MOLECULAR GENETICS OF FACTOR IX AND OTHER BLOOD PROTEINS

描述(改编自申请人的摘要)：本申请 代表首席调查员对 因子IX(FIX)基因的表达。在之前的研究中，他已经证明 内含子序列以及位于5‘端的消音器元件 和一个称为LS的5‘端小区域参与了 该基因的发育调节。拟议的研究将会有更多 使用缺失和特定于位点的方法清楚地定义这些序列 诱变。此外，还将进行凝胶流动性和足迹研究 用来描述调控蛋白和DNA之间的相互作用。 然后，这些蛋白质将被分离和鉴定。首字母 研究将使用细胞培养进行，但随着研究的进行， 转基因小鼠的测试将被用来确定这一规定是否 包括已识别的顺式作用元件和反式作用因子 在整个动物身上都可以操作。在这些研究中，一个微小的基因结构将是 被利用了。 首席调查员还提出了一种新的研究方法 对Fix-Leyden等突变因子IX基因的调控。一 然而，困难在于这些基因是在肝脏中表达的， 从患者那里获取这样的样本即使不是不可能，也是困难的。 因此，人们建议从患者身上提取的淋巴细胞携带 将改变的基因融合到肝癌细胞系中，使细胞永生化 并提供肝脏特异性表达。通过这种方式，有可能 详细研究改变后基因的表达情况。最后，FVII 基因将使用相同的方法进行研究，以表征修复 吉恩。

DESCRIPTION (Adapted from the applicant's abstract): This application represents a continuation of the Principal Investigator's studies on the expression of the factor IX (FIX) gene. In previous studies, he has shown that an intron sequence as well as a silencer element located at the 5' end of the gene and a small 5' region called LS are involved in the developmental regulation of this gene. The proposed studies will more clearly define these sequences using both deletion and site-specific mutagenesis. In addition, gel mobility and footprinting studies will be used to describe the interaction between regulatory proteins and the DNA. These proteins will then be isolated and characterized. The initial studies will be carried out using cell cultures but as the studies proceed, transgenic mouse assays will be utilized to determine if the regulation including the identified cis-acting elements and trans-acting factors are operable in whole animals. In these studies, a mini-gene construct will be utilized. The Principal Investigator has also proposed a novel approach for studying regulation of the mutant factor IX genes such as FIX-Leyden. One difficulty is that these genes are expressed in the liver, however, obtaining such samples from patients is difficult if not impossible. Therefore, it is proposed that lymphocytes from patients, which carry the altered gene, be fused to hepatoma cell lines to both immortalize the cells and provide liver specific expression. In this way, it may be possible to study in detail, the expression of the altered genes. Finally, the FVII gene will be studied using the same approach used to characterize the FIX gene.