| After hibernation, the fully-grown oocytes of a kind of common toad in China (Bufo bufo gargarizans) acquire potency to resume meiosis after progesterone stimulation, named as LTE-oocytes; however, those toads maintained at a relatively high temperature for 2-3 months whose oocytes cannot undergo GVBD and resume meiosis after progesterone treatment, nominated as HTE-oocytes. Therefore, the acquisition of maturation competence in oocyte is dependent on a suitable temperature.To address the molecular mechanism of temperature-dependence in acquiring maturation competence in oocyte, we constructed the subtractive cDNA library between LTE-oocytes and HTE-oocytes and identified 18 ESTs, which will probably preferentially expressed in LTE-oocytes. These 18 ESTs were all novel ESTs, which had been submitted to GenBank and dbEST with the accession numbers of GenBank CD347660. CD644751- CD644767 and dbEST ID 18335746. 18760438-18760454, respectively. Of these 18 ESTs, TS1-4, shared high similarity with human and mouse NF-YB genes (>90%). NF-YB is the B subunit of NF-Y complex (NF-YA+ NF-YB+ NF-YC), while NF-Y is a CCAAT-box binding nuclear transcription factor which regulates many genes including some cell cycle related key genes such as p34cdc2, cyclin Bl, cyclin B2, cdc25. Therefore, it is very necessary to clone the full-length gene of TS1-4, i.e. toad NF-YB gene (i:NF-YB), to further investigate the molecular mechanism of temperature- dependence in acquiring maturation potential in oocyte.By using RACE, we identified three alternatively spliced transcripts of tNF-YB derived from LTE-oocytes, which were all full-length genes and nominated as tNF-YB 1, tNF-YB2 and tNF-YB3 respectively. These genes had been submitted to GenBank with the accession numbers of AY442015, AY442016 and AY442017, respectively. The full-length tNF-YB 1 cDNA consisted of 1278 bp and encoded 206 amino acids. The theoretical molecular weight of the deduced tNF-YB 1 protein was 23 kDa and ProtParam analysis predicted a theoretical p/ of 4.40. In addition, ScanProsite tool predicted many glycosylation sites, phophorylation sites and myristoylation sites in tNF-YB 1 protein. The alternatively spliced tNF-YB2 cDNA consisted of 1262 bp, with a 12 bp section replaced by a 96 bp segment in the middleof its ORF. The replacement did not change its ORF, aside from substituting 32 amino acids residues. Splice variant tNF-YB2 encoded a protein of 234 amino acids and had a theoretical molecular weight of 26 kDa. The third variant tNF-YB3 cDNA comprised 1277 bp, extended from nt 131 to the end of tNF-YBl, and possessed an insert of 129 bp upstream of the ORF start codon. The insert did not change its ORF, so the deduced tNF-YB3 protein sequence was identical to tNF-YBl protein sequence. The tNF-YBl and tNF-YB3 proteins contained three conserved domains : histone-fold motif (aa 56-124), TBP-binding domain (aa 124-133) and Q-rich domain (aa 183-206). The histone-fold motif corresponds to the conserved domain CBFD NF-YBHMF (GenBank pfam 00808) of the histone-like transcription factor "CBF/NF-Y". The secondary structure of histone-fold motif consisted of three a -helices and two loops: a 1, L1, a 2, L2 and a 3. However, tNF-YB2 protein has an insert in the L1 loop, which may change its secondary structure and reduce its CCAAT-binding activity and/or biological function.Northern Blot analysis indicated that tNF-YB was influenced obviously by temperature at the level of transcription, i.e. high expression in LTE-oocytes versus low expression in HTE-oocytes. Western Blot analysis also suggested that there are differential expression of toad NF-YB between LTE-oocytes and HTE-oocytes at the level of translation. Therefore, these results indicated that the expression of toad NF-YB in HTE-oocytes was repressed/inhibited remarkably, leading to alterability of NF-Y complex composition, in turn might change its CCAAT box binding activity and its target genes transcriptional expression.Among NF-Y target genes, we are more interested in...
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