membrane of both sperm flagella and gill cilia of the scallop Aequipecten irradians may be selectively solubilized in 1% Triton X-100, 30 mM tris(hydroxymethyl)-aminomethane hydrochloride (Tris-HCl), pH8, and 3 mM MgCl2, leaving the axoneme totally intact. This membrane fraction represents about 20% of the total protein of the respective organelle. Analysis of the flagellar membrane by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis revealed one principal protein component, periodic acid-Schiff (PAS) positive and migrating with an apparent molecular weight of 250,000. The remaining minor proteins, none of them PAS positive, accounted for less than one-third of the total flagellar membrane fraction. Analysis of the ciliary membrane also revealed one major protein component, weakly PAS positive and migrating with an apparent molecular weight of 55,000. The remaining minor proteins represented about one-third of the total ciliary membrane fraction; two components with molecular weights of 100,000 and 40,000 predominated. The latter could be substantially reduced by purification of the cilia on a sucrose density gradient and was assumed to be actin, derived by vesiculation of the brush border during deciliation. The principal ciliary membrane protein, that of 55,000 daltons, was resolved into two equimolar components on NaDodSO4-Tris-glycine-polyacrylamide gels, comigrating with the alpha and beta chains of outer fiber tubulin. S-carboxymethylation caused increased splitting of the two components and concomitant migration with similarly treated ciliary tubulin. Preparative gel electrophoresis yielded separate components whose cyanogen bromide cleavage products were virtually identical in size distribution with those obtained from outer fiber alpha and beta chains; tryptic peptides corresponded almost exactly to those of authentic tubulin subunits but certain positional differences indicated possible side chain modification. At 25 degrees C both whole cilia and its solubilized membrane fraction bound colchicine while whole flagella and the 9 + 2 axoneme from either organelle did not. Thus certain molluscan flagellar membranes primarily contain a 250,000-dalton glycoprotein but ciliary membranes have a modified tubulin as the major protein component. At an electron microscopic level, flagellar membranes have a distinct trilamellar "unit membrane" structure while ciliary membranes appear thinner and considerably less distinct, perhaps reflecting the protein compositional differences in the membranes of these other wise morpholobically identical organelles.
Animals↗
Studies of human histone messenger RNA. I. Methods for the isolation and partial characterization of RNA fractions containing human histone message from HeLa S3 polyribosomes.
Large quantities of nonpolyadenylated [poly(A(-))] 4 to 18 S RNA were isolated from the polyribosomes of S phase HeLa S3 cells and were fractionated into multiple discrete RNA components by continuous elution preparative electrophoresis. Previous studies have shown that treatment os S phase HeLa cells with cytosine arabinoside inhibits DNA replication and causes translatable histone messenger RNA (mRNA) species to disappear from cytoplasmic polyribosomes (Borun, T. W., Scharff, M.D., and Robbins, E. (1967) Proc. Natl. Acad. Sci. U.S.A. 58, 1977-1983; Gallwitz, D., and Mueller, G. C. (1969) J. Biol. Chem. 244, 5948-5952; Borun, T. W., Gabrielli, F., Ajiro, K., Zweidler, A., and Baglioni, C. (1975) Cell 4, 59-67; Gallwitz, D. (1975) Nature 257, 247-248). In the present study it was found that cytosine arabinoside treatment does not appreciably affect major 7.5 to 8 S RNA species but does cause the disappearance of 8.6 to 13 S RNA components from preparative electrophoresis elution profiles of S phase polyribosomal 4 to 18 S RNA. Base ratio analysis of the 8.6 to 13 S putative histone mRNA species indicates that they are GC-rich but not like the HeLa 18 or 28 S rRNA in base composition.
Chromatography, Affinity↗
Studies of human histone messenger RNA. II. The resolution of fractions containing individual human histone messenger RNA species.
