Lee RM et al. (2011), Isolation and Expression Profile of the Ca-Acti...

XB-ART-43686

Lab Anim Res 2011 Jun 01;272:109-16. doi: 10.5625/lar.2011.27.2.109.

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Isolation and Expression Profile of the Ca-Activated Chloride Channel-like Membrane Protein 6 Gene in Xenopus laevis.

Lee RM , Ryu RH , Jeong SW , Oh SJ , Huang H , Han JS , Lee CH , Lee CJ , Jan LY , Jeong SM .

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To clone the first anion channel from Xenopus laevis (X. laevis), we isolated a calcium-activated chloride channel (CLCA)-like membrane protein 6 gene (CMP6) in X. laevis. As a first step in gene isolation, an expressed sequence tags database was screened to find the partial cDNA fragment. A putative partial cDNA sequence was obtained by comparison with rat CLCAs identified in our laboratory. First stranded cDNA was synthesized by reverse transcription polymerase-chain reaction (RT-PCR) using a specific primer designed for the target cDNA. Repeating the 5' and 3' rapid amplification of cDNA ends, full-length cDNA was constructed from the cDNA pool. The full-length CMP6 cDNA completed via 5'- and 3'-RACE was 2,940 bp long and had an open reading frame (ORF) of 940 amino acids. The predicted 940 polypeptides have four major transmembrane domains and showed about 50% identity with that of rat brain CLCAs in our previously published data. Semi-quantification analysis revealed that CMP6 was most abundantly expressed in small intestine, colon and liver. However, all tissues except small intestine, colon and liver had undetectable levels. This result became more credible after we did real-time PCR quantification for the target gene. In view of all CLCA studies focused on human or murine channels, this finding suggests a hypothetical protein as an ion channel, an X. laevis CLCA.

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Species referenced: Xenopus laevis
Genes referenced: actl6a camp clca1.1 clca1.2 clca1.3 clca2 clca3p clca4.1 clca4.2 cyp26a1

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	Figure 1. CMP6 cDNA isolation. (A) Sequence alignment of DC066238. Amino acid (aa) alignment was deduced by querying the DC066238 amino acid sequence using GENETYX. The amino acid homology is indicated as follows: identical (asterisk), very similar (double dot) and similar (dot). (B) The strategy is shown for generating full-length cDNA. First strand cDNA (dotted) and PCR products (thick) are shown with black lines. CMP6: 5'-RACE and 3'-RACE products represent Round 1 and Round 2, respectively. 3'-RACE products are shown in panel (C) lane 1, and 5'-RACE PCR products in (D) lane 1. PCR products were electrophoresed in a 1% agarose gel. Black arrowheads on panels (C) and (D) indicate the 1,700 and 1,296 bp of target products of CMP6. Molecular weights are shown for 1 kb Plus size markers (G&P, Anyang, Korea).
	Figure 2. Comparison of X. laevis CMP6 primary structure. (A) CMP6 amino acids were aligned by querying with rat CLCAs on CLUSTAL W. The homology of amino acids of CMP6 is indicated as identical (asterisk) very similar (double dot) and similar (dot). Consensus sites are marked as follows: N-linked glycosylation (●), N-myristoylation sites (▾), phosphorylation by PKC (#), by casein kinase II (◇) and by cAMP-dependent protein kinase (+). The predicted signal sequence (SS) and the hydrophobic carboxyl terminus (H) are overlined. An arrow indicates the predicted cleavage site. (B) Hydropathy analysis. The hydropathy plot was analyzed by the Kyte-Doolittle method. Hydrophobic domains are given as positive values. The proposed hydrophobic domains are four segments.
	Figure 3. Tissue distribution of CMP6 expression in X. laevis. (A) RT-PCR products in various tissues. The amplified PCR products were electrophoresed in a 2% agarose gel. The specific RT-PCR products are a 252 bp CMP6 (C415 and C416) and a 159 bp β-actin gene as an internal control (XHRMbA1 and xbA3). Expression ratios of CMP6 are shown as relative calculated values for β-actin. (B) CMP6 showed high expression levels in small intestine intestine (sm int) and colon. Each graph represents at least three independent experiments. The bars shown in the graph are mean±SD. *P<0.05 compared to control group.
	Figure 4. CMP6 quantification by real-time PCR. Relative quantification is shown as a bar graph. The relative CMP6 expression ratios for β-actin are shown. This result confirms the idea of CMP6 being largely expressed in liver, small intestine (sm int), and colon. The graph represents three independent experiments with each bar as mean±SD. *P<0.05 compared to control group.

References [+] :

Agnel, Identification of three novel members of the calcium-dependent chloride channel (CaCC) family predominantly expressed in the digestive tract and trachea. 1999, Pubmed