GENETIC EXPRESSION OF NONCOLLAGENOUS BONE PROTEINS

骨连接素（Osteonectin，ON）和骨钙素（boneglaprotein，BGP）是骨形成过程中 在骨和牙本质的细胞外基质中发现， 在矿化中发挥作用。 第三种蛋白质最初被鉴定为 作为Saos-2抗原（SAOS-P80），目前结构未知， 功能，似乎是特定的成骨细胞。 触目所及尽是些 在壁内胚层的细胞外发现的小鼠胚胎的种同源物 和牛43 K蛋白首次在内皮细胞培养物中鉴定。 的 后一种蛋白质被认为与细胞迁移和基底有关 膜细胞外基质合成和建模。 近日有 发现人类血小板含有大量的ON， 它可通过凝血酶活化而释放。 血小板活化可能在 维持止血和血管床的作用， 组织损伤后的修复和发病机制。 氨基酸序列 对于血小板ON，将通过合成、克隆和DNA 人红白血病（HEL）和正常人中编码ON的cDNA的测序 巨核细胞 抗骨和血小板抗体的表位定位 ON将使用PDagt 11缺失/表达系统进行。 的5' 还将分离和表征人ON基因的末端， 基因调控元件 5'端转录上游区域 起始位点以及5'端非翻译区中的大内含子的部分 将cDNA区域测序并放入pSV 2-CAT/哺乳动物细胞中 表达系统以评估基因的调控元件。 Saos- 将用单克隆抗体筛选2/PDdagt 11表达文库。 SAOS-P80 cDNA的抗体。 阳性噬菌体的特征在于： 酶图谱和DNA测序以及由此获得的氨基酸序列 推导 SAOS-P80 cDNA将用作探针以分离人SAOS-P80。 P80基因来自人基因组/cDNA EMBL 3文库。 基因将是 其特征在于，以类似于针对ON提出的方式，并且与 其他成骨细胞蛋白包括ON、BGP、碱性成骨细胞蛋白 磷酸酶和I型胶原蛋白。 来自这些研究的cDNA将用作 探针原位杂交和研究mRNA水平在各种 正在经历成骨细胞分化和发育或相关的 骨骼和结缔组织疾病

Osteonectin (ON) and bone gla protein (BGP) are proteins predominantly found in the extracellular matrix of bone and dentin where they are thought to play a role in mineralization. A third protein, originally identified as an Saos-2 antigen (SAOS-P80), is currently unknown structure and function and appears to be specific to osteoblastic cells. ON is the species homologue of mouse SPARC found extracellularly in parietal endoderm and bovine 43K protein first identified in endothelial cell cultures. The latter proteins are believed to be involved in cell migration and basement membrane extracellular matrix synthesis and modeling. Recently, it was discovered that human platelets contain a significant amount of ON and that it is releasable by thrombin activation. Platelet ON may play an important role in maintaining hemostasis and the vascular bed, and in bringing about repair following tissue injury and pathogenesis. The amino acid sequence for platelet ON will be determined from synthesis, cloning, and DNA sequencing of cDNAs encoding ON in human erythroleukemic (HEL) and normal megakaryocytic cells. Epitope mapping of antibodies to bone and platelet ONs will be performed with a lambdagt11 deletion/expression system. The 5' end of the human gene for ON will also be isolated and characterized as to genetic regulatory elements. The region upstream of the 5' transcriptional start site as well as portions of a large intron in the 5' non-translated region of the cDNA will be sequenced and put into a pSV2-CAT/mammalian cell expression system to evaluate regulatory elements of the gene. Saos- 2/lambdagt11 expression libraries will be screened with monoclonal antibodies for SAOS-P80 cDNAs. Positive phage will be characterized by enzyme mapping and DNA sequencing and the amino acid sequence thereby derived. SAOS-P80 cDNA will be used as a probe to isolate the human SAOS- P80 gene from a human genomic/lambdaEMBL3 library. The gene will be characterized in a manner similar to that proposed for ON and compared to those of other osteoblastic proteins including ON, BGP, alkaline phosphatase, and type I collagen. cDNAs from these studies will be used as probes for in situ hybridization and to study mRNA levels in a variety of cells undergoing osteoblastic differentiation and development or associated with bone and connective tissue disease.