cDNA Cloning and Expression Analysis of Mest Gene in the Bufo gargarizans

： The Mest （ mesoderm-specific transcript ） gene has been considered an imprinting gene in human and mouse, and was also confirmed in other mammals and flowering plants. To investigate the function and evolution of this gene, the cDNA of full length Mest gene was obtained using 5 ' - and 3 ' -RACE from the Chinese Large Toad ( Bufo gargarizans ). The transcript is 1 325 bp in length which contains a complete open reading frame (ORF) encoding a polypeptide of 326 amino acids (GenBank accession number: ABQ10905). There is a typical α / β hydrolase fold domain in the putative gene product, and it shows high similarity to sequence of homologous protein of Xenopus tropicali (86%), mammlian (70% − 80%). RT-PCR (reverse transcriptase-polymerase chain reaction) analysis demonstrated that the Bufo gargarizans Mest ( BgMest ) gene is expressed widely in testis, ovary, liver, kidney, spleen, brain, stomach and lung. The conservation of the BgMest gene sequences, protein secondary structure of the BgMest protein, in addition to the expression pattern of the BgMest gene, suggested that the function of BgMest was conserved in amphibians. However, the phylogenetic tree of the imprinting gene of the mammals and other vertebrates examined in this study indicated their divergent origins.

Genomic imprinting refers to the parent-of-origin specific gene modification resulting in monoallelic expression of a gene dependent on its parental origin (Brannan & Bartolomei, 1999). The molecular mechanism of genomic imprinting appears to share aspects of epigenetic regulation with X inactivation in human and mouse, since both phenomena involve DNA methylation and asynchronous replication of DNA (Saitoh & Wada, 2000). This phenomenon has been also observed in mammals and flowering plants (Scott & Spielman,2004). To date, there are approximately 100− 200 imprinting gens in the mammal genome, such as H19, IGF2, IGF2R, XIST, etc…. (Looijenga et al, 1997). These special genes in mammals are imprinted so that only one of the parental alleles is actually expressed in target cells. Mest (mesoderm-specific transcript)/Peg1 (paternally expressed gene 1) is an important maternally imprinted mammalian gene which is predominantly expressed in the mesoderm and its derivatives (Reule et al, 1998;Isles et al, 2006). Only the paternal copy of the gene is expressed in interspecific hybrids. This gene has been assigned to 7q32 in human and mapped to chromosome 6 in mouse (Lefebvre et al,1997;Kosaki et al,2000) The Mest orthologs were studied mainly in mammals, but there were also reports in non-mammalian species like pufferfish (Fugu rubripes), zebrafish, Xenopus tropicalis and tammar wallaby (Suzuki et al, 2005). The widespread Bufo gargarizans is a representative of Bufonidae, Anura, Amphbia, and it plays an important role in the ecological balance of nature. The amphibians represent a bridge in the evolution of vertebrates from aquatic to terrestrial. They have highly specialized morphological and functional characteristics to adapt to different environments. In this paper, we isolated and analyzed the expression pattern of BgMest. To understand the function and evolution of this gene, the phylogenic tree was also constructed.

Animals
Two male and two female adult B. gargarizans were obtained from the suburb of Wuhu city, Anhui province, China. Tissue from the testis, ovary, liver, kidney, spleen, brain, stomach and lung were dissected from the toads. Samples were snap frozen in liquid nitrogen, and stored at -80℃ until use.

Cloning of Mest cDNA
To amplify the conservative fragment of BgMest cDNA (424 bp), a pair of primers (M1: GATTGCCCT-GGACTTTAT, M2: TACACCTGTCGGGAGTCC) were designed according to the conserved amino acid sequences that have been found in human (Accession number: NP_803490), and mouse (Accession number: AAM78507). To obtain the complete BgMest cDNA, rapid amplification of cDNA ends (RACE) technique was performed (Invitrogen, Tokyo, Japan) according to instructions.
Total RNA was extracted from toad testis with TRIzol (Invitrogen) according to the manufacturer provided protocol. Single-stranded cDNA was prepared with SuperScript TM III reverse transcriptase, following the operating instruction. For the 3'-RACE, M1 and anchor primers (5'-CGTCTAGAGGTACCGGATCCA-ACA-3') were used. The PCR reaction consisted of 30 cycles of 95℃ (30 s), 65℃ (30 s) and 72℃ (3 min), ending with a 7 min extension step at 72℃. After cDNA fragments had been obtained by 3'-RACE, they were inserted into the PCR2.1 vector and sequenced. For the 5 ' -R A C E , s i n g l e -s t r a n d e d ( s s ) c D N A w a s reverse-transcribed from total RNA according to the manufacturer's protocol (Invitrogen, Tokyo, Japan). Oligo dA was added to the ss cDNA by terminate deoxynucleotidyl transferase according to the protocol (TOYOBO, Tokyo, Japan), which used as a template for the PCR. The PCR was performed by using M2 and oligo dT anchor (5'-CGTCTAGAGGTACCGGATC-CAACAT(17)V-3') primers for 30 cycles under the following conditions: denaturation at 95 ℃ for 30 s, annealing at 65℃ for 30 s, extension at 72℃ for 2 min, and ending with an extension step at 72℃ for 7 min. After cDNA fragments had been obtained by this procedure, they were inserted into the PCR2.1 vector and sequenced.

