Leptin is a 16 kD protein, synthesized by adipose tissue and is involved in regulation of feed intake, energy balance, fertility and immune functions. Present study was undertaken with the objectives of sequence characterization and studying the nucleotide variation in leptin gene in Murrah buffalo. The leptin gene consists of three exons and two introns which spans about 18.9kb, of which the first exon is not transcribed into protein. In buffaloes, the leptin gene is located on chromosome eight and maps to BBU 8q32. The leptin gene was amplified by PCR using oligonucleotide primers to obtain 289 bp fragment comprising of exon 2 and 405 bp fragment containing exon 3 of leptin gene. The amplicons were sequenced to identify variation at nucleotide level. Sequence comparison of buffalo with cattle reveals variation at five nucleotide sequences at positions 983, 1083, 1147, 1152, 1221 and all the SNPs are synonymous resulting no in change in amino acids. Three of these eight nucleotide variations have been reported for the first time in buffalo. The results indicate conservation of DNA sequence between cattle and buffalo. Nucleotide sequence variations observed at leptin gene between Bubalus bubalis and Bos taurus species revealed 97% nucleotide identity

A.M.J.B. Adikari

Anuj Chauhan

Archana Verma

I. D. Gupta

Indrasen Chauhan

Mandeep S. Azad

Sanjoy Datta

Yathish H.M.

