DIFFERENTIAL EXPRESSION AND 3D STRUCTURAL ANALYSIS OF THE MHC CLASS-II beta CHAIN FROM SEA BASS (Dicentrarchus labrax

The major histocompatibility complex class I and II molecules (MHC-I and MHC-II) plays a pivotal role in vertebrate immune response to antigenic peptides. In this paper we report the cloning and sequencing of the MHC class II b chain from sea bass (Dicentrarchus labrax L.). The six obtained cDNA sequences (designated as Dila-DAB) code for 250 amino acids, with a predicted 21 amino acid signal peptide and contain a 28 bp 5'-UTR and a 478 bp 3'-UTR. A multiple alignment of the predicted translation of the Dila-DAB sequences was assembled together with other fish and mammalian sequences and it showed the conservation of most amino acid residues characteristic of the MHC class II b chain structure. The highest basal Dila-DAB expression was found in gills, followed by gut and thymus, lower mRNA levels were evidenced in spleen, peripheral blood leucocytes (PBL) and liver. Stimulation of head kidney leukocytes with LPS for 4 h showed very little difference in the Dila-DAB expression, but after 24 h the DilaDAB level decreased to a large extent and the difference was statistically significant. Stimulation of head kidney leukocytes with different concentrations of rIL-1b (ranging from 0 to 100 ng/ml) resulted in a dosedependent reduction of the Dila-DAB expression. Moreover, two 3D Dila-DAB*0101 homology models were obtained based on crystallographic mouse MHC-II structures complexed with D10 T-cell antigen receptor or human CD4: features and differences between the models were evaluated and discussed. Taken together these results are of interest as MHC-II structure and function, molecular polymorphism and differential gene expression are in correlation with disease resistance to virus and bacteria in teleost fish. Dear Prof. Tony Ellis, I am sending you a revised version of the paper entitled: "MOLECULAR CLONING, DIFFERENTIAL EXPRESSION AND 3D STRUCTURAL ANALYSIS OF THE MHC CLASS-II beta CHAIN FROM SEA BASS (Dicentrarchus labrax L.)" (Ref. n. FSIM-D-07-00013), author team: Francesco Buonocore, Elisa Randelli, Daniela Casani, Susan Costantini, Angelo Facchiano, Giuseppe Scapigliati, Renè J.M. Stet. I have taken into account most of the suggestions from the referees and I have attached a list of all the changes made in the paper (in the text you will find in bold the phrases that differ form the first version). I hope that now the paper will be accepted for the publication, but let me know if there is any problem. Best regards Dr. Francesco Buonocore Cover Letter