CD8 GENE REGULATION AND LYMPHOCYTE DIFFERENTIATION

我们的目标是克隆编码蛋白质的基因， 人T淋巴细胞抗原CD 8的表达 淋巴细胞的发育。 有了这个基因，我们可以研究 淋巴细胞特异性分子的表达在正常情况下受到调节， 细胞以及淋巴瘤和白血病。 调控异常 基因会导致致癌作用。 如果调节异常， 淋巴细胞特异性基因产物的检测，我们将研究 这是详细的。 我们将使用一种新的方法来克隆这些基因。 CD8 将分离和分析CD 8 + T细胞系的缺失突变体， 鉴定编码调节蛋白基因的突变体。 等 突变体将作为用cDNA转染的受体 直接表达文库，以弥补这一缺陷。 的 用新的Epstein巴尔病毒穿梭载体制备文库， 允许cDNA克隆的稳定附加体复制。 回收 可以容易地获得来自转化体的完整cDNA克隆 通过Hirt分离，然后转化大肠杆菌。 另 该载体的优点是稳定的高效率 可以实现人细胞的转化， 将整个高复杂性表达文库引入 受体细胞 因此，在转染后，变体重新- 将使用抗CD 8单克隆抗体选择表达CD 8的细胞。 抗体和荧光激活细胞分选仪（FACS）。 的 负责互补的cDNA质粒将通过以下方法分离： Hirt提取和转化E.杆菌 我们将试图确定CD 8在胸腺教育中的作用， T淋巴细胞借此学习辨别自身主要 组织相容性抗原（MHC）。 CD 8是一种 T淋巴细胞与MHC相互作用重要分子 这就是所谓的MHC限制。 CD 8通常 所需的相互作用与MHC I类分子的成熟T 细胞 然而，它以不同的方式表达在胸腺细胞上。 分子形式--它与 分化抗原CD 1。 这种关联是否增强了 与MHC I类分子相互作用或减少相互作用。 我们将CD 1基因导入人T细胞毒性细胞系 并确定对T细胞与其 靶细胞。

Our aim is to cloe genes encoding proteins that regulate the expression of the human T lymphocyte antigen CD8 during development of the lymphocyte. With the gene, we can study how expression of lymphocyte specific molecules is regulated in normal cells and in lymphomas and leukemias. Abnormal regulation of genes can lead to carcinogenesis. If abnormal regulation of lymphocyte specific gene products was detected, we would study this in detail. We will be using a novel approach for cloning such genes. CD8 loss mutants of CD8+ T cell lines will be isolated and analyzed to identify mutants in genes encoding regulatory proteins. Such mutants will serve as recipients for transfection with a cDNA direct expression library in order to complement the defect. The library is made with a new Epstein Barr Virus shuttle vector that allows stable episomal replication of cDNA clones. Recovery of intact cDNA clones from transformants can be readily achieved by Hirt isolation followed by transformation of E.coli. The other advantage of this vector is that high efficiences of stable transformation of human cells can be achieved which allows the introduction of entire high complexity expression libraries into recipient cells. Therefore, after transfection variants re- expressing CD8 will be selected using anti-CD8 monoclonal antibody and a fluorescence-activated cell sorter (FACS). The cDNA plasmid responsible for complementation will be isolated by Hirt extraction and transformation of E. coli. We will try to determine the role of CD8 in thymic education of the T lymphocyte whereby T cells learn to distinguish self major histocompatibility antigens (MHC) from non-self. CD8 is an important molecule for interaction of T lymphocytes with MHC molecules in what is known as MHC restriction. CD8 is usually required for interaction with MHC class I molecules by mature T cells. However, it is expressed on thymocytes in a different molecular form--it is covalently associated with the differentiation antigen CD1. Does this association enhance interaction with MHC class I molecules or diminish interaction. We will transfect the CD1 gene into a human T cytotoxic cell line and determine the effect on interaction of the T cell with its target cell.