Human blood group antigens represent polymorphisms of cell surface molecules to which transfusion recipients may make antibodies. These molecules may also have vital roles in cell integrity or function. Also, blood group-active molecules are often expressed by many nonerythroid tissues. Expression of several blood group antigen-bearing proteins-- namely CD44, Lutheran proteins, and CR1--is down-regulated by the In(Lu) gene in a dominant and erythroid-specific manner. These proteins are encoded by different chromosomes (11, 19, and 1, respectively) and are structurally distinct. In addition, the carbohydrate antigens i and P1 are also down-regulated by In(Lu). The In(Lu) gene is a rare autosomal dominant gene, but its hypothetical normal allele is also postulated to regulate expression of these target antigens. This project will use affinity chromatography to complete purification of the Lutheran protein, so that the cDNA encoding it can be cloned and sequenced. Further, the 5' flanking region containing promoter/enhancer elements will be cloned and their roles investigated, in order to understand what factors regulate expression of Lutheran proteins. The promoter/enhancer region of the CD44 gene will be similarly investigated. Further documentation of the effect of In(Lu) on CR1-related antigens (Knops/McCoy/SIa) will be obtained by studying CR1 copy number, the CR1 copy-number-related RFLP, and copy number of other In(Lu) target proteins in both normal individuals well as normal and Lu(a-b-) people in families with the In(Lu) gene. Finally, several methods will be used to create model systems in which the level and mechanism of action of the In(Lu) gene may be studied. These include transfection of CD44 cDNA with a viral promoter, CD44 cDNA accompanied by the CD44 promoter, and the CD44 promoter linked to a reporter gene. These constructs will be transfected into erythroid cells, which will initially be murine erythroleukemia (MEL) cells. Transfected cells will then be hybridized with EBV- transformed lymphocytes from In(Lu) individuals and cells with down- regulated CD44 expression selected. These cells will then be studied to determine what human chromosomes are retained, in order to map the In(Lu) gene. RNA expression and protein synthesis will be studied to determine the level at which the In(Lu) gene acts.
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