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gene organization, alternate splicing and expression

By Diane Carter,2014-11-15 23:24
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gene organization, alternate splicing and expression

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    gene organization, alternate splicing and expression

    pattern of porcine visfatin gene

     Zaiqing Yang, Huaping Chen, Tao Xia, Lei Zhou, Xiaodong Chen, Li Gan, Wenshu Yao, Yin

    Peng.

    College of Life Science and Technology, Huazhong Agricultural University, Wuhan city, Hubei

    province, 430070, P.R.China yangzq@mail.hzau.edu.cn,

    Abstract

    Visfatin is a newly discovered visceral fat-specific adipocytokine. It is upregulated in obesity and exerts insulin-mimetic effects in various tissues in human and mouse. We reported here the cloning and characterization of porcine visfatin, its three alternate splicing variants. Sequence analysis indicated that variant 1 is the predominant form among species, which contains an open reading frame of 1473 bp encoding a 52-kDa protein of 491 amino acids. While the other two variants were predicted to encode two 3’truncated proteins due to early termination. The nucleotide and amino acid sequences

    deduced from variant 1 were conservative across species. The porcine visfatin gene was composed of eleven exons at least and had exactly the same exon/intron structure as the human orthologs. Nested PCR showed that variant 1 and variant 3 were ubiquitously expressed in porcine tissues and that variant 2 was expressed in most tissues examined with exception of testis and liver. The discovery of the three variants of visfatin in porcine would be useful to the further investigation of the function of the visfatin gene.

Keywords: visfatin; Molecular cloning; alternate splicing; adipocytokine; porcine

1 INTRODUCTION

    As the life level has been improved in the developed world, a direct result is human being's increasing body weight, however, a similar rapid rise is the incidence of type 2 diabetes mellitus and other metabolic related disease which is closely linked to obesity[1]. There are two kinds of adipose tissues in human’s body, namely abdominal (visceral) adipose tissue and subcutaneous adipose tissue, Studies

    showed that abdominal adipose tissue is more closely relative with those diseases[2]. Visceral obesity could enhance the secretion of TNF- and other proinflammatory adipocytokines, and reduce the

    secretion of antidiabetic and anti-inflammatory adipocytokines from visceral adipose tissue [3].

    Recently, Fukuhara et al. identified visfatin as a new protein that was preferentially produced in the intra-abdominal adipose tissue of obese mice and humans, it could exert insulin-mimetic effects in various tissue in human and mouse[4]. Visfatin was first identified to be involved in the maturation of B-cell precursors, it can synergizes with IL-7 and stem cell factor to enhance the development of early stage B cell, and thus named Pre-B Cell Colony-Enhancing Factor (PBEF) [5]. Subsequently, it was found that the expression of PBEF was upregulated by both mechanical force and inflammatory stimuli[5, 6]. Its bacterial homolog has been identified as an enzyme, nicotinamide

    phosphoribosyltransferase, that is involved in the biosynthesis of nicotinamide adenine dinucleotide (NAD)[7], the activity recently also demonstrated for murine PBEF[8]. Several lines of evidences have suggested that visfatin played a fundamental role in the body.

    The pig is evolutionarily related to human and is considered to share many biological properties with human. An example is that resistin, an important metabolic molecule, has a similar expression pattern This work was supported by the High Education Doctorial Subject Research Program (No: 20010504003), the grants from General Program and Key Program of National Natural Science Foundation and 863 Program of China to Dr. Zaiqing Yang.

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     with human, which is, however different from the data collected from mouse experiments[9]. Since the pig offers the advantage of this compared with murine and rat, the pig is more suitable to serve as a metabolic disease model.

    In this paper, we reported the molecular cloning and characterization of the three variants of porcine visfatin mRNA, together with the deduced probable genomic structure and expression pattern of visfatin in pig.

2 MATERIALS AND METHODS

    2.1 RNA isolation

    Three Meishan pigs (three males, 3-month old, 25?1.5 kg in body weight, all the pigs given ad libitum

    access to a standard growing diet) were killed by cardiac puncture, Ten tissues (heart, liver, spleen, lung, kidney, stomach, subcutaneous adipose tissues, longissimus dorsi muscle, brain, testicle) were freshly collected and immediately frozen in liquid nitrogen pending RNA extraction. The RNA was extracted with Trizol (Sangon, China) according to the manufacturer’s protocol. Then all RNA was

    treated with RNase-free DNase I (Takara, Japan) to remove contaminating genomic DNA.

     2.2RT-PCR

    Two micrograms of total RNA from each tissue samples was used to synthesize cDNA using M-MLV reverse transcriptase (Promega, American) as described in protocol. Briefly, total RNA was combined with 0.2;!g oligo(dT)18 and H2O and preheated at 70 ?C for 5 min to denature secondary structures. The mixture was then cooled rapidly to 0 ?C and then 10;!l 5 RT buffer, 200 μM dNTPs, 50 U RNase

    Inhibitor (Promega, American) and 200 U M-MLVreverse transcriptase were added for a total volume of 50 μl. The RT mix was incubated at 42 ?C for 60 min.

