DNA-binding region of the simian virus 40 tumor antigen.
DNA-binding region of the simian virus 40 tumor antigen.
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猿猴病毒 40 肿瘤抗原的 DNA 结合区域。
DOI：
10.1128/jvi.57.3.776-785.1986
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发表时间：
1986
影响因子：
5.4
通讯作者：
Simmons,DT
中科院分区：
文献类型：
--
作者：
Simmons,DT
通过免疫亲和层析纯化猴病毒40（SV 40）肿瘤（T）抗原，并用少量胰蛋白酶切割，并将所得片段进行SV 40 DNA纤维素层析。来自分子左端的44，000分子量片段（44 K片段）和从大约Lys 131到Lys 371的30 K片段结合到柱上，并用1 M NaCl洗脱。在第二系列实验中，T抗原用仓鼠抗T血清或各种单克隆抗体免疫沉淀，并用胰蛋白酶部分消化。通过使用SV 40 DNA片段结合试验，检测通过该处理溶解的片段的DNA结合活性。一个17 K的片段，它起源于氨基末端区域的多肽没有明显的结合活性，在这个试验中。另一方面，较大的片段（76 K，46 K，和30 K），其氨基末端被映射到周围的赖氨酸131没有显示DNA结合活性。最后，由SV 40 DNA和T-抗原片段组成的复合物在DNA结合试验中沉淀，该试验用单克隆抗体识别蛋白质的中心区域;然而，对氨基或羧基末端区域具有特异性的抗体是无活性的。这些结果有力地表明，T抗原的DNA结合区位于大约Lys 131和Lys 371之间，对应于DNA上的0.51和0.37图单位。
The simian virus 40 (SV40) tumor (T) antigen was purified by immunoaffinity chromatography and cleaved with small amounts of trypsin, and the resulting fragments were subjected to SV40 DNA cellulose chromatography. A 44,000-molecular-weight fragment (44K fragment) from the left end of the molecule and a 30K fragment mapping from approximately Lys 131 to Lys 371 bound to the column and were eluted with 1 M NaCl. In a second series of experiments, T antigen was immunoprecipitated with hamster anti-T serum or various monoclonal antibodies and partially digested with trypsin. Fragments that were solubilized by this treatment were tested for DNA-binding activity by using an SV40 DNA fragment-binding assay. A 17K fragment which originated from the amino-terminal region of the polypeptide had no apparent binding activity in this assay. On the other hand, larger fragments (76K, 46K, and 30K) whose amino termini were mapped around Lys 131 did display DNA-binding activity. Finally, complexes consisting of SV40 DNA and T-antigen fragments were precipitated in the DNA-binding assay with monoclonal antibodies that recognize the central region of the protein; however, antibodies with specificities to the amino- or carboxy-terminal regions were inactive. These results strongly suggest that the DNA-binding region of T antigen lies approximately between Lys 131 and Lys 371, corresponding to 0.51 and 0.37 map units on the DNA.