MYELOID CELL EXPRESSED ONCOGENE

NIH-3 T3细胞转染方法的使用使得能够在细胞内进行转染。 分离几种新的癌基因。该项目的目标是 研究新人类的激活和功能机制， NIH-3 T3转染人白血病细胞获得lbc转化基因 DNA，并通过其在裸鼠中形成肿瘤的能力进行分离。lbc cDNA 序列与已知序列没有同一性，并编码预测的 亲水性蛋白质约47 kD。Lbc在人体内表达 骨髓和淋巴细胞以及肌肉、肺和心脏组织。转染 NIH-3 T3细胞的lbc cDNA导致形态学转化灶 在培养物中，证明lbc cDNA的生物活性，和 提供了一种用于分析LBC的功能性体外测定 转化活动。提出了以下目标。A.初步 研究表明LBC癌基因代表了一种新的转录， 来源于位于 15号染色体上编码的异源序列。为了 为了充分阐明激活机制，建议获得 正常造血细胞lbc原癌基因cDNA全序列 组织，以分析原癌基因的转化活性， 其突变形式，并通过基因组学方法精确定义断点， 序列分析B。来自造血系统的人样本/细胞系 恶性肿瘤和疾病将筛选激活的lbc基因，基于 在LBC表达式上。lbc基因在人类染色体上的确切位置 15、在鼠标中将被精确定位并与位置进行比较 已知与血液学和其他疾病有关的遗传位点， 人类和小鼠。C. LBC的转化活性将通过 集中于两个功能上相关的结构域：1. DBL域 与小G蛋白ras超家族的调节活性相关 控制细胞增殖和细胞骨架组织的蛋白质; 该结构域也在DBL和VAV癌基因、BCR基因和 酵母细胞周期基因CDC 24。2. PH（普列克底物蛋白同源）结构域 在几种细胞内信号转导分子中发现， 定义了一个新的蛋白质相互作用域。这些领域将受到 定点诱变，并使用3 T3焦点分析突变体 形成分析。D.为了研究LBC的功能， 衍生以鉴定LBC蛋白，以确定其细胞 定位和分析LBC磷酸化。基于活动 与其他癌基因编码的dbl结构域相关， 为了测试已经可用的重组LBC蛋白的调节活性 对抗一组ras样小G蛋白此外，LBC的作用 在p2 Lras信号转导途径中的作用将使用孔- 特征性显性负性ras突变体N17。这些拟议的实验 将阐明新型LBC的转化活性和功能 癌基因

The utilization of the NIH-3T3 cell transfection method has enabled the isolation of several novel oncogenes. The goal of this project is to investigate the mechanism of activation and function of the novel human lbc transforming gene obtained by NIH-3T3 transfection with human leukemic DNA and isolated by its ability to form tumors in nude mice. The lbc cDNA sequence shows no identity to known sequences, and encodes a predicted hydrophilic protein of approximately 47 kD. Lbc is expressed in human myeloid and lymphoid cells and muscle, lung and heart tissue. Transfection of NIH-3T3 cells with lbc cDNA results in morphologically transformed foci in culture, demonstrating the biological activity of the lbc cDNA, and presenting a functional in vitro assay for the analysis of lbc transforming activity. The following aims are proposed. A. Preliminary studies indicate that the lbc oncogene represents a novel transcription unit derived from fusion of a truncated lbc proto-oncogene located on chromosome 15 with heterologous sequence encoded on chromosome 7. In order to fully elucidate the mechanism of activation, it is proposed to obtain complete lbc proto-oncogene cDNA sequence from normal hematopoietic tissues, to analyze the transforming activity of the proto-oncogene and its mutant forms, and to precisely define the breakpoint by genomic sequence analysis. B. Human samples/cell lines from hematopoietic malignancies and disorders will be screened for activated lbc genes, based on lbc expression. The exact location of the lbc gene on human chromosome 15, and in the mouse will be pinpointed and compared with the position of genetic loci known to be involved in hematologic and other diseases in human and mouse. C. The transforming activity of lbc will be analyzed by focusing on two functionally implicated domains: 1. A dbl domain associated with regulatory activity for the ras superfamily of small G proteins which control cell proliferation and cytoskeletal organization; this domain is also encoded in the dbl and vav oncogenes, the bcr gene and the yeast cell cycle gene CDC24. 2. A PH (pleckstrin homology) domain found in several intracellular signal transducing molecules, thought to define a novel protein interaction domain. These domains will be subjected to site-directed mutagenesis, and mutants assayed using the 3T3 focus forming assay. D. In order to study lbc function, antibodies will be derived to identify the lbc protein, to determine its cellular localization and to analyze lbc phosphorylation. Based on the activity associated with the dbl domain encoded in other oncogenes, it is proposed to test already available recombinant lbc protein for regulatory activity against a panel of ras-like small G proteins. Furthermore, the role of lbc in the p2lras signal transduction pathway will be analyzed using a well- characterized dominant negative ras mutant Nl7. These proposed experiments will elucidate the transforming activity and function of the novel lbc oncogene.