碩士 / 國立臺灣大學 / 生化科學研究所 / 89 / One STAT homologue from zebrafish (Danio rerio ) has been isolated. The full-length cDNA of zebrafish STAT5 ( z-ST5 ) contains 2,844 bp and encodes a protein of 785 amino acids. From the amino acid sequence comparison, the z-ST5 is much closer to human STAT5 with an overall identity of 77% and 76% to human-STAT5b ( h-ST5b ) and human-STAT5a ( h-ST5a ), respectively.
Since there is no cytokine, interleukins, or growth factors available, at present, we decided to construct a constitutively active variant of z-ST5. To obtain this variant, we constructed the z-ST5, which has been demonstrated to be autophosphorylated, with the JH1 domain of carp JAK1. Using anti-phosphotyrosine mAb PY-99, z-STAT5-HA-JH1, but not z-STAT5-HA, was demonstrated to be tyrosine-phosphorylated.
The phophorylated z-STAT5-HA-JH1 was further used to investigate the DNA binding properties. As expected, z-STAT5-HA-JH1 exhibited high affinity binding activity with STAT5 consensus binding site, which was identified in the promoter of mammalian ß-casein gene and with Int16 binding site that was located in the intron 16 of puffer fish JAK1 gene. In addition, z-STAT5-HA-JH1 also exhibited moderate binding activity with ST4 probe, but no binding activity with other ST1, ST3, ST6, GAS, and SIE probes.
To determine the transactivation activity of z-STAT5-HA-JH1, mammalian COS-1 cells and carp CF cells were transiently cotransfected with a reporter plasmid containing the CAT gene driven by ß-casein gene promoter and activator z-STAT5-HA-JH1. z-STAT5-HA-JH1 shows the moderate, transcriptional activity toward ß-casein gene promoter in both mammalian cells and fish cells.
| Identifer | oai:union.ndltd.org:TW/089NTU00103008 |
| Date | January 2001 |
| Creators | Pei-Shan Li, 李佩珊 |
| Contributors | Chang-Jen Huang, 黃銓珍 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 0 |
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