We have identified, for the first time, a mouse Fmo6 gene. In addition, we describe a novel human FMO gene cluster on chromosome 1, located 4 Mb telomeric of the original cluster. The novel cluster contains five genes, all of which exhibit characteristics of pseudogenes. We propose the names FMO 7P, 8P, 9P, 10P and 11P for these genes. We also describe a novel mouse gene cluster, located approximately 3.5 Mb distal of the original gene cluster on Chromosome 1. The novel mouse cluster contains three genes, all of which contain full-length open-reading frames and possess no obvious features characteristic of pseudogenes. One of the genes is apparently a functional orthologue of human FMO9P. We propose the names Fmo9, 12 and 13 for the novel mouse genes. Orthologues of these genes are also present in rat. Sequence comparisons and phylogenetic analyses indicate that the novel human and mouse gene clusters arose, not from duplications of the known gene cluster, but via a series of independent gene duplication events. The mammalian FMO gene family is thus more complex than previously realised.
The absence of FMO1-mediated N-oxidation of imipramine results in enhanced central nervous system effects of the drug. The results provide insights into the metabolism of imipramine in the brain and may explain the basis of the adverse reactions to the drug seen in some patients. The knockout mouse line will provide a valuable resource for defining the role of FMO1 in the metabolism of drugs and other foreign chemicals.
In humans, expression of the FMO1 (flavin-containing mono-oxygenase 1) gene is silenced postnatally in liver, but not kidney. In adult mouse, however, the gene is active in both tissues. We investigated the basis of this species-dependent tissue-specific transcription of FMO1. Our results indicate the use of three alternative promoters. Transcription of the gene in fetal human and adult mouse liver is exclusively from the P0 promoter, whereas in extra-hepatic tissues of both species, P1 and P2 are active. Reporter gene assays showed that the proximal P0 promoters of human (hFMO1) and mouse (mFmo1) genes are equally effective. However, sequences upstream (-2955 to -506) of the proximal P0 of mFmo1 increased reporter gene activity 3-fold, whereas hFMO1 upstream sequences (-3027 to -541) decreased reporter gene activity by 75%. Replacement of the upstream sequence of human P0 with the upstream sequence of mouse P0 increased activity of the human proximal P0 8-fold. Species-specific repetitive elements are present immediately upstream of the proximal P0 promoters. The human gene contains five LINE (long-interspersed nuclear element)-1-like elements, whereas the mouse gene contains a poly A region, an 80-bp direct repeat, an LTR (long terminal repeat), a SINE (short-interspersed nuclear element) and a poly T tract. The rat and rabbit FMO1 genes, which are expressed in adult liver, lack some (rat) or all (rabbit) of the elements upstream of mouse P0. Thus silencing of FMO1 in adult human liver is due apparently to the presence upstream of the proximal P0 of L1 (LINE-1) elements rather than the absence of retrotransposons similar to those found in the mouse gene.
The steps required to delete genes from the mouse genome are illustrated by showing how a cluster of three flavin-containing monooxygenase (Fmo) genes (Fmo1, Fmo2, and Fmo4) were deleted from mouse chromosome 1. Such large deletions are accomplished using loxP/Cre recombinase technology. Genomic clones corresponding to the genes to be deleted are first isolated, and then appropriate genomic fragments are cloned into vectors containing a loxP site. This produces targeting vectors, which are electroporated into mouse embryonic stem (ES) cells to allow a homologous recombination event to take place between the mouse genomic fragment, present within the vector, and the homologous sequences in the ES cell genome. Screening of ES cells for recombinants in which loxP sites have been inserted on either side of the gene cluster to be deleted is described. Recombination by Cre recombinase to produce ES cell lines carrying the deletion on chromosome 1 is also described.
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