BIOCHEMICAL GENETICS OF CHILDHOOD IMMUNODEFICIENCY

遗传性缺乏腺苷脱氨酶（ADA）导致致命的童年 疾病、严重联合免疫缺陷病。 所依据 免疫缺陷是腺苷和脱氧腺苷的严重干扰 新陈代谢. 以ADA模式蓄积的异常代谢物 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，在大多数其他细胞中通常更低。 组织中 例如，胃和十二指肠的粘膜是一个例外 其表达水平也与胸腺相当。 的速率 ADA基因被转录的区域以细胞类型特异性方式变化， 与ADA活性水平密切相关 第一个内含子（15 kb） 包含一系列复杂的顺式活性元件，这些元件至少在 部分，ADA基因转录的速率。 只有一个次区域 的第一个内含子似乎含有T细胞特异性增强子活性。 由于骨髓移植校正的ADA缺乏患者的数据 表明淋巴重建单独纠正免疫缺陷， 重要的是要确定的分子机制， 水平胸腺细胞ADA表达。 我们的基本假设是， 第一内含子中更特异的DNA序列（核心增强子元件）， 人ADA基因与细胞类型特异性DNA结合蛋白相互作用， 调节胸腺细胞分化过程中ADA的表达， 淋巴细胞 对这一假设的检验将是本报告的主要目标。 建议的工作，并将采用以下具体目标：（1） 描述基因第一内含子内的顺式调控元件， 人ADA基因作为转录特异性增强子， 胸腺细胞和T淋巴细胞;（2）纯化和表征胸腺蛋白 结合核心增强子元件并评估其参与 调节T淋巴细胞增殖过程中人类基因的发育表达 分化;和（3）克隆和功能表征cDNA， 和编码人胸腺增强子结合蛋白的基因。 向推定的核心增强子元件和特异性增强子元件分配功能 增强子结合蛋白需要广泛使用转基因小鼠 和细胞培养物中的转染/瞬时表达测定。 预期 结果是对ADA调节的分子机制的理解， 在人T淋巴细胞及其前体中的表达。

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.