Mapping , Tissue Distribution and Polymorphism Study of the Porcine SOCS 2 and SOCS 3 Genes

Using the somatic cell hybrid panel (SCHP) and radiation hybrid (IMpRH) panel, porcine SOCS2 gene was mapped at SSC5 (1/2) q21-q24 and closely linked with SW1383 marker (47 cR in distance), while SOCS3 gene was assigned to SSC12p11- (2/3p13) and closely linked with SW2490 (43 cR). The reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to detect the expression of these two genes in the different tissues and the results showed that both SOCS2 and SOCS3 genes were widely expressed in tissues investigated (heart, liver, spleen, lung, kidney skeletal muscle, fat and brain), although some tissues showed lower gene expression. Moreover, SOCS2 and SOCS3 genes had different expression levels at different stages, in different tissues and in different breeds. A G/A substitution, which can be recognized by restriction enzyme of Cfr421, was observed in 5' untranslated region (5'-UTR) of SOCS2 gene. The allele frequencies was investigated by PCR-restriction fragment length polymorphism (PCR-RFLP) method and it showed that the allele frequency among Dahuabai, Erhualian, Yushan, Qingping, Large white and Landrace tested were different. Association analysis in a cross experimental populations revealed no significant association between the SOCS2 gene polymorphism and the economic traits investigated. The full-length coding regions (CDs) of porcine SOCS3 gene was obtained by RT- PCR. (Asian-Aust. J. Anim. Sci. 2006. Vol 19, No. 2 : 165-170)


INTRODUCTION
SOCS (suppressor of cytokine signaling) family is a kind of factors that play key roles in the negative regulation of cytokine signal transduction.They inhibit the cytokineactivated Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway through a negative feedback loop.The family currently comprises eight members: SOCS1-SOCS7 and CIS genes (Masuhara et al., 1997;Starr et al., 1997;Hilton et al., 1998).Proteins coded by this family have a similar domain structure, which consist of an N-terminal variable region, a central conserved SH2 domain, and a conserved C-terminal domain containing a SOCS box.It was reported that SOCS2 gene directly induced by GH while not by IGF-1 (Greenhalgh et al., 2002).Moreover, SOCS2 gene can bind to GH receptor and IGF-1 receptor and inhibit the binding of STAT, which might be the mechanism of SOCS2 gene inhibit GH and IGF-1 signaling pathway (Dey et al., 1998;Greenhalgh et al., 2002).SOCS2 gene deficient mice at twelve weeks after birth exhibited a 30-40% increase in body weight compared with wild-type littermates (Metcalf et al., 2000).SOCS3 gene deficient mice died in uterus due to placental defects, which suggested that SOCS3 gene might play a key role in the formation of the placenta (Roberts et al., 2001).Recent study found that SOCS3 gene was associated with inflammation reactions.SOCS3 gene negatively regulated signaling pathway of IL-6 (Lehmann et al., 2003;Yasukawa et al., 2003).In order to study the functions of porcine SOCS2 and SOCS3 genes and find some traits associated SNPs, we physically mapped porcine SOCS2 and SOCS3 genes, analyzed tissues expression distribution of these two genes at different stages and at different breeds, found a novel SNP in the 5'-UTR of porcine SOCS2 gene.Traits association analysis revealed no significant association between this SNP and the economic traits studied.Moreover, we have isolated porcine SOCS3 gene fulllength CDs.

Primer design
All the primers employed for porcine SOCS2 and SOCS3 genes study were designed using software Primer 5.0.Primers of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were synthesized according to the reported sequence (Janzen et al., 2000) (Table 1).All the PCR fragments were sequenced by a commercial service.

Somatic cell hybrid and radiation hybrid mapping
The somatic cell hybrid panel (SCHP) was used for chromosomal assignments and the radiation hybrid (IMpRH) panel was used for more precise mapping (Yerle et al., 1996;1998).PCR reaction was performed in a 10 µl reaction mixture containing 25 ng of cell hybrid line DNA, 1×PCR buffer (TaKaRa), 0.3 µM of each primer (P1L, P1R and P1'L, P1'R), 300 µM of each dNTPs, 1.5 mM MgCl 2

