Fuschiotti P, Harindranath N, Mage R G, McCormack W T, Dhanarajan P, Roux K H
Laboratory of Immunology, NIAID, Bethesda, MD 20892.
Mol Immunol. 1993 Aug;30(11):1021-32. doi: 10.1016/0161-5890(93)90127-w.
The recombination activating genes RAG-1 and RAG-2 appear to be necessary components of the machinery needed for the Ig or TCR gene rearrangements that occur in developing B and T lymphocytes. In addition RAG-2 has been implicated in the process of V-gene diversification by somatic gene conversion in the chicken. Because gene conversion may be an important mechanism for V-gene diversification in the rabbit, we cloned the rabbit RAG locus and characterized the coding regions of the genomic RAG-1 and RAG-2. In addition, we sequenced cDNAs encompassing the RAG-2 coding region, part of the RAG-2 5' untranslated region and a 967 bp fragment of cDNA from the RAG-1 coding region. Northern analysis revealed a RAG-1 mRNA of 6.6 kb which is similar in size to the RAG-1 mRNA reported previously for other species, and a major species of RAG-2 mRNA of 4.4 kb, which is larger than that from the mouse (2.2 kb). Analysis of the genomic clones showed that, as in other species, the RAG-1 and RAG-2 genes are oriented so as to be convergently transcribed. The DNA sequence analysis showed that the rabbit RAG-1 coding region is 91, 85 and 72% identical to human, mouse and chicken, respectively. The deduced RAG-1 protein sequence for rabbit is 93, 90 and 78% identical to human, mouse and chicken. Comparison of the rabbit RAG-2 coding region revealed 90, 87 and 71% identity to human, mouse and chicken, respectively, at the nucleotide level, and 91, 90 and 72% at the protein level. Although there is considerable conservation of sequence between species, we obtained evidence for allelic forms of the rabbit RAG locus both by Southern analyses and by sequencing. A remarkable degree of polymorphism was found in our rabbit colonies, particularly in the region 3' of the rabbit RAG-2 coding region. A 5' cDNA probe hybridized with one or more additional fragments that are not detected with the coding region probes, suggesting that the 5' cDNA sequence results from splicing of one or more upstream exons.
重组激活基因RAG-1和RAG-2似乎是发育中的B淋巴细胞和T淋巴细胞中发生Ig或TCR基因重排所需机制的必要组成部分。此外，RAG-2与鸡中通过体细胞基因转换实现的V基因多样化过程有关。由于基因转换可能是兔子中V基因多样化的重要机制，我们克隆了兔子的RAG基因座并对基因组RAG-1和RAG-2的编码区进行了表征。此外，我们对包含RAG-2编码区、RAG-2 5'非翻译区的一部分以及来自RAG-1编码区的967 bp cDNA片段的cDNA进行了测序。Northern分析显示，有一个6.6 kb的RAG-1 mRNA，其大小与先前报道的其他物种的RAG-1 mRNA相似，还有一种主要的4.4 kb的RAG-2 mRNA，其比小鼠的（2.2 kb）大。对基因组克隆的分析表明，与其他物种一样，RAG-1和RAG-2基因的方向是反向转录的。DNA序列分析表明，兔子RAG-1编码区与人类、小鼠和鸡的编码区分别有91%、85%和72%的同一性。推导的兔子RAG-1蛋白序列与人类、小鼠和鸡的分别有93%、90%和78%的同一性。对兔子RAG-2编码区的比较显示，在核苷酸水平上与人类、小鼠和鸡的分别有90%、87%和71%的同一性，在蛋白质水平上分别有91%、90%和72%的同一性。尽管物种间序列存在相当程度的保守性，但我们通过Southern分析和测序都获得了兔子RAG基因座等位基因形式的证据。在我们的兔子群体中发现了显著程度的多态性，特别是在兔子RAG-2编码区的3'区域。一个5' cDNA探针与一个或多个用编码区探针未检测到的额外片段杂交，这表明5' cDNA序列是由一个或多个上游外显子的剪接产生的。
