Two RFLPs in the 5' end of the human osteonectin (ON) gene.
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Carrier Proteins↗
Biomedical subjects
M F Young
Publications and source records attributed to M F Young.
Isolation of the osteonectin gene: evidence that a variable region of the osteonectin molecule is encoded within one exon.
A complementary DNA clone for bovine osteonectin was used to isolate the osteonectin gene from two libraries of bovine genomic DNA fragments. Two overlapping clones were obtained whose relationship was determined by restriction mapping and sequence analysis. The two clones contain the entire osteonectin coding region spanning approximately 11 kilobases of genomic DNA. The coding region of the gene was determined, by electron microscopy and DNA sequencing, to reside in nine exons. In addition, there is at least one 5' exon interrupted by an intron in the 5'-nontranslated sequence of the gene. Excluding this 5' exon and the 3'-terminal exon, the exons are small and approximately uniform in size, averaging 130 +/- 17 base pairs. Three of the exons at the 5' end of the gene were sequenced and appear to encode discrete protein domains. For example, the putative exon 2 contains the coding region for the leader peptide of the molecule. The amino-terminal protein sequence was determined for osteonectin extracted from human, rabbit, and chicken bone and compared with those for bovine, mouse, and pig osteonectin. These data suggest that osteonectin is highly conserved between species, interspecies changes being seen primarily at the amino terminus of the protein and specifically in the region encoded by putative exon 3 in the bovine gene.
Amino Acid Sequence↗
Osteonectin cDNA sequence reveals potential binding regions for calcium and hydroxyapatite and shows homologies with both a basement membrane protein (SPARC) and a serine proteinase inhibitor (ovomucoid).
Osteonectin is a prominent noncollagenous protein of developing bone. A 2150-base-pair cDNA coding for osteonectin, isolated from a bovine bone cell lambda gt11 expression library, was sequenced and identified by comparison with protein sequence data. The nucleotide sequence predicts that osteonectin contains 304 amino acids, including a 17-residue signal peptide. Analysis of the deduced protein sequence suggests that the secreted protein contains at least four distinct structural domains. An acidic region at the amino terminus of the protein appears to be a potential hydroxyapatite-binding site. This is followed by a second domain, rich in cysteine, that shows sequence homology with cysteine-rich domains in turkey ovomucoid and other serine proteinase inhibitors. Two sequences homologous with central calcium-binding loops of "EF hands" and thus having potential to be high-affinity calcium-binding sites are located in two other domains within the carboxyl-terminal half of the protein. Finally, the osteonectin sequence shows near identity (greater than 90%) with another protein, SPARC (secreted protein, acidic and rich in cysteine), secreted by mouse parietal endoderm. These data suggest that osteonectin, a protein present in bone and other selected tissues, is a multifunctional protein.
Amino Acid Sequence↗
Osteonectin/SPARC/BM-40 in human decidua and carcinoma, tissues characterized by de novo formation of basement membrane.
The identification and cDNA cloning of bone-enriched osteonectin and parietal endoderm-enriched SPARC indicated that these proteins share greater than 90% homology. The present study reports substantial expression of this Mr 43,000 protein in human decidua and carcinoma. Metabolic labeling of human decidua tissue in organ culture demonstrated significant osteonectin biosynthesis. Further, Northern analysis and in situ hybridization showed that the osteonectin mRNA level in human decidua is very high. Immunohistochemically, the large mature decidual cells exhibited faint cytoplasmic immunoreactivity with antibodies to osteonectin and a distinct immunoreactivity of the newly deposited basement membranes encircling these cells. The intermediate-sized decidual cells exhibited a strong cytoplasmic immunostaining with antibodies to osteonectin. The small elongated stromal cells were devoid of osteonectin immunoreactivity. Blood vessels exhibited variable positive immunoreactivity. The osteonectin expression in 38 cases of human carcinomas was examined. In well-differentiated carcinomas osteonectin immunoreactivity was located in the basement membrane area, codistributing with laminin. Cytoplasmic osteonectin immunoreactivity was found in poorly differentiated carcinomas. Stromal cells in the peritumoral tissue and some vessels were immunoreactive. Using in situ hybridization, it appeared that both the tumor cells and the stromal cells contained osteonectin transcripts. In normal steady state human nonosseus tissues investigated, osteonectin was found inconsistently in the basement membranes only, as based on the present immunohistochemical technique. These results suggest that the osteonectin/SPARC gene appears activated in certain human tissues characterized by de novo formation of basement membrane.
