br cDNA cloning gene structure and chromosomal localization

cDNA cloning, gene structure and chromosomal localization The cDNA for the leukocyte-type 12S-lipoxygenase was first cloned from porcine leukocytes [9], and later from mouse [10], [11], rat [12], [13], bovine [14] and rabbit [15] sources. The cDNA for the platelet-type 12S-lipoxygenase cDNA has been isolated from the human [16], [17], [18] and mouse [10]. The epidermis-type 12S-lipoxygenase cDNA was cloned from newborn mouse epidermis [19], [20], [21]. The open reading frames of cDNAs for the three isozymes predict 662 amino acids molecular weights of approximately 75,000. When the amino RS-1 sequences of the murine enzymes are compared, the epidermal 12S-lipoxygenase displays 60% identity with both platelet and leukocyte isozymes [10]. The murine platelet 12S-lipoxygenase is 58% identical to the leukocyte isozyme [10]. It should be noted that leukocyte-type 12S-lipoxygenases shows much higher identity (71–86%) to human and rabbit reticulocyte 15-lipoxygenases (now known as 15-lipoxygenase-1, see elsewhere), and therefore they are collectively called 12/15-lipoxygenases [22]. However, all of these 12S-lipoxygenases display only 40% identity to 5-lipoxygenase [6], [7], indicating that 12- and 5-lipoxygenases belong to distinct enzyme subfamilies. At the genomic level, all of the 12S-lipoxygenase genes have 14 exons and 13 introns that are organized in the same exon/intron format with boundaries in the same positions [6]. The genes for the platelet, leukocyte and epidermis isozymes are designated Alox12p, Alox12l and Aloxe, respectively [19], [20]. The platelet-type 12S-lipoxygenase gene (13–17kb) is approximately twice the size of the genes for the other two isozymes (7.2–7.5kb) [6]. All of the murine 12S-lipoxygenase genes map to the central region of mouse chromosome 11 in a region of homology with human chromosome 17 [6]. Human platelet 12S-lipoxygenase gene has been precisely mapped to chromosome 17p13.1 by fluorescence in situ hybridization [23]. Humans have an epidermal 12-lipoxygenase pseudogene designated Alox12p2 which has been localized to chromosome 17p13 by analysis of somatic hybrid DNA panels [24]. Recently, a second epidermal enzyme (Aloxe3) was isolated from mouse skin [25]. This novel lipoxygenase gene is also localized to the central region of mouse chromosome 11, but contains 15 exons and 14 introns, spanning 22.3kb of genomic DNA. The cDNA for this new isoform encodes a protein of 711 amino acids with a molecular mass of 80.6kDa. It is most closely related to mouse and human 12R-lipoxygenase (approximately 54% sequence identity), but the enzymatic properties have not yet been elucidated. The enzyme is expressed in the stratified epithelia of skin, tongue, and fore-stomach. It has recently been found that the 12S-lipoxygenases, 12R-lipoxygenases and 15-lipoxygenases form an approximately 100-kb gene cluster on human chromosome 17p13.1 [26].
Tissue distribution and subcellular localization The tissue distribution of the three 12S-lipoxygenase isoforms in various species is shown in Table 1. It can be seen that the distribution is substantially different from species to species. For example, in the rat the leukocyte-type 12S-lipoxygenase is most abundant in pineal gland, followed by lung, spleen, aorta, adrenal gland, spinal cord, and pancreas [27]. In the mouse, strong immunoreactive staining for the 12S-lipoxygenase was found with macrophages, pineal gland, pituitary and kidney, and weaker staining bands were present with spleen, aorta and intestine [6], [10]. It should be noted that rabbit reticulocytes have a leukocyte-type 12S-lipoxygenase in addition to a well-known 15-lipoxygenase, indicating the co-expression of two separate genes in the same species [15]. The platelet-type 12S-lipoxygenase is found primarily in platelets and epidermis [10], [28]. Bovine corneal epithelium also contains platelet-type 12S-lipoxygenase mRNA as detected by Northern blot analysis [29]. It is of interest that rainbow trout thrombocytes generate significant amounts of 12-lipoxygenase products [30]. The epidermal 12S-lipoxygenase is specifically expressed in differentiated keratinocytes of mouse epidermis and in the root sheath and bulb of hair follicles; enzyme expression is also detected in conjunctiva of the eyelid and Meibomian and preputial (sebaceous) glands [20]. These results indicate that the leukocyte-type 12S-lipoxygenase has a relatively broad tissue distribution as compared with the other isozymes.