Genomic structure, expression and characterization of a STAT5 homologue from pufferfish (Tetraodon fluviatilis)

Abstract

The STAT5 (signal transducer and activator of transcription 5) gene was isolated and characterized from a round-spotted pufferfish genomic library. This gene is composed of 19 exons spanning 11 kb. The full-length cDNA of Tetraodon fluviatilis STAT5 (TfSTAT5) contains 2461 bp and encodes a protein of 785 amino acid residues. From the amino acid sequence comparison, TfSTAT5 is most similar to mouse STAT5a and STAT5b with an overall identity of 76% and 78%, respectively, and has < 35% identity with other mammalian STATs. The exon/intron junctions of the TfSTAT5 gene were almost identical to those of mouse STAT5a and STAT5b genes, indicating that these genes are highly conserved at the levels of amino acid sequence and genomic structure. To understand better the biochemical properties of TfSTAT5, a chimeric STAT5 was generated by fusion of the kinase-catalytic domain of carp Janus kinase 1 (JAK1) to the C-terminal end of TfSTAT5. The fusion protein was expressed and tyrosine-phosphorylated by its kinase domain. The fusion protein exhibits specific DNAbinding and transactivation potential toward an artificial fish promoter as well as authentic mammalian promoters such as the b-casein promoter and cytokine inducible SH2 containing protein (CIS) promoter when expressed in both fish and mammalian cells. However, TfSTAT5 could not induce the transcription of b-casein promoter via rat prolactin and Nb2 prolactin receptor. To our knowledge, this is the first report describing detailed biochemical characterization of a STAT protein from fish.

Molecular characterization and IFN signal pathway analysis of Carassius auratus CaSTAT1 identified from the cultured cells in response to virus infection

“…2). However, three known fish STAT1 proteins, zebrafish STAT1 [14], rainbow trout STAT1 and pufferfish STAT1 [15], all have the complete TAD and include the site of serine phosphorylation (Fig. 2).…”

“…Among these domains, SH2 domain is the most characteristic region of STAT proteins and is identified as a modular unit that binds specifically to phosphotyrosine [13]. In addition to mammalian STATs, lower vertebrate STATs, such as zebrafish STAT1 and STAT3 [14] and pufferfish STAT5 [15], as well as invertebrate STATs in Drosophila [16,17], Anopheles gambiae [18], Caenorhabditis elegans [19] and even in Dictyostelium [20], have been identified. In fish, previous studies show that the zebrafish STAT1 protein can rescue interferon (IFN)-signaling functions in a STAT1-deficient human cell line [14].…”

Characterization of Two Mosquito STATs, AaSTAT and CtSTAT

“…Preparation of Whole Cell Lysates and Nuclear Extracts-Whole cell lysates were prepared as described previously (30). In brief, cells were washed twice with phosphate-buffered saline and lysed in the lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) containing 0.2 mM Na 3 VO 4 and 2 mM phenylmethylsulfonyl fluoride.…”

“…The sequences of upper strand of the normal and the corresponding mutant oligonucleotides are listed in Table 3 of Ref. 30. The probes were prepared by annealing the upper and lower strands of oligonucleotides, one of which was end labeled with [␥- Transactivation Assay-The plasmids used for transfections were purified by Qiagen plasmid purification kit (Qiagen).…”

“…AaSTAT-HA-cJH1 and CtSTAT-HA-cJH1 fusion proteins were generated by fusing the JH1 domain of the JAK from C. tritaeniorhynchus mosquito into the C-terminal end of AaSTAT and CtSTAT proteins, respectively, as described previously (30). In vitro transcription and translation products of AaSTAT-HA, AaSTAT-HA-cJH1, CtSTAT-HA, and CtSTAT-HA-cJH1 were all recognized by mAb against the HA tag, but only AaSTAT-HA-cJH1 and CtSTAT-HA-cJH1 were recognized by anti-pY99 mAb (Fig.…”