RIBOSOMAL DNA IN PHYSARUM POLYCEPHALUM

多头绒泡菌核糖体RNA编码基因是一种 约200个60 kb大小的染色体外DNA分子的集合。 这 rDNA可以很容易地纯化;它的大部分已经被克隆和测序。 我们 我建议继续研究这些基因的结构和功能。 I.这项工作的主要部分将用于研究两个具体的 我们已经部分纯化的DNA结合蛋白。 一个绑定到 一个序列正好在RNA起始位点的上游，另一个结合在 rDNA端粒。 如果可能的话，将进一步纯化蛋白质， 均匀性，并确定其精确的结合位点。 二.我们有 发现了第三个，菌株特异性内含子在26 s编码区的 rDNA。 一个单拷贝的染色体序列， 内含子存在于rDNA中不携带第三内含子的菌株中。 内含子将被测序，染色体同源克隆和 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， 和扩增的rDNA在生活史中，并通过测试核仁， 用于固定一种或另一种rDNA类型的杂合疟原虫。 四.的 复制起点的确切位置将由 生物化学技术应用于rDNA的复制形式丰富。 诉 本研究将研究绒泡菌rDNA在酵母中稳定复制的能力 特别是关于"复制从哪里开始？ 什么样的序列对于高效率的 转化？ 瞬时表达测定的初步实验 将做测试方法引入rDNA回 绒泡菌

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.