Widespread Divergence of the CEACAM/PSG Genes in Vertebrates and Humans Suggests Sensitivity to Selection

Chang, Chia Lin; Semyonov, Jenia; Cheng, Po Jen; Huang, Shang Yu; Park, Jae Il; Tsai, Huai-Jen; Lin, Cheng-Yung; Grützner, Frank; Soong, Yung Kuei; Cai, James J.; Hsu, Sheau Yu Teddy

To verify the zebrafish CEACAM family genes in zebrafish, EST clones were obtained from Open Biosystem and analyzed with dideoxy sequencing. Expression analysis was also performed using snap frozen tissues collected from four adult zebrafish D. rerio and an adult male platypus O. anatinus as well as three deceased adult pufferfish T. nigroviridis. Total RNA was extracted using an RNeasy Mini Extraction kit (Qiagen) or the standard Trizol method. One microgram of RNA was reverse-transcribed in a total reaction volume of 20 ml containing 5 mM MgCl2, 106 buffer, 10 mM dithiothreitol, deoxyribonucleotide triphosphates, Poly-T primers, RNAsin 20 U, and 200 U of MuLV enzyme. Samples were incubated for 45 min at 42uC and 3 min in boiling water. PCR-amplification was performed in 20-ml samples containing 2.0 ml of cDNA, 4.6 ml of water, 2 ml of 10x PCR buffer, 1.2 ml of 25 mM MgCl2, 2 ml of 2.0 mM dNTP, 4 ml of forward and reverse primers, and 0.2 ml of Takara Ex Taq polymerase (5 U/ ml). After 2 min at 94uC, the amplification was carried out with 35 cycles at 94uC for 30 s and 68uC for 240 s.