Buffalo Bulletin (September 2008) Vol.27 No.3 MOLECULAR CLONING AND CHARACTERIZATION OF THE BETA-CASEIN GENE IN AN INDIAN RIVERINE BUFFALO (BUBALUS BUBALIS) ABSTRACT The present study was carried out to characterize beta-casein cDNA of Indian riverine buffalo. This gene was cloned in plasmid vector and sequenced for molecular characterization of the cDNA. The whole buffalo beta-casein cDNA was 675 bp in length. We have submitted the buffalo cDNA sequences to the NCBI GenBank with the accession number DQ631829. This gene was composed of 23.85% A, 20.59% G, 24.89% T and 30.67% C indicating 48.74% as AT and 51.26% as GC. The similarity of the buffalo cDNA sequence with that of cattle was estimated as 98.08% while with the sequences of other species like sheep, pig, camel, human, rat and rabbit, it was 96.60%. As far as protein sequence is concerned, the similarity of buffalo sequence with its cattle counter part was estimated as 97.30% and with the sequences of all other species studied here, it was calculated as 93.30%. The molecular weight of buffalo beta- casein protein was estimated as 25.105 kDa. The secondary structure composition of buffalo beta- casein protein was prediced as having 14.3% helix (H), 1.3% strand (E) and 83.9% loop (L) whereas solvent accessibility composition was 79.02 % of “e” type (residues exposed with more than 16% of their surface) and 20.98 % of “b” type (others). Keywords : buffalo, beta-casein, homology, nucleotide, sequence INTRODUCTION The buffalo is famous for production of high milk fat and protein and high total solid content in milk. Besides, it is well known that buffaloes have a unique feed conversion efficiency using low grade roughages and are able to thrive under harsh climatic conditions with resistance to many diseases. Despite its huge potential and superiority to cows in many aspects, the buffalo has remained generally neglected. In ruminants, caseins are the major milk proteins, which constitute 80% of the total protein in milk (Dalgleish, 1993). There are four types of caseins, namely alpha s1-, alpha s2-, beta- and kappa-casein present in milk of which beta-casein constitutes about 36% of total casein (Davies and Law, 1980). Beta-casein protein is a calcium sensitive protein and is insoluble in milk, and its concentration is 9.3 gm/litre (Eigel et al., 1984). Beta-casein increases the firmness of curd from enzymically coagulated milk (Jimenez-Flores and Richardson, 1988). Mariani (1983) reported that the beta-casein B variant was superior to A variant in cheese making. Beta-casein helps in the absorption of minerals like Fe and Zn in the intestinal tract, besides, Fe complexed to beta-casein displayed a better bioavailability than gluconate Fe (Bouhallab et al., 2002). It has been found that the binding of Zn to beta-casein improved Zn absorption and prevented Fe from inhibiting its absorption (Peres et al., 1998). Various studies have been found with respect to correlation between beta-casein variants and type I diabetes. (A 1 and B) variants of beta- casein have correlation (r = +0.982) with type I insulin 222 Tarun K. Bhattacharya 1 , Pushpendra Kumar 2 and Arjava Sharma 2 1 Project Directorate on Poultry, Rajendranagar, Hyderabad, Andhra Pradesh-500030, India e-mail: [email protected]2 Animal Genetics Division, Indian Veterinary Research Institute, Izatnagar, Bareilly, UP - 243122, India
Transcript
Buffalo Bulletin (September 2008) Vol.27 No.3
MOLECULAR CLONING AND CHARACTERIZATION OF THE BETA-CASEIN GENEIN AN INDIAN RIVERINE BUFFALO (BUBALUS BUBALIS)
ABSTRACT
The present study was carried out tocharacterize beta-casein cDNA of Indian riverinebuffalo. This gene was cloned in plasmid vector andsequenced for molecular characterization of thecDNA. The whole buffalo beta-casein cDNA was675 bp in length. We have submitted the buffalocDNA sequences to the NCBI GenBank with theaccession number DQ631829. This gene wascomposed of 23.85% A, 20.59% G, 24.89% T and30.67% C indicating 48.74% as AT and 51.26% asGC. The similarity of the buffalo cDNA sequencewith that of cattle was estimated as 98.08% whilewith the sequences of other species like sheep, pig,camel, human, rat and rabbit, it was 96.60%. As faras protein sequence is concerned, the similarity ofbuffalo sequence with its cattle counter part wasestimated as 97.30% and with the sequences of allother species studied here, it was calculated as93.30%. The molecular weight of buffalo beta-casein protein was estimated as 25.105 kDa. Thesecondary structure composition of buffalo beta-casein protein was prediced as having 14.3% helix(H), 1.3% strand (E) and 83.9% loop (L) whereassolvent accessibility composition was 79.02 % of“e” type (residues exposed with more than 16% oftheir surface) and 20.98 % of “b” type (others).
