Research on collagen gene expression in chondrocyte and non-chondrocyte, and its function of molecular domains

1)我们鉴定了人COL11A1、小鼠COL5α1和人COL5A3基因的近端启动子。在COL11A1中，从-199到1的片段是激活基础转录调控所必需的。-147到-121之间的ATTGG序列对结合是关键的。我们鉴定了与该区域结合的CBF/NF-Y。在COL5α1中，核心启动子被定义在转录起始点上游231bp内。在这个区域，我们确定了三个核因子结合位点。SP1、CBF/NF-Y和Sp1相关蛋白与这些区域特异性结合。在这些因子中，CBF/NF-Y是该基因表达的关键因子。在COL5A3中，核心启动子被定义在转录起始点上游的-129bp内。在这个区域，我们鉴定了四个DNA-蛋白质复合体。同源寡核苷酸竞争分析和特异性抗体超移位分析表明，与-12 2~-117结合的复合体A也由CbF/NF-Y组成。2)采用免疫组织化学方法结合原位杂交技术检测了前α3(V)胶原基因的表达。在小鼠胎盘的绒毛和蜕膜中未见α3(V)链的表达。在小鼠胚胎中，转录本出现在与骨形成以及肌肉和新生韧带发育有关的组织中。然而，免疫组织化学显示，前α3(V)蛋白在小鼠胚胎发育的骨骼中积累。3)我们继续对XIX胶原基因缺失的小鼠进行了鉴定。电子显微镜下可见胃食道交界处SMC周围有较厚的骨髓基质细胞。

1)We have characterized the proximal promoter in the human COL11A1, mouse col5α1 and human COL5A3 gene. In COL11A1, a segment from -199 to +1 is necessary for the activation of basal transcriptional regulation. The ATTGG sequence within -147 to -121 is critical for the binding. We identified CBF/NF-Y that bound to the region. In col5α1, the core promoter was defined within the 231 bp upstream from the transcription start site. In this region, we identified three nuclear-factor binding sites. Sp1,CBF/NF-Y, and Sp1-related protein specifically bind to these regions. In these factors, CBF/NF-Y is a key factor in the expression of this gene. In COL5A3, the core promoter was defined within the -129 bp immediately upstream from the transcription start site. In this region, we identified four DNA-protein complexes. The competition assays using consensus oligonucleotides and supershift assays with specific antibodies showed that complex A, which binds to the -122 to -117, also consists of CBF/NF-Y.2)We examined pro-α3(V) collagen gene expression using immunohistochemical methods combined with in situ hybridization. The pro-α3(V) chain was seen in funis and amnion, but not chorionic villi and deciduas of mouse placenta. In mouse embryo, the transcripts were seen in tissues that were related to bone formation as well as developing muscle and nascent ligament. However, immunohistochemistry showed that pro-α3(V) protein accumulated rather in the developing bone of mouse embryo.3)We continued characterization of null mice of the collagen XIX gene. Electron microscopy showed thicker BMs around SMC in the gastroesophageal junction.