Rapid communication: Three new allelic sequences at the ovine MHC class II DQA1 locus 1 H. Zhou and J. G. H. Hickford 2 Animal & Food Sciences Division, Lincoln University, Canterbury, New Zealand Name of Sequences. Ovine major histocompatibility complex (MHC) class II DQA1 alleles. Genus and Species. Ovis aries, New Zealand Merino breed. Origin of Clones. The published ovine MHC Class II DQA1 sequences (Scott et al., 1991; Fabb et al., 1993; Wright and Ballingall, 1994; Snibson et al., 1998) were used to design two primers for amplifying the second exon of the ovine MHC class II DQA1 gene. These prim- ers were 5′-ACTGACTCAGCTGACCACA-3′ (forward) and 5′-ATATTGTTGGTAGCAGCAGTAG-3′ (reverse), corresponding to nucleotides 349–367 and 590–611 of the DQA1 gene (Scott et al., 1991; GenBank M33304). Primers were synthesised by Life Technologies (Rock- ville, MD). PCR products amplified by Taq DNA poly- merase (Qiagen GmbH, Hilden, Germany) (consisting of initial denaturation at 94°C for 90 s, followed by 32 cycles of 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C, with a final extension of 5 min at 72°C) were cloned into pGEM T-Easy vector (Promega, Madison, WI) and both DNA strands were sequenced by the DNA Se- quencing Facility at the University of Waikato, New Zealand using M13 primers. At least three independent PCR amplifications for each sequence were carried out to confirm sequence identity, and only identical se- quences from at least three independent amplifications were analyzed. Of a number of sequences obtained, three sequences showed differences from those pre- viously published. Comparison with Related Sequences. The three novel ovine sequences, excluding the primer binding regions, were compared to sequences in the NCBI GenBank databases using a BLAST program. The most homolo- gous sequences were from the ovine MHC Class II DQA1 gene, suggesting that they represent previously unidentified alleles of the ovine DQA1 gene. A homology tree between these newly identified alleles and those published is shown in Figure 1. 1 This work was supported by PGSF (LIN801) and MeatNZ (LU165). We thank Susan Leslie and Hayley Ridgway for providing some sheep DNA samples. 2 Correspondence: P.O. Box 84 (phone: (64) (03) 325 2811; fax: (64) (03) 325 3851; E-mail: hickford@lincoln.ac.nz). Received June 26, 2000. Accepted October 16, 2000. 2001 American Society of Animal Science. All rights reserved. 779 Figure 1. Homology tree of the new and published ovine MHC class II DQA1 alleles. Alleles A, B, C, D, E, F, and G correspond to the sequences with the following respective GenBank accession numbers: M33304 (Scott et al., 1991), M93430 (Fabb et al., 1993), Z28418 (Wright and Ballingall, 1994), Z28420 (Wright and Ballingall, 1994), Z28518 (Wright and Ballingall, 1994), L49463 (Snibson et al., 1998), and L49464 (Snibson et al., 1998). H, I, and J represent three newly identified alleles (marked with *). Sequence Data. These sequences contained the second exon of the ovine DQA1, encoding the α 1 external do- main. Most of the amino acid sequence polymorphisms were located in three distinct regions corresponding to the first strand of the β pleated sheet and the start and end of the α helix lining the antigen binding site (Figure 2). EMBL/GenBank Accession Numbers. AF276954 (al- lele H), AF276955 (allele I), and AF276956 (allele J). Comments. DQA1 is a member of Class II major histo- compatibility complex genes whose products play an important role in the generation of effective immune responses by binding peptides and presenting them to T-lymphocytes (Trowsdale, 1993). Sequence variation