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The human genome sequencing initiative is sure to be an expensive and time-consuming project. To maximize the quality of the initial thrust, it is necessary to concentrate on sequencing those genes that are actively involved in crucial biological processes, both normal and otherwise. To identify the protein products of these genes, we propose the use of two-dimensional acrylamide gel electrophoresis and powerful computer software. The two-dimensional gels can then be scaled to yield sufficient amounts of the protein for direct amino acid sequence analysis. The amino acid sequence then provides the direct link back to the gene.

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A service is now available for analysing 2-dimensional gels and comparing them to existing databases, broadening the number of laboratories which can study protein expression using the technique.

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The adenovirus early-region 1B 19,000-molecular-weight tumor antigen is required for oncogenic transformation of cells by adenovirus. We have demonstrated that this tumor antigen is located in the nuclear envelope of infected and transformed cells and that a fraction of the protein within the nuclear envelope is associated with the nuclear lamina. During cell division in the transformed cells, the nuclear envelope containing the tumor antigen dissociates at metaphase and then reforms around the separated daughter chromosomes at telophase. Adenovirus mutants carrying lesions in the gene encoding this tumor antigen cause degradation of host cell chromosomal DNA, and in these mutants, the intracellular localization of the 19,000-dalton protein is altered. These results demonstrate that components of the nuclear envelope function in the organization of chromatin in infected and transformed cells and that a virus-encoded protein plays a critical role in this process.

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Living mitotic HeLa cells were microinjected with sodium fluorescein, fluorescein isothiocyanate (FITC)-labelled IgG, and Lucifer Yellow-CH to determine if the intracellular bridge and midbody forms a barrier to the migration of these molecules. All three fluorescent molecules were found to pass across the intercellular bridge. The post-mitotic cells maintained a molecular patency across the intercellular bridge that persisted for considerable lengths of time, extending into the next cell cycle.

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Cells were microinjected with four mouse monoclonal antibodies that were directed against either alpha- or beta-tubulin subunits, one monoclonal with activity against both subunits, and a guinea pig polyclonal antibody with activity directed against both subunits, to determine the effects on the distribution of cytoplasmic microtubules and 10-nm filaments. The specificities of the antibodies were confirmed by Western blots, solid phase radioimmunoassay, and Western blot analysis of alpha- and beta-tubulin peptide maps. Two monoclonals DM1A and DM3B3, an anti-alpha- and anti-beta-tubulin respectively, and the guinea pig polyclonal anti-alpha/beta-tubulin antibody (GP1T4) caused the 10-nm filaments to collapse into large lateral aggregates collecting in the cell periphery or tight juxtanuclear caps; the other monoclonal antibodies had no effect when microinjected into cells. The filament collapsing was observed to be complete at 1.5-2 h after injection. During the first 30 min after injection a few cytoplasmic microtubules near the cell periphery could be observed by fluorescence microscopy. This observation was confirmed by electron microscopy, which also demonstrated assembled microtubules in the juxtanuclear region. By 1.5 h, when most of the 10-nm filaments were collapsed, the complete cytoplasmic array of microtubules was observed. Cells injected in prophase were able to assemble a mitotic spindle, suggesting that the antibody did not block microtubule assembly. Metabolic labeling with [35S]methionine of microinjected cells revealed that total protein synthesis was the same as that observed in uninjected cells. This indicated that the microinjected antibody apparently did not produce deleterious effects on cellular metabolism. These results suggest that through a direct interaction of antibodies with either alpha- or beta-tubulin subunits, 10-nm filaments were dissociated from their normal distribution. It is possible that the antibodies disrupted postulated 10-nm filament-microtubule interactions.

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Mitotic BHK21 cells were examined by immunofluorescence optical sectioning after staining with anti-vimentin. Throughout all stages of mitosis, intact 10-nm filaments were observed to form a cage around the mitotic spindle. These observations were confirmed by serial section electron microscopy which demonstrated assembled 10-nm filaments in the cytoplasm surrounding the spindle. Therefore, in contrast to previous reports, mitotic BHK21 cells show a similar cage-like 10-nm filament arrangement, as found in a variety of other cell types.

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I have used fluorescence microscopy and antibodies to 10nm filaments and tubulin labelled with contrasting fluorochromes to compare the distribution of these proteins in endothelial cells during cell division. During interphase the two filament systems have entirely different distributions: The bulk of the 10nm filaments form a ring that surrounds the cell center and nucleus and remains parallel to the substrate, while the microtubules radiate from the cell center to the cell's border. When the mitotic spindle replaces the radial microtubule pattern in mitosis, the spindle poles remain within--and in close proximity to--the ring of 10nm filaments. This was confirmed by electron microscopy which showed the ring and centrioles in the same plane separated by a distance of 300-400 nm.