The buffalo is famous for production of highmilk fat and protein and high total solid content inmilk. Besides, it is well known that buffaloes have aunique feed conversion efficiency using low graderoughages and are able to thrive under harsh climaticconditions with resistance to many diseases. Despiteits huge potential and superiority to cows in manyaspects, the buffalo has remained generallyneglected. In ruminants, caseins are the major milkproteins, which constitute 80% of the total proteinin milk (Dalgleish, 1993). There are four types ofcaseins, namely alpha s1-, alpha s2-, beta- andkappa-casein present in milk of which beta-caseinconstitutes about 36% of total casein (Davies andLaw, 1980).
Beta-casein protein is a calcium sensitiveprotein and is insoluble in milk, and its concentrationis 9.3 gm/litre (Eigel et al., 1984). Beta-caseinincreases the firmness of curd from enzymicallycoagulated milk (Jimenez-Flores and Richardson,1988). Mariani (1983) reported that the beta-caseinB variant was superior to A variant in cheese making.Beta-casein helps in the absorption of minerals likeFe and Zn in the intestinal tract, besides, Fecomplexed to beta-casein displayed a betterbioavailability than gluconate Fe (Bouhallab et al.,2002). It has been found that the binding of Zn tobeta-casein improved Zn absorption and preventedFe from inhibiting its absorption (Peres et al., 1998).
Various studies have been found withrespect to correlation between beta-casein variantsand type I diabetes. (A1 and B) variants of beta-casein have correlation (r = +0.982) with type I insulin
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Tarun K. Bhattacharya1, Pushpendra Kumar2 and Arjava Sharma2
1Project Directorate on Poultry, Rajendranagar, Hyderabad, Andhra Pradesh-500030, Indiae-mail: [email protected] Genetics Division, Indian Veterinary Research Institute, Izatnagar, Bareilly, UP - 243122, India
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dependent diabetes mellitus. They yield a bioactivepeptide beta-casomorphin-7 after in vitro digestion,which has an immunosuppression property whichcould account for diabetes incidence (Elliott et al.,1999). A significantly increased level of antibodiesto beta-casein is found in patients with type Idiabetes (Moretini et al., 2002).
The primary protein sequence of beta-caseinhas been elucidated by Ridadeau Dumas et al.(1972) and Grosclaude et al. (1972). It is the mosthydrophobic among all casein proteins and consistsof more proline residues than any other caseinresidue.
