Research Journal of Recent Sciences _________________________________________________ ISSN 2277-2502 Vol. 3(12), 1-8, December (2014) Res.J.Recent Sci. International Science Congress Association 1 Studies for Identification of Polymorphism in Promoter Sequence of growth hormone gene from microminipig (MMP) with direct sequencing PCR Shedage Kishor Ashok, Yasushi Kuwabara, Shunnosuke Abe and Eugene Hayato Morita1,3* Laboratory of Molecular Cell Physiology, Department of Applied Bioresources, Faculty of Agriculture, Ehime University, Matsuyama-790-8566, JAPAN Fuji Nojo Service, 5247-34 Kitayama, Fujinomiya, Shizuoka, 418-0012, JAPAN Venture Business Laboratory, Ehime University, Matsuyama-790-8577, JAPAN Available online at: www.isca.in , www.isca.me Received 11th November 2013, revised 3rd March 2014, accepted 9th June 2014Abstract Growth hormone (GH) is the main protein hormone released from anterior pituitary gland of hypothalamus region of brain in mammals. It is extensively studied in pig and it is encoded by GH gene. Microminipig (MMP) is the world’s smallest pig developed by Fuji Micra Inc, Japan. It weighs 6-7 kg at the age of 6 months. The transcription regulation is the key step in expression of eukaryotic gene expression and it depends on various cis and transacting elements. To our knowledge, the variation in sequence of GH promoter region in MMP, 1 kb upstream from 1st exon has studied first time. All consensus sites are conserved in MMPs except TATA box sequence which has another version such as TATATAAA. The Variations at -934, -713, -626, -574 and -499 sites are observed only in MMPs. These variations of MMPs could be used as possible genetic markers for pig production. Keywords: Growth hormone gene, microminipigs, single nucleotide polymorphism reaction; snp, single nucleotide polymorphism; 5’ UTR, 5’ untranslated region. IntroductionThe substance present in the anterior pituitary gland of hypothalamus region of brain that accelerates the growth of rat was studied1-3. The bovine pituitary having alkaline extract was injected in rat which grown faster and increased muscle and decreased fat content. However, the Bovine GH is the first GH isolated from anterior pituitary. Later it was cleared that GH is the multifunctional hormone released from the anterior pituatory gland of hypothalamus region of brain. It is an important growth regulator in animal. GH has many metabolic effects that are remain throughout the life. GH is an anabolic hormone that increases muscle content and induces nitrogen balance. Two neuropeptide and hypothalamic hormones, known as GHRH (growth hormone-releasing hormone) and GHIH (growth hormone-inhibiting hormone) stimulates and inhibits the GH secretion, respectively. It is extensively studied in pig and has 216 amino acid chain protein out of it first 26 amino residues encodes for signal peptide. This gene is found on chromosome 12 pl.2~p1.5 and consists of five exons, covering about 1.7 kb transcribed area. Pigs are being used in medical research because of its similarity in physiological and anatomical systems with human. Normal pig and minipigs (MPs) weigh about 200-250 kg and 30-40 kg respectively and this could be big obstacle for researcher to test the any of article for long term because it may cost more. Fuji Nojo Services, Japan has developed world’s smallest pig, Microminipig (MMP) which weighs about 6-7 kg at its maturity. These MMPs are born from the female minipig, “Catherin”. Catherin was developed by the mating of Pot bellied pig and another type of minipig10. MMP could be ideal experimental animal for life science research because it requires less test article as compare to normal pig and MPs. However the reason for smallness of MMP is still unknown. Our previous studies showed that, injection of GH increases the body weight in male and female MMP (215 % and 68.1 % of starting body weight respectively) when compared to control MMP. (Manuscript submitted to The protein Journal). Our another study related to sequence