Abstract: This study aimed to analysis the genetic polymorphisms of MHC class II DRB gene in the Egyptian goat using PCR-RFLP method. The amplified fragment with size of 285-bp was digested by two restriction enzymes TaqI and PstI. Restriction digestion of PCR product by TaqI enzyme represented two digested fragments at 122- and 163-bp (T restriction pattern) or undigested fragment at 285-bp (t restriction pattern). The frequencies of TT, Tt and tt patterns of MHC class II DRB in the Egyptian goats were 29.5, 61.4 and 9.1%, respectively. After the PstI digestion of the 285-bp PCR products amplified by Egyptian goat DNA, the results showed that the frequency of pp pattern (270- and 15-bp restricted fragments) was 29.5% and the frequency of heterozygous Pp pattern (270-, 226-, 44- and 15-bp restricted fragments) was 70.5%, while the PP pattern (226-, 44- and 15-bp restricted fragments) was not displayed in tested goat animals. The results declared that the p pattern (64.8%) is dominant over the P pattern (35.2%) in Egyptian goat.
INTRODUCTION
Cell surface proteins coded for gene families in the Major Histocompatibility Complex (MHC) are classified as class I or class II based on differences in cellular distribution, molecular weight and function. Class I genes of the MHC code for proteins found on almost all nucleated cells of the body. Class II proteins are primarily restricted to the surface of immune cells and are responsible for immune regulation (Andersson et al., 1986). The characteristic feature of some class I and II genes is the very high degree of genetic polymorphism which has been documented in a number of vertebrate species (Klein, 1975; Zinkernagel and Doherty, 1979; Nagy et al., 1981).
There are different kinds of MHC class II molecules, where the DQ and DR subtypes are the most polymorphic both in man and domestic species and probably play a major role in the development of MHC restricted immune responses (Amills et al., 1996).
In cattle, two DQA genes, two DQB genes, three DRB genes and one DRA gene have been cloned and sequenced (Groenen et al., 1990 and Van der Poel et al., 1990). In the goat, one DRB gene with 22 different sequences has been described (Schwaiger et al., 1993). The resemblance between MHC polymorphisms in different species has been reported by Hoang-Xuan et al. (1982); Chardon et al. (1983) and Cameron et al. (1990), which is related to the persistence of short ancestral motifs, convergent evolution and overdominant selection (Klein, 1980, 1987; Hughes and Nei, 1989; Andersson et al., 1991).
The present research focused on the analysis of the genetic polymorphisms for MHC class II DRB gene in the Egyptian goat using TaqI- and PstI-RFLP method.
MATERIALS AND METHODS
Genomic DNA extraction: Genomic DNA was extracted from whole blood of 44 goat animals by phenol-chloroform method (John et al., 1991). Ten ml of blood taken on EDTA was mixed with 25 ml cold sucrose-triton and the volume was completed to 50 ml by autoclaved double distilled water. The nuclear pellet was suspended in lysis buffer with sodium dodecyl sulfate and proteinase K and incubated overnight in a shaking water-bath at 37°C.
Nucleic acids were extracted once with phenol, saturated with Tris-EDTA buffer followed by extraction with phenol-chloroform-isoamyl alcohol (25:24:1) and this was followed by extraction with chloroform-isoamyl alcohol (24:1). To the final aqueous phase, sodium acetate and cold 95% ethanol were added. The tubes were agitated gently to mix the liquids and a fluffy white ball of DNA was formed. The DNA was finally dissolved in an appropriate volume of 1X TE buffer. DNA concentrations were determined and diluted to the working concentration of 50 ng μL-1, which is suitable for polymerase chain reaction.
Polymerase chain reaction (PCR): The amplification of the second exon of the caprine DRB gene was achieved using primers DRB1.1: TAT CCC GTC TCT GCA GCA CAT TTC and DRB1.2: TCG CCG CTG CAC ACT GAA ACT CTC. A PCR cocktail consists of 1.0 μM upper and lower primers and 0.2 mM dNTPs, 10 mM Tris (pH 9), 50 mM KCl, 1.5 mM MgCl2, 0.01 % gelatin (w/v), 0.1% Triton X-100 and 1.25 units of Taq polymerase. The cocktail was aliquot into tubes with 100 ng DNA of goat. The reaction ran in a Perkin Elmar apparatus. The reaction was cycled for 1 min at 94°C, 1.30 min at 60°C and 2 min at 72°C for 30 cycles.
RFLP and agarose gel electrophoresis: Twenty microliter of PCR product were digested with 10 units of each restriction enzyme used in this study in a final reaction volume 25 μL. The reaction mixture was incubated at 37°C for PstI and at 65°C for TaqI in water bath over night. The restricted fragments were analyzed by electrophoresis on 2.5% agarose/1X TBE gel stained with ethidium bromide. The 100 bp ladder was used as molecular size marker.
