Differential expression and characterization analysis of a new gene with WD domains in fish oogenesis

A new gene with WD domains is cloned and characterized according to its dif ferential transcription and expression between previtellogenic oocytes (pha se I oocytes) and fully-grown oocytes (phase V oocytes) from natural gynoge netic silver crucian carp (Carassius auratus gibelio) by using the combinat ive methods of suppressive subtraction hybridization, SMART cDNA synthesis and RACE-PCR. The full-length cDNA is 1870 bp. Its 5 ' untranslated region is 210 bp, followed by an open reading frame of 990 bp, which has the typic al vertebrate initiator codon of ANNATG. The open reading frame encodes a p rotein with 329 amino acids. It has 670 bp of 3 ' untranslated region and a n AATAAA polyadenylation signal. Because it has 92% homology to STRAP (seri ne-threonine kinase receptor-associated protein), a recently reported gene, we named it FSTRAP (fish STRAP). Virtual Northern blotting indicated that the FSTRAP was transcribed in fully-grown oocytes (phase V oocytes), but no t in previtellogenic oocytes (phase I oocytes). RT-PCR analysis showed that FSTRAP was transcribed in brain, heart, kidney, muscle, ovary, spleen and testis, but not in liver. And its mRNA could be detected in the oocytes fro m phase II to phase V. Western blotting also showed that FSTRAP protein cou ld be detected in brain, heart, kidney, muscle, ovary, spleen and testis ex cept liver. Results of Western blotting on various oocytes were also simila r to the RT-PCR data. FSTRAP protein was not expressed in the previtellogen ic oocytes. Its expression initiated from phase II oocytes after vitellogen esis, and was consistent with the mRNA transcription.