Wood D L, Coleclough C
Proc Natl Acad Sci U S A. 1984 Aug;81(15):4756-60. doi: 10.1073/pnas.81.15.4756.
In order to assess whether DNA rearrangement occurs with equal frequency at each of the several J (joining region) elements in the mouse kappa light chain locus, we set out to determine the relative frequency of usage of J kappa segments in populations of B lymphocytes unperturbed by antigenic selection or cloning. To obtain such a population, we exposed a suspension of spleen cells to a mixture of mitogens capable of activating most B cells independently of their specificity for antigen. We estimated the relative usage of the J kappa elements in unspliced kappa chain gene transcripts in total and poly(A)-containing nuclear RNA, using an S1 nuclease protection assay, and in mature kappa chain mRNA, using a specifically primed cDNA hybridization assay. Both types of assay reveal a marked difference in the frequency of J kappa elements and indicate that their relative usage is: J1 approximately J2 much greater than J4 approximately J3. Comparison of the 5' flanking regions of the mouse J kappa elements, including the conserved putative recombination target sequences, shows no obvious differences consistent with the variation in recombinational efficiency, so we conclude that, although the consensus heptamer and nonamer signals may be sufficient to identify a recombination site, the probability that that site will be used depends also on other determinants. A review of published data suggests a nonequivalence of usage also among human J kappa elements and between mouse J lambda I and J lambda III loci. Extending the comparison to include these sets of sequences indicates that one of the determinants of frequency of J use may be the proximity of the consensus heptamer to a T-G dinucleotide within the J coding sequence, perhaps revealing an Escherichia coli gyrase-like substrate preference within a recombination enzyme.
为了评估DNA重排是否在小鼠κ轻链基因座的几个J（连接区）元件中的每一个处均以相同频率发生，我们着手确定在未受抗原选择或克隆干扰的B淋巴细胞群体中Jκ区段的相对使用频率。为了获得这样的群体，我们将脾细胞悬液暴露于能够独立于其对抗原的特异性而激活大多数B细胞的有丝分裂原混合物中。我们使用S1核酸酶保护分析法估计了总核RNA和含poly(A)的核RNA中未剪接的κ链基因转录本中Jκ元件的相对使用情况，并使用特异性引物cDNA杂交分析法估计了成熟κ链mRNA中Jκ元件的相对使用情况。这两种分析方法均揭示了Jκ元件使用频率的显著差异，并表明它们的相对使用情况为：J1≈J2远大于J4≈J3。对小鼠Jκ元件5'侧翼区域（包括保守的假定重组靶序列）的比较显示，与重组效率的变化没有明显一致的差异，因此我们得出结论，尽管共有七聚体和九聚体信号可能足以识别重组位点，但该位点被使用的概率还取决于其他决定因素。对已发表数据的综述表明，人类Jκ元件之间以及小鼠JλI和JλIII基因座之间的使用情况也不相等。将比较范围扩大到包括这些序列集表明，J使用频率的决定因素之一可能是共有七聚体与J编码序列内的T-G二核苷酸的接近程度，这可能揭示了重组酶内类似大肠杆菌gyrase的底物偏好。
