BMP-1, MAMMALIAN TOLLOID AND RELATED DEVELOPMENTAL GENES

建议研究的结构，表达，和功能的一个 一小群结构相关的基因可能在基质中很重要 生产和在正常的，也许是不正常的，发展的哺乳动物 胚胎研究包括： 1)进一步表征基因（PCOLCE），其蛋白质产物， 前胶原蛋白C-蛋白酶增强蛋白，参与生物合成 基质的主要成分，L型胶原蛋白。研究将包括： A）确定PCOLCE表达的时间和空间分布 在发育过程中， 增强子蛋白可能与其产物相互作用的其他基因（例如， J型胶原基因COL 1A 1; BMP-1/mTld基因，可能编码 I型前胶原蛋白酶和最近发现的一种新的BMP-1/mTld样基因 在P.I的实验室中发现的）; B）对 增强子蛋白刺激C-蛋白酶的活性， 增强子是否也可以刺激TGF-β样 蛋白质：C）研究PCOLCE被TGF-β 1上调的可能性。 β的这些研究应进一步深入了解 增强子蛋白和可能与其他蛋白质的相互作用， 发育和生理过程。 2)选择性剪接基因BMP-1/mTld的初步研究 参与骨器官发生的蛋白质（BMP-1）的转录物， 对于在背/腹侧中重要的蛋白质的哺乳动物同源物（mTld）， 果蝇胚胎的图案化（tolloid）。研究将：A）确定 不同选择性剪接RNA的差异表达程度 在胚胎、胚外和成体组织中的形态和分布 中枢神经系统中的mTld：B）证明蛋白质 存在各种可变剪接RNA形式的产物， 这些蛋白质在发育和成年组织中的定位。和 确定它们是否与细胞膜相关：C）确认 并扩展了BMP-1的原始观察结果。或者说非常接近 与生理性I型前胶原C-蛋白酶相关：D）确认 并扩展了BMP-1可以直接激活TGF-β的原始观察， E）确定β-样分子表达的可能上调 F）研究TGF-β诱导的BMP-1/mTld的可能的酶活性; mTld. 3)一个新的编码具有结构域的蛋白质产物的基因的鉴定 结构相同但序列不同。mTld的。这 最近在P.I.从cDNA文库中 从+/-敲除小鼠胚胎的mRNA构建， 表达BMP-1/mTld，但似乎表达代偿性酶 活动表征将包括确定：A）全长编码 序列：B）小鼠和人类基因组中的染色体位置：C）是否 选择性剪接发生：D）空间和时间模式 在成体和发育中的组织中表达; E）所述表达的可能功能 F）表达水平和/或分布是否 改变以补偿+/-敲除胚胎中BMP-1/mTld的损失： G）新基因在发育中的作用，通过创造和 对新基因无效等位基因纯合子敲除小鼠的研究。

Proposed are studies into the structure, expression, and function of a small group of structurally related genes likely to be important in matrix production and in normal, and perhaps abnormal, development of mammalian embryos. Studies include: 1) Further characterization of the gene (PCOLCE), whose protein product, the procollagen C-proteinase enhancer protein, is involved in biosynthesis of type l collagen, the major component of matrix. Studies will include: A) Determining temporal and spatial distribution of expression of PCOLCE during development and comparison to the distribution of expression of other genes with whose products the enhancer protein might interact (eg. the type J collagen gene COL1A1; the BMP-1/mTld gene, which may encode the type I procollagen C-proteinase; and a novel BMP-1/mTld-like gene recently discovered in the P.I's lab); B) Functional study of the mechanism whereby the enhancer protein stimulates the activity of C-proteinase and assaying whether the enhancer may also stimulate activation of TGF-beta-like proteins: C) Studying the possibility that PCOLCE is up-regulated by TGF- beta. Such studies should provide further insight into the role(s) of the enhancer protein and possible interactions with other proteins in developmental and physiological processes. 2) Definitive study of a gene (BMP-1/mTld) encoding alternatively spliced transcripts for a protein involved in organogenesis of bone (BMP-1) and for a mammalian homologue (mTld) of a protein important in dorsal/ventral patterning of Drosophila embryos (tolloid). Studies will: A) Determine the extent of differential expression of the various alternatively spliced RNA forms in embryonic extraembryonic and adult tissues and distribution of mTld in the central nervous system: B) Demonstrate that the protein products of the various alternatively spliced RNA forms exist and localization of these proteins in developing and adult tissues. and determine whether they may be associated with cell membranes: C) Confirm and extend the original observation that BMP-1 is. or is very closely related to the physiological type I procollagen C-proteinase: D) Confirm and extend the original observation that BMP-1 can directly activate TGF- beta-like molecules: E) Determine possible up-regulation of expression of BMP-1/mTld by TGF-beta; F) Investigate possible enzymatic activities of mTld. 3) Characterization of a new gene encoding a protein product with a domain structure identical to but sequence different than. that of mTld. This gene was recently isolated in the P.I.'s lab from a cDNA library constructed from mRNA of +/- knockout mouse embryos which no longer express BMP-1/mTld but which seem to express compensatory enzymatic activity. Characterization will include determining: A) Full-length coding sequences: B) Chromosomal locations in mouse and human genomes: C) Whether alternative splicing occurs: D) Spatial and temporal patterns of expression in adult and developing tissues; E) Possible functions of the protein product(s): F) Whether levels and/or distribution of expression are changed to compensate for loss of BMP-1/mTld in +/- knockout embryos: G) Role(s) of the new gene in development, determined through creation and study of knockout mice homozygous for null alleles of the new gene.