Animals↗
Heterogeneity of amelogenin mRNA in the bovine tooth germ.
The amelogenins are a complex mixture of hydrophobic proteins that are the major organic component of developing enamel. To study the molecular mechanisms underlying the heterogeneity of the amelogenins we isolated cDNA clones encoding these proteins. The clones were definitively identified by hybrid-selected translation experiments and by comparison of the DNA sequence with the protein-derived amino acid sequence. Southern hybridization of bovine genomic DNA indicated that amelogenin is a single copy gene. However, Northern hybridization experiments distinctly showed two major species of mRNA, each of which were sufficiently large enough to encode the highest known molecular weight species of amelogenin proteins. Furthermore, immunoprecipitation of hybrid-selected translation products using isolated amelogenin cDNA showed multiple, translated protein products. These data are supportive of a differential mRNA processing mechanism involved in generating a heterogeneous family of amelogenin matrix proteins from a single gene.
Ameloblasts↗
Osteoblasts synthesize and respond to transforming growth factor-type beta (TGF-beta) in vitro.
Transforming growth factor-type beta (TGF-beta) has been identified as a constituent of bone matrix (Seyedin, S. M., A. Y. Thompson, H. Bentz, D. M. Rosen, J. M. McPherson, A. Conti, N. R. Siegel, G. R. Gallupi, and K. A. Piez, 1986, J. Biol. Chem. 261:5693-5695). We used both developing bone and bone-forming cells in vitro to demonstrate the cellular origin of this peptide. TGF-beta mRNA was detected by Northern analysis in both developing bone tissue and fetal bovine bone-forming cells using human cDNA probes. TGF-beta was shown to be synthesized and secreted by metabolically labeled bone cell cultures by immunoprecipitation from the medium. Further, TGF-beta activity was demonstrated in conditioned media from these cultures by competitive radioreceptor and growth promotion assays. Fetal bovine bone cells (FBBC) were found to have relatively few TGF-beta receptors (5,800/cell) with an extremely low Kd of 2.2 pM (high binding affinity). In contrast to its inhibitory effects on the growth of many cell types including osteosarcoma cell lines, TGF-beta stimulated the growth of subconfluent cultures of FBBC; it had little effect on the production of collagen by these cells. We conclude that bone-forming cells are a source for the TGF-beta that is found in bone, and that these cells may be modulated by this factor in an autocrine fashion.
Animals↗
A characterization of amelogenin messenger RNA in the bovine tooth germ.
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Amelogenin↗
Molecular cloning of bovine amelogenin cDNA.
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Amelogenin↗
Characterization of bone PG II cDNA and its relationship to PG II mRNA from other connective tissues.
Two cDNA clones encoding the small proteoglycan II (PG II) of bone were isolated from a lambda gt11 expression library. These clones expressed recombinant protein which was cross-reactive with polyclonal and monoclonal antisera to PG II molecules from several connective tissues. The longest clone, lambda Pg 20 was studied in detail. The clone was shown to encode PG II by hybrid selected translation and immunoprecipitation. Northern analysis showed two species of the PG II message of approximately 1.4 and 1.8 kb. Substantial amounts of PG II message were found in bone, tendon, articular cartilage, skin, smooth muscle and cornea. Trace amounts of message were also detected in liver and brain. Radiolabeled bovine PG II cDNA hybridized to RNA from several other species including the human, rat and chicken. The level of PG II mRNA in chick embryonic fibroblasts was sensitive to transformation by Rous sarcoma virus.
