NEW INTEGRATION LOCUS FOR MOUSE MAMMARY TUMOR VIRUS

小鼠乳腺肿瘤病毒的新整合位点

• 批准号: 3459998
• 负责人: JANICE E KNEPPER
• 金额: $ 11.26万
• 依托单位: VILLANOVA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-01 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3459998
• 关键词: breast neoplasms; cellular oncology; computer assisted sequence analysis; gene expression; gene rearrangement; genetic mapping; genetic transcription; laboratory mouse; mammary epithelium; molecular cloning; mouse mammary tumor virus; neoplasm /cancer genetics; nucleic acid sequence; protooncogene; provirus; restriction mapping; southern blotting; tissue /cell culture; transposon /insertion element; viral carcinogenesis

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.

拟议研究的长期目标是了解流程 从而导致乳腺上皮细胞发生肿瘤。 这个目标将是 通过研究病毒诱导的小鼠乳腺癌来解决这一问题 宿主基因，其异常表达可导致细胞失去对 它的扩散。 该基因可能通过控制转录激活 逆转录病毒小鼠乳腺肿瘤病毒（MMTV）的序列，可能是 整合到乳腺肿瘤中受影响的基因附近。 几个基因 以这种方式受到影响的地区已被隔离并定性为 乳腺原癌基因，但这些基因不作为插入位点 用于 MMTV 病毒诱发的所有乳腺肿瘤。 中央 拟议研究的假设是，除了基因之外还存在其他基因 那些先前确定的可能被转录激活的 MMTV诱导肿瘤发生。 与细胞相邻的 DNA 片段 MMTV 在病毒诱导的乳腺肿瘤中的整合位点已被克隆。 肿瘤获得了一种新的原病毒，该原病毒没有整合到附近 已知在乳腺中经常受 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 细胞 DNA 片段代表遗传位点的一部分，可能 作为另一种“乳腺原癌基因”。 具体目标是 1) 从正常组织中获得新整合区域的分子克隆 2) 确定其他病毒诱发的乳腺肿瘤是否利用新的 整合区域作为 MMTV 的插入位点 3) 映射结构 通过限制性内切酶分析确定新的整合区域 在此图上，MMTV 在乳腺肿瘤中发生整合的位置 4) 表征基因内的转录活性 正常组织和肿瘤组织中的整合区域 5) 确定 基因的核苷酸序列，并将其序列与已知的序列进行比较 基因和 6) 检查培养乳腺的表型特性 上皮细胞从引入的基因拷贝表达RNA。 这 实验策略是识别代表正常拷贝的 DNA 克隆 通过与 MMTV 相邻的片段杂交来确定基因座 原来的肿瘤。 将通过限制性分析绘制基因座图 Southern 印迹法可识别其他肿瘤中基因座的重排。 该区域转录的 RNA 将通过 Northern 分析进行鉴定。 转录区域的DNA序列将通过双脱氧来确定 测序反应并通过计算机辅助与已知基因进行比较 同源性搜索。 将构建一个载体，强制表达 基因；这将被引入培养的小鼠乳腺上皮细胞中 细胞以确定基因表达对生长的影响 细胞的分化潜能。 相似之处在于 人类和小鼠乳腺癌的特征预测基因 以这种方式确定的可能与人类癌症有关，并且 通过在该系统中工作获得的见解将具有价值 了解人类疾病。