     2.3 Coding sequence isolation

    Following cDNA synthesis, Three primers (P1, P2, P10) designed based on two EST sequences available in the TIGR Unique Gene Indices database (TC211514, TC204083) were used to amplify the coding sequence of visfatin about 1613 bp in length, (Table 1). P1 and P10 were used to perform the first amplification, and then 1;!l aliquots of the primary PCR products were used as the template for the second amplification with P2 and P10. PCR was performed in a total volume of 25;!l containing

    2.5;!l of standard buffer, 1.5 mM MgCl2, 0.1;!M of each primer, 200;!M dNTPs, and 0.5 U of Taq

    polymerase (Casarray, Shanghai, China). Cycling conditions consisted of 35 cycles of 94 ?C for 30 s, 55 ?C for 30 s and 72 ?C for 1 min. PCR products were analyzed by electrophoresis through 1.0% agrose gels in 1 TBE buffer. Then it was gel-purified using a DNA Extraction Kit (V-gene, China) and TA-cloned into pMD-18T vector (Takara, Japan). The clones with the correct size of insert were sequenced by Beijing AuGCT Biotechnology Company.

     2.4 3’-Race

    In order to obtain the full-length visfatin cDNA sequence, we carried out RACE technology to clone porcine visfatin 5’ and 3’ends. Briefly, for 3’RACE, total RNA from porcine tissues was

    reverse transcribed with AMV reverse transcriptase (Promega, American) using

    oligo(dT)18-containing anchor primer (See Table 1). Touchdown PCR (TD-PCR) was performed using a combination of the adaptor primer and a gene-specific primer (P7). The gene-specific primer P7 was designed based on the EST sequence of porcine visfatin available in the TIGR Unique Gene Indices database (TC204083). Then the product was further identified with primer

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    P8 that is located on the downstream of P7 in the same forward. The PCR condition was 94 ?C for 3 min, followed by 16 cycles of 94 ?C for 30 s, 62 ?C;; 47 ?C for 30 s, and 72 ?C for 1 min, then

    14 cycles of 94 ?C for 30 s, 47 ?C for 30 s, and 72 ?C for 1 min, and the final extension was at 72 ?C for 7 min.

     Table 1 Primers for pig visfatin cDNA cloning

Primer Sequence (5’-3’) Primer location in cDNA

     AGCGGTGACTTAAGCAACGGAGC -35--13 P1 ATTTTTCTCCTTCCTCGCAGCCG 1-23 P2

    -385 ATGAAAAGGGATGGAACTACATTCTTGAG 357P3

    TCCTGAGGGCTCTGTCATTC 424-443 P4

    TTCATGCCTTCTACAATCTC 1154-1173 P5

    GGGATCAGCAACTGGGTCCTTGAA 1306-1330 P6

    GAGGAAGGAAAAGGAGACCTTGAG 1397-1420 P7

    GCTGAATATTGAACTGGAAGCAGCAC 1510-1535 P8

    GCCTAATGAGGTGCTGCTTCCAGTTC 1520-1545 P9

    GCATGCACGTAAACACACACCCTA 1592-1615 P10

    Adaptor primer AGCGTCTAGAGGTACCGAATTC

    Anchor primer AGCGTCTAGAGGTACCGAATTCTTTTTTTTTTTTTTTTTT

     2.5 Sequence analysis

    Sequence alignment and analysis were carried out with the BLAST network service of the National Center for Biotechnology Information and the National Animal Genome Research Program and with the online services of Prosite (http://www.expasy.org/prosite). The nucleotide and amino acid sequences of other species’ visfatin used for comparison were downloaded from Genbank.

     2.6 Expression pattern analysis of the visfatin gene in porcine

    Nested PCR was carried out to analyze the expression pattern of the visfatin gene. For the variant 1 and the variant 2 transcript, The first round of PCR was performed using a combination of P3 and P6 (See Table 1), The nested PCR was performed using an aliquot of the first PCR reaction mixture as a template, with a combination of P4 and P5 (See Table 1). The procedures of the two rounds of PCR were identical: 94 ?C for 3 min, then 30 cycles of 94 ?C for 30 s, 58 ?C for 45 s, and 72 ?C for 40 s, followed by a final extension at 72 ?C for 7 min. As for the detection of variant 3, P2 and P10, P3 and P9 were adopted in the same way. As a template control, pig;,-actin was amplified by RT-PCR with

    primer pairs P11 (5-GGACTTCGAGCAGGAGATGG-3) and P12

    (5-GCACCGTGTTGGCGTAGAGG-3). To insure that no false positive PCR fragments were generated from the contaminating genomic DNA, all the primers were tested using porcine genomic DNA as a negative control. 2 μl of cDNA of each tissue was used for the amplification performed in 50

    μl of 1 PCR buffer. For robustness issues, each sample was performed in triplicate at least.

     2.7 Nucleotide sequence accession numbers

    The nucleotide sequences of 2.2 kb, 1.9 kb and 1.2 kb porcine visfatin mRNA have been submitted to Genbank under accession number DQ001974, DQ231167 and DQ231168 respectively.

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3 RESULTS

    3.1 Molecular cloning and characterization of the three variants of porcine visfatin mRNA. According to the EST sequences (TC211514TC204083), which represent the two terminal sequences

    of the porcine visfatin, primers (Table 1) were designed to obtain the full length of porcine visfatin cDNA. Through 3’RACE experiments, products of 800 bp sequences were yielded. Through nested

    PCR, a fragment of 1613 bp containing the coding sequence was amplified. By assembling these two overlapping fragments, a visfatin cDNA of 2184 bp was obtained, which contains 67 bp of 5’UTR,

    1473 bp of coding region that encodes a protein of 491 amino acid residues with an estimated molecular mass of 52-kDa, and 644 bp of 3’UTR (Fig. 1).

