FHL2转录激活结构域的定位

LIM蛋白家族成员FHL2 (fourandhalfLIMdomainprotein)在转录调节、细胞凋亡及肿瘤的发生发展中都起着重要作用。利用GAL4转录因子中的DNA结合结构域 (DBD)和含有与DBD结合序列的荧光素酶报告基因(GAL4 LUC)构建了哺乳动物细胞转录激活系统 ,并利用该系统定位了FHL2的转录激活结构域。首先将GAL4 DBD序列以正确读框插入到pcDNA3载体的多克隆位点中 ,构建成真核表达载体pDBD ,再将野生型FHL2及其不同片段以正确读框与pDBD中GAL4 DBD序列融合 ,构建成野生型FHL2及其缺失突变体表达载体。将这些表达载体分别瞬时转染 2 93T胚胎肾细胞 ,野生型FHL2及其缺失突变体都得到了表达。利用GAL4 荧光素酶报告基因对野生型FHL2及其不同突变体的转录激活活性检测表明 ,在 2 93T胚胎肾细胞和乳腺癌MCF 7细胞中 ,野生型FHL2具有转录激活活性 ,缺失N端半个LIM结构域使FHL2转录激活活性降低 ,缺失C末端第二个LIM结构域对FHL2的转录激活功能影响不大 ,缺失C末端最后一个LIM结构域则使FHL2的转录激活功能完全丧失 ,而C末端缺失 2个LIM结构域使FHL2转录激活活性又有所恢复。这说明FHL2C末端最后一个LIM结构域对其转录激活功能是必需的 ,而C末端第二个LIM结构域可能对FHL2的转录激活功能有负调控作用 ,这种负调控作用取决于     

呼吸道黏蛋白5AC基因转录表达的顺式调控元件分析

目的：探讨呼吸道黏蛋白（mucin,MUC）5AC基因5'上游序列顺式调控元件在中性粒细胞弹力酶(neutrophil elastase , NE)诱导MUC5AC基因转录表达的调控机制。方法：应用DNA重组技术,构建含萤光素酶报告基因和MUC5AC启动子不同长度片段的嵌合质粒。采用定点突变技术,在嵌合质粒的基础上构建MUC5AC启动子区特殊蛋白（specificity protein）-1和核因子(nuclear factor, NF)-κB结合位点单独突变体,并测定NE刺激的转染细胞荧光素酶相对活性。结果：成功构建了4种含有不同长度MUC5AC基因启动子序列的荧光索酶报告基因质粒。含有启动子序列-1330bp、-689bp、-324bp的嵌合质粒荧光素酶相对光强度较对照组均显著增加,而含有启动子序列-64bp的嵌合质粒荧光素酶相对光强度与对照组相比差异无统计学意义。NE可诱导含有MUC5AC启动子区NF-кB结合位点单独突变体（pGL3E-MUC5AC-NF-кB-MU）荧光素酶相对光强度增加,而NE不能诱导Sp-l结合位点单独突变体（pGL3E-MUC5AC-SP-1-MU）荧光素酶表达增加。结论：MUC5AC 5'上游序列中-324～-64位点存在参与NE诱导MUC5AC基因表达的重要调控元件,位于此区域的顺式作用元件Sp-1位点在NE诱导MUC5AC基因表达机制中起重要作用,该位点可能作为靶向性基因治疗的关键调控元件。     

Regulation of JC virus expression in B lymphocytes.

The etiologic agent of progressive multifocal leukoencephalopathy, a subacute demyelinating disease of the central nervous system, is the human polyomavirus JC virus (JCV), which causes a lytic infection of myelin-producing oligodendrocytes. In infected individuals the JCV genome can be detected in brain tissue and B lymphocytes isolated from the blood, bone marrow, or lymph nodes. Using mobility shift assays and a radiolabeled oligonucleotide from the JCV promoter-enhancer region (JCV bp 130 to 160), referred to as domain B, we were able to detect specific bands of the same mobility in nuclear extracts from human fetal glial cells, U-251 glioma cells, different B-cell lines, and in vitro-activated tonsillar B lymphocytes but not from T cells. In addition, a specific shift was detected when using nuclear extracts from freshly isolated tonsillar or lymph node B cells from five AIDS patients, two of whom later developed progressive multifocal leukoencephalopathy. Somewhat surprisingly, the above gel shift was partially inhibited by unlabeled oligonucleotides containing a kappa E2-binding site. UV cross-linking of the protein-DNA complex from either B cells or glial cells and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a 46-kDa band. Transient transfection of a reporter plasmid constructed by fusing a trimer of the domain B sequence to a minimal promoter revealed activity in B lymphocytes and glial cells but not in T cells. Mutational analysis of this region demonstrated that the core TGGC repeat was essential for enhancer activity. Thus, a similar protein in B lymphocytes and glial cells may account for the preferential replication of JCV in these two cell types.     

Binding of myc proteins to canonical and noncanonical DNA sequences.

Using an in vitro binding-site selection assay, we have demonstrated that c-Myc-Max complexes bind not only to canonical CACGTG or CATGTG motifs that are flanked by variable sequences but also to noncanonical sites that consist of an internal CG or TG dinucleotide in the context of particular variations in the CA--TG consensus. None of the selected sites contain an internal TA dinucleotide, suggesting that Myc proteins necessarily bind asymmetrically in the context of a CAT half-site. The noncanonical sites can all be bound by proteins of the Myc-Max family but not necessarily by the related CACGTG- and CATGTG-binding proteins USF and TFE3. Substitution of an arginine that is conserved in these proteins into MyoD (MyoD-R) changes its binding specificity so that it recognizes CACGTG instead of the MyoD cognate sequence (CAGCTG). However, like USF and TFE3, MyoD-R does not bind to all of the noncanonical c-Myc-Max sites. Although this R substitution changes the internal dinucleotide specificity of MyoD, it does not significantly alter its wild-type binding sequence preferences at positions outside of the CA--TG motif, suggesting that it does not dramatically change other important amino acid-DNA contacts; this observation has important implications for models of basic-helix-loop-helix protein-DNA binding.