analysis of MMP GHshowed that, there was change in signal peptide sequence but the mature GH protein was conserved when compared with normal pig and MPs (Manuscript submitted to ISRN molecular Biology). Eukaryotic gene expression is most important and it depends on transcription regulation. Most of the transcription binding sites are present in promoter region of gene. Polymorphism in these sites may leads to low expression of gene. To check the presence of any SNPs present in promoter region of GH gene, it is necessary to carry out sequencing of this region. In present study, the 5’UTR and promoter region of GH gene was analysed from normal pig, MPs and MMPs by using direct DNA sequencing technique. This technique has some advantage over RFLP techniques such as it enables to identify the single nucleotide polymorphisms (SNPs) in nucleotide sequence which is difficult to visualize in RFLP data. Material and Methods Animals and genomic DNA extraction.This study was carried out at Faculty of Agriculture, Ehime University, Japan. In this Research Journal of Recent Sciences _____________________________________________________________ ISSN 2277-2502Vol. 3(12), 1-8, December (2014) Res.J.Recent Sci. International Science Congress Association 2 study we used 13, 4 and 28 breeds of normal pigs, MPs and MMPs respectively (table-1). The tissue samples were provided by Fuji Nojo Services, Shizuoka prefecture of Japan. Qiagen DNeasy kit was used to extract the total Genomic DNA from obtained tissue. The Quality of extracted genomic DNA was checked on 1 % agarose gel by electrophoresis. Table-1 Total list of pig breeds used for polymorphism study in promoter region of GH gene Pig breeds Sample No. Name and Sex Normal Pig 1 Landrace  2 Landrace  3 Wild  4 Wild  5 Duroc 1  6 Duroc 2  7 Berkshire  8 Berkshire  9 Yorkshire  10 Yorkshire  11 LYB  12 LYB 13 Mansubuta  Minipigs (MPs) 14 Mini Pig  15 Mini Pig  16 Catherin 1  17 Catherin 2  Microminipigs (MMPs) 18 Aota - 407  19 Aota - 409  20 Aota - 405  21 Happy  22 Akane  23 370  24 418  25 Pokki  26 340  27 341  28 344  29 Akata 337  30 31  31 32  32 405  33 358  34 361  35 Aka 407  36 Aka 409  37 443  38 437  39 Aka 302  40 Aka 335  41 Sandbird - 8  42 259  43 426  44 329  45 Maron  Amplification of promoter region by PCR. A sense and antisense primers (Pr-fw and Pr-rv) for amplification of covering promoter region, 5’ untranslated region, first exon and some part of intron were designed based on NCBI database sequence of pig GH gene. The sequence of primers is mentioned in table-2. The GH gene was amplified by using total genomic DNA as a template and 25 µl reaction mixture contained 0.3 µM of each primer, 1 µl of template (50 ng/ µl), 0.2 mM dNTPs, 1× PCR buffer, 1.5 mM MgSO and 0.5 unit of the KOD-Plus Neo enzyme (Toyobo, Japan). The final volume made to 25 µl by adding sterile distilled water. The reactions were set in the GeneAmp® PCR System 9700 and programme was set as, Pre-denaturation at 94 °C for 2 min, and 40 cycles with denaturation at 98 °C for 10 sec, and extension at 68 °C for 45 sec. Table-2 PCR primers and Sequencing Primers usedPrimer pair Forward and reverse primer sequence (5’ - 3’) Tm (°C) Pr-fw and Pr-rv GCCTCTCTGAGCCTCAGTC GAAGGAGAAGGGACAGGG CTGGTG 68.0 Sequencing Primer Forward and reverse primer sequence (5’ - 3’) Tm (°C) Pr FwS and Pr RvS GCCTCTCTGAGCCTCAGT GAAGGAGAAGGGACAGGG CTGGTG 60.0 List of PCR and sequencing primers used for amplification of pig GH gene along with its melting temperature. Purification of PCR product.After PCR reaction over, the target product was subjected to purification by using E-Gel® CloneWell 0.8 % SYBR Safe gel provided by Invitrogen following the instructions. The Purified product was run on 1 % agarose gel to check its concentration for sequencing PCR. Direct sequencing PCR.The sequencing reaction was carried out with 10 µl total volume, which contains 2 µl of 5X Big Dye sequencing Buffer, 1.6 pmol/µl sequencing primer, 2 µl BigDye® Terminator version 3.1 and about 10-40 