The polymorphism of beta-casein gene hasbeen studied by several workers in various specieslike cattle (Pinder et al., 1991; Lein et al., 1992;Damiani et al.,1992; Janno et al., 2002), goat (Maheand Grosclaude, 1993; Langley-Danysz, 1993;Pappalardo et al., 1996; Pappalardo et al., 1997;Bonifacio, 2001), and sheep (Serrano et al., 1999).Bovenhuis et al. (1992) reported that in Holstein-Fresian cattle the beta-casein phenotypes ranked inthe order of decreasing milk production as follows:A2B > A1A3, A1A2, A1A1 > A1B > BB. In theAyrshire breed of cattle, 305 days of the firstlactation yield for A2 variant of beta-casein was 6077kg, compared to 5838 kg for A1 variant (Kim et al.,1996). The A1 variant of beta-casein was associatedwith higher milk protein than the A2 variant(Bovenhuis et al., 1992; Ng-kwai-Hang et al.,1986). Various studies have found differences in fatcontent among the different phenotypes of beta-casein (Ng-Kwai-Hang et al., 1986; McLean et al.,1984). But, to date, this important gene has not beencharacterized in buffaloes in detail. Therefore, thepresent study was carried out to characterize thisgene in buffalo at the molecular level.
MATERIALS AND METHODS
SampleMammary tissue (100 mg) of a Murrah
buffalo was collected from the MunicipalCorporation slaughterhouse, Bareilly, Uttar Pradesh,India, and carried to the laboratory on ice. The samplewas stored at -70 0C till further use.
Isolation of mRNAThe total RNA was extracted following the
method described by Sambrook and Russel (2001).The RNA was stored at -70 0C for further use. Thepurity of the RNA was verified by measuringabsorbance of the RNA solution in UV-Spectrophotometer at 260 nm and 280 nm. The RNAsample showing the OD260 : OD280 value between1.9-2.2 was of good quality. The integrity of theextracted RNA was checked using 2.2 Mformaldehyde denatured agarose gel electrophoresis(Sambrooke and Russel, 2001). The mRNA waspurified from total RNA using an oligotex mRNAisolation kit (Qiagen, Germany).
Designing of primerFor the amplification of the beta-casein gene
of buffalo, the primers were designed from thepublished cattle, sheep, pig and human sequencesavailable from the NCBI GenBank (Acc. No.NM_181008, X16482, NM214434 and NM001891)with the help of DNASIS MAX software (HitachiMiraibio Inc., USA). The Primer sequences wereForward, 5' ATGAAGGTCCTCATCCTTGCCTG3 ' and reverse, 5 ' TTAGACAATAATAGGGAAGGGTC 3'.
RT-PCRThe total RNA was reverse transcribed
using murine reverse transcriptase enzyme tosynthesize single strand cDNA. About 2 μg (2 μl)of total RNA was taken in a 0.2 ml PCR tube andincubated at 700C for 10 minutes and immediatelysnapped in ice. Then the master mix (MgCl2, 25 mM;Reverse Transcription 10x buffer; dNTP mixture,10 mM; Rnasin; AMV Reverse Transcriptase,15u;Oligo (dT)15 primer and Nuclease free water tomake a final volume of 20 μl ) was added to thePCR tube. The reaction mix with RNA wasincubated at 420C for 15-20 minutes for reversetranscription. Then the sample was heated to 950Cfor 5 minutes and incubated at 50C for 5 minutes.The synthesized cDNA was stored at -200C forfurther use. The cDNA was purified following themethod described by Sambrook and Russel (2001).PCR was carried out to amplify 675 bp fragment ofbeta-casein cDNA from single strand cDNA usingforward and reverse primers. The final volume of25 μl PCR reaction mixture was composed of 100
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ng cDNA, 100 μM of each dNTP, 50 ng of F1 andR1 primer, 2.0 mM MgCl2, 1U Taq DNA polymeraseand assay buffer (Imperial Biomedics, India). Theamplification conditions were 950C for 2 minutesfollowed by 35 cycles of 940C for 45 seconds, 640Cfor 1 minute and 720C for 1 minute and finalextension of 720C for 5 minutes.
Elution of PCR productThe amplified products of beta-casein genes
were run in 0.8% agarose gel having a long combedwell. The products were visualized under a trans-illuminator, the gels having DNA fragments ofinterest were cut using a scalpel, and the DNA wasisolated from gel using a MinElute Gel ExtractionKit (Qiagen) following the manufacturer ’sinstructions.
