Small Ruminant Research 64 (2006) 285–292

Nucleotide sequencing and DNA polymorphismstudies on IGFBP-3 gene in sheep and its comparison

with cattle and buffalo

Pushpendra Kumar ∗, V. Choudhary, K. Ganesh Kumar, T.K. Bhattacharya,B. Bhushan, Arjava Sharma, A. Mishra

Molecular Genetics Laboratory, Animal Genetics Division, Indian Veterinary Research Institute,Izatnagar, Bareilly-243 122, Uttar Pradesh, India

Received 7 September 2004; received in revised form 14 April 2005; accepted 4 May 2005Available online 24 June 2005

Abstract

The insulin-like growth factor binding protein-3 (IGFBP-3) gene is a structural gene associated with the growth and devel-opment of the animals. The present investigation was carried out to study nucleotide sequencing and DNA polymorphism byPCR-RFLP of IGFBP-3 gene in sheep and its comparison with cattle and buffalo. Genomic DNA was isolated from a total of 152

animals from different livestock farms of India belonging to five Indian breeds of sheep namely Marwari, Mandya, Madras Red,Muzaffarnagari and Banur. A fragment of IGFBP-3 gene, comprising of a part of exon 2, complete intron 2, exon 3, and a partof intron 3, was amplified. The amplified fragment was found to be 654 bp in sheep as compared to our earlier reports of 651 bpin cattle and 655 bp in buffalo. On digestion of 654 bp with HaeIII restriction enzyme yielded single restriction pattern of eightfragments of sizes 201, 201, 87, 67, 56, 19, 16 and 7 bp in all the animals studied revealing absence of polymorphism in sheep. Oncomparison, this fragment was reported to be polymorphic in cattle and monomorphic in buffalo. Nucleotide sequencing of theamplified fragment of IGFBP-3 gene of Banur sheep was done and submitted to the NCBI GenBank (accession no. AY306012).The nucleotide sequence analysis indicated that the percent similarity of IGFBP-3 gene fragment of sheep with cattle and buffalowas nearly 90%. There was approximately 93% similarity in the amino acid sequence of sheep with cattle and buffalo.© 2005 Elsevier B.V. All rights reserved.

Keywords: IGFBP-3 gene; PCR-RFLP; Sheep; Nucleotide sequence; Polymorphism

∗ Corresponding author. Tel.: +91 581 2303382;fax: +91 581 2303284.

E-mail addresses: pushpendra@ivri.up.nic.in,pushpendraivri@rediffmail.com (P. Kumar).

1. Introduction

India possesses 40 recognized breeds of sheep thatare used mainly for the production of meat and wool.As India has widely varying agro-climatic conditions,

0921-4488/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.smallrumres.2005.05.018

286 P. Kumar et al. / Small Ruminant Research 64 (2006) 285–292

breeding for native sheep breeds over the years hasmade them to thrive better under warm and harshclimatic conditions and resistant to many pathogensand diseases. However, their inherent potential forgrowth and meat production has not been exploiteddue to inadequate information about genetic basis andthe breeding strategies.

Insulin-like growth factor binding protein-3(IGFBP-3) gene is a structural gene responsible for themultiple effects of insulin-like growth factors (IGFs)(Bale and Conover, 1992). IGF-I and IGF-II are coupleof hormones involved in the process of mammaliangrowth and regenerative processes besides havingactive role in mammary gland development (Hossneret al., 1997).

The bovine IGFBP-3 gene has been cloned andcharacterized and its mRNA is 1.65 kb in length (Sprattet al., 1991). The total length of gene is 8.9 kb havingfive exons (Martin and Baxter, 1992). Polymorphicstudies and nucleotide sequencing of IGFBP-3 genehave been reported in cattle (Maciulla et al., 1997;Haegeman et al., 1999; Shukla, 2001; Sun, 2002) andbuffalo (Padma et al., 2004). No such information isavailable in sheep so far, however, mRNA expressionof IGFBP-3 gene has been studied (Hastie et al., 2004)and the same group have submitted mRNA partialsequence to GenBank (accession no. AF327651). Con-sidering the importance of sheep in meat production,the present study was undertaken to find out poly-morphism, if any, and to sequence the sheep IGFBP-3g

2

2

1vMaaIbMtT

Farm, IVRI, Izatnagar, Bareilly, UP, respectively. Theblood samples of Banur animals were taken from Live-stock research station, Bombay Veterinary College,Mumbai.

2.2. Blood collection and DNA isolation

Approximately, 10 ml venous blood was collectedfrom each animal using 0.5 ml of 2.7% EDTA asan anticoagulant. The samples were brought to thelaboratory in a double-walled icebox containing icewith cool pack and stored at −20 ◦C till the isolation ofDNA. Genomic DNA was isolated from blood follow-ing the protocol of Sambrook and Russell (2001). Thequality of DNA was checked by spectrophotometrytaking ratio of optical density (OD) value at 260and 280 nm. Good quality DNA having OD ratiobetween 1.7 and 1.9 was used for further work. Thepoor quality DNA was re-extracted with phenol–chloroform.

