Sequence Characterization , Expression Profile , Chromosomal Localization and Polymorphism of the Porcine SMPX Gene

The full-length cDNA of the porcine SMPX gene was obtained by the rapid amplification of cDNA ends (RACE). The nucleotide sequences and the predicted protein sequences share high sequence identity with both human and mouse. The promoter of SMPX was sequenced and then analyzed to find the promoter binding sites. The reverse transcriptase-polymerase chain reaction (RTPCR) revealed that SMPX has a high level of expression in heart and skeletal muscle, a very low expression in lung and spleen and no expression in liver, kidney, fat and brain. Moreover, SMPX has a differential expression level in skeletal muscle, the expression in 65day embryos being higher than other stages. The porcine SMPX was mapped to SSCXp24 by using a somatic cell hybrid panel (SCHP) and was found closely linked to SW1903 using the radiation hybrid panel IMpRH. An A/G single nucleotide polymorphism (PCRRFLP) in the 3’-untranslated region (3’-UTR) was detected in eight breeds. The analysis of allele frequency distribution showed that introduced pig breeds (Duroc and Large White) have a higher frequency of allele A while in the Chinese indigenous pig breeds (Qingping pig, Lantang pig, YushanBlack pig, Large Black-White pig, Small Meishan) have a higher frequencies of allele G. The association analysis using an experimental population (188 pigs), which included two cross-bred groups and three pure-blood groups, suggested that the SNP genotype was associated with intramuscular fat content. (


INTRODUCTION
The small muscle protein X-link (SMPX/Csl) gene was firstly identified from human striated muscle, and might play a role in the cardiac or muscular development (Patzak et al., 1999).SMPX, a muscle-specific gene, was dramatically up-regulated in response to passive stretch in vivo, so it could be involved with the regulation of muscle fiber development (Kemp et al., 2001).SMPX was prominently expressed in cardiac and skeletal muscle in embryonic and postnatal stage.Its down regulation in Csx/Nkx2-5-null embryonic heart suggests that it is a target gene for Csx/Nkx2-5.SMPX could enhance transcriptional activities of the myocyte-specific enhancer factor 2 (MEF2) and nuclear factor of activated T cells (NFAT) in an IGF-1 signal-dependent manner (Palmer et al., 2001).Both MEF2 and NFAT are important for the differentiation and hypertrophy of cardiac and skeletal muscles.When SMPX is expressed in C2C12 myoblasts, it associates with focal adhesion complexes and can modify cell shape and actin dynamics in a Rac1 and p38 kinase-dependent manner (Schindeler et al., 2005).Furthermore, it is interesting that SMPX is highly expressed in red muscles than white muscles (Bai et al., 2003).In brief, the function of SMPX encode protein was not clear, but the expression and mapping results indicated that SMPX could play a critical role in the regulation of muscle fiber and heart development (Ervasti, 2003).SMPX which affects muscle fiber development may be a candidate gene related to muscle growth and quality in domestic pigs.
In this study, we obtained the porcine SMPX full-length cDNA, and part of SMPX promoter sequence.We then mapped it and analyzed its expression distribution in different tissues and at different stages.We also report an association of nucleotide variation of this gene with intramuscular fat content.

Isolation of full-length cDNA of porcine SMPX
The porcine SMPX full-length cDNA was obtained by the rapid amplification of cDNA ends (RACE).BLAST analysis (http://www.ncbi.nlm.nih.gov/blast/) was performed using the human SMPX cDNA sequence (GenBank accession number: 014332.1) in order to obtain porcine ESTs which shared at least 80% sequence homology.Then these ESTs were assembled into a contig for primers design (Yang et al., 2005).Total RNA of a mature Landrace pig was extracted from skeletal muscle tissue with a TRIzol Reagent Kit (Life Technologies, Grand Island, NE, USA), then treated with RNase-free DNase I (Promega, Madison, WI, USA) and precipitated with ethanol.RACE was performed according to the instructions of the SMART TM RACE cDNA Amplification Kit (Clontech Inc, Palo Alto, CA, USA).The PCR products were purified with Gel Extraction Mini Kit (Waston Biotechnologies Inc, Shanghai, China) and cloned into the pMD18-T vector (TaKaRa, Dalian, China), then sequenced by commercial service.Finally, the sequence was submitted to NCBI (GenBank accession no.DQ104414).

