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: 3459995
• 负责人: JANICE E KNEPPER
• 金额: $ 8.96万
• 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/3459995
• 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 hybridization; nucleic acid sequence; protooncogene; provirus; restriction mapping; 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过程 导致乳腺上皮细胞的肿瘤发生。 这一目标将 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 其扩散。 该基因可能是转录激活的控制 逆转录病毒小鼠乳腺肿瘤病毒（MMTV）的序列，其可以是 整合在乳腺肿瘤中受影响的基因附近。 几个基因 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 MMTV在所有由病毒诱导的乳腺肿瘤中的表达。 中央 这项研究的假设是，存在基因以外的基因， 那些先前鉴定的可能被转录激活的基因， MMTV诱导肿瘤发生。 一个细胞DNA片段， MMTV在病毒诱导乳腺肿瘤中的整合位点已被克隆。 肿瘤获得了一个新的前病毒，它没有整合在肿瘤周围。 已知在乳腺癌中经常受MMTV影响的三个基因中的任何一个 肿瘤的 这项研究的工作假设是， 细胞DNA片段代表遗传基因座的一部分， 作为另一个“乳腺原癌基因”发挥作用。 具体目标是：（1） 从正常组织中获得新整合区的分子克隆 2)为了确定其他病毒诱导的乳腺肿瘤是否利用新的 整合区作为MMTV的插入位点3）为了映射MMTV的结构， 通过限制性内切酶分析确定新的整合区 在该图上，MMTV在乳腺肿瘤中的整合发生在4）至 表征基因的转录活性， 5）为了确定正常和肿瘤组织中的整合区， 基因的核苷酸序列，并将其序列与已知的 基因和6）检查培养的乳腺癌细胞的表型特性， 上皮细胞从引入的基因拷贝表达RNA。 的 实验策略是鉴定代表正常拷贝的DNA克隆 通过与MMTV的邻近片段杂交， 原来的肿瘤。 该基因座将通过限制性分析作图， Southern印迹以鉴定其他肿瘤中基因座的重排。 将通过北方分析鉴定从该区域转录的RNA。 转录区域的DNA序列将通过双脱氧核糖核酸测定。 测序反应，并通过计算机辅助与已知基因进行比较 同源搜索 将构建一个向量，该向量强制表达 该基因;这将被引入培养的小鼠乳腺上皮细胞 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 人类和小鼠乳腺癌的特征预测， 以这种方式确定的可能与人类癌症有关， 通过在这一系统中开展工作而获得的见解， 了解人类疾病。