ROLE OF MORPHOGENETIC PROTEINS IN EMBRYOGENESIS AND TISSUE REGENERATION

在过去的一年里，我们的努力集中在发现增长上 简并聚合酶链式反应分析骨形态发生蛋白相关因子。 这些蛋白质正在成为过程中的关键决定因素 胚胎发育过程中位置信息的规范 对生物技术社区的重视，因为他们几乎 在各种临床情况下的某些治疗作用，包括 骨不连骨折及其他骨科和重建指征 和牙槽骨隆起术。我们花了我们大部分的时间 原肠发育和神经分化过程中基因表达的研究 Xenopus和Brachydanio rerio，尽管我们也检测了基因组DNA 从这些物种中。我们已经发现了六个基因片段，很可能 编码迄今未知的蛋白质。我们决定将重点放在以下两个方面 这些与已知蛋白质最明显的区别。这其中的一个 除了非洲爪哇和非洲爪哇外，还在人和牛的DNA中发现了 该基因显示出95%的氨基酸同源性和80%的氨基酸同源性。 在所有四个成熟物种之间表达的核苷酸同源性 蛋白质产品。另一种则显示出类似的守恒程度 在非洲爪哇和Brachydanio之间；哺乳动物的基因还没有 已确认身份。我们通过与gt11cdna的杂交获得了gt11cdna克隆。 并预计在下一年对它们进行测序 几个星期。接下来，我们将确定空间和时间表达式 利用整株进行原位杂交的基因图谱 非洲爪哇胚胎，并通过注射mRNAs研究其功能 在体外转录以诱导它们的过度表达。我们还将 尝试表达这些蛋白质，以探索它们对 反式作用因子的分化和表达 同源异型盒基因叉头)在隔离的动物帽子中，并允许 特定受体的鉴定。最终，我们希望这一战略 将允许构建显性负基因，这些基因可以用来 确定功能丧失的影响。小灵通6040(1992年第5版) 完成或推进了几个蛋白质测序项目 意义重大。一种新的与紫外线损伤的DNA特异结合的蛋白质 进行了测序和克隆；初步证据表明， 在色素性干皮病发病机制中的作用。一位之前身份不明的人 哺乳动物大脑中的钙调蛋白结合蛋白被证明是 与C型磷脂酶同源，在病原菌和 原生动物；没有具有显著一级结构同源性的哺乳动物基因 都是已知的。约80%的序列是由Edman确定的 降解；该基因现已被克隆和表达。一种蛋白质 新描述的第二信使环的作用所需的 ADP核糖已被鉴定。有限的数据来自高度纯化的 具有强大的淋巴增殖活性的组份具有特征 它与任何已知的细胞因子的区别已经得到了解决。

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 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. PHS 6040 (Rev. 5/92) Several protein sequencing projects were completed or advanced significantly. A novel protein that binds UV damaged DNA specifically was sequenced and cloned; preliminary evidence suggests a possible role in the pathogenesis of Xeroderma pigmentosum. A previously unidentified calmodulin binding protein from mammalian brain was shown to be homologous to C-type phospholipases found in both pathogenic bacteria and protozoa; no mammalian genes with significant primary structure homology are known. Approximately 80% of the sequence was determined by Edman degradation; the gene has now been cloned and expressed. A protein required for the action of the newly- described second messenger cyclic ADP ribose has been identified. Limited data from a highly purified fraction with potent lymphoproliferative activity with characteristics distinguishing it from any known cytokine have been obtained.