ng of purified template DNA and made 10 µl by using double distilled water. The sequencing reaction was carried out by on GeneAmp® PCR System 9700 with an initial denaturation at 96 °C for 1 min then repeated the following conditions for 25 cycles, 96 °C for 10 sec, 50°C for 5 sec, 60°C for 4 min. The sequencing PCR product was purified by Ethanol/ EDTA/ Sodium acetate precipitation method. Resuspension of pellet were done in 12 µl of Hi-Di™ Formamide and then vortexed for 15 sec. After it samples were incubated at 95°C for 2 min and sent for sequencing to the Integrated Center for Sciences, Tarumi, Ehime University, Japan. Antisense sequencing was performed to minimize the possible errors occurring during sequencing. FASTA formatting and comparative analysis.The obtained sequences were converted to FASTA format by using the online tool. Then, converted sequences were compared with reference sequence by using clustalw2 multiple sequence alignment tool The reference sequence was retrieved from the NC_010454.3, Research Journal of Recent Sciences _____________________________________________________________ ISSN 2277-2502Vol. 3(12), 1-8, December (2014) Res.J.Recent Sci. International Science Congress Association 3 NCBI database for GH gene of pig. Results and Discussion Promoter region amplification: With the use of sense and antisense primers, the target region of GH promoter was amplified. The successful amplification of target region gave 1.3 kb band which covers the area of 5’ upstream region, 5’ UTR, st exon, and part of 1st intron of GH gene (figure-1). Further this fragment was purified and used for direct sequencing PCR. Figure-1 Amplification of promoter region of GH gene M represents 1 kb marker used in this study and number 1 to 45 indicates the amplification of 1.3 kb DNA fragment of promoter region of GH gene from pig breeds Sequence analysis of Promoter region of GH: The obtained sequences were checked on sequence scanner version 1.0 of applied biosystems, USA. The unclear samples were performed again with direct sequencing PCR to obtain clear data. Comparative sequence analysis have showed that, 43% (13/30) variation in between 5’ flanking promoter region -550 bp and the translational start site (+1 bp) in all studied animals. In case of MMPs, variation in this region was 61.53 % (8/13) which is considered as important region for transcription regulation as it contains various cis-elements for a variety of trans-factors such as USF, Sp1, Pit-1, Zn-15 and TATA-box binding proteins. Sequence variation in promoter region. TATA box is present at 5’ region of promoter. The variation at this box i.e. at -26 (“b” Table 3) is found. A has changed to T in this position. Sp1 box is located at the position from -135 to -126 and within this site; G at -132 position is changed to T in Wild and in LYB (“e” Table 3). All the MMPs were conserved for this site (Table 3). In case of AP-2 protein binding site, “C” has changed to “A” at -176 position only in Landraceand Yorkshire (“f” table-3). All other breeds did not show any change at this position (table-3). In case of GHF-3 binding site which is present from -239 to -219 position, we found G has changed to A at -219 position (“g” Table 3). The sequence polymorphism sites (b to n) were found near or in these ciselement. The variation site “b” and “e” falls directly within TATA box and Sp1 box respectively (figure-2). The other variations which are not found in protein binding site and other cis and trans acting elements of GH gene from normal pigs, minipigs and microminipigs are showed in table-4. The insertion sequence was also found and it is shown in table-3. Figure-2 Variation sites studied in 5’UTR and promoter region of GH gene of pig breeds used in this experiment. Letters a to z and $1, $2, $3 and $4 indicates variation sites. Numbers 1 and 2 indicates where insertion occurred. The consensus sites studied in this experiment are underlined along with its name Sample no. 1 to 13 are of normal pigs. Sample