    ATTTTTCTCCTTCCTCGCAGCCGCGCCGGGCAGCCCGCGGCGCGTGGCCCCCACACTCCGGCCCGAGATG 70 M AATGCTGCGGCAGAAGCCGAATTCAACATCCTCCTGGCCACCGACTCGTACAAGGTTACTCATTATAAAC 140 N A A A E A E F N I L L A T D S Y K V T H Y K AGTACCCACCCAACACAAGCAAAGTTTATTCCTACTTTGAATGCCGTGAAAAGAAGACAGAAAACTCCAA 210 Q Y P P N T S K V Y S Y F E C R E K K T E N S K AATAAGGAAAGTGAAATATGAGGAAACAGTATTTTATGGGTTGCAGTACATTCTTAATAAGTACTTAAAA 280 I R K V K Y E E T V F Y G L Q Y I L N K Y L K GGTAAAGTAGTGACCAAAGAGAAGATCCAAGAAGCCAAAGAGGTGTACAAAGAGCATTTCCAAGATGATG 350 G K V V T K E K I Q E A K E V Y K E H F Q D D TCTTTAATGAAAAGGGATGGAACTACATTCTTGAGAAATACGATGGGCATCTTCCAATAGAGGTAAAAGC 420 V F N E K G W N Y I L E K Y D G H L P I E V K A TGTTCCTGAGGGCTCTGTCATTCCCAGAGGAAATGTTCTCTTCACAGTGGAAAACACAGATCCAGAGTGT 490 V P E G S V I P R G N V L F T V E N T D P E C TACTGGCTTACAAATTGGATTGAGACTATTCTTGTTCAGTCCTGGTATCCAATCACAGTGGCCACAAATT 560 Y W L T N W I E T I L V Q S W Y P I T V A T N CTAGAGAGCAGAAGAAAATATTGGCCAAATATTTGTTAGAAACATCTGGTAACTTAGATGGTCTGGAATA 630 S R E Q K K I L A K Y L L E T S G N L D G L E Y CAAGTTACATGATTTTGGCTACAGAGGAGTCTCTTCCCAAGAGACTGCTGGCATAGGGGCATCTGCTCAT 700 K L H D F G Y R G V S S Q E T A G I G A S A H TTGGTTAACTTCAAAGGTACAGATACAGTAGCAGGAATTGCTTTAATTAAAAAATACTATGGGACGAAAG 770 L V N F K G T D T V A G I A L I K K Y Y G T K ATCCTGTTCCAGGCTATTCAGTGCCAGCAGCAGAACACAGTACCATAACAGCTTGGGGGAAGGACCATGA 840 D P V P G Y S V P A A E H S T I T A W G K D H E AAAAGATGCTTTTGAACATATAGTAACACAATTTTCATCAGTGCCTGTATCTGTGGTCAGCGATAGCTAT 910 K D A F E H I V T Q F S S V P V S V V S D S Y GACATTTATAATGCGTGTGAGAAAATATGGGGTGAAGATCTAAGACATTTAATAGTATCAAGAAGTACAG 980 D I Y N A C E K I W G E D L R H L I V S R S T AGGCACCACTAATAATCAGACCTGATTCTGGAAATCCTCTTGACACTGTATTAAAGGTTTTGGATATTTT 1050 E A P L I I R P D S G N P L D T V L K V L D I L AGGAAAGAAGTTCCCTGTTACTGAGAACTCAAAGGGCTACAAGTTGCTGCCACCTTATCTTAGAGTTATT 1120 G K K F P V T E N S K G Y K L L P P Y L R V I CAAGGAGATGGAGTAGATATTAATACCTTACAAGAGATTGTAGAAGGCATGAAGCAAAAAAAATGGAGTA 1190 Q G D G V D I N T L Q E I V E G M K Q K K W S TTGAAAATATTGCCTTTGGTTCTGGTGGAGCTTTGCTACAGAAGTTAACGAGAGATCTCTTGAACTGTTC 1260 I E N I A F G S G G A L L Q K L T R D L L N C S CTTTAAATGTAGTTATGTCGTAACCAATGGTCTTGGGATTAATGTCTTCAAGGACCCAGTTGCTGATCCC 1330 F K C S Y V V T N G L G I N V F K D P V A D P AACAAAAGGTCCAAAAAGGGCCGATTATCTTTACATAGGACACCAGGAGGGAATTTTGTTACACTTGAGG 1400 N K R S K K G R L S L H R T P G G N F V T L E AAGGAAAAGGAGACCTTGAGGAATACGGTCATGATCTTCTCCATACTGTCTTCAAGAATGGAAAGGTGAC 1470 E G K G D L E E Y G H D L L H T V F K N G K V T AAAAAGCTATTCATTTGATGAAGTAAGAAAAAACGCACAGCTGAATATTGAACTGGAAGCAGCACCTCAT 1540 K S Y S F D E V R K N A Q L N I E L E A A P H TAGGCTTTGTTTTATGACTGGATGTGTGTGTGTGTGTGTACCTGCGTGTGCTTGGGTGTGTGTTTACGTG 1610 CATGCATGCACGTATACACCTGTGTGTGTGAGTATGTAATGCATAATGTTTATTGTACAGATGTGTGGGG 1680 TTTCTTTGTGTTTTATGACACATTACAGCCAAATTATTTGTTGGTTTATGGACATACTACCCTTTCATTT 1750 TTTTGTTTGTTTTTTGTTTGTTTGTTTGGGGCCAGTGTTTAGGTGATCTCAAATTAGGAAATGCACTTAA 1820 CCATGTAAAAGGTAATTGCTAAAGTAAGCTTTTTAGGGCCCTTTGCCAATAGATAGTAATTCAATCTGGT 1890 ATTGATCTTTTCACAAACAGAACCAAGAAACTTTTATATATATAACTCAAGATCACATAAAACAGATTTG 1960 CATAAAATTATCATGATTGCTTTATGTTTATATTTAACTTGTATTTTTGTACAAACAAGATTGTGTAAGA 2030 TATATTTAAAGTTTCAGTGATTTAACAGTCTTTCCAACTTTTCATGATTTTTATGAGCACAGACTTTCAA 2100 GAAAATACTTGAAAATAATTACATTGCCTTTTGTCCATTAATCAGCAAATAAAACATGGCCTTAACAAAA 2170 AAAAAAAAAAAAAA Fig. 1 The full-length cDNA sequence and the deduced amino acid sequence of porcine visfatin variant 1 which is a predominantly form in different species. Amino acid residues were given in the single letter code. Nucleotides are numbered to the right. Initiation codons ATG and termination codons TAG are indicated in bold face. The polyadenylation signal (AATAAA) is underlined. The nucleotide sequence has been deposited in the GenBank under Accession No. DQ001974.

