POSITIONAL CLONING OF MURINE AIRWAY RESPONSIVENESS GENES

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In the past ten years data have accrued from a number of sources which indicate that airway responsiveness to bronchoconstrictor agonists. In the absence of specific sensitization or challenge, is under genetic control. For example, in humans genetic loci linked to airway hyperresponsiveness have been identified on chromosomes 5q31-q33 and 11q13. Although genetic loci linked to airway hyperresponsiveness have been identified, no human genes responsible for conferring this trait have been identified. Two of the difficulties in identifying such genes in humans are: 1) the inability to provide in human subjects tight control of environmental exposures such as to antigens or environmental pollutants, which could potentially modify airway responsiveness. 2) The diversity of the human genome, which makes identification of genes responsible for given trials quite difficult in outbred populations. Because of the potential importance of identifying genes for airway responsiveness, we, and others, have used inbred mice to identify quantitative trait loci (QTL)s linked to the degree for airway responsiveness that occurs in the absence of environmental intervention. In the research proposed in this application, these QTLs will first be confirmed in additional crosses, between C57BL/6J mice carrying mutations at the Kit/W or the Mgf/Sl loci, and A/J mice. These crosses will provide specific information about the role of genes which require mast cells for their phenotypic expression in conveying airway hyperresponsiveness in the absence of environmental or allergic sensitization. The analysis of these crosses will be followed by the application of a novel approach to identifying airway responsiveness, loc, phenotypically driven repetitive backcrossing. This approach takes advantage of the recently introduced technique of barometric plethysmography which allows mice to be phenotyped for airway resp9onsivess non-invasively. This approach will use two crosses A/JxC57/BL/6J and A/JxC3H/HeJ; in both cases A/J i the high responder strain while C57BL/6J or C3H/HeJ is the low responder strain. Loci which are identified by both the standard QTL approach and by the repetitive backcrossing approach in both the crosses, i.e. A/JxC57BL/6J and A/JxC3H/HeJ, will be searched for candidate genes. Potential candidate genes will be tested for sequence diversity between A/J and C47BL/6J or C3H/HeJ and if this exists the causative role of these genes asserted through appropriate complementation and deletion experiments. In the absence of candidate genes at the loci identified, BACs and YACs will be searched for novel genes in the regions identified.