The DEAD-box RNA helicase Dbp5 functions in translation termination

In eukaryotes, termination of messenger RNA (mRNA) translation is mediated by the release factors eRF1 and eRF3. Using Saccharomyces cerevisiae as a model organism, we have identified a member of the DEAD-box protein (DBP) family, the DEAD-box RNA helicase and mRNA export factor Dbp5, as a player in translation termination. Dbp5 interacts genetically with both release factors and the polyadenlyate-binding protein Pab1. A physical interaction was specifically detected with eRF1. Moreover, we show that the helicase activity of Dbp5 is required for efficient stop-codon recognition, and intact Dbp5 is essential for recruitment of eRF3 into termination complexes. Therefore, Dbp5 controls the eRF3-eRF1 interaction and thus eRF3-mediated downstream events.     

Suppression of mutations in mitochondrial DNA by tRNAs imported from the cytoplasm

Mitochondrial import of a cytoplasmic transfer RNA (tRNA) in yeast requires the preprotein import machinery and cytosolic factors. We investigated whether the tRNA import pathway can be used to correct respiratory deficiencies due to mutations in the mitochondrial DNA and whether this system can be transferred into human cells. We show that cytoplasmic tRNAs with altered aminoacylation identity can be specifically targeted to the mitochondria and participate in mitochondrial translation. We also show that human mitochondria, which do not normally import tRNAs, are able to internalize yeast tRNA derivatives in vitro and that this import requires an essential yeast import factor.     

Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)-adenosine triphosphatase

The TFP1 gene of the yeast Saccharomyces cerevisiae encodes two proteins: the 69-kilodalton (kD) catalytic subunit of the vacuolar proton-translocating adenosine triphosphatase (H(+)-ATPase) and a 50-kD protein. The 69-kD subunit is encoded by the 5' and 3' thirds of the TFP1 coding region, whereas the 50-kD protein is encoded by the central third. Evidence is presented that both the 69-kD and 50-kD proteins are obtained from a single translation product that is cleaved to release the 50-kD protein and spliced to form the 69-kD subunit.     

Movement of eukaryotic mRNAs between polysomes and cytoplasmic processing bodies

Eukaryotic cells contain nontranslating messenger RNA concentrated in P-bodies, which are sites where the mRNA can be decapped and degraded. We present evidence that mRNA molecules within yeast P-bodies can also return to translation. First, inhibiting delivery of new mRNAs to P-bodies leads to their disassembly independent of mRNA decay. Second, P-bodies decline in a translation initiation-dependent manner during stress recovery. Third, reporter mRNAs concentrate in P-bodies when translation initiation is blocked and resume translation and exit P-bodies when translation is restored. Fourth, stationary phase yeast have large P-bodies containing mRNAs that reenter translation when growth resumes. The reciprocal movement of mRNAs between polysomes and P-bodies is likely to be important in the control of mRNA translation and degradation. Moreover, the presence of related proteins in P-bodies and maternal mRNA storage granules suggests this mechanism is widely adapted for mRNA storage.     

The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme

Mutants in the gene CDC34 of the yeast Saccharomyces cerevisiae are defective in the transition from G1 to the S phase of the cell cycle. This gene was cloned and shown to encode a 295-residue protein that has substantial sequence similarity to the product of the yeast RAD6 gene. The RAD6 gene is required for a variety of cellular functions including DNA repair and was recently shown to encode a ubiquitin-conjugating enzyme. When produced in Escherichia coli, the CDC34 gene product catalyzed the covalent attachment of ubiquitin to histones H2A and H2B in vitro, demonstrating that the CDC34 protein is another distinct member of the family of ubiquitin-conjugating enzymes. The cell cycle function of CDC34 is thus likely to be mediated by the ubiquitin-conjugating activity of its product.     

Bakers' yeast, a model for fungal biofilm formation

Biofilms are formed by the aggregation of microorganisms into multicellular structures that adhere to surfaces. Here we show that bakers' yeast Saccharomyces cerevisiae can initiate biofilm formation. When grown in low-glucose medium, the yeast cells adhered avidly to a number of plastic surfaces. On semi-solid (0.3% agar) medium they formed "mats": complex multicellular structures composed of yeast-form cells. Both attachment to plastic and mat formation require Flo11p, a member of a large family of fungal cell surface glycoproteins involved in adherence. The ability to study biofilm formation in a tractable genetic system may facilitate the identification of new targets for antifungal therapy.     

