TYPE III COLLAGEN GENE EXPRESSION IN CARTILAGE

III型胶原蛋白存在于大多数结缔组织中，但没有发现 在透明软骨中。 然而，一种独特的，以前未描述的RNA 已在鸡软骨中检测到。 三型胶原蛋白- 相关RNA具有3800个核苷酸的表观大小，因此， 远小于真正的5700核苷酸III型胶原 mRNA。 这种独特RNA的3'端与DNA序列相似或相同。 真正的III型胶原mRNA。 然而，外显子1和2，以及 另外的外显子似乎从RNA的5 ′部分缺失。 RNA结构的这种差异表明， III型胶原基因中的选择性转录起始位点， 优选用于软骨。 因此，缺乏第三类 软骨中的胶原蛋白不是由于 III型胶原基因或III型胶原不稳定 在这个组织中的转录本。 相反，它必须是由于其他一些 可能与观察到的RNA改变有关 结构 在本申请中，提出实验来确定 软骨中III型胶原相关RNA的结构及其作用 阻止软骨中III型胶原蛋白的产生。 的 软骨RNA的结构将被确定，并与 真正的III型胶原mRNA。 第三型小鸡的5英尺半 还将分离胶原蛋白基因，并且启动子和任何 以前未描述的外显子将通过比较 外显子/内含子组织与cDNA序列。 这些实验将 表明软骨RNA的结构差异是否是由于 使用替代启动子，替代RNA加工事件，或 两者 它们还将定义RNA中的任何开放阅读框， 指示RNA是否可能编码蛋白质（非- 胶原蛋白或III型胶原的改变形式）。 的 软骨III型胶原相关RNA的亚细胞分布 将确定;这些实验将表明是否类型III 胶原蛋白相关的RNA主要位于细胞核或细胞核中。 细胞质，如果在细胞质中发现，它是否与 核糖体或核糖核蛋白颗粒。 这些实验， 结合上述序列数据， 防止软骨中III型胶原蛋白产生的可能机制。 如果基于序列和亚细胞分布数据，II型 胶原蛋白相关的RNA似乎编码一种蛋白质，该蛋白质将 分离并表征。 如果RNA似乎不太可能编码 蛋白质，RNA的其他可能的功能将被检查。 最后 顺式作用序列和反式作用因子，这是重要的 III型胶原基因启动子在细胞中的功能 将识别适当的组织。 这些实验将提供 增加对预防癌症的分子机制的了解 软骨中III型胶原蛋白的产生。 此外，他们将 提供III型胶原蛋白结构的更广泛分析 基因和它的调节比迄今为止可用的。

Type III collagen is found in most connective tissues, but is not found in hyaline cartilage. However, a unique, previously undescribed RNA species has been detected in chick cartilage. This type III collagen- related RNA has an apparent size of 3800 nucleotides, thus is considerably smaller than the authentic 5700 nucleotide type III collagen mRNA. The 3' half of this unique RNA is similar or identical to the authentic type III collagen mRNA. However, exons 1 and 2, as well as additional exons, appear to be missing from the 5' portion of the RNA. This difference in RNA structure suggests that there may be an alternative transcription start site in the type III collagen gene which is preferentially used in cartilage. Thus the absence of type III collagen in cartilage is not due to transcriptional repression of the type III collagen gene or to instability of the type III collagen transcripts in this tissue. Rather, it must be due to some other mechanism, presumably related to the observed alteration in RNA structure. In this application experiments are proposed to determine the structure of the type III collagen-related RNA in cartilage and the role of this RNA in preventing type III collagen production in cartilage. The structure of the cartilage RNA will be determined and compared with the authentic type III collagen mRNA. The 5' half of the chick type III collagen gene will also be isolated, and the promoter(s) and any previously undescribed exons will be identified by comparison of the exon/intron organization with the cDNA sequence. These experiments will indicate whether the structural differences in the cartilage RNA are due to use of an alternative promoter, alternative RNA processing events, or both. They will also define any open reading frames in the RNA, thus indicating whether the RNA is likely to encode a protein (either a non- collagen protein or an altered form of type III collagen). The subcellular distribution of the cartilage type III collagen-related RNA will be determined; these experiments will indicate whether the type III collagen-related RNA is localized primarily in the nucleus or in the cytoplasm, and, if it is found in the cytoplasm, whether it is associated with ribosomes or with ribonucleoprotein particles. These experiments, in conjunction with the sequence data described above, will suggest possible mechanisms preventing type III collagen production in cartilage. If, based on sequence and subcellular distribution data, the type II collagen-related RNA appears to encode a protein, the protein will be isolated and characterized. If the RNA seems unlikely to encode a protein, other possible functions of RNA will be examined. Finally, the cis-acting sequences and trans-acting factors that are important for the function of the promoter(s) of the type III collagen gene in the appropriate tissues will be identified. These experiments will provide increased understanding of the molecular mechanisms that prevent production of type III collagen in cartilage. Furthermore, they will provide more extensive analysis of the structure of the type III collagen gene and its regulation than has heretofore been available.