Isolation and characterization of a cDNA clone for the murine I-E beta polypeptide chain.

Self Cite

100

domain variability is clustered into three discrete regions, two of which divide the Ap chains into subgroups, suggesting an evolutionary history for the separation of alleles in inbred strains of mice. The amino acid sequences of these five chains are compared to each other and to previously published I-Ap chains. Correlations are made between the primary structural differences and the serologic and immune response characteristics mapping to the I-A subregion.The intimate relationship between I-region associated (Ia) antigens and immune responsiveness to a multitude of natural and synthetic antigens has been known for some time (1, 2).Recent studies using either anti-Ia antisera or monoclonal antibodies to block immune responses strongly suggest that Ia antigens are the products of immune response (Ir) genes (3-5), but how a limited number of Ia molecules differentially regulate responses to a myriad of antigens is yet to be resolved. It seems likely that their role in the presentation of antigen to imtnunocompetent cells is of fundamental importance and that primary structural alterations in the a and (3 chains of the class II heterodimer result in a failure to present antigen in a configuration appropriate for recognition. These alterations must also account for the multiple epitopes recognized by both anti-Ia antibodies and alloreactive T cells.In an effort to address the nature of primary structural differences among class II molecules and how these differences affect immune responses, we have undertaken the cloning and sequencing of cDNAs encoding I-Ap chains from several mouse haplotypes. This should facilitate the eventual assignment of particular serologic epitopes and restriction elements to specific amino acids in these chains. Such analysis is a first step in designing experiments in which immune responsiveness can be manipulated via structural alterations in Ia molecules and then, perhaps, understood. Clones hybridizing to the human DQp or the murine Al cDNA probes were tested for the presence of restriction enzyme sites consistent with AP but not Ed coding sequences (9). The longest such clones were subcloned into the EcoRI site ofpBR322 and from there into the EcoRI site of M13 mp8 for sequencing. Sequencing was performed by the dideoxysequencing method of Biggin et al. (10). All clones were sequenced using the M13 universal primer (UP) from the EcoRI ends, as shown in Fig. 1. In addition, five 18-bp synthetic oligonucleotide primers homologous to conserved regions of the known AP and DQO sequences (8,(11)(12)(13)(14) were constructed and used to obtain sequence information on portions of the clones inaccessible from the EcoRI ends (Fig. 1, B-H productive (8, 9, 11-17). MATERIALS AND METHODS

Section: Methodsmentioning

confidence: 85%

Sequence analysis and structure-function correlations of murine q, k, u, s, and f haplotype I-A beta cDNA clones.

Estess¹,

Begovich²,

Koo³

et al. 1986

Self Cite

100

“…The E/k gene was derived from a construct consisting of the leader exon from the Ed gene and the remainder of the molecule from the Ek gene (unpublished data). As the leader peptide is excised prior to cell surface expression (leaving three apparently silent amino acid substitutions in the Ek NH2-terminal region), and the E. and E. subunits are almost identical (15,16), the expressed Ed/k and EkEd molecules are referred to here as E and I-Ed, respectively.…”

Section: Methodsmentioning

confidence: 99%

Ia-transfected L-cell fibroblasts present a lysozyme peptide but not the native protein to lysozyme-specific T cells.

Shastri¹,

Malissen²,

Hood³

1985

“…A first step in this analysis has been the molecular cloning and nucleotide sequencing of several allelic forms of A, (3), AO (4)(5)(6), Ea (7,8) and Es (9)(10)(11) pgl-A-1. Positive colonies were used to prepare DNA and restriction-mapped to identify a recombinant clone with a single pkI exon in the correct orientation.…”

mentioning

confidence: 99%

"Exon-shuffling" maps control of antibody- and T-cell-recognition sites to the NH2-terminal domain of the class II major histocompatibility polypeptide A beta.

Germain¹,

Ashwell²,

Lechler³

et al. 1985

To investigate the role of the highly polymorphic amino-terminal (beta 1) domain of the class II major histocompatibility polypeptide A beta during recognition by T cells and antibodies, "exon-shuffling" was carried out between genomic recombinant DNA clones of Ak beta and Ad beta to generate a hybrid gene containing Ak beta exons for the amino-terminal domain followed by the Ad beta exons for the remainder of the molecule. L-cell gene transfectants expressing this hybrid A beta gene in combination with Ak alpha were compared to L cells expressing wild-type Ak beta Ak alpha dimers in tests of antigen-presentation to T-cell clones and hybridomas and for staining by a panel of anti-I-Ak-specific monoclonal antibodies. These antibodies were also tested for their reactivity with a B-lymphoma transfectant expressing Ak beta in the absence of Ak alpha. The results showed no qualitative differences in either T-cell or antibody-mediated recognition of I-Ak molecules containing either the exon-shuffled or wildtype Ak beta. Together with the data involving the B cell transfectant expressing only Ak beta, these results map control of the A beta contribution to the immunologically relevant determinants of I-Ak to the highly polymorphic amino-terminal domain and indicate little, if any, contribution to allele-specific recognition by amino acid sequence variations in the remaining portions of the A beta polypeptide.