Disrupted Iron Storage in Dental Fluorosis.

Enamel formation and quality are dependent on environmental conditions, including exposure to fluoride, which is a widespread natural element. Fluoride is routinely used to prevent caries. However, when absorbed in excess, fluoride may also lead to altered enamel structural properties associated with enamel gene expression modulations. As iron plays a determinant role in enamel quality, the aim of our study was to evaluate the iron metabolism in dental epithelial cells and forming enamel of mice exposed to fluoride, as well as its putative relation with enamel mechanical properties. Iron storage was investigated in dental epithelial cells with Perl's blue staining and secondary ion mass spectrometry imaging. Iron was mainly stored by maturation-stage ameloblasts involved in terminal enamel mineralization. Iron storage was drastically reduced by fluoride. Among the proteins involved in iron metabolism, ferritin heavy chain (Fth), in charge of iron storage, appeared as the preferential target of fluoride according to quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry analyses. Fluorotic enamel presented a decreased quantity of iron oxides attested by electron spin resonance technique, altered mechanical properties measured by nanoindentation, and ultrastructural defects analyzed by scanning electron microscopy and energy dispersive x-ray spectroscopy. The in vivo functional role of Fth was illustrated with Fth+/- mice, which incorporated less iron into their dental epithelium and exhibited poor enamel quality. These data demonstrate that exposure to excessive fluoride decreases ameloblast iron storage, which contributes to the defective structural and mechanical properties in rodent fluorotic enamel. They raise the question of fluoride's effects on iron storage in other cells and organs that may contribute to its effects on population health.

Higher order structure of Balbiani ring premessenger RNP particles depends on certain RNase A sensitive sites.

Specific premessenger ribonucleoprotein (RNP) particles, the Balbiani ring (BR) granules from Chironomus tentans salivary glands, were treated with RNase A to study the effect of RNA strand breaks on the higher order structure of the particles. Isolated, radioactively labeled BR granules, known to sediment at 300 S, were digested with RNase A and centrifuged in sucrose gradients. The fractionated particles were subsequently analyzed using electron microscopy and caesium chloride centrifugation. At a low RNase concentration, most of the 300 S particles disintegrated completely, and no metastable degradation products were observed. At intermediate RNase concentrations, no 300 S particles were left, but a minor fraction of the BR granules had unfolded and sedimented at 160 S. These granules could represent particles modified during the RNase treatment or represent a more slowly degrading subfraction of the particles. At a high RNase concentration, no RNP particles at all remained in the gradient. The rapid disintegration of the majority of the BR granules was investigated further by electrophoretic analysis of RNA in the remaining particles. During the RNase treatment BR granules, still sedimenting at 300 S, accumulated strand breaks; in fact, as many as 50 to 100 nicks in the 37 kb RNA could be tolerated. It was concluded from RNA analyses that the disintegration of the BR granules was not dependent on any single nick in the RNA, nor on the accumulation of a certain number of nicks, but rather on one or a few critical strand breaks. We propose that there are organizing sequences essential for particle integrity; once these sequences are nicked, the premessenger RNP particles are rapidly and completely degraded.

New cellular models for tracking the odontoblast phenotype.

