During Fas-mediated apoptosis, FADD binds to the Fas death domain and recruits procaspase 8, which is an apical protease for inducing apoptosis [14] and [15]. Fas is constitutively expressed by a broad range of normal epithelial cells and various haematopoietic cells. Notably, some tumour cells such as those of adult T-cell leukaemia, acute myelogenous leukaemia, chronic lymphocytic leukaemia, hepatocellular carcinoma and colon carcinoma click here abnormally over- and under-express Fas [16], [17], [18] and [19] and some of these virus-infected cells are sensitive to Fas-mediated apoptosis. The expression of Fas appears to be induced by interferon γ and CD40, which indicates

that the expression of Fas is controlled by certain mechanisms [20] and [21]. When CTLs recognise antigens derived from the virus by the MHC molecule, they express the FasL and induce apoptosis [22]. Thus, the FasL plays an important role in maintaining homoeostasis in PLX4032 solubility dmso the immune system by inducing apoptosis. Several members of the TNF superfamily and TNF superreceptors family have been cloned in fish [23],

[24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37] and [38]. Although a Fas molecule has been reported in zebrafish and medaka [29] and [30], expression analyses in rock bream has not been reported. Molecular cloning and characterisation of the Fas should contribute to elucidating the mechanism of innate immunity in rock bream. Here we report the molecular cloning and sequence analysis of a Fas gene from rock bream

and its expression in relation to infection by Streptococcus Proteasome inhibitor iniae or iridovirus. Fas cDNA was identified by analysing expressed sequence tags in the cold shock-stimulated rock bream erythrocytes library. The full-length cDNA of RbFas was obtained by 5′ RACE. The 5′ and 3′ ends of the RbFas cDNA were identified by the RACE cDNA amplification kit (Clontech, CA, USA) according to sequence information from the obtained fragment. A specific primer set (RbFas-5R and RbFas-3F) was designed according to the known EST sequences of RbFas (Table 1). Briefly, total RNA was extracted from red blood cells using Trizol Reagent (Invitrogen, USA) and first strand cDNA was synthesised using the protocol recommended by the SMART RACE cDNA Amplification Kit. The primer set of lRbFas-3F and a nested universal primer (supplied by Clontech) was used for 3′ RACE, and 5′ RACE was carried out with RbFas-5R and the nested universal primer. The generated PCR products were purified, cloned into pGEM T-easy vector system (Promega, USA) and subsequently sequenced. Thus the complete cDNA of RbFas was compiled by the 5′ and 3′ RACE DNA sequences. The determined nucleotide and deduced amino acid sequences and multiple sequence alignments were analysed with GENETYX ver. 8.0 (SDC Software Development Co. Ltd., Tokyo, Japan).