Molecular cloning, sequencing, and expression of equine interleukin-6

Equine interleukin-6 (IL-6) cDNA was amplified from mitogen-stimulated equine peripheral blood mononuclear cells (PBMC) using consensus sequence primers. The 727bp amplified cDNA contains the entire coding region for equine IL-6 and includes 118 bases in the 3' non-translated region. The coding sequence translates to a protein of 208 amino acids with a predicted 28 amino acid leader sequence. The mature protein of 180 amino acids has a predicted molecular mass of 20471Da without post-translational modifications. The amino acid sequence of equine IL-6 displays between 46 and 84% similarity to other mammalian IL-6 sequences. Expression of equine IL-6 in Chinese hamster ovary (CHO) cells yielded a supernatant that supported the proliferation of B9 cells in a dose-dependent manner. Treatment of B9 cells with an anti-IL-6 receptor antibody ablated the response to the recombinant equine IL-6.     

Bovine interleukin 2: regulatory mechanisms

A cDNA clone of the bovine interleukin-2 (IL-2) gene has been isolated and demonstrated to produce a functional bovine IL-2 protein when transfected into either CV-1 or COS-1 monkey cells. Homology comparisons of both the nucleotide and predicted amino acid sequences of bovine IL-2 with those of the human and mouse show extensive regions of sequence conservation between the species. The amino acid sequence of the mature bovine IL-2 protein shares about 60-63% homology with those of the human and mouse, but the 3' untranslated regions of the human and mouse gene share as much, if not greater, sequence homology with the 3' untranslated regions of the human and mouse genes. In particular, a tandemly repeated sequence (TATT), n, found in the 3' untranslated tail of the bovine IL-2 clone is also found in the 3' untranslated region of a large group of cytokine genes and other inducible genes of the lymphoid and immune response systems. This sequence may serve a specific regulatory function in the immune system.     

Expressed gene sequences of the equine cytokines interleukin-17 and interleukin-23

This report describes the initial cloning and characterization of the equine interleukin-17 (IL-17) expressed gene sequence from mRNA obtained from equine intestinal tissue and interleukin-23 (IL-23) expressed gene sequence from mRNA obtained from equine peripheral blood mononuclear cells. Equine IL-17 has 462 nucleotides in the translated region, determined by homology with known human and mouse sequences, and shares 84% and 75% identity, respectively. For the deduced amino acid sequences, the identity with human and mouse is 76% and 70%. Equine IL-23 has 579 nucleotides in the translated region. Homology with known human and mouse sequences was determined to be 89% and 77%. Deduced amino acid identities are 89% with the human sequence and 70% with the mouse sequence. The gene sequences were identified as part of the U.S. Veterinary Immune Reagent Network with a goal of developing reagents in order to aid veterinary researchers in the investigation of diseases in livestock species.     

Molecular cloning of feline interferon-gamma-inducing factor (interleukin-18) and its expression in various tissues

Interleukin-18 (IL-18) is a cytokine with potent interferon-gamma-inducing activity, and plays an important biologic role in the enhancement of the activity of natural killer cells and cytotoxic T-lymphocytes. In this study, feline IL-18 cDNA was cloned and characterized to establish a basis for the prospective cytokine therapy in small animal practice. The nucleotide sequence of feline IL-18 cDNA obtained in this study was 712bp long and contained its entire open reading frame encoding 192 amino acid residues. The predicted amino acid sequence of feline IL-18 cDNA showed 77.2, 84.8, 60.2 and 62.6% similarity with those of human, dog, rat and mouse counterparts, respectively. The feline IL-18 cDNA included a putative cleavage site of IL-1beta-converting enzyme (ICE) and IL-1 signature-like sequences identified in human and mouse IL-18 cDNAs. Expression of IL-18 mRNA was detected in various tissues including spleen, liver and cerebrum in the cat.     

Molecular cloning and phylogenetic analysis of inflammatory cytokines of Camelidae (llama and camel)

We cloned, sequenced and analyzed the cDNAs encoding Camelidae inflammatory cytokines, including llama (lama glama) interleukin (IL)-1alpha, IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha and camel (Camelus bactrianus) IL-6 and TNF-alpha. The similarity levels of the deduced amino acid sequences of IL-1alpha, IL-1beta, IL-6 and TNF-alpha from llama (camel) to those from other mammalian species, ranged from 60.7% to 87.7%, 52.8% to 75.3%, 41.4% to 98.6%, and 72.9% to 99.6%, respectively. Phylogenetic analyses based on nucleic acid sequences showed that llama IL-1alpha, IL-1beta, IL-6 and TNF-alpha were more closely related to those of camel, pig, cattle, sheep and horse than to those of human, dog, cat, mouse and rat.     