Odontoblasts and osteoblasts differ functionally and histologically. Because of their close relationship, mesenchymal cells derived from teeth and bone are difficult to distinguish ex vivo. Indeed, the main non-collagenous components of the odontoblastic extracellular matrix, dentin sialoprotein (DSP) or dentin matrix protein 1 (DMP1), have also been detected in osteoblasts. The need to develop cellular models of odontoblast differentiation and to identify markers specific for the odontoblast lineage, has led us to establish clonal cell lines from tooth germs of day 18 mouse embryos transgenic for an adenovirus-SV40 recombinant plasmid. In this study, we analyzed the phenotypes of three independent clones by RT-PCR and Western blot. These clones synthesised DSP, DMP1 and other extracellular matrix proteins typical of the odontoblast and are therefore likely to be derived from the pulp. Transcripts encoding a set of homeobox proteins involved in craniofacial development, such as Pax9, Msx1, Cbfa1, Dlx2 and 5 were also expressed albeit at a different level. These features of the pulpal clones are shared by the C1 mesodermal cells that are capable of differentiating along osteogenic, chondrogenic or adipogenic lineages In contrast, transcripts for two LIM-domain homeobox family genes (Lhx6 and Lhx7) were only detected in the dental clones. Since these genes are preferentially expressed in the mesenchyme of the developing tooth, this suggests that our transgenic-derived cell lines retain intrinsic properties of odontoblastic cells. They may help to characterise genes specifying the odontoblast phenotype and the signalling pathways underlying odontoblast differentiation.

Endochondral bone formation in toothless (osteopetrotic) rats: failures of chondrocyte patterning and type X collagen expression.

The pacemaker of endochondral bone growth is cell division and hypertrophy of chondrocytes. The developmental stages of chondrocytes, characterized by the expression of collagen types II and X, are arranged in arrays across the growth zone. Mutations in collagen II and X genes as well as the absence of their gene products lead to different, altered patterns of chondrocyte stages which remain aligned across the growth plate (GP). Here we analyze GP of rats bearing the mutation toothless (tl) which, apart from bone defects, develop a progressive, severe chondrodystrophy during postnatal weeks 3 to 6. Mutant GP exhibited disorganized, non-aligned chondrocytes and mineralized metaphyseal bone but without cartilage mineralization or cartilaginous extensions into the metaphysis. Expression of mRNA coding for collagen types II (Col II) and X (Col X) was examined in the tibial GP by in situ hybridization. Mutant rats at 2 weeks exhibited Col II RNA expression and some hypertrophied chondrocytes (HC) but no Col X RNA was detected. By 3rd week, HC had largely disappeared from the central part of the mutant GP and Col II RNA expression was present but weak and in 2 separate bands. Peripherally the GP contained HC but without Col X RNA expression. This abnormal pattern was exacerbated by the fourth week. Bone mineralized but cartilage in the GP did not. These data suggest that the tl mutation involves a regulatory function for chondrocyte maturation, including Col X RNA synthesis and mineralization, and that the GP abnormalities are related to the Col X deficiency. The differences in patterning in the tl rat GP compared to direct Col X mutations may be explained by compensatory effects.

A novel gene expressed in rat ameloblasts codes for proteins with cell binding domains.

Two variants of an mRNA sequence are identified that are expressed at high levels in rat ameloblasts during the formation of the enamel matrix. The sequences contain open reading frames for 407 and 324 amino acid residues, respectively. The encoded proteins, which we call amelins, are rich in proline, glycine, leucine, and alanine residues and contain the peptide domain DGEA, an integrin recognition sequence. The sequences coding for the C-terminal 305 amino acid residues, the 3' nontranslated part, and a microsatellite repeat at the nontranslated 5' region are identical in both mRNA variants. The remaining 5' regions contain 338 nucleotides unique to the long variant, 54 common nucleotides, and 46 nucleotides present only in the short variant. Eleven nucleotides have the potential to code for 5 amino acids of both proteins in different reading frames. The reading frame of the longer variant includes codons for a typical N-terminal signal peptide. The amelins are likely to be constituents of the enamel matrix and the only proteins that have so far been implicated in binding interactions between the ameloblast surface and its extracellular matrix.

Expression of amelogenin mRNA sequences during development of rat molars.

