BIOCHEMICAL GENETICS OF CARBONIC ANHYDRASE DEFICIENCIES

这项研究的总体目标是继续研究遗传的 人碳酸酐酶(CA)的缺陷以确定 这个基因家族的个体成员的生理作用， 破译它们的遗传和生理相互关系，并 了解他们在健康和疾病方面的个体重要性。 在本项目期间，有五个具体目标： 1)完整的定义生物化学和分子遗传学的研究 碳酸氢酶II缺乏症。(CA II缺乏是 导致骨化、肾小管畸形的先天错误新分类 酸中毒和脑钙化。) 2)完成碳酸酐酶的生化和分子遗传学研究 (CA IV是肾、肺和肺组织中的膜锚定CA 毛细血管内皮细胞的管腔表面)。 3)通过定向诱变建立CA IV缺乏症小鼠模型 在ES细胞中。 4)界定了CA V的生化和分子遗传学(CA V是 肝脏和肌肉中的线粒体CA是核编码的。) 5)通过靶向诱变建立CA V缺乏症小鼠模型 ES细胞。 碳酸酐酶被提纯，表征，经受 微测序，并用于制备代谢多克隆抗体 标签和生物合成研究。克隆的cDNA和基因组是 分离以确定基因组组织、内含子序列 围绕内含子/外显子边界、染色体定位和 碳水化合物表达改变背后的突变的性质 脱水酶基因。将利用ES细胞中的定向突变来 制作选定CA缺乏症的动物模型。

The broad goal of this research is to continue studies of inherited deficiencies of human carbonic anhydrases (CAs) in order to define the physiological roles of individual members of this gene family, to decipher their genetic and physiologic interrelationships, and to understand their individual importance in health and disease. For this project period there are five specific aims: 1) Complete studies defining the biochemical and molecular genetics of carbonic anhydrase II deficiency. (CA II deficiency is the basis of the newly classified inborn error producing osteopetrosis, renal tubular acidosis, and brain calcification.) 2) Complete the biochemical and molecular genetics of carbonic anhydrase IV. (CA IV is the membrane anchored CA in kidney and lung and on the luminal surface of capillary endothelial cells). 3) Produce a murine model for CA IV deficiency by targeted mutagenesis in ES cells. 4) Define the biochemical and molecular genetics of CA V. (CA V is the nuclear-encoded, mitochondrial CA in liver and muscle.) 5) Produce a murine model of CA V deficiency by targeted mutagenesis in ES cells. Carbonic anhydrases are purified, characterized, subjected to microsequencing, and used to prepare polyclonal antibodies for metabolic labeling and biosynthetic studies. The cDNA and genomic clones are isolated to determine the genomic organization, the intronic sequences surrounding intron/exon boundaries, the chromosomal localization, and the nature of the mutations underlying altered expression of the carbonic anhydrase genes. Targeted mutagenesis in ES cells will be exploited to produce animal models of selected CA deficiencies.