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130Exploratory Animal and Medical Research, Vol.2, Issue -II, December, 2012INTRODUCTIONLeptin, a 16 kD a protein that is synthesizedby adipose tissue is involved in regulation offeed intake, energy balance, fertility andimmune functions (Fruhbeck et al. 1998). Milkproduction trait is a quantitative trait andpolygenic in inheritance. Since milk productiontrait is directly related to feed intake and energybalance, it is obvious that there is effect ofleptin on milk production. Several singlenucleotide polymorphisms (SNP) have beenreported in the coding region of the gene(Konfortov et al. 1999,  Orrù et al.  2007,Liefers et al. 2002) found that some SNP areassociated to production, live weight, energybalance, feed intake and fertility in Holsteinheifers. Leptin is a 167-amino acid proteinproduced by the leptin gene (leptos means thin).NUCLEOTIDE SEQUENCE VARIATION IN LEPTIN GENE OFMURRAH BUFFALO (BUBALUS BUBALIS)Sanjoy  Datta, A.M.J.B. Adikari, Anuj Chauhan, Archana Verma, I. D. Gupta, IndrasenChauhan, Mandeep S. Azad and Yathish H.M.ABSTRACT : Leptin is a 16 kD protein, synthesized by adipose tissue and is involved in regulationof feed intake, energy balance, fertility and immune functions. Present study was undertaken withthe objectives of sequence characterization and studying the nucleotide variation in leptin gene inMurrah buffalo. The leptin gene consists of three exons and two introns which spans about 18.9kb, ofwhich the first exon is not transcribed into protein. In buffaloes, the leptin gene is located onchromosome eight and maps to BBU 8q32. The leptin gene was amplified by PCR using oligonucleotideprimers to obtain 289 bp fragment comprising of exon 2 and 405 bp fragment containing exon 3 ofleptin gene. The amplicons were sequenced to identify variation at nucleotide level. Sequencecomparison of buffalo with cattle reveals variation at five nucleotide sequences at positions 983,1083, 1147, 1152, 1221 and all the SNPs are synonymous resulting no in change in amino acids. Threeof these eight nucleotide variations have been reported for the first time in buffalo. The results indicateconservation of DNA sequence between cattle and buffalo. Nucleotide sequence variations observedat leptin gene between Bubalus bubalis and Bos taurus species revealed 97% nucleotide identity.Key words: Leptin, Sequence variation, Murrah.Molecular Genetics Lab, Dairy Cattle Breeding Division, National Dairy Research Institute, Karnal,Haryana, India, Pin - 132001.* Corresponding authorExplor. Anim. Med. Res.,  Vol.2, Issue - II, 2012, p.  130-136 ISSN  2277- 470X (Print)ISSN 2319-247X (Online)131Leptin is also responsible for the regulation ofbody weight and energy homeostasis and is oneof the potential biomarker for high performanceleading to better adaptability and productivity.(Friedman and Halaas 1998).  The Leptin geneconsists of three exons and two introns whichspans about 18.9 kb, of which the first exon isnot transcribed into protein. Leptin gene hasbeen mapped to chromosome 7 in human(Green et al. 1995) chromosome 4 (BTA4) inbovine (Stone et al.  1996) and chromosome 8(BBU 8q32) (Vallinato et al.  2004) in buffalo.Several SNPs have been previously identifiedin introns and exons of leptin among differentbreeds of cattle.Leptin plays a critical role in regulating andcoordinating energy metabolism (Friedman andHalaas  1998). It regulates the metabolism ofkey tissues involved in the storage anddissipation of energy (Banks et al.  2000).Therefore, leptin may be important inregulating metabolic adaptation of nutrientpartitioning during the energy-consumingprocesses of pregnancy and lactation (Moschoset al.  2002). Since buffalo contributes about54 per cent of the total milk produced in India.Although the economic importance ofbuffaloes has always been known, yet very littlework has  been  carried out  to  exploit  thegenetic  potentials  of  this  animal. Hence thepresent study was undertaken with the objectiveto identify sequence variation in leptin gene ofbuffalo.MATERIALS  AND  METHODSSample Collection and DNA preparationBlood samples were collected in vacutainers(Bacton-Dickinson vacutainer system)containing sodium EDTA as an anticoagulantfrom Murrah buffaloes maintained at NationalDairy Research Institute, Karnal. GenomicDNA was isolated from blood samplesfollowing phenol-chloroform extractionmethod described by Sambrook and Russel(2001) with slight modifications. DNA wasdissolved in TE buffer at 60°C in water bathfor 2 hrs to dissolve pellet properly in bufferand was kept in refrigerator for further use.Quality of DNA was checked throughspectrophotometry. DNA samples with