Coding potential of the Q6 gene from the Qa-2a region of BALB/c Crgl mice was analyzed by a combination of hybrid class I gene construction and DNA-mediated gene transfer. Recombinant genes were created by exon shuffling of the 5' coding region of the Q6 gene and the 3' coding region of a gene encoding a transplantation antigen (K(d), D(d), or L(d)), or the inverse. Some of these hybrid class I genes were expressed in the transfected mouse fibroblasts (L cells). The hybrid class I molecules encoded by the 5' end of the Q6 gene and the 3' end of the L(d) gene precipitated as 45,000 mol wt molecules associated with β2-microglobulin. The expression of the hybrid proteins indicates that 926 basepairs of the 5' flanking region upstream of the structural Q6 gene contain a promoter that functions as a transcription initiation site in L cells. The 3' portion of the Q6 gene appears to be responsible for the lack of cell surface expression of the intact Q6 and the hybrid L(d)/Q6 genes in mouse fibroblasts. Accordingly, this portion of the Q6 class I gene may play a regulatory role in tissue-specific expression. Serological analyses of hybrid Q6 proteins suggested that Q6 may be a structural gene for CR (H-2 crossreactive) antigen found normally on subpopulations of lymphocytes. If this identification is correct, Q6 gene will define a new category of class I genes encoding ~40,000 mol wt molecules and carrying a characteristic truncated cytoplasmic tail. Analysis of L cells transfected with Q6 hybrid genes demonstrated also that the cytotoxic T cells specific for Qa-2a region-coded antigens recognize the amino-terminal α1-α2 domain of Q6 fusion products. This recognition can be blocked by anti-Qa-2a alloantiserum and monoclonal antibodies reactive with the α3-β2-microglobulin portion of the Q6 hybrids. We propose that the structural requirements for the anti-Qa-2a cytotoxic T lymphocyte-specific epitopes on target molecules are the same as for anti-H-2-alloreactive cytotoxic T lymphocyte determinants on transplantation antigens and that the mechanism of target recognition is similar in both cases. This interpretation is consistent with the following structural similarities found in both categories of class I molecules: (a) K(d) and Q6 α1-α2 domains share serologically defined epitopes. (b) Cytotoxic T lymphocyte-specific and antibody-specific determinants on Q6- and H-2-defined α1-α2 domains are not distorted in hybrid proteins carrying different polymorphic α3-β2-microglobulin carboxyl termini. (c) Amino acid comparisons between H-2 and Qa molecules indicated that the two groups are almost as closely related to each other as the individual members within each group.
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