DNA STRUCTURE AND GENE REGULATION

分子水平上的遗传调控机制将 在体外和体内进行研究，特别强调 DNA特性和构象作为特异性的决定因素。 不寻常的DNA构象（左手Z-DNA，十字形， 寡嘌呤、寡嘧啶序列所采用的结构）将 用合成的寡核苷酸、限制性内切酶 片段和重组质粒。 构象特征 将用各种探针进行分析，包括核酸酶， 化学修饰，超螺旋松弛测定， 交联研究、电子显微镜和免疫学 方法。 不寻常的DNA构象的结构和性质将 在体外用寡核苷酸本身和 重组质粒。 一系列的结构，热力学， 动力学、分子模拟和流体动力学特性将 评估。 将在联合体的基础上进行模式研究 用于评估这些探针在碱基对处的特性 水平 特定蛋白质与不寻常DNA结构的相互作用 将在体外和体内进行研究。 的结合特性 急诊大肠杆菌recA蛋白和T4基因32蛋白在体内和在 将使用几种不同的方法建立Z-DNA的体外模型。 测定。 邻近左手Z-DNA束对 E. colilac阻遏物及其操纵子， 相邻启动子的影响将作为一个 负超螺旋作用。 不寻常的类型 SV 40和JC遗传控制区所采用的构象 病毒在存在和不存在特定蛋白质的情况下， 研究了 不寻常的DNA构象在体内的存在和功能， 包括Z-DNA在内的新系统将得到评估 为此目的。 质粒不相容性试验出现 在初步调查中特别有希望。 HhaI和 将建立EcoRI甲基化酶并在体内利用， 构象探针 Z-DNA在同源重组中的作用 重组和体内基因表达测定将是 进行。 对基因表达细节的理解 对于我们最终理解正常细胞 生长和分裂以及疾病综合征， 不受控制的生长，如癌症。

The mechanism of genetic regulation at the molecular level will be studied in vitro and in vivo with special emphasis on the role of DNA properties and conformations as determinants of specificity. Unusual DNA conformations (left-handed Z-DNA, cruciforms, structures adopted by oligopurine.oligopyrimidine sequences) will be investigated with synthetic oligonucleotides, restriction fragments, and recombinant plasmids. Conformational features will be analyzed with a variety of probes including nucleases, chemical modifications, supercoil relaxation determinations, crosslinking studies, electron microscopy, and immunological approaches. The structure and properties of unusual DNA conformations will be studied in vitro with oligonucleotides per se and with recombinant plasmids. A range of structural, thermodynamic, kinetic, molecular modeling, and hydrodynamic properties will be evaluated. Model studies will be undertaken on a consortium basis for evaluating the characteristics of these probes at the base pair level. The interactions of specific proteins with unusual DNA structures will be investigated in vitro and in vivo. The binding properties of the E. coli recA protein and the T4 gene 32 protein in vivo and in vitro with Z-DNA will be established using several different assays. The influence of neighboring left-handed Z-DNA tracts on the interaction of the E. coli lac repressor with its operator and the influence of the neighboring promoter will be studied as a function of negative supercoiling. The types of unusual conformations adopted by genetic control regions of SV40 and JC virus in the presence and absence of specific proteins will be studied. The in vivo existence and function of unusual DNA conformations, including Z-DNA, will be evaluated with new systems developed for this purpose. A plasmid incompatibility assay appears particularly promising in preliminary investigations. The HhaI and EcoRI methylases will be established and utilized in vivo as conformational probes. The role of Z-DNA in homologous recombination and in in vivo gene expression assays will be undertaken. An understanding of the details of genetic expression is fundamental to our ultimate comprehension of normal cellular growth and division as well as disease syndromes characterized by unregulated growth such as cancer.