RIBOSOMAL DNA IN PHYSARUM POLYCEPHALUM

多头绒泡菌中编码核糖体RNA的基因以 收集了大约200个染色体外DNA分子，大小为60kb。这 RDNA可以很容易地提纯；大部分已经被克隆和测序。我们 建议继续我们对这些基因的结构和功能的研究。 一、工作的一大部分将致力于研究两个具体的 我们已经部分纯化了DNA结合蛋白。与某人捆绑在一起 序列正好在RNA起始点的上游，另一个结合在 RDNA端粒。如果可能的话，这些蛋白质将进一步纯化，以 均一性，并确定了它们的精确结合部位。二、我们有 在该菌株的26S编码区发现了第三个菌株特有的内含子 RDNA。看起来像是一个与 内含子存在于rDNA中没有携带第三内含子的菌株中。 将对内含子进行测序，并对染色体同源基因克隆和 也进行了测序，以获得它们进化关系的线索。三. RDNA单拷贝非孟德尔遗传的机制 将通过分析孢子中的rDNA，通过寻找破坏来进行研究 和rDNA在生命周期中的扩增，并通过检测核仁 用于固定一种或另一种rDNA类型的杂合性疟原虫。IV. 复制源的确切位置将通过以下方式确定 生物化学技术应用于富含复制形式的rDNA。五. 绒毛霉菌rDNA在酵母中稳定复制的能力将被研究 请特别参考这些问题，复制是在哪里启动的？ 以及，哪些序列对于高效的 转型？瞬时表达分析的初步实验 将测试将rDNA重新引入到 绒毛霉属。

The gene coding for ribosomal RNA in Physarum polycephalum exist as a collection of about 200 extrachromosomal DNA molecules 60 kb in size. This rDNA can be purified readily; most of it has been cloned and sequenced. We propose to continue our work on the structure and function of these genes. I. A major fraction of the effort will be devoted to studying two specific DNA binding proteins that we have purified partially. One binds to sequences just upstream of the RNA start site, and the other binds close to the rDNA telomeres. The proteins will be purified further, if possible to homogeneity, and their precise binding sites determined. II. We have discovered a third, strain-specific intron in the 26s coding region of the rDNA. What appears to be a single copy chromosomal sequence homologous to the intron exists in strains not carrying the third intron in their rDNA. The intron will be sequenced, and the chromosomal homologue clond and sequenced as well, to gain clues to their evolutionary relationship. III. The mechanism underlying the single copy, non-Mendelian inheritance of rDNA will be studied, by analysis of rDNA in spores, by a search for destruction and amplification of rDNA in the life cycle, and by testing nucleoli in heterozygous plasmodia for fixation of one or the other rDNA type. IV. The exact locations of the replication origins will be determined by biochemical techniques applied to rDNA enriched for replicating forms. V. The ability of Physarum rDNA to replicate stably in yeast will be studied with particular reference to the questions, Where is replication initiated? and, What sequences are important for the high efficiency of transformation? Preliminary experiments with transient expression assays will be done to test methods for introduction of the rDNA back into Physarum.