Nucleotide Sequencing and PCR-RFLP of Insulin-like Growth Factor Binding Protein-3 Gene in Riverine Buffalo ( Bubalus bubalis )

Insulin-like growth factor binding protein-3 (IGFBP-3) gene is a structural gene associated with the growth and development of the animals. The present investigation was carried out to unravel nucleotide sequence and polymerase chain reactionrestriction fragment polymorphism (PCR-RFLP) of IGFBP-3 gene in buffalo. Genomic DNA was isolated from a total of 157 animals belonging to Murrah, Surti, Jaffarabadi and Nagpuri breeds of Indian riverine buffalo. A 655 bp of IGFBP-3 gene was amplified in all the breeds and amplicons were digested with Hae III, Taq I and Msp I restriction enzymes. On digestion with Hae III yielded single restriction pattern of 8 fragments of sizes 201, 165, 154, 56, 36, 19, 16 and 8 bp in all the animals studied. Similarly Taq I and Msp I also revealed single restriction pattern yielding fragments of sizes 240 and 415 bp and 145 and 510 bp, respectively. This shows nonpolymorphic nature of restriction sites in buffalo. Nucleotide sequencing of 587 bp of IGFBP-3 gene in Murrah buffalo was done and submitted to the GenBank (Accession No. AY304829). Nucleotide sequencing revealed an addition of 4 bases in the intronic region as compared to cattle. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 7 : 910-913)


INTRODUCTION
India possesses more than half of the world buffalo population and account for 55% of the country's total milk production.Indian buffaloes are hardy, thrive well on poor quality nutrition and are also good converter of inputs into milk as compared to cattle (Tomer, 1999;Biswas et al., 2003).Buffalo is also emerging as a good source of meat apart from its better milk production.However, their inherent potentials for growth and production have not been exploited due to inadequate information about genetic basis and the breeding strategies.
Insulin-like growth factor binding protein-3 (IGFBP-3) gene is a structural gene responsible for the multiple effects of insulin-like growth factors (IGFs) (Bale and Conover, 1992).IGF-I and IGF-II are a couple of hormones involved in the process of mammalian growth and regenerative processes besides having active role in mammary gland development (Hossner et al., 1997) and also has a role in muscle protein synthesis in young chickens (Kita et al., 2002).
Cattle IGFBP-3 gene has been cloned and sequenced and found to be 8.9 kb in length (Spratt et al., 1991).Polymorphic studies and nucleotide sequencing of IGFBP-3 gene have been reported in cattle (Maciulla et al., 1997;Haegeman et al., 1999;Sun et al., 2002).However, no such study has been reported in buffalo so far.Considering the importance of buffaloes in Indian context, there is a need to identify a molecular marker for growth performance of the animals.Based on above observations, present study has been undertaken to find out polymorphism and sequence the buffalo IGFBP-3 gene.

Experimental animals
This study was conducted on four breeds of riverine buffalo involving 54 animals of Murrah breed maintained at Cattle and Buffalo farm, IVRI, Izatnagar (UP); 25 of Surti breed maintained at Gujarat Agricultural University, Anand (Gujrat); 32 animals of Jaffarabadi breed maintained at cattle breeding farm, Gujarat Agricultural University, Junagarh (Gujarat) and 46 animals of Nagpuri breed from the adjoining fields of Nagpur district in Maharastra which is the breeding tract of this breed.

Collection of blood samples
About 15 ml of venous blood was collected from the jugular vein into a sterile 15 ml polypropylene centrifuge tube containing 0.5 ml of 2.7% EDTA solution as an anticoagulant.It was then brought to the laboratory in ice box and stored at 4°C till the isolation of DNA.

Isolation of genomic DNA
The genomic DNA was isolated by phenol-chloroform extraction method as described by Sambrook et al. (1989).
The purity of genomic DNA was assessed by spectrophotometry.Samples showing OD ratio (260 nm/280 nm) between 1.7 and 1.9 were retained for further analysis and others were reprocessed.Concentration of DNA was calculated by using OD value at 260 nm by the following formula: DNA concentration (µg/ml)= OD 260 ×dilution factor×50.
The amplification was carried out using a preprogrammed thermal cycler (PTC-200, M J Research) with the following conditions: initial denaturation of 5 min at 94°C followed by 35 cycles of denaturation at 94°C, annealing at 60°C and extension at 37°C each of 1 min and finally the final extension of 5 min at 72°C.The PCR products were checked by agarose gel electrophoresis using 1.5% agarose gel in 1×TAE buffer at 6 volts/cm for one hour.The amplified product was visualized under UV transilluminator.

Restriction enzyme analysis
The amplified PCR products were digested separately with three restriction enzymes viz.Hae III, Taq I and Msp I.About 15 µl of PCR product was digested with 5 units of restriction enzyme using suitable 10×RE buffer.Hae III and Msp I digestion was carried out at 37°C and Taq I digestion at 65°C for 3 h.The RE digested PCR products were electrophoresed in agarose gel containing ethidium bromide as staining agent in 1×TAE buffer for 2-3 h at 6 volts/cm.Hae III digests were electrophoresed in 4% agarose and Taq I and Msp I digests were electrophoresed in 2% agarose gel.The digested products were visualized and documented under gel documentation system.

