Yin Y et al. (2011), Molecular cloning and gene expression analysis ...

XB-ART-43453

PLoS One 2011 Jan 01;66:e20929. doi: 10.1371/journal.pone.0020929.

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Molecular cloning and gene expression analysis of Ercc6l in Sika deer (Cervus nippon hortulorum).

Yin Y , Tang L , Zhang J , Tang B , Li Z .

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One important protein family that functions in nucleotide excision repair (NER) factors is the SNF2 family. A newly identified mouse ERCC6-like gene, Ercc6l (excision repair cross-complementing rodent repair deficiency, complementation group 6-like), has been shown to be another developmentally related member of the SNF2 family.In this study, Sika deer Ercc6l cDNA was first cloned and then sequenced. The full-length cDNA of the Sika deer Ercc6l gene is 4197 bp and contains a 3732 bp open reading frame that encodes a putative protein of 1243 amino acids. The similarity of Sika deer Ercc6l to Bos taurus Ercc6l is 94.05% at the amino acid sequence level. The similarity, however, is reduced to 68.42-82.21% when compared to Ercc6l orthologs in other mammals and to less than 50% compared to orthologs in Gallus gallus and Xenopus. Additionally, the expression of Ercc6l mRNA was investigated in the organs of fetal and adult Sika deer (FSD and ASD, respectively) by quantitative RT-PCR. The common expression level of Ercc6l mRNA in the heart, liver, spleen, lung, kidney, and stomach from six different developmental stages of 18 Sika deer were examined, though the expression levels in each organ varied among individual Sika deer. During development, there was a slight trend toward decreased Ercc61 mRNA expression. The highest Ercc6l expression levels were seen at 3 months old in every organ and showed the highest level of detection in the spleen of FSD. The lowest Ercc6l expression levels were seen at 3 years old.We are the first to successfully clone Sika deer Ercc6l mRNA. Ercc6l transcript is present in almost every organ. During Sika deer development, there is a slight trend toward decreased Ercc61 mRNA expression. It is possible that Ercc6l has other roles in embryonic development and in maintaining the growth of animals.

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Species referenced: Xenopus
Genes referenced: actl6a ercc6 ercc6l smarca4

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	Figure 1. The cDNA sequences of Sika deer were obtained from heart mRNA using 5′ RACE and 3′ RACE.(A) Products obtained during the first 3′ RACE were 446 bp in length. (B) Products obtained during the second 3′ RACE were 445 bp in length. (C) Products obtained during a third 3′ RACE were 546 bp in length. (D) 5′ RACE products were 444 bp in length.
	Figure 2. Nucleotide and deduced amino acid sequences of Ercc6l.The ORF is presented in upper case, whereas the 5′-UTR and 3′-UTR sequences are presented in lower case. The putative amino acid sequence (total 1243 a.a.) is shown under the triplet codon. The stop codon is marked by an transverse line.
	Figure 3. ClustalX alignment of the Ercc6l protein sequence with other members of SNF2 family as reported in other mammals.The conserved amino acid residues in these sequences are shaded to show homology.
	Figure 4. A phylogenic tree showing the similarity between Sika deer Ercc6l protein amino acid sequences and those of cattle, panda, dog, horse, fowl, human, monkey, mouse, pongo, rat, xenopus and marmoset.The number at each node indicates the percentage of bootstrapping after 1000 replications. The bar at the bottom indicates 5% amino acid divergence in the sequences. These proteins are highly conserved throughout evolution; high similarity matches exist between Ercc6l sequences across species.
	Figure 5. Organs distribution of fetal Sika deer (labeled FSD) and adult Sika deer Ercc6l (labeled ASD).mRNAs in the Sika deer were detected by RT-PCR. (a) After 25 cycles, FSD was detected in the heart (H), liver (L), spleen (S), lung (LU), kidney (K), stomach (ST), brain (B), intestines (I), ear (E), lymph (LY), leg (LE) and negative (N); (b) FSD and ASD were detected in organs such as heart (H), liver (L), spleen (S), lung (LU), kidney (K), stomach (ST) and negative (N).
	Figure 6. Organs distribution of the Sika deer Ercc6l transcript as measured by SYBR Green RT-PCR.Quantitative analysis was performed on Ercc6l gene expression relative to β-actin in different Organs and different developmental stages. Ercc6l gene expression was analyzed in the following Organs: heart, liver, spleen, lung, kidney and stomach. Expression data for each Organ were analyzed from three individual Sika deer. Vertical bars represent the mean±S.E.

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