Localization of non-Mhc collagen-induced arthritis susceptibility loci in DBA/1j mice

One approach to understanding common human diseases is to determine the genetic defects responsible for similar diseases in animal models and place those defective genes in their corresponding biochemical pathways. Our laboratory is working with an animal model for human rheumatoid arthritis called collagen-induced arthritis (CIA). We are particularly interested in determining the location of disease-predisposing loci. To that end, we performed experiments to localize susceptibility loci for CIA in an F2 cross between the highly susceptible mouse strain DBA/1j and the highly resistant mouse strain SWR/j. Specifically, a quantitative trait locus analysis was performed to localize regions of the mouse genome responsible for susceptibility/severity to CIA. One susceptibility locus, Cia1 in the major histocompatibility locus, had been identified previously. Two additional loci were detected in our analysis that contribute to CIA severity (Cia2, Cia3) on chromosomes 2 and 6. A third locus was detected that contributes to the age of onset of the disease. This locus (Cia4) was located on chromosome 2 and was linked to the same region as Cia2. Determining the identity of these loci may provide insights into the etiology of human rheumatoid arthritis.

Chrysanthemyl diphosphate synthase: Isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium

Chrysanthemyl diphosphate synthase (CPPase) catalyzes the condensation of two molecules of dimethylallyl diphosphate to produce chrysanthemyl diphosphate (CPP), a monoterpene with a non-head-to-tail or irregular c1′-2-3 linkage between isoprenoid units. Irregular monoterpenes are common in Chrysanthemum cinerariaefolium and related members of the Asteraceae family. In C. cinerariaefolium, CPP is an intermediate in the biosynthesis of the pyrethrin ester insecticides. CPPase was purified from immature chrysanthemum flowers, and the N terminus of the protein was sequenced. A C. cinerariaefolium λ cDNA library was screened by using degenerate oligonucleotide probes based on the amino acid sequence to identify a CPPase clone that encoded a 45-kDa preprotein. The first 50 aa of the ORF constitute a putative plastidial targeting sequence. Recombinant CPPase bearing an N-terminal polyhistidine affinity tag in place of the targeting sequence was purified to homogeneity from an overproducing Escherichia coli strain by Ni2+ chromatography. Incubation of recombinant CPPase with dimethylallyl diphosphate produced CPP. The diphosphate ester was hydrolyzed by alkaline phosphatase, and the resulting monoterpene alcohol was analyzed by GC/MS to confirm its structure. The amino acid sequence of CPPase aligns closely with that of the chain elongation prenyltransferase farnesyl diphosphate synthase rather than squalene synthase or phytoene synthase, which catalyze c1′-2-3 cyclopropanation reactions similar to the CPPase reaction.