Tissue distribution of porcine SOCS2 and SOCS3 genes
Gene expression patterns were determined by RT-PCR.Total RNAs were extracted from 90-day embryo of Tongcheng pig, adult Tongcheng pig and adult Landrace.Eight tissues, heart, liver, spleen, lung, kidney, skeletal muscle, fat and brain, were selected to study the expression patterns.All RNAs were isolated using TRIzol reagent (Invitrogen), treated with RNAse-free DNase I (Promega) and precipitated with ethanol.Reverse transcription was performed as described by Pan et al. (2003).PCR was performed in 20 µl reactions containing 2 µl of each reverse transcription products, 1×PCR buffer (TaKaRa), 1.5 mM MgCl 2 , 300 µM dNTPs, 0.3 µM of each primer (P2L, P2R and P2'L, P2'R), and 2 U Taq DNA polymerase (TaKaRa).PCR amplification conditions of SOCS2 were 4 min at 94°C, followed by 27 cycles of 94°C 40 s, 65°C 40 s, 72°C 40 s and a final extension step of 5 min at 72°C.The PCR amplification conditions of SOCS3 were same to SOCS2 except for 69°C anneal temperature and 30 cycles.GAPDH was used as internal control, which was annealing at 60°C and 27 cycles.Eight microlitre PCR products were used to detect the expression profile on 2.0% agarose gels stained with 0.5 µg/ml ethidium bromides.

Porcine SOCS3 gene full-length coding regions isolation
Two fragments of porcine SOCS3 gene were isolated by RT-PCR.The two fragments were cloned to PGEM-T (promega) clone vector and sequenced.There was a 225 bp overlap region between these two fragments.Therefore, the two fragments were assembled to one contig of 2,122 bp.An open reading frame (ORF) was found and the amino acid sequence was deduced with the program Seqman (DNA star, Madison, WI, USA).Homologous between human and pig was analyzed by BLAST tool available on the web site (http://www.ncbi.nlm.nih.gov/BLAST/).

SCHP and RH mapping of the porcine SOCS2 and SOCS3 genes
Porcine SOCS2 and SOCS3 genes were assigned to SSC5 (1/2) q21-q24 and SSC12p11-(2/3p13) by SCHP analysis respectively.RH mapping allowed the locations of these two genes to be defined more precisely.RH analysis showed that SOCS2 was closely linked with SW1383 marker (distance = 47cR, LOD = 8.3) and SOCS3 was closely linked with SW2490 marker (distance = 43cR, LOD = 8.05).The mapping results were displayed on Table 2.

Tissue distribution of SOCS2 and SOCS3 genes
RT-PCR analysis indicated the expression patterns of SOCS2 and SOCS3 genes.There were at least five points need noticing.Firstly, all six tissues (heart, liver, spleen, lung, kidney and skeletal muscle) of Tongcheng pig 90-day embryo expressed SOCS2 and SOCS3 genes.Secondly, the expression level of SOCS2 gene at 90-day embryo stage was relatively higher than at adulthood.Thirdly, there were weak or no expressions of SOCS2 and SOCS3 genes in many tissues at adulthood.Fourthly, there was higher expression in the liver of adult Tongcheng pig than adult Landrace.In addition, SOCS3 gene expressed high in spleen at adulthood (Figure 1).

Genetic variation identification and association analysis
The 349 bp PCR fragment of porcine SOCS2 gene was sequenced and revealed a G to A transition at position of 72    bp.Restriction enzyme analysis revealed a polymorphic Cfr421 site.The two allele-specific patterns obtained after Cfr421 digestion were an uncut 349 bp fragment for allele A and two fragments of 276 bp and 73 bp (the 73 bp band was too weak to see) for allele G (Figure 2).The analysis of allele frequency distribution revealed that exotic breeds (Large white and Landrace) had higher frequency of the A allele whereas Chinese native breeds (Dahuabai, Erhualian, Yushan, Qingping) had higher frequency of the G allele (Table 3).The statistical analysis indicated a significant difference between Chinese native breeds and exotic breeds (Table 4).Traits association analysis was performed in an experimental population.There were no significant associated between the polymorphism and the three traits (ADG, BF, LC) (Table 5).

Porcine SOCS3 gene coding regions isolation
The 2,122 bp fragment includes a 690 bp ORF, which codes 229 amino acids, was obtained by RT-PCR.The blast results indicated that there was 94% identity homologous between the ORF and human SOCS3 gene ORF (Genbank: NM_003955) and 96% homologous of amino acid sequence.Porcine SOCS3 gene has been submitted to the genebank database (AY944571).

