SL12 T-LYMPHOMA: A NEW MODEL FOR GENE CONTROL IN TUMORS

从SL12.4小鼠T淋巴瘤细胞系中分离新的cDNA克隆 通过针对缺乏的姐妹细胞克隆的消减杂交， 这些基因的表达。 该提案侧重于三个最 有趣的克隆，具有意想不到的和惊人的特点。 所有这三个都是新颖的，因为它们与已知的没有显著的相似性。 基因或基因产物。 它们在胚胎发育期间受到调节， 在成年动物中显示组织特异性表达，并且可以在 明确定义的体外系统。 其中一个新基因的特征是 一个致癌基因 另一种仅在T淋巴谱系中表达 并且转录物在T细胞活化期间被强烈诱导。 的 第三种是在经历成熟的SL 12.4细胞中可诱导的， 在卵巢组织和人卵巢癌细胞系中表达。 基因表达调控。 每个基因的调控将是 在诱导和非诱导条件下，在相关和 不相关的谱系，并在体细胞杂交之间形成表达 和非表达细胞。 将评估转录率。 的5' 每个基因的调控区将被分离并测试其功能， 瞬时转染测定。 评估DNA-蛋白质的体外实验 将检查控制区域中的相互作用。 蛋白质产品的鉴别。 研究建议分离和 表征蛋白质产品。 其中一个基因预测了一种蛋白质 具有4个假定的跨膜结构域，并且可能是 位于细胞表面。 另外两个可能在细胞内 proteins. 将针对预测的蛋白质制备抗体， 评估蛋白质的物理性质，潜在的糖基化， 亚基结构，并确定每个亚细胞定位。 新基因表达的分析。 基因的表达将是 在淋巴瘤发展的不连续阶段，使用 Esther Hays博士开发的模型系统（他将提供细胞）。 将在选定的成人组织中检查基因的表达， 在胚胎发生过程中使用原位杂交。

Novel cDNA clones were isolated from the SL12.4 murine T-lymphoma cell line by subtraction hybridization against a sister cell clone which lacks expression of these genes. This proposal focuses on the three most interesting clones which possess unexpected and striking characteristics. All three are novel in that they have no significant similarity with known genes or gene products. They are regulated during embryonic development, show tissue specific expression in adult animals, and can be induced in well defined in vitro systems. One of the novel genes has the hallmarks of an onco-fetal gene. Another is expressed only in the T-lymphoid lineage and the transcripts are strongly induced during T cell activation. The third is inducible in SL12.4 cells undergoing maturation and is highly expressed in ovarian tissues and human ovarian carcinoma cell lines. Regulation of gene expression. The regulation of each of the genes will be examined under inducing and non-inducing conditions, in related and unrelated lineages, and in somatic cell hybrids formed between expressing and non-expressing cells. Rates of transcription will be assessed. The 5' regulatory region of each gene will be isolated and tested for function in transient transfection assays. In vitro experiments to assess DNA-protein interactions in the control regions will be examined. Identification of the protein product. Studies are proposed to isolate and characterize the protein products. One of the genes predicts a protein with 4 putative transmembrane spanning domains and is likely to be localized on the cell surface. The other two likely to be intracellular proteins. Antibodies will be prepared against the predicted proteins to assess the physical properties of the protein, potential glycosylation, subunit structure and to determine the subcellular localization of each. Analysis of novel gene expression. The expression of the genes will be examined in AKR animals at discrete stages of lymphoma development using a model system developed by Dr. Esther Hays (who will provide the cells). Expression of the genes will be examined in selected adult tissue and during embryogenesis using in situ hybridization.