NEW INTEGRATION LOCUS FOR MOUSE MAMMARY TUMOR VIRUS

拟议研究的长期目标是了解过程 从而导致乳腺上皮细胞的肿瘤发生。这个目标将是 通过研究病毒诱导的小鼠乳腺癌来确定 一种宿主基因，其异常表达可导致细胞失去对 它的扩散。该基因可能被对照转录激活。 逆转录病毒小鼠乳腺肿瘤病毒(MMTV)的序列，其可能是 在乳腺肿瘤中与受影响基因相邻整合。几个基因 以这种方式受到影响的病毒已被分离出来，并被描述为 乳腺原癌基因，但这些基因不作为插入位点 所有由病毒引起的乳腺肿瘤中的MMTV。中环 这项拟议研究的假设是，存在除 那些先前发现的可能被转录激活的基因 MMTV可诱导肿瘤发生。细胞DNA的片段，与一个 MMTV在病毒诱导的乳腺肿瘤中的整合位点已被克隆。 肿瘤已经获得了一个新的前病毒，它不是近整合的 已知的在乳腺中经常受MMTV影响的三个基因中的任何一个 肿瘤。拟议研究的工作假设是，克隆的 细胞DNA片段代表遗传位点的一部分，该基因位点可能 发挥另一种“乳腺原癌基因”的作用。具体目标是：1) 从正常组织中获得新整合区的分子克隆 2)确定其他病毒诱导的乳腺肿瘤是否利用新的 整合区域作为MMTV的插入位点3)绘制结构图 用限制性内切酶分析确定新的整合区域 在这张地图上，MMTV在乳腺肿瘤中的整合发生在哪里 表征基因在基因中的转录活性 正常组织和肿瘤组织中的整合区域5)以确定 基因的核苷酸序列，并与已知的序列进行比较 基因和6)检测培养乳腺的表型特性 表达来自引入的基因拷贝的RNA的上皮细胞。这个 实验策略是识别代表正常拷贝的DNA克隆 通过与MMTV相邻的片段杂交获得该基因座 原来的肿瘤。该轨迹将通过限制分析和 Southern blotting以确定其他肿瘤中该基因的重排。 从该区域转录的RNA将通过Northern分析进行鉴定。 转录区的DNA序列将通过双脱氧来确定 用计算机辅助测序反应并与已知基因进行比较 同源搜索。将构造一个载体，以强制表达 基因；这将被导入培养的小鼠乳腺上皮细胞 细胞，以确定基因表达对生长和 细胞的分化潜能。它们之间的相似之处 人类和小鼠乳腺癌的特征预示着基因 以这种方式鉴定可能与人类癌症有关，而且 通过在这个系统中的工作获得的洞察力将在 了解人类疾病。

The long term objective of the proposed research is to understand processes which lead to tumorigenesis of breast epithelial cells. This goal will be addressed by studying virus-induced mammary cancer in the mouse to identify a host gene whose abnormal expression can cause a cell to lose control of its proliferation. This gene may be transcriptionally activated by control sequences of the retrovirus mouse mammary tumor virus (MMTV), which may be integrated adjacent to the affected gene in mammary tumors. Several genes which are affected in this way have been isolated and characterized as mammary proto-oncogenes, but these genes do not serve as insertion sites for MMTV in all mammary tumors induced by the virus. The central hypothesis for the proposed research is that there exist genes other than those previously identified which may be transcriptionally activated by MMTV to induce tumorigenesis. A fragment of cellular DNA adjacent to an integration site for MMTV in a virus-induced mammary tumor has been cloned. The tumor had acquired a single new provirus which was not integrated near any of three genes known to be frequently affected by MMTV in mammary tumors. The working hypothesis for the proposed studies is that the cloned cellular DNA fragment represents a portion of a genetic locus which may function as another "mammary proto-oncogene". The Specific Aims are 1) To obtain a molecular clone of the new integration region from normal tissue 2) To determine if other virus-induced mammary tumors utilize the new integration region as an insertion site for MMTV 3) To map the structure of the new integration region by restriction enzyme analysis and to determine where on this map integrations of MMTV in mammary tumors occur 4) To characterize the transcriptional activity of the gene within the integration region in normal and neoplastic tissues 5) To determine the nucleotide sequence of the gene and compare its sequence with that of known genes and 6) To examine phenotypic properties of cultured mammary epithelial cells expressing RNA from an introduced copy of the gene. The experimental strategy is to identify DNA clones representing normal copies of the locus by hybridization to the fragment which was adjacent to MMTV in the original tumor. The locus will be mapped by restriction analysis and Southern blotting to identify rearrangements of the locus in other tumors. RNA transcribed from the region will be identified by Northern analysis. The DNA sequence of the transcribed region will be determined by dideoxy sequencing reactions and compared to known genes by computer assisted homology searches. A vector will be constructed which forces expression of the gene; this will be introduced into cultured mouse mammary epithelial cells to determine the effect of expression of the gene on growth and differentiation potential of the cells. Similarities in the characteristics of breast cancer in humans and mice predict that genes identified in this way may be involved in human cancer, and that the insight gained through work in this system will be of value in understanding the human disease.