The expression of amelogenin mRNA in growing rat molars was studied. Northern blotting and the analysis of cDNA isolates revealed two predoninant variants. One group of cDNA inserts contained sequences of a long mRNA version and the other group contained mRNA sequences of the shorter leucin-rich amelogenin polypeptide (LRAP). The LRAP group was deficient in an internal stretch which coded for a peptide with a high potential for beta turns. Northern blot experiments showed that most amelogenin RNA in rat teeth was represented by two bands of 1.1 and 0.8 kb. Two oligonucleotide probes were designed that were specific for the long version and for the LRAP variant. The probes were used for in situ hybridization experiments on sections of developing maxillar teeth of rats between day 2 and day 15 after birth. Both RNA species were accumulated concomitantly and exclusively in cells of the inner enamel epithelium. Expression was first observed at the mesial cusp sides and finally involved the whole ameloblast layer except for the cells adjacent to the enamel-free region at the tip of the cusps. The early amelogenin RNA expression occurred adjacent to the initial deposition of the dentin matrix. Low amounts of amelogenin RNA persisted after the differentiation of ameloblasts into the maturative stage. The sequence of events was similar in all three molars.

Immunocytochemical evidence for a stepwise assembly of Balbiani ring premessenger ribonucleoprotein particles.

In the active Balbiani ring (BR) genes of the dipteran Chironomus tentans, the assembly of a specific pre-mRNP particle can be analyzed in situ, and the incorporation of hnRNP proteins into the nascent pre-mRNP can be directly visualized by immunoelectron microscopy. In the present study we have shown that hrp36, one of the major hnRNP proteins in Chironomus tentans, is continuously added to the nascent BR pre-mRNP particle throughout transcription and is localized along the entire BR RNP fiber. Interestingly, hrp36 becomes concealed during the structural transition that occurs during the formation of the mature BR RNP particle. This conclusion is based on the observation that hrp36 can be revealed by a monoclonal antibody during the initial assembly of the BR RNP fiber but becomes almost undetectable in the final packaging stage. The hrp36 protein, however, is not removed from the BR RNP particle since the ability of the monoclonal antibody to reveal hrp36 is restored by artificial relaxation of mature BR RNP particles. Another major hnRNP protein, hrp45, is also incorporated in a continuous manner into the nascent pre-mRNP fiber but remains accessible in mature BR RNP particles. Our results provide immunocytochemical evidence for drastic structural changes occurring in the final stage of BR pre-mRNP packaging, and suggest that different hnRNP proteins might be differently involved in the pre-mRNP assembly process.

Collagen mRNA expression during tissue development: the temporospacial order coordinates bone morphogenesis with collagen fiber formation.

Bone formation of the maxilla and premaxilla of rats was studied by in situ hybridization, using probes for fibrillar collagen mRNAs. Chondroblasts, osteoblasts, fibroblasts and peripheral bone cells differed in their expression patterns. Prospective nasal chondroblasts expressed collagen alpha1(II) and alpha1(XI) RNA from day 15 post coitum. Bone formation in the adjacent maxilla and premaxilla started around day 17: groups of osteoblasts, representing ossification centers, expressed collagen alpha1(I) RNA strongly, and alpha1(V), alpha2(V) and alpha1(XI) RNA weakly, but they were deficient in collagen alpha1(III) RNA. As the centers expanded, osteoblasts in the resulting bone domains expressed collagen alpha1(I) RNA in abundance, whereas collagen alpha1(III) RNA was absent. Bone domains were surrounded by fibroblasts containing collagens alpha1(I), alpha1(III) and alpha2(V) RNA. Widely separated fibroblasts underwent condensation into densely packed periosteum and sutural soft tissues. Cells at the periphery of fast-growing bone domains also displayed, apart from collagen alpha1(I) RNA, collagens alpha2(V) and alpha1(XI) RNA. Given the continuous recruitment of cells from the periosteum, peripheral bone cells represent differentiating osteoblasts synthetizing collagens alpha2(V) and alpha1 (XI) RNA transiently. Thus, gene expression during osteoblast differentiation reflects synthesis of fiber components during bone growth, since collagen V is located in the center of fibers consisting primarily of collagen I.

