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ATP5O, NDUFA12 and UQCRH from Muscle Full-Length Cdna Library of Black-Boned Sheep

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ATP5O, NDUFA12 and UQCRH from Muscle Full-Length Cdna Library of Black-Boned Sheep Mol Biol Rep (2012) 39:5767–5774 DOI 10.1007/s11033-011-1387-9 Molecular characterization and tissue expression profile of three novel ovine genes: ATP5O, NDUFA12 and UQCRH from muscle full-length cDNA library of black-boned sheep R. S. Ye • H. B. Pan • G. F. Yin • Y. Huang • S. M. Zhao • S. Z. Gao Received: 9 October 2010 / Accepted: 17 December 2011 / Published online: 3 January 2012 Ó Springer Science+Business Media B.V. 2012 Abstract Three novel ovine genes were obtained from no strand in UQCRH. One potential signal peptide structure muscle full-length cDNA library of black-boned sheep. in ATP5O protein were found. NDUFA12 and UQCRH Sequence analysis revealed that nucleotide sequences of have the extremely low possibility of signal peptides. these genes were not homologous to any of the known sheep Meanwhile, RasMol was used for visualizing the PDB files or goat genes, but these genes have high similarity to ATP generated by Swiss-Model in cartoon or three-dimensional synthase subunit O (ATP5O), NADH dehydrogenase (ubi- format. ATP5O and UQCRH protein were modeled by quinone) 1 alpha subcomplex, 12 (NDUFA12) and ubi- Swiss-Model. Tissue expression profile indicated that the quinol-cytochrome c reductase hinge protein (UQCRH) ovine ATP5O, NDUFA12 and UQCRH genes could be genes of other mammal animals (accession number: expressed in all detected tissues including muscles, heart, FJ546085, FJ546078 and FJ546083). The alignment anal- liver, spleen, lung, kidney and adipose tissues, but the ysis showed that the ovine ATP5O, NDUFA12 and expression abundance of these genes were various in the UQCRH genes and proteins have closer genetic relation- different tissues. Our experiment supplied the primary ships with the ATP5O, NDUFA12 and UQCRH genes and foundation for further researches on these three ovine genes. proteins from cattle. Conserved domain prediction showed that these three genes included OSCP, NDUFA12 super- Keywords Sheep Á ATP5O Á NDUFA12 Á UQCRH Á family and UCR-hinge superfamily domains respectively. Structure predication Á Tissue expression The deduced sequence of ATP5O, NDUFA12 and UQCRH protein had 213, 145 and 91 amino acid residues, with a molecular weight of approximately 23419.66, 17089.50 and Introduction 10657.75 Da and a theoretical isoelectric point of 9.90, 9.68 and 4.45. The secondary structure prediction revealed that Construction of a full-length cDNA library is an effective 60% helix structure in ATP5O, 60% coils in NDUFA12 and way to understand the functional expression of genes in muscle tissue, and in addition, novel genes for further R. S. Ye and H. B. Pan have contributed equally to this work and research can be found in the library. Muscle full-length could be regarded as the first authors. cDNA library of black-bone sheep was constructed in our previous study [1]. In the subsequent study, many new Electronic supplementary material The online version of this ovine genes were founded by conducting large-scale article (doi:10.1007/s11033-011-1387-9) contains supplementary material, which is available to authorized users. sequencing of colonies. A novel gene PSAM6 of black- bone sheep was reported in 2009 [1]. Meanwhile, three R. S. Ye Á H. B. Pan Á G. F. Yin Á Y. Huang Á other novel ovine genes-ATP synthase subunit O (ATP5O), & & S. M. Zhao ( ) Á S. Z. Gao ( ) NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, Yunnan Key Laboratory of Animal Nutrition and Feed Science, Yunnan Agricultural University, Kunming 650201, China 12 (NDUFA12) and ubiquinol-cytochrome c reductase e-mail: [email protected] hinge protein (UQCRH) were isolated and characterized S. Z. Gao from the same muscle full-length cDNA library of black- e-mail: [email protected] boned sheep. 123 5768 Mol Biol Rep (2012) 39:5767–5774 Defects in mitochondrial biogenesis are the most fre- containing 10 units of MMLV reverse transcriptase (Pro- quent cause of mitochondrial disorders [2]. ATP5O, mega, Belgium), 1 mM dNTP mixture (Promega, Bel- NDUFA12 and UQCRH are nuclear encoded mitochondrial gium), 40 units of recombinant RNasin ribonuclease genes [3–5], which are involved in oxidative phosphoryla- inhibitor (Promega, Belgium) and 0.5 ll of oligo (dT) 18 tion (OXPHOS). OXPHOS is a process where electrons are (Promega, Belgium) in sterilized water and buffer supplied passed from NADH and FADH2 along a series of carrier by the manufacturer. After incubation at 42°C for 60 min, molecules [2, 6]. Protons are pumped across the inner the mixture was heat treated at 95°C for 5 min. cDNA mitochondrial membrane to produce a proton gradient and samples were kept in -20°C for Real-time PCR detection. in a final step ATP is produced from ADP and phosphate. This process occurs in the respiratory chain, which consists Sequence analysis of five multiprotein complexes [7]. ATP5O is a nuclear encoded subunit of complex