    In the process of the nest PCR amplification, we encountered two additional shorter variants of visfatin, which are 1958 bp (variant 2) and 1258 bp (variant 3), respectively. Sequence comparison revealed that variant 2 and variant 3 are resulted from the deletion from variant 1 (Fig. 2).

     1 90

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     variant_1 (1) AAATACGATGGGCATCTTCCAATAGAGGTAAAAGCTGTTCCTGAGGGCTCTGTCATTCCCAGAGGAAATGTTCTCTTCACAGTGGAAAAC variant_2 (1) AAATACGATGGGCATCTTCCAATAGAGGTAAAAGCTGTTCCTGAGGGCTCTGTCATTCCCAGAGGAAATGTTCTCTTCACAGTGGAAAAC variant_3 (1) AAATACGATGGGCATCTTCCAATAGAG--------------------------------------------------------------- Consensus (1) AAATACGATGGGCATCTTCCAATAGAGGTAAAAGCTGTTCCTGAGGGCTCTGTCATTCCCAGAGGAAATGTTCTCTTCACAGTGGAAAAC 91 variant_1 (91) 180 variant_2 (91) ACAGATCCAGAGTGTTACTGGCTTACAAATTGGATTGAGACTATTCTTGTTCAGTCCTGGTATCCAATCACAGTGGCCACAAATTCTAGA variant_3 (28) ACAGATCCAGAGTGTTACTGGCTTACAAATTGGATTGAGACTATTCTTGTTCAGTCCTGGTATCCAATCACAGTGGCCACAAATTCTAGA Consensus (91) ------------------------------------------------------------------------------------------ ACAGATCCAGAGTGTTACTGGCTTACAAATTGGATTGAGACTATTCTTGTTCAGTCCTGGTATCCAATCACAGTGGCCACAAATTCTAGA variant_1 (181) 181 variant_2 (181) 270 variant_3 (28) GAGCAGAAGAAAATATTGGCCAAATATTTGTTAGAAACATCTGGTAACTTAGATGGTCTGGAATACAAGTTACATGATTTTGGCTACAGA Consensus (181) GAGCAGAAGAAAATATTGGCCAAATATTTGTTAGAAACATCTGGTAACTTAGATGGTCTGGAATACAAGTTACATGATTTTGGCTACAGA ------------------------------------------------------------------------------------------ variant_1 (271) GAGCAGAAGAAAATATTGGCCAAATATTTGTTAGAAACATCTGGTAACTTAGATGGTCTGGAATACAAGTTACATGATTTTGGCTACAGA variant_2 (271) 271 variant_3 (28) 360 Consensus (271) GGAGTCTCTTCCCAAGAGACTGCTGGCATAGGGGCATCTGCTCATTTGGTTAACTTCAAAGGTACAGATACAGTAGCAGGAATTGCTTTA GGAGTCTCTTCCCAAGAGACTGCTGGCATAGGGGCATCTGCTCATTTGGTTAACTTCAAAGGTACAGATACAGTAGCAGGAATTGCTTTA variant_1 (361) ------------------------------------------------------------------------------------------ variant_2 (361) GGAGTCTCTTCCCAAGAGACTGCTGGCATAGGGGCATCTGCTCATTTGGTTAACTTCAAAGGTACAGATACAGTAGCAGGAATTGCTTTA variant_3 (28) 361 Consensus (361) 450 ATTAAAAAATACTATGGGACGAAAGATCCTGTTCCAGGCTATTCAGTGCCAGCAGCAGAACACAGTACCATAACAGCTTGGGGGAAGGAC variant_1 (451) ATTAAAAAATACTATGGGACGAAAGATCCTGTTCCAGGCTATTCAGTGCCAGCAGCAGAACACAG------------------------- variant_2 (426) ------------------------------------------------------------------------------------------ variant_3 (28) ATTAAAAAATACTATGGGACGAAAGATCCTGTTCCAGGCTATTCAGTGCCAGCAGCAGAACACAG Consensus (451) 451 540 CATGAAAAAGATGCTTTTGAACATATAGTAACACAATTTTCATCAGTGCCTGTATCTGTGGTCAGCGATAGCTATGACATTTATAATGCG ------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------

