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Sequencing studies of HLA class II molecules have focused almost exclusively on exon 2. In this study the complete cDNA sequence of the DRB1*09012 allele is reported for the first time. This sequence was previously only partially published. In the DR9 antigen, two synonymous allelic variants (DRB1*09011 and 09012) were officially recognized, though it was later found that the first one contained an error and both sequences were, thus, identical. Major histocompatibility complex (MHC) class II molecules are highly polymorphic cell surface glycoproteins that play a central role in the immune response by binding and presenting peptides to helper T lymphocytes (Brown et al., 1993). The use of DNA-based typing methods for the analysis of class II antigens has resulted in the continuous detection of new variants. DNA sequencing of these alleles has, however, focused mainly on exon 2, which codes for the most polymorphic β1 domain of the molecule, and complete cDNA sequences are scarce. A complete cDNA sequence (including all six exons) of the DRB1*09012 allele is reported in this paper for the first time; only its exon 2 sequence has been previously published elsewhere (Bell et al., 1987). HLA-DRB DNA typing by Amplicor low-resolution test (Roche, NJ, USA) of an infant born to an HIV-infected mother yielded an HLA-DR3, DR9 phenotype. To further assess the results, cDNA sequencing of all HLA-DRB alleles was carried out. RNA was obtained from PBLs using the NP-40 protocol. Cells were pelleted and resuspended in 250 μL of lysis buffer (10 mM Tris pH 8.6, 140 mM NaCl, 1.5 mM MgCl2 0.5% NP-40) with RNAsin 1000 U mL–1 (final concentration) (Promega, Madison, WI). After 1 minutes’ incubation at 4 °C, lysates were centrifuged and supernatants were transferred to 250 μL of proteinase K buffer 2X (200 mM Tris pH 7.5, 25 mM EDTA, 300 mM NaCl, 2% SDS) with 200 μg of proteinase K. These samples were vortexed and incubated for 30 min at room temperature. A standard phenol-chloroform extraction was done to obtain whole RNA. cDNA synthesis was performed using a reverse transcription system (Promega, Madison, WI) according to the manufacturer’s protocols, using an oligo (dT)15 primer. The cDNA was subjected to polymerase chain reaction (PCR) amplification using the DRB5'UT and DRB3'UT primers (Corell et al., 1991), in order to obtain the full cDNA sequence. The PCR products from two different amplifications were purified using Qiaquick gel extraction Kit (Qiagen, Germany) and inserted into the pMOSBlue T-vector (Amersham Life Science, UK). Ten clones for each of the two PCR amplifications were sequenced in an Applied Biosystem 373 A DNA sequencer (Foster City, CA), as previously described (Gomez-Casado et al., 1995). To assess base identification, direct (5'-3') and reverse (3'-5') sequencing was done. The complete cDNA sequence (exons 1–6) of the DRB1*09012 allele is described for the first time in the present work (Fig. 1). In the DR9 antigen two synonymous allelic variants, DRB1*09011 and DRB1*09012, were officially recognized. It was later found that the original DRB1*09011 allele, whose sequence was obtained from the DR9 homozygous cell line ISK, contained an error and was finally confirmed as being identical to the DRB1*09012 allele (Naruse et al., 1997). The DRB1*09012 allele presents a GTC codon, which codes for valine, at position 57 of this DRB1 molecule. Aspartate is commonly found at position 57 in most DRB1 molecules and forms a salt bridge with a conserved arginine residue at position 76 of the DRα chain (Brown et al., 1993). Furthermore, this same residue at the HLA-DQβ chain has been found to correlate with protection against insulin-dependent diabetes mellitus (IDDM) in different populations, including the Spanish population (Vicario et al., 1992). The residue 57 lies in the terminal helical β region forming part of the binding site of HLA-DR1 molecules (Brown et al., 1993). Furthermore, residue 57 belongs to pocket 9, and is directly in contact with the foreign peptide during antigen presentation (Stern et al., 1995). Thus, a change in it could influence the repertoire of the peptides bound to the DRB1 molecule. As a comparison, an alignment of the HLA-DRB1*09012 sequence with that of a close HLA-DRB allele (HLA-DRB1*0701) was carried out (see Fig. 1). It was observed that, besides the already well-known changes affecting exon 2, only one further change is detected outside this exon. In codon 217 (exon 4), CTG is found in HLA-DRB1*09012, whereas TTG is present in HLA-DRB1*0701. Both codons, however, code for leucine. |
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