Isolation of the porcine SMPX promoter
Comparative anchor tagged sequences (CATS) is an important method to isolate the unknown sequences of genes (Kenealy et al., 1998).By using CATS, the forward (P1L) and reverse (P1R) primers were designed according to the human promoter and porcine SMPX exon 1 sequences respectively (Table 1).PCR was performed in 20 µl reaction mixture containing 1×PCR buffer, 1.5 mM MgCl 2 , 75 µM dNTP, 0.3 µM of each primer (P1L, P1R), and 1 unit Taq DNA polymerase (TaKaRa).PCR amplification conditions were 95°C for 4 min, followed by 35 cycles of 94°C for 40 s, 59°C for 40 s, 72°C for 40 s, and a final extension step of 5 min at 72°C.Then PCR product were purified and sequenced.Finally, the sequence was submitted to NCBI (GenBank accession no.DQ104415).

Sequence analysis
The ORF of porcine SMPX gene were found and the amino acid sequences were deduced with the EditSeq program (DNA Star, Madison, WI, USA).The motifs of the putative protein were analyzed with PSORTII (http://www.nibb.ac.jp) and ScanProsite (http://au.expasy.org/tools/scanprosite/) programs.The putative promoter motifs were analyzed with the tools available on the web site, Transcription Element Search System (http://www.cbil.upenn.edu/cgi-bin/tess/tess?RQ=WELCOME) (Wingender et al., 2000).

Expression profile analysis
Total RNAs were extracted from adult porcine heart, skeletal muscle, lung, liver, spleen, kidney, fat and brain respectively.In addition RNAs were extracted from skeletal muscles at different stages: 33-day, 65-day, 90-day embryos, birth, 28-day and adult Tongcheng pigs.Reverse transcriptions were performed as described by Pan et al (2003) using the primer pairs P2L and P2R (Table 1).PCR conditions were 4 min at 95°C followed by 27 cycles of 30 s at 94°C, 30 s at 61°C, 30 s at 72°C, and a final extension of 5 min at 72°C.Amplification of GAPDH was performed as an internal control in the same conditions, and GAPDH primers were synthesized according to the reported sequence (Janzen et al., 2000).Finally 8 µl of each PCR products were used for the expression profile analysis on 2.0% agarose gels.

Somatic cell hybrid and radiation hybrid mapping
The somatic cell hybrid panel SCHP (Yerle et al., 1996) was used to cytogenetically map SMPX on porcine chromosomes and its position was then refine using the radiation hybrid panel IMpRH (Yerle et al., 1998).PCR reactions were performed in a volume of 10 µl of 1×PCR buffer (TaKaRa), containing 20 ng of hybrid DNA (either SCHP or IMpRH samples), 0.2 µM of each primer (P3L, P3R) (Table 1), 100 µM of each dNTP, 1.5 mM MgCl 2 and 2 units Taq DNA Polymerase (TaKaRa).The PCR conditions were 3 min at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 65°C, 15 s at 72°C, and an extra 5 min extension at 72°C.In addition, PCR reactions were carried out with pig genomic DNA (as positive control) and without DNA (as negative control).Mapping results were analyzed using the tools available at (http://www.toulouse.inra.fr/lgc/pig/hybrid.htm)(Chevalet et al. 1997) and (http://imprh.toulouse.inra.fr/)(Milan et al., 2000) for SCHP and RH mapping respectively.

Genetic variation analysis and traits association analysis
A 158 bp genomic fragment was amplified using the primers P4L and P4R.Polymorphism was detected in SMPX by PCR-RFLP and analysis on 3.0% agarose gels.PCR fragments from different genotypes were cloned and sequenced.DNA samples of 199 unrelated pigs from eight breeds (Duroc, Large White, Min pig, Qingping pig, Lantang pig, YushanBlack pig, Large Black-White pig and Small Meishan) were genotyped for genetic variation analysis and χ 2 test on the allele frequencies was performed with population genetic structure comparison performed using SAS version 8.1.An experimental population (188 pigs) including two cross-bred groups and three pure-blood groups, Large White×(Landrace×Tongcheng) (43 individuals), Landrace×(Large White×Tongcheng) (51 individuals), Tongcheng breed (33 individuals), Landrace (29 individuals) and Large white (32 individuals), were selected for association analysis.The association between genotype and traits (carcass traits and meat quality) was performed with the least square method (GLM procedure, SAS version 8.1).The model used to analyze the data was assumed to be: Y = µ+B i +P j +G k +e ijk .Where, Y is the observation of the trait; µ is the least square mean; B i is the effect of breed, P j is the effect of batch (j = 1 to 8); G k is the effect of genotype (k = AA, AG, GG); e ijk is the random residual.

