During the past five years of this project, these investigators have been analyzing mammalian development by positional cloning of embryo lethal genes residing in the T/t complex on mouse chromosome 17. They have recently succeeded in cloning a second Brachyury gene (T2) which, together with the original Brachyury gene (T1) represents a model of the developmental abnormality (birth defect), spina bifida, common in humans. The original Brachyury mutation (T) turns out to be a large deletion encompassing both T1 and T2. The phenotype in heterozygotes is a short tail. The phenotype in homozygous mutants is embryonic death with no notochord development and no organized axis distal to the forelimb bud. The tct mutation is common to all t haplotypes (which is a variant form of chromosome 17 consisting of multiple inversions spanning 15-20 centiMorgans of the proximal part of the chromosome). The tct mutation in heterozygotes and in homozygous mutants has no phenotype. However, when heterozygous with the T mutation, mice have no tails. There are many t haplotype alleles (that is, multiply inverted chromosomes but with non-identical mutations in recessive lethal genes), and any one t haplotype in homozygous form is lethal due to the presence of recessive, embryonic lethal mutations. In this application, it is hypothesized that T2 is actually a mutation at the tct locus. (So that the original T deletion/t haplotype = homozygous null for T2 and hemizygous for T1). Since a mutant allele of T1 (TLAF) does not complement the T2 mutation (meaning they are non-complementing, non-allelic genes), it is further postulated that T1 and T2 act together in determining axial development. Because the phenotype of the T1LAF/T2 heterozygotes is death (as compared to short tails for T/+ heterozygotes), it is postulated that the T2 mutant is a dominant negative allele. These are likely not the only genes important in axial development. A third candidate to be explored is t-int, an-unlinked, genetically defined (modifier of T and tct), but not-yet-cloned locus. Plans include: 1. Genetic studies to determine whether T2 is an allele of the 'tail factor' gene (tct). 2. Yeast two-hybrid studies to try to identify the t-int protein. 3. Attempts to correct the T mutation (deletion) with transgenic T1 and T2 genes into the appropriate mouse strains. 4. Generation and use of antibodies to T1 and T2, as well as mutant and wild-type constructs to both test the notion that they interact with one another and to assess their function. 5. Clone and sequence T2 from a distant species to help identify its conserved domains. A second project is to clone the tw5 lethal gene. The tw5 homozygote never develops the embryonic ectoderm. The investigators believe it may affect a decision of cells in the embryo to either differentiate or die. The tw5-homozygous mutant cells can be rescued by extrinsic factors (supporting a model in which programmed cell death can be averted by alternate signals - not depending upon tw5). Five YACS have been identified from the relevant region of the chromosome. These will be mapped further and tested for ability to rescue the tw5 mutation. Rescuing YACS will be subdivided into BACS and candidate genes encoded in a rescuing region will be identified by cDNA selection and exon trapping. A tw5-revertant has been recovered which should help in confirming identity of this gene.
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