Analysis of functienal domain and expression in bone, cartilage and muscle of minor collagen

1)首先采用寡核苷酸方法确定小鼠α3(V)基因的转录起始点。该基因位于ATG密码子上游约100bp处。我们利用核心启动子区域的结构-功能分析来阐明该基因的转录特征。通过瞬时转染实验，将核心启动子定义在转录起始点上游约300bp的范围内。在该区域，我们通过凝胶迁移率改变分析(EMSA)确定了两个核因子结合位点：BS1(-130到-110)和BS2(-190到-170)。寡核苷酸竞争和超移位分析表明，CbF-3(V)链的N-末端球状结构域含有23个氨基酸组成的高度碱性的片段，与α-3(V)链上的BS1特异结合。该多肽对骨肉瘤细胞有特异的粘附性，但对软骨肉瘤细胞无粘附性。这一特征来源于该片段中的四个肝素结合位点。当多肽…更多的黏附在细胞上，形成肌动蛋白纤维，这是被抑制的Rho-Kinase抑制剂，Y27632.2)我们已经鉴定了人α1(III)胶原基因(COL3A1)的近端启动子。瞬时转染实验表明，-96至-34片段是激活转录所必需的。EMSA表明，根据细胞类型的不同，多种蛋白质以不同的组合形成DNA-蛋白质复合体。-79~-63bp是DNA蛋白质复合体形成的关键区域。3)XXIV胶原蛋白是一个新发现的胶原纤维形成家族成员，主要在骨组织中表达。细胞转染实验和DNA结合实验表明，Co124a1启动子的活性受上游顺式作用元件的控制，该元件可被c jun、CREB1、atf1和ATF2二聚体特异性结合识别。较少

1) Initially, to determine the transcription initiation site of mouse α3 (V) gene, Oligo-Cap methods was used. It was located about 100 bp upstream of ATG codon. We used structure-function analysis of the core promoter region to elucidate the transcriptional features of the gene. The core promoter was defined within about 300 bp immediately upstream from the transcription start site by transient transfection experiments. In this region, we identified two nuclear-factor binding sites by electrophoretic mobility shift assay (EMSAs): BS1 (-130 to -110), BS2 (-190 to -170). Oligonucleotide competition and supershift assays revealed that CBF,/NF-Y specifically bind to BS1.The N-terminal globular domain of the pro-α3 (V) chain has a unique structure that contains a highly basic segment of 23 amino acids. The peptide showed a specific adhesive feature to osteosarcoma cells but not to chondrosarcoma cells. This feature was derived from four heparin binding sites in the segment. When the peptid … More e adhere to the cell, actin fiber was formed, which was inhibited Rho-kinase inhibitor, Y27632.2) We have characterized the proximal promoter of the human α1 (III) collagen gene (COL3A1). Transient transfection assays indicated that the segment from -96 to -34 was necessary to activate transcription. EMSAs showed that the multiple proteins form the DNA-protein complex in different combinations depending on the cell types. The region of -79 to -63 bp was critical for DNA protein complex formation. This sequence was contained in the B element of mouse al (III) collagen gene (Col3a1) reported by other researcher.3) Collagen XXIV is a newly discovered and poorly characterized member of the fibril-forming family of collagen molecules, which is expressed predominantly in bone tissue. Cell transfection experiments in combination with DNA binding assay demonstrated that Co124a1 promoter activity was under control of an upstream cis-acting element, which was recognized by specific combination of c jun, CREB1, ATF1, and ATF2 dimers. Less