no. 14 to 17 are of Minipigs (MPs) including Catherin . Sample no. 18 to 45 are of Microminipigs MMPs. Research Journal of Recent Sciences _____________________________________________________________ ISSN 2277-2502Vol. 3(12), 1-8, December (2014) Res.J.Recent Sci. International Science Congress Association 4 Table-3 Variations found in important consensus sites of promoter region of GH gene b e f g 1 2 Positions by reference number NC_010454.3 - 26 TATA Box - 132 Sp1 box - 176 AP-2 site - 219 GHF-3 site Insertion After -628 Insertion After - 822 Reference A G C G Landrace  T G A A GGCAAAGTGTAG Landrace  A G C G GGCAAAGTGTAG Wild  T G C A GGCAAAGTGTAG Wild  T T C G Duroc1  T G C G Duroc2  A G C G Berkshire  T G C A Berkshire  A G C G GGCAAAGTGTAG Yorkshire  T G C A GGCAAAGTGTAG G Yorkshire  T G A A GGCAAAGTGTAG LYB  T T C G GGCAAAGTGTAG LYB T G C G GGCAAAGTGTAG Mansubuta  A G C G Mini Pig  A G C G GGCAAAGTGTAG Mini Pig  A G C G GGCAAAGTGTAG Catherin gilt  A G C A GGCAAAGTGTAG Catherin sow  A G C A GGCAAAGTGTAG Aota - 407  T G C A GGCAAAGTGTAG Aota - 409  A G C A GGCAAAGTGTAG Aota - 405  A G C A GGCAAAGTGTAG Happy  A G C A GGCAAAGTGTAG Akane  A G C A GGCAAAGTGTAG 370  A G C A GGCAAAGTGTAG 418  T G C A GGCAAAGTGTAG Pokki  T G C A GGCAAAGTGTAG 340  T G C A GGCAAAGTGTAG 341  T G C A GGCAAAGTGTAG 344  T G C A GGCAAAGTGTAG Akata337  T G C A GGCAAAGTGTAG 31  T G C A GGCAAAGTGTAG 32  T G C A GGCAAAGTGTAG 405  T G C A GGCAAAGTGTAG 358  A G C A GGCAAAGTGTAG 361  T G C A GGCAAAGTGTAG Aka 407  T G C A GGCAAAGTGTAG Aka 409  A G C A GGCAAAGTGTAG 443  A G C A GGCAAAGTGTAG 437  T G C A GGCAAAGTGTAG Aka 302  T G C A GGCAAAGTGTAG Aka 335  T G C A GGCAAAGTGTAG Sandbird8  T G C A GGCAAAGTGTAG 259  A G C A GGCAAAGTGTAG 426  A G C A GGCAAAGTGTAG 329  A G C A GGCAAAGTGTAG Maron  T G C A GGCAAAGTGTAG Capital and bold letters indicates the reference sequence (Genbank: NC_010454.3) along with its respective position. Red color nucleotide indicates the variations found in respected pig breeds. Positions b, e, f and g are the mentioned protein binding sites in promoter region where variation is found. 1 and 2 shows the insertion of GGCAAAGTGTAG and G respectively has been found inserted in promoter region after -628 and after -822 position respectively. Research Journal of Recent Sciences _____________________________________________________________ ISSN 2277-2502Vol. 3(12), 1-8, December (2014) Res.J.Recent Sci. International Science Congress Association 5 Table -4 Variations found in other than consensus sites of promoter region of GH gene a c d h i j k l m n o p q r s t u v W x y z $1 $2 $3 $4 Positions by reference number NC_010454.3 +22 -62 -119 -291 -380 -385 -391 -497 -499 -514 -574 -626 -661 -713 -797 -835 -869 -879 -881 -891 -905 -923 -934 -938 -943 -950 Reference C G A C C T C C C C C C C G T C G A C T A T G T A C Landrace  Normal pigs T A A C C A T C C T C C C G T C G A C T A C G T A C Landrace  C G A C C T C C C C C C C G T C G A C T A T G T A C Wild  T G A C T A C T C C C C C G T G G C C T A T G T A C Wild  T G A C C A T C C C C C C G T C G A C T A T G T A C Duroc1  T G A C C T C C C C C C C G T C G A C T A T G T A C Duroc2  C G A C C T C C C C C C C G T C G A C T A T G T A C Berkshire  T G A T T A C T C C C C C G T C G A C T A T G T G A Berkshire  T G A C C T C C C C C C C G T C G A C T A T G T A C Yorkshire  T G A T T A C T C C C C C G C C A A G T A T G T A C Yorkshire  T A A C C A T C C T C C C G T C G A C T A C G T A C LYB  T G A C C A T C C C C C C G T C G A C T A T G T A C LYB T G A T C T C T C C C C C G T C G A C C C T G T A C Mansubuta  C G A C C T C C C C C C C G T C G A C T A T G T A C Mini Pig  Minpigs C G A C C T C C C C C C C G T C G A C T A T G T A C Mini Pig  C G T C C A T C A C C A C G T C G A C C A T G T A C Catherin gilt  T G A T C A C C C C C A C G T C G A C T A T G T A C Catherin sow  T G A T C A C C A C C A C A T C G A C T A T G T A C Aota - 407  Microminipigs (MMPs) T G A T T A C T C C C A C A T C G A C T A T G T A C Aota - 409  C G A C C A T C A C C A C G T