IMMUNOGLOBULIN-RELATED T-LYMPHOCYTE SURFACE RECEPTORS

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This research is directed towards obtaining detailed information on the structural properties of the antigen-specific surface component of defined murine and primate T-cell lines. The overall goal is to obtain detailed information regarding the biochemical and molecular biological properties of T-cell surface components which are serologically related to immunoglobulin-VH region markers (VT), which will allow direct comparison with other possible T-cell receptors such as surface complexes restricted by the major histocompatibility complex. The specific aims are: (1) to characterize the VT-related T-cell molecules structurally. The major approach here will be to begin with large quantities of homogenous, in vitro-grown T-cell lines (greater than 50 grams) and to prepare the molecules by immune affinity chromatography. Conditions for specific cleavage of peptide bonds will be used and the fragments will be separated by high performance liquid chromatography. (2) The restricted VT-related determinant expressed and synthesized by the T-cell line 70-N2 is inherited as an autosomal dominant within human populations. We will determine the genetic linkage of the gene specifying this marker. (3) In order to ensure that complete sequence information is obtained, we will approach the problem of the VT-bearing t-cell receptor of our defined lines using the techniques of molecular biology. We will use our specific antisera to isolate intact polyribosomes bearing nascent polypeptide chains from our cell lines, isolate mRNA and produce cDNA followed by cloning and sequencing of the cDNA. An alternative strategy will be the construction of a cDNA library using total mRNA and the expression vector pUC8 which allows synthesis of antigenic products. We will screen the cDNA library with nicktranslated DNA probes directed against putative T-cell surface receptors. The positive clones will then be screened with our antisera to determine whether the antisera we have generated to VT-related products react with the products of the putative T-cell receptor genes recently sequenced. (CS)