BMP-1, MAMMALIAN TOLLOID AND RELATED DEVELOPMENTAL GENES

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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.