ИСТИНА

Ascidians belong to the subphylum Tunicata, which is one of the most ancient groups of chordates. A characteristic feature of these animals is their integument – tunica. Tunica is composed of polysaccharides and proteins sclerotized by quinon tanning. Sclerotization process is executed by means of phenoloxidase (PO) system (Chaga, Solovey, 1986), which is also involved in animal defense (Smith, 1996) and allorecognition (Akita, Hoshi, 1995). Components of phenoloxidase system, such as phenoloxidase, polyphenolic compounds, cationic proteins, are localized in morular blood cells (Smith, 1970). Two major proteins p48 and p26 kDa expressed in morular cells of Styela rustica were shown to participate in tunic repair. They are supposed to be the part of phenoloxidase system (Podgornaya, Shaposhnikova, 1998). Specific antibodies raised against p48 and p28 also stain granules inside test cells, surrounding oocytes of S. rustica. Ascidians’ oocytes are surrounded by both test and morular cells. Latter participate in adult tunic formation. Whereas, test cells are present only during embryogenesis. Some authors believe that they are involved in formation of larval tunic. Thus we can assume that two major proteins of Stiela rustica's morular cells, p26 and p48, play an important role at the larval and adult stages of ascidians. However their primary structure and function remain unclear. We held MS-MS analysis, which allowed us to determine the amino acid sequences of several short peptides – tryptic digestion products of two proteins. One of these peptides, prepared from p26 protein, had a high probability of precise amino acid sequence. Sequence of this peptide was used to design degenerate primers. Then we synthesized cDNA on the basis of poly(A)RNA from ascidian blood cells and used it as a matrix in RACE-PCR with degenerate primers. The product of RACE-PCR was cloned and sequenced. In this way we obtained a sequence which contained open reading frame 567 nucleotides long. Comparison of this sequence with known sequences using online databases showed that our sequence is unique/new and was not described previously. Open reading frame of 567 nucleotides must correspond to the part of p26 gene. In the near future we want to get full-length gene coding for p26 kDa protein.