Zeff R A, Geier S S, Nathenson S G
J Immunol. 1986 Aug 15;137(4):1366-70.
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.
分离出小鼠主要组织相容性复合体的体细胞变体，以研究H-2K基因表达所需的分子特征。在杂合的[H-2b（Kb，Db）×H-2d（Kd，Dd，Ld）]前B淋巴母细胞系（R8）上进行体外筛选，以获得失去H-2Kb基因产物膜表达的变体。用针对Kb、Kd、Db和Ld抗原的单克隆抗体通过细胞荧光术分析多个独立分离的变体细胞系，揭示了多种H-2表型。变体被分类为仅失去K抗原表达的分子缺失型，或不再表达整个H-2b单倍型（即Kb和Db均为阴性）的单倍型缺失型。用K基因特异性寡核苷酸进行DNA杂交分析表明，Kb基因存在于所有分子缺失变体中，这表明基因缺失不是Kb抗原表达丧失的原因。相比之下，在单倍型缺失变体中未检测到Kb基因。为了在RNA水平分析突变体，用H-2特异性合成寡核苷酸进行杂交提供了一种确定的方法，以鉴定H-2杂合细胞系中的特定I类基因转录本。对分子缺失变体进行了此类分析，结果显示在一部分变体（R8.2、R8.96、R8.116、R8.178）中不存在Kb mRNA。除一个Kb mRNA缺陷细胞系（R8.2）外，所有其他细胞系均鉴定出Kd mRNA，这一发现与分配给每个细胞系的血清型一致。相连的Db基因转录正常。这些数据表明，在几个独立选择的细胞系中，K基因的特定改变导致了H-2表型的改变。此类变体为分析负责控制主要组织相容性复合体多基因家族单个成员表达的哺乳动物基因组特性提供了潜力。