    541

    630 variant_1 (541) TGTGAGAAAATATGGGGTGAAGATCTAAGACATTTAATAGTATCAAGAAGTACAGAGGCACCACTAATAATCAGACCTGATTCTGGAAAT variant_2 (426) ------------------------------------------------------------------------------------------ variant_3 (28) ------------------------------------------------------------------------------------------ Consensus (541) 631 720 variant_1 (631) CCTCTTGACACTGTATTAAAGGTTTTGGATATTTTAGGAAAGAAGTTCCCTGTTACTGAGAACTCAAAGGGCTACAAGTTGCTGCCACCT variant_2 (426) ---------------------GTTTTGGATATTTTAGGAAAGAAGTTCCCTGTTACTGAGAACTCAAAGGGCTACAAGTTGCTGCCACCT variant_3 (28) ------------------------------------------------------------------------------------------ Consensus (631) GTTTTGGATATTTTAGGAAAGAAGTTCCCTGTTACTGAGAACTCAAAGGGCTACAAGTTGCTGCCACCT 721 810 variant_1 (721) TATCTTAGAGTTATTCAAGGAGATGGAGTAGATATTAATACCTTACAAGAGATTGTAGAAGGCATGAAGCAAAAAAAATGGAGTATTGAA variant_2 (495) TATCTTAGAGTTATTCAAGGAGATGGAGTAGATATTAATACCTTACAAGAGATTGTAGAAGGCATGAAGCAAAAAAAATGGAGTATTGAA variant_3 (28) ------------------------------------------------------------------------------------------ Consensus (721) TATCTTAGAGTTATTCAAGGAGATGGAGTAGATATTAATACCTTACAAGAGATTGTAGAAGGCATGAAGCAAAAAAAATGGAGTATTGAA 811 900 variant_1 (811) AATATTGCCTTTGGTTCTGGTGGAGCTTTGCTACAGAAGTTAACGAGAGATCTCTTGAACTGTTCCTTTAAATGTAGTTATGTCGTAACC variant_2 (585) AATATTGCCTTTGGTTCTGGTGGAGCTTTGCTACAGAAGTTAACGAGAGATCTCTTGAACTGTTCCTTTAAATGTAGTTATGTCGTAACC variant_3 (28) ------------------------------------------------------------------------------------------ Consensus (811) AATATTGCCTTTGGTTCTGGTGGAGCTTTGCTACAGAAGTTAACGAGAGATCTCTTGAACTGTTCCTTTAAATGTAGTTATGTCGTAACC 901 976 variant_1 (901) AATGGTCTTGGGATTAATGTCTTCAAGGACCCAGTTGCTGATCCCAACAAAAGGTCCAAAAAGGGCCGATTATCTT variant_2 (675) AATGGTCTTGGGATTAATGTCTTCAAGGACCCAGTTGCTGATCCCAACAAAAGGTCCAAAAAGGGCCGATTATCTT variant_3 (28) -----------------------------------------------------GTCCAAAAAGGGCCGATTATCTT Consensus (901) AATGGTCTTGGGATTAATGTCTTCAAGGACCCAGTTGCTGATCCCAACAAAAGGTCCAAAAAGGGCCGATTATCTT

    Fig. 2 Comparison of the sequences of the three variants (just shown the different part).

    Through searching the National Animal Genome Research Program (NAGRP) Blast Server using the sequence of porcine visfatin cDNA as query, partial genomic fragments porcine visfatin gene were identified, after compared with the genomic sequence of Homo sapiens visfatin. By comparing cDNA sequence and the genomic sequences, predicted genomic structure (Fig. 3) and the exon-intron boundaries (Table 2) of visfatin gene was obtained, except for the regions covering the first exon and the eighth exon. The splice junction sequences of all the analyzed introns conform to the GT-AG rule[10]. Sequence analysis revealed that variant 2 has skipped exon 7, while variant 3 has skipped exon 5, 6, 7, 8, 9 and parts of exon 4 and 10, these two short variants are predicted to encode two 3’

    truncated proteins due to early termination.

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     (A)

    (B)

    Fig. 3 The probable gene structure and alternate splicing model of porcine visfatin. (A) The probable gene structure; (B) Alternate splicing model of porcine visfatin. Through the comparison of eight porcine genomic sequences containing fragment of visfatin gene with Homo sapiens counterpart, we deduced the probable gene structure of porcine visfatin gene, variant 1 has skipped exon 7, while variant 2 has skipped exon 5, 6, 7, 8, 9 and part of exon 4 and 10.

    Table 2 Exonintron boundaries of the porcine visfatin gene

Exon No. Size (bp) TI number 5’ splice donor 3’ splice acceptor

     1 >124 unavailable ?

    2 157 848156682 AAGgtattatttt tttcatatagGTT

    3 104 857276627(partial overlap) ? atccttttagGTA

    4 129 847951690 GAGgtgaattgtt ttcttaccagAAT

    773985486 5 159 GAGgtaagaaact cttattgcagACT

    744325675 6 137 CAGgtaaaattct tattttccagACT

    226 781228608 7 AAGgtaaatttta tgttttctagTAC

    8 120 unavailable ? ?

