CD8 GENE REGULATION AND LYMPHOCYTE DIFFERENTIATION

我们的目标是关闭编码蛋白质的基因，这些蛋白质调节 人T淋巴细胞抗原CD8在慢性粒细胞白血病中的表达 淋巴细胞的发育。有了这个基因，我们就可以研究 正常人群中淋巴细胞特异性分子的表达受到调节 细胞以及淋巴瘤和白血病中。不正常的调节 基因可以导致癌症的发生。如果不正常的调节 检测到淋巴细胞特异性基因产物，我们将研究 这一点很详细。 我们将使用一种新的方法来克隆这些基因。CD8 CD8+T细胞株的缺失突变株将被分离并分析 确定编码调控蛋白的基因中的突变。是这样的 突变体将作为cdna转基因的受体。 直接表达文库以弥补这一缺陷。这个 图书馆由一种新的爱泼斯坦-巴尔病毒穿梭载体制成， 允许稳定的cdna克隆的异体复制。恢复 从转化子中获得完整的cDNA克隆是容易的 通过Hirt分离，然后转化E.coli.另一个 这种载体的优点是稳定的高效 可以实现人类细胞的转化，这使得 将整个高复杂性表达式库引入到 受体细胞。因此，在转染变异体后， 将用抗CD8单抗选择表达CD8 抗体和荧光激活细胞分类器(FACS)。这个 负责互补的cDNA质粒将通过以下方法分离 大肠杆菌Hirt的提取和转化。 我们将尝试确定CD8在胸腺教育中的作用。 T淋巴细胞--T细胞学习如何区分自己的专业 异体组织相容性抗原(MHC)。CD8是一种 T淋巴细胞与MHC相互作用的重要分子 分子在所谓的MHC限制中。CD8通常是 成熟T细胞与MHC I类分子相互作用所需 细胞。然而，它在胸腺细胞上表达的方式不同 分子形式--它与 分化抗原CD1。这种联系是否增强了 与MHC I类分子相互作用或减弱相互作用。 我们将CD1基因导入人T细胞毒细胞系 并确定对T细胞与其相互作用的影响 目标单元格。

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.