Sequence of an HLA-DR α-chain cDNA clone and intron-exon organization of the corresponding gene

Three families of human Ia molecules, DP, DQ, and DR, have previously been defined. A cDNA clone, pDSH-9.1, encoding the a chain of a DQ molecule derived from an HLA-DRw9 homozygous cell line has been isolated and sequenced. Comparison of the nucleotide and predicted protein sequence to those of other DQ a subunits reveals that DQ a subunits derived from DR4, -7, and -9 cells are very similar to each other but quite different from a DQ a subunit derived from a DRw6 cell line. These studies suggest that certain Ia haplotypes have a common evolutionary history. Furthermore, in the context of current serologic and biochemical knowledge, they suggest that the gene encoding the DQ a subunit is in strong linkage disequilibrium with the DR locus.The Ia region of the major histocompatibility complex (MHC) consists of a series of closely linked loci encoding cell surface molecules that regulate the immune response. These Ia molecules may be divided into three groups, DP (formerly known as SB) (1), DQ (formerly known as DS) (2), and DR (3), that are structurally distinct. Ia molecules consist of two subunits, a and ,3, with molecular weights of approximately 28,000 and 33,000. Within each group, DP, DQ, and DR, multiple a-and /8-chain gene loci have been described; at present seven P3 chain (two DP, two DQ, and three DR) and five a chain (two DP, two DQ, and one DR) gene loci have been identified (4-9). Although their precise genetic order is not yet known, the DP locus is known to be centromeric to both DQ and DR, which in turn are centromeric to the class I loci, HLA-A, -B, and -C (10). Recombinational analysis indicates that both DP and the class I loci are approximately 1 centimorgan from the DQ and DR loci (10,11). No recombinational event between DQ and DR has yet been confirmed, suggesting a relatively low frequency of recombination. Biochemical studies (2,(12)(13)(14)(15)(16)(17)(18) and analysis of cDNA clones (19)(20)(21)(22)(23)(24)(25)(26) indicate that a majority of the Ia genes are expressed as products. Ia gene products belonging to the same group are highly related (greater than 95% protein homology) while those belonging to different groups are only approximately 60% related.The Ia system is highly polymorphic. At least 11 alleles at the DR locus (designated numerically-i.e., DR1-DR11) have been defined serologically (27). Similarly, at least 6 alleles at the DQ locus and 6 at the DP locus have been defined biochemically and by cellular assays, respectively (27,28 (29). Similarly, cells typing as DR1, -2, and -6 frequently display the MB1 antigenic determinant (30).Although the molecular basis for these antigenic determinants is not known, in some instances, the gene products expressing them have been identified. Thus, MB1, which is expressed on Drl, -2, and -6 cells, and MB3, which is expressed on DR4 and DR5 cells, are both present on different allelic forms of DQ molecules (28,31,32). In other instances, the molecular assignment has been more controversial; MT3 has been localized to DR-like mol...

Section: Introductionmentioning

confidence: 99%

Nucleotide sequence of an HLA-DQ alpha chain derived from a DRw9 cell line: genetic and evolutionary implications.

Moriuchi¹,

Moriuchi²,

Silver³

1985

“…In man three class II molecules designated DC, DR, and SB, each consisting of an a and a f chain, have been identified (9,10). The amino acid sequences of some of these chains have been determined either directly (11) or deduced by the analysis of cDNA clones (12)(13)(14)(15)(16)(17)(18)(19). This sequence information suggests that the mouse I-A and I-E molecules correspond to the human DR and DC molecules, respectively (20).…”

mentioning

confidence: 99%

Complete primary structures of the E beta chain and gene of the mouse major histocompatibility complex.