Animals↗
Osteonectin mRNA: distribution in normal and transformed cells.
Overlapping cDNA clones encoding bovine osteonectin were isolated from a lambda gt11 expression library constructed from bovine bone cell mRNA. The longest clone, lambda On 17 (insert size 2.0 kb) was studied in detail. The clone was shown to encode osteonectin by hybrid select translation experiments and by DNA sequence analysis. Northern analysis of bone cell RNA showed the length of the osteonectin mRNA to be 2.0 kb. Osteonectin message was found in bone but not in soft tissue (liver and brain) preparations consistent with the distribution of the protein in these tissues. On the other hand, osteonectin message was observed in tendon, a tissue in which little or no osteonectin protein is found in vivo. Hybridization of osteonectin cDNA was detected in cells from a number of species including human, rat, mouse and chick. The level of osteonectin mRNA was drastically decreased in chick embryo fibroblasts transformed by Rous sarcoma virus.
Animals↗
Effects of diflubenzuron on the mouse liver.
Diflubenzuron (DFB), a potent inhibitor of insect chitin synthesis, was administered to Swiss Webster mice in a 30-day oral intubation study. Animal groups received either no treatment, vehicle control (Polyethylene glycol 400), or DFB suspensions at doses of 125, 500, and 2,000 mg/kg body weight. Hepatic glutathione S-transferase activity as well as morphological characteristics were studied. DFB was shown to elicit hepatocellular changes at all dose levels. The activities of three glutathione S-transferases (S-aryl, S-aralkyl, and S-epoxide) were all altered after DFB administration. Light microscopy revealed radial arrays of hepatocellular vacuolization between the portal and central vein areas. Electron-microscopic examination, verified by morphometric analysis, revealed degenerative changes as well as an increased volume density of the endoplasmic reticulum.
Animals↗
Isolation and partial characterization of genomic clones coding for a human pro-alpha 1 (II) collagen chain and demonstration of restriction fragment length polymorphism at the 3' end of the gene.
We have isolated two clones containing 19 kilobases (kb) of the human gene coding for a pro-alpha 1 (II) collagen chain from human lambda genomic DNA libraries. A 3' clone, HC2A, was selected by cross-hybridization with a cDNA clone containing sequences coding for the carboxy propeptide of chick type II procollagen. A second clone, HC2B, was obtained by screening the library with the 5' part of HC2A. The sequence analysis of exon 3 corresponding to the C propeptide reveals the presence of stretches of conserved nucleotides between the human and the chick type II procollagen genes. On Northern blots, the human collagen clone hybridizes strongly to a 5.5-kb RNA for the rat type II procollagen chain. Finally, studies of genomic DNAs from normal individuals reveal the presence of a HindIII and a BamHI polymorphic site at the 3' end of the gene.
Amino Acid Sequence↗
Methylation of type II and type I collagen genes in differentiated and dedifferentiated chondrocytes.
The methyl-sensitive restriction endonucleases HpaII and HhaI as well as the methyl-insensitive enzyme MspI were used to examine the methylation status of the pro-alpha 1(II) collagen gene of cartilage. Five different cell types with varying abilities to express type II collagen were studied. Chick embryo chondrocytes express type II collagen, while 5-bromodeoxyuridine-treated chondrocytes, retinoic acid-treated chondrocytes, chick embryo fibroblasts, and erythrocytes do not synthesize type II collagen. Both cDNA and genomic probes for the pro-alpha 1(II) collagen gene were used, covering the complete 3' end of the gene and its flanking sequences. The pro-alpha 1(II) collagen DNA was undermethylated in chondrocytes, compared to either fibroblasts or erythrocytes. However, the methylation of the 5-bromodeoxyuridine-treated and retinoic acid-treated chondrocytes was identical to that of control chondrocytes. The methylation pattern of two regions of the gene of the pro-alpha 2(I) collagen chain was identical in all cell types tested, whether or not the gene was expressed. Our results indicate that genes for these collagen chains differ in their methylation pattern. The type II collagen gene shows reduced methylation in expressing cartilage, but does not acquire an increase in methylation in "dedifferentiated" chondrocytes. The changes in DNA methylation that occur during cell differentiation do not appear to be sufficient to explain gene activation and deactivation.
