We request renewal of support for our studies of genes and gene products of the murine major histocompatibility complex encoded telomeric to H-2D. The work will focus on two specific antigens of the TL region, Qa-1 and Mta, epitopes of which are best defined by cloned cytolytic T cells (CTL). Mta is a unique maternally transmitted cell surface antigen that has immunological properties of a class I H-2 molecule. However, expression of Mta is determined by both nuclear and non-nuclear genes. One gene, Hmt, maps to the TL region, but antigenic polymorphism is controlled by a maternally transmitted genetic element, Mtf. Our previous studies strongly implicate mitochondria in Mtf expression. We will investigate the gene encoding Mtf in somatic cell hybrids constructed with mitochondria from two parents and codominantly expressing alternative forms of the antigen, as well as in cell lines into which mitochondria have been introduced by microinjection. These experiments will directly test the hypothesis that Mta expression is linked to the mitochondrial genome. In order to identify the nuclear gene, Hmt, we will utilize retrovirus-mediated insertional mutagenesis coupled with selection protocols based on loss of susceptibility to Mta-specific CTL clones. A strategy is proposed for identification of mutants in Hmt disrupted by the integrated retrovirus and for recovery, cloning and characterization of the Hmt gene. In contrast to Mta, Qa-1 is a typical class I H-2 molecule. However, the gene for Qa-1 has not yet been identified. This will similarly be approached employing retrovirus-mediated insertional mutagenesis in Qa-1 heterozygous cell lines. Recent data demonstrate that Qa-1 expression is regulated by an additional locus or loci. One locus, probably in the H-2D region, is associated with low expression of Qa-1 and a second dominant gene restores expression. Breeding studies to map these genes are proposed with an eventual goal of molecular characterization of these class I regulatory elements. Together, the proposed studies will employ novel approaches and techniques to explore fundamental issues in cell biology and immunology. We believe they will substantially enhance our understanding of the structure and function of these poorly understood class I molecules of the 17th chromosome, as well as our appreciation of the biological functions of the mitochondrial genome.
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