MOLECULAR MECHANISMS REGULATING ALFA2 COLLAGEN GENE E

胶原蛋白，是高等结缔组织中含量最丰富的蛋白质 真核生物在许多组织器官中发挥着重要的结构作用。在… 至少有13种胶原蛋白已被描述，由于它们的 多亚基结构，代表至少23个基因的产物。多数 的胶原蛋白基因是受发育调节的，因此给定的 组织或细胞类型只表达胶原基因的一个子集。第I类 本提案将讨论的胶原蛋白已在 鸡胚胎最早发育4.5天，主要在 推测为真皮、体细胞中胚层和皮瘤肌层板。 在胚胎发育的后期，在成人中，I型胶原 几乎在所有结缔组织中都有发现，但一般不存在于 软骨。令人惊讶的是，编码I型胶原的mRNAs 在胚胎发育早期(大约发育第一天)观察到 在软骨中也有。这些I型胶原mRNAs早期的功能 胚胎发生和软骨中，在没有检测到I型的情况下 胶原蛋白的产生，目前尚不清楚。然而，分子机制 这似乎阻止了早期胚胎中α2(I)胶原的产生 在软骨中也已被发现。 此前未描述的启动子和外显子(外显子A)已在 鸡α2(I)胶原蛋白基因内含子2。成绩单开始于 该启动子包含外显子A而不是外显子1和2，并且不包含 外显子7；在mRNA的其他区域也可能存在差异。 在胚胎发育和软骨转录的早期 α2(I)胶原基因几乎完全在这个新的 已确定的启动子。外显子A包含启动开放阅读的AUG 具有胶原蛋白编码序列的帧外的帧；因此 胚胎/软骨α2(I)胶原mRNA显然不能编码 α2(I)胶原蛋白。然而，在细胞质中发现了mrna，并且 与平均三个可释放的嘌呤霉素核糖体有关(12个)。 这些结果表明该信使核糖核酸被主动翻译，但提示 这个开放阅读框架可能相对较小。一种制备的抗血清 与预测开放阅读框架的前22个氨基酸进行比较 识别存在于早期胚胎细胞核中的35kd和33kd蛋白质 软骨细胞，但不在其他类型的结缔组织细胞的核中。 这项申请建议进行实验以确定分子机制。 胚胎/软骨启动子在胚胎发育早期的应用 在软骨中，以及骨/肌腱启动子在所有其他 结缔组织。它还提出了实验来确定 胚胎/软骨型α2(I)胶原基因的结构 并对其蛋白质产物进行鉴定和表征。这些信息 这些实验提供的信息将使我们更深入地了解 这一重要基因家族的发育调控表达。在……里面 特别是，它们将通过以下方式增加对机制的理解 哪种I型胶原的生产被限制在适当的 胚胎发育的组织和阶段。

Collagens, the most abundant proteins in connective tissues of higher eukaryotes, play important structural roles in many tissues and organs. At least 13 types of collagen have been described, which, due to their multisubunit structure, represent the products of at least 23 genes. Most of the collagen genes are developmentally regulated, such that a given tissue or cell type expresses only a subset of the collagen genes. Type I collagen, which this proposal will discuss, has been demonstrated in the chicken embryo as early as 4.5 days of development, primarily in presumptive dermis, somatic mesoderm, and the dermatomal myotomal plate. At later times in embryonic development, and in the adult, type I collagen is found in almost all connective tissues, but is generally excluded from cartilage. Surprisingly, the mRNAs encoding type I collagen have been observed both early in embryonic development (about day 1 of development) and in cartilage. The function of these type I collagen mRNAs early in embryogenesis and in cartilage, in the absence of detectable type I collagen protein production, is unknown. However, the molecular mechanism that appears to prevent production of alpha2(I) collagen in early embryos and in cartilage has been identified. A previously undescribed promoter and exon (exon A) have been found within intron 2 of the chick alpha2(I) collagen gene. Transcripts initiating at this promoter contain exon A instead of exons 1 and 2, and do not contain exon 7; there may be differences in other regions of the mRNA as well. Early in embryonic development and in cartilage transcription of the alpha2(I) collagen gene initiates almost exclusively at this newly identified promoter. Exon A contains an AUG initiating an open reading frame that is out of frame with the collagen coding sequence; thus the embryonic/cartilage alpha2(I) collagen mRNA apparently cannot encode alpha2(I) collagen. However, the mRNA is found in the cytoplasm and is associated with an average of three puromycin-releasable ribosomes (12). These results indicate that the mRNA is actively translated, but suggest that open reading frame may be relatively small. An antiserum prepared against the first 22 amino acids of the predicted open reading frame recognizes 35 and 33 kd proteins present in nuclei of early embryos and chondrocytes, but not in nuclei of other types of connective tissue cells. This application proposes experiments to identify the molecular mechanisms dictating use of the embryonic/cartilage promoter early in embryogenesis and in cartilage, and the use of the bone/tendon promoter in all other connective tissues. It also proposes experiments to determine the structure of the embryonic/cartilage form of the alpha2(I) collagen mRNA and to identify and characterize its protein product. The information provided by these experiments will give us increased insight into the developmentally regulated expression of this important gene family. In particular, they will provide increased understanding of the mechanisms by which production of type I collagen is restricted to the appropriate tissues and stages of embryonic development.