Molecular cloning and sequencing of feline CD7

Human CD7 is one of the earliest molecules to appear in T cell development. In this study, putative feline CD7 cDNA was identified based on its similarities with human and mouse CD7 genes. The feline CD7 cDNA contained an open reading frame consisting of 630 nucleotides. The amino acid sequence of feline CD7 had 47.7% identity with that of human CD7, and 52.9% with that of mouse CD7. In addition, the feline CD7 protein fused with histidine tag was expressed in 293T cells. The expression was confirmed by indirect immunofluorescence assay.     

Nucleotide sequence and expression of the feline vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is an angiogenic factor which targets vascular endothelial cells. In this study, cDNA encoding a feline VEGF (fVEGF) isoform was cloned from a feline lymphoid tumor cell line and sequenced. The fVEGF cDNA contained an open reading frame of 567 nucleotides coding for a polypeptide of 163 amino acids with a putative signal peptide of 26 amino acids. The predicted fVEGF amino acid sequence shared 98.4, 94.2 and 94.2% homology with the sequences of canine, bovine and human VEGF, respectively. Though predicted fVEGF polypeptide was two amino acid residues shorter than human VEGF165, a potential glycosylation site and regions critical for receptor binding were conserved in all the species examined. Transient expression of fVEGF in mammalian cells resulted in secretion of VEGF which could be detected by antibodies against human VEGF165. Furthermore, wide expression of fVEGF mRNA was observed in various feline tissues using RT-PCR methods.     

Antigens expressed in feline enteroepithelial-stages parasites of Toxoplasma gondii

In an investigation aimed to identify Toxoplasma gondii antigens expressed in feline enteroepithelial-stages parasites, a cDNA library was constructed and fourteen positive clones were isolated by immunoscreening using sera from cats immunized with feline enteroepithelial-stages parasites. By DNA sequence homology analysis, these fourteen isolated clones were classified into four groups: hypoxanthine-guanine phosphoribosyl transferase (HGPRT) cDNA, heat shock protein 70 (HSP70) cDNA, 14-3-3 protein homologue cDNA, and cDNA encoding an unknown product. In an indirect immunofluorescence antibody test, sera from mice immunized with the recombinant protein encoded by the cDNA for HGPRT, HSP70, 14-3-3 protein, or the unknown product each showed a relatively high level of immunoreactivity with feline enteroepithelial-stages parasites.     

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.     

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.     

Expression of recombinant feline serum amyloid A (SAA) protein.

Feline serum amyloid A (SAA) cDNA clone was isolated from a hepatic mRNA of a mixed-breed cat. The feline SAA cDNA clone contains 333 nucleotides and deduced amino acid sequence shows 87.4%, 73.9%, and 65.8% homology with dog, human and mouse SAA respectively. Relative to the human and mouse SAA proteins, an additional peptide of eight amino acids is specified in the feline cDNA clone. Recombinant feline SAA (rfSAA) was expressed at high levels using pGEX bacterial expression system. Cleavage from the fusion moiety, and purification using glutathione-sepharose yielded pure soluble form of rfSAA. Antibodies generated against rfSAA were specific for feline SAA and showed no cross-reactivity with human SAA. Furthermore, antibodies against human SAA did not react with feline SAA. These results indicate that antigenicity of feline SAA is totally different from human SAA.     

Expression of Bcl-2 in feline lymphoma cell lines

BACKGROUND: The Bcl-2 gene is a member of the rapidly expanding Bcl-2 family of genes that regulate apoptosis. Bcl-2 has been shown to repress cell death triggered by a diverse array of stimuli, including chemotherapy and gamma irradiation. OBJECTIVE: The purpose of this study was to determine feline Bcl-2 expression level in feline lymphoma cells using an immunoblot assay with anti-human and anti-canine Bcl-2 monoclonal antibodies. METHODS: About 708 base pairs containing the coding sequence of the feline Bcl-2 gene were transformed into Escherichia coli. The recombinant Bcl-2 was used as a positive control for an immunoblot assay using mouse monoclonal antibodies against human and canine Bcl-2. An immunoblot assay using the monoclonal antibodies was carried out to determine the level of feline Bcl-2 expression in lymphoma and lymphocytic leukemia cell lines. RESULTS: The recombinant feline Bcl-2 protein produced in E. coli had a molecular weight of about 26 kDa and was detected by immunoblot assay by using anti-human Bcl-2 mouse monoclonal antibody. Feline Bcl-2 expression was high in lymphoma cell lines (FL-74-UDC-1 and FT-1) and low in the cell line from peripheral blood mononuclear cells from a healthy cat (FeTJ-1) but not low in freshly isolated peripheral blood mononuclear cells from a healthy cat. The anti-human Bcl-2 mouse monoclonal antibody was found to cross-react with feline Bcl-2. CONCLUSIONS: These results confirm the expression of Bcl-2 in T-cell lymphoma cell lines and indicate that it is suitable to detect feline Bcl-2 using an immunoblot assay. Pending further evaluation, Bcl-2 expression might be useful in the differential diagnosis of feline tumors.     