SMPX full-length cDNA isolation
Based on bioinformatics analysis, 10 porcine ESTs (Acc.No: BX664815, BX667327, BX666546, BX664976, BE014221, AJ654762, CO938466, AJ656431, BE013988 and CA778404) were obtained and these overlapping ESTs were assembled into one contig covering 842 bp.The primers for RACE were designed in the contig.The size of the 5'RACE and 3'RACE products were 405 bp and 571 bp respectively.Bioinformatics analysis of the combined nucleotide sequence revealed that there was a 261 bp ORF flanked by a 209 bp 5'-UTR and a 450 bp 3'-UTR.A polyadenylation signal (AATAAA) was detected in the 3'-UTR region.Sequence comparisons revealed that the porcine SMPX sequence is respectively 85% and 79% identical to the human (NM_014332) and mouse (NM_025357) corresponding sequences.PSORTII analysis showed that the SMPX gene encoded 86 amino acids with a calculated molecular mass of 9.34 kDa and isoelectric point of 9.16.The deduced porcine protein sequence is 94% and 82% identical to human and mouse corresponding sequences respectively.Cytoplasmic/nuclear discrimination (PSORTII) predicted that SMPX might exist predominantly in the nuclear with a probability of 94.1%.Prediction of protein sorting signals (PSORTII) identified a pat 7 nuclear localization motif (PPRRKECT) (Hicks and Raikhel, 1995).Conservation of the casein kinase phosphorylation site and of the pat 7 nuclear localization signal was also observed in pig, mouse and human sequences, which suggest that these putative sites were genuine.

Analysis of the 5' SMPX promoter
By the means of comparative anchor tagged sequences (CATS), the SMPX upstream sequence (381bp) was obtained (Figure 1).The PCR product was obtained using genomic DNA as template.Analysis of the 5'promoter region revealed that there were no typical TATA box and cap motifs.However, the upstream sequence contain putative binding sites for several muscle-specific transcription factors, such as MyoD, E12, E47, MEF-2, RSRFC4 and Nkx2.5 which also existed in human SMPX promoter.Sequence comparisons between pig and human SMPX upstream sequences showed that the sequences shared 88% identity which was higher than that observed between the porcine and human coding sequences even in the ORF region.The result showed the conservation of SMPX cis-site is very high.

Expression profile analysis
RT-PCR was performed to detect the porcine SMPX expression profile in eight different tissues and six different stages (Figure 2).The result showed that the expression was predominant in heart and skeletal muscles, very low in lung and spleen, and no expression was detected in liver, kidney, fat and brain.Moreover, SMPX presented different expression levels in skeletal muscle in these different stages.The expression levels of 33-day embryo and adulthood were lower than other four stages.

SCHP and RH mapping
Using the somatic cell hybrid panel, SMPX was assigned to porcine Chr Xp24 (probability of regional localization 0.8875, Correlation 1.0000 and error risk <0.1%).The mapping was further precised by using the radiation hybrid panel IMpRH.The statistical analysis revealed that SMPX is significantly linked to microsatellite SW1903 with a distance of 55cR and LOD score of 7.04.

Analysis of polymorphism in different breeds and association analysis
An A/G (611) single nucleotide polymorphism (PCR-RFLP) was found by sequence analysis in 3'-untranslated region (3'-UTR).No restriction enzyme was available to directly genotype the SNP.So a TaaI RFLP test was developed by using a mismatch forward primer.The TaaI enzyme recognizes "ACNGT" sequence and the genomic sequences have "CCCGT" or "CCCAT".The forward primer sequence was designed to include the C↔A substitution at the position 608 and ended at the position 610 (sequence: 5'-CGGAATGCCTGAGACTCTAGCAG AAATGTACC-3').After TaaI digestion, allele A was characterized by an uncut fragment of 158 bp and allele G by two fragments of 126 bp and 32 bp (the 32 bp fragment was not detected on agarose as it produced a too weak band) (Figure 3).The analysis of allele frequency distribution revealed that introduced pig breeds (Duroc and Large white ) present a high frequency of A allele while the G allele is more frequently presented in the Chinese indigenous pig breeds (Min pig, Qingping pig, Lantang pig, YushanBlack pig, Large Black-White pig, Small Meishan) (Table 2).The x 2 test results for the allele frequency distribution indicates that there is a significant difference between Chinese native breeds and exotic breeds Duroc (p<0.01) and Large white (p<0.05)(Table 3).Traits association analysis was done in an experimental population.In the association analysis, the TaaI-RFLP genotype was significantly associated with intramuscular fat content.The intramuscular fat content of pigs with GG genotype was significantly higher than those of pigs with the AA genotypes (p = 0.0141) and the AG genotypes (p = 0.0244) (Table 4).