Cloning of cDNABeta-casein cDNA amplified from the
Murrah buffalo was cloned using the principle ofUA ligation. The amplified products were ligatedwith the pDRIVE Cloning Vector following themanufacturer’s instructions (Qiagen) and it wastransformed into E.coli DH5α strain. The ligationreaction was set up in a 0.5 ml PCR tube with 2XRapid Ligation buffer, pDRIVE-cloning Vector (50ng/μl), A- tailed PCR product (template DNA) andT4 DNA Ligase (3 units/μl). The ligation mix wasbriefly mixed and incubated at 190C for 3 h. Thecompetent cells were prepared following theinstructions of Sambrook and Russel (2001). Theligation mix (1.0 μl) was added to freshly thawedcompetent cells (200 μl) using a pre-chilled pipettetip and mixed gently. Then the transformation wascarried out following the method described bySambrook and Russel (2001). After transformation,the plates were screened for the presence of blue/white colonies. The recombinant clones wereidentified by white color on indicator plates. In orderto minimize the number of clones to be handled,clones were initially checked by colony PCR andlater confirmed by plasmid-PCR. The positivesamples were reconfirmed by restriction enzymedigestion of the plasmid DNA with EcoR1 enzymeas EcoRI enzyme has cutting sites on either side ofmultiple cloning site in pDRIVE cloning vector.
SequencingSequencing was performed by an
automated sequencer (ABI prism) using Sanger’sdideoxy chain termination method. The cloned PCRproducts from the Murrah buffalo were submittedin the form of the stab culture. The T7F and SP6Rprimers (position of T7F and SP6R primers bindingsite in pDRIVE cloning vector are 239-258 and 400-418 respectively) were used for the sequencing ofthe clones.
AnalysisThe sequence obtained was first blasted(www.ncbi.nlm.nih.gov/BLAST) to ascertain thatthe sequence was of beta-casein. Nucleotides aswell as derived amino acid sequences were thenaligned with those of the reported beta-casein genesequences of different species using the clustermethod of MegAlign Programme of LasergeneSoftware (DNASTAR). Secondary structure ofprotein was predicted (Rost and Sander, 1993) andsolvent accessibilities were determined (Rost et al.,1996).
RESULTS AND DISCUSSION
The whole cDNA of buffalo beta-caseingene was cloned in plasmid vector and sequencedto determine the nucleotide sequence of the gene.
Sequence analysisThe whole buffalo beta-casein cDNA was
675 bp in length whereas the length of its cattlecounterpart was similar in length. The sheep, pig,human, camel, rat and rabbit beta-casein cDNA was669 bp in length. We used beta-casein sequence ofcattle, sheep, pig, camel, human, rat and rabbitavailable at NCBI data bank. We submitted thebuffalo cDNA sequences to the NCBI GenBankand the accession number was obtained asDQ631829. While analyzing buffalo cDNA, it wasobserved that the percentage of A, G, T and C in thegene itself were 23.85, 20.59, 24.89 and 30.67%showing 48.74% as AT and 51.26% as GC. In cattle,the nucleotide organization revealed that the AT andGC% in the gene were 49.04 and 50.96, respectively,whereas in sheep, pig, camel, human, rat and rabbit,the AT and GC% were 48.88 and 51.12, respectively.
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Bovine cDNA sequence of beta-casein gene wasfirst reported by Stewart et al. (1987). The Davis,Botstein, Roth melting temperature of this gene inbuffalo was calculated as 85.18; which was foundto be slightly higher than that of cattle, where it was85.050C. In other species like sheep, pig, camel,human, rat and rabbit, the temperatures were foundas 85.10C.
