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

Abstract

A new gene with WD domains is cloned and characterized according to its differential transcription and expression between previtellogenic oocytes (phase I oocytes) and fully-grown oocytes (phase V oocytes) from natural gynogenetic silver crucian carp (Carassius auratus gibelio) by using the combinative 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 typical vertebrate initiator codon of AN-NATG. The open reading frame encodes a protein with 329 amino acids. It has 670 bp of 3 untranslated region and an AATAAA polyadenylation signal. Because it has 92% homology to STRAP (serine-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 not 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 from phase II to phase V. Western blotting also showed that FSTRAP protein could be detected in brain, heart, kidney, muscle, ovary, spleen and testis except liver. Results of Western blotting on various oocytes were also similar to the RT-PCR data. FSTRAP protein was not expressed in the previtellogenic oocytes. Its expression initiated from phase II oocytes after vitellogenesis, and was consistent with the mRNA transcription. The studies on functional roles of genetic factors and maternal RNAs in oogenesis have been mainly focused on the human being and cultured animals. In fish, however, only a few of recent attempts have been made to identify the genetic factors [1] , but the knowledge about regulative mechanism underlying gene transcription and translation in oogenesis is very little. Recently, along with advances of developmental biology, fish oogenesis has been recognized as an important research field. For instance, Selman et al. investigated different stages of oocyte development in zebrafish (Brachydanio rerio) [2] . Tyler and Sumpter reviewed oocyte growth and development in teleosts [3] . Nagahama et al. initiated the studies on molecular biology of oocyte growth and maturation in fish [4] . However, numerous basic questions in fish oogenesis remain unanswered.

Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp

“…Then, the secondstrand cDNA was synthesized with SMARTTM Oligonucleotide, which has an oligo(G) sequence at its 3' end. SMART cDNA can replace RNA in analysis of virtual northern hybridization and the information given by virtual northern hybridization was the same as provided by standard northern hybridization [12], [31]. This character is valuable for studying embryonic development of animal that can only spawn one time.…”

“…Suppression subtractive hybridization (SSH) [7] has been widely demonstrated to be a very successful technique for studying differential gene expression [8][9][10][11][12]. By the way of hybridization and PCR of mRNA or cDNA of different tissues, organs, development stages or individuals, the general genes are removed and low abundant specific genes can be enriched.…”

Comparative studies on in vitro sperm decondensation and pronucleus formation in egg extracts between gynogenetic and bisexual fish

“…Therefore, we propose that some regulative factors might exist in the gibel carp egg cytoplasm. A series of molecular and biochemical studies on identification of the candidate factors have been initiated in our laboratory [32][33][34][35][36], and the studies will help us to elucidate the molecular basis underlying the reproduction modes.…”

cDNA cloning and characterization of a novel SNX gene differentially expressed in previtellogenic oocytes of gibel carp

“…Differentially expressed genes were identified using the Clontech PCR-Select cDNA Subtraction Kit as described previously (Fan et al, 2000;Xie et al, 2001Xie et al, , 2003Wen et al, 2001). In brief, double-strand cDNAs synthesized from 2 mg polyA RNA of phase I and phase V oocytes q were subjected to blunt-end digestion by the restrictive enzyme RsaI.…”

“…Several PCR-based techniques for differential gene screening have been established (Wang and Brown 1991;Liang and Pardee, 1992). A suppression subtractive hybridization (SSH) technique, owing to its high efficiency and rapidity, has been widely applied to many molecular cloning studies for the identification of disease, development and tissuespecific, or other differentially expressed genes (Diatchenko et al, 1996;Chu and Mckinsey, 1997;Davis and Benzer, 1997;Wong et al, 1997;Evans and Wheeler, 1999;Shimono and Behringer, 1999;Xie et al, 2001Xie et al, , 2003Wen et al, 2001). Using the suppression subtractive hybridization (SSH), we have cloned a lot of differentially transcribed genes between different stages of gibel carp oogenesis .…”