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.
Department of Veterinary Science, Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546, USA. rfcook1@uky.edu
R Frank
RF
Cook
Sheila J
SJ
Cook
Deborah L
DL
Even
Catherine
C
Schaffer
Charles J
CJ
Issel
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