BIOCHEMICAL GENETICS OF THE CARBONIC ANHYDRASES

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We plan to investigate five aspects of the carbonic anhydrase (CA) multigene family in humans and other organisms: (1) the characterization of the structure, organization, and evolution of the CA gene family, (2) definition of DNA sequences important in the tissue specific and developmental regulation of mammalian CA genes, (3) examination of the molecular bases of the mutations responsible for inherited deficiencies of CA isozymes, (4) investigation of the role of active site residues in the activities of CA isozymes with different properties, and (5) structural and functional analysis of a CA-like vaccinia virus gene and its product. In the first area, we will complete the characterization of the CA genes we have already cloned (human CA II, macaque CA I, human CA Z, and two different but as yet unidentified human genes) in addition to screening for and characterizing new CA genes. Regulatory sequences in CA genes will be investigated by comparative sequence analysis, construction of deletion mutants, transfection studies and transgenic mice experiments to elucidate those sequences which direct CA gene expression in healthy, functioning organisms. Conversely, in the third area we will study the molecular lesions which cause deficiencies in expression of the CA isozymes such as the human CA II deficiency syndrome, CA II deficient mouse model and the macaque CA I deficiency. The human CA II deficiency results in a syndrome characterized by dysfunction of bone (osteopetrosis), kidney (renal tubular acidosis), and brain (cerebral calcification) and suggest that deficiencies in other CA isozymes may be causative factors in undefined genetic diseases. In the fourth area, we will construct mutations by site-directed mutagenesis in the codons for residues believed to be important in the catalytic mechanisms of different CA isozymes, express them in E. coli, and carry out kinetic studies on the purified altered enzymes. Finally, we will study a vaccinia virus gene and its product, the derived sequence of which is highly homologous to the CAs, to gain insights into its origin, function, and evolutionary relationship to CA genes in other organisms.