This proposal has as its long-term objective the elucidation of molecular mechanisms of normal and abnormal expression of specialized genes in human erythroid cells. The studies focus on the genes for membrane proteins that are specifically expressed in erythroid cells, in particular the genes for spectrin, the major membrane skeletal protein of the red blood cell. Study of these genes is of scientific interest and importance for a number of reasons: 1) A number of human diseases (hereditary hemolytic anemias) are associated with abnormalities in the amount, structure and/or function of different membrane skeleton proteins, and these abnormalities result in abnormal membrane function and shortened red cell survival. Elucidation of the molecular basis for many of these disorders is one of the major specific aims of this proposal; 2) Membrane proteins analogous to those expressed in red blood cells, but having a somewhat different structure, are expressed in non- erythroid cells. Elucidation of the molecular mechanisms responsible for the generation of these different protein isoforms and for the erythroid- specific pattern of expression of the erythroid isoforms constitute another major specific aim of the proposed research: 3) The structure/function relationships of the interacting membrane skeleton proteins are not completely understood. The availability of cloned genes for these proteins combined with genetic engineering technology will allow the elucidation of these relationships, as a third specific aim of these studies. The general methodology to be utilized includes: cloning and structural analysis of the cDNAs and genomic DNA segments of the relevant genes by use of recombinant DNA technology; study of the DNA from individuals with different hereditary disorders of these genes by use of the recently developed polymerase chain reaction (PCR) technique; studies cis-acting sequences by gene manipulation followed by gene transfer/expression studies in tissue culture cells; studies of trans- acting factors by gel retardation assays, DNase I footprinting and methylation interference techniques; studies of structure/function relationships by site directed and/or insertional or deletional mutagenesis, followed by production of recombinant proteins in various expression systems and analysis of their function in various biochemical and/or biological test systems. These studies should lead to a more thorough understanding of the molecular and cellular events associated with the biogenesis of membrane skeleton proteins, as well as to new information on mechanisms of tissue-specific gene expression and genetic mechanisms causing human hereditary disorders.
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