MOLECULAR GENETIS OF FACTOR IX AND OTHER BLOOD PROTEINS

描述（改编自申请人摘要）：本申请 代表了主要研究者关于 因子IX（FIX）基因的表达。 在以前的研究中，他已经表明， 内含子序列以及位于5'端的沉默元件 和一个小的5'区域称为LS参与了 这个基因的发育调控。 拟议的研究将更多 使用缺失和位点特异性 诱变 此外，还将进行凝胶流动性和足迹研究。 用于描述调节蛋白和DNA之间的相互作用。 然后将这些蛋白质分离和表征。 初始 将使用细胞培养物进行研究，但是随着研究的进行， 将利用转基因小鼠试验来确定调节是否 包括已鉴定的顺式作用元件和反式作用因子， 可在整个动物中操作。 在这些研究中，将构建一个微型基因构建体， 利用。 首席研究员还提出了一种新的研究方法， 调节突变因子IX基因，如FIX-莱顿。 一 困难在于这些基因在肝脏中表达，然而， 从病人身上获得这样的样品即使不是不可能，也是困难的。 因此，提出了来自患者的淋巴细胞，其携带 改变的基因，与肝癌细胞系融合，使细胞永生化， 并提供肝脏特异性表达。 通过这种方式， 详细研究改变的基因的表达。最后，FVII 将使用用于表征FIX的相同方法研究基因 基因

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.