Structure of the gene of tum- transplantation antigen P198: a point mutation generates a new antigenic peptide.
Structure of the gene of tum- transplantation antigen P198: a point mutation generates a new antigenic peptide.
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DOI：
10.1084/jem.172.1.35
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发表时间：
1990-07-01
期刊：
影响因子：
--
通讯作者：
Boon T
中科院分区：
文献类型：
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作者：
Sibille C;Chomez P;Wildmann C;Van Pel A;De Plaen E;Maryanski JL;de Bergeyck V;Boon T
小鼠肿瘤细胞系P815的诱变剂处理产生被同基因小鼠排斥的tum-变体，因为它们表达新的移植抗原。这些tum抗原被细胞毒性T淋巴细胞（CTL）识别，但不诱导可检测的抗体应答。通过用tum-变体P198的DNA转染P815细胞系P1.HTR，我们获得了表达tum-抗原P198的转染子，可以根据它们刺激抗P198 CTL的能力来鉴定。用衍生自变体P198的粘粒文库的DNA重复这一过程，并从转染子中回收携带编码抗原P198的序列的粘粒。P198基因长3kb，包含8个外显子。它与先前鉴定的tum基因P91A没有同源性，也与数据库中目前记录的任何基因没有同源性。长开放阅读框编码23.5 kD蛋白。P198基因的抗原等位基因与正常等位基因的区别在于位于外显子7的点突变。该突变导致Ala至Thr的改变，并且通过定点诱变显示该突变负责抗原的表达。一个11个氨基酸的合成肽覆盖的序列周围的tum突变，使P815细胞敏感的裂解抗P198 CTL。对应于该基因正常序列的同源肽没有，但它能够竞争结合主要组织相容性复合体分子Kd。我们的结论是，逆转突变P198产生了一个新的表位识别的同源T细胞。如对P91A基因所观察到的，我们发现，将仅包含外显子3 - 7的P198基因片段克隆到非表达载体中，有效地转移了抗原的表达。
Mutagen treatment of mouse tumor cell line P815 produces tum- variants that are rejected by syngeneic mice because they express new transplantation antigens. These tum- antigens are recognized by cytotoxic T lymphocytes (CTL) but induce no detectable antibody response. By transfecting P815 cell line P1.HTR with DNA of tum- variant P198, we obtained transfectants expressing tum- antigen P198 that could be identified on the basis of their ability to stimulate anti-P198 CTL. This was repeated with DNA of a cosmid library derived from variant P198, and a cosmid carrying the sequence encoding antigen P198 was recovered from a transfectant. Gene P198 is 3 kb long and contains eight exons. It shows no homology with previously identified tum- gene P91A, nor with any gene presently recorded in the data banks. The long open reading frame codes for a 23.5-kD protein. The antigenic allele of gene P198 differs from the normal allele by a point mutation located in exon 7. This mutation causes an Ala to Thr change, and was shown by site-directed mutagenesis to be responsible for the expression of the antigen. An 11-amino acid synthetic peptide covering the sequence surrounding the tum- mutation rendered P815 cells sensitive to lysis by anti-P198 CTL. The homologous peptide corresponding to the normal sequence of the gene did not, but it was able to compete for binding to major histocompatibility complex molecule Kd. We conclude that tum- mutation P198 generates a new epitope recognized by syngeneic T cells. As observed with gene P91A, we found that a fragment of gene P198 that contained only exons 3-7, cloned in nonexpression vectors, transferred efficiently the expression of the antigen.