Genomic structure, characterization, and identification of the promoter of the human IL-8 receptor A gene.
Two unique but homologous receptors for the neutrophil chemoattractant, IL-8 have been cloned (designated IL-8RA and IL-8RB), each of which binds IL-8 with high affinity. IL-8RA mRNA expression was found to be regulated by granulocyte-CSF and LPS. In an attempt to understand the tissue-specific expression and to identify transcriptional regulatory elements, we have cloned, sequenced, and characterized the human IL-8RA gene. A lambda-DASH clone encoding the entire human IL-8RA gene was isolated by screening a genomic library with a PCR-generated cDNA. After mapping, subcloning, and sequencing several restriction fragments, a 9.2-kb continuous DNA sequence was obtained. As the sizes of the published cDNA (1.9 kb) and the mRNA determined by Northern blot analysis (2.1 kb) were not in agreement, a full-length cDNA was cloned by using a modified rapid amplification of cDNA ends technique. We identified a 5'-untranslated region of 119 bp. After comparison with the genomic sequence, we found the gene consisted of two exons interrupted by an intron of 1.7 kb. A 1050-bp ORF was encoded entirely in the second exon together with a 834-bp 3'-untranslated region. The immediate GC-rich 5'-flanking region upstream of exon 1 could serve as a constitutively active promoter in chloramphenicol-acetyl-transferase-expression assays. Expression analysis of additional upstream regions suggested the presence of silencer elements between positions -841 and -280. In conclusion, cloning a full-length cDNA permitted us to clone the human IL-8RA gene, identify the genomic structure, and characterize the promoter region.
Sprenger
H
H
Division of Cancer Treatment, National Cancer Institute, Frederick Cancer Research and Development Center, MD 21702.
Lloyd
A R
AR
Meyer
R G
RG
Johnston
J A
JA
Kelvin
D J
DJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons; D015698:Genomic Library; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D018123:Receptors, Interleukin / Q000096:biosynthesis / Q000235:genetics*; D023062:Receptors, Interleukin-8A
D018076:DNA, Complementary; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A