Molecular cloning and expression analysis of the STAT1 gene from olive flounder, Paralichthys olivaceus

Abstract

Background: Signal transducer and activator of transcription 1 (STAT1) is a critical component of interferon (IFN)-alpha/beta and IFN-gamma signaling. Although seven isoforms of STAT proteins have been reported from mammals, limited information is available for the STAT genes in fish. We isolated complementary DNA with high similarity to mammalian STAT1 from the olive flounder, Paralichthys olivaceus.

Results: A DNA fragment containing the conserved SH2 domain was amplified by RT-PCR using degenerate primers designed based on the highly conserved sequences in the SH2 domains of the zebrafish and mammalian STAT1. The complete cDNA sequence was obtained by 5' and 3' RACE. The flounder STAT1 transcript consisted of 2,909 bp that encoded a polypeptide of 749 amino acids. The overall similarity between flounder STAT1 and other STATs was very high, with the highest amino acid sequence identity to snakehead (89%). Phylogenetic analyses reveal that flounder STAT1 is in the same monophyletic group with snakehead STAT1. Quantitative real time RT-PCR and in situ hybridization revealed that STAT1 was expressed in almost all examined organs and tissues, with high expression in gill, spleen, kidney, and heart. The accumulation of STAT1 mRNA in different developmental stages, as determined by real time RT-PCR, increased with development.

Conclusion: Recent cloning of various cytokine genes and the STAT1 gene of olive flounder here suggest that fish also use the highly specialized JAK-STAT pathway for cytokine signaling. Identification of other STAT genes will elucidate in detail the signal transduction system in this fish.

Figure 1

Cloning of flounder STAT1 cDNA. (A) Comparison of the domain structure of human STAT1 to zebrafish STAT1. ND, N-domain responsible for dimer-dimer interactions; COILED-COIL, coiled-coil domain responsible for protein-protein interactions; DNA, DNA-binding domain; SH2, src homology 2 domain responsible for receptor binding and dimerization. (B) The degenerative PCR primers used in the experiments. (C) PCR strategies used to clone flounder STAT1. The STAT5'-1 and STAT3' primers were used for first-round PCR, and the STAT5'-2 and STAT3' primers were used for nested PCR.

Figure 2

Amino acid sequence alignments of STAT1 proteins. Conserved amino acid residues are highlighted.

Figure 3

Phylogenetic tree of the STAT family transcription factors based on amino acid sequences. A phylogenetic tree of the aligned sequences was constructed using the neighbor-joining algorithm in MEGA (version 3.0). The confidence for each node was determined by bootstrap analysis (1000 repetitions).

Figure 4

Northern blot analysis of flounder STAT1. (A) Zebrafish mRNA was used as a positive control that showed STAT1 (upper band) and STAT3 (lower band) mRNA. (B) Flounder mRNA detected with a flounder STAT1 SH2 domain-specific probe.

Figure 5

Quantitative real-time PCR analysis of the STAT1 gene expression (A) Expression analysis of STAT1 mRNA at different developmental stages. EG, egg; LV, larva; 7D, 7 day post-hatch; 14D, 14 day post-hatch; 27D, 27 day post-hatch; 34D, 34 day post-hatch. (B) Expression of STAT1 mRNA in various tissues of the flounder. MS, muscle; LV, liver; IT, intestine; ST, stomach; KD, head kidney; SK, skin; FN, fin; SP, spleen; GI, gill; EY, eye; HR, heart. Data are averages from three replications with standard deviations.

Figure 6

In situ hybridization of flounder tissues to detect STAT1 mRNA. Flounder tissues fixed in neutral buffered formalin were probed with a SH2 domain-specific cRNA probe. The left panels in each tissue hybridized with the probe show blue color, which indicates the presence of the mRNA. Right panels in each tissue showing no blue region are the same tissue probed with a sense probe as a negative control.