    847887227 9 141 GGGgtatgtcctt tttaatctagATT

    10 135 774255925 CATgtatgtatct tggattccagATT

    11 >734 860474869 tattgtgcagGAT

    4 (within) GAGgtaaaagctg 10(within) ccaacaaaagGTC

? denotes the region has not been determined yet, the Exonintron boundaries of the porcine

    visfatin gene are deduced from the comparison of porcine visfatin cDNA with its genomic sequences, and then identified by the comparison of porcine genomic sequences with the visfatin genomic sequence of Homo sapiens, the sequences can be found in Genbank by the corresponding numbers. This table also includes the 5’ splice donor and 3’ splice acceptor

    located in exon 4 and exon 10 of variant 1 respectively. Exon sequences are in uppercase letters, and intron sequences are in lowercase.

    3.2 Alignment and phylogenetic tree analysis of porcine visfatin with other sources. Comparison of the coding sequences and the deduced amino acid sequences of visfatin between porcine and other species shows highly similarity, the coding sequences comparison shows that the normal porcine visfatin sharing 95.5 similarity with Homo sapiens, 91.6 with Mus musculus,

    90.0 with Rattus norvegicus, 84.1 with Gallus gallus, 71.4 with Tetraodon nigroviridis, 61.8

    with Danio rerio, and 48.9 with Haemophilus ducreyi. The amino acid sequences comparison

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    Mus musculus, 97.4 with Rattus norvegicus, 93.9 with Gallus gallus, 85.8 with Tetraodon

    nigroviridis, 57.0 with Danio rerio, and 29.1 with Haemophilus ducreyi (Fig. 4). Several modification sites were also observed in the deduced amino acid sequence (Fig. 4).

