HGM2002 Poster Abstracts: 4. Functional Genomics
POSTER NO: 180
Mouse Fliih is a gelsolin-related cytoskeletal regulator essential for early development: rescue of embryonic lethality by the human FLII gene
1Hugh D. Campbell, 1Shelley Fountain, 2Ian S. McLennan, 3Leise A. Berven, 4Michael F. Crouch, 3Deborah A. Davy, 1Jane A. Hooper, 1Stuart K. Archer, 1Kynan Waterford, 5Ken-Shiung Chen, 5James R. Lupski, 3Ian G. Young, 3Klaus I. Matthaei
The Drosophila melanogaster flightless I (fliI) gene is essential for early fly development. In fliI null mutant embryos, in the absence of maternal product, defects are first observed during cellularization of the syncytial blastoderm and gastrulation fails. Genes encoding conserved homologues of the 1256 amino acid residue fliI protein are present in Caenorhabditis elegans, mouse (Fliih) and human (FLII). Recently, we have identified a related gene in sequence data from the genome project for the slime mould Dictyostelium discoideum and we are now characterizing this homologue also. The gene is not present in Saccharomyces cerevisiae, although genes encoding proteins related to the individual modules from which the fliI protein is made up are present. The human and mouse proteins are 95% identical to each other, and both are 58% identical to the D. melanogaster protein. The fliI-related proteins contain a domain similar to the actin-binding protein gelsolin, as well as a leucine-rich-repeat (LRR) domain involved in protein-protein interactions. A variety of evidence indicates that the fliI protein and its homologues may play an important role in linking signal transduction pathways involving Ras- related GTPases with cytoskeletal control mechanisms.
The human FLII and mouse Fliih genes both contain 30 exons covering 14 kb of genomic DNA. We have generated a null mutant allele of the mouse Fliih gene by homologous recombination in ES cells. While heterozygous Fliih mutant mice develop normally, the homozygous mutant exhibits early embryonic lethality. In culture, mutant blastocysts hatch, attach, and form an outgrowing trophoblast cell layer, but egg cylinder formation fails and the embryos degenerate. Degeneration of the mutant embryos appears to correlate with the loss of maternal Fliih protein. Mutant embryos initiate uterine implantation in vivo, but then degenerate rapidly. The mouse Fliih mutation is much more severe than mutations in the genes for the related gelsolin family members gelsolin, villin and Cap G, where the homozygous null mutant mice are viable and fertile. We have constructed transgenic mice carrying a human genomic cosmid that contains the 14 kb human FLII gene and 20 kb of 5' flanking sequence. By performing appropriate crosses, we were able to generate homozygous Fliih mutant mice carrying the human FLII transgene. These mice are viable and fertile. The results establish that the regulatory elements of the human FLII gene are active in mice and that the human FLII protein is capable of functionally substituting for the mouse Fliih protein.
Other abstracts in same session