Three projects concerning regulation of the expression of class I major histocompatibility genes in human lymphoblastoid cell lines (LCL) will be undertaken. (1) Which DNA sequences regulate transcription of the HLA-A, -B and -C genes? The class I genes in gamma ray-induced HLA antigen-loss mutants that have an apparently normal gene but lack the corresponding mRNA will be analyzed in order to answer this question. (2) The rate of transcription of class I genes in B-LCLS is approximately 20 times greater than that in T-LCLS. Which DNA sequences and proteins are responsible for this cell type-specific difference in transcription? Modified class I genes cloned in non-integrating (pHeBo) vectors will be tested for expression by transferring them into a class I-null B-LCL mutant and into class I-poor T-LCLs. Nuclease protection an migration retardation in gels will also be used to identify cell type-specific transcription regulatory sequences. Regulatory proteins will be sought on episomally maintained class I genes. (3) """"""""5.4"""""""", """"""""6.0"""""""" and """"""""6.2"""""""" are cloned, apparently normal, non-A, B, C class I genes for which corresponding antigens are unknown. They will be tested for expression, before and after in vitro modification,, by transfer into class I-null B-LCLs. If antigens appear antibodies will be raised against them and used to study their distribution in the body. Immunoprecipitation and electrophoresis will be used to study the biochemical abnormality in B-LCL mutants that have a post- transcriptional defect in class I antigen expression. Transfer of cloned class II genes into class II-null mutant B-LCLS will be used to create cells that have defined class II expression. These cells will then be used to study aspects of antigen presentation and T cell recognition. Some human B-LCLS are deficient in surface expression of the lymphocyte common antigen T200. The T200 DNA, RNA and protein of these mutants will be studied in order to determine the cause of T200 deficiency. Other T200 mutants will be isolated in ways to be informative about the genetic regulation of T200 expression.
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