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11.
Genetic tailoring of N-linked oligosaccharides: the role of glucose residues in glycoprotein processing of Saccharomyces cerevisiae in vivo 总被引:1,自引:0,他引:1
In higher eukaryotes a quality control system monitoring the folding state
of glycoproteins is located in the ER and is composed of the proteins
calnexin, calreticulin, glucosidase II, and UDP-glucose: glycoprotein
glucosyltransferase. It is believed that the innermost glucose residue of
the N- linked oligosaccharide of a glycoprotein serves as a tag in this
control system and therefore performs an important function in the protein
folding pathway. To address this function, we constructed Saccharomyces
cerevisiae strains which contain nonglucosylated (G0), monoglucosylated
(G1), or diglucosylated (G2) glycoproteins in the ER and used these strains
to study the role of glucose residues in the ER processing of
glycoproteins. These alterations of the oligosaccharide structure did not
result in a growth phenotype, but the induction of the unfolded protein
response upon treatment with DTT was much higher in G0 and G2 strains as
compared to wild-type and G1 strains. Our results provide in vivo evidence
that the G1 oligosaccharide is an active oligosaccharide structure in the
ER glycoprotein processing pathway of S.cerevisiae. Furthermore, by
analyzing N- linked oligosaccharides of the constructed strains we can
directly show that no general glycoprotein glucosyltransferase exists in S.
cerevisiae.
相似文献
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13.
Branchini BR Ablamsky DM Murtiashaw MH Uzasci L Fraga H Southworth TL 《Analytical biochemistry》2007,361(2):253-262
Light emission from the North American firefly Photinus pyralis, which emits yellow-green (557-nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. We present studies on the production of a set of thermostable red- and green-emitting luciferase mutants with bioluminescent properties suitable for dual-color reporter assays, biosensor measurements with internal controls, and imaging techniques. Starting with the luciferase variant Ser284Thr, we introduced the mutations Thr214Ala, Ala215Leu, Ile232Ala, Phe295Leu, and Glu354Lys to produce a new red-emitting enzyme with a bioluminescence maximum of 610 nm, narrow emission bandwidth, favorable kinetic properties, and excellent thermostability at 37 degrees C. By adding the same five changes to luciferase mutant Val241Ile/Gly246Ala/Phe250Ser, we produced a protein with an emission maximum of 546 nm, providing a set of thermostable enzymes whose bioluminescence maxima were separated by 64 nm. Model studies established that the luciferases could be detected at the attomole level and six orders of magnitude higher. In microplate luminometer format, mixtures containing 1.0 fmol total luciferase were quantified from measurements of simultaneously emitted red and green light. The results presented here provide evidence that it is feasible to monitor two distinct activities at 37 degrees C with these novel thermostable proteins. 相似文献
14.
人肺腺癌细胞分化相关基因cDNAs的克隆 总被引:2,自引:0,他引:2
在用10-5 mol/L全反式维甲酸(RA)诱导人肺腺癌细胞系GLC-82分化的基础上,以M13噬菌粒pSPORT1为载体,应用定向克隆技术,分别构建了未经RA诱导和RA诱导1d及4d细胞的3个cDNA文库.以含重组子的诱导文库单链DNA为靶标(Target)同未诱导文库的cDNA驱除子(Driver)进行消减杂交,富集RA特异性单链DNA,将富集的单链DNA回复为双链后转化感受态菌,建立细胞诱导分化过程中活化表达基因的cDNA消减文库,得到124个cDNA消减克隆.经同源性分析和与文库总cDNA作Southern印迹杂交,进而与RA诱导前后细胞的RNA作Northern印迹杂交,筛选出2个(RA5,RA28)诱导后呈早期瞬时表达和1个(RA42)呈早期并持续表达的cDNA克隆,cDNA全长分别为1.8,1.5和0.7kb.序列测定及初步功能分析结果表明,RA5,RA28和RA42这3个首次报道的序列,可能是人肺腺癌细胞分化相关基因的cDNA克隆. 相似文献
15.
