ROLE OF MORPHOGENETIC PROTEINS IN EMBRYOGENESIS AND TISSUE REGENERATION

在过去的一年里，我们的工作重点是发现增长 简并PCR检测骨形态发生蛋白相关因子。 这些蛋白质正在成为这一过程中的关键决定因素， 在胚胎发生过程中的位置信息的规范， 生物技术社区的重要性，因为他们几乎 在各种临床环境中具有一定的治疗效用，包括 骨不连骨折和其他骨科和重建适应症， 牙槽嵴增高术 我们已经投入了我们的大部分时间 关注原肠胚形成和神经胚形成过程中表达的基因， 爪蟾和斑马鱼，虽然我们也检查了基因组DNA， 从这些物种。 我们发现了六个基因片段， 编码迄今未知的蛋白质。 我们决定把重点放在两个 这些蛋白质与已知的蛋白质截然不同。 其中一 除了非洲爪蟾和Brachydanio外，在人和牛的cDNA中也发现了; 该基因显示95%的氨基酸同一性和80%的核苷酸同一性 在成熟表达的蛋白质产物中所有四种物种之间。 的 另一个则显示了爪蟾和 Brachydanio;哺乳动物基因尚未确定。 我们有 通过与PCR杂交，分离出的Gt11 cDNA克隆 扩增产物，并预计在接下来的几年中对其进行测序 周 接下来，我们将确定空间和时间表达 通过原位杂交，使用完整的 非洲爪蟾胚胎，并研究其功能，通过注射mRNA 在体外转录以诱导其过表达。 我们还将 尝试表达蛋白质，以探索它们对 分化和表达的反式作用因子（如 同源框基因叉头），并允许 识别特定受体。 最终，我们希望这个策略 将允许构建显性负基因， 确定功能丧失的影响。

During the past year, our efforts have focused on discovery of growth factors related to the bone morphogenetic proteins using degenerate PCR. These proteins are emerging as key determinants in the process of specification of positional information during embryogenesis and have assumed importance to the biotechnology community owing to their almost certain therapeutic utility in a variety of clinical contexts, including nonunion fractures and other orthopedic and reconstructive indications and alveolar ridge augmentation. We have devoted the majority of our attention to genes expressed during gastrulation and neurulation in Xenopus and Brachydanio rerio, though we have also examined genomic DNA from these species. We have found six gene fragments which probably encode heretofore unknown proteins. We have decided to focus on two of these which are most clearly distinct from known proteins. One of these was found in human and bovine cDNA in addition to Xenopus and Brachydanio; this gene demonstrates 95% amino acid identity and 80% nucleotide identity between all four species in the mature expressed protein product. The other shows a similar degree of conservation between Xenopus and Brachydanio; mammalian genes have not yet been identified. We have isolated lambdal gt11 cDNA clones by hybridization to the PCR amplification products and anticipate sequencing them during the next few weeks. Next, we will determine the spatial and temporal expression patterns of the genes by hybridization in situ using whole mounts of Xenopus embryos, and investigate their functions by injecting mRNAs transcribed in vitro to induced their over-expression. We will also attempt expression of the proteins in order to explore their effects on differentiation and the expression of trans acting factors (such as the homeobox gene forkhead) in isolated animal caps and to allow identification of specific receptors. Ultimately, we hope this strategy will allow construction of dominant negative genes that can be used to determine the effects of loss of function.