BIOCHEMICAL GENETICS OF CHILDHOOD IMMUNODEFICIENCY

遗传性腺苷脱氨酶(ADA)缺陷导致儿童死亡 疾病，严重的联合免疫缺乏症。潜在的 免疫缺陷是腺苷和脱氧腺苷的深层紊乱 新陈代谢。ADA模式中积聚的异常代谢物 表情。在人类胸腺细胞中有非常高水平的表达， 在成熟的T细胞中要低得多，而且通常在大多数其他细胞中更低 纸巾。例如，胃和十二指肠的粘膜是一个例外。 这也表现出了与胸腺相当的表达水平。利率为 ADA基因的转录方式因细胞类型而异， 与ADA的活动水平非常接近。第一个内含子(15kb) 包含顺活动元素的复杂数组，这些元素至少在 部分，ADA基因转录的速度。只有一个子区域 的第一个内含子似乎包含T细胞特异性增强子活性。 由于来自骨髓移植矫正的ADA缺陷患者的数据 表明仅淋巴重建就能纠正免疫缺陷， 重要的是要确定不充分高的分子机制。 胸腺细胞中ADA的表达水平。我们的基本假设是 更具体的DNA序列(核心增强子元件)在第一内含子 人ADA基因与细胞型特异性DNA结合蛋白相互作用 调节胸腺细胞分化过程中ADA和T细胞的表达 淋巴细胞。对这一假设的检验将是 拟议的工作，并将采用以下具体目标：(1) 描述He基因第一内含子中的顺式调控元件(S) 作为特异性转录增强子的人ADA基因 胸腺细胞和T淋巴细胞；(2)胸腺蛋白的纯化和鉴定(S) 与核心增强子元件(S)结合并评估其参与 T淋巴系发育过程中人类基因表达的调控 分化；(3)克隆和功能鉴定(S) 和编码人胸腺增强子结合蛋白的基因(S)(S)。 将功能分配给假定的核心增强子元件和特定 增强子结合蛋白(S)将需要转基因小鼠的广泛使用 细胞培养转染法和瞬时表达法检测。预期中的 结果是对ADA调节的分子机制有了理解 人T淋巴细胞及其前体细胞的表达。

Inherited deficiency of adenosine deaminase (ADA) causes a fatal childhood illness, severe combined immunodeficiency disease. Underlying the immunodeficiency are profound disturbances of adenosine and deoxyadenosine metabolism. Abnormal metabolites that accumulate in ADA pattern of expression. In humans there is very high level expression in thymocytes, much lower in mature T-cells, and generally lower yet in most other tissues. One exception, for example, is the mucosa of stomach and duodenum that also exhibits a level of expression comparable to thymus. The rate at which the ADA gene is transcribed varies in a cell-type specific fashion, and closely parallels ADA activity levels. The first intron (15 kb) contains a complex array of cis-active elements that determine, at least in part, the rate at which the ADA gene is transcribed. Only one sub-region of the first intron appears to contain T-cell specific enhancer activity. Since data from bone-marrow transplant-corrected ADA deficient patients indicate that lymphoid-reconstitution alone corrects the immunodeficiency, it is important to determine the molecular mechanisms that underly high level ADA expression in thymocytes. Our basic hypothesis is that one or more specific DNA sequences (core enhancer elements) in the first intron of the human ADA gene interact with cell-type specific DNA binding proteins to regulate ADA expression during differentiation of thymocytes and T- lymphocytes. Testing of this hypothesis will be the major objective of the proposed work and will employ the following specific aims: (1) characterize the cis-regulatory elements(s) within the first intron of he human ADA gene that act as specific enhancers of transcription in thymocytes and T-lymphocytes; (2) purify and characterize thymic protein(s) that bind to the core enhancer element(s) and assess their involvement in regulating the developmental expression of the human gene during T-lymphoid differentiation; and (3) clone and functionally characterize the cDNA(s) and gene(s) that encode the human thymic enhancer binding protein(s). Assignment of functions to putative core enhancer elements and specific enhancer binding protein(s) will require extensive use of transgenic mice and transfection/transient expression assay in cell culture. The expected outcome is an understanding of the molecular mechanism of regulation of ADA expression in human T-lymphocytes and their precursors.