C G A C T A C G T A C Aota - 405  T G A C C A C C C C C C C G T C G A C T A T G G A C Happy  C G A C C A T C A C C A C G T C G A C T A C G T A C Akane  C G A C C A T C A C C A C G T C G A C T A C G T A C 370  T G A T C A C T A C C A C G T C G A C T A T G T A C 418  T G A T T A C T C C A C C A T C G A C T A T G T A C Pokki  T G A T C A C C A C C A C A T C G A C T A T G T A C 340  T G A T C A C C A C C A C G T C G A C T A T G T A C 341  T G A T C A C C A C C A C G T C G A C T A T A T A C 344  T G A T C A C C A C C A C G T C G A C T A T G T A C Akata337  T G A T C A C C A C C A C G T C G A C T A T A T A C 31  T G A T C A C C A C C A C G T C G A C T A T G T A C 32  T G A T C A C C A C C A C G T C G A C T A T A T A C 405  T G A T T A C T C C C A C A T C G A C T A T G T A C 358  C G A C C A T C A C C A C G T C G A C T A C A T A C 361  T G A T T A C T C C C A C A T C G A C T A T A T A C Aka 407  T G A T T A C T C C C A C A T C G A C T A T G T A C Aka 409  C G A C C A T C A C C A C G T C G A C T A C G T A C 443  C G A C C A T C A C C A C G T C G A C T A C G T A C 437  T G A T T A C T C C C A C A T C G A C T A T A T A C Aka 302  T G A T T A C T C C C A A A T C G A C T A T G T A C Aka 335  T G A T T A C T C C C A C A T C G A C T A T G T A C Sandbird8  T G A T T A C T C C C A C A T C G A C T A T G T A C 259  C G A C C A T C A C C A C G T C G A C T A C G T A C 426  C G A C C A T C A C C A C G T C G A C T A C G T A C 329  C G A C C A T C A C C A C G T C G A C T A C G T A C Maron  T G A T T A C T C C A C C A T C G A C T A T G T A C Capital and bold letters indicates the reference sequence (Genbank: NC_010454.3) along with its respective position. Red color nucleotide indicates the variations found in Normal pigs, minipigs and Microminipigs (MMPs). All the positions are other than mentioned protein binding sites. Research Journal of Recent Sciences _____________________________________________________________ ISSN 2277-2502Vol. 3(12), 1-8, December (2014) Res.J.Recent Sci. International Science Congress Association 6 Inside a single cell, gene transcription depends on presence of cis-elements and trans-acting factors11-17. Direct or indirect interaction of transcription factors with basic transcription machinery may occur within the promoter or enhancer region containing binding sites18-21. The sequence 5’ of transcription start site is important for tissue specific promoter activity in human or rat pituitary cells22. This point suggested us to search all binding sites of transcription factors present in proximal promoter region through direct DNA sequencing technique. The TATA-box consensus sequence site TATAAA23 is present in upstream of transcription binding site of GH gene (Fig 2). In present study it is located -26 bp upstream of transcription binding site. The TATA binding protein (TBP) binds to 8 bp sequence of AT rich base pairs24,25. However porcine GH TATA-box has 2 types of sequences. TATAAAAA (TATA1) and TATATAAA (TATA2) are two types of TATA boxes reported by Kirkpatrick and his co workers26. In present study, variation “b” found within TATA box and it showed that TATA1 and TATA2 boxes are present in studied animal. In case of Catherin 1 and Catherin 2 has only TATA1 type of box and most of the MMPs showed TATA2 box. GH promoter contains conserved pair of Pit-1 binding site within the 5’ flanking sequence27,28. The transcription factor Pit-1 is essential for anterior pituitary development, function and for activation of the GH gene29. This Pit-1 transcription factor is also known as GHF-1. Pig GH promoter also has two Pit-1 binding sites. First site is ATGCATAA prox Pit-1 (From -87 to -80) and second site is TTATCCAT dist Pit-1 (From -118 to -109). In present study both the Pit-1 consensus sequences are conserved in all the pig breeds (figure-2). The transcription factor, Sp1 has a zinc finger protein motif that binds directly to DNA sequence and activates gene transcription. It is also known as GC-box-binding protein16. In case of hGH1 promoter, Sp1 consensus site