DIRECTOR REARRANGEMENT OF THE MAMMALIAN GENOME IN VIVO

分子遗传学方法将被用来检查是否有 酿酒酵母FLP重组酶(或大肠杆菌噬菌体P1 Cre 重组酶)可以介导位点特异性重组事件 哺乳动物细胞。我们将构建四种不同的融合 哺乳动物对FLP结构基因的启动子(带有紫胶 位于预测起点的运算符序列 每个启动子的转录)，并检测FLP mRNA 在哺乳动物的细胞核中转录并活跃，当这些 融合基因被引入小鼠和人类成纤维细胞中 多瘤病毒和逆转录病毒载体。缺陷型逆转录病毒载体 将构建携带FLP位点(FSV)并将其导入 小鼠和人类成纤维细胞。我们将确定是否有几个 所催化的重组和重排事件类 哺乳动物细胞中存在野生型或突变型FLP重组酶 携带一个或多个FSV。包括FLP是否可以调解 (1)相互易位；(2)环状整合 携带FLP位点的DNA进入FSV，(3)染色体丢失，和(4) 有效的环(或缺失)染色体的形成。我们会 试图调节小的人类着丝粒的形成 环状或缺失染色体，分离(或纯化)一小环 有着丝粒的(或缺失的)染色体，也许要克隆 此(环)染色体在酵母中作为线性染色体，并 克隆并开始解剖人类着丝粒的结构。

A molecular genetic approach will be used to examine whether the yeast Saccharomyces cerevisiae FLP recombinase (or coliphage P1 Cre recombinase) can mediate site-specific recombination events in mammalian cells. We will construct four different fusions of mammalian promoters to the FLP structural gene (with the lac operator sequence located at the predicted startpoint of transcription of each of the promoters) and test whether FLP mRNA is transcribed and active in the mammalian nucleus when these fusions are introduced into mouse and human fibroblasts on polyomaviral and retroviral vectors. Defective retroviral vectors carrying FLP sites (FSV's) will be constructed and transfected into mouse and human fibroblasts. We will determine whether several classes of recombination and rearrangement events catalyzed by wild-type or mutant FLP recombinases occur in mammalian cells carrying one or more FSV's. These include whether FLP can mediate (1) reciprocal translocations, (2) the integration of a circular DNa carrying a FLP site into an FSV, (3) chromosome loss, and (4) efficient ring (or deficiency) chromosome formation. We will attempt to mediate the formation of small, human centromere-bearing ring or deficiency chromosomes, to isolate (or purify) a small ring (or deficiency) chromosome with its centromere, perhaps to clone this (ring) chromosome in yeast as a linear chromosome, and to clone and begin to dissect the structure of the human centromere.