Biomedical subjects
Publications and source records attributed to L H Rome.
We have isolated a cDNA clone encoding the 104-kDa major vault protein (MVP) from Rattus norvegicus. The complete nucleotide sequence was determined. Comparison of the deduced amino acid (aa) sequence to that of MvpA from Dictyostelium discoideum revealed that the proteins share about 57% aa identity. Southern blot analysis indicates that the rat MVP is a single-copy gene.
A novel gene transcribed by RNA polymerase (pol) III has been recently identified that produces an RNA component of a large cytoplasmic ribonucleoprotein complex (Kickhoefer, V. A., Searles, R. P., Kedersha, N. L., Garber, M. E., Johnson, D. L., and Rome, L. H. (1993) J. Biol. Chem. 268, 7868-7873). Since sequence analysis revealed that this gene contains promoter elements from two different classes of RNA pol III gene promoters, we examined the function of the 5'-flanking type-3 and internal type-2 sequences on transcription activity and the production of stable transcription complexes. We find that the vRNA gene contains a novel RNA pol III promoter, where both the external and internal sequences are essential for template activity and for the productive interaction of TFIIIC with the internal elements. Thus, the vRNA gene represents the first example of a template that requires both type-2 and type-3 promoter elements that appear to function synergistically in the formation of productive transcription complexes. We have further examined the function of the unique arrangement of an internal A box and two B box elements. We find that at least one B element is required for template activity. In the absence of the 5'-flanking sequence the presence of both B elements inhibits transcription and the binding of TFIIIC. The formation of active complexes is restored when either the B2 element is inactivated or the distance separating the two B elements is increased. Therefore, the B2 element appears to negatively regulate template activity in the absence of the upstream sequences. This unique RNA pol III promoter arrangement may provide a novel mechanism for the regulation of vRNA gene activity.
Central nervous system myelin is elaborated by oligodendrocytes, which have been studied extensively in cell culture. Dissociated brain cultures allow in vitro analysis of events in myelinogenesis, including cell-cell interactions. Microglia, the primary phagocytic cell of the central nervous system, appear in developing fiber tracts prior to the onset of myelination in vivo. To gain insight into potential oligodendrocyte-microglial interactions during development, these cells were co-cultured and various parameters of myelin synthesis were measured. In co-culture, microglia stimulated the synthesis of sulfatide, a myelin-specific galactolipid, in oligodendrocytes, as well as the expression of the myelin-specific proteins myelin basic protein and proteolipid protein. Activity of the oligodendrocyte cytoplasm-specific enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase was not elevated, suggesting that the effects of microglia were not due to stimulation of oligodendrocyte proliferation. This was confirmed by the inability of microglia to induce significant DNA synthesis. Conditioned medium from cultured microglia provided a similar stimulatory activity, suggesting that the increase in myelin synthesis does not require contact between oligodendrocytes and microglia. These findings suggest a stimulatory role for microglia during myelinogenesis.
A direct role for neurons in CNS myelination has yet to be demonstrated. CNS myelination can be examined in cerebellar slice cultures, which faithfully reproduce both synthesis and wrapping of myelin. In an attempt to demonstrate a role for axolemma in this process, we generated more than 2000 axolemma-reactive monoclonal antibodies. One clone, G21.3, repeatedly blocked myelination in cerebellar slices, as documented by both biochemistry and morphology. The antibody caused a dramatic reduction in myelin lipid and protein synthesis. CNS white matter, sciatic nerve, and neuronal cultures were positively stained with G21.3, whereas oligodendrocytes and myelin were fully negative. The antibody identified a restricted number of proteins in purified axolemma. These results suggest a direct involvement of axons in CNS myelination.
We have investigated the expression of integrins by rat oligodendroglia grown in primary culture and the functional role of these proteins in myelinogenesis. Immunochemical analysis, using antibodies to a number of alpha and beta integrin subunits, revealed that oligodendrocytes express only one detectable integrin receptor complex (alpha OL beta OL). This complex is immunoprecipitated by a polyclonal anti-human beta 1 integrin subunit antibody. In contrast, astrocytes, the other major glial cell type in brain, express multiple integrins including alpha 1 beta 1, alpha 3 beta 1, and alpha 5 beta 1 complexes that are immunologically and electrophoretically indistinguishable from integrins expressed by rat fibroblasts. The beta subunit of the oligodendrocyte integrin (beta OL) and rat fibroblast beta 1 have different electrophoretic mobilities in SDS-PAGE. However, the two beta subunits appear to be highly related based on immunological cross-reactivity and one-dimensional peptide mapping. After removal of N-linked carbohydrate chains, beta OL and beta 1 comigrated in SDS-PAGE and peptide maps of the two deglycosylated subunits were identical, suggesting differential glycosylation of beta 1 and beta OL accounts entirely for their size differences. The oligodendrocyte alpha subunit, alpha OL, was not immunoprecipitated by antibodies against well characterized alpha chains which are known to associate with beta 1 (alpha 3, alpha 4, and alpha 5). However, an antibody to alpha 8, a more recently identified integrin subunit, did precipitate two integrin subunits with electrophoretic mobilities in SDS-PAGE identical to alpha OL and beta OL. Functional studies indicated that disruption of oligodendrocyte adhesion to a glial-derived matrix by an RGD-containing synthetic peptide resulted in a substantial decrease in the level of mRNAs for several myelin components including myelin basic protein (MBP), proteolipid protein (PLP), and cyclic nucleotide phosphodiesterase (CNP). These results suggest that integrin-mediated adhesion of oligodendrocytes may trigger signal(s) that induce the expression of myelin genes and thus influence oligodendrocyte differentiation.