Cloning and sequence analysis of the complementary DNA for feline preproparathyroid hormone

OBJECTIVES: To clone and sequence the cDNA for feline preproparathyroid hormone (preproPTH) and to compare that sequence with other known parathyroid hormone (PTH) sequences. SAMPLE POPULATION: Parathyroid glands from 1 healthy cat. PROCEDURES: A cDNA library was constructed in lambda phage from feline parathyroid gland mRNA and screened with a radiolabeled canine PTH probe. Positive clones were sequenced, and nucleic acid and deduced amino acid sequences were analyzed and compared with known preproPTH and PTH sequences. RESULTS: Screening of approximately 2 X 10(5) recombinant plaques revealed 3 that hybridized with the canine PTH probe; 2 clones comprised the complete sequence for feline preproPTH. Feline preproPTH cDNA consisted of a 63-base pair (bp) 5'-untranslated region (UTR), a 348-bp coding region, and a 326-bp 3'-UTR. The coding region encoded a 115-amino acid peptide. Mature feline PTH consisted of 84 amino acids. Amino acid sequence analysis revealed that feline PTH was > 83% identical to canine, bovine, swine, equine, human, and macaque PTH and 69, 71, and 44% identical to mouse, rat, and chicken PTH, respectively. Within the region responsible for hormonal activity (amino acids 1 to 34), feline PTH was > 79% identical to other mammalian PTH sequences and 64% identical to the chicken sequence. CONCLUSIONS AND CLINICAL RELEVANCE: The amino acid sequence of PTH is conserved among mammalian species. Knowledge of the cDNA sequence for feline PTH may be useful to investigate disturbances of calcium metabolism and alterations in PTH expression in cats.     

Molecular cloning and identification of full-length cDNA encoding high affinity Fc receptor for bovine IgG (Fc gamma RI)

The receptor I for the Fc region of immunoglobulin G (Fc gamma RI) is a member of the Ig superfamily with a high affinity, and it mediates antibody-dependent cellular cytotoxicity and immune complex clearance. In this study, a cDNA encoding the bovine Fc gamma RI was cloned. The full-length cDNA sequence is 1050 bp long with a short 5'- and long 3'-untranslated end regions, which codes for 349 amino acids and contains a signal peptide, an extracellular region with three Ig-like domains, and transmembrane and intracytoplasmic domains. Five potential N-linked glycosylation sites are recognized in this sequence. Compared with the sequences of human and mouse Fc gamma RI, the homologies of nucleotide sequences are 80 and 69% and homologies of deduced amino acid sequences are 66 and 55%, respectively. It is shown that the sequences of the monomeric IgG binding domain in these three species of Fc gamma RI are highly conserved.     

Molecular cloning and characterization of beluga whale (Delphinapterus leucas) interleukin-1beta and tumor necrosis factor-alpha.

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are cytokines produced primarily by monocytes and macrophages with regulatory effects in inflammation and multiple aspects of the immune response. As yet, no molecular data have been reported for IL-1beta and TNF-alpha of the beluga whale. In this study, we cloned and determined the entire cDNA sequence encoding beluga whale IL-1beta and TNF-alpha. The genetic relationship of the cytokine sequences was then analyzed with those from several mammalian species, including the human and the pig. The homology of beluga whale IL-1beta nucleic acid and deduced amino acid sequences with those from these mammalian species ranged from 74.6 to 86.0% and 62.7 to 77.1%, respectively, whereas that of TNF-alpha varied from 79.3 to 90.8% and 75.3 to 87.7%, respectively. Phylogenetic analyses based on deduced amino acid sequences showed that the beluga whale IL-1beta and TNF-alpha were most closely related to those of the ruminant species (cattle, sheep, and deer). The beluga whale IL-1beta- and TNF-alpha-encoding sequences were thereafter successfully expressed in Escherichia coli as fusion proteins by using procaryotic expression vectors. The fusion proteins were used to produce beluga whale IL-1beta- and TNF-alpha-specific rabbit antisera.     

