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

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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.