A new sequence (Mhc-BJ ) showing similarity both to Mhc-B alleles and to the HLA-J pseudogene in Macaca mulatta

Class I major histocompatibility complex (Mhc) genes in humans have been divided into two groups: 1) classical (HLA-A, -B, and -C) that encode polymorphic membrane glycoproteins implicated in the presentation of intracellular derived peptides to the cytotoxic T cells, and 2) nonclassical that include a group of genes (HLA-E, -F, and -G) which code for molecules whose functions have not yet been defined (and certainly are not antigen presenting molecules in the case of -G and Macaca mulatta family; Castro et al. 1996) and also a set of several pseudogenes [(HLA-H, -J, -K, -L) (Bodmer et al. 1995)]. The mechanisms implicated in generating antigen site diversity of the presenting Mhc molecules remain unclear. It has been suggested that this diversity is generated by point mutation, segmental exchange, and recombination events, and then selection maintains or deletes the new products generated in a population (Klein et al. 1990). An Mhc class I sequencing study in M. mulatta (rhesus monkeys) was carried out to define possible new alleles or genes and investigate the mechanisms responsible for the generation of diversity of the Mhc alleles in apes. A new Mhc class I sequence was found, characterized by a 75 nucleotide deletion at the beginning of exon 3, which resembled Mhc-B alleles and possibly HLA-J sequences. Whole RNA was extracted from kidney cell lines (107 cells per line) for two M. mulatta individuals (rhesus monkeys, Rh-8, Rh-14) using NP-40 standard protocol. cDNA synthesis was performed using a reverse transcription system (Promega, Madison, WI) according to the manufacturer’s protocols. Partial exon 1, exon 2, exon 3 and partial exon 4 from this new Mamu-Mhc nucleotide sequence gene was obtained by polymerase chain reaction (PCR) using 5E1m (59-CGCTCCTCCTGCTGCTCTCGGC) and 3E4m (59-AGATGGGGTGGTGGGTCACG) primers. Random sequencing of the amplified products was carried out and PCR products were also purified and inserted into the pGEM-T vector (Promega). Doublestranded DNA stretches were automatically sequenced in an Applied Biosystems (Foster City, CA) machine as previously described (Castro et al. 1996). Exonic phylogenetic trees were made using the neighbor-joining method (Saitou and Nei) and the computer program NJDRAW, designed by J. W. H. Ferguson. A DNA phylogenetic tree was based on the number of nucleotide substitutions per site, and was corrected for multiple hits by the Jukes and Cantor (1969) method using the computer program NAG (version 2.0), designed by T. Ota and M. Nei. Five clones cDNA sequences obtained from two different M. mulatta (Rh-8 and Rh-14) were identical and corresponded to this new sequence, previously not found in M. mulatta (Boyson et al. 1995; Castro et al. 1996; Fig. 1). A comparison of its exons with those of classical and non-classical class I alleles showed that exon 2 is more similar to HLA-J and HLA-G*0101 and Mamu-Mhc-G*I and exon 3 is closer to Mamu-Mhc-B*07. These data suggested that this new sequence could be generated by a recombination event between a Mamu-Mhc-B gene and an analogue of HLA-J (see below) postulating a segmental exchange between neighboring genes. It has been tentatively named Mamu-Mhc-BJ and is not a PCR artefact, since it comes from two different individuals, five clones, and different PCR reactions; it has a 75 nucleotide deletion at the begining of exon 3, which would not change the reading frame. A possible protein resulting from a hypothetical transcription and translation would not bear residues Tyr 96 and Tyr 101, which are necessary for the inderdomain dysulphide bond and also lack residues between positions 91– 116 that are implicated on the overall interdomain contacts between α2 and α1 domain and β2-microglobulin (Bjorkman et al. 1987). Thus, a putative MamuMhc-BJ protein, if translated, would not be an antigen The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accesion number L41828 (Mamu-Mhc-BJ)