The goal of this project is to clone a gene that complements the mouse pallid mutant. This mutant is one of a group of eleven genetically distinct mutants affecting coat color and bleeding time. These effects are mediated through alterations in melanosomes and platelet dense granules, both of which are present, but defective in the mutant mice. Some of the mutants, including pallid, also have some effects on the function of a third organelle, the lysosome. Pallid is one of three mutants that affect the inner ear, and the mutants display ataxia because the otoliths, required for balance, are missing. Cloning the normal gene will allow analysis of the normal function of the gene in all parts of its complex phenotype, both at the molecular and cellular level, and analysis of the mutant phenotype. The proposed approach to cloning this gene involves identifying the molecular marker closest to the pallid mutation on mouse chromosome 2. This will be done using an interspecific backcross (C57BL/6J-pa a1/pa a1 X M. spretus-+ A/+ A)F1 X C57BL/6J-pa a1/pa a1, that has already produced 123 progeny. Preliminary data suggest that beta2-microglobulin is a very close marker. A YAC clone containing the selected marker will be obtained and used to start the walk to pallid. The ends of the YAC will be cloned and sequenced to identify overlapping YACs. Backcross animals containing recombination breakpoints will be identified and used first to determine the direction of the walk and later to monitor progress between the starting point and the target. YAC clones overlapping the interval between the recombination breakpoints flanking the pallid mutation will be candidates for the desired clones. To determine whether any of the genes on the YAC are expressed in megakaryocytes (platelet precursors) or in skin, cDNA libraries from skin and bone marrow cells will be made in a 'fragmenting vector' which contains a selectable yeast marker not present in the YAC and a yeast telomere. The libraries will be transformed into yeast cells containing the candidate YAC and recombinants between individual cDNAs and genes on the YAC will be selected. This will allow identification of one or more candidate genes, as regions between the candidate gene and the telomere will be deleted in the recombinant YACs, being replaced by vector sequences. Experiments to detect the function of the locus involve transformation of DNA from the candidate YAC or its cosmid subclones into bone marrow cells and reintroduction of these cells into mice to determine whether the fraction of platelets with filled dense granules is increased. To determine whether the pigment dilution can be corrected, transgenic mice will be made by injecting the same constructs into fertilized eggs homozygous for the mutant.