O.D.ratio between 1.7 and 1.9 were considered asgood and used for further study. The samplesbeyond this range were re-extracted by Phenol-chloroform extraction method. DNA qualitywas also checked by running the sample in 0.8percent agarose gel electrophoresis. The qualityof DNA was checked on 0.8% agarose andquantity by UV spectrophotometer at A260/A280nm.DNA AmplificationThe PCR amplification using primers(Adikari  2006) for exon 2 and 3 of leptin genewas carried out in a programmable thermalcycler (PTC 200, MJ Research) using thefollowing program: For the amplification of 2ndexon, 25 ml of PCR reaction mix contained3.000 ml genomic DNA (50 ng/ml), 0.600 mlprimers each (100 pM/ml), 12.5 ml FermentasMaster MixTM (2X) and 8.3 ml double distilledH2O. , Initial denaturation at 93°C for 1 min,denaturation at 93°C for 1 min, annealing at59°C for 30 sec, extension at 72°C for 1.3 minwere carried out for 36 cycles.For the amplification of 3rd exon, 25 ml ofPCR reaction mix contained 3.000 ml genomicDNA (50 ng/ml), 0.600 ml primers each (100pM/ml), 12.5 ml Fermentas Master MixTM (2X)and 8.3 ml double distilled H2O, Initialdenaturation at 93°C for 1 min, denaturationNucleotide sequence variation in leptin gene of murrah buffalo (Bubalus bubalis)132Exploratory Animal and Medical Research, Vol.2, Issue -II, December, 2012at 93°C for 1 min, annealing at 56°C for 30sec, extension at 72°C for 1.3 min  were carriedout for 36 cycles. The PCR products wereloaded on 1.5% agarose to confirm theamplification of target region using 100 bpladder as a marker and were visualized byethidium bromide staining, which was addedto the agarose gel @ 1 ml /100 ml of gel. Theagarose gel electrophoresis was performed in1X buffer at 100 volts for 45 minutes and PCRproducts were visualized on UVtransilluminator and photographed with geldocumentation system. The primers designed,regions amplified, annealing temperatures andproduct sizes are given in Table1.Nucleotide SequencingThe PCR products were concentrated to 50ng/m by pooling several tubes to precipitate bythe isopropanol procedure.  In order to obtainclean fragment for sequencing, the PCRproducts were separated by electrophoresis in aTAE agarose gel containing ethidium bromideusing standard protocols. The desired PCRproduct band was excised using a clean, sterilerazor blade or scalpel (band was visualized in amedium or long wavelength (e.g.  300 nm) UVlight, and excised quickly to minimize exposureof the DNA to UV light).  The minimum agaroseslice was transferred to a 1.5 ml microcentrifugeor screw cap tube and then purified by usingcommercially available gel extraction kits(Qiagen). Quantification was done by loadingone µl of eluted sample in 1% Agarose gel andcomparing with standard molecular marker (PhiX 174 DNA ladder or 100 bp DNA ladder). Onlysamples with good concentration (>50 ng/ml)were selected and subjected to sequencing.Amplified PCR products from each set ofprimers were subjected to custom DNAsequencing from both ends (5' and 3' ends). fromM/s. Chromous biotech, INDIA.Sequence Data AnalysisSequence data were analyzed by usingChromas (Ver. 1.45, http://www.technelysium.com.au/chromas.html). Sequence data fromvariants of different regions were subjected tomultiple alignments (DNASTAR, Clustal W)for identifying the SNPs.Database SearchThe database search of sequences for apossible match to the DNA sequence of leptingene was conducted using the BLASTalgorithm available at the National Center forTable 1:Description of primers used and the amplified product of different loci studied133Biotechnology Information (NCBI). Translatedprotein sequences of leptin genes in differentspecies namely Bos taurus, Bos indicus, Ovisaries, Capra hircus  and Homo sapiens werealso subjected to BLAST algorithm.RESULTS AND DISCUSSIONSequence data were analysed using chromas(Ver.1.45, http://www.technelsium.com.au /chromas.html). ClustalW multiple alignmentswith Bubalus bubalis sequence referencesequence NCBI GI 46254758)  revealednucleotide changes at 5 positions in the gene.These variations are found in nucleotidesequences at positions G983A in intron 1 whichis heterozygous; at C1083T-homozygous ,T1147G- heterozygous, A1152G-homozygousand C1221T- heterozygous mutations in exon2; at T1371C in intron 2 homozygous mutation;(Fig.I). However, all these nucleotide changesare synonymous  i.e. there is no change in aminoacids. (Table 1) Nucleotide sequence data andannotation details are given in Fig.2 .Orru  et al. (2007) while investigating  RiverBuffalo and two Egyptian River Buffalo reported12 new single nucleotide polymorphism (SNP);two polymorphic sites G3333A and G3441Apreviously identified in exon 3 by Vallinoto