Analysis of hypermutation in immunoglobulin heavy chain passenger transgenes.
Analysis of hypermutation in immunoglobulin heavy chain passenger transgenes.
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免疫球蛋白重链过客转基因的超突变分析。
DOI：
10.1002/eji.1830260515
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Carroll,WL
中科院分区：
文献类型：
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作者：
Johnston,JM;Ihyer,SR;Smith,RS;Tai,KF;Farmer,T;Korsmeyer,SJ;Nadon,NL;Carroll,WL
免疫球蛋白（Ig）基因的体细胞超突变在人类抗体反应的成熟中起着关键作用。然而，这一重要过程的分子基础尚不清楚。为了鉴定启动和靶向超突变的顺式作用序列，我们制作了三个含有Ig重链（IgH）位点不同部分的微转基因。每个转基因都是一个乘客，携带着无意义的突变，阻止了它的翻译；因此，转基因突变反映的是内源性突变过程，不受亲和选择的影响。为了在体内研究转基因通过与高突变相关的隔室循环后，我们用半抗原4 -羟基- 3 -硝基苯基乙酰（NP）过度免疫小鼠后，将B细胞作为杂交瘤保存。采用聚合酶链反应扩增杂交瘤转基因区和内源可变区，进行亚克隆和测序。内源性抗NP VDJ区域显示出预期的，有时广泛程度的碱基取代。在携带最小结构的小鼠中，包括2.4 kb的5 ‘ IgH序列，重排的VDJ区，5 ’基质附着区和内含子增强子，四个可评估杂交瘤中的一个在一个转基因拷贝的V片段上显示两个碱基替换。这两个较大的结构包括额外的3 ‘ IgH序列（α恒定区和3 ’增强子）和原始的VDJ片段或替代的T细胞受体β片段。尽管在所有的杂交瘤中都有明显的转基因转录，但来自具有这些较大结构的小鼠的10个杂交瘤没有显示出靶向突变的证据。在我们的系统中，重排的VDJ片段和周围的启动子/增强子区域的突变不会因为恒定区域片段和IgH 3 '增强子的并置而增加。
Somatic hypermutation of immunoglobulin (Ig) genes plays a critical role in the maturation of the human antibody response. The molecular basis of this important process is, however, unknown. To identifycis‐acting sequences that initiate and target hypermutation, we have made three minitransgenes containing different portions of an Ig heavy chain (IgH) locus. Each transgene is a passenger, bearing a nonsense mutation preventing its translation; thus, transgene mutations reflect the endogenous mutational process and are not subject to affinity selection. To study transgenes after their circulation through the compartment associated with hypermutationin vivo, we rescued B cells as hybridomas after hyperimmunizing mice with the hapten 4‐hydroxy‐3‐nitrophenyl acetyl (NP). Hybridoma transgene and endogenous variable regions were amplified by polymerase chain reaction, subcloned, and sequenced. Endogenous anti‐NP VDJ regions show the expected, at times extensive degree of base substitution. In mice bearing the smallest construct, which includes 2.4 kb of 5′ IgH sequences, a rearranged VDJ region, the 5′ matrix attachment region, and the intron enhancer, one of four evaluable hybridomas demonstrates two base substitutions in the V segment of one transgene copy. The two larger constructs include additional 3′ IgH sequences (an α constant region and the 3′ enhancer) and either the original VDJ segment or a substituted T cell receptor β segment. Ten hybridomas derived from mice bearing these larger constructs demonstrate no evidence of targeted mutation, despite demonstrable transgene transcription in all hybridomas. In our system, mutation of a rearranged VDJ segment and surrounding promoter/enhancer regions is not increased by the juxtaposition of a constant region segment and the IgH 3′ enhancer.