Animals↗
Isolation of cDNA and genomic DNA clones encoding type II collagen.
A cDNA library constructed from total chick embryo RNA was screened with an enriched fraction of type II collagen mRNA. Two overlapping cDNA clones were characterized and shown to encode the COOH propeptide of type II collagen. In addition, a type II collagen clone was isolated from a Charon 4A library of chick genomic fragments. Definitive identification of the clones was based on DNA sequence analysis. The 3' end of the type II collagen gene appears to be similar to that of other interstitial collagen genes. Northern hybridization data indicates that there is a marked decrease in type II collagen mRNA levels in chondrocytes treated with the dedifferentiating agent 5-bromodeoxyuridine. The major type II collagen mRNA species is 5300 bases long, similar to that of other interstitial collagen RNAs.
Amino Acid Sequence↗
Uptake and distribution of exogenous serum proteins in the cultured rat embryo.
Head fold stage rat embryos were cultured on a medium consisting of adult rat serum for 48 hr. During the final 10 or 20 hr of culture [35S]-methionine was added to the medium. Sera from visceral yolk sac blood vessels, exocoelomic cavity fluid, and amniotic sac fluid were analyzed by SDS-polyacrylamide gel electrophoresis followed by Amido Black staining and fluorography. Amido Black staining showed bands in all three embryonic fluid compartments which migrated at rates comparable to transferrin, alpha-fetoprotein, albumin, IgG, and hemoglobin. Fluorography, however, showed that radioactivity was restricted to alpha-fetoprotein, IgG, and hemoglobin. This suggested that at least some transferrin and albumin were transferred intact from the exogenous medium to the embryonic fluid compartments.
Amniotic Fluid↗
Synthesis of serum proteins by cultures of chick embryo yolk sac endodermal cells.
Endodermal cells were isolated from yolk sacs of 3-day chick embryos and cultured for 6 days in Eagle's minimal essential media plus 10% fetal calf serum. During this period cells rapidly lost their ability to synthesize DNA as judged by [3H]thymidine incorporation into DNA. In spite of this loss of DNA synthesis serum protein synthesis and secretion remained at a constant 45% of total protein synthesis and secretion. This was determined by immunoprecipitation of culture media using antibodies directed against embryonic chick serum proteins. Media were also analyzed for the synthesis and secretion of specific serum proteins using polyacrylamide gel electrophoresis. The relative synthesis and secretion of the individual serum proteins followed that previously observed in ovo with the exception of alpha-globulin-a which became undetectable. When culture media were supplemented with ovalbumin or insulin the relative synthesis and secretion of certin specific serum proteins were altered. However, analysis of these same media samples showed that the total amounts of serum protein synthesis and secretion were unaffected.
Animals↗
Case studies of juvenile leukosis.
Clinical signs, hematologic values and gross pathologic changes in two calves with juvenile leukosis were similar to those described in other reports. Although there was considerable variation of hematologic values between the two calves, both were within the range previously determined for leukosis animals. Hematologic variations were observed in sequential samples obtained from one animal in this study during the course of the disease. These data would indicate that the extreme variations observed between animals in juvenile leukosis hemograms may be the result of aberrations caused by differences in relative sampling times. It is suggested that if hematologic parameters are to be meaningfully used as an aid in the diagnosis of juvenile bovine leukosis, multiple hematologic samplings should be taken and correlated with the disease state of the animal and time before death.
Animals↗