International Mammalian Genome Society

The 13th International Mouse Genome Conference
October 31-November 3, 1999

Table of Contents * Structure * Bioinformatics * Sequence * Mapping * New Tools * Gene Discovery * Developmental * Mutagenesis * Functional Genomics

F2 Positional Cloning of the Gunmetal Mouse Mutation

John C. Detter, Stephen Kingsmore. Molecular Staging, Inc. Guiford, CT 06437

Few molecular events important in platelet biogenesis have been identified. Mice homozygous for the recessive mutation gunmetal (gm) have platelet and megakaryocyte defects that cause prolonged bleeding and high mortality; gm platelets are increased in size, decreased in abundance, lack a- and d-granule proteins, and contain abnormal small GTPases; gm megakaryocytes are increased in abundance and have abnormal intracellular membrane accumulations. The main goal of this dissertation work was to identify the gene responsible for the gm mouse mutant using a positional cloning strategy. Positional cloning is an approach to disease gene identification based primarily on genetic location. The steps involved include: (1) genetic localization; (2) physical contig assembly; (3) candidate gene identification; (4) mutation analysis; and (5) characterization of gene mutation.

Using 2462 gm backcross mice, we localized gm to a 0.64 centiMorgan (cM) region of mouse chromosome (Chr) 14. Using direct cDNA selection, the -subunit of Rab geranylgeranyl transferase (Rabggta) was isolated from contiguous yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) clones that contained this region. In gm mice, DNA sequence analysis of Rabggta identified a G A substitution at residue -1 of the intronic splice acceptor site preceding exon 1 (which contains the start codon). Most gm Rabggta mRNAs lacked the start codon due to exon 1 skipping, while a minute amount retained the start codon due to the utilization of cryptic splice sites. Rab geranylgeranyl transferase (Rab GGTase, composed of a, ß, and REP-1 components) covalently attaches lipid moieties to the C-terminus of Rab GTPases, enabling reversible attachment to membranes. Rabs regulate intracellular vesicular transport by acting as molecular switches that oscillate between membrane-attached, GTP-bound and cytosolic, GDP-bound states. Levels of Rabggta protein and Rab GGTase activity were reduced 4-fold in gm platelets. Prenylation and membrane association of a Rab GGTase substrate, Rab27a, was also decreased in gm platelets. These data indicate that prenylation of Rab GTPases is important for platelet synthesis and delivery of proteins to platelet granules, and suggest Rab GGTase as a therapeutic target for thrombocytosis and clotting disorders.


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