BIOCHEMICAL GENETICS OF THE CARBONIC ANHYDRASES

我们计划从五个方面研究碳酸酐酶 (CA)人类和其他生物体的多基因家族：（1） 的结构、组织和演变的表征 CA基因家族，（2）DNA序列的定义， 哺乳动物组织特异性和发育调控 CA基因，（3）检测CA基因的分子基础， 导致CA同工酶遗传缺陷的突变， (4)活性位点残基在蛋白质合成中的作用 不同性质的CA同工酶活性;（5） CA样痘苗病毒基因结构与功能分析 及其产品。 在第一个领域，我们将完成 我们已经克隆的CA基因的表征（人类 CA II，猕猴CA I，人类CA Z，以及两种不同的但迄今为止 未鉴定的人类基因）， 表征新CA基因。 CA基因中的调控序列 将通过比较序列分析进行研究， 缺失突变体的构建、转染研究和 转基因小鼠实验来阐明那些序列， 在健康的、功能正常的生物体中直接表达CA基因。 相反，在第三个领域，我们将研究分子病变， 其引起CA同工酶表达的缺陷， 人CA II缺乏综合征，CA II缺乏小鼠 模型和猕猴CA I缺乏。 人类CA II 缺乏导致以功能障碍为特征的综合征， 骨（骨硬化症）、肾（肾小管酸中毒）和脑 （脑钙化），并表明其他CA的缺陷 同工酶可能是不明遗传病的致病因素。 在第四个领域，我们将通过定点突变构建突变。 在密码子中突变被认为是重要的残基 不同CA同工酶的催化机制，表达它们 在大肠大肠杆菌，并进行动力学研究，对纯化的改变 内切酶 最后，我们将研究牛痘病毒基因及其 产物，其衍生序列与 CA，以深入了解其起源，功能和进化 与其他生物体中CA基因的关系。

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.