人Elongator复合物Elp3亚基基因可显著补偿酵母elp3缺失菌株的生长缺陷

从HeLa细胞中分离的人的Elongator复合物在组成及与RNAPⅡ的作用方式上与酵母的Elongator复合物十分相似．但对其功能研究极少。为了研究人的Elongator复合物催化亚基Elp3的功能,将人elp3等基因转入酵母elp3基因缺失的突变菌株（elp3△菌株）,并对转化菌株进行功能互补实验和ssa和pho5基因表达分析,结果表明人elp3基因可显著恢复突变菌株对高温和Caffeine的敏感性．在低磷条件下显著补偿了突变株ph05基因表达延迟的缺陷．并可在热激条件下提高ssa3基因的表达。含酵母elp3非HAT区和人elp3 HAT区的融合yhelp3对上述缺陷有着更强的补偿能力。而HAT区催化结构域缺失的yhelp3HAT-没有任何补偿能力．表明人Elp3亚基可能与酵母的该亚基功能相似．人Elp3的HAT活性也为其行使功能所必需。     

Elongator function in tRNA wobble uridine modification is conserved between yeast and plants

Based on studies in yeast and mammalian cells the Elongator complex has been implicated in functions as diverse as histone acetylation, polarized protein trafficking and tRNA modification. Here we show that Arabidopsis mutants lacking the Elongator subunit AtELP3/ELO3 have a defect in tRNA wobble uridine modification. Moreover, we demonstrate that yeast elp3 and elp1 mutants expressing the respective Arabidopsis Elongator homologues AtELP3/ELO3 and AtELP1/ELO2 assemble integer Elongator complexes indicating a high degree of structural conservation. Surprisingly, in vivo complementation studies based on Elongator‐dependent tRNA nonsense suppression and zymocin tRNase toxin assays indicated that while AtELP1 rescued defects of a yeast elp1 mutant, the most conserved Elongator gene AtELP3, failed to complement an elp3 mutant. This lack of complementation is due to incompatibility with yeast ELP1 as coexpression of both plant genes in an elp1 elp3 yeast mutant restored Elongator's tRNA modification function in vivo. Similarly, AtELP1, not ScELP1 also supported partial complementation by yeast–plant Elp3 hybrids suggesting that AtElp1 has less stringent sequence requirements for Elp3 than ScElp1. We conclude that yeast and plant Elongator share tRNA modification roles and propose that this function might be conserved in Elongator from all eukaryotic kingdoms of life.     

RNA polymerase II elongator holoenzyme is composed of two discrete subcomplexes.

Elongator is a histone acetyltransferase complex that associates with the elongating form of RNA polymerase II. We purified Elongator to virtual homogeneity via a rapid three-step procedure based largely on affinity chromatography. The purified factor, holo-Elongator, is a labile six-subunit factor composed of two discrete subcomplexes: one comprised of the previously identified Elp1, Elp2, and Elp3 proteins and another comprised of three novel polypeptides, termed Elp4, Elp5, and Elp6. Disruption of the yeast genes encoding the new Elongator proteins confers phenotypes indistinguishable from those previously described for the other elp mutants, and concomitant disruption of genes encoding proteins in either subcomplex does not confer new phenotypes. Taken together, our results indicate that holo-Elongator is a functional entity in vitro as well as in vivo. Metazoan homologues of Elp1 and Elp3 have previously been reported. We cloned the human homologue of yeast ELP4 and show that this gene is ubiquitously expressed in human tissues.     

Subunit communications crucial for the functional integrity of the yeast RNA polymerase II elongator (gamma-toxin target (TOT)) complex

In response to the Kluyveromyces lactis zymocin, the gamma-toxin target (TOT) function of the Saccharomyces cerevisiae RNA polymerase II (pol II) Elongator complex prevents sensitive strains from cell cycle progression. Studying Elongator subunit communications, Tot1p (Elp1p), the yeast homologue of human IKK-associated protein, was found to be essentially involved in maintaining the structural integrity of Elongator. Thus, the ability of Tot2p (Elp2p) to interact with the HAT subunit Tot3p (Elp3p) of Elongator and with subunit Tot5p (Elp5p) is dependent on Tot1p (Elp1p). Also, the association of core-Elongator (Tot1-3p/Elp1-3p) with HAP (Elp4-6p/Tot5-7p), the second three-subunit subcomplex of Elongator, was found to be sensitive to loss of TOT1 (ELP1) gene function. Structural integrity of the HAP complex itself requires the ELP4/TOT7, ELP5/TOT5, and ELP6/TOT6 genes, and elp6Delta/tot6Delta as well as elp4Delta/tot7Delta cells can no longer promote interaction between Tot5p (Elp5p) and Tot2p (Elp2p). The association between Elongator and Tot4p (Kti12p), a factor that may modulate the TOT activity of Elongator, requires Tot1-3p (Elp1-3p) and Tot5p (Elp5p), indicating that this contact requires a preassembled holo-Elongator complex. Tot4p also binds pol II hyperphosphorylated at its C-terminal domain Ser(5) raising the possibility that Tot4p bridges the contact between Elongator and pol II.     

Mammalian and viral DNA sequences which interfere with the maintenance of a centromeric vector in yeast.

