MOLECULAR BIOLOGY OF HUMAN ERYTHROCYTE ALPHA-SPECTRIN

Yale University, New Haven CT

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Abstract

DESCRIPTION: (Investigator's abstract) The long-term goals of this proposal are to elucidate the molecular mechanisms involved in normal and abnormal expression of the erythrocyte membrane protein a-spectrin. Erythrocyte a-spectrin is an important component of the erythrocyte membrane skeleton. The first aim of this proposal is to identify a-spectrin mutations in patients with recessively inherited hereditary spherocytosis and hereditary pyropoikilocytosis, severe hemolytic anemias, and to analyze the structural and/or functional significance of these abnormalities. The second aim of this proposal is to identify and characterize the promoter and other key regulatory factors that control expression of the erythrocyte a spectrin gene. These results will be applied to the study of the role of a-spectrin gene transcription in erythropoiesis and membrane biogenesis and to the genetic study of patients with hemolytic anemia who have been found to have mutations in the putative a-spectrin gene promoter. The third aim of this proposal is to correct the defect in an a spectrin deficient murine erythroleukemia cell line and in the erythroid cells of sph/sph mice, an a-spectrin deficieny model of inherited hemolytic anemia, via retroviral transduction of the a-spectrin cDNA. The general methodology tc be utilized in this research includes: study of genomic DNA from patients with a-spectrin linked rHS and HPP using PCR-based single stranded conformational polymorphism (SSCP) analysis, followed by nucleotide sequence analysis cloning and structural analysis of the cDNA and genomic fragments of the a-spectrin gene relevant to its expression and regulation by the use of recombinant DNA technology; study of cis-acting sequences by gene manipulation followed b, gene transfer/expression studies in tissue culture cells; studies of trans-acting factors by electrophoretic mobility shift assays, DNAse-I footprinting, methylation interference techniques and site-directed mutagenesis followed by in vitrc and in vivo analyses, and guanine-adenine ligation-mediated PCR (GA-LMPCR) dimethyl sulfate in vivo footprinting developmental and tissue-specific studies of the regulatory sequences of the a-spectrin gene promoter in transgenic mice; In vitro transduction of a-spectrin deficient MEL cells with a retrovirus containing the a-spectrin cDNA transduction of hematopoietic stem cells (HSCs) from a-spectrin deficient sph/sph mice with an ecotropic retrovirus containing the a-spectrin cDNA, followed by HSC-retroviral gene transplant into the sph/sph mice. These studies will provide important insights into our studies of the role of a-spectrin in normal and disease states.