DNA-PROTEIN INTERACTIONS IN LYMPHOCYTE DIFFERENTIATION

B和T淋巴细胞的抗原受体编码在离散的DNA中 在发育过程中由特定部位的DNA连接的片段 重新安排。抗原受体基因组装，或V(D)J重排，是 这是脊椎动物中唯一已知的定点DNA重组的例子。 异常的V(D)J重组很可能参与了 细胞原癌基因与抗原之间的染色体易位 在高比例的淋巴系统恶性肿瘤中出现的受体基因座。 对V(D)J重组及其调控的理解仍在继续 这是这个项目的一个长期目标。 两种蛋白质，rag-1和rag-2是激活所必需且足够的。 V(D)J重排。我们过去的研究提供了以下几点 RAG-2蛋白的表达和V(D)J重组是 细胞周期调控：(1)RAG-2蛋白表达受限 通过转录后机制进入G0/G1；(2)RAG-2磷酸化 在体外通过特定部位的细胞周期蛋白依赖性激酶(CDK)；(3) 体内该位点的磷酸化与快速降解有关。 RAG-2；和(4)在V(D)J处的位点特异性双链DNA断裂 重组信号序列也优先在G0/G1中累积。 我们的工作假设是RAG-2被一个或多个 CDK调节RAG-2的积聚，进而调节V(D)J 重组。其他观察结果表明，这种联系 RAG-2的磷酸化和降解之间可能反映出更一般的 机制。 在下一个资金阶段，我们希望研究RAG-2的表达情况 与细胞周期和这一调控与 V(D)J复合的时间。为此，我们提出如下建议 具体目标：(1)确定RAG-2的结构决定因素 不稳定性与细胞周期调控；(2)确定相对分子质量 蛋白质合成和降解在RAG-2调控中的作用 (3)检测RAG-2在细胞周期中的磷酸化。 细胞周期并测试RAG-2的降解是否被 磷酸化；(4)确定细胞周期依赖于 V(D)J复合中间体的积累是 调节RAG-2的表达；(5)检测RAG-2的生理后果 在培养细胞和转基因小鼠中非程序性表达RAG-2；以及 (6)建立酵母RAG-2降解分析系统， 在基因水平上研究这一过程的长期目标。

The antigen receptors of B and T lymphocytes are encoded in discrete DNA segments that are joined during development by site-specific DNA rearrangements. Antigen receptor gene assembly, or V(D)J rearrangement, is the only known example of site-specific DNA recombination in vertebrates. Aberrant V(D)J recombination is likely to be involved in generating the chromosomal translocations between cellular protooncogenes and antigen receptor loci that are seen in a high proportion of lymphoid malignancies. An understanding of V(D)J recombination and its regulation continues to be a long-term goal of this project. Two proteins, RAG-1 and RAG-2, are necessary and sufficient for activation of V(D)J rearrangement. Our past studies have provided the following evidence that expression of the RAG-2 protein and V(D)J recombination are regulated in the cell cycle: (1) expression of RAG-2 protein is restricted to G0/G1 by a posttranscriptional mechanism; (2) RAG-2 is phosphorylated by a cyclin-dependent kinase (cdk) at a specific site in vitro; (3) phosphorylation of this site in vivo is associated with rapid degradation of RAG-2; and (4) site-specific double-strand DNA breaks at V(D)J recombination signal sequences also accumulate preferentially in G0/G1. Our working hypothesis is that phosphorylation of RAG-2 by one or more cdk's regulates accumulation of RAG-2, in turn regulating V(D)J recombination. Additional observations suggest that the association between RAG-2 phosphorylation and degradation may reflect a more general mechanism. In the next funding period, we wish to examine how RAG-2 expression is coupled to the cell cycle and the relationship of this regulation to the timing of V(D)J recombination. To this end, we propose the following specific aims: (1) to define the structural determinants of RAG-2 instability and cell cycle regulation; (2) to determine the relative contributions of protein synthesis and degradation to regulation of RAG-2 accumulation in the cell cycle; (3) to assess phosphorylation of RAG-2 in the cell cycle and to test whether RAG-2 degradation is targeted by phosphorylation; (4) to determine whether cell cycle-dependent accumulation of V(D)J recombination intermediates is a consequence of regulated RAG-2 expression; (5) to examine the physiologic consequences of unscheduled RAG-2 expression in cultured cells and in transgenic mice; and (6) to develop a system for analysis of RAG-2 degradation in yeast, with the long-term goal of examining this process at the genetic level.