Nottenburg C, St John T, Weissman I L
J Immunol. 1987 Sep 1;139(5):1718-26.
Allelic exclusion of immunoglobulin gene products results in the expression of only one of two possible alleles in normal B lineage cells. Attempts to define the role of heavy chain gene rearrangements in this process have revealed the nonexpressed allele to be rarely in its germline context, but rather incompletely rearranged (D/J rearrangements) or completely rearranged (V/D/J rearrangements) and in a context that cannot be expressed as a protein. Nearly all DNA sequences of heavy chain genes from the nonexpressed allele have originated from plasmacytomas, virus-induced pre-B leukemias, and hybridomas--all clonal cell lines perhaps altered by the neoplastic process. In this communication, we present the first examples of isolation and DNA sequence analysis of heavy chain gene rearrangements from the nonexpressed allele of normal B lymphocytes. Splenic B lymphocytes from allotype heterozygous (BALB/c X C57BL/6J)F1 mice expressing the BALB/c IgD allotype were detected with a monoclonal antibody, H10-4.22, to the BALB/c delta chain genetic marker and were isolated with a FACS (fluorescence-activated cell sorter). lambda phage clones containing the JH gene region from the sorted cells were isolated, and those containing sequences derived from the C57BL/6J nonexpressed chromosome were identified by a restriction fragment length polymorphism. DNA sequences of seven rearranged clones from the nonexpressed allele are presented. Five of these rearrangements have unexpected compositions. Four of the five clones contain V/D/J rearrangements with no obvious impediments to expression generated by the rearrangements. The novel variable region gene in one of these V/D/J rearrangements is a member of a newly described VH gene family. The fifth clone has a 3.5 kb deletion removing all of the JH gene segments. The remaining two clones contain D/J rearrangements that are typical of the initial stage of variable region assembly. These findings suggest that the mechanisms that generate nontranslatable heavy chains do not exclusively account for allelic exclusion. Rather, several different mechanisms may contribute to the establishment of allelic exclusion in normal B lymphocytes.
免疫球蛋白基因产物的等位基因排斥导致正常B淋巴细胞系细胞中仅表达两个可能等位基因中的一个。为确定重链基因重排在这一过程中的作用所做的尝试表明，未表达的等位基因很少处于其种系状态，而是不完全重排（D/J重排）或完全重排（V/D/J重排），且处于无法表达为蛋白质的状态。几乎所有来自未表达等位基因的重链基因DNA序列均源自浆细胞瘤、病毒诱导的前B白血病和杂交瘤——所有这些克隆细胞系可能都因肿瘤形成过程而发生了改变。在本通讯中，我们展示了从正常B淋巴细胞未表达等位基因中分离重链基因重排并进行DNA序列分析的首个实例。用针对BALB/c δ链遗传标记的单克隆抗体H10 - 4.22检测来自表达BALB/c IgD同种异型的同种异型杂合（BALB/c×C57BL/6J）F1小鼠的脾B淋巴细胞，并用荧光激活细胞分选仪（FACS）进行分离。从分选细胞中分离出含有JH基因区域的λ噬菌体克隆，并通过限制性片段长度多态性鉴定出含有源自C57BL/6J未表达染色体序列的克隆。展示了来自未表达等位基因的7个重排克隆的DNA序列。这些重排中有5个具有意外的组成。5个克隆中有4个包含V/D/J重排，重排未产生明显的表达障碍。这些V/D/J重排中的一个新可变区基因是新描述的VH基因家族的成员。第5个克隆有一个3.5 kb的缺失，去除了所有JH基因片段。其余两个克隆包含可变区组装初始阶段典型的D/J重排。这些发现表明，产生不可翻译重链的机制并非等位基因排斥的唯一原因。相反，几种不同机制可能有助于正常B淋巴细胞中等位基因排斥的建立。