百脉根离子通道蛋白Pollux与翻译延伸因子EF1A的相互作用

利用酵母双杂交技术,以百脉根离子通道蛋白Pollux胞内结构域为诱饵,从百脉根AD-cDNA文库中筛选到与Pollux相互作用的多个阳性克隆,经DNA序列测定及NCBI数据库BLAST分析,其中有6个阳性克隆编码翻译延伸因子EF1A。进一步在大肠杆菌中表达与纯化Pollux和EF1A融合蛋白,通过Pull-down及Western杂交证实Pollux胞内结构域与EF1A的C-末端相互作用。     

Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1

Control of cyclin levels is critical for proper cell cycle regulation. In yeast, the stability of the G1 cyclin Cln1 is controlled by phosphorylation-dependent ubiquitination. Here it is shown that this reaction can be reconstituted in vitro with an SCF E3 ubiquitin ligase complex. Phosphorylated Cln1 was ubiquitinated by SCF (Skp1-Cdc53-F-box protein) complexes containing the F-box protein Grr1, Rbx1, and the E2 Cdc34. Rbx1 promotes association of Cdc34 with Cdc53 and stimulates Cdc34 auto-ubiquitination in the context of Cdc53 or SCF complexes. Rbx1, which is also a component of the von Hippel-Lindau tumor suppressor complex, may define a previously unrecognized class of E3-associated proteins.     

The structure of the eukaryotic ribosome at 3.0 Å resolution

Ribosomes translate genetic information encoded by messenger RNA into proteins. Many aspects of translation and its regulation are specific to eukaryotes, whose ribosomes are much larger and intricate than their bacterial counterparts. We report the crystal structure of the 80S ribosome from the yeast Saccharomyces cerevisiae--including nearly all ribosomal RNA bases and protein side chains as well as an additional protein, Stm1--at a resolution of 3.0 angstroms. This atomic model reveals the architecture of eukaryote-specific elements and their interaction with the universally conserved core, and describes all eukaryote-specific bridges between the two ribosomal subunits. It forms the structural framework for the design and analysis of experiments that explore the eukaryotic translation apparatus and the evolutionary forces that shaped it.     

Microinjection of a conserved peptide sequence of p34cdc2 induces a Ca2+ transient in oocytes

The product of the yeast cell cycle control gene cdc2, and its homologs in higher eukaryotes (p34cdc2), all contain a perfectly conserved sequence of 16 amino acids that has not been found in any other protein sequence. Microinjection of this peptide triggers a specific increase in the concentration of intracellular free Ca2+ that originates from intracellular stores in both starfish and Xenopus oocytes. Thus, p34cdc2 might interact through its conserved peptide domain with some component of the Ca2(+)-regulatory system.     

拟南芥中一个与ABA信号途径相关未知蛋白质的研究初报

利用酵母双杂交系统在拟南芥cDNA文库中筛选出1个与ABI2有相互作用的未知蛋白质（AC）。在酵母系统中的进一步研究表明，AC与ABI1、ABI2有相互作用，其作用依赖于ABI1、ABI2的PP2C活性。在大肠杆菌中表达和纯化了与预测结果一致的AC蛋白质。构建了真核过量表达载体，转化拟南芥后筛选和鉴定出转基因AC植株。转基因植株的生理性状分析表明，AC基因的过量表达降低了植物对ABA敏感性。研究初步证明AC可能是脱落酸信号传导途径中的1个新信号分子。     

饲料酵母对大菱鲆生长及肠道显微结构的影响

试验旨在研究不同比例的饲料酵母替代鱼粉对大菱鲆生长及肠道显微结构的影响。设计5种等氮、等能的试验饲料(B1(对照组)、B2、B3、B4和B5),分别用0、15%、30%、45%、60%的饲料酵母替代饲料中0、17%、34%、51%、68%的鱼粉。经过56 d的饲养,结果表明:B2的增重率(WGR)和特定生长率(SGR)最高(B2的WGR是275.30%,SGR是1.91%;对照组WGR为235.66%,SGR为1.82%),饲料系数(FCR)最低(B2的FCR为1.34,而对照组为1.53)。B2(97.44%)和B4(94.87%)的成活率(SR)均显著高于对照组(84.62%)(P<0.05),而B5(74.36%)显著低于对照组(P<0.05)。鱼体的粗蛋白含量随着饲料酵母替代比例的增加而逐渐降低(P<0.05),粗灰分则逐渐升高(P<0.05)。从切片观察肠道皱襞高度显著下降,当饲料酵母替代68%鱼粉时,前肠肠道组织结构完整性被破坏,部分肠绒毛脱落,中肠和后肠的肠绒毛都变短,且后肠固有层变宽,粘膜层与肌层结缔组织连接疏松。综合考虑大菱鲆的生长指标、形体指标、鱼体成分和显微结构等因素,要获得最佳的养殖效...     