Molecular cloning and sequencing of the cDNA encoding the feline FcgammaRIIIA (CD16) homologue

We amplified the cDNA encoding the feline FcgammaRIIIA (CD16) homologue from peripheral blood mononuclear cells by polymerase chain reaction and cloned two forms of FCGR3A cDNA. Sequencing analysis revealed that the open reading frame of feline FCGR3A cDNA consists of 750 or 747 base pairs encoding 250 or 249 amino acid residues, respectively. Comparison of the predicted amino acid sequence of feline FCGR3A cDNA with those of other mammalians' homologues revealed that the extracellular domain has a relatively low homology. However, the cytoplasmic domain contained an 8-amino acid motif, Leu-Phe-Val-Val-Asp-Thr-Gly-Leu, which was considered to interact with an accessory molecule such as the gamma chain of Fc receptors for IgE to form heterodimeric complexes.     

Molecular cloning and sequence analysis of feline interferon-stimulated gene 15

The interferon-stimulated gene 15 (ISG15) is induced by type I interferon (IFN). Recent studies have revealed that like ubiquitin, ISG15 is conjugated with target proteins. In this study, the feline ISG15 (FeISG15) gene was cloned from feline IFNomega (FeIFNomega)-stimulated feline kidney epithelial (CRFK) cells. According to gene sequence results, cDNA was 474bp long and encoded a protein of 157 amino acids. The putative amino acid sequences showed 62.5-72.1% identity with those of other mammalian ISG15s. Similar to human and mouse ISG15, FeISG15 included tandem ubiquitin-like domains; its homology with feline ubiquitin was 36.3-39.5%. The LRLRGG conjugating motif was located only in the carboxyl terminal ubiquitin-like domain. FeISG15 also lacked the carboxyl terminal extension after the LRLRGG motif, which is present in mouse and human ISG15. Recombinant FeISG15 protein was expressed as a His-tagged fusion protein in Escherichia coli and purified by ion-exchange chromatography followed by affinity chromatography. Monoclonal anti-FeISG15 antibodies revealed free FeISG15 and FeISG15 conjugated with target proteins in cells after IFNomega stimulation by Western blotting analysis. Furthermore, mRNA of IFNgamma was detected from peripheral blood mononuclear cells (PBMCs) after stimulation with rFeISG15 extracellularly by RT-PCR. Taken together, these results suggested that FeISG15 had ubiquitin- and cytokine-like activity, as in other species.     

Molecular cloning and expression of feline CD28 and CTLA-4 cDNA

Feline CD28 and CTLA-4 (CD152) cDNA were cloned from Con-A stimulated feline peripheral blood mononuclear cells (PBMC) by rapid amplification of cDNA end-PCR (RACE-PCR). Both CD28 and CTLA-4 proteins belong to the immunoglobulin superfamily (Ig SF) and are composed of a signal sequence, an extracellular domain, a transmembrane domain and a cytoplasmic domain. The open reading frame (ORF) of CD28 cDNA encoded a predicted protein of 221 amino acids and that of CTLA-4 cDNA encoded a predicted protein of 223 amino acids. The B7 ligands binding motif MYPPPY hexamer was found on the extracellular Ig V-like domains of both receptors and phosphatidylinositol 3-kinase (PI 3-kinase) binding motifs pYMNM for CD28 and pYVKM for CTLA-4 were identified in the cytoplasmic domains. Comparisons of amino acid sequences of feline proteins with known sequences of other species indicated that rabbit CD28 and CTLA-4 were most closely related and mouse molecules were the least conserved with feline molecules. Comparison of each domain of both molecules with that of other animals showed that the cytoplasmic domain of CTLA-4 was 100% conserved and that of CD28 was the most conserved domain. The cloned CD28 and CTLA-4 cDNA could be expressed in transfected mammalian cells. Expression of feline CD28 and CTLA-4 mRNA in freshly isolated feline PBMC was demonstrated by RT-PCR. Stimulation of PBMC with Con-A similarly increased the expression of both CD28 and CTLA-4 mRNA.     

