TRANSFORMATION OF CELLS BY THE V-REL ONCOGENE

Rev-T是一种禽类逆转录病毒， 淋巴样细胞在体内和体外。 Rev-T的癌基因是v- rel; v-rel编码转化蛋白p59 v-rel。 v-rel是 来源于正常细胞基因c-rel，与 果蝇背部基因 我们感兴趣的是 v-rel转化淋巴样细胞的途径， 不转化成纤维细胞。 使用连接器插入和 我们打算分离V-rel突变体， 对淋巴细胞转化温度敏感。 这些突变体将用于实验， 如果p59 v-rel是蛋白激酶活性或与蛋白激酶活性相关 重要的是它的转换功能。 如果激酶活性 对于v-rel变换函数， 将试图确定一个相关的生化功能， 使用从表达p59 v-rel的细菌细胞纯化的蛋白质， v-rel蛋白质。 我们还将确定磷酸化位点 以及p59 v中第二个核导向功能的身份- rel，以及这些序列对于转换的重要性。 使用定点诱变功能。 禽类蛋白p59 v- REL在鼠成纤维细胞中是有毒的。 我们将确定v-rel 负责这种毒性的序列，以及是否无毒 禽REL蛋白可转化鼠淋巴样细胞。 否则我们 将尝试分离小鼠c-rel cDNA克隆，并通过体外 诱变，我们将尝试创建一个显性鼠相关基因， 转化基因 利用DNA转移技术，我们将 试图从淋巴细胞中分离出基因 转化成纤维细胞。 最后，通过使背侧 在逆转录病毒载体的杂交基因，我们将确定是否 这两个基因之间的序列同源性也反映了 两种蛋白质之间的功能同源性。 拟议 这些研究旨在加深我们对逆转录病毒的理解 转化过程，并可能与淋巴 肿瘤发展和其他正常的发展过程， 将军

Rev-T is an avian retrovirus that specifically transforms early lymphoid cells in vivo and in vitro. The oncogene of Rev-T is v- rel; v-rel encodes the transforming protein p59v-rel. v-rel is derived from a normal cellular gene c-rel, and is highly related to the Drosophila dorsal gene. We are interested in the mechanism by which v-rel transforms lymphoid cells, and the reason why it does not transform fibroblast cells. Using linker insertion and in vivo mutagenesis we intend to isolate v-rel mutants that are temperature-sensitive for transformation of lymphoid cells. These mutants will be used in experiments designed to determine if p59v-rel is, or is asssociated with, a protein kinase activity important for its transforming function. If kinase activity does not seem to be important for the v-rel transforming function we will attempt to determine a pertinent biochemical function of p59v-rel using protein purified from bacterial cells expressing the v-rel protein. We will also determine the sites of phosphorylation and the identity of a second nuclear directing function in p59v- rel, and the importance of these sequences for the transforming function using site-directed mutagenesis. The avian protein p59v- rel is toxic in murine fibroblasts. We will determine the v-rel sequences responsible for this toxicity, and whether a non-toxic avian rel protein can transform murine lymphoid cells. If not, we will attempt to isolate a murine c-rel cDNA clone, and by in vitro mutagenesis we will attempt to create a dominant murine rel transforming gene. Using DNA transfer techniques, we will attempt to isolate genes from lymphoid cells that will allow transformation of fibroblast cells. Finally, by making rel-dorsal hybrid genes in retroviral vectors we will determine whether the sequence homology between these two genes also reflects a functional homology between the two proteins. The proposed studies are intended to further our understanding of retroviral transforming processes, and may have relevance to lymphoid tumor development and other normal developmental processes in general.