ISOLATION OF AMPLIFIED GENE IN GERM CELL TUMORS

我们的首要目标是分离该基因并对其进行分子特征分析(S) 在性腺外和转移的人类生殖细胞中被发现扩增 肿瘤(GCT)及其基因(S)在正常生殖细胞中的作用 在发展和肿瘤发生中。男性人类生殖细胞肿瘤发生在 睾丸和性腺外部位(主要在中线部位) 后者是原发的或转移性的。这些肿瘤，由于 从生殖细胞的转化，可以呈现出组织学上的 合子前和合子后的组织均可见。白粉病的核型分析 GCTS揭示了非随机染色体标记的存在和 为GCTS的性腺外部位基因扩增提供了证据。 利用DNA在分子水平上证实了基因扩增 凝胶内复性试验；然而，扩增出的基因(S)的同源性 仍然不为人所知。基因产物可能在体内起到信号的作用 原始生殖细胞在中线定居的早期胚胎发生 在随后的性腺发育过程中。我们的具体目标将涉及 以下是几种方法。1.在性腺外扩增的S基因的分离 以及使用凝胶内复性技术的转移性GCTS。准备工作 凝胶内复性分析将是。对从GCT提取的DNA执行 具有均匀染色区域(HSR)的细胞系。扩增的DNA将 从凝胶中分离出来，然后克隆到载体上，进一步 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。细胞系DNA的基因组文库 将用扩增片段进行筛选以分离重叠基因 序列。克隆的进一步表征将涉及限制 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 最终可能过度表达的基因产物。3.GCTS的筛选 以扩增出的基因(S)为探针。Southern杂交分析将是 用从所有组织的GCTS中提取的DNA作为探针 扩增出上述分离的DNA序列。这将决定流行率 该基因在性腺和性腺外GCTS中的扩增(S) 演示文稿。这样的研究可能具有临床意义，如果模式 在临床预后较差的肿瘤中可观察到扩增。4. 分离基因在正常细胞和正常细胞中作用的鉴定 肿瘤发生学。将进行实验研究，以调查 基因(S)结构和拷贝数(Southern杂交分析) 水平(Northern杂交分析和S1核酸酶分析)和 蛋白质水平(Western blotting和免疫组织化学技术) 各种人体组织、肿瘤和细胞系。亚细胞定位 将通过免疫荧光检测蛋白质的含量。

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.