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BMC Mol Biol
2008 Jan 30;9:19. doi: 10.1186/1471-2199-9-19.
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Molecular characterization and expression analysis of five different elongation factor 1 alpha genes in the flatfish Senegalese sole (Solea senegalensis Kaup): differential gene expression and thyroid hormones dependence during metamorphosis.
Infante C
,
Asensio E
,
Cañavate JP
,
Manchado M
.
Abstract
Eukaryotic elongation factor 1 alpha (eEF1A) is one of the four subunits composing eukaryotic translation elongation factor 1. It catalyzes the binding of aminoacyl-tRNA to the A-site of the ribosome in a GTP-dependent manner during protein synthesis, although it also seems to play a role in other non-translational processes. Currently, little information is still available about its expression profile and regulation during flatfish metamorphosis. With regard to this, Senegalese sole (Solea senegalensis) is a commercially important flatfish in which eEF1A gene remains to be characterized. The development of large-scale genomics of Senegalese sole has facilitated the identification of five different eEF1A genes, referred to as SseEF1A1, SseEF1A2, SseEF1A3, SseEF1A4, and Sse42Sp50. Main characteristics and sequence identities with other fish and mammalian eEF1As are described. Phylogenetic and tissue expression analyses allowed for the identification of SseEF1A1 and SseEF1A2 as the Senegalese sole counterparts of mammalian eEF1A1 and eEF1A2, respectively, and of Sse42Sp50 as the ortholog of Xenopus laevis and teleost 42Sp50 gene. The other two elongation factors, SseEF1A3 and SseEF1A4, represent novel genes that are mainly expressed in gills and skin. The expression profile of the five genes was also studied during larval development, revealing different behaviours. To study the possible regulation of SseEF1A gene expressions by thyroid hormones (THs), larvae were exposed to the goitrogen thiourea (TU). TU-treated larvae exhibited lower SseEF1A4 mRNA levels than untreated controls at both 11 and 15 days after treatment, whereas transcripts of the other four genes remained relatively unchanged. Moreover, addition of exogenous T4 hormone to TU-treated larvae increased significantly the steady-state levels of SseEF1A4 with respect to untreated controls, demonstrating that its expression is up-regulated by THs. We have identified five different eEF1A genes in the Senegalese sole, referred to as SseEF1A1, SseEF1A2, SseEF1A3, SseEF1A4, and Sse42Sp50. The five genes exhibit different expression patterns in tissues and during larval development. TU and T4 treatments demonstrate that SseEF1A4 is up-regulated by THs, suggesting a role in the translational regulation of the factors involved in the dramatic changes that occurs during Senegalese sole metamorphosis.
Figure 1. Comparison of the primary structure of SseEF1As and various others eEF1A proteins (see Table 3). The alignment was performed using MegAlign software. Dots represent identity with SseEF1A1, and dashes represent gaps. G1 to G4 indicate the critical regions involved in GDP/GTP exchange and GTP hydrolysis. The consensus sequence composed of the three consensus elements GXXXXGK (G14-K20), DXXG (D91-G94), and NKXD (N153-D156) present in the GTP-binding domain is shaded in grey. The GTP-binding elongation factor signature corresponding to amino acids 61 to 76 is boxed.
Figure 2. Phylogenetic relationships among SseEF1As and a wide range of vertebrate eEF1As (see Table 3) using the maximum likelihood method. D. melanogaster and Artemia sp elongation factor 1 alpha proteins were used as outgroups to root tree. Only bootstrap values higher than 50% are indicated on each branch. The scale for branch length (0.1 substitutions/site) is shown below the tree.
Figure 3. Phylogenetic relationships among SseEF1As and a wide range of vertebrate eEF1As (see Table 3) using the neighbor-joining method. D. melanogaster and Artemia sp elongation factor 1 alpha proteins were used as outgroups to root tree. Only bootstrap values higher than 50% are indicated on each branch. The scale for branch length (0.1 substitutions/site) is shown below the tree.
Figure 4. A) Relative expression levels in tissues of the five SseEF1A genes. Expression values were normalized to those of Ubiquitin. Data were expressed as the mean fold change (mean ± SEM, n = 3) from the calibrator group (liver). Values marked with an asterisk are significantly different from liver values (P < 0.05). L: liver, Sp: spleen, I: intestine, St: stomach, Hk: head-kidney, G: gills, M: skeletal muscle, B: brain, H: heart, Sk: skin. B) Comparison of the relative levels of SseEF1A transcripts in tissues. Data are expressed as the ratio (calculated using 2-(ÎCt)) of target mRNA to Ubiquitin mRNA.
Figure 5. A) Relative SseEF1A expression levels during larval development (from 2 to 22 DPH) in Senegalese sole. Expression values were normalized to those of GAPDH2. Data are expressed as the mean fold change (mean ± SEM, n = 3) from the calibrator group (2 DPH). Different letters denote days that are significantly different (P < 0.05) analyzed by ANOVA followed by a Tukey test. The interval for the metamorphic process is shaded. B) Comparison of the relative levels of SseEF1A transcripts during larval development. Data are expressed as the ratio (calculated using 2-(ÎCt)) of target mRNA to GAPDH2 mRNA.
Figure 6. Relative SseEF1A expression levels as determined by real-time quantitative PCR in the untreated (grey) and TU-treated (black) groups. Untreated and TU-treated samples were collected for RNA isolation at four different times: 8 hours, and 6 days, 11 days, and 15 days after treatment starting on 7 DPH. Expression values were normalized to those of GAPDH2. Data are expressed as the mean fold change (mean ± SEM, n = 3) from the calibrator group (control 8 hours). Values marked with an asterisk are significantly different (P < 0.05 or less) from the corresponding untreated control group values.
Figure 7. Relative SseEF1A4 expression levels as determined by real-time quantitative PCR in the untreated control (grey), and in TU (black) and TU+T4 (white) treated groups. Larvae samples were collected for rRNA isolation at 8 and 13 days after T4 treatment starting 7 DPH. Expression values were normalized to those of GAPDH2. Data are expressed as the mean fold change (mean ± SEM, n = 3) from the calibrator group (untreated control day 8). Values marked with an asterisk are significantly different (P < 0.05 or less) from the corresponding untreated control group values.
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