2.3. PCR-RFLP and nucleotide sequencing

A region of IGFBP-3 gene spanning over a part ofexon 2, complete intron 2, exon 3 and a part of intron3 was amplified by using a set of forward (P3: 5′-CCAAGC GTG AGA CAG AAT AC-3′) and reverse (P4:5′-AGG AGG GAT AGG AGC AAG AT-3′) primers(Maciulla et al., 1997). For amplification, 25 �l ofPCR reaction was prepared by adding 10 pM of eachpPToMddaowtptbvs

1

ene and compare it with those of cattle and buffalo.

. Materials and methods

.1. Experimental animals

The present study was conducted on a total of52 animals belonging to five Indian sheep breedsiz. Marwari (30), Mandya (30), Madras Red (30),uzaffarnagari (30) and Banur (32) maintained

t different livestock farms of India. The Marwarinimals are maintained at Central Arid Zone Researchnstitute, Jodhpur, Rajasthan whereas Mandya animalselonged to Madras Veterinary College, Chennai. Theadras Red and Muzaffarnagari animals were main-

ained at Livestock Research Station, Kattupakkam,NUVAS, Chennai, Tamilnadu and Sheep and Goat

rimer, 100 �M of each dNTPs, 1.5 mM MgCl2, 10×CR assay buffer, 100 ng DNA template and 0.5 Unitaq DNA polymerase. The amplification was carriedut using a pre-programmed thermal cycler (PTC-200,

J Research) with the following conditions: initialenaturation of 5 min at 94 ◦C followed by 35 cycles ofenaturation at 94 ◦C, annealing at 60 ◦C and extensiont 72 ◦C each of 1 min and lastly the final extensionf 5 min at 72 ◦C. An aliquot of 20 �l of PCR productas digested overnight with 5 Units of HaeIII restric-

ion enzyme. The restriction enzyme digested PCRroducts were electrophoresed in 4% agarose gel con-aining ethidium bromide as staining agent in 1× TAEuffer for 2–3 h at 6 V/cm. The digested products wereisualized and documented under gel documentationystem.

For sequencing, the PCR products were run in.5% agarose gel and the product band was eluted

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Fig. 1. A 654 bp sequence of IGFBP-3 gene of sheep (NCBI accession no. AY306012).

using gel elution kit (GIBCO BRL) for purification.The purified PCR products were sequenced usingautomated dye-terminator cycle sequencing methodwith Ampli Taq DNA polymerase in ABI PRIZM377 DNA sequencer (Perkin-Elmer). The sequenceanalysis was performed using DNASTAR programme.

3. Results and discussion

Nucleotide sequencing of amplified fragment ofIGFBP-3 gene of sheep was done and submitted to theNCBI GenBank (accession no. AY306012) (Fig. 1).

FIgl

Although Hastie et al. (2004) has first reported thecDNA sequence of this gene but this is the first report ofnucleotide sequencing of IGFBP-3 gene of sheep usinggenomic DNA. The length of the amplified productof IGFBP-3 gene was 654 bp in sheep (Fig. 2). How-ever, in our earlier work we have reported the length ofamplified product as 651 bp in cattle (Fig. 3) (Shukla,2001; Choudhary, 2004) and 655 bp in buffalo (Kumaret al., 2004) (Fig. 4).

FILm

ig. 2. Representative HaeIII restriction fragment pattern of 654 bpGFBP-3 gene of sheep. Lanes 1–5, AA genotype; Lane U, undi-ested PCR product; Lane M, molecular size marker (100 bp DNAadder).

ig. 3. Representative HaeIII restriction fragment pattern of 651 bpGFBP-3 gene in cattle. Lane 1, AA genotype; Lane 2, AB genotype,ane 3, BB genotype; Lane U, undigested PCR product; Lane M,olecular size marker (100 bp DNA ladder).

288 P. Kumar et al. / Small Ruminant Research 64 (2006) 285–292

Fig. 4. Representative HaeIII restriction fragment pattern of 655 bpIGFBP-3 gene in buffalo. Lanes 1–4, AA genotype; Lane U, undi-gested PCR product; Lane M, molecular size marker (100 bp DNAladder).

The amplified 654 bp fragment of sheep IGFBP-3 gene is comprised of last part of exon 2, completeintron 2, exon 3 and a part of intron 3 (Fig. 5a). Theexon–intron regions were assigned on the basis of thepublished reports of this gene in cattle (Maciulla et al.,1997).