Saito¹,

Maki²,

Clayton³

et al. 1983

152

Using the cross-hybridization with plasmid pDC(-1, containing the cDNA coding for the DCfi chain of the human major histocompatibility complex class II molecules, we have cloned and subjected to sequence analysis both the cDNA and genomic gene for the Efi chain of the BALB/c (d haplotype) mouse. The nucleotide sequences of the cDNA and genomic DNA clones permitted us to deduce the entire primary structure of the Efi chain and the complete exon-intron structure of the Efi gene. Unlike a chain genes that contain five exons, the EP gene consists of six exons corresponding to the six functional domains-the leader, f31and .82 domains, transmembrane peptide, intracytoplasmic peptide, and 3' untranslated region. In addition, two short blocks of sequences common to a and fl chain genes were identified in the 5' flanking regions. We propose that these sequences are involved in the coordinate expression of a and ,B chains. MATERIALS AND METHODSEscherichia coli strains K803 and X1776 and phage A Charon 4A are from our laboratory collection. BALB/c B-cell lymphoma L10A2J (I-Ad and I-Ed positive) was obtained from Philippa Marrack (National Jewish Hospital, Denver, CO). Preparations of mouse embryo (BALB/c) EcoRl partial and EcoRI* libraries by using Charon 4A as a vector were described previously (21,22). A cDNA library of L10A2J mRNA was constructed according to Okayama and Berg (23), except that we used X1776 as a host strain. These libraries were screened by the methods of Benton and Davis (24) and Hanahan and Meselson (25). The human DCl3 cDNA clone used as the hybridization probe was previously designated as pDRB1 (14). Because we now know that the clone in fact contains DCB rather than DR/3 cDNA (18), we refer to this clone as pDCB1 to avoid any possible confusion. Nucleotide sequences were determined according to Maxam and Gilbert (26). RESULTS AND DISCUSSIONIsolation and Sequence Analysis of E. cDNA Clones. We screened the L1OA2J cDNA library by using plasmid pDCJ31 as the probe. Independently isolated cDNA clones fell into two distinctive groups on the basis of their restriction maps. The clones of the first group hybridized to the human DC(3 probe much more strongly than the clones of the second group. Because the human DC/3 gene corresponds to mouse AB gene (20) and because AP3 and EP3 genes are expected to cross-hybridize (27), we tentatively concluded that clones in the first group contained the mouse AB cDNA and those in the second group, the mouse EB cDNA. Among the five putative E13 cDNA clones, one (pEB24) contained an insert of about 1.3 kilobases (kb), which was almost as long as the E/3 mRNA. Because the original isolate of pEB24 tends to spontaneously produce a small deletion in the relatively long poly(dA) tail, we subcloned a plasmid (pE,324-1) with a shorter poly(dA) tail. We then constructed the restriction map of this clone and determined its nucleotide sequence according to the strategy shown in Fig. 1.

“…In contrast, DQa is the only markedly polymorphic a chain. DRa is constant between individuals, while DPa shows a low level of variation and only two alleles have so far been identified (2,3). Alloantisera, mainly produced by fetal maternal stimulation, were the original basis for the definition of the HLA-DR and -DQ region products and their polymorphism.…”

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confidence: 99%

Monoclonal antibodies to HLA-DP-transfected mouse L cells.

Heyes¹,

Austin²,

Bodmer³

et al. 1986

Mouse L cells transfected with human HLA-DP (DPw4) a and f3 genes were used to make monoclonal antibodies in C3H mice. A polymorphic antibody, DPl1. Alloantisera, mainly produced by fetal maternal stimulation, were the original basis for the definition of the HLA-DR and -DQ region products and their polymorphism. It is clear that most of the serologically detected variation is in the DR and DQ 83 chains. The DP specificities were identified by cellular typing techniques and there is, so far, no corresponding serological identification. One monoclonal antibody, IRL1, has been identified, which appears to react preferentially to DPw2 and DPw3 (4). Monoclonal antibodies that are monomorphic have made a major contribution to the biochemical analysis of the HLA-D region products (5, 6), but useful polymorphic antibodies have been harder to obtain (7). A wider range of polymorphic HLA-D monoclonal antibodies, particularly for the definition of HLA-DP types, would be extremely useful both for serological typing and for biochemical and functional studies.The production by transfection of mouse L cells expressing a single human HLA-D region product, in this case HLA-DP (8), provides the basis for a novel approach to making anti-HLA class II monoclonal antibodies. If an HLA-DPexpressing L-cell transfectant is used to immunize C3H mice, the strain from which L cells were derived, then the only product the mice should react to, apart from L-cell-specific determinants, is HLA-DP. This principle is an extension of that used to make human-specific antisera (9) and monoclonal antibodies (10) by immunizing mice with human-mouse somatic cell hybrids containing a single human chromosome. In this paper, we describe the recognition of polymorphic specificities on L-cell DP transfectants and the use of these transfectants to make a polymorphic monoclonal anti-DP antibody with, surprisingly, specificity for the a chain of DPw4 and to an apparently lesser extent, DPw2.