RT-PCR analysis and tissue expression of Mest gene
To analyze the tissue specificity of Mest gene expression, total RNA was extracted from several tissues including testis, ovary, liver, kidney, spleen, brain, stomach, lung of male and female specimens. It was prepared for homogenizing the tissue in 100 uL Trizol reagent (Takara, China) and 50 uL chloroform. The aqueous phase was precipitated in 150 uL isopropanol at 4℃ overnight. The RNA pellet was rinsed in 75% ethanol and resuspended in 12 uL DEPC-treated ddH 2 O and immediately used for RT-PCR. Reverse transcriptase polymerase chain reaction (RT-PCR) amplifications were performed using two degenerated primers (M1 and M2) The nucleotide sequences of the primers used were EFIα, H1: TCCACCACCACCGGCCACCT; H2:CTCCCA-CACCAGCAGCAACAAT.
RT-PCR was carried out using 3 uL total RNA per reaction, 1×buffer reaction mix and 0.2 µmol/L b-actin primers or 0.3 µmol/L of Mest primers in separate tubes, in a total volume of 20 µL. All reactions for Mest contained 6% DMSO. cDNA synthesis was performed at 50℃ for 30 min and amplification conditions were: 94℃ /5 min for initial denaturation; 94℃/30 s for denaturation, 56℃/60 s for annealing, 72℃/60 s for extension, 35 cycles; 72 ℃ /10 min for final extension. Each PCR product was detected by electrophoresis on 1.2% agarose gel.

Sequence analysis of BgMest gene
To isolate the BgMest, the complete cDNA were amplified by 5' and 3'-RACE technique, a 1 325 bp Mest cDNA encoding a predicted protein of 336 amino acids was obtained. The cDNA sequence included 34 nucleotide acids in 5'-UTR, 310 nucleotide acids in 3'-UTR, and a complete open reading frame (ORF) which conformed to several characteristics that GC content of cDNA sequence is high but the sequence is short in 5'-UTR. Fig.1 showed the full-length Mest cDNA of B. gargarizans. This BgMest sequence was determined by comparison with orthologous proteins of the amino acid sequences using BLAST (Accession number is ABQ10905 in the NCBI database).
The putative amino acid sequences of BgMest were aligned with orthologous proteins from Homo species, Mus musculus, Macaca mulatta, Gallus gallus, Xenopus tropicalis, Takifugu rubripes and Danio rerio. The BgMest showed a significant sequence homology with those of other vertebrate Mest proteins. It exhibited the highest similarity to Xenopus tropicalis Mest (86% identities) and the BgMest is similar to other vertebrates with 70% − 80%nt identities (Fig.2).
Additionally, based on amino acid sequence of the Mest protein, they contained a conserved domain named α/β-hydrolase fold domain. It conformed to the 3-D model of the Mest protein (Fig. 3). According to analysis via Phyre program, the model belonged to the α/β-hydrolase family. Fig. 2 showed that this domain was located at amino position 88 − 320 in mammal, at 105 − 337 in zerbrafish and at 64 − 296 in Gallus gallus. The domain of BgMest protein was located at position 107 − 321. This conserved domain was determined to be prominently expressed in mesodermal derivatives during embryogenesis only from the paternally inherited allele, and was detected in a number of proteins of several animals, plants and microorganism (Kamei et al, 2007;Yun et al, 2002).
Analysis of protein secondary structure (Fig. 4) and 3-D model of BgMest protein can understand its function and recognize the interactions within proteins, which is important in biological and medical research. Protein secondary structure of BgMest gene is composed of helix (h), strand (s) and coil (c) like the Mest gene of mammals. It includes six helixes, six strands and sixteen coils within the BgMest protein (Fig. 4). It was shown that the BgMest protein also contains a conserved α/β-hydrolase fold domain by analysis 3-D model of BgMest protein (Fig.3).