V of the respiratory chain and The cDNA sequence prediction was conducted using Gen- influences the transmission of conformational changes and Scan software (http://genes.mit.edu/GENSCAN.html). The proton conductance [3]. NDUFA12 encodes an accessory protein prediction and analysis were performed using the subunit of complex I which is required either at a late step in Conserved Domain Architecture Retrieval Tool of BLAST the assembly of complex I, or in the stability of complex I at the National Center for Biotechnology Information [4]. The gene coding for mitochondrial hinge protein (NCBI) server(http://blast.ncbi.nlm.nih.gov/), the ClustalW UQCRH gene is a subunit of complex III [5]. software (http://www.ebi.ac.uk/Tools/clustalw2/index.html) In the present experiment, we will analyze the coding and DNAstar software. sequences of ovine ATP5O, NDUFA12 and UQCRH genes Isoelectric point and molecular weight (pI/Mw) of ovine basing on the conserve sequence information of human, three novel gene-coding proteins (ATP5O, NDUFA12 and mouse or cattle, compared the homology of ovine EST UQCRH) was analyzed in compute pI/Mw (http://web. sequence information with coding sequences of these three expasy.org/compute_pi/)[8]. Secondary structure of these genes of cattle or other mammals, subsequently perform proteins was predicted using PSIPRED (http://bioinf.cs.ucl. some necessary protein physical and chemical character- ac.uk/psipred/)[9]. DNAstar software was employed to istics as well as structure predication of deduced proteins, predict these proteins’ physiology characteristics which and finally detect the gene expression distribution of these include hydrophobicity, amphilicity, flexibility and genes in different tissues. The study will establish the antigenecity. primary foundation for understanding these three ovine Signal peptides prediction (neural networks signal pep- genes. tides prediction, signalP-NN and hidden Markov model signal peptides prediction, signalP-HMM) was accom- plished by SignalP 3.0 Server (http://www.cbs.dtu.dk/ Materials and methods services/SignalP/)[10]. Transmembrane helices prediction was conducted in TMpred analysis (http://www.ch.embnet. Sample preparation, total RNA extraction and reverse org/software/TMPRED_form.html)[11]. Swiss-Model (http:// transcript swissmodel.expasy.org/) was employed to homology mod- eling of three ovine proteins, and RasMol V2.7.5 was used to Twelve black-bone sheep (six females and six males) at the visualize the PDB files generated by Swiss-Model [12]. age of 12 weeks were slaughtered, respectively. The lon- gissimus dorsi muscles and other tissue samples including heart, liver, spleen, lung, kidney and adipose tissue of all Tissue expression profile animals were collected. Total RNA was extracted from all the samples using the Total RNA Extraction Kit (Invitro- Twelve samples of each tissue in black-boned sheep were gen, America) as per the manufacturer’s instructions. Total used. Real-time PCR was performed using the IQTM RNA concentration was then quantified by measuring the SYBR Green Supermix (Bio-Rad) following the manu- absorbance at 260 nm in a photometer (Eppendorf Bio- facturer’s instructions. The 18S rRNA (195 bp) gene was photometer). Ratios of absorption (260/280 nm) of all regarded as an internal control. The specific primers were: preparations were between 1.8 and 2.0. Aliquots of RNA 18S rRNA, 50-GCGGCTTTGGTGACTCTA-30 (forward samples were subjected to electrophoresis through a 1.4% primer) and 50-CTGCCTCCTTGGATGTG-30 (reverse agarose formaldehyde gel to verify their integrity. RNA primer); ATP5O gene, 50-GGTGATTTCCGCATTT-30 was stored at -80°C prior to use. (forward primer) and 50-TTCAGTCAGAGTGGCTTC-30 Reverse transcription was performed using the RNA (reverse primer); NDUFA12 gene, 50-GCAAATGATGT (2 lg) described above in a final volume of 25 ll GAGGGTT-30 (forward primer) and 50-TTACGAGCAG 123 Mol Biol Rep (2012) 39:5767–5774 5769 TAGGTGGTT-30 (reverse primer); UQCRH gene, 50-AG cattle (100%), human (89%) and mouse (90%). The ovine GACGAGCAAAGGATG-30 (forward primer) and 50-GC UQCRH protein has high homology with UQCRH protein CCAGGTGATGAAGG-30 (reverse primer). Conditions for from cattle (98%), human (94%) and mouse (93%).The cycling were 94°C for 4 min, followed by 40 cycles of ovine ATP5O, NDUFA12 and UQCRH have common denaturation at 94°C for 1 min; annealing at 50°C for conserved domains with highly homologous proteins from 1 min for ATP5O gene; 61°C for 1 min for NDUFA12 other mammals (Table 1; Fig. S2 in Supplementary gene; 60°C for 1 min for UQCRH gene; 55°C for 1 min for material). 18S rRNA gene; extension at 72°C for 1 min; and fluo- From the results obtained above, it can be concluded rescence reading at 82°C for 10 s. The results were that these genes belong to the sheep-ATP5O, NDUFA12 expressed as fold changes described previously by Zhao and UQCRH genes. Based on the results of the alignment et al. [13]. of ATP5O, NDUFA12 and UQCRH proteins in four ani- mals, phylogenetic trees were constructed using the DNAstar software (Fig.
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