Molecular cloning of the gene for human leukotriene C-4 synthase - Organization, nucleotide sequence, and chromosomal localization to 5q35

Leukotriene C-4 (LTC(4)) synthase catalyzes the conjugation of LTA(4) with reduced GSH to form LTC(4), the parent of the receptor active cysteinyl leukotrienes implicated in the pathobiology of bronchial asthma. Previous cloning of the cDNA for human LTC(4) synthase demonstrated significant homology of its amino acid sequence to that of 5-lipoxygenase activating protein (FLAP) but none to that of the GSH S-transferase superfamily. Genomic cloning from a P1 library now reveals that the gene for LTC(4) synthase contains five exons (ranging from 71 to 257 nucleotides in length) and four introns, which in total span 2.52 kilobase pairs in length. The intron/exon junctions of LTC(4) synthase align identically with those of FLAP; however, the small size of the LTC(4) synthase gene contrasts with the >31-kilobase pair size reported for FLAP. Confirmation of the LTC(4) synthase gene size to ensure that no deletions had occurred during the cloning was obtained by two overlapping polymerase chain reactions from genomic DNA, which provided products of the predicted sizes. Primer extension analysis with poly(A)(+) RNA from culture-derived human eosinophilic granulocytes or the KG-1 myelogenous cell line revealed multiple transcriptional start sites with prominent signals at 66, 69, and 96 base pairs 5' of the ATG translation start site. The 5'-flanking region revealed a GC-rich promotor sequence consistent with an SP-1 site and consensus sequences for AP-1 and AP-2 enhancer elements, 24, 807, and 877 bp, respectively, 5' from the first transcription initiation site. Southern blot analysis of a genomic DNA (with full-length cDNA as well as 5' and 3' oligonucleotide probes) confirmed the size of the gene and indicated a single copy gene in normal human genomic DNA. Fluorescent in situ hybridization mapped LTC(4) synthase to chromosomal location 5q35, which is in close proximity 60 the cluster of genes for cytokines and receptors involved in the regulation of cells central to allergic inflammation and implicated in bronchial asthma.