ISOLATION OF AMPLIFIED GENE IN GERM CELL TUMORS

生殖细胞肿瘤中扩增基因的分离

• 批准号: 2095366
• 负责人: Jane Houldsworth
• 金额: $ 11.72万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-02-01 至 1995-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2095366
• 关键词: DNA replication; complementary DNA; cytogenetics; gel filtration chromatography; genetic library; genome; germ cell neoplasms; gonads; human tissue; immunocytochemistry; immunofluorescence technique; messenger RNA; metastasis; molecular cloning; natural gene amplification; neoplasm /cancer genetics; nucleic acid probes; nucleic acid sequence; restriction mapping; southern blotting; tissue /cell culture; transfection

Our primary goal is to isolate and molecularly characterize the gene(s) found to be amplified in extragonadal and metastatic human germ cell tumors (GCTs) and to determine the role of the gene(s) in normal germ cell development and in tumorigenesis. Male human germ cell tumors occur in the testis and at extragonadal sites (primarily at midline locations) with the latter being of either primary or metastatic origin. These tumors, arising from the transformation of germ cells, can take on the histological appearance of both pre- and post-zygotic tissues. Karyotypic analysis of GCTs has revealed the presence of nonrandom chromosome markers and provided evidence for gene amplification in GCTs at extragonadal sites. Gene amplification has been confirmed at the molecular level using the DNA in-gel renaturation assay; however, the identity of the gene(s) amplified remain unknown. It is possible that the gene product acts as a signal in early embryogenesis for primordial germ cells to settle in the midline during subsequent gonad development. Our specific aims would involve the following approaches. 1. Isolation of gene(s) amplified in extragonadal and metastatic GCTs using the In-gel renaturation technique. Preparative in-gel renaturation assays will be. performed on DNA extracted from a GCT cell line with a homogeneously staining region (HSR). Amplified DNA will be isolated from the gel and subsequently cloned into vectors for further identification of sequences uniquely amplified in this cell line. 2. Isolation and characterization of complete gene sequences from human genomic and CDNA libraries. Genomic libraries of DNA from the cell line will be screened with amplified fragments to isolate overlapping gene sequences. Further characterization of clones will involve restriction mapping, identification of transcribed sequences, CDNA isolation, sequencing, and examination of sequences for possible coding regions of gene products that may be ultimately overexpressed. 3. Screening of GCTs with amplified gene(s) as probes. Southern hybridization analysis will be performed on DNA isolated from GCTs of all histologies using as probes the amplified DNA sequences isolated above. This will determine the prevalence of amplification of this gene(s) in GCTs of both gonadal and extragonadal presentation. Such a study has possible clinical implications if patterns of amplification are observed in tumors with poor clinical outcome. 4. Identification of the role of Isolated genes In normal cells and In tumorigenesis. Experimental studies will be undertaken to investigate the gene(s) structure and copy number (Southern hybridization analysis), MRNA levels (Northern hybridization analysis and Sl nuclease assays) and protein levels (Western blotting and immunohistochemical techniques) in a variety of human tissues,tumors and cell lines. Subcellular localization of the protein will be examined by immunofluorescence.

我们的主要目标是分离和分子特征的基因（S） 发现在性腺外和转移性人类生殖细胞中扩增 肿瘤（GCTs），并确定该基因在正常生殖细胞中的作用 发育和肿瘤发生。男性生殖细胞肿瘤发生在 睾丸和性腺外部位（主要在中线位置）， 后者是原发性的或转移性的。这些肿瘤， 从生殖细胞的转变，可以采取组织学上的 合子前和合子后组织的外观。核型分析 GCT显示存在非随机染色体标记， 提供了性腺外部位GCT基因扩增的证据。 基因扩增已在分子水平上得到证实， 凝胶内复性试验;然而，扩增的基因的同一性 仍然未知。基因产物有可能作为一种信号， 原始生殖细胞在胚胎发生早期定居在中线 在随后的性腺发育中。我们的具体目标将涉及 遵循方法。1.性腺外扩增基因的分离 和转移性GCTs。制备型 凝胶内复性试验。对从GCT中提取的DNA进行 具有均匀染色区域（HSR）的细胞系。扩增的DNA将 从凝胶中分离，随后克隆到载体中， 鉴定在该细胞系中唯一扩增的序列。2. 人全基因序列的分离与鉴定 基因组和cDNA文库。来自细胞系的DNA的基因组文库 将用扩增片段筛选以分离重叠基因 序列的克隆的进一步表征将涉及限制性内切酶， 作图，转录序列的鉴定，cDNA分离， 测序，并检查可能的编码区的序列 基因产物可能最终被过度表达。3. GCT筛选 用扩增的基因作为探针。Southern杂交分析将 对从所有组织学的GCT中分离的DNA进行，使用 上面分离的扩增的DNA序列。这将决定 该基因在性腺和性腺外GCT中的扩增 演示文稿.这样的研究可能具有临床意义，如果模式 在临床结果较差的肿瘤中观察到扩增。4. 分离基因在正常细胞中的作用的鉴定 肿瘤发生将进行实验研究， 基因结构和拷贝数（Southern杂交分析），mRNA 水平（北方杂交分析和Sl核酸酶测定）和 蛋白质水平（蛋白质印迹和免疫组织化学技术） 各种人类组织、肿瘤和细胞系。亚细胞定位 将通过免疫荧光检查蛋白质的量。