Elongation Factor ELL (Eleven-Nineteen Lysine-rich Leukemia) Acts as a Transcription Factor for Direct Thrombospondin-1 Regulation

The eleven-nineteen lysine-rich leukemia (ELL) gene undergoes translocation and fuses in-frame to the multiple lineage leukemia gene in a substantial proportion of patients suffering from acute forms of leukemia. Studies show that ELL indirectly modulates transcription by serving as a regulator for transcriptional elongation as well as for p53, U19/Eaf2, and steroid receptor activities. Our in vitro and in vivo data demonstrate that ELL could also serve as a transcriptional factor to directly induce transcription of the thrombospondin-1 (TSP-1) gene. Experiments using ELL deletion mutants established that full-length ELL is required for the TSP-1 up-regulation and that the trans-activation domain likely resides in the carboxyl terminus. Moreover, the DNA binding domain may localize to the first 45 amino acids of ELL. Not surprisingly, multiple lineage leukemia-ELL, which lacks these amino acids, did not induce expression from the TSP-1 promoter. In addition, the ELL core-response element appears to localize in the -1426 to -1418 region of the TSP-1 promoter. Finally, studies using zebrafish confirmed that ELL regulates TSP-1 mRNA expression in vivo, and ELL could inhibit zebrafish vasculogenesis, at least in part, through up-regulating TSP-1. Given the importance of TSP-1 as an anti-angiogenic protein, our findings may have important ramifications for better understanding cancer.

Toll-like receptor 4 signaling pathway can be triggered by grass carp reovirus and Aeromonas hydrophila infection in rare minnow Gobiocypris rarus

Toll-like receptor 4 (TLR4) is critical for LPS recognition and cellular responses. It also recognizes some viral envelope proteins. Detection mostly results in the inflammation rather than specific antiviral responses. However, it's unclear in fish. In this report, a TLR4 gene (named as GrTLR4b) was cloned and characterized from rare minnow Gobiocypris rarus. The full length of GrTLR4b cDNA consists of 2766 nucleotides and encodes a polypeptide of 818 amino acids with an estimated molecular mass of 94,518 Da and a predicted isoelectric point of 8.41. The predicted amino acid sequence comprises a signal peptide, six leucine-rich repeat (LRR) motifs, one leucine-rich repeat C-terminal (LRRCT) motif, followed by a transmembrane segment of 23 amino acids, and a cytoplasmic region of 167 amino acids containing one Toll - interleukin 1 - receptor (TIR) motif. It's closely similar to the zebrafish (Danio rerio) TLR4b amino acid sequence with an identity of 77%. Quantitative RT-PCR analysis showed GrTLR4b mRNA was constitutive expression in gill, heart, intestine, kidney, liver, muscle and spleen tissues in healthy animals and up-regulated by viruses and bacteria. After being infected by grass carp reovirus or Aeromonas hydrophila, GrTLR4b expressions were up-regulated from 24 h post-injection and lasted until the fish became moribund (P < 0.05). These data implied that TLR4 signaling pathway could be activated by both viral and bacterial infection in rare minnow. (C) 2009 Elsevier Ltd. All rights reserved.

Enhanced grass carp reovirus resistance of Mx-transgenic rare minnow (Gobiocypris rarus)

In the interferon-induced antiviral mechanisms, the Mx pathway is one of the most powerful. Mx proteins have direct antiviral activity and inhibit a wide range of viruses by blocking an early stage of the viral genome replication cycle. However, antiviral activity of piscine Mx remains unclear in vivo. In the present study, an Mx-like gene was cloned, characterized and gene-transferred in rare minnow Gobiocypris rarus, and its antiviral activity was confirmed in vivo. The full length of the rare minnow Mx-like cDNA is 2241 bp in length and encodes a polypeptide of 625 amino acids with an estimated molecular mass of 70.928 kDa and a predicted isoelectric point of 7.33. Analysis of the deduced amino acid sequence indicated that the mature peptide contains an amino-terminal tripartite GTP-binding motif, a dynamin family signature sequence, a GTPase effector domain and two carboxy-terminal leucine zipper motifs, and is the most similar to the crucian carp (Carassius auratus) Mx3 sequence with an identity of 89%. Both P0 and F1 generations of Mx-transgenic rare minnow demonstrated very significantly high survival rate to GCRV infection (P < 0.01). The mRNA expression of Mx gene was consistent with survival rate in F1 generation. The virus yield was also concurrent with survival time using electron microscope technology. Rare minnow has Mx gene(s) of its own but introducing more Mx gene improves their resistance to GCRV. Mx-transgenic rare minnow might contribute to control the GCRV diseases. (C) 2008 Published by Elsevier Ltd.

Identification of a novel gene K23 over-expressed in fish cross-subfamily cloned embryos

A novel gene-K23, differentially expressed in cross-subfamily cloned embryos, was isolated by RACE-PCR technique. It had 2580 base pairs (bp) in length, with a 1,425 bp open reading frame (ORF) encoding a putative protein of 474 amino acids (aa). Bioinformatic analysis indicated that K23 had 22 phosphorylation sites, but it had no signal peptides. Developmental expression analysis in zebrafish showed that K23 transcripts were maternally expressed in ovum and the amount of K23 transcripts increased gradually from zygote to pharyngula period. Subcellular localization analysis revealed that K23 protein was homogeneously distributed both in nuclei and cytoplasm. Taken together, our findings indicate that K23 gene is a novel gene differentially expressed in fish cross-subfamily cloned embryos.

Molecular characterization and expression profiles in response to bacterial infection of Chinese soft-shelled turtle interleukin-8 (IL-8), the first reptilian chemokine gene

In this study, an IL-8 homologue has been cloned and identified from a reptile, Chinese soft-shelled turtle for the first time. The full-length cDNA of turtle IL-8 was 1188 bp and contained a 312 bp open reading frame (ORF) coding for a protein of 104 amino acids. The chemokine CXC domain, which contained Glu-Leu-Arg (ELR) motif and four cysteine residues, was well conserved in turtle IL-8. The 4924 bp genomic DNA of turtle IL-8 contained four exons and three introns. Phylogenetic analysis showed that the amino acid sequence of turtle IL-8 clustered together with birds. RT-PCR analysis showed that turtle IL-8 mRNA was constitutively expressed liver, spleen, kidney, heart, blood and intestine tissues of control turtles. Real-time quantitative PCR analysis further indicated that the turtle IL-8 mRNA expression was apparent in various tissues at 8 h and up-regulated significantly during 8 h-7 d after Aeromonas hydrophila infection. The present studies will help us to understand the evolution of IL-8 molecule and the inflammatory response mechanism in reptiles. (C) 2009 Elsevier Ltd. All rights reserved.