ALDOLASE GENES IN NORMAL AND FRUCTOSE INTOLERANT HUMANS

拟议的研究将集中于了解 正常和突变型人果糖二磷酸的生物化学 醛缩酶同工酶 这些酶在糖酵解中起作用， 异源发生 建议的研究将导致更大的 了解新陈代谢和遗传性人类疾病。 的 遗传性果糖不耐症（HFI）是一种人类疾病， 涉及肝脏中碳水化合物代谢的消化紊乱。 对这些同工酶基因的研究将最终导致一个 更好地理解酶的机制，蛋白质结构， 蛋白质折叠、进化和基因表达控制 在组织特异性发育期间。 初步提案的范围利用了一套 导致上述消化系统疾病的人类突变 （HFI）。 重组DNA技术将用于 研究突变的确切性质， 自己在这种疾病中。导致这种情况的突变 疾病将被识别和表征。 这些研究将 从而产生了携带者检测和产前诊断方法， 有疾病史的家庭。 实验方法 也将从传统意义上研究这种疾病的遗传学 使用更现代的反向遗传学方法。 具体目标包括分离和表征 编码醛缩酶的完整的人类基因组。 则 将分离来自HFI疾病患者的突变基因 和表征了 将构建基因组文库， 筛选携带这些基因的克隆。 的DNA序列 基因将被确定。 体外转录实验将 确定启动子所必需的侧翼区 活动 来自HFI患者的基因将与来自HFI患者的基因进行比较。 正常基因 这些DNA将来自确诊为 疾病通过活检和/或家庭分析。 他们将被要求 知情同意参与提供血液样本。 的 发现的突变将进行反向遗传学分析， 对基因表达或酶活性的影响。 正常基因 将通过体外试验改变以模拟HFI突变基因， 诱变 最后，在体内进行竞争实验， 将研究跨监管控制的要素和模式 参与了这个基因家族。

The proposed studies will concentrate on understanding the biochemistry of both normal and mutant human fructose diphosphate aldolase isozymes. These enzymes function in glycolysis and gluconeogenesis. The studies proposed will lead to a greater understanding of metabolism and hereditary human disease. The human disease, hereditary fructose intolerance (HFI), is a digestive disorder involving carbohydrate metabolism in the liver. Studies on the genes for these isozymes will eventually lead to a better understanding of enzyme mechanism, protein structure, protein folding, evolution, and the control of gene expression during tissue specific development. The scope of the initial proposal takes advantages of a set of human mutations which lead to the aforementioned digestive disease (HFI). Recombinant DNA technology will be employed for investigation of the precise nature of the mutations which manifest themselves in this disease. The mutations which lead to this disease will be identified and characterized. These studies will lead to methods of carrier detection and prenatal diagnosis in families with a history of the disease. The experimental approach will study the genetics of the disease in a classical sense as well as use the more modern approach of reverse genetics. The specific aims involve the isolation and characterization of the complete set of human genes encoding the aldolases. Then the mutant genes from patients with the HFI disease will be isolated and characterized. Genomic libraries will be constructed and screened for clones harboring these genes. The DNA sequence of the genes will be determined. In vitro transcription experiments will determine the flanking regions necessary for the promoter activities. The genes from HFI patients will be compared to the normal genes. The DNA will be from patients confirmed for the disease by biopsy and/or family analysis. They will be asked to participate by informed consent to provide blood samples. The mutations found will be analyzed be reverse genetics for their effect on the gene expression or enzyme activity. The normal gene will be changed to mimic the HFI mutant gene by in vitro mutagenesis. Finally, competition experiments performed in vivo will investigate the elements and modes of trans-regulatory control involved in this family of genes.