PATHOLOGICAL AND NORMAL FUNCTION OF A NOVEL HOMEBOX GENE

这个应用程序集中在一个新的基因，Pem，我们最近分离。 cDNA克隆自AKR T淋巴瘤细胞系。 cDNA序列 含有同源框基序，预测的蛋白质具有一些独特的 功能. Pem是第一个定位于X染色体的同源异型盒基因 染色体。 Pem在胚胎发生中表达，但在 正常成人组织或淋巴样细胞;它大量表达于 许多转化的和永生化的细胞系。 Pem表示为 成熟卵母细胞，并在胚胎外以高度调节的方式 在小鼠发育的早期。 现场分析表明， 表达Pem的细胞侵入母体子宫组织， 置入 Pem在致瘤性中起作用的可能性， 将研究转移性侵袭。 从细胞系中， Pem克隆是孤立的原因突出的双侧卵巢肿瘤在95% 的IV注射的雌性AKR小鼠。 Pem基因的功能分析 提供了一个机会来调查一个可能的作用， Pem在转化和/或侵入过程中的表达。 的 将检查Pem基因的生理和病理功能 使用两种基本方法：“功能丧失”和“功能获得” 实验 功能丧失的研究将通过基因 同源重组（homologous recombination）。 缺乏Pem的动物的后代 表达将产生。 缺乏Pem基因功能的细胞， 在多种体外测定中测试。 Pem函数的增益为 通过将Pem cDNA转染到相关的SL 12 T淋巴瘤细胞中完成 并进入不表达该基因的正常AKR和人成纤维细胞 并评估其致瘤潜力。 Pem基因的表达将 在胚胎发生过程中，在多能干细胞中， 通过原位杂交和/或 免疫组化 Pem消融干细胞的成熟状态 将进行实验性改变，以评估是否需要Pem功能 用于体外分化。 我们将评估Pem表达是否是 与细胞形成卵巢肿瘤的能力有关。 的事实 Pem蛋白含有一个同源结构域，位于细胞核内， 区室表明Pem作为转录调节因子发挥作用。 我们的亲和纯化的抗Pem抗血清将是一个有用的辅助， 确定Pem蛋白1）是否被转录后修饰; 2） 在复合物中发挥作用或与其他蛋白质协同作用; 3）结合 DNA和/或与DNA结合蛋白的复合物以抑制DNA结合。

This application focuses upon a novel gene, Pem, we recently isolated. The cDNA was cloned from an AKR T lymphoma cell line. The cDNA sequence contains a homeobox motif and the predicted protein has some unique features. Pem is the first homeobox gene to be localized to the X chromosome. Pem is expressed in embryogenesis but not detectably in normal adult tissues or lymphoid cells; it is abundantly expressed in numerous transformed and immortalized cell lines. Pem is expressed in mature oocytes and in a highly regulated manner in the extraembryonic cells early in murine development. In situ analysis shows that some cells which express Pem invade the maternal uterine tissue during implantation. the possibility that Pem has a role in tumorigenicity or metastatic invasion will be investigated. The cell line from which the Pem clone was isolated causes prominent bilateral ovarian tumors in 95% of IV injected female AKR mice. the functional analysis of the Pem gene presents an opportunity to investigate a possible role of disregulated Pem expression in processes of transformation and/or invasion. The physiological and pathological function of the Pem gene will be examined using two basic approaches: "loss of function" and "gain of function" experiments. the loss of function studies will be accomplished by gene targeting (homologous recombination). Progeny of animals lacking Pem expression will be produced. Cells lacking Pem gene function will be tested in a variety of in vitro assays. Gain of Pem function will be accomplished by transfecting Pem cDNA into related SL12 T lymphoma cells and into normal AKR and human fibroblasts which do not express the gene and assessed for their tumorigenic potential. Pem gene expression will be further examined during embryogenesis, in pluripotent stem cells and in selected adult stem cell tissues by in situ hybridization and/or immunohistochemistry. The maturation status of Pem ablated stem cells will be experimentally altered to assess whether Pem function is required for in vitro differentiation. We will assess whether Pem expression is associated with the capacity of cells to form ovarian tumors. The fact that the Pem protein contains a homeodomain and is located in the nuclear compartment indicates that Pem functions as a transcriptional regulator. Our affinity purified anti-Pem antisera will be a useful adjunct to determine whether Pem protein 1) is post-transcriptionally modified; 2) functions in a complex or cooperatively with other proteins; 3) binds to DNA and/or complexes with DNA binding proteins to inhibit DNA binding.