Distribution of protein kinase C alpha and accumulation of extracellular Ca2+ during early dentin and enamel formation.

Activation of the protein kinase C (PKC)-related signal transduction system has been associated with phenotypic expression in a wide variety of cell types. In in vitro studies, it has often been activated by relatively small increases in the Ca2+ concentration ([Ca2+]) in the medium. The studies reported here explored the hypothesis that localized increases in the extracellular [Ca2+] and activation of the PKC-related pathway may be involved in early dentin and enamel formation. Whole-head, freeze-dried sections through the developing molars of 5-day-old rats were evaluated by methods that localized non-crystalline Ca2+. Immunohistochemical methods were adapted for use with the freeze-dried sections, and two monoclonal antibodies were used to localize PKC alpha in the formative cells of the developing teeth. Low concentrations of extracellular Ca2+ were observed in the early, unmineralized dentin in the area of ameloblast differentiation. Increased concentrations occurred at the point of initial dentin mineralization, immediately before the beginning of enamel matrix deposition. PKC alpha was localized in the differentiating odontoblasts, at the beginning of dentin matrix deposition. It was intensely localized in the distal borders of the pre-ameloblasts, and appeared to redistribute in the cells during ameloblast differentiation. These observations suggest that local increases in the extracellular [Ca2+] and the PKC signal transduction pathway may be involved in key inductions in the early stages of dentin and enamel formation.

Expression of collagen alpha1(I) mRNA variants during tooth and bone formation in the rat.

Collagen alpha1(I) mRNA is composed of two variants of 5 and 6 kb, differing in the length of the 3' untranslated region. In this work, the nucleotide sequences of the two rat mRNA variants were compared, and their expression pattern in cells forming bone, dentin, and cementum were analyzed. The sequences were determined from cDNA inserts of tooth and bone libraries plus directly from PCR fragments, obtained from bone. A total of 5721 bases of the rat collagen alpha1(I) sequence from cDNA of tooth and bone was determined. All sequences of the short variant were represented in the long variant. Only the alternatively poly-A additions gave rise to the variants in hard tissue. Two oligonucleotides were chosen as probes, one of which recognized, on Northern blots, the two bands of 5 and 6 kb, and the other the 6-kb variant only. The oligonucleotides were used in in situ hybridization experiments, for study of the distribution of the variants in different extracellular matrix-forming cells. Osteoblasts, odontoblasts, and cementum-associated cells were closely examined in sections from rat maxillae from 2 to 25 days of age. A similar or identical pattern of mRNA expression was observed with both oligonucleotides, indicating that the two mRNA variants were co-expressed in all cases.

Sutures, growth plates and the craniofacial base--experimental studies in the toothless (tl-osteopetrotic) rat.

The craniofacial skeleton develops from a base in which coordinated growth at sutures and growth centres assures the development of normal form. In this report we describe features of retarded postnatal craniofacial development in the osteopetrotic mutation, toothless (tl), in the rat in which bone growth in both the nasal area and the cranial base is reduced, suggesting that the mutation affects bone formation in sutures and growth plates. We began a systematic search for potential mechanisms by analysing the expression in time and intensity of RNA coding for collagens type I (Col I) and type III (Col III) analysed by in situ hybridisation of cells in the premaxillary-maxillary suture (PMMS). In the centre of the PMMS of tl rats, cells expressing Col I and Col III appeared later than in normal littermates and exhibited lower signal. During osteoblast recruitment from the suture centre into the bone domains, Col III RNA expression is switched off. Osteoblasts expressing Col I in abundance, but no Col III, appeared in the flanking bone regions of tl rats later than in normal littermates. It is proposed that the tl mutation restricts the number of available osteoblast progenitor cells, and that the shortage of these cells affects bone growth in the PMMS and in the cranial base. Additional analyses are needed to test this hypothesis and to understand the developmental dynamics in the cranial base.

Rat enamel contains RP59: a new context for a protein from osteogenic and haematopoietic precursor cells.

