Determination of the cDNA sequence and mRNA expression of interleukin-1 receptor antagonist in horses

Abstract

To determine the complementary DNA (cDNA) sequence of interleukin-1 receptor antagonist (IL-1ra) in horses and compare messenger RNA (mRNA) expression of IL-1ra among horses of various breeds. Blood samples from neonatal and adult horses examined for a variety of diseases. A polymerase chain reaction procedure was used to amplify a 220 base pair (bp) portion of the genomic DNA. The upstream and downstream regions of the cDNA sequence were determined by means of 5' and 3' rapid amplification of cDNA ends (RACE) procedures. Northern blot hybridization was used to examine steady-state mRNA expression of IL-1ra. The consensus sequence of the cDNA obtained with the 5'-RACE procedure and the sequence for the 220 bp portion of the genomic DNA represented the putative sequence for secreted IL-1ra. The predicted secreted IL-1ra amino acid sequence contained 176 residues with an in-frame stop codon; the N-terminal 25 amino acid residues resembled the signal peptide reported for human secreted IL-1ra. An approximately 1.3 kilobase pair (kb) band that represented a portion of the 3' end of the coding region and the 3' untranslated region was obtained by use of the 3' -RACE procedure. Northern blot hybridization detected a 1.6 kb transcript in blood RNA from adult Arabian, Belgian, Thoroughbred, and Standardbred horses. Results suggest that the DNA for equine secreted IL-1ra has a short (29 bp) 5' untranslated region, a 534 bp coding region, and a long (approximately 1,080 bp) untranslated region.