The porcine SMPX full-length cDNA and promoter sequence
The porcine SMPX was identified, cloned and characterized.It generated a mRNA of 920 bp and expressed prominently in skeletal and cardiac muscles.SMPX mRNA contains A/T-rich regions which are associated with instability.And there are several ATTTA or ATTTT motifs in the 3'-UTR which were identified as the highly conserved motif in several short-lived lymphocytes (Shaw and Kamen, 1986).These results suggest that SMPX mRNA is probably unstable and should have a short-life within skeletal muscle.The porcine SMPX transcript codes for an 86 amino acid protein and there are two overlapping casein kinase II (CK2) phosphorylation sites.It is interesting that the two CK2 phosphorylation sites are conserved among human, mice and pig.CK2 is a serine/ threonine kinase that has been implicated in cell growth and proliferation.And CK2 is required for Myf-5 activity though  CK2-mediated phosphorylation of Myf-5 (Bai et al., 2003).It suggests that the function of SMPX is possibly related to CK2 and Myf-5 in muscle growth.
We obtained the SMPX promoter sequence.Although the porcine SMPX doesn't contain TATA or CCAAT boxes, but there are several banding sites of muscle-specific transcription factors (MyoD, E12, E47, MEF-2, RSRFC4 and Nkx2.5) which generally found in muscle-related gene promoters.MyoD can activate the muscle-specific genes that play an important role in the myoblast proliferation (Valdez et al., 2000).The MEF2 are members of the MADS (MCM1, agamous, deficiens, and serum response factor) gene family and specific transcription factors which cooperate with MyoD in the process of muscle-specific gene transcription (Jeffery et al., 1996).RSRFC4 (related to SRF) protein can bind to AT-rich promoter regions which were recognized by other muscle-specific transcription factors (Pollock and Treisman, 1991).Nkx2-5 is an interrelated factor of cardiac chamber formation and development which been shown to increase during adrenergic and pressure-induced cardiac hypertrophy (Saadane et al., 1999).The basic helix-loop-helix (bHLH) transcription factors E12 and E47 regulate cell type-specific transcription and growth by binding to the E box (CANNTG) on target genes (Kho et al., 1997).The result suggested that SMPX might be implicated in muscle development and growth.

Expression pattern and mapping of porcine SMPX gene
RT-PCR analysis demonstrated that porcine SMPX gene expressed mainly in skeletal and cardiac muscles, while at a weak level in spleen and lung.It was similar to the situation observed in human and mouse.Moreover, it is interesting that the expression is stage-dependant.It is up-regulated from 33-day embryo to 65-day embryo and down-regulated from 28-day to adult.As report, there are three climaxes of the generations of myogenic cells, 35 days of foetal life, 55 days of foetal life and 0 to 15 days of postnatal life (Lefaucheur et al., 1995).Our result was consistent with the study.So we suggested SMPX maybe involve with generations of myogenic cells.The results also are consistent with the corresponding reports in human and mouse (Patzak et al., 1999;Palmer et al., 2001).
SMPX was mapped to porcine chromosome Xp24 and was linked to SW1903.SMPX have been mapped to chromosome X in mouse and to chromosome Xp22.1 in human (http://www.ncbi.nlm.nih.gov).The information is in agreement with comparative mapping data between porcine chromosome X and human chromosome X (McCoard et al., 2002).There are several of papers reported quantitative trait loci (QTL) on porcine chromosome X.Cepica et al. (2003) identified QTL for carcass traits and meat quality in the proximal part of SSCX between SW949 and SW2126(0-30 cM).And SW1903 is located between SW949 and SW2126.

Polymorphism and association analysis
An A→G transition was found within the 3'-UTR of SMPX PCR specific fragments which were subjected to RFLP analysis.Allele frequency studies indicated that all the breeds present a polymorphism except the Duroc breed.The allele distribution revealed that introduced pig breeds present a high frequency of allele A whereas the Chinese indigenous breeds have a high frequency of allele G except Min pig.
As reported, SMPX links to the cardiac developing and myoblast proliferation.Overexpression of SMPX in C2C12 myblasts induced lamellipodia formation and differentiation into large myosacs (Kemp et al. 2001).And there was a high correlation between the muscle fiber and intramuscular fat content (Larzul et al., 1997).Moreover Cepica et al. (2003) discovered there was a QTL for carcass traits and meat quality in the region nearby SMPX gene.Here, we provide evidence that a polymorphism in the SMPX gene is associated with intramuscular fat content.Although data sets for some of the individual SMPX genotypes were limited, these results suggest that SMPX or a closely linked gene to SMPX may be important in intramuscular fat content in swine.And it is necessary to repeat this association analyses in other population of pigs.

Figure 1 .
Figure 1.Part of putative promoter sequence 381 bp and putative transcription factor binding sites are underlined and in boldface type.The MEF-2 binding site is in italics and boldface type.

Table 1 .
Primer pairs for porcine SMPX fragments isolation Primer purpose Primer name

Table 2 .
Allele frequencies of the porcine SMPX gene in different breeds

Table 3 .
x 2 test results for the allele frequency distribution among different populations of TaaI-RFLP for the porcine SMPX gene

Table 4 .
T-test results for the association analysis of TaaI-RFLP for the porcine SMPX gene in the experimental population