Characterization of transactivation domain and developmental expression of pituitary specific transcription factor, Pit-1 of ayu (Plecoglossus altivelis).

Pit-1 is a pituitary-specific transcription factor, which regulates the expression of growth hormone, prolactin, and thyroid stimulating hormone-beta genes. We previously reported the expression of a Pit-1 gene from ayu (Plecoglossus altivelis), which is an important cultivated food fish in Taiwan and Japan. Comparison of ayu Pit-1 with that of salmon, turkey, and rodent, revealed that the Pit-1 structure is highly conserved through vertebrates, especially in POU-specific and POU-homeo domains. The variation among fish, bird, and mammal are mainly found in transactivation domain by alternative splicing and initiation. Three insertions were found. The gamma-insert in fish Pit-1 is homologous to the exon 2a of avian Pit-1, which is not found in mammals. The beta-insert of fish Pit-1 is homologous to the 28 amino acids (a.a.) and 26 a.a. insert of avian Pit-1 beta(*) and mammalian Pit-1 beta, respectively. An additional similarity was noticed between fish and bird, as both of them contain 7 a.a. insert that is not present in mammalian Pit-1. By site directed mutagenesis, we demonstrated that the beta, gamma, and the 7 a.a. inserts of ayu Pit-1 are critical for activation of zebrafish growth hormone promoter. The ayu Pit-1 protein was found to be expressed specifically in pituitary gland, and its mRNA was first detected at embryonic day 4, significantly increased at embryonic day 5, then sustained to time of hatching at day 8.

Expression of ayu (Plecoglossus altivelis) Pit-1 in Escherichia coli: its purification and immunohistochemical detection using monoclonal antibody.

The pituitary-specific transcription factor Pit-1 belongs to the family of POU-domain proteins and is known to play an important role in the differentiation of pituitary cells. Here we report the complete nucleotide sequence of cDNA encoding Pit-1 from the brackish water fish, ayu (Plecoglossus altivelis). Nucleotide sequence analysis of 1910 bp of ayu Pit-1 cDNA revealed an open reading frame of 1074 bp that encodes a protein of 358 amino acids containing a POU-specific domain, POU homeodomain, and an STA (Ser/Thr-rich activation) transactivation domain. We inserted the coding region of Pit-1 cDNA, obtained by PCR, into a pET-20b(+) plasmid to produce recombinant Pit-1 in Escherichia coli BL21 (DE3) pLysS cells. Upon induction with isopropyl beta-D-thiogalactopyranoside, Pit-1 was expressed and accumulated as inclusion bodies in E. coli. The protein was then purified in one step by affinity chromatography on a nickel-nitrilotriacetic acid agarose column under denaturing conditions. This method yielded 0.7 mg of highly pure and stable protein per 200 ml of bacterial culture. A band of 40 kDa, resolved as recombinant ayu Pit-1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, agrees well with the molecular mass calculated from the translated cDNA sequence. The purified recombinant Pit-1 was confirmed in vitro through Western blot analysis, using its monoclonal antibody. This monoclonal antibody detected Pit-1 in the nuclei of ayu developing pituitary by immunohistochemical reaction. It serves as a good reagent for the detection of ayu Pit-1 in situ.