| Summary: | 博士 === 國立清華大學 === 生命科學系 === 90 === 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 and regulates the expression of growth hormone, prolactin and thyroid stimulating hormone- genes. The complete nucleotide sequence of cDNA encoding Pit-1 from amphidromous water fish, ayu (Plecoglossus altivelis) is reported here. Nucleotide sequence analysis of the 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-homeo domain and a STA (Ser/Thr-rich Activation) transactivation domain. Insertion of the coding region of Pit-1 cDNA, obtained by PCR, into a pET-20b(+) plasmid produce recombinant Pit-1 in E. coli BL21 (DE3) pLysS cells. Upon induction with isopropyl β-D-thiogalactopyranoside, Pit-1 was expressed and accumulated as inclusion bodies in E. coli. The protein was then purified in one-step purification 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 kilodalton, resolved in 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. 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 -insert in fish Pit-1 is homologous to the exon 2a of avian Pit-1, which is not found in mammals. The -insert of fish Pit-1 is homologous to the 28 amino acids (a.a.) and 26 a.a. insert of avian Pit-1* and mammalian Pit-1, 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, I demonstrated that the and the 7 a.a. inserts of ayu Pit-1 are critical for activation of zebrafish growth hormone promoter. The tissue specificity and expression pattern of ayu Pit-1 is also interesting. The tissue specific expression of Pit-1 was detected in pituitary gland of adult ayu by Western blot analysis. To study the expression pattern of Pit-1 during development, ayu embryos from fertilization (day 1) through hatching (day 8) were collected daily for Western blot analysis and RT-PCR. Pit-1 transcripts could be detected as early as embryonic day 4, and significantly increased at embryonic day 5, then sustained to the time of hatching at day 8.
|
|---|
