Summary 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 syn- onymous 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 glycopro- teins that play a central role in the immune response by binding and presenting peptides to helper T lympho- cytes (Brown et al., 1993). The use of DNA-based typing methods for the analy- sis 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 pub- lished 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 MgCl 2 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 tempera- ture. A standard phenol-chloroform extraction was done to obtain whole RNA. cDNA synthesis was per- formed using a reverse transcription system (Promega, Madison, WI) according to the manufacturer’s proto- cols, using an oligo (dT) 15 primer. The cDNA was sub- jected 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 synony- mous 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, con- tained an error and was finally confirmed as being iden- tical 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 corre- © 1998 Blackwell Science Ltd, European Journal of Immunogenetics 25, 307–309 Complete cDNA sequence of the HLA-DRB1*09012 allele* N. Martínez-Quiles, † J. M. Martín-Villa, † S. Ferre-López, M. A. Moreno-Pelayo, J. Martínez-Laso, M. Pérez-Blas, R. Alegre & A. Arnaiz-Villena 307 Department of Immunology, Hospital ‘12 de Octubre’, Universidad Complutense, 28041 Madrid, Spain. †NMQ and JMM-V contributed equally to the work and the order of authorship is arbitrary. *The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and the accession number U66826 assigned. The name DRB1*09012 has been officially assigned by the WHO Nomenclature Committee in October 1996. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al., 1997), names will be assigned to new sequences as they are identified. A list of such new alleles was published in the last WHO Nomenclature Report (Bodmer et al., 1997). Received 28 January 1998; revised 2 March 1998; accepted 4 March 1998 Correspondence: Dr A. Arnaiz-Villena, Departamento de Inmunología, Hospital ‘12 de Octubre’, Universidad Complutense, Ctra de Andalucía, 28041 Madrid, Spain. Short Communication