Sequence homologyOur buffalo sequence was subjected to
BLAST analysis at the NCBI website to retrievesimilar sequences of mammalian origin. The multiplealignment study revealed that the similarity of buffalocDNA sequence with that of cattle was estimatedas 98.08% while with other sequences like sheep,pig, camel, human, rat and rabbit, it was 96.60%.When protein sequence was considered, thesimilarity of buffalo sequence with its cattle counterpart was estimated as 97.30%. The similarity ofbuffalo protein with that of all other species studiedhere was calculated as 93.30%. When comparingsequence of sheep with that of other species likepig, camel, human, rat and rabbit, all the species werefound to be 100% similar at both nucleotide andamino acid level.
Sequence variabilityA number of changes of nucleotides were
observed between buffalo and other species, andthey have been depicted in Figure 1. Whencomparing the buffalo sequence with that of cattle,nucleotide changes were detected at severallocations of which some changes at position 119th,168th, 249th, 319th, 363rd and 488th showed functionalchanges while others remained as silent in nature.The sequence alignment study revealed that a blockof six nucleotides, viz ATC CCC was deleted fromposition 584-589th of buffalo cDNA compared toother species, such as sheep, pig, camel, human, ratand rabbit.
The molecular weight of buffalo beta-caseinprotein was estimated as 25.105 kDa whereas cattleprotein was 25.097 kDa. In other species, it wasquite a bit less: approximately 24.874 kDa. Bothbuffalo and cattle proteins were composed of 224amino acids whereas sheep, pig, camel, human, ratand rabbit protein was constituted of 222 aminoacids. In the buffalo protein, we found 16 stronglybasic, 23 strongly acidic, 78 hydrophobic, 53 polar
and 54 neutral amino acids. In cattle, 16 stronglybasic, 23 strongly acidic, 78 hydrophobic, 56 polarand 51 neutral amino acids were found, which wasquite different from its buffalo counterpart. Thesheep, pig, camel, human, rat and rabbit proteins werecomposed of 16 strongly basic, 23 strongly acidic,78 hydrophobic, 54 polar and 51 neutral amino acids.The differences of polar and neutral amino acidsbetween the buffalo and other species suggest thatthis protein in several species will have its own uniquestructure for conferring differential biologicalactivities. The primary protein sequence in cattlewas elucidated by Ridadeau-Dumas et al. (1972)and Grosclaude et al. (1972). However, thedifferences of amino acids between buffalo andcattle were determined at 4th (H/R), 56th (M/T), 83rd
(K/N), 107th (I/V), 121st (H/Q) and 163rd (P/H)locations (Figure 2). The variability of amino acidsbetween buffalo and cattle were observed fromneutral to strongly basic at 40th, neutral to polar at56th, strongly basic to polar at 83rd and neutral topolar at 121st position of the polypeptide. The aminoacid changes between buffalo and other species likesheep, pig, camel, human, rat and rabbit weredetected at 18th(L/Q), 24th(P/V), 27th(I/T), 56th(M/T), 70th(T/A), 78th(P/T), 83rd(K/N), 90th(P/L),111th(S/P), 116th(A/T), 118th(A/V), 147th(N/K),155th(L/V) and 183rd(S/P) position of the polypeptidechain. While comparing the amino acid profilesamong sheep, pig, camel, human, rat and rabbit, therewere no differences found in the protein. But, dueto deletion of a block of nucleotides from the position584-589th of buffalo sequence, the amino acidsproline and tyrosine were lacking from the position195th and 196th of polypeptide chain of sheep, pig,camel, human, rat and rabbit beta-casein protein.