We have recently identified a protein, RP59, in bone marrow cells and young osteoblasts, in cells involved in bone repair and in young erythroblasts and megakaryocytes. Here, we report immunohistochemical data at the light- and electron-microscope level indicating that RP59 is also present in newly secreted tooth enamel of the rat and in ameloblasts, the formative cells. In enamel matrix, RP59 was located proximal to secretory ameloblasts only, i.e. in newly secreted material. Distal enamel and enamel in association with maturation stage ameloblasts were unlabelled. Secretory ameloblasts contained RP59 in the matrix-proximal region including Tomes' processes, post-secretory ameloblasts in the cell-matrix interface. Western blotting of proteins from tooth germs identified RP59 as a band at 90 kD, co-migrating with RP59 from bone marrow and spleen. Antisera versus a chemically synthesised RP59 peptide and versus a bacteria-synthesised protein fragment reacted in the same manner. In situ hybridisation of tooth tissue revealed RP59 RNA specifically in ameloblasts. The reverse transcription/polymerase chain reaction method identified tooth RNA coding for RP59. Sequence analysis indicated that RP59 RNA from tooth and marrow had the same sequence. An internal sequence motif was found in rat RP59 resembling a signal implicated in secretion of the chicken "engrailed" gene product. The findings indicate that RP59 is a genuine product of ameloblasts and that it is secreted in the course of enamel formation together with other matrix components.

Events in glucocorticoid hormone action. A correlation of histone H1 variant pattern changes, hormone binding to cell nuclei, and induction of mouse mammary tumor virus RNA.

To approach experimentally changes of chromatin structure introduced by glucocorticoids, the histone H1 compositions of hormone-treated and non-treated mouse mammary tumor cells of the GR line [Ringold, G., Lasfargues, E. Y., Bishop, J. M. and Varmus, H. E. (1975) Virology 65, 135-147] were compared. To define the biologically important hormone concentration range, the cells were exposed to different concentrations of triamcinolone, a synthetic glucocorticoid. The induction of mouse mammary tumor virus (MMTV) RNA was measured by cDNA excess hybridization, and the amount of hormone bound to nuclei was determined by a filter-binding assay. Between 0.3 nM and 30 nM triamcinolone the relative increase in nuclear bound hormone corresponded well with the relative induction of MMTV RNA. The half-life of triamcinolone in nuclei of growing cells was 1 h, as measured by a pulse-chase experiment. Reversed-phase high-performance liquid chromatography of histone H1 resulted in its separation into four subfractions. The treatment of cells with biologically active glucocorticoid, 3 nM or 30 nM triamcinolone or 1 microM dexamethasone, resulted in changes in the relative amounts of two subfractions and to a positional shift of two subfractions as compared to untreated cells. No changes were observed after exposure to 3 nM dexamethasone, a concentration which does not induce MMTV RNA [Ringold, G. M., Yamamoto, K. R., Tomkins, G. M., Bishop, J. M. and Varmus, H. E. (1975) Cell 6, 299-305].

Isolation and characterisation of a transcribing polynucleosomal chromatin fraction.

A method is described for preparation of a fraction of chromatin enriched in transcribing regions from nuclei of mouse GR cells. This fraction, released by mild staphylococcal nuclease digestion of isolated nuclei, contains 2 to 10% of the DNA as polynucleosomal chromatin together with 50-70% of pulse-labelled RNA and about 90% of all template-engaged RNA polymerase B molecules, titrated with (3H)-alpha-amanitin. Hybridisation of DNA from this chromatin fraction to total nuclear RNA in excess shows that it is enriched in frequently-transcribed DNA sequences. A modification of the Miller technique, allowing the spreading of the active chromatin fraction for electron microscopy, has been developed. Examination of the spreads reveals that this chromatin fraction contains 20-100 nucleosome-long polynucleosomal chains bearing lateral RNP fibrils interpreted as nascent transcripts. The average length of the DNA fragments in the fraction is greater than that of average transcribed regions, suggesting that the transcribed regions are linked to flanking segments whose chromatin conformation probably contributes to the selective release of transcribing chromatin.

