Genomic cloning and identification of a novel murine Cyp4a gene
The mouse cytochrome P450 4a family of genes is poorly characterised. This thesis describes a detailed study of these genes. Using primers derived from exon 1 of the mouse cyp3a10 cDNA sequence, a 1.4kb genomic fragment was cloned which contained the promoter region of the gene. Additionally, a lambda genomic library was screened with probes derived from the partial cDNA clones of the mouse cyp4a10 and cyp4a12. Originally, two classes of phage lambda clones were isolated. One clone, lambda 16, was partially sequenced and was found to contain the 3' end of the cyp4a12 gene. The second clone, lambda 4, was subcloned and found to be distinct from previously known murine cyp4a genes. This novel gene has been designated cyp4a14. To obtain the full cyp4a14 gene, the genomic library was rescreened. Lambda clone 12 was subsequently isolated from the library, which was found to contain exons 1 to 5 of Cyp4a14. In order to study the promoter region of cyp4a14, a pcr-based approach was undertaken to clone 4.4 kb upstream of exon 1 of the gene and a 1.2kb fragment has been sequenced. Cyp4a14 RNA was also found to be highly induced by a peroxisome proliferator, methylclofenapate. Primer extension analyses were performed to determine the start of transcription of Cyp4a14, which was mapped to a single T nucleotide 26bp upstream of the putative start site of protein translation. The promoter region of Cyp4a14 is highly similar to the corresponding regions of the rat CYP4A2 genes. however, similarity is dramatically reduced about 350bp upstream of the start of transcription, which coincides with the presence of two 358bp repeats in teh CYP4A2 gene. The promoter also contains a highly conserved 19bp element which is also found in the promoter regions of the CYP4A1 and CYP4A2 genes. Cyp4a14 is a novel member of the murine Cyp4a subfamily. It contains 12 exons and is highly similar to the rat CYP4A2/3 genes. However, there is high conservation in exon 3 between Cyp4a14 and cyp4a3, which makes it marginally more related to this gene. In addition, cyp4a14 shows very high similarity in exons 4,8, 11 and 12 with other known CYP4A genes. Correspondingly, the peptide sequences encoded by these exons are highly conserved. Exon 8 encodes a 16 amino acid motif, LRAEVDTFMGEGHDTT, which is highly conserved in the CYP4 family. Exons 22 and 12 encode the well known RNCIG motif, containing the haem-binding domain, which is the proposed signature for a P450 protein. Exon 4 in the CYP4A genes encodes for a highly conserved, but previously unreported motif, HRRMLLTPGFHYDIL. Primary sequence alignments indicate that these motifs map very close to or within predicted substrate recognition sites and thus could have an important role in the enzyme activities of the CYP4A enzyme. The identification of Cyp4a14 also enables the analysis of the evolution of the murine Cyp4a subfamily. The mouse has 4 genes in the Cyp4a subfamily; however, the closely related rat has four CYP4A members. As the rat CYP4A2, which is very similar to CYP4AA3, was not to have a homologue in the mouse, it must have arisen after the mouse lineage had diverged from the rat in evolutionary history. As all three mouse Cyp4a genes are significantly more similar to their equivalents in the rat than to other murine Cyp4a genes, the gene duplication events giving rise to these three genes must have occurred before the mouse had diverged from the rat. Phylogenetic analyses of the CYP4A, CYP4B and CYp4F subfamilies demonstrate that CYP4A genes are more similar to the CYP4B family than to the CYP4F subfamily. This suggests that the gene duplication event giving rise to the CYP4A and CYP4B genes must be a more recent event than that giving rise to the CYP4F subfamily. In addition, the CYP4A subfamily of genes is more divergent than the CYP4B subfamily across species.