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The distribution of flourescently labeled alpha-actinin after microinjection into fibroblasts has been determined in both living and fixed cells. We have found that the distribution of the injected tetramethylrhodamine isthiocyanate-labeled protein (TMRITC-alpha-actinin) in living cells, which is in ruffling membranes, actin microfilament bundles, and polygonal microfilament networks (Feramisco, 1979, Proc. Natl. Acad. Sci. U. S. A. 76:3967-3971), was virtually unaffected by the fixation (3.5 percent formaldehyde) and extraction (absolute acetone) used for the preparation of the cells for immunoflourescence. Also, these patterns were found to coincide with the alpha-actinin revealed by immunoflourescence. Also, these patterns were found to coincide with the alpha-actinin revealed by immunoflourescence. These findings offer, for the first time, evidence indicating the validity of the immunoflourescence technique in the localization of alpha-actinin in cultured cells. With the combination of the injection procedure and the immunoflourescence localization of endogenous structural proteins, it was determined that nearly all of the actin stress fibers were decorated in a periodic manner with the injected alpha-actinin. Endogenous tropomyosin in the injected cells was found to be distributed with a periodic pattern along the stress fibers that was antiperiodic to the pattern observed for the microinjected alpha-actinin. The tropomyosin antibody stained the polygonal microfilament networks and was excluded from the foci, whereas the microinjected alpha-actinin was incorporated into the foci of the networks. Thus, the microinjected fluorescent derivative of alpha-actinin appears to be incorporated into the functional pools of alpha-actinin within the living cell and to be utilized by the cell with fidelity.

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By indirect immunofluorescence the behavior of the 10-nm filaments was studied at various stages of mitosis in guinea pig vascular endothelial cells. Interphase cells contain a ring of 10-nm filaments that encircles the nucleus and is maintained in a plane parallel to the substrate. During prophase and metaphase the cells round up and the 10-nm filament ring becomes wavy though still a closed structure. As anaphase progresses the ring then elongates into a rectangle that contains the spindle apparatus and chromosomes. In late telophase, cytokinesis cleaves the 10-nm filaments into crescents at the site of the contractile ring. These crescents then close into rings in the daughter cells. If cytokinesis is inhibited with 5 microgram of cytochalasin B per ml, then cleavage of the 10-nm filaments is blocked and the daughter nuclei remain surrounded by the parent ring. At no point during mitosis does the array of 10-nm filaments undergo major disassembly. These results indicate that, in contrast to the other major cytoplasmic structures, ventral microfilament bundles and cytoplasmic microtubules, which disassemble and reassemble during mitosis, 10-nm filaments remain intact throughout this process. The possibility is discussed that these filaments may function in transport of organelles and structural proteins, and provide the daughter cells with topological information about placement and assembly of these elements within the microtrabecular lattice.

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Guinea pig vascular endothelial cells contain naturally occurring rings of intermediate filaments that completely encircle the nucleus. Indirect immunofluorescence staining showed that these perinuclear rings bound antibody prepared against protein from bovine brain 9-nm filaments. In endothelial cells grown in the presence of 1 muM demecolcine (Colcemid) the perinuclear ring "coils" into a juxtanuclear "cap". Throughout this process we could demonstrate staining of the intermediate filaments. Chick cardiac muscle cells in culture stained diffusely with the antibody. After treatment for 24 hr with 1 muM demecolcine the cardiac cells accumulated large bands of intermediate filaments. These bands stained intensely with the antibody. Our findings suggest that intermediate filaments in guinea pig endothelial cells and those induced in chick cardiac muscle cells are antigenically similar to bovine brain filaments. The staining of these filaments is not affected by treatment with demecolcine.

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Vascular endothelial cells cultured from guinea pig aorta or portal vein contain naturally occurring bundles of 100 A (diameter) filaments that completely encircle the nucleus. These rings are phase lucent and birefringent when examined with the light microscope. Perinuclear bundles of 100 A filaments were also seen in endothelial cells in vivo, indicating that they are a normal cytoplasmic component. These filaments did not decorate with S-1, and were not disrupted by glyceination. With these cells, experiments were designed to answer the following questions: (a) does Colcemid have an effect on these naturally occuring bundles? And (b) do these filaments remain during cell division? Endothelial cells grown in the presence of Colcemid were followed over 24 h. The perinuclear ring coiled into a juxtanuclear cap that consisted of disorganized arrays of 100 A filaments. This "coiling" effect was not blocked by cycloheximide, an inhibitor of protein synthesis. In another experiment, dividing cells were examined. During division the bundle of filaments is passively pulled in half into the daughter cells. These bundles did not disappear during the mitosis when mitotic spindle microtubules assemble. These studies suggest that Colcemid may exert a direct effect on 100 A filaments, independent of microtubules. Since these filaments do not disappear during mitosis, it is possible that in these cells the 100 A filaments and tubulin do not share a common pool of precursor proteins.

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