TEMPORAL EXPRESSION OF DNP-SPECIFIC V-GENES

以前的工作已经证实，在体内的反二硝基苯基(DNP)反应 新生Balb/c小鼠仅限于几个独特型，而 成年人的反应相当不同。这种行为大致类似于 Balb/c免疫系统的全面扩展：新生小鼠表达a 总共约10(4)种不同的特异性(克隆类型)和 成虫的克隆型逐渐增加到10(7)个以上。其目的是 是为了在DNA水平上表征时间上的 Balb/c小鼠抗DNP反应的研究进展与 斯克里普斯诊所和研究基金会的诺曼·克林曼博士，我们将 制备表达不同抗DNP独特型的杂交瘤细胞 利用Klinman脾病灶对这些独特型的时间表达 化验。表达共同Balb/c新生儿克隆型的三株杂交瘤 已经在克林曼的实验室里建造好了。使用重组DNA 方法，我们将确定DNP特异的V、D和J基因片段 这些杂交瘤细胞中表达的序列及其相对位置 这些基因片段在Balb/c生殖系DNA中。然后我们将尝试 了解每个基因产物在时间上的表达 序列和侧翼序列，以及它在生殖系基因组中的位置。 我们将把这项工作扩展到其他近交系小鼠；这将使我们能够 为了测试我们对Balb/c数据的解释，以及探索 不同菌株抗DNP反应差异的分子基础。

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.