Studio di geni coinvolti nella proliferazione cellulare, nell'apoptosi, e nelle risposte allo stress ossidativo durante il ciclo coloniale blastogenetico dell'ascidia coloniale Botryllus schlosseri

In the last ten years, an increasing number of publications has highlighted the most interesting aspects of the biology of the colonial ascidian Botryllus schlosseri, indicating this non-vertebrate chordate as a reliable model organism for the study of gene expression, function and interaction pathways. The study of important cellular processes such as, for example, cell proliferation and death or stress and immune responses, is facilitated, in B. schlosseri, by its particular colonial life-cycle in which a continuos interchange from an apoptotic to a regenerative condition occurs. Each colony includes three different blastogenetic generations: adult filter-feeding zooids, primary buds on the adults and secondary buds on the primary buds. At the beginning of each cycle, new secondary buds originate from the atrial wall of primary buds and start to develop and grow until they become primary buds and, finally, zooids of the new adult generation replacing the old one. During the phase of generation change, also defined as take-over, these old zooids undergo a massive apoptosis and are gradually resorbed. The primary buds of the newly formed adults will then generate secondary buds. Although a morphological classification of the Botryllus blastogenetic cycle has been established, the complete lack of information on the type of genes involved, their role and level of expression remains a hard challenge. In order to add some new data, we used a library of Botryllus ESTs and considered specific sequences that showed a great BLAST similarity towards well-known vertebrate genes involved in apoptosis, proliferation and stress responses. Next, we were able to isolate their complete transcripts and we also analysed their site of synthesis and the differential expression between the main phases of the blastogenetic cycle, mid-cycle and take-over. We especially concentrated our work on the probable Botryllus orthologs of some key factors that are largely studied in vertebrates like the apoptotic BAX, AIF (Apoptosis Inducing Factor), PARP1 (Poly ADP-Ribose Polymerase 1), BIR Containing Proteins of the IAP family (Inhibitor of Apoptosis) and the transcription factor NF-kB1 (Nuclear Factor Kynase B 1); markers of cell proliferation such as PCNA (Proliferating Cell Nuclear Antigen), the Antigen KI67 and Nanos; antioxidant agents sensitive to stress conditions: the GCLM subunit of GCL (Glutathione Cysteine Ligase), GS (Glutathione Sintethase), GPx (Glutathione Peroxidase) and SOD (Superoxide Dismutase). Preliminary results obtained employing cDNA samples of two different colonies at the same blastogenetic stages of mid-cycle and take-over, indicate for five of the genes analyzed an unexpected general underexpression in the level of transcription in correspondence of the take-over phase. This work represents a first, detailed study of gene expression during the colonial blastogenetic cycle of B. schlosseri