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

1）我们对人COL 11 A1、小鼠COL 5 α1和人COL 5A 3基因的近端启动子进行了鉴定。在COL 11 A1中，从-199到+1的片段对于激活基础转录调节是必需的。在-147至-121范围内的ATTGG序列对于结合是关键的。我们鉴定了与该区域结合的CBF/NF-Y。在col 5 α1中，核心启动子被限定在转录起始位点上游231 bp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相关蛋白特异性结合这些区域。在这些因子中，CBF/NF-Y是该基因表达的关键因子。在C 0 L5 A3中，核心启动子被限定在转录起始位点上游紧邻的约129 bp内。在这个区域，我们确定了四个DNA-蛋白质复合物。竞争性寡核苷酸分析和特异性抗体的超位移分析表明，复合物A与-122 ~-117结合，也由CBF/NF-Y组成。2）采用免疫组织化学结合原位杂交的方法检测了前-α3（V）胶原基因的表达。原α3（V）链在小鼠胎盘的脐部和羊膜中可见，而在绒毛膜绒毛和蜕膜中未见。在小鼠胚胎中，这些转录物在与骨形成以及发育中的肌肉和新生韧带相关的组织中可见。免疫组化结果显示，pro-α3（V）蛋白在小鼠胚胎骨发育过程中大量积累。电镜下可见胃食管交界处SMC周围BM增厚。

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.