REGULATORY ELEMENTS OF INTERLEUKIN-2 GENE EXPRESSION

我们的长期利益是更好地理解分子机制 涉及T淋巴细胞的活化和增殖。在这份提案中，我们 特别关注定义5‘-侧翼区的序列 人白介素2基因参与白介素2基因表达的研究 正常T淋巴系和白血病T淋巴系及其反式作用因子 在这些反应中起作用。特别是，我们将检测IL2基因 丝裂原和佛波酯的激活和随后的下调 (PHA/PMA)，T细胞表面抗原T3、T11和9.3的抗体，以及 离子载体；2)环孢素A对IL-2表达的抑制；3)细胞 IL-2表达对某些T淋巴样细胞的特异性限制；4) 放线菌亚胺介导的IL-2mRNA的超诱导；5)是否有 种特异性IL2在小鼠和长臂猿淋巴系中的表达，以及6) 反式活动因子在明显的正常缺乏中的潜在作用 IL-2在长臂猿白血病细胞系MLA144中的表达 基本的方法将是测试细菌的表达 《记者》酶基因氯霉素乙酰转移酶融合到 IL2的5‘-侧翼区(PIL2CAT)。我们已经证明了监管 在这些结构中的行为与内源性IL2基因在 丝裂原诱导型人T细胞株J32。IL-2中构建的突变 PIL2CAT的5‘侧翼序列可用于比较 正常和突变的质粒来定义重要的调控序列。5‘ 缺失、连接扫描器和定点突变体将允许精确 责任残留物的划定。CAT表达式数据将为 补充S1核酸酶保护CAT的mRNAs证实 CAT活性的变化反映了转录调控的正确 启动了“IL2成绩单”。此外，竞赛实验使用 正常或改变的pIL2CAT与其他与之相关的基因的共转染 正常或改变的IL2 5‘侧翼序列应该允许我们确定 如果反式作用因子在任何IL2基因反应中发挥作用，第一步 对细胞内可能至关重要的物质的分离和进一步研究 一系列受调控的基因事件中的分子。 IL-2在T淋巴细胞活化、扩增等过程中的中枢作用 淋巴细胞反应以及对同种异体移植物滞留的显著影响 IL-2抑制剂环孢素A对IL-2基因表达的研究 对于理解和潜在地操纵免疫极其重要 回应。

Our long-term interest is a better understanding of molecular mechanisms involved T lumphocyte activation and proliferation. In this proposal we focus specifically on defining sequences in the 5'-flanking region of the human interleukin-2 (IL2) gene that participate in IL2 gene expression in normal and leukemic T lymphoid lines and whether trans-acting factors function in these responses. In particular we will examine 1) IL2 gene activation and subsequent down regulation by mitogenes and phorbol esters (PHA/PMA), antibodies to T cell surface antigens T3, T11 and 9.3, and ionophores; 2) inhibition of IL2 expreession by cyclosporina A; 3) cell specific restriction of IL2 expression to certain T lymphoid cells; 4) cycloheximide-mediated superinduction of Il2 mRNA; 5) whether there is species specific IL2 expression in murine and gibbon lymphoid lines, and 6) the potential role of trans-active factors in the apparent lack of normal IL2 expression in the gibbon leukemia line, MLA144. The basic approach will be to test the expression of the bacterial "reporter" enzyme gene chloramphenicol acetyl transferase (CAT) fused to 5'-flanking regions of IL2 (pIL2CAT). We have demonstrated regulatory behavior in these constructs which parallels the endogenous IL2 gene in the mitogen-inducible human T cell line, J32. Mutations constructed in the Il2 5'-flanking sequences of pIL2CAT would permit a comparison of transfected normal and mutant plasmids to define important regulatory sequences. 5' deletions, linker-scanner and site-directed mutants will allow precise delineation of responsible residues. CAT expression data will be supplemented by S1 nuclease protection of CAT mRNAs to confirm that alterations in CAT activity reflect transcriptional regulation of properly initiated "IL2 transcripts." Moreover, competition experiments using cotransfection of normal or altered pIL2CAT with other genes linked to normal or altered IL2 5'-flanking sequences should permit us to determine if trans-acting factors play a role in any Il2 gene responses, a first step toward the isolation and further study of what may be crucial intracellular molecules in a host of regulated gene events. The central role of Il2 in T lymphocyte activation, expansion and other lyumphocyte responses, along with the marked effect on allograft retention of the Il2 inhibitor, cyclosporin A, make the study of Il2 gene expression extremely important for understanding and potentially manipulating immune responses.