ALDOLASE GENES IN NORMAL AND FRUCTOSE INTOLERANT HUMANS

拟议的研究将集中于了解 正常和突变人二磷酸果糖的生物化学 醛缩酶同工酶。这些酶在糖酵解和 糖异生作用。建议的研究将导致更大的 对代谢和遗传性人类疾病的理解。这个 人类疾病遗传性果糖不耐受(HFI)是一种 涉及肝脏碳水化合物代谢的消化障碍。 对这些同工酶基因的研究最终将导致 更好地理解酶的作用机制、蛋白质结构、 蛋白质折叠、进化与基因表达调控 在组织特定的发育过程中。 最初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 对表现出的突变的确切性质的调查 他们自己在这种疾病中。导致这一现象的突变 疾病将被识别和表征。这些研究将 导致携带者检测和产前诊断的方法 有该病病史的家庭。实验方法 也将研究经典意义上的疾病遗传学 因为使用了更现代的反向遗传学方法。 具体目标包括隔离和刻画 编码醛缩酶的全套人类基因。然后是 来自HFI疾病患者的突变基因将被分离 并以此为特征。将构建基因组文库并 筛选出含有这些基因的克隆。该病毒的DNA序列 基因将被确定。体外转录实验将 确定启动子所需的侧翼区域 活动。来自HFI患者的基因将与 正常基因。DNA将来自确诊为 疾病通过活组织检查和/或家庭分析。他们将被要求 以知情同意的方式参与提供血样。这个 将对发现的突变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 将在体外改变为模拟HFI突变基因 诱变。最后，在活体内进行了竞争实验 将调查跨监管控制的要素和模式 与这个基因家族有关。

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.