    1 80 Su (1) ----MNAAAEAEFNILLATDSYKVTHYKQYPPNTSKVYSYFECREKKTENSKIRKVKYEETVFYGLQYILNKYLKG---- Ho (1) ----MNPAAEAEFNILLATDSYKVTHYKQYPPNTSKVYSYFECREKKTENSKLRKVKYEETVFYGLQYILNKYLKG---- Mu (1) ----MNAAAEAEFNILLATDSYKVTHYKQYPPNTSKVYSYFECREKKTENSKVRKVKYEETVFYGLQYILNKYLKG---- Ra (1) ----MNAAAEAEFNILLATDSYKVTHYKQYPPNTSKVYSYFECREKKTENSKVRKVKYEETVFYGLQYILNKYLKG---- Ga (1) ---MECAAAGAEFNILLATDSYKVTHYKQYPPNTSKVYSYFECREKKTENSKLRKVKYEETVFYGLQYILNKYLKG---- Te (1) -----MEHRDSDFNILLATDSYKVTHYKQYPPNTSKVYSYFECREKRTDPSKSRKVTYDKTVFYGLQYILHKYLKG---- Da (1) -------MAAQDFNFLLATDSYKITHYKQYPPNINKVYSYFECRHKKG-------AQFSEVVFFGLQYLLKKYLTG---- Ha (1) MDNLLNYSSRASAIPSLLCDFYKTSHRIMYPECSQIIYSTFTPRSNEQ------APYLTQVVSFGFQAFIIKYLIHYFND (1) Consensus MNAAAEAEFNILLATDSYKVTHYKQYPPNTSKVYSYFECREKKTENSKLRKVKYEETVFYGLQYILNKYLKG (A) (B)(A) (C) (D) Su (73) 81 Ho (73) 160 Mu (73) KVVTKEKIQEAKEVYKEHFQDDVFNEKGWNYILEKYDGHLPIEVKAVPEGSVIPRGNVLFTVENTDPECYWLTNWIETIL Ra (73) KVVTKEKIQEAKDVYKEHFQDDVFNEKGWNYILEKYDGHLPIEIKAVPEGFVIPRGNVLFTVENTDPECYWLTNWIETIL Ga (74) KVVTKEKIQEAKEVYREHFQDDVFNERGWNYILEKYDGHLPIEVKAVPEGSVIPRGNVLFTVENTDPECYWLTNWIETIL Te (72) KVVTKEKIQEAKEVYREHFQDDVFNERGWNYILEKYDGHLPIEVKAVPEGSVIPRGNVLFTVENTDPECYWLTNWIETIL Da (63) KVVTKEKIKEAKEVYREHFQDDVFNEKGWNYILEKYDGHLPIEIKAVPEGSVIPRGNVLFTVENTDPECYWLTNWIETIL Ha (75) KVVTPEKIQEAKDVYREHFQDDVFNEKGWTYILEKYNGHLPIEIKAVPEGSVIPRGNVLFTVESTDPECYWLTNWVETIL (81) Consensus PVITEEKIQEAKVFYQMHFKQAVFDEEGWRKVLEKYDGRLPIRIKAVPEGKIIPRGNVLFTVENTDADFFWLTNYIETML NFFSRDKHDVVTEYSAFIEKTLQLEDTGEHIAKLHELGYLPIRIKAIPEGKTVAIKVPVMTIENTHSDFFWLTNYLETLI KVVTKEKIQEAKEVYREHFQDDVFNEKGWNYILEKYDGHLPIEIKAVPEGSVIPRGNVLFTVENTDPECYWLTNWIETIL Su (153) (C) Ho (153) 161 Mu (153) 240 Ra (153) -VQSWYPITVATNSREQKKILAKYLLETSGNLDGLEYKLHDFGYRGVSSQETAGIGASAHLVNFKGTDTVAGIALIKKYY Ga (154) -VQSWYPITVATNSREQKKILAKYLLETSGNLDGLEYKLHDFGYRGVSSQETAGIGASAHLVNFKGTDTVAGLALIKKYY Te (152) -VQSWYPITVATNSREQKRILAKYLLETSGNLDGLEYKLHDSGYRGVSSQETAGIGASAHLVNLKGTDTVAGIALIKKYY Da (143) -VQSWYPITVATNSREQKKILAKYLLETSGNLDGLEYKLHDFGYRGVSSQETAGIGASAHLVNFKGTDTVAGIALIKKYY Ha (155) -VQSWYPITVATNSREQKKILAKYLLETSGSLEGLEYKLHDFGYRGVSSQETAGIGASAHLVNFKGTDTVAGIALIKKYY (161) Consensus -VQIWYPITVATNSREQKKILAQYLLETSGSLEGLEYKLHDFGYRGVSSQETAGIGASAHLVNFKGTDTVAGIGVIKKFY -VQMWYPITVATISREFKKILAKHLKATSGNLESLDLKLHDFGYRGVSSQESAALGGAAHLVNFCSTDTVAGLLMAQRYY NVSLWQPMTSASIAFAYRTALIKFANETCDNQEHVPFQSHDFSMRGMSSLESAETSGAGHLTSFLGTDTIPALSFVEAYY Su (232) VQSWYPITVATNSREQKKILAKYLLETSGNLDGLEYKLHDFGYRGVSSQETAGIGASAHLVNFKGTDTVAGIALIKKYY Ho (232) (E) (D) Mu (232) (C) (E) Ra (232) 241 Ga (233) 320 Te (231) GTKDPVPGYSVPAAEHSTITAWGKDHEKDAFEHIVTQFSSVPVSVVSDSYDIYNACEKIWGEDLRHLIVSRSTEAPLIIR Da (222) GTKDPVPGYSVPAAEHSTITAWGKDHEKDAFEHIVTQFSSVPVSVVSDSYDIYNACEKIWGEDLRHLIVSRSTQAPLIIR Ha (235) GTKDPVPGYSVPAAEHSTITAWGKDHEKDAFEHIVTQFSSVPVSVVSDSYDIYNACEKIWGEDLRHLIVSRSTEAPLIIR (241) Consensus GTKDPVPGYSVPAAEHSTITAWGKDHEKDAFEHIVTQFSSVPVSVVSDSYDIYNACEKIWGEDLRHLIVSRSTEAPLIIR GTKDPVPGYSVPAAEHSTITAWGKDHEKDAFEHIVTQFSSVPVSVVSDSYDIYNACEKIWGDDLRHIIEGRSPEAPLIIR GTKDPVPGFSLPAAEHSTITAWGKDHEKDAFEHIVKQFPSVPVSVVSDSYDIYNACEKIWGEDLRSLIETRSADAPLVVR Su (312) G--CPMAGFSIPAAEHSTIISWGRSREKDAFECLLDQFPSGPVAVVSDSYDIFKACKHIWGDKLKERVMERSEDSALIIR Ho (312) GS-SSLIGTSIPASEHSVMSSHGVD-ELSTFRYLMAKFPHNMLSIVSDTTDFWHNIT-VNLPLLKQEIIARPENARLVIR Mu (312) GTKDPVPGYSVPAAEHSTITAWGKDHEKDAFEHIVTQFSSVPVSVVSDSYDIYNACEKIWGEDLRHLIVSRSTEAPLIIR Ra (312) (F) Ga (313) 321 Te (311) 400 Da (300) PDSG------------NPLDTVLKVLDILGKKFPVTENSKGYKLLPPYLRVIQGDGVDINTLQEIVEGMKQKKWSIENIA Ha (312) PDSG------------NPLDTVLKVLEILGKKFPVTENSKGYKLLPPYLRVIQGDGVDINTLQEIVEGMKQKMWSIENIA (321) Consensus PDSG------------NPLDTVLKVLDILGKKFPVTENSKGYKLLPPYLRVIQGDGVDINTLQEIVEGMKQKKWSIENVS PDSG------------NPLDTVLKVLDILGKKFPVSENSKGYKLLPPYLRVIQGDGVDINTLQEIVEGMKQKKWSIENVS PDSG------------NPLDTVLKVLEILGKKFPITENSKGYKLLPPYLRVIQGDGVDINTLQEIVEGMKKNKWSIENIA Su (380) PDSG------------NPLDTVLKVLEILGKKFIPVENSKGYKVLPPYIRVIQGDGVDINTLQEIVEGMKEHKWSIENIA Ho (380) PDSG------------DPAETLLEVIKILEECFGCSLNSVGYKVLPSYLRIIQGDGIDLNSINEILQKLSDEGWSAENVF Mu (380) PDSGNFFAIICGDPTADTEHERKGLIECLWDIFGGTVNQKGYKVINPHIGAIYGDGVTYEKMFKILEGLQAKGFASSNIV Ra (380) PDSG NPLDTVLKVLEILGKKFPVTENSKGYKLLPPYLRVIQGDGVDINTLQEIVEGMKQKKWSIENIA Ga (381) (G) (E) (C) (H) Te (379) 401 Da (368) 480 Ha (392) FGSGGALLQKLTRDLLNCSFKCSYVVTNGLGINVFKDPVADP-NKRSKKGRLSLHRTPGGNFVTLEEGKGDLEEYGHDLL (401) Consensus FGSGGGLLQKLTRDLLNCSFKCSYVVTNGLGINVFKDPVADP-NKRSKKGRLSLHRTPAGNFVTLEEGKGDLEEYGQDLL FGSGGALLQKLTRDLLNCSFKCSYVVTNGLGVNVFKDPVADP-NKRSKKGRLSLHRTPAGNFVTLEEGKGDLEEYGHDLL FGSGGALLQKLTRDLLNCSFKCSYVVTNGLGVNVFKDPVADP-NKRSKKGRLSLHRTPAGTFVTLEEGKGDLEEYGHDLL Su (459) FGSGGALLQKLTRDLLNCSFKCSYVVTNGLGVNVFKDPVADP-NKRSKKGRLSLHRTPAGDFVTLEEGKGDLEEYGQDLL Ho (459) FGSGGALLQKLTRDLLNCSFKCSYVVTNGLGVNVFKDPVADP-NKRSKKGRLSLHRTQSGDFVTLEEGKGDLEEYGADLL Mu (459) FGCGSALLQKINRDTLNCAFKCSYVETCGKGMDVYKQPVTDP-SKGSKRGRLSLRRNSDGFIETVERGAGKPEE---DML Ra (459) FGVGAQTYQRNTRDTLGFALKATSITINGEEKAIFKNPKTDDGFKKSQKGRVKVLSR-----DTYVDGLTSADDFSDDLL Ga (460) FGSGGALLQKLTRDLLNCSFKCSYVVTNGLGINVFKDPVADP NKRSKKGRLSLHRTPAGNFVTLEEGKGDLEEYG DLL Te (458) (E) (B)(A) (E) Da (444) Ha (467) (A) (E) (C) (F) (481) Consensus 481 534 HTVFKNGKVTKSYSFDEVRKNAQLN-IELEAAPH-------------------- HTVFKNGKVTKSYSFDEIRKNAQLN-IELEAAHH-------------------- HTVFKNGKVTKSYSFDEVRKNAQLN-IEQDVAPH-------------------- HTVFKNGKVTKSYSFDEVRKNAQLN-MEQDVAPH-------------------- HTVFKNGKVTKSYSFDEVRQNARLKNSELETASH-------------------- HTVFQNGKIVKTYTFDEVRDNAKLKESELQEL---------------------- VTVFENGTILQEYTLDEIRKAAQLREEDLSPSLHNREDGTTLDIHQKHIMNGVH ELLFEDGKLLRQTDFDEIRQNLLVSRTTL------------------------- HTVFKNGKVTKSYSFDEVRKNAQLN IELE A H (C)