Sequence analyses of feline B7 costimulatory molecules

Using RT-PCR amplifications with mRNA from mitogen-stimulated feline peripheral blood mononuclear cells, cDNA of feline B7-1 (CD80) and B7-2 (CD86) were cloned. The cDNA were sequenced and putative translated protein sequences compared with known counterpart sequences. Hydrophilicity patterns of the feline CD80 and CD86 which were only 26.8% identical at the amino acid sequence were very distinct from each other, but similar to the putative human CD80 and CD86 proteins, respectively. The feline CD80 gene encoded a protein of 292 amino acids and the CD86 gene encoded a protein of 329 amino acids. Amino-terminal signal sequences, extracellular Ig V- and Ig C-like domains, transmembrane domains, and carboxyl cytoplasmic domains were identified in both molecules. Although the most conserved domain among the CD80 sequences was the Ig C-like domain, the most conserved domain among the CD86 sequences was the Ig V-like domain. Among the known sequences, the bovine CD80 and the porcine CD86 sequences available for comparisons were identified as most closely related to the feline CD80 (63.3%) and CD86 (67.5%), respectively. The mouse molecules were the least identical (43.6 and 43.6%, respectively) with the feline CD80 and CD86 proteins. The human CD80 and CD86 molecules were 56.3 and 57.0% identical with the feline molecules.     

Expression of RANTES mRNA in skin lesions of feline eosinophilic plaque

Abstract One of the mechanisms of eosinophil infiltration is its induction by chemoattractants such as regulated upon activation, normal T-expressed and secreted (RANTES) which is a cysteine–cysteine chemokine that mediates chemotaxis and activation of eosinophils in humans and mice. Skin lesions of feline eosinophilic plaque are characterized by a predominant infiltration of eosinophils. The mechanism(s) of eosinophilic infiltration in the skin and/or mucosa of cats is unknown. It is possible that RANTES is involved. To investigate the presence of RANTES in the skin of cats with eosinophilic plaques and nonaffected skin, we cloned and sequenced the full-length feline RANTES cDNA gene, in order to determine whether it is present in the skin of cats with eosinophilic plaques and/or if it is present in normal adjacent skin. We were able to document the the expression of RANTES mRNAs in skin with feline eosinophilic plaque as well as in normal cat skin. The full-length cDNA sequence of the RANTES gene (742 bp) contained a single open reading frame of 276 bp encoding a protein of 92 amino acids. The amino acid sequence of feline RANTES shared 67 and 74% sequence identity with that of bovine and mouse RANTES genes, respectively. RT–PCR analysis on RANTES mRNA in the skin of cats with eosinophilic plaque revealed that its expression was higher in the eosinophilic plaque skin lesions than in the normal skin. The result suggested that RANTES might play a role to induce eosinophil infiltration in feline eosinophilic plaque lesions.     

Molecular cloning of feline hepatocyte growth factor (HGF) cDNA

Hepatocyte growth factor (HGF) is a pleiotropic cytokine responsible for regeneration, development and maintenance of various organs, and growth, invasion and metastasis of tumor cells. A full-length feline HGF cDNA was cloned and sequenced by RT-PCR from cat liver. Feline HGF consists of 728 amino acid and contains alpha- and beta-chains encoded in a single open reading frame. The predicted amino acid sequence of feline HGF showed 93.2, 93.3 and 93.3% homology with those of human, mouse and rat HGF, respectively. The putative proteolytic processing site, all cysteine residues, and four potential glycosylation sites are conserved in all species. Therefore, feline HGF is expected to have a similar three-dimensional structure to human, mouse and rat HGF.