| Members of the Sox gene family are characterized by a HMG-box which shows sequence similarity with that of the mouse testis-determining gene SRY. Using degenerate primers PCR, seven and eight HMG-box motifs of SRY-related genes from respective genomic DNA of Trionyx sinensis and Alligator sinensis with TSD were cloned and sequenced, which were called TS41-TS47 in Trionyx sinensis and AS41-AS48 in Alligator sinensis respectively. TS41-TS47 form three major gene classes, which are not completely overlapped with those from AS41-AS48. TS43-TS46 and ,AS43-AS46 were demonstrated to be the mouse Sox12 homologues. ,AS48 was identified to be the mouse Sox4 homologue. TS47 may be a Sox21 orthologue of Trionyx sinensis, and ,AS47 may be a Sox4 orthologue of Alligator sinensis based on Blast search. Blast analyses showed that the percentage of identity at the amino acid level in the HMG-box region between TS-41, -42, AS-41, -42 and the non-reptilian Sox-1, -2, -3 was lower than that among the non-reptilian Sox-l, -2, -3. The molecular phylogenetic analysis indicated that the clustering of TS-41, -42 and AS-41, -42 was distant to the clustering of the non-reptilian Sox-l, -2, -3 homologues identified previously in species, including fish, amphibian, bird and mammalian. This suggests that TS-41, -42 and ,AS-41, -42 may represent the species-specific evolution and share a unique function. The unexpected sequence change in TS-41, -42 and ,AS-41, -42 may have occurred after divergence of amniotes. It is the first report that the sequence homologues of the progenitor of the mammalian testis-determining gene SRY were identified in reptile. These findings might provide molecular evidences that SRY-related genes may be involved into TSD.In order to clone new genes expressed during early embryonic development of Trionyx sinensis, we constructed and characterized a cDNA expression library from poly (A+) mRNA isolated from 250 mg of cranial/kidney/gonad complex tissues of one-week-old embryos of Trionyx sinesis using the SMART (Switching MechanismAt 5' end of RNA Transcript) cDNA synthesis and LD-PCR amplification strategy. The measured complexity of the cDNA library is 4.134X 10s directional clones. The insert lengths of 192 randomly selected clones ranged from 400 to 3800 bp. In all, 12 clones were randomly picked and sequenced, all showing typical cDNA characteristics. All sequences had the first codon of the ORFs. These sequences represent five groups: including ribosomal protein gene(tsRPL4, tsRPL6, tsRPL26), tissue-specific gene (tsFTH, tsHBAD), mitochondria! gene (tsCOXI), structure protein gene (tsDCN, tsMMIP, tsB-Actiri), transcriptional factor gene (tsHMG1, tsDAP5). The cDNA library can be used to provide expressed sequence tags (ESTs). The stage-specific cDNA library will be a useful resource for the study of gene structure, expression and regulatory during the early process of embroygensis of Trionyx sinensis.Using Random Primed Gene Walking PCR (RPGW-PCR), CtSox2 gene was cloned and sequenced from the digested genomic DNA of Trionyx sinensis. Compared with the mouse and chicken Sox2, CtSox2 shared 86% and 91% nucleotide homology respectively, and 93% and 97% amino acid identity respectively. The predicated amino acid sequences contain two conserved functional domains: a DNA-binding domain (HMG-box) and a transactivation domain. The conserved motif of group B homology lies immediate adjacent to C-proximal region of the HMG-box. Similar structure and identity of Sox2 gene among mammals, birds, amphibians and reptiles suggest that Sox2 gene have evolutionary conserved roles. Cloning of CtSox2 gene shows the validation of RPGW-PCR used for isolating genes from the digested genomic DNA.
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