Akamatsu Y, Tsurushita N, Nagawa F, Matsuoka M, Okazaki K, Imai M, Sakano H
Department of Genetics and Molecular Biology, Kyoto University, Japan.
J Immunol. 1994 Nov 15;153(10):4520-9.
Recombination signal sequences for V(D)J joining consist of a conserved heptamer (CACAGTG) and a nonamer (ACAAAAACC) separated by a spacer of a constant length (12 bp or 23 bp). In the present study, we have analyzed various recombination signal mutations for their effects in V(D)J joining. Using a retroviral vector, we introduced mutant substrates stably into pre-B cells, and assayed recombination using the lacZ gene as a reporter. This method allowed us to study recombination in a single copy within the context of the host cell chromosome. Because this assay did not show any detectable background, it was quite useful in the analysis of low level recombinations. In the heptamer, mutations in the first three residues severely dropped the joining rates. Among them, the first residue adjacent to the recombination site was found to be most essential. Although mutations in the heptamer reduced the joining rate to various extents, they did not lower the site-specificity of recombination. With regard to the nonamer, the presence of three consecutive A residues was necessary for efficient recombination. Furthermore, the nucleotides flanking the A-rich core needed to be other than A residues, probably marking the border of the A-stretch. This may be important when the recombinase measures the distance between the heptamer and the nonamer to satisfy the 12/23-bp spacer rule.
V(D)J 重排的重组信号序列由一个保守的七聚体（CACAGTG）和一个九聚体（ACAAAAACC）组成，二者被一段固定长度（12 bp 或 23 bp）的间隔序列隔开。在本研究中，我们分析了各种重组信号突变对 V(D)J 重排的影响。利用逆转录病毒载体，我们将突变底物稳定地导入前 B 细胞，并以 lacZ 基因作为报告基因检测重排情况。这种方法使我们能够在宿主细胞染色体背景下研究单拷贝中的重排。由于该检测未显示任何可检测到的背景，因此在分析低水平重排时非常有用。在七聚体中，前三个残基的突变严重降低了连接率。其中，发现与重组位点相邻的第一个残基最为关键。尽管七聚体中的突变在不同程度上降低了连接率，但它们并未降低重排的位点特异性。关于九聚体，三个连续 A 残基的存在对于有效重排是必需的。此外，富含 A 的核心两侧的核苷酸需要不是 A 残基，这可能标志着 A 序列延伸的边界。当重组酶测量七聚体和九聚体之间的距离以满足 12/23 bp 间隔规则时，这可能很重要。
