In this study, the full-length cDNA sequence of the p8 gene was cloned from Atlantic halibut (H. hippoglossus). It contained an ORF of 231 nucleotides encoding a putative protein http://www.cusabio.com/catalog-13-1.html of 76 amino acids. The Atlantic halibut p8 protein, consistent with other p8 proteins, has no signal peptide or transmembrane regions but contains a bHLH motif and a nuclear localization signal motif. Comparison of the full-length and bHLH domains indicates that only the bHLH domain is well conserved among all the species (Table 1; Fig. 2). The amino-terminal region was more variable than the bHLH domain. The p8 amino acid sequence from vertebrates was longer than p8 from invertebrates. The longer amino terminal residues of the p8 protein from Atlantic halibut and other vertebrates are enriched in proline (P), glutamate (E), serine (S), and threonine (T), dubbed PEST domains, which are thought to promote ubiquitin-dependent protein degradation by proteasomes (Stuart et al. 1999; Goruppi and Kyriakis 2004).
Most nucleoprotein functions are regulated by phosphorylation/dephosphoryla- tion. Human p8 is a phosphoprotein, with DNA-binding activity enhanced by phosphorylation (Encinar et al. 2001). The primary results obtained by incubating COS-7 cells with P32 indicate that human p8 is indeed a phosphoprotein, but the sites of phosphorylation and the kinases involved remain to be determined (Vasseur et al. 1999a). Several predicted targets for phosphorylation of conserved serine, threonine, and tyrosine residues were distributed in the full-length p8 sequence (Fig. 2). Despite a relatively low level of conservation in these regions (except the bHLH region) of the p8 protein, these phosphorylation sites are conserved in all the species, consistent with functional importance. These observations are compatible with a role in some phosphorylation/dephosphorylation signaling pathways involving translocation to the nucleus and speci?c binding to DNA (Mallo et al. 1997).
In humans, p8 mRNA is highly expressed in liver, prostate, pancreas, colon, ovary, spinal cord, thyroid, trachea, and adrenal gland, with moderate expression in heart, lung, kidney, testis, placenta, skeletal muscle, small intestine, stomach, and lymph node, and low expression in brain, bone marrow, thymus, and spleen. It remained undetectable in peripheral blood leukocytes (Vasseur et al. 1999a). In the common sea urchin, it was reported that p8 was shown to have the strongest expression in intestine (Wang et al. 2009). Recently, in amphioxus, p8 was detected in muscle, gill, foregut including hepatic caecum, hindgut, ovary, testis, and notochord, with the highest expression in gut, gill, and ovary. In this study, the Atlantic halibut p8 gene was expressed in 9 of the 12 tissues tested. This p8 transcript is constitutively expressed in the pancreas, intestine, stomach, gill, head kidney, heart, liver, ovary, and spleen, whereas its expression remained undetectable in brain, muscle, and skin (Fig. 3), suggesting that p8 has a nonubiquitary and nonvital function under physiological conditions. Expression in Atlantic halibut and human or rat p8 correlates well for all tissues, except for the pancreas in the rat. Whereas normal rat pancreas expresses low levels of p8 mRNA, human and Atlantic halibut pancreas seem to express high levels (Mallo et al. 1997; Vasseur et al. 1999a). Rat p8 mRNA was discovered because of its strong activation in the pancreas during the acute phase of pancreatitis, but activation of p8 mRNA expression is not restricted to the pancreas with acute pancreatitis (Mallo et al. 1997). In fact, p8 mRNA is strongly activated in response to injury (such as pancreatitis in the pancreas, transient ischemic episode in the brain) or during development, indicating that p8 mRNA overexpression may be part of a ubiquitous defense program against cellular injury or cellular stress (Vasseur et al. 1999a). In these cases, we can speculate that p8 gene expression is regulated through a very complex system.
MicroRNAs are small noncoding RNA. They play a role in gene expression regulation by inhibiting translation of their target mRNAs (Bartel 2004; Calin and Croce 2006). Their target predictions show that three human miRNAs (hsa-miR-1, hsa-miR-206, and hsa-miR-613) were found to have the same target site in the 30UTR of Atlantic halibut p8. Further investigation is needed to con?rm whether corresponding miRNA molecules exist in Atlantic halibut.
In summary, we succeeded in obtaining the full-length p8 cDNA from Atlantic halibut. Atlantic halibut p8 had the strongest expression in the pancreas, intestine, and stomach; intermediate in the gill, head kidney, heart, liver, and ovary; and low in the spleen. No signal was detected in the brain, muscle, and skin. The topology of the dendrogram of the p8 gene http://www.cusabio.com/catalog-15-1.html is basically in agreement with the classic taxonomic structure of the animal kingdom. Furthermore, three human miRNA were found to have the same target site in the 30UTR of Atlantic halibut p8 by theoretical prediction.