G21.3, a monoclonal antibody previously shown to block central nervous system (CNS) myelination, is now demonstrated to recognize an extracellular matrix (ECM) component. The antigen is present on the surface of neurons but not oligodendrocytes and is highly abundant in the white matter of the adult rat brain; however, it is not found in isolated myelin. Double immunostaining studies indicate a neuronal and ependymal cell source of the G21.3 antigen and a developmental expression pattern distinct from known markers of CNS myelination. The antigen is found at low levels in non-neuronal tissue and is mainly localized to basement membranes. G21.3 immunoreactive proteins are upregulated by retinoic acid-induced differentiation of SK-N-SHF neuroblastoma cells. These results suggest that the G21.3 antigen is an axolemma-associated ECM component with roles in postnatal CNS development and cell-matrix interactions during morphological differentiation of neuroblastoma cells.
Myelin deposition and maintenance are critical to proper function of the mammalian nervous system. Previous investigations of myelination in the central nervous system (CNS) were hampered by the lack of an in vitro system that can faithfully reproduce in vivo events yet is amenable to biochemical investigation. We have developed a procedure, based on organotypic cultures, which permits efficient preparation of large numbers of cerebellar slice cultures that can be easily manipulated. Cultures have been examined to document myelination biochemically (by incorporation of [35S]sulfate into sulfolipids), immunohistochemically (by labeling the myelin components myelin basic protein and galactocerebroside), and morphologically (by both light and electron microscopy). We tested the effects of biologically active peptides and antibodies on myelination in the thin slices. The results indicate that the cultures provide an in vitro system that can be used to examine specific cellular events that occur during CNS myelination.
Vaults are large cytoplasmic ribonucleoprotein particles found in nearly all eukaryotic cells. Dictyostelium vaults contain two major proteins, MVP alpha (94.2 kDa) and MVP beta (approximately 92 kDa). Using an anti-rat vault antibody, we screened a Dictyostelium cDNA expression library and isolated a 2.8-kilobase pair clone that contained a single full-length reading frame. The identity of the clone was established by the presence of a predicted 20-amino acid sequence identical to that found in a peptide sequenced from purified MVP alpha. We have disrupted the single copy gene using homologous recombination and have demonstrated a loss of MVP alpha. Although the cells still produce MVP beta, they do not contain characteristic vault particles, suggesting that MVP alpha is required for normal vault structure. These cells should be a valuable tool for elucidating the function of vaults.
Vaults are large cytoplasmic ribonucleoprotein particles with a sedimentation value of about 150 S. These particles contain a unique small RNA (vault RNA (vRNA)). We have determined the sequence of the RNA associated with vaults purified from both rat and bullfrog. The rat vRNA is 141 bases in length, whereas the bullfrog vRNA is present as two highly related species of 89 and 94 bases. Despite the differences in length the predicted secondary structures of the three vRNAs are clearly related. All of the vRNAs contain sequences related to the internal promoter elements necessary for transcription by RNA polymerase III. The gene for the rat vRNA was isolated and sequenced from a rat genomic library, and its transcription by RNA polymerase III was verified using an in vitro transcription assay. The rat vRNA gene was efficiently transcribed in vitro, producing a single transcript of about 140 bases. Unlike most RNA polymerase III genes, the rat vRNA is present as a single copy gene and has a distinct tissue-specific expression pattern.
Vaults are cytoplasmic ribonucleoprotein organelles that are highly conserved among diverse eukaryotic species. Their mass (12.9 MDa), diameter (26-35 nm) and shape (two halves, each with eightfold radial symmetry) have recently been determined and are similar to those ascribed to the central plug (or transporter) of the nuclear pore complex (NPC). The size and eightfold symmetry of the vault particle make it conducive to interacting physically in a complementary manner with NPCs. The present study demonstrates that vaults specifically associate with nuclei by both immunoblotting and immunofluorescence. Immunogold EM confirmed that vaults associate with the nuclear envelope in tissue sections and with NPCs of isolated nuclei.
