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upstream regions compared, and their promoters transcribed in a cell-free system derived from liver. The CYP2A1 gene was completely sequenced and spanned 12,835 bp. The CYP2A2 gene was sequenced except for 1.5 and 12 kbp in the second and fifth introns, respectively. This gene was about 10 kbp longer than CYP2A1. Both genes possess nine exons that displayed overall 93% nucleotide similarity. DNA 4544 and 5529 bp upstream from the CYP2A1 and CYP2A2 genes, respectively, was also sequenced, and the transcription start sites were determined. Both genes had typical TATA boxes but did not contain CCAAT boxes within -100 bp of their polymerase start sites. CYP2A1, however, contained a reverse CCAAT box between -85 and -90. Search of the Gene Bank revealed a 255 bp region that lies -3 kbp upstream from the transcription start site of CYP2A1 displaying similarity with retrovirus polymerase. Two regions upstream of CYP2A2 were also found that displayed 90% sequence similarity with the consensus long interspersed middle repetitive element (LINE). In addition, an unusual 1.6 kbp inserted sequence was detected between -165 and -1779 bp upstream of the CYP2A2 gene that appears to be a retropseudogene. A nuclear extract derived from adult hepatocytes was used to direct in vitro transcription of the CYP2A1 and CYP2A2 gene promoters. Both genes were accurately transcribed in extracts derived from livers of male and female rats. This result is surprising in view of the fact that the CYP2A1 gene is expressed in adult female rats while the CYP2A2 gene is expressed exclusively in adult males. The CYP2A1 promoter was more actively expressed in both extracts than that of CYP2A2. By analyzing the segregation pattern of CYP2A genes in backcross offspring from an interspecies cross between the laboratory strain NFS/N and the wild mouse Mus musculus musculus, the Cyp2a subfamily was mapped proximal to the Gpi-1 locus near the centromere on chromosome 7.