Molecular cloning and sequencing of a cattle DRA cDNA

Abstract

To date there is full-length sequence information for only two cattle MHC class 11 genes, the DQB gene on the genomac clone Y1 (Groenen et al. 1990) and the DRB3 cDNA clone (Burke et al. 1991). It is known that most sequence polymorphisms are located m the peptide binding domain, especially in exon 2 of the chain. However, proper correlanon of immune fanctaon with sequence polymorphimn necessarily involves studying the complete expressed molecule. In thas commumcation we report the cloning and sequencing of the entire coding region of a cattle DRA gene. Peripheral blood monneytas were purified from whole blood of Fries~an (Bos taurus) cattle with class 1I types DRBF 6,3. Reverse mmscriptmn-polymerase chain reaction (RT-PCR) was carried out on total RNA, using oligo-dT and primers derived from the pubhshed full-length sheep DRA cDNA sequence (Fabb et al 1993), m a reacuon volume of 50 gl comprising 10 mM Trts-Hcl pH 8.3, 50 mM KC1, 100 pg/ml gelatin, 2 mM MgCh, and 1 umt Taq polymerase (Boehrmger Mannhetm~ Lewes, UK). Amplificataon was primed wath 25pmolas of forward (5'-CACCAAAGAAGAAAATGGCC-Y) and reverse (5'-TGAGACCCACTI~AAGTITACTGTATTC-Y) primers and conststed of 20 cycles of 94 ° C for ] man, 55 ° C for 2 min, and 72 ° C for 3 mm, followed by a 7 mm extension at 72 ° C. ~ products were cloned into the TA vector pCR 11 (Invitrogen, San Dtego, CA) and sequenced in both directions, using Sequenase Vermon 2 0 enzyme (US Biochemacah, Cleveland, OH). We present here (Fig. 1) the sequence of a cailie DRA cDNA. The 1195 base pairs (bp) sequence contains a 762 bp reDon coding for a 253 residue polypeptade. Companr, on with the sequences of exons 2, 3, and 4 from the truncated genomic clone W3 (van der Pcel et al. 1990) shows that the two genes are idenUcal over the regton aligned, thus confirming the idenUty of the DRA cDNA clone and transcription of the W3-encoded gone. The degree of identaty between the translated cattle DRo~ chain sequence and the human DRa sequence ts 80%, and conserved residues of functional sigmficance are highhghted in Figure 1. Interestingly, these residues include E88 and K l l l wlmch may be important m the observed dimerisanon of I-ILA DRI ct/~ he~erodimers (Brown et al. 1993). However, It IS unclear whether this dimerisafion occurs m wvo or if It is ftmclaonally significant.

DOI: 10.1007/BF00189981

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@article{Fraser1994MolecularCA, title={Molecular cloning and sequencing of a cattle DRA cDNA}, author={Douglas C. Fraser and Susan C Craigmile and George C Russell}, journal={Immunogenetics}, year={1994}, volume={40}, pages={311-311} }