Abstract: The complete coding sequences of three sheep genes-ARF3, ARF4 and ARF5 were amplified using the Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). The nucleotide sequences of these three genes revealed that sheep ARF3 gene encodes a protein of 181 amino acids that shares high homology with the ADP-Ribosylation Factor 3 (ARF3) proteins of five species human (99%), zebrafish (99%), African clawed frog (99%), pig (99%) and atlantic salmon (98%). The sheep ARF4 gene encodes a protein of 180 amino acids that shares high homology with the ADP-Ribosylation Factor 4 (ARF4) proteins of third species-cattle (100%), platypus (99%), green anole (99%), pig (99%), rabbit (98%), African clawed frog (98%), red jungle fowl (98%), mouse (98%), zebrafish (96%), human (97%), atlantic salmon (92%), rainbow trout (92%) and Caligus rogercresseyi (89%). The sheep ARF5 gene encodes a protein of 180 amino acids that shares high homology with the ADP-Ribosylation Factor 5 (ARF5) proteins of seven species human (100%), mouse (100%), rat (100%), gray short-tailed opossum (99%), chicken (98%), channel catfish (93%) and zebrafish (93%). Finally, these three novel sheep genes were assigned to GeneIDs; 100302303, 100302304 and 100302305. The phylogenetic analysis indicated that the sheep ARF3 gene has closer genetic relationship with the ARF3 gene of human. The sheep ARF4 gene has closer genetic relationship with the ARF4 gene of cattle and the sheep ARF5 gene has closer genetic relationship with the ARF5 genes of human, mouse and rat. Tissue expression profile analysis was also carried out and results demonstrated that sheep ARF3, ARF4 and ARF5 genes were differentially expressed in detected tissues.

INTRODUCTION

ADP-Ribosylation Factor 3 (ARF3), ADP-Ribosylation Factor 4 (ARF4) and ADP-Ribosylation Factor 3 (ARF5) are three members of the ARF gene family. These genes encode small guanine nucleotide-binding proteins that stimulate the ADP-ribosyltransferase activity of cholera toxin and play a role in vesicular trafficking and as activators of phospholipase D. The gene products include 6 ARF proteins and 11 ARF-like proteins and constitute 1 family of the RAS superfamily. The ARF proteins are categorized as class 1 (ARF1-ARF3), class 2 (ARF4 and ARF5) and class III (ARF6) and members of each class share a common gene organization (Bailey et al., 2010; Manolea et al., 2010; Woo et al., 2009; Kimura et al., 2006; Kim et al., 2003; Shiba et al., 2006; Brandenberger et al., 2004).

However, latest studies have shown that ARF3 plays a unique function at the Trans-Golgi Network (TGN) that likely involves recruitment by a specific receptor and is associated with protein-protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration (Lim et al., 2006; Manolea et al., 2010). Recent researches also showed that ARF4 participates in the regulation of glioblastoma apoptosis through the inhibition of stress-mediated apoptotic signals (Woo et al., 2009; Kim et al., 2003). What’s more, ARF5 had been identified to be involved in controlling tumor proliferation and metastasis (Boulay and Claing, 2009).

As mentioned before, ARF3, ARF4 and ARF5 genes are three genes which have important functions. Until today, ARF3, ARF4 and ARF5 genes had been reported in human and other animals but the sheep ARF3, ARF4 and ARF5 genes have not been reported yet.

In present experiment, we will isolate the coding sequences of sheep ARF3, ARF4 and ARF5 genes based on the coding sequence information of ARF3, ARF4 and ARF5 genes from human or other mammals and their highly homologous sheep ESTs sequence information, subsequently perform some necessary sequence analysis and tissue expression profile analysis for these genes. These will establish the primary foundation of understanding these three sheep genes.

MATERIALS AND METHODS

Animals and sample preparation: The five adult Yunnan local sheep were slaughtered. Spleen, skin, lung, fat, muscle, heart, liver, kidney and ovary samples were collected, frozen in liquid nitrogen and then stored at -8°C. The total RNA was extracted using the total RNA Extraction kit (Gibco, USA). First-strand cDNA synthesis was performed as that described by Liu et al. (2004). These first-strand cDNA samples were used to perform RT-PCR for the isolation of sheep ARF3, ARF4 and ARF5 genes and tissue expression profile analysis.