Protein parametersThe isoelectric point of buffalo and all other
species except cattle were observed as 5.264 whileits magnitude in cattle was 5.118. The charge ofthis protein in buffalo was -6.256 at pH 7.0 while incattle it was found as -6.421 at neutral pH. In sheep,pig, camel, human, rat and rabbit, the charge of thisprotein was observed as -6.255 at pH 7.0. Predictedsecondary structure composition of buffalo beta-casein protein showed 14.3% helix (H), 1.3% strand(E) and 83.9% loop (L). Predicted solventaccessibility composition of buffalo beta-caseinprotein showed 79.02% of “e” type (residues
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exposed with more than 16% of their surface) and20.98% of “b” type (others). In the case of cattle,predicted secondary structure was composed of14.29% helix, 0.89% strand, 84.82% loop and thepredicted solvent accessibility was determined as20.09% b and 79.91% e . In sheep, pig, camel, human,rat and rabbit, the predicted structure was composedof 16.22% helix, 0.90% strand and 82.88% loop,and the predicted solvent accessibility was estimatedas 20.72% as b and 79.28% as e.
Phylogenetic treeFigures 3 and 4 depict the phylogenetic tree
constructed on the basis of nucleotide and aminoacid sequence of the beta-casein protein. The trendsof evolutionary relationship among differentmammalian species were the similar in nature.Buffalo and cattle form one cluster having closestrelationship while maintaining a certain distantrelation with sheep. The rabbit was to some extentrelated with this group but maintained a distantrelationship from camel and pig which formedanother cluster. The rat and human were the mostdistant ones from buffalo and were located inseparate branches. As the buffalo and cattle fallunder the same Bovidae family, they were expectedto have the closest relationship in the evolutionarypathway. But other species, although they weremammals, fall in different family or genera andconsequently, formed separate clusters. Our studysuggests that various mammalian species althoughsecreting beta-casein protein in their milk, havedifferent nucleotide/amino acid combinations in theirgenes, which was reflected in this dendogram.
Thus, in the present study, the organizationof cDNA of buffalo beta-casein gene and predictedprotein has been presented and compared with otherspecies, which not only enables dairy scientists tounderstand the characteristics and property of thisprotein, but also helps scientists to explorebiochemical dynamics of the protein.
ACKNOWLEDGEMENT
The authors are thankful to ADG (AnimalBreeding and Production), ICAR for providingfinancial help to carry out research work underICAR Ad-hoc scheme.
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..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
1
pig.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
1
rabbit.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
1
rat.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
1
sheep.SEQ
GT
GA
GG
AA
TC
TA
TT
AC
AC
AC
AT
CA
AT
AA
GA
AA
AT
TG
AG
AA
GT
TT
CA
AA
GT
GA
GG
AA
CA
AC
AG
CA
AA
CA
GA
GG
AT
GA
AC
TC
CA
GG
AT
AA
AA
TC
CA
CC
CC
TT
Majority
110
120
130
140
150
160
170
180
190
200
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
G.
..
..
..
..
..
G.
..
..
..
TG
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
101
buffalo.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
101
camel.SEQ
..
..
..
..
..
..
..
..
..
G.
..
..
..
..
..
..
..
..
..
..
..
..
..
G.
..
..
..
..
..
G.
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
101
cattle.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
101
human.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
101
pig.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
101
rabbit.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
101
rat.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
101
sheep.SEQ
TG
CC
CA
GG
CA
CA
GT
CT
CT
AG
TC
TA
TC
CC
TT
CA
CT
GG
GC
CC
AT
CC
CT
AA
CA
GC
CT
CC
CA
CA
AA
AC
AT
CC
TG
CC
TC
TT
AC
TC
AA
AC
CC
CT
GT
GG
TG
GT
GC
CG
Majority
210
220
230
240
250
260
270
280
290
300
..
..
..
.A
..
..
..
..
..
..
..
..
..
..
..
.C
..
..
..
..
..
..
..
..
G.
..
..
..
..
..
..
..
..
..
C.
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
201
buffalo.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
201
camel.SEQ
..
..
..
.A
..
..
..
..
..
..
..
..
..
..
..