RESULTS AND DISCUSSION
MHC class II molecules are glycoproteins composed of two non-covalently linked α and β chains. Several domains can be distinguished in each one: two extracellular domains (α1, α2 and β1, β2), a transmembrane segment and a cytoplasmatic tail (Amills et al., 1995). MHC class II molecules are mainly found at the surface of B cells and antigen presenting cells such as macrophages, dendritic cells and Langerhans cells. Their molecules are involved also in antigen presentation to CD4+ T cells which help B cells to produce appropriate immunoglobulins (Andersson, 1990).
The second exon of the caprine MHC class II DRB gene encodes the β1 domain of DR molecule which is in close contact with foreign antigen, displaying a very high degree of polymorphism with 22 different sequences identified (Schwaiger et al., 1993). This highly polymorphism correlated the functional ability of MHC II molecules to form complexes with very large array of different antigen derived peptides.
Our PCR product amplified the caprine MHC class II DRB23 allele (Caae DRB23) containing a 285-bp which is very similar to the caprine DRB5 allele with 96.5% nucleotide identify (Amills et al., 1995). Caae-DRB5 and caae-DRB23 diverged in five amino acid substitutions that are very polymorphic in goat. Four amino acids substitutions have been found at position 70 (Glu, Ser, Arg and Asp), three at position 71 (Arg, Ser and Lys), two at position 73 (Ala and Thr), three at position 74 (Ala, Glu and Asn) and four at position 78 (Tyr, Val, Cys and Phe) (Schwaiger et al., 1993).
Restriction analysis of the caprine second exon of MHC class II by TaqI enzyme represents two digested fragments at 122- and 163-bp (T restriction pattern) or undigested fragment at 285-bp (t restriction pattern). Also, the presence of the TaqI site could be associated with TTC codon (Phe) at position 40 which its absence associated with TAC codon (Tyr) at the same position (Amills et al., 1995).
Our results showed that the appearance of the TT homozyogous restriction pattern in 13 of 44 tested animals (29.5%), the Tt pattern was displayed in 27 animals (61.4%) and the homozyogous tt pattern was found in 4 animals (9.1%) (Fig. 1). This result declared that the T pattern is dominant over the t pattern in the Egyptian goat breeds where the frequency of T pattern was 60.2%, while the frequency of t pattern was 39.8% in tested animals.
Also, the results declared the absence of TaqI restriction pattern at position 40 bp, indicating the presence of the TAC codon (Tyr) at this position. Tyr is considered as one of the amino acids play an important role in the HLA-DR molecule, which involved in the formation of the antigen-binding site (Brown et al., 1993). In cattle, Tyr substitution at position 78 of the bovine DR molecule has been correlated with a tendency towards susceptibility to persistent lymphocytosis in cattle infected with bovine leukemia virus (Lewin, 1994).
The PstI polymorphism sites expressed at bands 226-, 44- and 15-bp (P restriction pattern) or 270- and 15-bp (p restriction pattern). The PstI site at position 15-bp reported as a non-polymorphic site.
| Fig. 1: | DNA electrophoretic pattern obtained after digestion of PCR amplified goat MHC class II products with TaqI | ||||||||
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| Fig. 2: | DNA electrophoretic pattern obtained after digestion of PCR amplified goat MHC class II products with PstI | ||||||
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The presence of PstI site could be associated with TTC or TAC codon at position 78, while its absence associated with GTG (Val) or TGT (Cys) codon at the same position (Amills et al., 1995).
After the PstI digestion of PCR, we found the appearance of homozyogous pp pattern in 13 animals (29.5%). The heterozygous Pp pattern was displayed in 31 animals (70.5%) whereas PP pattern was not displayed in tested animals (Fig. 2). Present results showed that the frequency of P allele in Egyptian goat was 35.2% and the allele p was predominant in our breeds where its frequency was 64.8%. Also, the result revealed the absence of PstI restriction site at position 78 bp, indicating the presence of GTG (Val) or TGT (Cys) codon at this position.
The genetic polymorphisms of the caprine MHC class II DRB gene were studied in three Spanish breeds (Amills et al., 1995). Their frequencies patterns were 65%, (T), 35% (t), 41% (P) and 59% (p). This result nearly close to our results where the pattern frequencies in the Egyptian goats were 60.2, 39.8, 35.2 and 64.8% for T, t, P and p patterns, respectively.
In conclusion, the structural organization and polymorphisms of the caprine MHC provided the possibility for existence of close associations between restriction sites and amino acid substitutions at position with disease resistance. Therefore, improving of the caprine quantitative traits in different breeds require further studies for detecting the polymorphisms of the Antigen-Recognition Site (ARS) of the caprine MHC molecules and their values in disease resistance.