Isolation of the sheep ARF3, ARF4 and ARF5 genes: The primers for sheep ARF3 gene isolation were designed based on the coding sequence information of human ARF3 gene and its highly homologous sheep EST sequences: DY513665 and EE747569. Similarly, the primers for sheep ARF4 gene isolation were designed based on the coding sequence information from human ARF4 gene and its highly homologous sheep EST sequences: DY485622 and DY489964. The primers for sheep ARF5 gene isolation were designed based on the coding sequence information from human and mouse ARF5 genes and their highly homologous sheep EST sequences: EE794299 and FE033388. These primer sequences and their annealing temperature for RT-PCR reaction were shown in Table 1. The RT-PCR was performed to isolate these three sheep genes using the pooled cDNAs from different tissues above. The 25 μL reaction system was; 2.0 μL cDNA, 2.5 μL 2 mM mixed dNTPs, 2.5 μL 10xTaq DNA polymerase buffer, 2.5 μL 25 mM MgCl₂, 2.0 μL 10 μM forward primer, 2.0 μL 10 μM reverse primer, 2.0 units of Taq DNA polymerase (1U 1 μL^-1) and 9.5 μL sterile water. The PCR program initially started with a 94 denaturation for 4 min followed by 35 cycles of 94°C/50 sec, Ta°C/50 sec, 72°C/50 sec then, 72°C extension for 10 min, finally 4 to terminate the reaction. These PCR products for sheep ARF3, ARF4 and ARF5 genes were then cloned into PMD18-T vector and sequenced bidirectionally with the commercial fluorometric method. At least five independent clones were sequenced for every gene.

RT-PCR for tissue expression profile analysis: RT-PCR for tissue expression profile analysis was performed as previously described elsewhere (Liu and Gao, 2009; Liu, 2009). We selected the housekeeping gene β-actin (Accession No.: NM_001009784) as a positive control.

Table 1:	Primers for sheep ARF3, ARF4, ARF5 and β-actin genes and their annealing temperatures

The primers of sheep ARF3, ARF4 and ARF5 genes which were used to perform the RT-PCR for tissue expression profile analysis were same as the primers for isolation RT-PCR above. The PCR reactions were optimized for a number of cycles to ensure product intensity within the linear phase of amplification. The 25 μL reaction system was 1 μL cDNA (100 ng μL^-1), 5 pmoles each oligonucleotide primer, 2.5 μL 2 mmol L^-1 mixed dNTPs, 2.5 μL 10HTaq DNA polymerase buffer, 2.5 μL 25 mmol L^-1 MgCl₂, 1.0 unit of Taq DNA polymerase and finally add sterile water to volume 25 μL. The PCR program initially started with a 94 denaturation for 4 min, followed by 25 cycles of 94/50, Ta/50, 72/50 sec then 72 extension for 10 min, finally 4 to terminate the reaction.

Sequence analysis: The cDNA sequence prediction was conducted using GenScan software (http://genes.mit.edu/GENSCAN.html). The protein prediction and analysis were performed using BLAST tool at the National Center for Biotechnology Information (NCBI) server (http://www.ncbi.nlm.nih.gov/BLAST) and the ClustalW software (http://www.ebi.ac.uk/ clustalw).

RESULTS AND DISCUSSION

RT-PCR results for sheep ARF3, ARF4 and ARF5 genes: Through RT-PCR with pooled tissue cDNAs for sheep ARF3, ARF4 and ARF5 genes, the resulting PCR products were 546, 543 and 543 bp (Fig. 1).

Sequence analysis: These cDNA nucleotide sequence analysis using the BLAST software at NCBI server (http://www.ncbi.nlm.nih.gov/BLAST) revealed that these three genes were not homologous to any of the known sheep genes and they were then deposited into the GenBank database (Accession No.: FJ969413, FJ969400 and FJ969399 ). The sequence prediction was carried out using the GenScan software and results showed that the 546, 543 and 543 bp cDNA sequences represent three single genes which encoded 181, 180 and 180 amino acids, respectively. Finally, these three novel sheep genes were assigned to GeneIDs; 100302303, 100302304 and 100302305.

Fig. 1:	RT-PCR results for sheep ARF3, ARF4 and ARF5 genes. M: DL2000 DNA Markers; 1: PCR product for sheep ARF3 gene; 2: PCR product for sheep ARF4 gene and 3: PCR product for sheep ARF5 gene

Fig. 2:	The alignment of the protein encoded by sheep ARF3 gene and five other kinds ARF3 proteins

Further BLAST analysis of these proteins revealed that the sheep ARF3 protein has high homology with the ADP-Ribosylation Factor 3 (ARF3) proteins of five specie-human (Accession No.: NP-001650; 99%), zebrafish (Accession No.: NP-001003441; 99%), African clawed frog (Accession No.: NP-001086694; 99%), pig (Accession No.: NP-001153898; 99%) and Atlantic salmon (Accession No.: NP-001133306; 98%) (Fig. 2).

The sheep ARF4 protein has high homology with the ADP-Ribosylation Factor 4 (ARF4) proteins of 3rd species-cattle (Accession No.: NP-001029702; 100%), platypus (Accession No.: XP-001511094; 99%), green anole (Accession No.: XP-003217762; 99%), pig (Accession No.: NP-001041537; 99%), rabbit (Accession No.: XP-002713309; 98%), African clawed frog (Accession No.: NP-001082540; 98%), red jungle fowl (Accession No.: XP-001232784; 98%), mouse (Accession No.: NP-031505; 98%), zebrafish (Accession No.: NP-956170; 96%), human (Accession No.: NP-001651; 97%), Atlantic salmon (Accession No.: ACM08502; 92%), rainbow trout (Accession No.: NP-001154084; 92%) and caligus rogercresseyi (Accession No.: ACO10868; 89%) (Fig. 3). The sheep ARF5 protein has high homology with the ADP-Ribosylation Factor 5 (ARF5) proteins of seven species-human (Accession No.: NP-001653; 100%), mouse (Accession No.: NP-031506; 100%), rat (Accession No.: NP-077063; 100%), gray short-tailed opossum (Accession No.: XP-001366170; 99%), chicken (Accession No.: NP-990656; 98%), channel catfish (Accession No.: NP-001188198; 93%) and zebrafish (Accession No.: NP-954969; 93%) (Fig. 4).

