Human Leukotriene B₄Ω- Hydroxylase (CYP4F3) Gene: Molecular Cloning and Chromosomal Localization

Abstract

Leukotriene B4 (LTB4) omega-hydroxylase catalyzes the conversion of LTB4 into a biologically less active product, 20-hydroxy-LTB4. In a preceding paper (Kikuta et al., 1993), we showed human polymorphonuclear leukocyte (PMN) LTB4 omega-hydroxylase to be a novel form of cytochrome P450, designated CYP4F3, on the basis of its cDNA cloning and expression in yeast cells. Here, we have isolated the gene encoding CYP4F3 and determined its genomic organization and chromosomal localization. The CYP4F3 gene contained 13 exons and spanned approximately 22.2 kb. The cDNA of CYP4F3 contained 5050 nucleotides excluding the poly(A) tail. The translation initiation codon (ATG) was present in exon II. Primer extension and S1 mapping analyses indicated that the transcription initiation site is 49 nucleotides upstream from the 3' end of exon I, and no other initiation sites were detected. A TATA-box-like sequence (TACAT) and 120-b GC-rich sequence were observed just before transcription initiation site. Several putative regulating elements recognized by the GATA family, MZF-1, CACCC binding protein, and C/EBP, were identified in its 5' flanking region. Genomic DNA screening for CYP4F3 and Southern blot analysis suggested the existence of other CYP4F genes in addition to CYP4F3 and CYP4F2 in the human genome. Fluorescence in situ hybridization demonstrated that the CYP4F3 gene is located at 19p13.2.

Expression of the CYP4F3 Gene

“…All introns conform to the gt-ag rule (24), and a summary of the splice junctions is shown in Table II. These data are in agreement with a recent description of the gene (19), but include a previously unrecognized exon, designated exon 3, which is not incorporated into the characterized cDNA of neutrophil CYP4F3 (16). Examination of the sequence of exons 3 and 4 and the genomic flanking regions (Fig.…”

“…The sequence matched the previously reported cDNA sequence of neutrophil CYP4F3 with an additional 17 bp at the 5Ј-end. These data are in agreement with conclusions derived from S1 nuclease protection analysis of neutrophil RNA (19). The presence of a TATA box-like sequence and a consensus site for the myeloid-specific factor MZF-1 upstream of this initiation site has been noted previously (19).…”

“…The CYP4F subfamily utilizes LTB 4 as a substrate. Two members of this subfamily have been identified in humans and are designated CYP4F3 (16,19) and CYP4F2 (20). Studies of neutrophil microsomes or recombinant CYP4F3 have determined the specificity of CYP4F3 for LTB 4 (15,21).…”

“…They were cloned into pcDNA3 (Invitrogen) and expressed in COS-7 cells. CYP4F3A has been purified and characterized previously (19,21) but was included as a control in expression and kinetic studies to provide a direct experimental comparison with CYP4F3B. Total RNA was isolated from the COS-7 cells and analyzed by RT-PCR using the same four primer combinations as before ( Fig.…”

“…3. A revised map of the genomic organization, originally suggested to comprise 13 exons and 12 introns (19), is included.…”