CHROMATIN STRUCTURE AND FUNCTION

我们继续研究了表达基因附近的染色质结构。鸡红系细胞中的珠蛋白基因家族作为一个模型系统，其中有可能研究红系发育过程中与家族的簇和个体成员的调节相关的机制。我们将注意力集中在鸡β-珠蛋白基因座5端的1.2 kb绝缘子DNA序列上，当它位于增强子-启动子之间时，它能够阻断增强子-启动子相互作用。我们先前已经将这种活性缩小到1.2 kb序列内的一个小DNA片段。我们纯化了负责这种活性的蛋白质，并将其鉴定为CTCF，CTCF是脊椎动物中普遍存在的已知因子，对多种基因的表达具有刺激和抑制作用。我们已经表明，CTCF网站是存在于其他脊椎动物基因位点，在网站上的其他人报道有增强子阻断活性。在平行的工作中，我们已经检查了鸡β珠蛋白基因座的3端的组织，并通过核酸酶敏感性的变化确定了基因座的3个边界。我们发现在这个边界的组成过敏网站非常相似的标志5边界的轨迹。它具有与上游元件类似的增强子阻断特性，并含有CTCF位点。除此之外，我们发现一个完整的开放阅读框架的气味受体（OR）。在同时工作中（与Fred哈钦森癌症研究中心的Mark Groudine和哥伦比亚大学的Richard阿克塞尔的实验室合作）我们在小鼠β珠蛋白基因座附近鉴定了相同OR家族的成员。鸡OR不表达在红系细胞中，提高的可能性，3边界元件，以防止两个基因系统之间的相互作用，就像5边界可能会防止相互作用的珠蛋白基因座与叶酸受体基因，我们最近发现只有16 kb 5的珠蛋白基因座。我们还继续研究RNA聚合酶转录染色质模板的机制。在早期的工作中，我们使用单个核小体作为模板，并表明组蛋白八聚体能够转移出聚合酶的路径而不释放DNA。我们提出了一个模型的中间体在这个过程中。我们现在已经与Chris Woodcock（马萨诸塞州大学）合作获得了这些中间体的电子低温显微照片，这些照片证实了溶液观察结果，并提供了有关其结构的更详细信息。- 染色质结构，珠蛋白基因，红系细胞，绝缘子，增强子阻断，叶酸受体，嗅觉受体

We have continued our studies of chromatin structure in the neighborhood of expressed genes. The globin gene family in chicken erythroid cells serves as a model system in which it is possible to study the mechanisms associated with regulation of the cluster and individual members of the family during erythroid development. We have focused attention on the 1.2 kb insulator DNA sequence at the 5 end of the chicken beta-globin locus, which is capable of blocking enhancer- promoter interaction when it lies between them. We had earlier narrowed down this activity to a small DNA fragment within the 1.2 kb sequence. We purified the protein responsible for this activity and identified it as CTCF, which is a known factor ubiquitous in vertebrates, with both stimulatory and inhibitory effects on expression of a variety of genes. We have shown that CTCF sites are present in other vertebrate gene loci, at sites reported by others to have enhancer blocking activity. In parallel work we have examined the organization of the 3 end of the chicken beta globin locus, and identified by changes in nuclease sensitivity the 3 boundary of the locus. We find at this boundary a constitutive hypersensitive site very similar to the one that marks the 5 boundary of the locus. It has enhancer blocking properties like those of the upstream element and contains a CTCF site. Beyond this we find a complete open reading frame for an odorantreceptor (OR). In simultaneous work (in collaboration with the laboratories of Mark Groudine, Fred Hutchinson Cancer Research Center and Richard Axel, Columbia Univ.) we identified members of the same OR family near the mouse beta globin locus. The chicken OR is not expressed in erythroid cells, raising the possibility that the 3 boundary element serves to prevent interaction between the two gene systems, just as the 5 boundary may prevent interaction of the globin locus with the folate receptor gene that we have recently located only 16 kb 5 of the globin locus. We have also continued studies of the mechanism by which RNA polymerase is able to transcribe chromatin templates. In earlier work we used single nucleosomes as templates and showed that the histone octamer was capable of transferring out of the path of the polymerase without letting go of the DNA. We proposed a model for the intermediates in this process. We have now obtained in collaboration with Chris Woodcock (Univ. Massachusetts) electron cryo- micrographs of these intermediates, which confirm the solution observations, and provide more detailed information about their structure. - chromatin structure, globin gene, erythroid cells, insulator, enhancer blocking, folate receptor, olfactory receptor