Fig. 3:	The alignment of the protein encoded by sheep ARF4 gene and third other kinds of ARF4 proteins

Based on the results of the alignment of ARF3, ARF4 and ARF5 proteins, three phylogenetic trees were constructed using the Dendrogram procedure of ClustalW software (http://align.genome.jp/) as shown in Fig. 5-7. The phylogenetic analysis revealed that the sheep ARF3 gene has a closer genetic relationship with the ARF3 gene of human.

The sheep ARF4 gene has a closer genetic relationship with the ARF4 gene of cattle. The sheep ARF5 gene has a closer genetic relationship with the ARF5 genes of human, mouse and rat.

Fig. 4:	The alignment of the protein encoded by sheep ARF5 gene and seven other kinds of ARF5 proteins

Fig. 5:	The phylogenetic analysis for six kinds of ARF3 genes

Fig. 6:	The phylogenetic analysis for fourteen kinds of ARF4 genes

Fig. 7:	The phylogenetic analysis for eight kinds of ARF5 genes

Tissue expression profile: Tissue expression profile analysis was carried out and results revealed that the sheep ARF3 gene was highly expressed in liver, moderately expressed in skin, lung, muscle, spleen, heart and fat and weakly expressed in kidney and ovary. The sheep ARF4 gene and ARF5 gene displayed similar tissue expression distribution. They were both highly expressed in spleen, lung, muscle, kidney and ovary, moderately in skin, liver and heart and hardly expressed in fat (Fig. 8).

In the current study, we firstly get the coding sequences of sheep ARF3, ARF4 and ARF5 genes by RT-PCR.

Fig. 8:	Tissue expression distribution of sheep ARF3, ARF4 and ARF5 genes. The β-actin expression is the internal control.1: Spleen; 2: Skin; 3: Lung; 4: Muscle; 5: Heart; 6: Fat; 7: Liver; 8: Kidney and 9: Ovary

With the development of modern bioinformatics and specific sheep NCBI EST database established along with different convenient analysis tools, researchers can easily find the useful ESTs which were highly homologous to the coding sequences of human genes. Based on these sheep EST sequences, we can obtain the complete coding sequences of some novel sheep genes through the some experimental methods such as RT-PCR. From the clone and sequence analysis of sheep ARF3, ARF4 and ARF5 genes, it could be seen that this is an effective method to isolate some novel sheep genes.

Through sequence analysis, we found that the encoding protein of the sheep ARF3, ARF4 and ARF5 genes are highly homologous with ARF3, ARF4 and ARF5 proteins of human and some other animals. This implied that the ARF3, ARF4 and ARF5 genes were highly conserved in some species and the sheep ARF3, ARF4 and ARF5 genes will have similar functions as the ARF3, ARF4 and ARF5 genes of human and other animals. We also found that the sheep ARF3, ARF4 and ARF5 proteins do not show complete identity to human or other animals. This implied that the sheep ARF3, ARF4 and ARF5 genes will have some differences in functions to those of human or other mammals.

The phylogenetic analysis revealed that the sheep ARF3 gene has a closer genetic relationship with the ARF3 gene of human. This implied that we can use sheep as a model organism to study the human ARF3 gene or use human as a model organism to study the sheep ARF3 gene. Similarly, the sheep ARF4 gene has a closer genetic relationship with the ARF3 gene of cattle and the sheep ARF5 gene has a closer genetic relationship with the ARF5 genes of human, mouse and rat so that we can use cattle as a model organism to study the sheep ARF3 gene and use human, mouse and rat as model organisms to study the sheep ARF5 gene. From the tissue distribution analysis in the experiment, it can be seen that the sheep ARF3, ARF4 and ARF5 genes were obviously differentially expressed in some tissues. As we did not study functions at protein levels yet there might be many possible reasons for differential expression of sheep ARF3, ARF4 and ARF5 genes. The suitable explanation for this under current conditions is that at the same time those biological activities related to the mRNA expression of sheep ARF3, ARF4 and ARF5 genes were presented diversely in different tissues.

CONCLUSION

In this study, we 1st isolated the sheep ARF3, ARF4 and ARF5 genes and performed necessary sequence analysis and tissue transcription profile analysis. This established the primary foundation for further insight into these novel sheep genes.

ACKNOWLEDGEMENTS

This study was supported by grants from the Natural Science Foundation Key Project of Yunnan Province (No. 2009CC015), National Nature Science Foundation of China (No. 30800810) and the Candidates of the Young and Middle Aged Academic and Technical Leaders of Yunnan Province (2009CI055).