Abstract
Leukotriene C4 (LTC4) synthase catalyzes the conjugation of LTA4 with reduced GSH to form LTC4, the parent of the receptor active cysteinyl leukotrienes implicated in the pathobiology of bronchial asthma. Previous cloning of the cDNA for human LTC4 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 super-family. Genomic cloning from a P1 library now reveals that the gene for LTC4 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 LTC4 synthase align identically with those of FLAP; however, the small size of the LTC4 synthase gene contrasts with the > 31-kilobase pair size reported for FLAP. Confirmation of the LTC4 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 promoter 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 LTC4 synthase to chromosomal location 5q35, which is in close proximity to the cluster of genes for cytokines and receptors involved in the regulation of cells central to allergic inflammation and implicated in bronchial asthma.
Highlights
Leukotriene C4 (LTC4) synthase catalyzes the conjugation of LTA4 with reduced GSH to form LTC4, the parent of the receptor active cysteinyl leukotrienes implicated in the pathobiology of bronchial asthma
Characterization of Genomic Clone for Human LTC4 Synthase—A 5.5-kbp SacI-digested fragment liberated from the P1 plasmid hybridized with the full-length cDNA for LTC4 synthase and with oligonucleotide primers from the 5Ј end and the 3Ј end of the cDNA, indicating that the full-length gene was contained within this fragment
The cloning and sequencing of the gene for human LTC4 synthase (Fig. 1) have revealed that its intron size and chromosomal location are prominently different from the gene for FLAP, which encodes the only known homologous protein
Summary
Cell Culture—KG-1 cells, (American Type Culture Collection, Rockville, MD) were cultured in RPMI 1640 medium (JRH Biosciences, Lenexa, KS), supplemented with 10% fetal calf serum (Sigma), 100 units/ml penicillin, and 100 g/ml streptomycin (Sigma) at 37 °C under 5% CO2. For identification of the specific nucleotides at the transcription initiation sites, a genomic sequencing ladder was generated with the same primer as in the extension reaction to sequence the LTC4 synthase genomic clone according to the dideoxy chain termination method of Sanger et al [29]. These reaction products and 32P-labeled molecular weight standards were run in parallel lanes of the same gel.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
