Genotyping of Feline MHC (FLA) Class II DRB by PCR-RFLP Method Using Group-Specific Primers.

Abstract

ABSTRACT. For genotyping of feline major histocompatibility complex (FLA) class II DRB, the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method using group-specific primers was tried. Sixty-six DRB genes were classified into 8 groups according to differences in the first 5' amino acid sequences. The group-specific primers were designed as forward ones, which were specific for 5' base sequences of genes in each group. Three to 7 appropriate restricted enzymes were selected by computer analysis for RFLP typing of the genes divided into each group. In 6 out of 9 cats, the results of DRB typed by direct sequence method agreed with results of the PCR-RFLP method using group-specific primers. In the other 3 cats, the number of genes amplified by group-specific primers was 1 or 2 more than those detected by direct sequence method. The direct sequence method in 9 cats identified 5 new FLA-DRB genes. The PCR-RFLP method using group-specific primers could divide 66 genes into 37 genes and 10 subgroups from the RFLP pattern. One to 6 genes in each cat, and a total of 203 genes and subgroups were detected in 68 domestic cats. The genes detected might be biased to the subgroup G1-1a (28.8%), DRB*0501 (10.3%), G1-2a (9.4%) and G6b (7.4%). The PCR-RFLP method using group-specific primers may be useful in typing FLA class II DRB. KEY WORDS: feline, FLA class II DRB genotyping, group-specific primer, PCR-RFLP method.J. Vet. Med. Sci. 62(12): 1283-1289, 2000 Major histocompatibility complex (MHC) class II molecules are heterodimeric glycoproteins involved in the regulation of the immune responses [21]. Human leukocyte antigen (HLA) class II genes consist of various components, DR, DP, DQ and etc. [11]. However, the structure of feline MHC (FLA) class II has not been well established, and only DR genes have been detected at the present time [29]. The DRB gene which encodes the β chain of DR molecule is the most polymorphic in class II genes in humans [13], pigs [8] and dogs [3]. Polymorphism of DRB gene accumulates at the second exon, which encodes the first extracellular domain, β 1 domain of the DR β chain [10]. The type of DRB gene is associated with the result of mixed lymphocyte reaction (MLR) [1], percent of 1-year renal graft survival [22], as well as susceptibility to autoimmune diseases [21,25] in humans. Polymerase chain reaction (PCR)-sequence specific oligonucleotide probe (SSOP) method or PCR-restriction fragment length polymorphism (RFLP) method has been used commonly to distinguish the genotypes of MHC class II [9,11]. It has also been reported that digestions of PCR products by some restriction endonucleases after the PCR for grouping of alleles by group-specific primers (modified PCR-RFLP method combined with group-specific primers) were used for genotyping of human DRB1 [16].Chronic renal failure (renal sclerosis) is one of the frequent and important diseases in feline medicine. The basic therapies for this failure are fluid therapy, and administrations of antibiotics, steroids,...

Effects of Genotype Matching of Feline Major Histocompatibility Complex (FLA) Class II DRB on Skin-allograft Transplantation in Cats.

“…An RT reaction for synthesizing cDNA from RNA was performed with a thermal cycler (TaKaRa TP2000, Takara Shuzo Co., Ltd., Shiga); 42°C for 30 min and 99°C for 5 min with a commercial kit (RNA PCR Kit, Takara Shuzo Co., Ltd., Shiga) including AMV Reverse Transcriptase XL (Life Sciences, Inc., FL, U.S.A.). The second exon of FLA-DRB gene was amplified from cDNA by PCR using groupspecific primers (G1-1, G1-2, G2, G3, G4, G5, G6 and G7) [10] as forward primers, and oligonucleotides for human DRB61A (5'-CCGCTGCACTGTGAAGCT-3') [5] as a common reverse primer [24]. Thermal cycles were performed under the same conditions described in another paper [10].…”

“…The second exon of FLA-DRB gene was amplified from cDNA by PCR using groupspecific primers (G1-1, G1-2, G2, G3, G4, G5, G6 and G7) [10] as forward primers, and oligonucleotides for human DRB61A (5'-CCGCTGCACTGTGAAGCT-3') [5] as a common reverse primer [24]. Thermal cycles were performed under the same conditions described in another paper [10]. PCR amplified products were digested with some restriction endonucleases (G1-1: Alu I, Hae III, Bpm I, BstU I and Eae I; G1-2: BssH II, EcoR II, Taq I, Fnu4H I and Eae I; G5: Mae III, BssH II, Bsr I and Dde I; G6: Bsr I, BssH II, Eae I, Ava II and Xmn I) [10].…”

“…Thermal cycles were performed under the same conditions described in another paper [10]. PCR amplified products were digested with some restriction endonucleases (G1-1: Alu I, Hae III, Bpm I, BstU I and Eae I; G1-2: BssH II, EcoR II, Taq I, Fnu4H I and Eae I; G5: Mae III, BssH II, Bsr I and Dde I; G6: Bsr I, BssH II, Eae I, Ava II and Xmn I) [10]. The restriction enzyme-cleaved DNAs were subjected to electrophoresis in 3% agarose gel (Mupid II, Cosmo Bio Co., Ltd., Tokyo), and visualized by staining with ethidium bromide.…”

“…The feline MHC (FLA)-DRB gene also displays an extensive degree of genetic polymorphism [10,23,24]. Sixty-six FLA-DRB sequences have been found by PCRsequence-based typing in 45 cats [10,24], and those have been divided into 37 genes and 10 subgroups according to the RFLP pattern by us [10].…”

“…Sixty-six FLA-DRB sequences have been found by PCRsequence-based typing in 45 cats [10,24], and those have been divided into 37 genes and 10 subgroups according to the RFLP pattern by us [10]. To date, 1 to 6 kinds of FLA-DRB genes per head have been identified [10,24], so that cats may have more than 3 loci in the DRB gene. However, it remains unknown what functions the expressed DRB genes have in cats.…”