sequence is TGGGAGGAGC30The similar sequence (from -135 to -126) was found in porcine promoter region except last “C” is changed to “G” at -126 position (figure-2). In present study, breeds Wild and LYB has TGGTAGGAGG consensus site and at -132 “G” has changed to “T” (Table 3) which was found to be similar with haplotype GH-H4 studied by Larsen and Nielsen31. It might be playing role in lower affinity of Sp1 protein to this site in Wild and LYB . The Sp1 site and Pit 1 site overlaps and binding of these sites is considered as mutually exclusive32. Remaining breeds showed no change in this site. The Zn-15 represents a major binding activity to Pit-1(prox Pit-1) site and it suggests that, this factor may be an important component of GH gene expression. The consensus sequence studied in rat GH promoter for Zn-15 is AGCACAAGCTGTCAGTGG33. In this study, Zn-15 consensus sequence is located from -109 to -91 (figure-2) and as compare to rat Zn-15 consensus sequence, “A” at -103 has changed to “T” and it is conserved in all pig breeds. Lower levels of trans-acting factor AP-2 is expressed in GH producing GC pituitary tumor cells34. Several viral and cellular genes having GC rich cis-regulatory regions on which the AP-2 protein binds. The variation in this site -176 C/A (“f” table-3) is only observed in Landrace and Yorkshire . All the MMPs are conserved for this sequence. This result is consistent with the GH-H3 haplotype studied by studied by Larsen and Nielsen31. According to rat GH promoter sequence, the sequence between -239 and -219 is responsible for binding of GHF-3 protein (figure-2) which act as transcription factor and plays role in transcription activity35. By comparing porcine GH promoter sequence for rat GHF-3 binding site, we have found -219 G�A (“g” Table 3) is changed in all MMPs including in Catherin 1 and 2 from MPs. This may be playing role in low transcription activity by lowering affinity of GHF-3 protein to this site. The insertion of sequence “GGCAAAGTGTAG” was found in all the Mini Pigs and MMPs except in Wild , Duroc1 , Duroc2 , Berkshire and Manshubuta . This suggests that, these insertions were found only in MPs and MMPs and was completely absent in Normal pigs. Further studies with exact role of this inserted sequence are needed. All other variations including changes in various sites described here, is summarized in table-4. ConclusionAll of the previously studied and mentioned here transcription factors are almost conserved in studied pigs. We cannot exclude that, changes in other positions other than consensus binding site and located distantly away may be playing important role in MMPs by means of some secondary structural changes in promoter region. To our knowledge, the variation in sequence of promoter region 1 kb upstream from 1st exon of MMPs has studied first time. All variations found in GH promoter of normal pig and MPs were also observed in Catherin 1 and 2 and all the MMPs. We cannot eliminate the possibility of some combinational effects of these variations on GH gene expression of MMP. This study is the first step in developing a sequence map for promoter region of GH in Catherin and MMPs, which will provide more information about the link for growth performance in MMPs. These variations in MMPs could be used as possible markers for pig production through MMP as a parent. Acknowledgments Shedage Kishor Ashok is a Ph.D student in the Ehime University, Japan and currently he is supported by The Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) during his course of research work. We also appreciate the help of Mrs. Kana Hondo and staff of Integrated Center for Sciences, Tarumi, Ehime University, Japan for providing sequencing facility. Research Journal of Recent Sciences _____________________________________________________________ ISSN 2277-2502Vol. 3(12), 1-8, December (2014) Res.J.Recent Sci. International Science Congress Association 7 References 1.Evans H.M. and Long J. 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