International Mammalian Genome Society

The 14th International Mouse Genome Conference (2000)

B17. Comparative Sequencing of the Cytochrome P-450 Cyp1a2 Locus Between Inbred Mouse Strains with Normal and Low Expression of the Gene

William L. Casley, Michele Lemieux, Lori Lavigne, Jennifer McCaig, J. Allan Menzies and Larry W. Whitehouse.
Research Division, Therapeutic Products Programme, Health Canada
Sir F.G. Banting Research Centre, Tunney's Pasture, Ottawa, ON. Canada K1A 0L2

The APN inbred mouse strain, derived from Swiss Webster mice in our laboratory, had previously been demonstrated to have decreased expression of the cytochrome P-450 Cyp1a2 gene, relative to C3H/HeJ mice. A genome-wide scan for quantitative trait loci influencing caffeine metabolism identified a 5 cM QTL interval which encompassed the known map position of the Cyp1a2 locus. Since CYP1A2 is the principal enzyme of caffeine metabolism, this gene was a strong candidate to explain the variance in the phenotype attributable to this QTL.

In the present study, the Cyp1a2 locus from both the APN and C3H/HeJ strains, including 5' flanking and 3' untranslated regions, was sequenced in an effort to identify variations which may account for the observed differences in steady state CYP1A2 mRNA levels between these strains. No variations in amino acid coding sequences were observed between APN and either C3H/HeJ or the Genbank reference sequence (accession # X00479) derived from a C57BL/6N mouse. Two short tandem repeat (STR) length polymorphisms were detected. A deletion of 7 (GT) dinucleotides from the APN sequence, relative to C3H/HeJ was found at approximately -650 from the transcription start site. This dinucleotide repeat region is highly variable between strains and underlies the genetic marker D9Mit21, from the M.I.T. STR map. A second STR variation was identified in intron 6, consisting of a deletion of 4 (GGAA) tetranucleotide repeats from the APN strain relative to C3H/HeJ. An analysis of this STR region in other inbred strains identified this region as polymorphic, with at least 4 alleles. Two single nucleotide variants were also identified. A T to G transversion was identified in the APN sequence in the 3' UTR at + 57 after the end of the coding sequence. A G to A transition was also identified at -76 in the 5' flanking region. This variant was unique to APN among the 8 strains sequences for this region. This transition alters a GATA transcription factor consensus binding sequence. Electrophoresis mobility shift assays indicated that the presence of this sequence variation affected the ability of oligonucleotides to bind an, as yet unidentified, protein or proteins in nuclear extracts. The functional significance of this sequence variation in influencing basal transcription of the gene is under investigation.

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