Nucleotide and deduced amino acid sequence of equine retinal and pineal gland phosducin

OBJECTIVES: To determine the full-length complementary DNA (cDNA) sequence of equine retinal and pineal gland phosducin (PHD) and to clone these sequences. SAMPLE POPULATION: Samples of equine retinal RNA. PROCEDURE: A primer set was designed for use in identifying a fragment of the equine PHD nucleotide sequence, derived from retinal RNA samples, and subsequently for use to deduce specific primers for additional examination. The full-length cDNA was determined by the method of rapid amplification of cDNA ends (RACE). For full-length cDNA, newly designed primers were used. Nucleotide sequences were analyzed by use of computer software. The deduced amino acid sequence was compared with sequences of PHD reported for other species. In addition, the sequence of equine pineal PHD was cloned. RESULTS: The cDNA nucleotide sequence for equine PHD was 1,209 base pairs (bp) in length with an open-reading frame encoding a protein of 245 amino acids and a calculated molecular mass of 28.214 kd. Similarity with amino acid sequences of PHD from other species was 89 to 93%. Sequences of equine PHD from retina and pineal gland were identical. Equine PHD contained a peptide sequence with 100% homology to an uveitopathogenic peptide reported for rat PHD. CONCLUSIONS: Equine PHD is a highly conserved protein that has homology of immunologic interest with rat PHD. These results establish a basis for studying the role of PHD in ocular inflammation of horses.     

Cloning and nucleotide sequence of the equine and elk pituitary pre-prolactin cDNA

We report the equine (Equs equs) and elk (Cervus elaphus) pituitary pre-prolactin (PRL) cDNA cloning, and their nucleotide and deduced amino acid sequences. Pre-PRL cDNA was obtained by RNA ligation mediated-rapid amplification of cDNA ends (RLM-RACE) and polymerase chain reaction (PCR). The elk pre-PRL cDNA exhibits two polymorphisms at positions 96 and 672, which are silent since they encode for the same amino acids, proline and isoleucine, respectively. We found no polymorphisms in the equine pre-PRL cDNA. The deduced amino acid sequence of the equine pre-PRL is 99% identical to the previously reported protein sequence. Pre-PRL mRNA is <1 kb in length and is highly expressed in the anterior pituitary gland, as demonstrated by Northern hybridization analysis. In summary, we cloned and sequenced the equine and elk pre-PRL cDNAs. The deduced amino acid sequence of elk and equine pre-PRL appears to be moderately conserved among other mammalian species. The polymorphic sites found in the elk cDNA could potentially be used in parentage testing and gene mapping.     

Cloning of bovine CD69

CD69 is rapidly inducible on various hematopoietic cells upon stimulation and is detectable as an early activation antigen. Although CD69 is well characterized in human and mouse, no information is available on bovine CD69. We report here that, bovine CD69 was cloned from a cDNA expression library prepared from activated peripheral blood lymphocytes. The full-length cDNA contained an 80bp 5' untranslated region, followed by a 600bp coding region and AU-rich motifs in a 3' untranslated region (GenBank accession number AF272828). Comparison of the bovine CD69 coding sequence reveals 69.4 and 78.2% nucleotide sequence identities with mouse and human CD69, respectively. The predicted amino acid sequence of bovine CD69 shares 56.3 and 62.3% sequence identity when compared with mouse and human CD69, respectively. Bovine CD69 has the highly conserved amino acid sequences found in the C-type lectin family, suggesting that the conserved residues may be important for conformation and binding to the, as yet unidentified ligand. In addition, the cytoplasmic tail of bovine CD69 has two casein kinase-2 (CK-2) phosphorylation sites. These data suggest that bovine CD69 plays an important role in the activation of lymphocytes.     

Molecular cloning of canine IL-13 receptor alpha chain (alpha1 and alpha2) cDNAs and detection of corresponding mRNAs in canine tissues