We constructed a recombinant plasmid by inserting into the pRS314 yeast centromeric plasmid vector the mouse DNA sequence responsible for the maintenance in transgenic mice of plasmid p12B1 (1). Such constructs could constitute convenient shuttle vectors between yeast and mouse cells. However, the recombinant molecule could not be established as a stable plasmid in Saccharomyces cerevisiae. A region with a limited similarity to the yeast centromere (CEN element) is present in this mouse sequence as well as in two other sequences subsequently identified in a data bank search using the CEN consensus. One of them is localized in Bovine Papillomavirus Type 1 DNA, and the other one in the human beta-globin locus. Once inserted in pRS314, these two sequences showed the same inhibitory effect on plasmid maintenance as the p12B1 mouse DNA fragment. This effect appears to depend on the simultaneous presence in the construct of one of the "CEN-like regions" and of an authentic CEN element. Non-centromeric yeast plasmids carrying one of the three sequences could replicate autonomously, and were even stabilized to a significant extent. These results identify in the genomes of higher eukaryotes and their viruses a family of sequences which cannot be simply cloned in centromeric yeast vectors.     

含2A片段的重组黄热病毒17D疫苗表达载体的构建

黄热疫苗是一种减毒的黄热病毒17D(YF-17D)活疫苗,是现有疫苗中最安全、最有效的疫苗之一,适于发展为疫苗载体。用RT-PCR法扩增出覆盖YF-17D全长基因组的3个cDNA片段:5′cDNA(A)、3′cDNA(B)和中间cDNA(C),同时引入SP6增强子序列、酶切位点和重复序列。顺序将A和B同E.coli-yeast穿梭质粒pRS424连接,再与C共转染酵母菌,利用缺少色氨酸和尿嘧啶的选择性固体培养基筛选出含YF-17D全长基因组的cDNA质粒。以该质粒为模板,经过DNA重组和酵母同源重组,获得含有口蹄疫病毒蛋白水解酶2A片段的重组YF-17D表达载体。将该表达载体体外转录后,电击转染BHK-21细胞。间接免疫荧光检测结果表明,RNA转录体在BHK-21细胞中进行了稳定的表达;滴度测定与形态学观察结果表明,重组病毒在细胞中的生长曲线等特征同母本YF-17D十分相似。结果提示,利用酵母菌同源重组在2A部位引入异种抗原基因,重组YF-17D表达载体pRS-YF-2A1具有成为高效活疫苗表达载体的潜力。     

A multiprotein complex that interacts with RNA polymerase II elongator

A three-subunit Hap complex that interacts with the RNA polymerase II Elongator was isolated from yeast. Deletions of genes for two Hap subunits, HAP1 and HAP3, confer pGKL killer-insensitive and weak Elongator phenotypes. Preferential interaction of the Hap complex with free rather than RNA polymerase II-associated Elongator suggests a role in the regulation of Elongator activity.     

System of centromeric, episomal, and integrative vectors based on drug resistance markers for Saccharomyces cerevisiae

Integrative, centromeric, and episomal plasmids are essential for easy, fast, and reliable genetic manipulation of yeast. We constructed a system of shuttle vectors based on the widely used plasmids of the pRS series. We used genes conferring resistance to Geneticin (kanMX4), nourseothricin (natNT2), and hygromycin B (hphNT1) as markers. The centromeric and episomal plasmids that we constructed can be used the same way as the traditional auxotrophic marker-based shuttle vectors (pRS41x and pRS42x series). Additionally, we created a set of nine yeast integrative vectors with the three dominant markers. These plasmids allow for direct integration in the LEU2, URA3, and HIS3 locus of any yeast strain and the concomitant partial deletion of the gene. This prevents multiple integrations and allows for the rapid identification of correct integrants. The set of new vectors considerably enhances the flexibility of genetic manipulations and gene expression in yeast. Most notably, the new vectors allow one to work with natural yeast isolates, which do not contain auxotrophic markers.     

The Elp2 subunit of elongator and elongating RNA polymerase II holoenzyme is a WD40 repeat protein

A novel yeast gene, ELP2, is shown to encode the 90-kDa subunit of the Elongator complex and elongating RNA polymerase II holoenzyme. ELP2 encodes a protein with eight WD40 repeats, and cells lacking the gene display typical elp phenotypes, such as temperature and salt sensitivity. Generally, different combinations of double and triple ELP gene deletions cause the same phenotypes as single ELP1, ELP2, or ELP3 deletion, providing genetic evidence that the ELP gene products work together in a complex.     

Molecular analysis of KTI12/TOT4, a Saccharomyces cerevisiae gene required for Kluyveromyces lactis zymocin action

TOT, the putative Kluyveromyces lactis zymocin target complex from Saccharomyces cerevisiae, is encoded by TOT1-7, six loci of which are isoallelic to RNA polymerase II (RNAPII) Elongator genes (ELP1-6). Unlike TOT1-3 (ELP1-3) and TOT5-7 (ELP5, ELP6 and ELP4 respectively), which display zymocin resistance when deleted, TOT4 (KTI12) also renders cells refractory to zymocin when maintained in multicopy or overexpressed from the GAL10 promoter. Elevated TOT4 copy number results in an intermediate tot phenotype, which includes mild sensitivities towards caffeine, Calcofluor white and elevated growth temperature, suggesting that TOT4 influences TOT/Elongator function. Tot4p interacts with Elongator, as shown by co-immunoprecipitation, and cell fractionation studies demonstrate partial co-migration with RNAPII and Elongator. As Elongator subunit interaction is not affected by either deletion of TOT4 or multicopy TOT4, Tot4p may not be a structural Elongator subunit but, rather, may regulate TOT/Elongator in a fashion that requires transient physical contact with TOT/Elongator. Consistent with a regulatory role, the presence of a potential P-loop motif conserved between yeast and human TOT4 homologues suggests capability of ATP or GTP binding and P-loop deletion renders Tot4p biologically inactive.