SJL/J lymphomas can be used as a model of tumor progression from a slow-growing malignancy to an aggressive tumor. We have previously shown that one of the class I MHC antigens, D?s?, is not expressed in some SJL/J lymphomas. The D?s?-negative tumors are not rejected and grow rapidly. The goal of this research is to determine the structural defect(s) that prevents expression of the D?s? antigens. Loss of expression could be due to gross deletion or rearrangement of the gene or small alterations such as point mutations or gene conversion. We will identify and sequence genomic D?s? genes from normal tissue and from several malignant SJL/J lymphoma lines. A comparison of the sequences will locate the gene(s). Since the amino acid sequence of the D?s? glycoprotein is not known, the D?s? gene(s) must be identified from the other 33-35 cross-hybridizing class I genes. A combination of two approaches will be used to identify and determine the number of D?s? genes: (1) several cDNA probes will be made from mRNA isolated by immunopurification of polysomes. Size-selected, double-stranded cDNA will be used for insertion into pBR322. These cDNA clones will be tested for their ability to cross-hybridize with a class I probe and then will be sequenced; and (2) the genomic D?s? gene(s) will be identified from the other 33-35 cross-hybridizing class I genes on a Southern blot by comparing the DNA from various H-2 congenic and intra-MHC recombinant mouse strains as well as from many SJL/J lymphoma lines. The D?s? gene(s) will be identified by its """"""""unique"""""""" restriction enzyme site polymorphism. This gene(s) will be cloned into a cosmid vector for sequence analysis. The identify of the genomic D?s? gene(s) will be confirmed by cloning the appropriate restriction fragment(s) containing the D?s? gene(s) and comparing the sequence of the genomic gene(s) with the D?s? cDNA sequence(s). The D?s? genes will be subcloned into the pSV2-neo vector for both sequence and immunochemical analyses. Immunochemical analyses of the glycoproteins produced in transformed mouse L cells will confirm the production of clones containing the D?s? gene(s). Defective D?s? genes will then be cloned from various SJL/J lymphoma lines and sequenced. Such alterations in gene structure may be detected by restriction site analyses on Southern blots followed by sequencing of the appropriate segment of the tumor D?s? gene. These studies will, therefore, identify the genetic mechanisms that prevent the expression of cell surface antigens in malignancies. (AG)
