ABSTRACT. Using polymerase chain reaction, bottle-nosed dolphin (Tursiops truncatus) major histocompatibility complex (DoLA) class Iα chain cDNA was cloned and sequenced. Predicted amino acid sequences of DoLA class Iα chain have cystein residues for intradomain disulfide bond formation and N-linked glycosylation sites, suggesting that DoLA class Iα chain molecules construct α1, α2, and α3 domain structures. Similarity of DoLA class Iα chain cDNA to land mammal MHC class Iα chain cDNA was 90.4% in cattle, 90.2% in horse, 89.4% in sheep, and 87.8% in human. This investigation suggests that DoLA is closely related to land mammals. -KEY WORDS: bottle-nosed dolphin, MHC class I cDNA.

ABSTRACT. Using polymerase chain reaction (PCR), a bottle-nosed dolphin (Tursiops truncatus) interleukin-4 (IL4) cDNA was cloned and sequenced. IL4 specific primers were based on the 5' and 3' untranslated regions of the human and murine IL4 gene. The dolphin IL4 cDNA is 528 base pairs in length and contains an open reading frame of 402 nucleotides coding an IL4 precursor of 133 amino acids, with the putative signal peptide of 24 amino acids. Analysis of the mature amino acid sequence shows three potential N-linked glycosylation sites and three disulfide bonds. Comparison of the predicted amino acid sequence shows that dolphin IL4 shares 77, 74, 58 and 41% identity with the bovine, ovine, human and mouse IL4s, respectively.-KEY WORDS: bottle-nosed dolphin, cDNA, IL-4.

ABSTRACT. The bottle-nosed dolphin (Tursiops truncatus) interferon-γ (IFN-γ) cDNA was cloned from mitogen stimulated peripheral blood mononuclear cells (PBMC) RNA utilizing the reverse transcription-polymerase chain reaction (RT-PCR). The dolphin IFN-γ cDNA was 585 base pairs in length and contained an open reading frame of 498 nucleotides encoding an IFN-γ precursor of 166 amino acids, with cleavage of the putative signal peptide of 19 amino acids. Analysis of the mature amino acid sequences revealed two potential N-linked glycosylation sites. Comparison of the predicted amino acid sequence showed that dolphin IFN-γ shares 86, 85, 63 and 42% similarity with the bovine, ovine, human and mouse IFN-γ s , respectively.-KEY WORDS: cDNA, dolphin (bottle-nosed), IFN-γ.

ABSTRACT. The bottle-nosed dolphin (Tursiops truncatus) IL-1α and IL-1β cDNA were cloned from mitogen stimulated peripheral blood mononuclear cells (PBMC) RNA utilizing the reverse transcription-polymerase chain reaction (RT-PCR). The sequences of these cDNAs showed that dolphin IL-1α and IL-1β clones contained open reading frames encoding 265 and 266 amino acids, respectively. Comparison of the deduced amino acid showed that dolphin IL-1α sequence shared 77, 77, 77, 74, 71, 65 and 57% similarity with the bovine, ovine, porcine, equine, feline, human and mouse IL-1α sequences, respectively. Similarly, the amino acid sequence showed that dolphin IL-1β shared 77, 77, 74, 69, 65, 64 and 63% similarity with the bovine, ovine, porcine, equine, feline, human and mouse IL-1β sequences, respectively. The relatedness of dolphin IL-1α and IL-1β were relatively distant with 21% amino acid homology.-KEY WORDS: bottlenosed dolphin, IL-1α, IL-1β.

ABSTRACT. A monoclonal antibody, 6E9 established from mice injected with dolphin peripheral blood lymphocytes (PBLs) was characterized. In addition to its reactivity against 89.4% of dolphin PBLs, 6E9 reacted with 33.1% of bovine PBLs of which 22% were CD5 + , 11.1% were CD5 . 6E9 recognized a 34 kD protein on the surface of dolphin and bovine PBLs. Analysis of the protein's Nterminal amino acid sequence indicated that 6E9 recognizes bovine major histocompatibility complex (MHC) class II antigens. These results suggested that 6E9 recognized MHC class II antigens on bovine PBLs. As we have already produced an anti-dolphin MHC class I monoclonal antibody, analysis of immune system using these monoclonal antibodies will advance our understanding of the evolution of the mammalian immune system.-KEY WORDS: dolphin, MHC class II antigen, monoclonal antibody.J. Vet. Med. Sci. 60(3): 291-293, 1998 mice were purchased from Clea Japan Inc. (Tokyo, Japan).Cows were maintained at Nihon University. Production of 6E9 mAb: BALB/c mice were immunized with dolphin peripheral blood lymphocytes (PBLs) isolated by the Ficoll-Paque gradient (1.077) centrifugation method. The immunization schedule consisted of intraperitoneal injections of live 10 7 cells at 2-month intervals. After three injections, splenocytes from immune mice were fused with P3-X63-Ag8.653 myeloma cells [6]. The screening of hybridoma supernatants was based on the reactivity to dolphin PBLs, as analyzed by immunofluorescence staining. A hybridoma, termed 6E9 selected by this screening procedure, was further subcloned by limiting dilution and was cultured in RPMI 1640 (Gibco, Grand Island, New York) containing 10% heat-inactivated fetal calf serum (FCS), 1% L-glutamine, 1% nonessential amino acids, 1% Na-pyruvate, and 5 × 10 5 M 2-mercaptoethanol (ME), referred to as complete medium.Immunofluorescence tests: For immunofluorescence staining, cells were incubated at 4°C for 30 min with 30 µl of hybridoma supernatant. The cells were then washed with cold PBS (pH 7.2) containing 2% heat-inactivated newborn calf serum (NCS) and 0.05% NaN 3 , and mixed with 10 µl of fluorescein isothiocyanate (FITC)-conjugated rabbit antimouse Ig mAb (Dakopatts, Cophenhagen, Denmark) diluted in PBS containing 2% NCS and 0.05% NaN 3 . The expression of the 6E9 surface antigens on bovine PBLs was compared with that of anti-bovine CD4 (CC30), CD5 (CC17), and CD8 (CC63) antibodies using two-color immunofluorescence tests. Namely, cells were stained with these mAbs followed by FITC-conjugated rabbit anti-mouse Ig mAb, and then, cells were stained with biotin-conjugated 6E9 and then vizualized with phycoerythrin (PE)-conjugated avidin (Caltag Laboratories, San Francisco, CA). Background staining was assessed using FITC-conjugated anti-mouse Ig mAb and PE-conjugated avidin. The Dolphins are very interesting mammals because they have returned to the sea from the land, after acquiring pulmonary respiration system. We are attempting to characterize their immune functions and as the first step t...