Full-length and internally deleted forms of interleukin-7 are present in horse (Equus caballus) lymph node tissue.
Horse IL-7 (HIL-7) cDNA was isolated from adult lymph node tissue by reverse transcription polymerase chain reaction (RT-PCR) using oligonucleotide primers based on horse genomic sequences (The Broad Institute). In addition, to the full-length (FL) 531bp reading frame encoding 176 amino acids, shorter open-reading frames of 477, 396 and 264bp were also amplified. Nucleotide sequence analysis of these RT-PCR products demonstrated they were homologous except the shorter species were missing internal sequences consistent with multiple RNA splicing events. Consequently, the shorter open-reading frames were re-named splice variant (SV) 1 (477bp), 2 (396bp) and 3 (264bp). Organization of the horse IL-7 is predicted to be similar to that in humans with exon 5 deleted from SV1, exons 3, 5 deleted from SV2 and exons 3, 4, and 5 missing from SV3. Each of these open-reading frames has the potential to be stably expressed as demonstrated using a polyclonal antiserum against human IL-7 to visualize the protein products produced when the FL HIL-7 and each SV were molecularly cloned into pCI and transfected in brefeldin A treated HEK 293 cells. Furthermore, addition of supernatants to horse PBMC from HEK cells transfected (without brefeldin A treatment) with pCI HIL-7 FL, pCI HIL-7SV1, pCI HIL-7SV2 and pCI IL-7SV3 all induced significant incorporation of (3)H-thymidine in the presence of sub-stimulatory amounts of concanavalin A compared to supernatants from mock-transfected cells. Therefore, all isoforms of horse IL-7 described in this report have the ability to stimulate proliferative responses in ex vivo horse PBMC cultures.
Cook
R Frank
RF
Department of Veterinary Science, Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546, USA. rfcook1@uky.edu
Cook
Sheila J
SJ
Even
Deborah L
DL
Schaffer
Catherine
C
Issel
Charles J
CJ
D000595:Amino Acid Sequence; D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D006736:Horses / Q000235:genetics* / Q000276:immunology; D015851:Interleukin-7 / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008198:Lymph Nodes / Q000276:immunology*; D008969:Molecular Sequence Data; D020033:Protein Isoforms; D012313:RNA / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment; D014162:Transfection / Q000662:veterinary
D018076:DNA, Complementary; D015851:Interleukin-7; D020033:Protein Isoforms; D012313:RNA