Morphogenesis in Mucor mucedo: mutations affecting gamone response and organ differentiation.

Mutants of Mucor mucedo minus strain that are affected in their trisporic acid (TA) mediated zygophore formation have been isolated. We have found mutants with cold sensitive (cs), with temperature sensitive (ts) and without zygophore formation as well as mutants with unstable zygophores (Zst-). From the appearence of certain pleiotropic phenotypes we deduce a one-dimensional sequence of states of competence of the mycelium to form different organs. TA appears to be a growth substance for zygophores acting on one transient state of competence. The fact that all isolates with lowered response to TA also have a lowered response to the mating type specific TA-precursor P strongly suggests that P has to be converted into TA before inducing zygophore growth. Furthermore, one mutant with lowered sensitivity to TA exhibits an excess zygophore formation in the presence of high TA-concentrations, while high concentrations of P cause a depressed zygophore formation (Fig. 6). Our interpretation of this behaviour is that P acts as an antagonist to TA in the regulation of zygophore growth.

The complexity of 75S premessenger RNA in balbiani ring granules studied by a new RNA band retardation assay.

Under normal growth conditions, Balbiani ring granules constitute premessenger ribonucleoprotein (RNP) particles synthesized in two chromosomal puffs, Balbiani ring (BR) 1 and 2, in the larval salivary glands of Chironomus tentans. At least three genes encoding 75S RNA are present in these two BRs: one in BR1 and two in BR2 (BR2.1 and BR2.2). The complexity of BR granule 75S RNA was studied by agarose gel electrophoresis under non-denaturing conditions. We recorded three main bands, designated I, II and III. Experiments with denaturing gels demonstrated that the differences in migration reflected mainly, but not exclusively, conformational differences. Northern blotting experiments showed that band I contained BR1 sequences, band II contained BR2.1 sequences, and band III contained BR2.2 sequences. To study whether additional genes contributed to the BR granule 75S RNA, an RNA band shift assay was developed. When an oligodeoxyribonucleotide complementary to repetitive BR1 and BR2.2 sequences was hybridized to 75S RNA prior to electrophoresis, bands I and III were retarded but not band II. An oligonucleotide complementary to a repetitive BR2.1 sequence only shifted band II. Since no detectable 75S RNA remained unchanged in these experiments, and all bands were identified by Northern blotting, all the BR granules are likely to originate from the BR1, BR2.1 and BR2.2 genes; no additional genes have to be invoked. Possible applications of the new RNA band shift assay are discussed.

Influence of titanium ion on mineral formation and properties of osteoid nodules in rat calvaria cultures.

The effect of the addition of titanium ion (Ti) on osteoblast function and the mineralization of osteoid nodules in rat calvaria cultures was characterized. Concentrations of 10 ppm of Ti or more inhibited cell proliferation; 5 ppm or less either had no effect or stimulated proliferation. The number of nodules formed was not influenced by 5 ppm of Ti, but mineral deposition in nodules was suppressed, as revealed by von Kossa staining. Likewise, 5 ppm of Ti inhibited the incorporation of [(45)Ca] in cultures during nodule formation even if the Ti was withdrawn from the medium when mineralization was initiated. In order to test whether the synthesis of osteoid components was affected, the expression of osteonectin (OSN), osteopontin (OPN), osteocalcin (OSC), and alkaline phosphatase (ALP) mRNAs as well as ALP enzyme activity was analyzed. The expression of OSN and OPN mRNAs was reduced dramatically, but OSC mRNA was little affected by 5 ppm of Ti. Ti delayed the development of ALP mRNA expression and enzyme activity relative to the controls. Thus Ti treatment changed the proportional composition of cellular mRNA contributing the osteoblast phenotype.

