scholarly journals A cell-free yellow lupin extract containing activities of pseudouridine 35 and 55 synthases.

1998 ◽  
Vol 45 (3) ◽  
pp. 745-754 ◽  
Author(s):  
J Pieńkowska ◽  
J Wrzesiński ◽  
Z Szweykowska-Kulińska

Plant cytoplasmic tyrosine tRNA was pseudouridylated at three different positions: 35, 39 and 55. These pseudouridines were introduced by three different enzymes--pseudouridine synthases. Variants of the Arabidopsis thaliana pre-tRNA(Tyr) were constructed that allow to monitor specifically pseudouridylation at different nucleotide positions. Using such RNAs to assay pseudouridine synthesis we have prepared an extract from Lupinus luteus cv. Ventus seeds containing activities of at least psi35 and psi55 synthases. This is the first report describing the preparation of the lupin seed extract that specifically modifies plant pre-tRNA(Tyr) transcribed by T7 RNA polymerase. U35 is converted to psi35 only in an intron-dependent manner, while pseudouridylation of U55 is insensitive to the presence or absence of an intron.

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Wan-Wen Ting ◽  
Shih-I Tan ◽  
I-Son Ng

Abstract Background Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter is a powerful genetic element to mediate protein expression in different cells. Among all, Escherichia coli possess advantages of fast growth rate, easy for culture and comprehensive elements for genetic engineering. As E. coli W3110 owns the benefits of more heat shock proteins and higher tolerance to toxic chemicals, further execution of T7-based system in W3110 as cell factory is a conceivable strategy. Results Three novel W3110 strains, i.e., W3110:IL5, W3110::L5 and W3110::pI, were accomplished by chromosome-equipped T7RNAP. At first, the LacZ and T7RNAP with isopropyl-β-D-thiogalactopyranoside (IPTG) induction showed higher expression levels in W3110 derivatives than that in BL21(DE3). The plasmids with and without lacI/lacO repression were used to investigate the protein expression of super-fold green fluorescence protein (sfGFP), carbonic anhydrase (CA) for carbon dioxide uptake and lysine decarboxylase (CadA) to produce a toxic chemical cadaverine (DAP). All the proteins showed better expression in W3110::L5 and W3110::pI, respectively. As a result, the highest cadaverine production of 36.9 g/L, lysine consumption of 43.8 g/L and up to 100% yield were obtained in W3110::pI(−) with plasmid pSU-T7-CadA constitutively. Conclusion Effect of IPTG and lacI/lacO regulator has been investigated in three chromosome-based T7RNAP E. coli strains. The newly engineered W3110 strains possessed similar protein expression compared to commercial BL21(DE3). Furthermore, W3110::pI displays higher production of sfGFP, CA and CadA, due to it having the highest sensitivity to IPTG, thus it represents the greatest potential as a cell factory.


Plant Disease ◽  
2014 ◽  
Vol 98 (8) ◽  
pp. 1158-1158 ◽  
Author(s):  
K. S. Han ◽  
B. S. Kim ◽  
I. Y. Choi ◽  
J. H. Park ◽  
H. D. Shin

