TEMPORAL EXPRESSION OF DNP-SPECIFIC V-GENES

以前的工作已经确定，抗二硝基苯基（DNP）的反应， 新生Balb/c小鼠仅限于少数独特型， 成年人的反应是多种多样的。 这种行为大致类似于 Balb/c免疫库的全面扩展：新生小鼠表达一种 总共约10（4）种不同的特异性（克隆型）， 逐渐增加到成人中大于10（7）个克隆型。 目的 的拟议计划是表征，在DNA水平，时间 Balb/c小鼠中抗DNP应答的发展。 协同 博士斯克里普斯诊所和研究基金会的诺曼·克林曼， 制备表达不同抗-DNP独特型的杂交瘤，并确定 使用Klinman脾病灶的这些独特型的时间表达 比色法 三个表达常见Balb/c新生儿克隆型的杂交瘤具有 已经在克林曼的实验室里完成了 使用重组DNA 方法，我们将确定DNP特异性V-，D-和J-基因片段 在这些杂交瘤的每一个中表达的序列和相对位置 Balb/c种系DNA中的这些基因片段。 然后我们将尝试 理解每个基因产物的时间表达， 序列和侧翼序列，及其在种系基因组中的位置。 我们将把这项工作扩展到其他近交系小鼠品系;这将使我们能够 为了测试我们对Balb/c数据的解释， 抗DNP应答中菌株依赖性差异的分子基础。

Previous work has established that the anti-dinitrophenyl (DNP) response in neonatal Balb/c mice is restricted to only a few idiotypes whereas the adult response is quite diverse. This behavior roughly parallels the overall expansion of the Balb/c immune repertoire: newborn mice express a total of about 10(4) different specificities (clonotypes) and this gradually increases to greater than 10(7) clonotypes in the adult. The aim of the proposed program is to characterize, at the DNA level, the temporal development of the anti-DNP response in Balb/c mice. In collaboration with Dr. Norman Klinman here at Scripps Clinic and Research Foundation, we will prepare hybridomas expressing distinct anti-DNP idiotypes and determine the temporal expression of these idiotypes using the Klinman splenic focus assay. Three hybridomas expressing common Balb/c neonatal clonotypes have already been constructed in Klinman's laboratory. Using recombinant DNA methods, we will determine the DNP-specific V-, D-, and J-gene segment sequences expressed in each of these hybridomas and the relative locations of these gene segments in Balb/c germline DNA. We will then try to understand the temporal expression of each gene product in terms of its sequence and flanking sequences, and its location in the germline genome. We will extend this work to other inbred mouse strains; this will allow us to test our interpretations of the Balb/c data as well as to probe the molecular basis for strain-dependent differences in the anti-DNP response.