The physical interaction between LdPLCs and Arabidopsis G beta in a yeast two-hybrid system

Phosphoinositide-specific phospholipase C plays pivotal roles in a host of physiologic processes in both animals and plants. Animal PI-PLC is regulated by heterotrimeric G-protein. Plant PI-PLCs are structurally close to the mammalian PI-PLC-ζ isoform, and it is not testified what regulated this isoform enzyme. In this paper, two isoform genes of LdPLC (Pan, 2005) and three subunits of heterotrimeric G-protein in Arabidopsis were amplified and recombinated with plasmids of a yeast two-hybrid system. Using this system, we provided the evidence that LdPLC1 and Gβ subunit could be able to interact with each other. This result indicated that LdPLC1 might be regulated by G-protein.     

An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site

Aminoacyl-transfer RNA (tRNA) synthetases, which catalyze the attachment of the correct amino acid to its corresponding tRNA during translation of the genetic code, are proven antimicrobial drug targets. We show that the broad-spectrum antifungal 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), in development for the treatment of onychomycosis, inhibits yeast cytoplasmic leucyl-tRNA synthetase by formation of a stable tRNA(Leu)-AN2690 adduct in the editing site of the enzyme. Adduct formation is mediated through the boron atom of AN2690 and the 2'- and 3'-oxygen atoms of tRNA's3'-terminal adenosine. The trapping of enzyme-bound tRNA(Leu) in the editing site prevents catalytic turnover, thus inhibiting synthesis of leucyl-tRNA(Leu) and consequentially blocking protein synthesis. This result establishes the editing site as a bona fide target for aminoacyl-tRNA synthetase inhibitors.     

Effects of feeding yeast(Saccharomyces cerevisiae), organic selenium and chromium mixed on growth performance and carcass traits of hair lambs

Yeasts and organic minerals are used in diets to improve health, productive performance and some carcass characteristics of ruminants and non-ruminants. Thirty-two lambs(Pelibuey×Katahdin; BW=(30.55±1.67) kg; n=8) were used in a 56-d feeding experiment to study the effects of different levels of live yeast(Saccharomyces cerevisiae; yeast), selenium(Se) and chromium(Cr) mixed(Se-Cr), and a mixture of yeast-Se-Cr on growth performance and carcass traits. Animals were stratified by body weight(BW) and randomly assigned to one of four treatments: 1) control group(0.0 g kg–1 yeast); 2) yeast(1.50 g kg–1 dry matter intake(DMI) d–1); 3) Se-Cr premix(1.5 mg kg–1 DMI d–1 for each mineral); and 4) yeast-Se-Cr mixture. There were no treatment effects on final BW; whereas lambs fed Se-Cr or yeast-Se-Cr had higher(P0.05) DMI than animals supplemented with only yeast. Average daily gain(ADG), gain:feed ratio, chop area, dorsal fat and carcass yield were similar(P0.05) among treatment groups. In conclusion, supplementation with yeast, Se-Cr mixed or yeast-Se-Cr did not improve ADG, final BW, back fat content and carcass yield of growing of Pelibuey×Katahdin lambs. Supplementation with Se-Cr and yeast-Se-Cr increased DMI, and approximately 250 g ADG animal–1 d–1 was produced with no negative effects on growth and health of the animals.     

雌激素调控的绿色荧光蛋白在酵母细胞中的表达

采用绿色荧光蛋白(GFP)作为报告基因,用酵母细胞构建环境雌激素(EEH)的评价体系,可敏感、快速、方便地筛选出EEH化合物。通过分子生物学技术,将GFP基因插入到pM P 206/ERE启动子CYC 1的下游,用GFP取代L ac Z基因,并转化于酵母细胞。DNA测序发现ERE-CYC 1-GFP框架正确。对转化子进行PCR鉴定,发现有雌激素受体基因(hER cDNA)和GFP基因特异条带,说明hER cDNA和GFP已重组于酵母细胞。荧光显微镜下发现大量的绿色荧光颗粒,表明GFP基因在酵母细胞中成功表达。酵母细胞经不同浓度雌二醇作用后,与GFP表达量有剂量效应关系。与其他酵母评价体系相比,以GFP为报告基因评价EEH不需要破坏细胞壁,也不需要底物,可在96孔板中完成,这为高通量筛选EEH化合物奠定了基础。