JOSEP PIÑOL XAVIER ESPADALER NÚRIA CAÑELLAS JORDI MARTÍNEZ‐VILALTA JOSÉ A. BARRIENTOS DANIEL SOL 《Ecological Entomology》2010,35(3):367-376
1. Predation‐exclusion experiments have highlighted that top‐down control is pervasive in terrestrial communities, but most of these experiments are simplistic in that they only excluded a single group of predators and the effect of removal was evaluated on a few species from the community. The main goal of our study was to experimentally establish the relative effects of ants and birds on the same arthropod assemblage of canopy trees. 2. We conducted 1‐year long manipulative experiments in an organic citrus grove intended to quantify the independent effects of bird and ant predators on the abundance of arthropods. Birds were excluded with plastic nets whereas ants were excluded with sticky barriers on the trunks. The sticky barrier also excluded other ground dwelling insects, like the European earwig Forficula auricularia L. 3. Both the exclusion of ants and birds affected the arthropod community of the citrus canopies, but the exclusion of ants was far more important than the exclusion of birds. Indeed, almost all groups of arthropods had higher abundance in ant‐excluded than in control trees, whereas only dermapterans were more abundant in bird‐excluded than in control trees. A more detailed analysis conducted on spiders also showed that the effect of ant exclusion was limited to a few families rather than being widespread over the entire diverse spectrum of spiders. 4. Our results suggest that the relative importance of vertebrate and invertebrate predators in regulating arthropod populations largely depends on the nature of the predator–prey system. 相似文献
16.
Barbara Piotrkowski Osvaldo R. Koch Elena M. V. De Cavanagh César G. Fraga 《Free radical research》2013,47(4):390-399
Renal and cardiac benefits of renin-angiotensin system inhibition exceed blood pressure (BP) reduction and seem to involve mitochondrial function. It has been shown that RAS inhibition prevented mitochondrial dysfunction in spontaneously hypertensive rats (SHR) kidneys. Here, it is investigated whether a non-antihypertensive enalapril dose protects cardiac tissue and mitochondria function. Three-month-old SHR received water containing enalapril (10 mg/kg/day, SHR+Enal) or no additions (SHR-C) for 5 months. Wistar-Kyoto rats (WKY) were normotensive controls. At month 5, BP was similar in SHR+Enal and SHR-C. In SHR+Enal and WKY, heart weight and myocardial fibrosis were lower than in SHR-C. Matrix metalloprotease-2 activity was lower in SHR+Enal with respect to SHR-C and WKY. In SHR+Enal and WKY, NADH/cytochrome c oxidoreductase activity, eNOS protein and activity and mtNOS activity were higher and Mn-SOD activity was lower than in SHR-C. In summary, enalapril at a non-antihypertensive dose prevented cardiac hypertrophy and modifies parameters of cardiac mitochondrial dysfunction in SHR. 相似文献
17.
Tania González Felipe Eng Reinaldo Fraga Jennifer Fonseca Isis Amores 《Journal of applied genetics》2013,54(4):495-499
The glyceraldehyde-3-phosphate dehydrogenase promoter of the food yeast Candida utilis strain NRRL Y-660 was cloned to create a novel integrative vector for Agrobacterium tumefaciens-mediated transformation. The new binary vector harbors β-glucuronidase activity as reporter and kanamicin/geneticin resistance as selection marker. Recombinant clones of A. tumefaciens show kanamycin resistance and high β-glucuronidase activity under the control of the C. utilis promoter. This finding can be explained by the presence of a prokaryotic core in the yeast promoter, predicted by in silico analysis of the sequence. This is the first report about functionality of a yeast promoter in A. tumefaciens. 相似文献
18.
Henar Hernando Claire Shannon-Lowe Abul B Islam Fatima Al-Shahrour Javier Rodríguez-Ubreva Virginia C Rodríguez-Cortez Biola M Javierre Cristina Mangas Agustín F Fernández Maribel Parra Henri-Jacques Delecluse Manel Esteller Eduardo López-Granados Mario F Fraga Nuria López-Bigas Esteban Ballestar 《Genome biology》2013,14(1):R3
19.
20.
Leonardo P. Farias Greice Krautz-Peterson Cibele A. Tararam Bogar O. Araujo-Montoya Tatiana R. Fraga Henrique K. Rofatto Floriano P. Silva-Jr Lourdes Isaac Akram A. Da'dara R. Alan Wilson Charles B. Shoemaker Luciana C. C. Leite 《PLoS neglected tropical diseases》2013,7(10)