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

1.从酿酒酵母中克隆了3个与35 SrRNA基因转录相关的基因RRN 3、RRN 6和RRN 7。从推导的氨基酸序列来看，RRN 3编码627个氨基酸，RRN 6编码894个氨基酸，RRN 7编码514个氨基酸。标准基因破坏实验表明，RRN 3、RRN 6和RRN 7是必需基因。通过免疫亲和纯化和生化分离，我们获得了高度纯化的Rrn 6/Rrn 7复合物，其由Rrn 6p、Rrn 7 p和另一个表观分子量为66，000的蛋白质组成。体外转录实验表明，Rrn 6/Rrn 7复合物是形成具有转录能力的前起始复合物所必需的。Rrn 3 p在转录过程中的作用尚不清楚。然而，酿酒酵母中Rrn 3 p的过量产生抑制细胞生长，表明Rrn 3 p捕获一些必需蛋白。 关于我们 酿酒酵母表达载体中的粟酒裂殖酵母cDNA库和乳酸克鲁维酵母基因组DNA库分别用于补充酿酒酵母rrn 3、rrn 6和rrn 7突变体以分离功能同源物。我们从粟酒裂殖酵母cDNA文库中获得了SRN 31和SRN 32，发现它们可以互补温度敏感的rrn 3突变，并测定了SRN 31的全核苷酸序列，发现SRN 31编码434个氨基酸的蛋白，计算分子量为50，200。Rrn 3 p和Srn 31 p之间没有明显的同源性。来源于乳酸克雷伯氏菌的SRN 33也可以补充温度敏感的rrn 3。3.在粟酒裂殖酵母RNA聚合酶II启动子的基础上，尝试转录35 S核糖体RNA，将nmt 1启动子与rDNA融合，构建了nmt 1 - 35 S rDNA融合基因。将多拷贝载体上的融合基因转化到粟酒裂殖酵母的温度敏感的RNA聚合酶I突变体（nuc 1）中。然而，在高温下没有观察到转化体的生长，表明nmt 1启动子提供的rRNA量不足以补充nuc 1突变。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