Collaborative Project: Molecular Cloning of mRNA Encoding Latent Ig Allotypes

在家兔中，大多数免疫球蛋白（Ig）名义同种异体被认为是通过共显性等位基因分离遗传的。然而，Ig kappa轻链和γ重链基因位点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等位型的存在。分子机制的作用，在核酸水平上允许潜伏同种异体的表达尚未确定。研究潜伏Ig同种异体表达的一个主要障碍是无法重复地诱导潜伏Ig分子的表达。利用布氏锥虫抗原刺激诱导兔表达潜伏的Ig κ pa轻链和γ重链序列，克服了这一障碍。从感染家兔身上提取的淋巴结已被证明含有编码潜在kappa轻链和gamma重链同种异体的poly (A)阳性RNA序列。这是首次在核酸水平上检测到潜伏同种异体。这项研究的长期目标是阐明产生编码潜在Ig同种异体的核酸序列表达的分子和细胞机制。为了实现这一目标，将追求以下具体目标：多(A)阳性RNA序列的分离和结构分析：1)潜在的b5和b6 kappalight链，2)潜在的a1-e14单倍型，3)兔-小鼠杂杂瘤细胞系H158分泌的标称的b6 kappalight链。这些RNA序列的分离将通过采用标准的双工cDNA克隆技术和新的同种异体特异性筛选方法来实现。分离编码潜在Ig同种异型的cDNA序列将在核酸水平上验证其存在，并允许与其名义同种异型对应体进行直接结构比较。获得的信息将用于未来的研究，旨在分离编码这些潜在序列的活性转录的Ig kappa和γ基因。这些研究将有助于我们对Ig基因表达调控的全面理解。

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.