Molecular Cloning And Expression Analysis Of Ostf And β-actin Genes, And Primary Construction Of The Rice Field Eel BAC Library

Sexual development in vertebrates is a complex process. Several mechanisms were used in determination of the development of the male or female of vertebrates. The genes for determination of sequential hermaphrodite sex are unknown. We identified a homologue of human osteoclast-stimulating factor (OSF) from the rice field eel, a teleost with characteristic of naturally sex transformation from female, via intersex to male during its life. The rice field eel OSF-like cDNA encoded a peptide of 214 amino acids that contains a c-Src homology 3 domain, proline-rich region and ankyrin repeats, suggesting that it was potentially involved in cell signaling. The gene was clustered into the OSF gene group of all the other vertebrates. Although expressed in the three kinds of gonads and also in other tissues, in gonads of all the three sexes, OSF-like expression was restricted to the gonadal germinal epithelium, from where bipotential gonia (oogonia or spermatogonia) will differentiate, suggesting that the OSF-like may be potentially involved in sexual differentiation, besides other roles as a regulator in development.β-actin is a member of the actin family of genes, which play important roles in maintaining cytoskeletal structure, cell motility, cell division, intracellular movements and contractile processes. We report here the identification of a β-actin cDNA of the rice field eel, a teleost fish with a characteristic of natural sex reversal. The cDNA sequence of this gene was 1860 bp in length, which codes a 375 amino acid protein. Amino acid identities of the 6-actin between the rice field eel and other vertebrates including human, chicken, and other fishes, were more than 98%. Phylogenetic trees including all β-actin cds or 3' UTR of teleost fishes were constructed respectively, which indicate that teleost β-actin can be classified into four types, and the β-actin cloned from the rice field eel here is type II. RT-PCR showed expression of the rice field eel β-actin in testis, ovotestis, ovary, heart, liver, spleen and brain, suggesting ubiquitous expression pattern.BAC (bacterial artificial chromosome) library is a crucial tool for modern structural, functional, and comparative genomics analysis of large and complex-genome organisms. Here we try to construct a BAC library of rice field eel genome, which is only 600 Mb. This genome DNA was partially digested withenzyme BamH I, subjected to twice size selections, electroeluted and cloned into the pIndigoBAC-5 which is derived from pBeloBACll. Finally, we obtained about 3000 clones with an average insert size 110kb and 12% empty clones, which provides technique base for further construction of the BAC library of the rice field eel.