.C
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
CT
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
201
cattle.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
201
human.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
201
pig.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
201
rabbit.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
201
rat.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
201
sheep.SEQ
CC
TT
TC
CT
TC
AG
CC
TG
AA
AT
AA
TG
GG
AG
TC
CC
CA
AA
GT
GA
AG
GA
GA
CT
AT
GG
TT
CC
TA
AG
CA
CA
AG
GA
AA
TG
CC
CT
TC
CC
TA
AA
TA
TC
CA
GT
TG
AG
CC
CT
Majority
310
320
330
340
350
360
370
380
390
400
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
T.
..
..
..
..
..
..
.G
..
..
..
C.
..
..
..
..
..
.A
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
301
buffalo.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
301
camel.SEQ
..
..
..
..
..
..
..
..
..
G.
..
..
..
..
..
T.
..
..
..
..
..
..
.G
..
..
..
C.
..
..
..
..
A.
.A
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
301
cattle.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
301
human.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
301
pig.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
301
rabbit.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
301
rat.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
301
sheep.SEQ
TT
AC
TG
AA
AG
CC
AG
AG
CC
TG
AC
TC
TC
AC
TG
AT
GT
TG
AA
AA
GC
TG
CA
CC
TT
CC
TC
TG
CC
TC
TG
GT
CC
AG
TC
TT
GG
AT
GC
AC
CA
GC
CT
CC
CC
AG
CC
TC
TT
CC
Majority
410
420
430
440
450
460
470
480
490
500
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
T.
..
..
..
..
..
..
..
..
..
..
C.
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
.G
..
401
buffalo.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
401
camel.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
T.
..
..
..
..
..
..
..
..
..
..
C.
..
..
..
..
..
..
..
..
..
..
..
.A
..
..
..
..
..
..
401
cattle.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
401
human.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
401
pig.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
401
rabbit.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
401
rat.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
401
sheep.SEQ
TC
CA
AC
CG
TC
AT
GT
TT
CC
TC
CT
CA
GT
CC
GT
GC
TG
TC
CC
TT
TC
TC
AG
CC
CA
AA
GT
TC
TG
CC
TG
TT
CC
CC
AG
AA
AG
CA
GT
GC
CC
CA
GA
GA
GA
TA
TG
CC
CA
TC
Majority
510
520
530
540
550
560
570
580
590
600
..
..
..
T.
..
..
..
..
..
C.
..
..
..
..
..
..
..
..
..
..
..
..
..
T.
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
T.
TC
CC
C.
G.
GA
GA
T.
.G
501
buffalo.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
501
camel.SEQ
..
..
..
T.
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
T.
..
..
..
C.
..
..
..
..
..
..
..
..
..
..
..
..
..
T.
TC
CC
C.
G.
GA
GA
T.
.G
501
cattle.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
501
human.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
501
pig.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
501
rabbit.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
501
rat.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
501
sheep.SEQ
CA
GG
CC
TT
TC
TG
CT
GT
AC
CA
GG
AG
CC
TG
TA
CT
TG
GT
CC
TG
TC
CG
GG
GA
CC
CT
TC
CC
TA
TT
CT
TG
TC
TA
AX
XX
XX
XMajority
610
620
630
640
650
660
670
.C
CA
TT
CA
GG
CC
T.
TC
TG
.T
.T
.C
.A
G.
AG
.C
..
TA
.T
..
GT
.C
T.
TC
.G
GG
GA
..
CT
.C
.C
.A
.T
AT
TG
TC
TA
A
601
buffalo.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
.
601
camel.SEQ
.C
CA
TT
CA
GG
CC
T.
TC
TG
.T
.T
.C
.A
G.
AG
.C
..
TA
.T
C.
GT
.C
T.
TC
.G
GG
GA
..
CT
.C
.C
.A
.T
AT
TG
TC
TA
A
601
cattle.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
.
601
human.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
.
601
pig.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
.
601
rabbit.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
.
601
rat.SEQ
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
.
601
sheep.SEQ
Decoration 'Decoration #1': Hide (as '.') residues that match the Consensus exactly.