The elementary RNP fiber--not the higher order structure--determines the all-or-none disintegration behaviour of Balbiani ring pre-messenger RNP particles upon RNase A treatment.

Balbiani ring premessenger ribonucleoprotein (RNP) particles are built from a 7-nm RNP fiber which is tightly folded into a ring-shaped RNP ribbon. Isolated particles are known to disintegrate in all-or-none fashion upon RNase A treatment. In the present study we investigated whether this mode of disintegration is dependent on an intact particle structure or is inherent in the 7-nm fiber. When treated at low ionic strength, the Balbiani ring (BR) particles lost their higher order structure and the 7-nm fiber was unpacked, as evidenced by sucrose gradient sedimentation and electron microscopy. When treated with RNase A, unfolded as well as intact particles disintegrated in the all-or-none fashion, with similar kinetics and without apparent intermediates. Proteinase K treatment, however, obliterated this pattern: the protein-free particle RNA degraded progressively. As the typical disintegration pattern of the particles was not altered by unfolding, but was lost by deproteinization, the all-or-none mode of disintegration is likely to be a property of the 7-nm RNP fiber.

Assembly and disassembly of spliceosomes along a specific pre-messenger RNP fiber.

Transcriptionally active Balbiani ring (BR) genes in the salivary glands of the dipteran Chironomus tentans were studied by immunoelectron microscopy to establish the distribution of spliceosome components along a specific pre-messenger ribonucleoprotein (pre-mRNP) fiber. The BR genes are 35-40 kb in size with three introns close to the 5' end and one close to the 3' end; a very large middle portion lacks introns. As a rule the 5' introns are spliced concomitant with transcription in the promoter proximal third of the gene, while the 3' intron is spliced post-transcriptionally. The BR genes with growing pre-mRNPs were visualized in situ, while completed and released pre-mRNPs were isolated from the nucleoplasm and studied unfolded on a grid surface. An anti-snRNP antibody (Y12) bound mainly to the promoter proximal third of the BR gene (86%) and only to a minor extent to the middle and distal thirds (7 and 7% respectively). An antibody to an hnRNP protein reacted with the proximal, middle and distal regions to an increasing extent (17, 38 and 45% respectively), reflecting the increase in size of the growing transcription product. In the nucleoplasmic pre-mRNP particle only one end of the RNP fiber was labeled by Y 12, presumably the 3' end; the anti-hnRNP antibody decorated the entire RNP fiber. Thus, the snRNPs do not associate along the whole pre-mRNP fiber but rather bind to the 5' and 3' ends, i.e. the regions containing the introns. The results also imply that the spliceosomes both assemble and disassemble rapidly on the pre-mRNP fiber.

A new protein expressed in bone marrow cells and osteoblasts with implication in osteoblast recruitment.

To study osteoblast recruitment from bone marrow cells, a rat femur cDNA library was screened by in situ hybridization for novel mRNA sequences that are frequently expressed in both marrow cells and osteoblasts. One isolated clone, called RP59, is described here. Northern blots indicated two bands of 2.6 and 2.8 kb in femur and spleen, tissues containing high amounts of immature mesenchymal cells, and no or little expression in other tissues. The cDNA sequence revealed a reading frame for a repetitive protein composed of arrays of 14-mers and phased phosphorylation sites. Antisera versus RP59 detected a single band of 90 kDa by Western blotting of femur extract. Immunohistochemistry indicated strong RP59 presence in the cytoplasm of bone marrow cells and weaker presence in nuclei of osteoblasts. Intermediate stages were found between strongly labeled, round, free bone marrow cells and weaker labeled, fibroblast-like young osteoblasts associated with bone matrix. These data indicated that marrow cells with high RP59 content were recruited into growing bone tissue. RP59 may help to study the transition of bone marrow cell to osteoblast in more detail.