A. Yilmaz , M. E. Davis1 , and H. C. Hines
The Ohio State University Department of Animal Sciences
The objective of this study was to search for mutations in the ovine IGF-I gene that may be related to production traits such as live weight and number of multiple births using Polymerase Chain Reaction (PCR) and Single-Strand Conformation Polymorphism (SSCP). A fragment of approximately 265 bp in the 5’ flanking region of the ovine IGF-I gene, about 530 to 795 bp upstream from the 3’ end of exon 1, was amplified for SSCP analysis. Sequences of primers that were used in PCR were 5'-ATTACAAAGCTGCCTGCCCC-3' (forward primer) and 5'-CACATCTGCTAATACACCTTACCCG-3' (reverse primer). A PCR-SSCP polymorphism was detected in the 5’ flanking region of the ovine IGF-I gene. Relationships of this polymorphism with production traits in sheep will be investigated.
Insulin-like growth factors are proteins that are bound to carrier proteins in the blood. The name insulin-like growth factor was given to these molecules due to their structural similarities to insulin. They are currently the subject of much livestock research due to their influence on various metabolic processes in the body. Insulin-like growth factor-I (IGF-I) is a polypeptide that increases cell proliferation (Svoboda and Van Wyk, 1983) and sugar uptake (Poggi et al., 1979) by cells.
Correlations of IGF-I concentrations with weaning weight, postweaning weight, and postweaning weight gain in heifers (Davis and Bishop, 1994; Davis and Simmen, 1997), with growth rate in pigs (Buonomo et al., 1987), with fetal growth in sheep (Gluckman et al., 1983), with litter size, fetal weight, total placental weight, and mammary gland weight in mice (Kroonsberg et al., 1989), and with growth in humans (Merimee et al., 1982) have been demonstrated.
Since DNA contains genetic information that maintains the cell, it would be interesting to search for mutations in DNA that may have the potential to change the expression of IGF-I protein. Previous research has shown the presence of two SSCP-detected alleles in the 5’ flanking region of the IGF-I gene in cattle (Ge et al., 1997). They detected a significant difference in frequency of two IGF-I alleles in cattle selected for high or low blood serum IGF-I concentration, indicating that these alleles could be used as markers for blood serum IGF-I concentration in cattle. The objective of this study was to search for the same marker in sheep that was found in cattle using the primers of Ge et al. (1997) and a mutation detection method called Single-Strand Conformation Polymorphism (SSCP).
DNA was obtained from sheep blood using common procedures (ethanol precipitation). Blood samples were collected from sheep located at the Ohio Agricultural Research and Development Center’s Wooster campus. The total number of samples was 240, 46 of which were genotyped at the time this manuscript was written. The sheep breeds were crossbreds or purebreds of Polypay, Hampshire, Targhee, Rambioullet, Dorset, and Suffolk.
PCR Conditions
A fragment of approximately 265 bp in the 5’ flanking region of the ovine IGF-I gene, about 530 bp to 795 bp upstream from the 3’ end of exon 1, was amplified for SSCP analysis. Primer sequences were 5'-ATTACAAAGCTGCCTGCCCC-3' (forward primer) and 5'-CACATCTGCTAATACACCTTACCCG-3' (reverse primer). A total of 30 ml of PCR mixture containing 1 ml of the forward primer (5'-ATTACAAAGCTGCCTGCCCC-3') and reverse primer (5'-CACATCTGCTAATACACCTTACCCG-3'), 2 ml of genomic DNA, 3 ml of dNTPs, 1 ml of Taq polymerase (Gibco-BRL), and 3 ml of 10X reaction buffer (Gibco-BRL), containing 1.5 mM MgCl2, was used. The amplification procedure was conducted in a DNA Thermal Cycler (Perkin Elmer Cetus) and consisted of 31 cycles of the conditions listed after denaturation at 95°C for two minutes: 94°C for 45 s, 58°C for one minute, and 72°C for one minute. The final extension step was carried out at 72°C for five minutes.
Polymorphism
Eight microliters of PCR product diluted with 16 ml of distilled water and 8 ml of diluting dye (95% formamide, 0.5% bromophenol, 0.5% xylene cyanol) were denatured at 95°C for 10 min and immediately plunged into ice for 5 min. The dilution was loaded onto a 12% polyacrylamide (37.5:1 Acrylamide/bis, 16 x 20 x 0.15 cm) gel with 10% glycerol. The gel was run in 0.5X TBE buffer (using Bio-Rad Protein II xi) at a constant voltage of 250 V at 20°C for 24 hours. Gels were silver stained (Bio-Rad Kit) for visualization of DNA bands.
Twenty mixed-breed sheep were examined, and three distinct genotypes were observed (AA, 0.70; BB, 0.05; and AB, 0.25; Figure 1). Calculated allele frequencies were A = 0.82 and B = 0.18. In addition, genotypic frequencies were determined in 22 purebred Polypay sheep (AA, 0.77 and AB, 0.23). Calculated allele frequencies in Polypays were: A = 0.89; B = 0.11. Observed and expected numbers of the respective genotypes in mixed-breed and Polypays indicated the existence of Hardy-Weinberg equilibrium (chi-square test statistic = 0.47, P > 0.10, df = 2).
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| Figure 1. SSCP analysis of amplified IGF-I 5’ flanking segments from 15 mixed-breed sheep. Three distinct phenotypes corresponding to A/A, A/B, and B/B genotypes are evident. |
Mendelian Inheritance
Codominant segregation of alleles was observed in six paternal half-sib progeny.
Chromosomal Location
The ovine IGF-I gene has been mapped to chromosome 3 (Imam-Ghali et al., 1991).
The mutation in the 5’ flanking region of the IGF-I gene that resulted in three distinct genotypes in sheep will be further investigated to determine if it influences production traits of sheep.
We thank Judy Riggenbach for her excellent technical support.
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