DNA sequencing
PCR products were run in 1% agarose gel and the product band was eluted using gel elution kit (GIBCO BRL) for purification.The purified PCR products were sequenced using the automated dye-terminator cycle sequencing method with Ampli Taq DNA polymerase in ABI PRIZM 377 DNA sequencer (Perkin-Elmner).

RESULTS AND DISCUSSION
Nucleotide sequencing of 587 bp of IGFBP-3 gene in Murrah buffalo was done and submitted to the GenBank (Accession No. AY304829) (Figure 1).This was the first report of sequencing of buffalo IGFBP-3 gene.The length of the amplified product of IGFBP-3 was 655 bp in all the breeds of buffalo studied (Figure 2).In contrast, a 651 bp of amplified product was found in cattle using the same set of primers in our laboratory (Accession No. AY306011) (Shukla, 2001).There were some insertions of nucleotides in the intronic region of buffalo IGFBP-3 gene causing an increase in the length of PCR product to 655 bp as compared to 651 bp of cattle.These insertions could be utilized as a maker for species differentiation/ identification.
The 655 bp of amplified fragment of buffalo IGFBP-3 gene comprised of last part of exon 2, complete intron 2, 1 tcttgtggat gtgggggtgg ggccacctg g ccctgggtat ccagagatca cagggtc acc 61 attactcaag agcccagcag t t a c t c c a g t g g t c c t g c t g a t g c a c t g a g cagctg tgag 121 cccctgctta cagaagggat a t t g a c c c t c ccctatggca gagatcccag gagaatcagt 181 gcactgctct ccaggcctcg gctgggcaga g c a g t g t t c t cacaaagctg g c c t c t t t t t 241 gttcacttgg c c t c t g a g t g t c c t g g c c t g t g t a t c c c t g t c c c a g t c c t gtagc ttgcc 301 ctggggaatc acaagagaga cagggggctg t g g t t g g c a t ctgcacagga acagtgacaa 361 ctaaatcaga caaaagatac tcgaggagca c g t g g t c a g t c c c c t g g g t g t t a c a g g g t t 421 ttatcagaca cagagttccc agg ta a c cc a t g c c t c c t t c ccaggggccc tgccgccggg 481 aaatggaaga cacgctgaac c a c c t g a a g t t c c t g a a c a t gctcagcccc aggggcatcc 541 acattcccaa ctgcgacaag a a g g g c t t c t acaagaaaaa gcaggtg exon 3 and a part of intron 3 (Figure 3).The exon-intron regions were assigned on the basis of the available reports in cattle (Spratt et al., 1991).To study the polymorphism, the sequence was then analyzed for restriction sites of various enzymes.The restriction sites of Hae III, Taq I and Msp I enzymes in IGFBP-3 gene are presented in Figure 3. Digestion of the PCR product with Hae III revealed only one type of restriction pattern yielding 8 fragments of sizes 201,165,154,56,36,19,16 and 8 bp (Figure 4).This pattern was assigned as AA genotype.However, three genotypes were identified in exotic cattle with 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) (Maciulla et al., 1997).All the buffaloes have intact Hae III restriction site at the base no.266 (Figure 1) indicating absence of polymorphism at this site.However, the corresponding site in cattle showed polymorphism due to absence of this site.
Since Hae III showed absence of polymorphism, other enzymes i.e.Taq I and Msp I were tried.The reason behind selecting Taq I was its position in the intron 2 of 655 bp fragment of IGFBP-3 gene.It was assumed that Taq I site could show polymorphism as mutations are relatively more common in introns than exons.Though introns do not code for any protein, it may play a role in splicing of mRNA and its stability.All the animals screened showed the presence of a Taq I site, characterized by a single homozygous genotype possessing two fragments of sizes 415 and 240 bp (Figure 5).No homozygote possessing only 655 bp fragment or heterozygote with three fragments of sizes 655, 415 and 240 bp were found.
The Msp I site (C ↓ CGG) on 655 bp fragment is located in the exon 3. The site involves two adjacent codons, both coding for Arginine.Since four bases of the site comprise two wobble bases (1 st C and 2 nd G of C ↓ CGG) of the two   codons, there is a high possibility of occurrence of mutation for this site at the wobble base.So the Msp I enzyme was employed to check for the presence or absence of the site and thereby to determine the genotypes.All the animals revealed presence of Msp I site yielding two fragments of sizes 145 and 510 bp (Figure 5).Thus the Msp I site was also non-polymorphic as was in the case of Hae III and Taq I.The findings of present study with Taq I and Msp I could not be compared as this aspect has not been reported in any species of livestock.
In conclusion, the PCR-RFLP studies with Hae III, Taq I and Msp I indicated single restriction pattern in all the breeds of buffalo.The nucleotide sequencing revealed an addition of four bases in the intronic region of buffalo IGFBP-3 gene as compared to cattle.These findings may be utilized as a marker for species differentiation/ identification.

Figure 3 .
Figure 3. Diagrammatic representation of exon-intron regions and Hae III, Msp I and Taq I restriction sites on IGFBP-3 gene.