Primers used for PCR amplification were osgshsp-F TACCTgTTTgAgTgCggTgACT and osgshsp-R gATACgACgCATggACTggA; they were designed

from four peptide sequences obtained by mass spectrometry of protein (Supplementary Fig. 2A, boxed region) and matched those of crystallin. Putative crystallin sequences were examined for homogeneity by published crystallin sequences with Blast (http://www.ncbi.nlm.nih.gov/BLAST). RACE-PCR was carried AZD2281 purchase out as previously described [29]. Based on the verified sequence of the 979 bps fragment of crystallin cDNA, two primers crystallin-5RACE, osgshsp-a387 gCggTAggAgTTgCTCC, osgshsp-a516 CCAgAgggTgggCACATC, and crystallin-3RACE, osgshsp-s300 TCgCTgggATACCTggAgC and osgshsp-s600, gATCTgCCTgTATgAgCTCTCCg were PCR-amplified and cloned for the 5′ and 3′ ends, respectively. Two adapter primers for both ends were provided in the Marathon cDNA Amplication Kit (Clontech, Mountain View, CA, USA). The (RACE)-PCR thermal cycle profile was as follows: 94 °C for 1 min; 30 s at 94 °C, 4 min at 72 °C, and 10 min at 72 °C for 30 cycles; followed by an extension at 72 °C for 10 min. The amplified

fragment was verified with subcloning into a pCR II vector for sequencing. To minimize polymerization errors during PCR, a proofreading polymerase was employed in PCR reactions. Real time reverse transcription (RT)-PCR was used to quantify the Selleckchem Gemcitabine expression of mRNA for crystallin with expression of actin as reference. The primers osgshsp-F TACCTgTTTgAgTgCggTgACT and osgshsp-R gATACgACgCATggACTggA were used to amplify a 130 bp fragment of grouper crystallin. A standard curve was constructed by

serially diluting a linearized plasmid containing the open reading frame of grouper crystallin. Grouper beta actin primers were applied to normalize the starting quantity of RNA. Typical profile times used were initial step, 95 °C for 15 min, followed by a second step at 94 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s for 40 cycles with melting curve analysis. The level of target mRNA was normalized to the level of actin and compared to control (healthy grouper) and the values were calculated by the 2−ΔΔCT method. To elucidate the evolutionary history of crystallin, novel crystallin sequences were identified Mannose-binding protein-associated serine protease in zebrafish and eight mammalian species, and used to explore the phylogenetic relationships of the crystallin gene family. A neighbor-joining phylogenetic tree was produced by the MEGA3.1 program [30] on the basis of a ClustalW alignment of the nucleotide sequences of the open-reading frames along with all known complete sequences of crystallin cDNA with the complete deletion of gaps for 1000 bootstrap replications. Numbers indicated bootstrap confidence values through 1000 replications; only bootstrap values over 70% are exhibited. RAW 264.