This communication reports the cloning of cDNAs encoding two canine IL-13 receptor alpha chains (caIL-13Ralpha1 and caIL-13Ralpha2). As described for the members of type-I cytokine receptors, both caIL-13Ralpha1 and caIL-13Ralpha2 were found to contain the highly conserved motifs, such as cysteine and tryptophan residues in their N-terminal portion and the WSXWS at C-terminus. The isolated caIL-13Ralpha1 cDNA contains 1547 nucleotides with an open reading frame that encodes 405 amino acid residues. Canine IL-13Ralpha1 is 82.0 and 69.3% identical to human and mouse IL-13Ralpha1s, respectively, at the amino acid level. Canine IL-13Ralpha1 has an almost identical cytoplasmic domain to its human and mouse counterparts. The isolated caIL-13Ralpha2 cDNA contains 1454 nucleotides and encodes an open reading frame of 386 amino acid residues. Canine IL-13Ralpha2 is 62.6 and 47.5% identical to its human and mouse counterparts, respectively, at the amino acid level. Using RT-PCR with caIL-13Ralpha1 and caIL-13Ralpha2 specific primers, mRNAs of caIL-13Ralpha1 and caIL-13Ralpha2 were detected in most dog tissues. In addition, RT-PCR detected caIL-13Ralpha1 mRNA in one of two canine mastocytoma (C2 but not Br) cell lines and in a canine macrophage-derived cell line (DH82). CaIL-13Ralpha2 mRNA was detected in all three canine cell lines.     

Molecular cloning and expression analysis of pig CD79alpha

The CD79alpha (immunoglobulin alpha, Igalpha), a part of B cell receptor (BCR) complex, forms a heterodimer with CD79beta (Igbeta) and plays an important role in the B cell signaling. In this study, we have cloned pig Cd79a cDNA using RT-PCR and determined the complete cDNA sequence of pig Cd79a. Pig Cd79a cDNA contains an open reading frame (672bp) encoding 223 amino acids. The putative amino acid identity of pig CD79alpha with those of human, cattle and mouse are 70.4, 81.4, and 67.7%, respectively. Alignment of the CD79alpha amino acid sequence with those of mammalian species showed that the extracellular domain is the most divergent, whereas transmembrane region and cytoplasmic tail including immunoreceptor tyrosine-based activation motif (ITAM) are largely conserved. Pig Cd79a mRNA was detected mainly in lymphoid tissues by RT-PCR. The highest level of Cd79a mRNA expression was observed in mesenteric lymph node and spleen. Relatively low level of Cd79a mRNA expression was observed in lung, thymus and small intestine. The lowest level of Cd79a mRNA expression was observed in large intestine. Flow cytometry analyses demonstrated that human CD79alpha antibody recognizes a CD79alpha in pig B cells. Further, immunohistochemistry analysis using human CD79alpha antibody on pig spleen was revealed that CD79alpha is strongly expressed in the follicular mantle zone rather than in the germinal center. Future study will be focused on defining the functional role of CD79alpha during the course of pig infectious diseases and the formation of neoplasm.     

Molecular cloning and sequencing of equine interleukin 4

We have cloned equine interleukin 4 (IL-4) cDNA using the polymerase chain reaction (PCR) and primers based on the human IL-4 sequence. The cDNA was amplified from mitogen-stimulated equine peripheral blood mononuclear cells (PBMC). The cloned PCR product shares extensive homology with IL-4 sequences from other species.     

Molecular cloning of equine chromogranin A and its expression in endocrine and exocrine tissues

Chromogranin A (CGA) is a member of a family of highly acidic proteins co-stored and co-released with catecholamines in the adrenal medullary cells as well as in other neurons and paraneurons. The nucleotide sequence encoding equine CGA was determined using RT-PCR and rapid amplification of complementary DNA (cDNA) ends (RACE) techniques. A total 1,828 bp of the nucleotide sequence reveals that equine CGA is a 448-residue protein preceded by an 18-residue signal peptide. Comparison of the amino acid sequence of equine CGA with those of human, porcine, bovine, mouse, rat and frog CGA showed high conservation at the NH2-terminal 1-77 amino acids regions (94.8%, 93.5%, 92.2%, 81.8%, 83.1% and 66.2%, respectively) and COOH-terminal 314-430 amino acids regions (90.6%, 81.4%, 90.6%, 80.5%, 83.3% and 39.0%, respectively), as well as a potential dibasic cleavage site, whereas the middle portion showed marked sequence variation (52.5%, 49.1%, 38.9%, 26.6%, 27.9% and 6.2%, respectively). Northern blot analysis and RT-PCR elucidated the tissue distribution of equine CGA mRNA. Its expression was confirmed not only in the adrenal medullary cells but also in other organs (cerebrum, cerebellum, pituitary gland, spinal cord, liver, thyroid gland, striated muscle, lung, spleen, kidney, parotid gland and sublingual gland). Further, in adrenal chromaffin cells and pituitary cells of the anterior-intermediate lobe, the expression was confirmed by in situ hybridization with anti-sense CGA cRNA probe.