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We have investigated the expression of integrins in C6 glioma, a chemically-induced glial tumor cell line from rat brain. Immunochemical analysis revealed that C6 cells express sets of integrin receptor complexes which immunologically and electrophoretically are indistinguishable from those expressed by normal rat skin fibroblasts. These include the well-characterized fibronectin (alpha 5 beta 1) and the multi-specific laminin, collagen and fibronectin (alpha 3 beta 1) receptors. Assay of cell adhesion indicated that C6 cells adhere to fibronectin-coated surfaces or matrix deposited by the C6 glioma cells (CGM) in an RGD- and divalent cation-dependent fashion. However, anti-fibronectin antibodies, which are able to inhibit fibroblast adhesion to fibronectin, did not inhibit adhesion of the C6 cells to fibronectin or CGM. This may reflect differences in functional properties and/or distribution patterns of integrins in C6 cells and normal fibroblasts.
The structure of rat liver vault ribonucleoprotein particles was examined using several different staining techniques in conjunction with EM and digestion with hydrolytic enzymes. Quantitative scanning transmission EM demonstrates that each vault particle has a total mass of 12.9 +/- 1 MD and contains two centers of mass, suggesting that each vault particle is a dimer. Freeze-etch reveals that each vault opens into delicate flower-like structures, in which eight rectangular petals are joined to a central ring, each by a thin hook. Vaults examined by negative stain and conventional transmission EM (CTEM) also reveal the flower-like structure. Trypsin treatment of vaults resulted exclusively in cleavage of the major vault protein (p104) and concurrently alters their structure as revealed by negative stain/CTEM, consistent with a localization of p104 to the flower petals. We propose a structural model that predicts the stoichiometry of vault proteins and RNA, defines vault dimer-monomer interactions, and describes two possible modes for unfolding of vaults into flowers. These highly dynamic structural variations are likely to play a role in vault function.
The developmental appearance of ameboid and ramified microglia in the rat brain has been examined by immunofluorescent localization of vaults, recently described ribonucleoprotein particles (Kedersha and Rome, 1986a). Vaults are distinct, multiarched structures of unknown function expressed by higher and lower eukaryotic species. Although vaults have been detected in all mammalian cells examined to date, they are highly enriched in macrophages. In the brain, vault antisera is highly specific for both ameboid and ramified microglia. The developmental profile of vault immunoreactivity in rat brain slices suggests that microglia enter the brain at 2 locations, with different time scales for each. The first migration, which begins before embryonic day 15 and subsides between postnatal days 7 and 14, was identified by vault immunoreactivity and Bandeiraea simplicifolia B4-isolectin (a microglia marker) staining. The cells appear to enter from blood vessels and display a ramified morphology as soon as they are detected in the brain. The second microglial migration occurs in the first postnatal week, when ameboid microglia appear in the corpus callosum and other large fiber tracts. Ameboid microglia appear to differentiate into ramified microglia between postnatal days 4 and 14. Vault immunoreactivity, as a very early microglial marker, provides new insight regarding the much-debated origin of the ramified microglia. It is quite clear that ameboid cells are not the sole source of ramified microglia because ramified cells can be detected before the influx of ameboid microglia. Colocalization studies with monocyte/macrophage markers ED1 and OX42 demonstrate that both ramified and ameboid microglia originate from monocyte lineage.
A system was developed to analyze early events in the process of myelination. Primary cultures of rat oligodendrocytes were maintained in the presence of glass micro-fibers which served as artificial axons. A culture chamber was constructed which allowed the close apposition of fibers and cells in a three-dimensional arrangement designed to resemble an in vivo environment. Cells cultured in the presence of glass micro-fibers coated with a glial cell matrix extract were induced to organize into clusters around the fibers. Examination of oligodendrocyte-fiber sandwiches by SEM revealed the presence of a number of cell contacts with the fibers. TEM images showed that, in most cases, fibers were surrounded by the cells and not multiply wrapped. Only occasionally was a loose wrapping of cell membrane observed around the fibers. Cells cultured in the presence of matrix-coated glass micro-fibers showed an increased production of sulfolipids that was at least partially dependent on the presence of the matrix coating. Coating of these "artificial axons" may aid in the identification of signal molecules produced by neurons which enable them to be myelinated.
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Vaults are cytoplasmic ribonucleoprotein structures that display a complex morphology reminiscent of the multiple arches which form cathedral vaults, hence their name. Previous studies on rat liver vaults (Kedersha, N. L., and L. H. Rome. 1986. J. Cell Biol. 103:699-709) have established that their composition is unlike that of any known class of RNA-containing particles in that they contain multiple copies of a unique small RNA and more than 50 copies of a single polypeptide of 104,000 Mr. We now report on the isolation of vaults from numerous species and show that vaults appear to be ubiquitous among eukaryotes, including mammals, amphibians (Rana catesbeiana and Xenopus laevis), avians (Gallus Gallus), and the lower eukaryote Dictyostelium discoideum. Electron microscopy reveals that vaults purified from these diverse species are similar both in their dimensions and morphology. The vaults from these various species are also similar in their polypeptide composition; each being composed of a major polypeptide with an approximate mass of 100 kD and several minor polypeptides with molecular masses similar to those seen in the rat. Antibodies raised against rat vaults recognize the major vault protein of all species including Dictyostelium. Vaults therefore appear to be strongly conserved and broadly distributed, suggesting that their function is essential to eukaryotic cells.