Isolation and characterization of transcription factors of ribosoma DNA in lower eukaryotes

1.从推导的氨基酸序列中克隆了酿酒酵母中参与RNA聚合酶I转录35S rRNA基因的三个基因：RRN3、RRN6和RRN7，分别编码627个氨基酸的蛋白质、894个氨基酸的蛋白质和514个氨基酸的蛋白质。标准基因阻断实验表明，RRN3、RRN6和RRN7是必需基因。通过免疫亲和纯化和生化分级，我们得到了一种高纯度的制剂(Rrn6/Rrn7复合体)，它由Rrn6p、Rrn7p和另一种衣物相对分子质量为66000的蛋白质组成。体外转录实验表明，Rrn6/Rrn7复合体是形成具有转录活性的预起始复合体所必需的。Rrn3p在转录过程中的作用尚不清楚。然而，Rrn3p在酿酒酵母中的过量生产抑制了细胞的生长，这表明Rrn3p捕获了一些必要的蛋白质。2.裂殖酵母…分别用酿酒酵母表达载体中较多的pombe cDNA库和乳酸克鲁维酵母基因组DNA库来补充酿酒酵母rrn3、rrn6和rrn7突变体，以分离功能同源物。我们发现从S.pombe文库中获得的SRN31和SRN32可以互补温度敏感的rrn3突变，并测定了SRN31的全核苷酸序列，发现SRN31编码一个434个氨基酸的蛋白质，计算的相对分子质量为50,200。Rrn3p和Srn31p之间没有明显的同源性。来源于K.lactic的SRN33也可以补充对温度敏感的rrn3。3.我们尝试在pombe链霉菌中从RNA聚合酶II启动子中转录出具有功能的35S核糖体RNA，将nmt1启动子融合到一个单位的rDNA中，构建了nmt1-35S rDNA融合基因。将多拷贝载体上的融合基因转化到pombe菌株的温度敏感型RNA聚合酶I突变体(Nuc1)中。然而，在高温下没有观察到转化子的生长，这表明nmt1启动子提供的rRNA不足以补充nuc1突变。较少

1.We have cloned three genes, RRN3, RRN6, and RRN7 involved in the transcription of 35S rRNA genes by RNA polymerase I in Saccharomyces cerevisiae, From deduced amino acid sequences, RRN3 appcars to encode a protein of 627 amino acids, RRN6 to encode a protein of 894 amino acids, and RRN7 to encode a protein of 514 amino acids. Standard gene disruption experiments showed that RRN3, RRN6, and RRN7 are essential genes. By use of immunoaffinity purification and biochemical fractionation, we obtained a highly purified preparation (Rrn6/Rrn7 complex), which consisted of Rrn6p, Rrn7p, and another protein with an apparet molecular weight of 66,000. In vitro transcription experiments showed the Rrn6/Rrn7 complex is required for the formation of a transcription -competent preinitiation complex. The role of Rrn3p in the transcription process ia still unclear. However, a overproduction of Rrn3p in S.cerevisiae inhibits cell growth, suggesting that Rrn3p traps some essential protein.2.Schizosaccha … More romyces pombe cDNA bank in S.cerevisiae expression vector and Kluveroyces lactis genomic DNA bank, respectively, were used to complement S.cerevisiae rrn3, rrn6, and rrn7 mutants to isolate functional homologues. We have found SRN31 and SRN32 obtained from S.pombe cDNA bank can complement temperature-sensitive rrn3 mutation, determined the complete nucleotide sequence of SRN31 and found that SRN31 encodes a protein of 434 amino acids with a calculated molecular weight of 50,200. There is no apparent homology between Rrn3p and Srn31p. SRN33 derived from K.lactic can also complement temperature-sensitive rrn3. We have not analyzed SRN32 and SRN33 yet.3.We attempted to transcribe the functional 35S ribosomalRNA from RNA polymerase II promoter in S.pombe, We fused nmt1 promoter to one unit of rDNA to construct a nmt1-35S rDNA fusion gene. The fusion gene on a multicopy vector was transformed into a temperature-sensitive RNA polymerase I mutant (nuc1) of S.pombe. However, the growth of transformants was not observed at a high temperature, indicating that the amount rRNA supplied from nmt1 promoter is not enough to complement nuc1 mutation. Less