Collaborative Project: Molecular Cloning of mRNA Encoding Latent Ig Allotypes

在兔中，大多数免疫球蛋白（IG）名义同种异型被认为是通过共显性等位基因分离遗传的。然而，IG κ轻链和γ重链基因座的潜在或非等位基因激活已被证实。血清学和结构分析证实了这些潜在的IG同种异型的存在。在核酸水平上允许潜在同种异型表达的分子机制尚未确定。研究潜伏性IG同种异型表达的主要障碍是不能可重复地诱导潜伏性IG分子的表达。通过使用布氏锥虫的抗原刺激来诱导兔中潜伏的IG κ轻链和γ重链序列的表达，已经绕过了该障碍。已显示从感染的兔中取出的淋巴结含有编码潜在的κ轻链和γ重链同种异型的poly（A）阳性RNA序列。这是第一次在核酸水平上检测到潜在的同种异型。本研究的长期目标是阐明产生编码潜在IG同种异型的核酸序列表达的分子和细胞机制。为了实现这一点，将追求以下具体目标：分离和结构分析poly（A）阳性RNA序列，其编码：1）潜伏性b5和b6 κ轻链，2）潜伏性a1-e14单倍型，3）由兔-小鼠杂交瘤细胞系H158分泌的标称b6 κ轻链。这些RNA序列的分离将通过采用标准的双链cDNA克隆技术和新的同种异型特异性筛选方法来实现。分离编码潜伏性IG同种异型的cDNA序列将证实它们在核酸水平上的存在，并允许与它们的标称同种异型对应物进行直接结构比较。所获得的信息将用于未来的研究，旨在分离积极转录的IG κ和γ基因编码这些潜在的序列。这些研究将有助于我们对IG基因表达调控的全面了解。

In the rabbit, most immunoglobulin (Ig) nominal allotypes are presumed to be inherited via codominant allelic segregation. However, latent or non-allelic gene activation has been documented for the Ig kappa light and gamma heavy chain gene loci. Serological and structural analyses have confirmed the existence of these latent Ig allotypes. Molecular mechanisms acting at the nucleic acid level which allow the expression of latent allotypes have yet to be determined. A major obstacle in studying the expression of latent Ig allotypes has been the inability reproducibly to induce the expression of latent Ig molecules. This obstacle has been circumvented by antigenic stimulation using Trypanosoma brucei to induce the expression of latent Ig kappa light and gamma heavy chain sequences in the rabbit. Lymph nodes taken from infected rabbits have been shown to contain poly (A) positive RNA sequences encoding latent kappa light and gamma heavy chain allotypes. This represents the first time latent allotypes have been detected at the nucleic acid level. The long term objective of this investigation is to elucidate molecular and cellular mechanisms which generate the expression of nucleic acid sequences encoding latent Ig allotypes. To achieve this the following specific aims will be pursued: Isolation and structural analysis of poly (A)-positive RNA sequences encoding: 1) latent b5 and b6 kappa lightchains, 2) the latent a1-e14 haplotype, 3) the nominal b6 kappalight chain secreted by the rabbit-mouse hybridoma cell line, H158. Isolation of these RNA sequences will be achieved by employing standard duplex cDNA cloning technology and novel allotype-specific screening methodologies. Isolation of cDNA sequences encoding latent Ig allotypes will verify their existence at the nucleic acid level and allow direct structural comparisons to be made with their nominal allotypic counterparts. Information obtained will be used in future studies designed to isolate the actively transcribed Ig kappa and gamma genes encoding these latent sequences. These studies will contribute to our overall understanding of the regulation of Ig gene expression.