Use of a conservation-of-linkage strategy to identify a candidate for the rat Lymphopenia gene

Abstract

The rat Lymphopenia gene, Lyp, is absolutely required for the development of Insulin-Dependent Diabetes Mellitus (IDDM) in the BioBreeding (BB) rat. In order to overcome the lack of positional cloning resources available in the rat to clone the Lyp gene, we developed and successfully implemented a conservation-of-linkage (COL) cloning strategy. Using the COL between the rat Lyp region and its murine counterpart, we developed large-scale YAC and fine-scale P1/BAC physical maps of the mouse Lyp region, and integrated these maps with a fine-scale genetic map of the rat Lyp region. Using cDNA fragments obtained through direct cDNA selection, we established a transcript map of the mouse Lyp region. One of these transcripts, PA, maps in the region fully-linked with Lyp and its transcript level is severely reduced in Lyp/LLyp homozygous animals. It is expressed chiefly in immune system organs, most strongly in the thymus, where Lyp is thought to act; within the thymus, PA is restricted to punctate expression in the thymic medulla--again, the inferred site of Lyp action in late T-cell thymic maturation. Although no functional mutation has yet been identified in the Lyp haplotype allele of PA, it remains the leading candidate gene for Lyp. The successful cloning of the rat Lyp region validates the COL cloning strategy and suggests that this strategy may be widely applicable among mammalian species.