etal. (2004) and originally referred to as 276-SNP,384-SNP  and  one of the new SNP, C1221Toccur in exon 2 and three G3195A, G3318Aand G3434A in exon 3 while SNPs T1015C,C1071T, G1072A, T1081C, T1131G, T1143C,T1145G and A1151G are all in intronic region.They also reported a mutation at G3434A in theexon 3, resulting in Arg to Gln substitution inthe codon 159 but it has been observed onlyone Italian Buffalo while all other animals werehomozygote GG genotype. In exon 3, the SNPG3318A was found polymorphic only in theEgyptian Buffaloes, while it resultedmonomorphic in Italian Buffaloes. PositionG3441A was found monomorphic in bothEgyptian and Italian Buffalo.Fitzsimmones et al. (1998) reported 4 SNPsin exon 2 in Bos taurus. Whereas, Haegman etal. (2002) found 2 SNPs in exon 2 in Bostaurus. Adikari (2006) has also reportednucleotide changes at position 11, 365, 369 and371 in intron 2 region. Six SNPs in exon 2 ofTable 2:  SNPs Identified in Leptin Gene (Murrah buffaloes)Nucleotide  sequence variation in leptin gene of murrah buffalo (Bubalus bubalis)134Exploratory Animal and Medical Research, Vol.2, Issue -II, December, 2012Fig.1: Clustal W Alignment with Reference sequence of Leptin Gene (Exon-2) in Murrah BuffaloFig. 2: NCBI BLAST data (Query: Reference Exon 2 sequence data, Subject:Aligned sequence data of the sample)135Jersey cattle were reported by Konfortovet al. (2006). Recent research in India with  Bosgrunniens revealed 4 SNPs in both exon 2 and3 as reported  by Dongre et al. (2008). Amongall these results, there was change of aminoacids in protein product in some cases (i.e. non-synonymous mutation).  Whereas, in few cases,no changes in amino acids were observed (i.e.synonymous mutation )Jhala et al. (2009) have reported three pointmutations at 42, 44 and 250 nucleotide positionsin Mehsana buffalo. Orru et al. (2011) reporteda total of 10 SNP: 2 in intronic region, 2 in exon2 and 6 in exon 3 out of which three SNP(C978T; C3100T; A3157G) have been reportedto affect muscle fat in Simmental bulls. Scatà etal. (2012) reported eight SNPs in 5' flanking andexon 1 of  Bubaline leptin gene :A83G,  A90G,A121G,  G256T, A283G,  G959T,  A1010C andG1254A. Tanpure et al. (2012) reported fivesingle nucleotide polymorphic sites 98, 111, 172,209 and 266 in intron 1, which correspond tothree haplotypes in Mehsana buffalo.Sequence Homology of buffalo leptin genewith other speciesThe nucleotide sequences deduced for therespective exons of leptin gene in Murrahbuffaloes were arranged to represent the codingregion and were compared with other leptinsequences of the related species available inNCBI Gene Bank viz Bovine, caprine  usingNCBI web site Basic Local Alignment SearchTool (BLAST). There exists 97%, 97%, 99%,98% and 80% sequence homology with Bostaurus, Bos indicus, Ovis aries, Capra hircusand Homo sapiens respectively. The highersequence similarity of buffaloes with sheep andgoat than cattle might be due to lower querycoverage (47%) in these two species.CONCLUSION Bovine leptin gene specific primersamplified the buffalo leptin gene and PCRamplification yielded an amplified product ofexon 2 and exon 3 of bubaline leptin gene.  EightSNPs were identified while comparing bubalineleptin gene with that of other species, none ofwhich resulted in change in amino acid sequencei.e. synonymous mutations. There exists 97%,97%, 99%, 98% and 80% sequence homologywith  Bos taurus, Bos indicus, Ovis aries, Caprahircus and Homo sapiens  respectively. Becausethe leptin gene plays an important role ininfluencing economic traits in cattle, the noveldetected single nucleotide polymorphisms mightbe used in association studies to assess theirpotential of being genetic markers for selection.REFERENCESAdikari AMJB.(2006). Molecularcharacterization of leptin gene in Murrahbuffaloes. M.Sc. Thesis submitted to N.D.R.I.(Deemed University). Karnal. India.Banks AS,  Davis SM,  Sarah HB  andMyers MG.(2000).  Activation of  DownstreamSignals by the Long Form of the Leptin Receptor.J. Biol. Chem.  275(19): 14563-14572.Dongre VB,  Singh A,  Dubey PP,  GourDS,  Prashant,  Mondal G,  Sharma A,  JoshiBK and  Kumar D.(2008).  Molecularcharacterization of leptin (obese) gene indomesticated Yak (Bos grunniens).  African J.Biotech.  8(5): 760-762.Fitzsimmones CJ, Schmutz  SM, BergenRD  and McKinnon JJ.(1998). A potentialassociation between the BM 1500 microsatelliteand fat deposition in beef cattle.  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NUCLEOTIDE SEQUENCE VARIATION IN LEPTIN GENE OF MURRAH BUFFALO (BUBALUS BUBALIS)

NUCLEOTIDE SEQUENCE VARIATION IN LEPTIN GENE OF MURRAH BUFFALO (BUBALUS BUBALIS)

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