The overall theme of this proposal is the correlation of structural features of Class II molecules with their role in cell surface expression of Ia. The approach chosen is the molecular and functional analysis of Class II mutant B lymphoma cell lines produced by immunoselection on the basis of cell-surface expression or the transfection of in situ mutagenized Class II genes into Ia-negative B lymphoma cells or into non-lymphoid cells. Ia variant cell lines which have structural defects which result in the complete lack of expression of cell surface Ia despite the presence of intracytoplasmic Ia polypeptides will be used to identify sites in the Class II genes key for post-transcriptional and post-translational regulation of Class II gene expression. These cell lines will be analyzed by two dimensional gel electrophoresis, immunoelectronmicroscopy and DNA sequencing of the relevant mutant gene. Site-directed mutagenesis and recombinant DNA technology will be used to produce Class II molecules which have truncated cytoplasmic regions. These tailless mutants will be used to examine the role of the cytoplasmic domain of the Ia in chain-pairing, assembly, transport, association with invariant chain and transmembrane signalling. The role of the invariant chain in cell surface expression of Ia will be examined by the production and analysis of invariant chain molecules with truncated cytoplasmic and transmembrane regions by site directed mutagenesis to determine whether the targeting and transport of Ii affects the transport and expression of Ia. Exon- shuffling experiments between the Ii gene and the Class I Q10 gene will be performed to map the sites on Ii which mediate the contact between Ii and Ia. The central theme of this proposal, therefore, is the identification, by a mutational approach, of structural features of Class II genes including the Ii gene which affect the expression of these proteins at the post-translational level.
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