Digestion of the PCR product with HaeIII revealedonly one type of restriction pattern yielding eightfragments of sizes 201, 201, 87, 67, 56, 19, 16 and7 bp in sheep (Fig. 2). However, three genotypes wereidentified in exotic (Holstein Friesian and Jersey) cattlewith restriction fragments of sizes 199, 164, 154, 56,36, 18, 16 and 8 bp (AA genotype); 215, 164, 154, 56,36, 18 and 8 bp (BB genotype) and 215, 199, 164, 154,56, 36, 18, 16 and 8 bp (AB genotype) (Shukla, 2001;Choudhary, 2004). The present results showed onlyone genotype (AA) in sheep, similar to the findingsreported in buffalo (Padma et al., 2004). However, thesizes of restriction fragments were differed as 201, 165,154, 56, 36, 19, 16 and 8 bp (Padma et al., 2004). Allthe sheep have intact HaeIII restriction site (GG ↓ CC)at the base no. 300 (Fig. 1) indicating the absence ofpolymorphism at this site. However, the correspondingsite in cattle showed polymorphism due to absence of

Fig. 5. Diagrammatic representation of (a) exon–intron regions of sheep anin sheep, cattle and buffalo.

d (b) HaeIII restriction sites on amplified IGFBP-3 gene fragment

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Fig. 6. Nucleotide sequence comparison of amplified IGFBP-3 gene of sheep with cattle and buffalo.

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Fig. 6. (Continued ).

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Fig. 7. Phylogenetic tree based on nucleotide sequence of IGFBP-3gene of sheep, cattle and buffalo.

this site. The polymorphism in the cattle was due toC → A (GG ↓ CC to GG AC) transition in intron 2 ofthe gene at 299th base position of HF sequence, whichalters a HaeIII restriction site (Choudhary, 2004). Insheep the GG ↓ CC is present in all the animals andthus the site of HaeIII is maintained yielding only‘AA’ genotype. A comparative analysis of HaeIIIrestriction sites in IGFBP-3 gene fragment of sheep,cattle and buffalo is depicted in Fig. 5b. The presentfindings of sheep could not be compared with otherstudies, as no such report on sheep is available in theliterature.

3.1. Nucleotide sequence comparison

The sequences of same region of IGFBP-3 gene ofcattle (NCBI GenBank accession no. AY306011) andbuffalo (NCBI GenBank accession no. AY304829)submitted by us were used to compare with sheepsequence. There were five insertions and two dele-tions of nucleotides in the intronic region of sheepIGFBP-3 gene causing an increase in the lengthof PCR product to 654 bp as compared to 651 bpof cattle (Fig. 6). There was one insertion and twodeletions of nucleotide in sheep sequence in com-parison to 655 bp fragment of buffalo (Fig. 6). Theseinsertions/deletions could be utilized as a marker for

Table 1Nucleotide sequence distances: percent similarity (above diagonal);percent divergence (below diagonal) of IGFBP-3 gene of sheep, cattleand buffalo

Breeds Sheep Cattle Buffalo

Sheep – 89.7 89.9Cattle 6.2 – 93.9Buffalo 7.3 2.8 –

species differentiation/identification. Comparison ofthe nucleotide sequence from this study with that ofalready published cDNA sequence (GenBank acces-sion no. AF327651) of sheep showed 100% homologyat the exon 3 (base pair 498–617) of the present studysequence.

The nucleotide sequence analysis indicated that thepercent similarity of IGFBP-3 gene fragment of sheepwith cattle and buffalo was nearly 90% (Table 1).Based on respective IGFBP-3 gene sequences thephylogenetic tree was constructed among sheep, cattleand buffalo showing their relative distance (Fig. 7).

3.2. Protein sequence comparison

The partial sequence of exon 2 and exon 3 present inthe amplified IGFPB-3 gene fragment of sheep, cattleand buffalo was translated using standard translationmethod of DNASTAR programme (Fig. 8). The pro-tein sequence of sheep is different from the cattle andbuffalo sequence by three amino acids. The changedamino acids were aspartic acid, threonine and ala-nine in sheep as compared to histidine, methionine andglycine, respectively, in cattle and buffalo. Hence, therewas approximately 93% similarity in the amino acidsequence between sheep and the two bovine speciesviz. cattle and buffalo.

ce of IG

Fig. 8. Comparative analysis of protein sequen FBP-3 gene of sheep with cattle and buffalo.

292 P. Kumar et al. / Small Ruminant Research 64 (2006) 285–292

4. Conclusion

In conclusion, the HaeIII-RFLP studies indicatedsingle restriction pattern in all the breeds of sheep. Thelength of amplified product of IGFBP-3 gene variedwith that of cattle and buffalo. The nucleotide sequenceanalysis indicated that the percent similarity of IGFBP-3 gene fragment of sheep with cattle and buffalo wasnearly 90%. Three amino acids were different in theprotein of IGFBP-3 of sheep as compared to the twobovine species.

Acknowledgement

The authors are thankful to the Director, IndianVeterinary Research Institute, Izatnagar, Bareilly forproviding necessary facilities to carryout this researchwork.

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