BIOCHEMICAL GENETICS OF THE CARBONIC ANHYDRASES

我们计划从五个方面研究碳酸酐酶 (Ca)人类和其他生物中的多基因家族：(1) 结构、组织和演变的特征 CA基因家族，(2)DNA序列的定义在 哺乳动物的组织特异性和发育调控 CA基因，(3)分子碱基的检测 导致CA同工酶遗传性缺陷的突变， (4)活性中心残基在环境中的作用 不同性质CA同工酶的活性，以及(5) 一株类CA痘苗病毒基因的结构和功能分析 以及它的产品。在第一个领域，我们将完成 我们已经克隆的CA基因的特征(人 CA II，猕猴CA I，人类CA Z，以及两个不同但尚不相同的 未知的人类基因)除了筛查和 新的CA基因的特征。CA基因中的调控序列 将通过比较序列分析进行调查， 缺失突变体的构建、转染性研究和 转基因小鼠实验以阐明哪些序列 在健康、功能正常的生物体中直接表达CA基因。 相反，在第三个区域，我们将研究分子损伤 导致CA同工酶表达不足，如 人CA II缺乏综合征、CA II缺陷小鼠 模型和猕猴的CA缺乏症。人类的CA II 缺乏导致的综合征的特点是功能障碍 骨(骨化症)、肾(肾小管性酸中毒)和脑 (脑钙化)，并提示其他CA的缺陷 同工酶可能是不明原因遗传病的致病因素。 在第四个区域，我们将通过定点定向构建突变 密码子中残基的突变被认为是重要的 不同CA同工酶的催化机理及其表达 在大肠杆菌中表达，并对纯化后的改变蛋白进行动力学研究 酵素。最后，我们将研究一种牛痘病毒基因及其 其衍生序列高度同源的产物 CA，以深入了解其起源、功能和演变 与其他生物中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.