Polyribosomal 4 to 18 S RNA from S phase HeLa S-3 cells has been fractionated by chromatography on oligo(dT)-cellulose and resolved into multiple discrete components by continuous elution preparation electrophoresis. The human histone messenger RNA (mRNA) species associated with various polyadenylated [poly(A(+))] and nonpolyadenylated [poly(A(-))] components of 4 to 18 S RNA were determined by translation of these RNA fractions in vitro using a Krebs II ascites cell-free system followed by resolution of histones synthesized in vitro on polyacrylamide gels containing Triton X-100. The results of these studies indicate that poly(A(-)) 4 to 18 S RNA from S phase HeLa polyribosomes contains: (a) large quantities of discrete 7.4 and 8 S RNA species which are not functional histone mRNA; (b) a discrete 8.6 S RNA fraction which contains the templates of human histone H4; (c) 9.2 to 10.7 S RNA which contains mixtures of incompletely resolved histone H2B, H2A, and H3 mRNA (These mRNA species do not closely correspond to discrete RNA subfractions resolvable by our techniques.); (d) discrete 12 and 13 S RNA fractions which contain templates of human histone H1 polypeptides. The present studies also indicate that the mRNA templates of histone variants H3.2 and H3.3 have a slightly lower electrophoretic mobility than H3.1 mRNA and that H2A.2 mRNA has a slightly lower electrophoretic mobility than H2A.1 mRNA. In addition, appreciable quantities of H3.2, H3.3, and H2A.2 mRNA are bound to oligo(dT)-cellulose in 0.5 M KCl. These results indicate that mRNA species of the same histone class differ slightly in primary structure and are consistent with the hypothesis that some histone mRNA species contain short tracts of poly(A).
Chromatography, Affinity↗
Connective tissue synthesis by cultured scleroderma fibroblasts. II. Incorporation of 3h-glucosamine and synthesis of glycosaminoglycans.
Fibroblasts from normal and scleroderma skin, grown tissue culture, were incubated with 3H-glucosamine for 24 hours. No definite trends could be established in 3H-glucosamin incorporation or glycosaminoglycan synthesis. Over 90% of the total 3H activity as well as the glycosaminoglycans synthesized were secreted into the medium. Characterization of glycosaminoglycans showed that in both the medium and cells, hyaluronic acid ins the major glycosaminoglycan in normal and scleroderma fibroblasts. In the medium, hyaluronic acid represented 87% of the total glycosaminoglycans; it was slightly decreased in the cells. Fibroblasts were compared from the upper and lower areas of the affected dermis and an uninvolved dermal area of the same scleroderma patients.
Adult↗
Differential protein synthesis and utilization during cilia formation in sea urchin embryos.
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Animals↗
Effect of cell proliferation on levels and diversity of poly(A)-containing mRNA.
The relationship between cell proliferation and the amount and diversity of polyribosome-associated poly(A)-containing messenger RNA [poly(A)+mRNA]has been investigated using a cloned AKR-mouse embryo cell culture system. The following results were obtained. First, an early response to the stimulation of proliferation of AKR-2B cells in culture is a rapid increase in the rate of accumulation of polyribosomal poly(A)+ mRNA. This results in a large increase in the total poly(A)+ mRNA content of rapidly proliferating cells compared to that found in resting cells. Second, the total amount of unique DNA sequence contributing to the poly(A)+ mRNA populations of both growing and resting cells is not detectably different. This corresponds to 9000-11,000 diverse gene equivalents of DNA and represents the transcription of 0.8-0.9% of the haploid mouse genome. Third, most of the increased poly(A)+ mRNA content of growing cells (greater than 90%) reflects an increased rate of production of polysomal mRNA species which are also found in resting cells. Fourth, growing cells appear to contain some species of poly(A)+ mRNA which are either absent or present in very low concentrations in non-growing cells. Within the limits of detection, however, all species of poly(A)+ mRNA present in non-growing cells are also present in growing cells.
Cell Division↗
RNA precursor pool metabolism and RNA synthesis in X-irradiated Tetrahymena.
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Animals↗