Molecular biological analysis of animal behavior : Generation of genetically insomniac animals

前列腺素(PG)D2是大鼠和人类中枢神经系统产生的一种主要的前列腺素，是大鼠体内最有效的内源性睡眠促进物质，也被认为与非洲昏睡病或肥大细胞增多症患者的深度睡眠有关。在这项研究中，我们试图通过基因工程技术培育出遗传性失眠动物。我们分离了小鼠和人类两种不同类型的PGD合成酶的cDNA和基因，即定位于中枢神经系统和男性生殖器官的Lipocalin型酶，以及分布于周围组织的抗原提呈细胞和肥大细胞的造血酶。对这两种酶的氨基酸序列和基因结构的分析表明，这两种PGD合成酶在氨基酸序列上没有明显的同源性，它们是从不同的独特祖先进化而来的。Lipocalin型PGD合成酶是Lipocalin基因家族的一员，由多种分泌蛋白组成，结合和运输小分子亲脂性物质。或者，造血型PGD合成酶是第一个被发现的谷胱甘肽S转移酶Sigma类脊椎动物同源物。因此，这两种不同类型的PGD合酶被认为是功能融合的新例子。我们制备了重组的Lipocalin型和造血酶，并对这些酶进行了结晶，并通过X射线晶体分析确定了造血酶的结构。我们还培育了过量生产每种人类酶的转基因小鼠，以及缺乏Lipocalin类型酶基因的敲除小鼠。然后，我们开发了用于这些突变小鼠的睡眠监测系统。

Prostaglandin (PG) D2 is a major prostanoid produced in the central nervous system of rats and humans, characterized as the most potent endogenous sleep-promoting substance in rats, and also considered to be involved in deep sleep of patients with African sleeping sickness or mastocytosis. In this sutdy, we try to produce genetically insomniac animals by using gene-engineering techniques. We isolated mouse and human cDNAs and genes for two distinct types of PGD synthase, i.e., the lipocalin-type enzyme localized in the central nervous system and male genital organs, and the hematopoietic enzyme distributed in the antigen-presenting cells and mast cells in the peripheral tissues. Analyzes of the amino acid sequences of the enzymes and their gene structres revealed that these two PGD synthases did not possess any significant homology in the amino acid sequence with each other and have evolved from the unique ancestors distinct from one another. The lipocalin-type PGD synthase is a member of the lipocalin gene family composed of a variety of secretory proteins which bind and transport small lipophilic substances. Alternatively, the hematopoietic PGD synthase is the first recognized vertebrate homolog of the sigma class of glutathione S-transferase. These two distinct types of PGD synthase are, therefore, considered to be a new example of functional convergence. We produced the recombiant lipocalin-type and hematopoietic enzymes, crystallized these enzymes, and determined the tartiaty structure of the hematopoietic enzyme by X-ray crystallographic analysis. We also generated transgenic mice which overproduced each of those human enzymes and knockout mice lacking the gene for the lipocalin-type enzyme. We then developped the sleep-monitoring system used for those mutant mice.