Mapping of porcine SOCS2 and SOCS3 genes
In human being, SOCS2 gene was located at 12q21.3-q23 (Yandava et al., 1999) and SOCS3 gene was located at 17q25.3 (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db =gene&cmd=Retrieve&dopt=Graphics&list_uids=9021).Therefore, porcine SOCS2 and SOCS3 genes mapping results were consistent with human-pig comparative mapping results and the location of porcine SOCS3 was confirmed by Zhao et al. (in press).Porcine SOCS3 gene had been located at the same region by Zhao et al. (in press).Moreover, porcine SOCS2 gene was linked to the important porcine growth factor IGF-1 gene, which was located at 5q23 or 5q25 (http://www.toulouse.inra.fr/lgc/pig/cyto/gene/chromo/SSCG5.htm).Porcine SOCS3 gene was linked to the important porcine growth factor GH gene, which was located at 12q14 (Chowdhary et al., 1994).The information of gene mapping indicated that porcine SSC5 long arm and porcine SSC12 short arm may closely related to the porcine growth.On the other hand, SOCS2 and SOCS3 genes link  with important growth genes.Therefore, the result of traits association analysis may influence by these linked genes.

Tissue distribution of porcine SOCS2 and SOCS3 genes
All the tissues studied at 90-day embryo expressed SOCS2 and SOCS3 genes indicated that they expressed broadly at swine embryo middle-late period.SOCS2 gene expressed higher at 90-day embryo then at adult period, which may because that GH expressed higher at embryo then at adulthood.In addition, SOCS2 gene expressed higher in liver of adult Tongchen pig then in liver of adult Landrace.It is well known that liver is the major target organ of growth regulation of GH.Therefore, further study to confirm whether the low growth speed of Tongcheng pig dues to the high expression of SOCS2 gene in liver would be necessary.At spleen tissue of both adult Tongcheng pig and Landrace, SOCS3 gene had higher expression.This implied that SOCS3 gene might play an important role in immunity of pig.In rat, SOCS3 gene had relatively high expression at Lung and spleen tissues (Tollet-Egnell et al., 1999).

Polymorphism and association analysis
An A/G transition was found at 5'-UTR of porcine SOCS2 gene.The allele distribution revealed that the Chinese indigenous breeds had higher frequency of the G allele whereas Landrace had higher frequency of the A allele.The frequency of A allele in Large white was much higher than in Chinese native breeds, Although it was lower than 50%.The difference between domestic breeds and foreign breeds may be mainly responsible for the difference of the allele frequencies.In addition, this site may link with some locus which associated with some traits.Therefore, the allele frequencies changed under the selection (Zeng et al., 2005).As a result, the alleles frequencies differ between domestic breeds and foreign breeds.It is well known that 5'-UTR is the regulation regions of expression.Mutation in this region might affect the expression level of gene, which result in change of the function of gene then influence some phenotype traits.It is a pity that there were no significant associated between the novel SNP and traits studied.We will continue to detect SNPs of SOCS2 and SOCS3 genes in the future in order to find some traits associated SNPs.
Porcine SOCS3 gene full-length coding regions were isolated.SOCS3 gene has very important function, but the complete CDs of porcine SOCS3 gene have not been cloned.According to the conserve regions of mouse SOCS3 gene (Genbank: NM_007707) and human SOCS3 gene (Genbank: NM_003955), a primer (5'-CTCCGTGCGCCA TGGTCA-3') was designed for porcine SOCS3 gene isolation.This primer includes the start code (ATG) of porcine SOCS3 gene.Other primers were designed using porcine ESTs information (NCBI).Porcine SOCS3 gene full-length CDs were finally been cloned.The high homologous between human SOCS3 gene and porcine SOCS3 gene indicated that this result is reliable.

Figure 2 .
Figure 2. PCR-RFLP (Cfr421) analysis and genotypes of porcine SOCS2 gene.The genotypes were indicated on the top of the lanes (M stands for 100 bp DNA ladder).
This research was supported by the Key Project of National Natural Science Foundation of China (30330440), the key Project of National Basic Research and Developmental Plan of China (G2000016103), National High Science and Technology Foundation of China (2004AA222170), Doctorate Foundation of Ministry of Education of China and National Natural Science Foundation of China (30270952).

Table 1 .
Primer pairs for Porcine SOCS2, SOCS3 and GAPDH genes fragments isolation

Table 3 .
Genotypes and alleles frequency of porcine SOCS2 gene in different breeds, based on the genotyping of the PCR-PFLP

Table 4 .
Significance chi-squared test results for the allele frequency distribution among different populations of Cfr421-RFLP for the

Table 5 .
Association analyses of SOCS2 gene cfr421-RFLP genotypes with average day gain, backfat thickness and lipid content