The long term objectives are to elucidate the function of the erythroid cytoskeletal proteins and to determine the pathological consequences of their absence. A heritable deficiency of ankyrin, Ank1, provides a mouse model for the human disease, Hereditary Spherocytosis. The murine disease manifestations, as in the human, are not limited to red blood cells but involve other tissues, such as brain, where erythroid transcripts are normally localized. For example, humans with Ank1 deficits have neurological symptoms and we have shown that latent degeneration of the cerebellar Purkinje cells in the Ank1 deficient, nb/nb, mice occurs consonant with onset of a tremor. The remarkable diversity of tissue specific and developmentally regulated isoforms suggests alternate and as yet unknown functional roles for ankyrin. Focus here is on the role of specific motifs at the 3' end of Ank1 and on the identification of the Ank1 transcripts in Purkinje cells. Our recent findings target research to the following. (1) A 17.5 kD muscle isoform (skAnk1) with a unique functional membrane anchor has it's own first exon and promoter in the distal end of the Ank1 gene. (2) Two COOH-terminal motifs of Ank1 are expressed in a tissue and developmental specific fashion. (3) Ank1 3'UTR sequences are highly conserved (greater than 70 percent) between mouse and human and vary due to use of alternate sites of polyadenylation. Specific Aims are to: Determine the function of the skAnk1 isoform and its membrane anchor by deleting the skeletal muscle first exon through homologous recombination in embryonic stem cells (ESC); Determine the functional significance of the alternative COOH-termini by using homologous recombination in ESC to delete the specific exon segments that code for each terminus; Define the functional significance of sequences within the 3'UTR by in situ hybridization and transfection of reporter constructs into cells in culture: and Characterize the process of Purkinje cell degeneration in the nb/nb mice and identify the role of Ank1 proteins in this process.