MOLECULAR GENETICS OF THE CARBONIC ANHYDRASES

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The carbonic anhydrase (CA) and CA-related genes comprising the mammalian CA multigene family represent a remarkably diverse group of eight genes. These genes range from in their expression single tissues (e.g., CA VI in salivary glands) to their expression in certain cells of virtually all tissues (e.g., CA II). In the cell, they are found in: the cytoplasm (CA I, II, III), associated with membranes (CA IV), the secretory granules of salivary glands (CA VI),. or the inner matrix of mitochondria. Because so little is known about the specific cellular roles of these different CA isozymes, the technique of gene targeting will be used to investigate the functions of the different gene products by observing the behavioral, developmental, morphological or physiological effects produced by specific gene inactivations in mice. Initially, those genes will be studied whose inactivations are most likely to produce informative results. These are: CA III (expressed primarily in skeletal muscle), CA IV (membrane- associated), CA V (mitochondria), and the highly evolutionarily-conserved CA-related protein, CARP. The following specific aims are proposed: (l) to make targeting constructs and generate ES cell lines with mutations in the four selected mouse genes, (2) to generate homozygous mice deficient for each of the genes, (3) to map the chromosomal location of the CA genes that are not already mapped (CA IV, CA V and CARP), and (4) to generate mice with combined deficiencies for multiple CA genes in order to detect overlapping functions. If time permits, similar studies will be carried out on the other CA genes, CA I, CA VI and CA VII. In addition to elucidating the functions of the gene products, other aspects of the CA deficient mice will be highly useful. For example, if the phenotype of a homozygous CA deficient mouse is similar to an inherited human disease of unknown genetic etiology, it is possible that the same CA deficiency is responsible for the human disorder thereby providing a useful animal model for the human disease. Also, an important reason for determining the chromosomal locations of these genes is the possibility that a spontaneous mutation in a specific CA gene may be responsible for one of the classical phenotypic mutants of the mouse. Such spontaneous mutations, whether complete deficiencies or partial losses of activity, can provide insight into the biological function of the affected gene.