Yellow lupin (Lupinus luteus L.) is native to the Mediterranean region of southern Europe. In Korea, yellow lupins are cultivated for ornamental purposes. In May 2013, hundreds of yellow lupins that were grown in pots for 7 weeks in polyethylene-film-covered greenhouses were observed severely damaged by a previously unknown disease with about 30% disease incidence in a flower farm in Yongin City, Korea. Voucher specimens were deposited in the Korea University Herbarium (KUS). Early symptoms on petioles and stems appeared as small, slightly sunken, water-soaked, and circular spots. Lesions increased in size (4 to 12 μm in diameter), became more depressed, with a darkened central portion. As the disease progressed, affected areas sometimes girdled the stem and killed the shoot. Leaves were partly blighted, but less damaged. The darkened areas contained blackish acervuli from which masses of pale salmon-colored conidia were released in moist weather. Acervuli were circular to ellipsoid, 80 to 400 μm in diameter. Acervular setae were not observed. Conidia (n = 30) were long obclavate to oblong-elliptical, aguttulate, hyaline, and 10 to 18 × 3.6 to 5.2 μm with a length/width ratio of 2.6 to 3.6. Appressoria were single or occasionally in small dense clusters, medium brown, elliptical to round in outline with a smooth to lobate margin, and 8 to 14 × 6 to 9 μm. These characters were consistent with the description of Colletotrichum lupini (Bondar) Damm, P.F. Cannon & Crous (1,3). An isolate was deposited in the Korean Agricultural Culture Collection (Accession No. KACC47254). Fungal DNA was extracted with DNeasy Plant Mini DNA Extraction Kits (Qiagen Inc., Valencia, CA). The complete internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced. The resulting 545-bp sequence was deposited in GenBank (Accession No. KJ447119). The sequence showed 100% identity with sequences of C. lupini (e.g., GenBank AJ301968, JN943480, JQ948162, and KF207599). To confirm pathogenicity, inoculum was prepared by harvesting conidia with sterile distilled water from 3-week-old cultures on potato dextrose agar. A conidial suspension (2 × 105 conidia/ml) was sprayed until runoff onto the aerial parts of five healthy plants. Control plants were sprayed with sterile water. The plants were covered with plastic bags to maintain a relative humidity of 100% for 48 h and then transferred to a greenhouse. Typical symptoms of necrotic spots appeared on the inoculated leaves 6 days after inoculation, and were identical to the ones observed in the field. C. lupini was re-isolated from symptomatic leaf tissues. No symptoms were observed on control plants. The pathogenicity test was repeated twice. Anthracnose associated with C. lupini on lupins has been known from Europe (Germany, Ukraine, Austria, and Netherlands), North America (Canada and the United States), South America (Bolivia and Brazil), and Oceania (Australia and New Zealand) (2,4). To our knowledge, this is the first report of C. lupini on yellow lupins in Asia as well as in Korea. The presence of C. lupini on lupins in Asia can be considered as a potentially new and serious threat to this ornamental plant. References: (1) U. Damm et al. Stud. Mycol. 73:37, 2012. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab., Online publication, ARS, USDA, Retrieved February 17, 2014. (3) H. I. Nirenberg et al. Mycologia 94:307, 2002. (4) E. Rosskopf et al. Plant Dis. 98:161, 2014.


2006 ◽  
Vol 73 ◽  
pp. 85-96 ◽  
Author(s):  
Richard J. Reece ◽  
Laila Beynon ◽  
Stacey Holden ◽  
Amanda D. Hughes ◽  
Karine Rébora ◽  
...  

The recognition of changes in environmental conditions, and the ability to adapt to these changes, is essential for the viability of cells. There are numerous well characterized systems by which the presence or absence of an individual metabolite may be recognized by a cell. However, the recognition of a metabolite is just one step in a process that often results in changes in the expression of whole sets of genes required to respond to that metabolite. In higher eukaryotes, the signalling pathway between metabolite recognition and transcriptional control can be complex. Recent evidence from the relatively simple eukaryote yeast suggests that complex signalling pathways may be circumvented through the direct interaction between individual metabolites and regulators of RNA polymerase II-mediated transcription. Biochemical and structural analyses are beginning to unravel these elegant genetic control elements.


2020 ◽  
Vol 64 (2) ◽  
pp. 383-396
Author(s):  
Lara K. Krüger ◽  
Phong T. Tran

Abstract The mitotic spindle robustly scales with cell size in a plethora of different organisms. During development and throughout evolution, the spindle adjusts to cell size in metazoans and yeast in order to ensure faithful chromosome separation. Spindle adjustment to cell size occurs by the scaling of spindle length, spindle shape and the velocity of spindle assembly and elongation. Different mechanisms, depending on spindle structure and organism, account for these scaling relationships. The limited availability of critical spindle components, protein gradients, sequestration of spindle components, or post-translational modification and differential expression levels have been implicated in the regulation of spindle length and the spindle assembly/elongation velocity in a cell size-dependent manner. In this review, we will discuss the phenomenon and mechanisms of spindle length, spindle shape and spindle elongation velocity scaling with cell size.


1987 ◽  
Vol 262 (9) ◽  
pp. 3940-3943
Author(s):  
M. Yamagishi ◽  
J.R. Cole ◽  
M. Nomura ◽  
F.W. Studier ◽  
J.J. Dunn

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