The Evolution of Lymphocyte Recognition

本研究的长期目标是了解金鱼免疫球蛋白(Ig)重链基因的结构、组织和表达。在Lambda噬菌体中构建了基因组文库，完成了三个VH相关基因的初步测序，并通过寡核苷酸探针杂交鉴定了可能的JH区域。这些研究将在三个方面扩大。首先，对具有代表性的VH、DH、JH和CH区进行鉴定和测序，以阐明它们的生殖系组织。将使用标准的DNA克隆、测序和测序策略。为了识别CH区，将对金鱼IgM重链的多肽进行显微测序，并合成与其编码序列相对应的特异性混合寡核苷酸探针。其次，将对金鱼VH基因序列进行计算机辅助分析，并将它们彼此进行比较，并与其他脊椎动物的VH序列进行比较，试图推断选择、突变、漂移和非互惠交换在这些基因进化中的相对贡献。第三，将研究金鱼Ig重链基因可能的调控区的功能。将构建合适的表达载体来测试金鱼重链启动子(与哺乳动物Ig增强子区域相关联)是否允许在哺乳动物淋巴细胞中表达基因。哺乳动物以外的其他动物的免疫功能机制目前还知之甚少。对鱼类免疫球蛋白基因的结构、组织和表达调控的基本知识不仅对鱼类的内在兴趣具有重要意义，而且因为了解鱼类的免疫功能对于鱼类在商业养殖中的生长和维持能力以及了解鱼类在自然环境中生存成败的因素是基本的。从这些研究中获得的关于免疫功能进化的知识也可能有助于揭示高等脊椎动物的免疫系统功能。

The long term goals of this research are to understand the structure, organization, and expression of immunoglobulin (Ig) heavy chain genes in the goldfish, Carassius auratus. A genomic library has been constructed in lambda phage; initial sequencing of three VH.related genes has been completed, and putative JH regions have been identified by oligonucleotide probe hybridization. These studies will be expanded in three areas. First, representative VH, DH, JH, and CH regions will be identified and sequenced to elucidate their germline organization. Standard DNA cloning, mapping, and sequencing strategies will be used. To identify CH regions, peptides of goldfish IgM heavy chains will be microsequenced, and specific mixed oligonucleotide probes will be synthesized to correspond to their coding sequences. Second, computer assisted analyses of goldfish VH gene sequences and comparison of these with each other and with published VH sequences for other vertebrates will be undertaken in an attempt to infer the relative contributions of selection, mutation, drift, and non.reciprocal exchange in the evolution of these genes. Third, the function of putative regulatory regions of the goldfish Ig heavy chain genes will be studied. Appropriate expression vectors will be constructed to test whether the goldfish heavy chain promoter (in association with a mammalian Ig enhancer region) permits gene expression in mammalian lymphoid cells. The mechanisms of immune function in animals other than mammals are as yet poorly understood. Basic knowledge of the structure, organization and regulation of expression of immunoglobulin genes in fish is of importance not only for its intrinsic interest, but also because understanding of immune function in fish is fundamental for the capability to grow and maintain of fish in commercial culture and for understanding the factors involved in the success or failure of fish to survive in natural environments. Knowledge of the evolution of immune function obtained from these studies may also shed light on the function of immune systems in higher vertebrates.