Elastin is a unique, rubbery protein with an unusual amino acid composition, a low turnover rate and a limited window of developmental expression. Defective elastin is implicated in several human genetic diseases, including cutis laxa and Marfan syndrome, and abnormal elastic fibers are one hallmark of degenerative diseases of the arteries, lungs, and skin. Our initial studies of the cellular and molecular biology of elastin in animals (sheep, pig) have now been extended to the biology of the human gene. This laboratory discovered that human skin fibroblasts contain significant quantities of elastin mRNA (mRNAe) and produce elastin in vitro. Further studies have shown that genetic and developmental variation in elastin gene expression is readily studied in a human diploid cell culture system. Elastin synthesis is induced in fetal skin cells and may be reduced by aging. In at least two cases of cutis laxa there appears to be a pretranslational defect in elastin synthesis. The sheep elastin gene has been partially characterized and used both to quantify mRNA levels in a number of systems and to isolate a human elastin genomic clone. Because of the dilute nature of the elastin gene, cDNA cloning of mRNAe becomes essential to finalizing the primary structure of the protein and to the positive identification of exons in the gene. Since mRNAe is GC-rich, random priming of sheep mRNAe will be used to produce overlapping cDNA clones. Using the present genomic isolates, the remainder of the human and sheep genes will be isolated in systems which reduce the chances of internal recombination--possibly a frequent event in elastin clones. Normal regulatory processes will be studied in both sheep and human cells, particular attention being given to the roles of prolyl hydroxylation, glucocorticoids, methylation, and matrix. Regulatory elements within or near the elastin gene will be examined. Defects in elastin in human genetic diseases (cutis laxa, Marfan syndrome, progeria) will be studied at the level of protein production, gene expression, and genomic polymorphism. Structural protein abnormalities will be analyzed by peptide mapping procedures, while pretranslational defects will be investigated by studies of mRNA metabolism. The combined experimental manipulation of normal cells and the detailed examination of abnormal cells can move our knowledge of the biology of this matrix protein forward.
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