Brigati C, Kurtz S, Balderes D, Vidali G, Shore D
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Mol Cell Biol. 1993 Feb;13(2):1306-14. doi: 10.1128/mcb.13.2.1306-1314.1993.
A yeast gene encoding a DNA-binding protein that recognizes the telomeric repeat sequence TTAGGG found in multicellular eukaryotes was identified by screening a lambda gt11 expression library with a radiolabeled TTAGGG multimer. This gene, which we refer to as TBF1 (TTAGGG repeat-binding factor 1), encodes a polypeptide with a predicted molecular mass of 63 kDa. The TBF1 protein, produced in vitro by transcription and translation of the cloned gene, binds to (TTAGGG)n probes and to a yeast telomeric junction sequence that contains two copies of the sequence TTAGGG separated by 5 bp. TBF1 appears to be identical to a previously described yeast TTAGGG-repeat binding activity called TBF alpha. TBF1 produced in vitro yields protein-DNA complexes with (TTAGGG)n probes that have mobilities on native polyacrylamide gels identical to those produced by partially purified TBF alpha from yeast cells. Furthermore, when extracts are prepared from a strain containing a TBF1 gene with an antigen tag, we find that the antigen copurifies with the predominant (TTAGGG)n-binding activity in the extracts. The DNA sequence of TBF1 was determined. The predicted protein sequence suggests that TBF1 may contain a nucleotide-binding domain, but no significant similarities to any other known proteins were identified, nor was an obvious DNA-binding motif apparent. Diploid cells heterozygous for a tbf1::URA3 insertion mutation are viable but upon sporulation give rise to tetrads with only two viable spores, both of which are Ura-, indicating that the TBF1 gene is essential for growth. Possible functions of TBF1 (TFB alpha) are discussed in light of these new results.
通过用放射性标记的TTAGGG多聚体筛选λgt11表达文库，鉴定出了一个编码DNA结合蛋白的酵母基因，该蛋白可识别多细胞真核生物中发现的端粒重复序列TTAGGG。这个基因，我们称之为TBF1（TTAGGG重复序列结合因子1），编码一个预测分子量为63 kDa的多肽。通过克隆基因的转录和翻译在体外产生的TBF1蛋白，可与(TTAGGG)n探针以及一个酵母端粒连接序列结合，该序列包含两个由5个碱基对隔开的TTAGGG序列拷贝。TBF1似乎与先前描述的一种名为TBFα的酵母TTAGGG重复序列结合活性相同。体外产生的TBF1与(TTAGGG)n探针形成的蛋白质-DNA复合物，在天然聚丙烯酰胺凝胶上的迁移率与从酵母细胞中部分纯化的TBFα产生的复合物相同。此外，当从含有带有抗原标签的TBF1基因的菌株中制备提取物时，我们发现该抗原与提取物中主要的(TTAGGG)n结合活性共纯化。测定了TBF1的DNA序列。预测的蛋白质序列表明TBF1可能含有一个核苷酸结合结构域，但未发现与任何其他已知蛋白质有显著相似性，也没有明显的DNA结合基序。对于tbf1::URA3插入突变杂合的二倍体细胞是有活力的，但在孢子形成时会产生只有两个有活力孢子的四分体，这两个孢子都是尿嘧啶营养缺陷型，表明TBF1基因对生长至关重要。根据这些新结果讨论了TBF1（TFBα）的可能功能。
