PATHOLOGICAL AND NORMAL FUNCTION OF A NOVEL HOMEBOX GENE

这项应用集中在我们最近分离的一个新基因Pem上。 该基因是从AKR-T淋巴瘤细胞系中克隆的。全长cDNA序列 包含一个同源框基序，预测的蛋白质具有一些独特的 功能。PEM是第一个定位于X的同源框基因 染色体。PEM在胚胎发育中表达，但在 正常成人组织或淋巴样细胞；它在 无数转化和永生化的细胞系。PEM用来表示 成熟的卵母细胞，并以高度调控的方式在胚胎外 小鼠发育早期的细胞。现场分析表明，一些 在孕期，表达Pem的细胞侵入母体子宫组织 植入。Pem在肿瘤发生或发展中起作用的可能性 将对转移性侵袭进行调查。细胞系，从该细胞系 分离的PEM克隆导致95%的双侧卵巢肿瘤 静脉注射的雌性AKR小鼠。Pem基因的功能分析 提供了一个机会来调查不受监管的 PEM在转化和/或侵袭过程中的表达。这个 将对Pem基因的生理和病理功能进行检测 使用两种基本方法：“功能的丧失”和“功能的获得” 实验。功能丧失的研究将由基因完成 靶向(同源重组)。缺乏Pem的动物的后代 就会产生表情。缺乏Pem基因功能的细胞将是 在各种体外测试中进行了测试。Pem函数的增益将为 将Pem基因导入相关的SL12 T淋巴瘤细胞 并转化为正常的AKR和不表达该基因的人成纤维细胞 并对它们的致癌潜力进行评估。PEM基因的表达将 在胚胎发育过程中，在多能干细胞和 在选定的成人干细胞组织中进行原位杂交和/或 免疫组织化学。Pem消融后干细胞的成熟状态 将在实验中更改以评估是否需要PEM函数 用于体外分化。我们将评估Pem的表达是否 与细胞形成卵巢肿瘤的能力有关。事实是 Pem蛋白含有一个同源结构域，位于细胞核中 间隔室表明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工具 确定Pem蛋白1)是否转录后修饰；2) 在复合体中或与其他蛋白质协同发挥作用；3)结合 DNA和/或与DNA结合蛋白的复合体以抑制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.