Molecular loss variants of the murine major histocompatibility complex: nonexpression of H-2K antigens associated with marked reduction in H-2K mRNA as determined by oligonucleotide hybridization analysis.
Somatic cell variants of the murine major histocompatibility complex were isolated to study the molecular features required for H-2K gene expression. In vitro selection was performed on a heterozygous [H-2b (Kb,Db) X H-2d (Kd, Dd, Ld)] pre-B lymphoblastoid cell line (R8) for variants that had lost membrane expression of the H-2Kb gene product. Analysis of a number of independently isolated variant cell lines by cytofluorometry with monoclonal antibodies to the Kb, Kd, Db, and Ld antigens revealed a variety of H-2 phenotypes. Variants were classified as either molecular loss for those that had lost K antigen expression only or as haplotype loss for those that no longer expressed the entire H-2b haplotype (i.e., negative for Kb and Db). DNA hybridization analysis with a K gene-specific oligonucleotide indicated that the Kb gene was present in all of the molecular loss variants, suggesting that gene deletion was not responsible for the loss of Kb antigen expression. In contrast, the Kb gene was not detected in haplotype loss variants. For analyzing the mutants at the RNA level, hybridization with H-2-specific synthetic oligonucleotides provided a definitive procedure to identify specific class I gene transcripts in the H-2 heterozygous cell lines. Such analyses were performed on the molecular loss variants and revealed that in a subset of variants (R8.2, R8.96, R8.116, R8.178) there was an absence of Kb mRNA. Kd mRNA was identified for all but one (R8.2) of the Kb mRNA-deficient cell lines, a finding consistent with the serotype assigned to each. Transcription of the linked Db gene was normal. These data demonstrate that a specific alteration for the K gene in several independently selected cell lines gave rise to the altered H-2 phenotype. Such variants offer the potential to analyze the properties of the mammalian genome responsible for controlling expression of individual members of the major histocompatibility complex multigene family.
D000818:Animals; D004247:DNA / Q000032:analysis; D004587:Electrophoresis, Agar Gel; D005786:Gene Expression Regulation; D005796:Genes; D006183:H-2 Antigens / Q000032:analysis / Q000235:genetics*; D064532:Histocompatibility Antigen H-2D; D051379:Mice; D009154:Mutation; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D006183:H-2 Antigens; D064532:Histocompatibility Antigen H-2D; D009841:Oligonucleotides; D012333:RNA, Messenger; D004247:DNA