Phylogenetic tree of Mest gene
The relationships between the proteins encoded by BgMest gene and the corresponding proteins from other species were analyzed using neighbour-joining (NJ) method (Fig. 5). The tree showed that all mammalian proteins cluster together and the fish and amphibian cluster into another branch. This may suggest the divergent origins separating mammalians from the other vertebrates including teleosts, avian and amphibians. Mycobacterium tuberculosis C belonged to an actinomyces, which was separated from the vertebrate. The results were identical with traditional classification, which supports the Mest protein monophyly.

Expression of BgMest
To determine the multiple tissue expression of this gene, RT-PCR was carried out on various tissues (including testis, ovary, liver, kidney, spleen, brain, stomach and lung) in adult B. gargarizans. As a control, cytoplasmic EFI-a gene from B. gargarizans was used. As seen in Fig. 6, all the aforementioned tissues were expressed.

Discussion
The genomic structure of Mest gene has been shown to include a C P G island in human, mouse and zerbrafish (Reule et al, 1998;Kosaki et al, 2000;Hahn et al, 2005). A characteristic of imprinted genes is that maternal and paternal alleles show differences in methylation position Nucleotides are numbered up and deduced amino acids are below. ATG: initiation codon; TAG: termination codon. (Georgiades et al, 2001;Edwards et al, 2007). In the researches of huamn and mouse, imprinted role of Mest gene was related to the methylation of C P G island (Reule et al, 1998;Kosaki et al, 2000). However, unlike the C P G island of normal and biallelically expressed genes, the C P G island of Mest is exclusively unmethylated on the expressed paternal allele, but fully methylated on the silenced maternal allele (Reule et al, 1998). In human and mouse, Mest gene was expressed in amnion, brain, heart, lung, stomach, kidney and liver. Mammalian Mest gene is widely expressed throughout the embryo, mostly in mesodermal tissues (Reule et al, 1998;Kosaki et al, 2000). In our study, the Mest gene was also expressed in testis, ovary, liver, kidney, spleen, brain, stomach and lung of the adult B. gargarizans. And there is high similarity of the protein sequences and evolutionary  Hahn et al (2005) concluded that zebrafish Mest gene is not imprinted, at least in the larval stage. Thus, whether the genomic structure of BgMest contains a typical C P G island and structure of DNA methylation needs to be further dertermined. According to Fig. 5, the tree may indicate the evolutionary relationships of the potential imprinting genes of mammals and other vertebrates examined in the study. The classical Mendel's law of inheritance suggested that both alleles are actively transcribed and functionally equivalent. However, imprinted genes represent an exception to this rule, as they are expressed   from one of the two parentally inherited chromosome homologues and repressed on the other (Kaneko et al, 2002;Hou et al, 2005). The Mest genes of human and mouse were considered as maternal imprinted gene, and imprinting role was for methylation of C P G island (Reule et al, 1998;Kosaki et al, 2000). In mice, normal expression of Mest gene was very important for development. Knocking out and low expression of this gene may result in restraining growth and decreasing prenatal and postnatal survival (Reule et al, 1998). Nishita et al(1996)suggested that human Mest gene, the first imprinted gene to be identified on chromosome 7, was involved in the causation of Silver-Russell syndrome (SRS) (Nishita et al, 1996). Frequent loss of imprinting of the Mest gene in humans may cause cancer tissues, such as breast cancer or lung cancer and additionally, this gene regulates placental and fetal growth (Pedersen et al, 1999;Kosaki et al, 2000;Nakanashi et al, 2004;Suzuki et al, 2005). The zebrafish Mest has been found to be biallelic expressed (Hahn et al, 2005). In addition, ASCL2/CASH4 and INS were imprinted genes in mammals, but shown biallelic expression in the chicken (Yokomine et al, 2005). In the opossum (marsupial) IGF2 and M6P were imprinted as in placental mammals, but these genes are biallelically expressed in monotremes and birds (Suzuki et al, 2005). These observations may suggest that the gene imprinting occurs in mammals except monotremes (Hahn et al, 2005). Hahn et al (2005) considered that species, or clade, specific imprinted genes may have survived the selection for diluted imprinting in the evolution of non-mammalian species. Does the BgMest gene conform to the opinion of Hahn et al (2005)? Is BgMest an imprinted gene? Further research is required to uncover the structure and function of this gene.