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     http:///week114

    Fig. 4 Comparison of the deduced porcine visfatin protein with other species’ counterparts and

    sequence analysis. Highly conserved regions are shaded. Modification sites are underlined or boxed. (A) Protein kinase C phosphorylation site; (B) N-glycosylation site; (C) Casein kinase II phosphorylation site; (D) Tyrosine kinase phosphorylation site; (E) N-myristoylation site; (F) Tyrosine sulfation site; (G) Amidation site; (H) cAMP- and cGMP-dependent protein kinase phosphorylation site. Su, Sus scrofa; Ho, Homo sapiens; Mu, Mus musculus; Ra, Rattus norvegicus; Te, Tetraodon nigroviridis; Ga, Gallus gallus; Da, Danio rerio; Ha, Haemophilus ducreyi NAPRTase; the corresponding accession number are as follows respectively: DQ001974, NM_005746, NM_021524, NM_177928, DQ002887,

    NM_001030728, NM_212668, AY434675.

Phylogenetic analysis suggests that sus scrofa visfatin is more closely linked to Homo sapiens’ than

    compared with Rattus norvegicus’ and Mus musculus’ (Fig. 5).

     Sus_scrofa

    Mus_musculus

    Rattus_norvegicus

    Homo_sapiens

    Gallus_gallus

    Tetraodon_nigroviridis

    Danio_rerio

    Haemophilus_ducreyi

    Fig. 5 The evolution of visfatin amino acid sequences among different species.

    3.3 Tissue distributions of the three variants.

    RT-nested PCR analysis of total cDNA showed that variant 1 and variant 3 of visfatin were expressed in all tissues examined, including heart, liver, spleen, lung, kidney, stomach, subcutaneous adipose tissues, longissimus dorsi muscle, brain, testicle. Variant 2 was expressed in almost all tissues examined except testis and liver (Fig. 6), a band located closely to the band of variant 1 was also shown, we had experimentally demonstrated it to be a product due to non-specific amplification (data not shown).

     Fig. 6 Expression analysis of the three variants in tissue samples of Meishan pigs by nested PCR. He, heart; Br, brain; Mu, muscle; Ad, adipose; St, stomach; Lu, lung; Te, testis; Gu, gut; Ki, kidney; Li, liver.

4 DISCUSSION

    4.1 The visfatin gene is conserved among different organisms.

    We have successfully cloned the porcine visfatin gene. The nucleotide and amino acid sequences of porcine visfatin were compared with published sequences of seven other representative organisms. The comparison results indicate that visfatin is highly conserved among species, suggesting that it may play an important role in biological function.

    As figure 5 indicated, visfatin is more closely related to the human orthologs compared with other species, the porcine visfatin gene was mapped on SSC9[11], while the human visfatin was located in HSA 07, the localization of visfatin to porcine chromosomes is consistent with the comparative map

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