scholarly journals Expression of tombusvirus open reading frames 1 and 2 is sufficient for the replication of defective interfering, but not satellite, RNA

2004 ◽  
Vol 85 (10) ◽  
pp. 3115-3122 ◽  
Author(s):  
Luisa Rubino ◽  
Vitantonio Pantaleo ◽  
Beatriz Navarro ◽  
Marcello Russo
Keyword(s):  
Plant Cells ◽  
Open Reading Frames ◽  
Ringspot Virus ◽  
Yeast Cells ◽  
Deletion Mutants ◽  
Replication Proteins ◽  
Satellite Rna ◽  
Replicase Proteins ◽  
Replication Strategy ◽  
Reading Frames

Yeast cells co-expressing the replication proteins p36 and p95 of Carnation Italian ringspot virus (CIRV) support the RNA-dependent replication of several defective interfering (DI) RNAs derived from either the genome of CIRV or the related Cymbidium ringspot virus (CymRSV), but not the replication of a satellite RNA (sat RNA) originally associated with CymRSV. DI, but not sat RNA, was replicated in yeast cells co-expressing both DI and sat RNA. Using transgenic Nicotiana benthamiana plants constitutively expressing CymRSV replicase proteins (p33 and p92), or transiently expressing either these proteins or CIRV p36 and p95, it was shown that expression of replicase proteins alone was also not sufficient for the replication of sat RNA in plant cells. However, it was also shown that replicating CIRV genomic RNA deletion mutants encoding only replicase proteins could sustain replication of sat RNA in plant cells. These results suggest that sat RNA has a replication strategy differing from that of genomic and DI RNAs, for it requires the presence of a cis-replicating genome acting as a trans-replication enhancer.

Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica

1990 ◽  
Vol 10 (9) ◽  
pp. 4795-4806
Author(s):  
J W Xuan ◽  
P Fournier ◽  
N Declerck ◽  
M Chasles ◽  
C Gaillardin
Keyword(s):  
Yarrowia Lipolytica ◽  
Stop Codon ◽  
Open Reading Frames ◽  
Yeast Cells ◽  
Lacz Gene ◽  
Galactosidase Activity ◽  
Phenotypic Effect ◽  
Frame Fusion ◽  
Beta Galactosidase ◽  
Reading Frames

Mutants affected at the LYS5 locus of Yarrowia lipolytica lack detectable dehydrogenase (SDH) activity. The LYS5 gene has previously been cloned, and we present here the sequence of the 2.5-kilobase-pair (kb) DNA fragment complementing the lys5 mutation. Two large antiparallel open reading frames (ORF1 and ORF2) were observed, flanked by potential transcription signals. Both ORFs appear to be transcribed, but several lines of evidence suggest that only ORF2 is translated and encodes SDH. (i) The global amino acid compositions of Saccharomyces cerevisiae SDH and of the putative ORF2 product are similar and that of ORF1 is dissimilar. (ii) An in-frame translational fusion of ORF2 with the Escherichia coli lacZ gene was introduced into yeast cells and resulted in a beta-galactosidase activity regulated similarly to SDH; no beta-galactosidase activity was obtained with an in-frame fusion of ORF1 with lacZ. (iii) The introduction of a stop codon at the beginning of ORF2 prevented SDH expression in yeast cells, whereas no phenotypic effect was observed when ORF1 translation was blocked.

scholarly journals Complete Nucleotide Sequence and Genome Organization of Hibiscus Chlorotic Ringspot Virus, a New Member of the Genus Carmovirus: Evidence for the Presence and Expression of Two Novel Open Reading Frames

2000 ◽  
Vol 74 (7) ◽  
pp. 3149-3155 ◽  
Author(s):  
Mei Huang ◽  
Dora Chin-Yen Koh ◽  
Li-Juan Weng ◽  
Min-Li Chang ◽  
Yun-Kiam Yap ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Genome Organization ◽  
Complete Nucleotide Sequence ◽  
Full Length ◽  
Open Reading Frames ◽  
Ringspot Virus ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
High Amino Acid ◽  
Reading Frames

ABSTRACT The complete nucleotide sequence of hibiscus chlorotic ringspot virus (HCRSV) was determined. The genomic RNA (gRNA) is 3,911 nucleotides long and has the potential to encode seven viral proteins in the order of 28 (p28), 23 (p23), 81 (p81), 8 (p8), 9 (p9), 38 (p38), and 25 (p25) kDa. Excluding two unique open reading frames (ORFs) encoding p23 and p25, the ORFs encode proteins with high amino acid similarity to those of carmoviruses. In addition to gRNA, two 3′-coterminated subgenomic RNA (sgRNA) species were identified. Full-length cDNA clones derived from gRNA and sgRNA were constructed under the control of a T7 promoter. Both capped and uncapped transcripts derived from the full-length genomic cDNA clone were infectious. In vitro translation and mutagenesis assays confirmed that all the predicted ORFs except the ORF encoding p8 are translatable, and the two novel ORFs (those encoding p23 and p25) may be functionally indispensable for the viral infection cycle. Based on virion morphology and genome organization, we propose that HCRSV be classified as a new member of the genus Carmovirus in familyTombusviridae.

scholarly journals Distinct types of short open reading frames are translated in plant cells

2019 ◽  
Vol 29 (9) ◽  
pp. 1464-1477 ◽  
Author(s):  
Igor Fesenko ◽  
Ilya Kirov ◽  
Andrey Kniazev ◽  
Regina Khazigaleeva ◽  
Vassili Lazarev ◽  
...  
Keyword(s):  
Plant Cells ◽  
Open Reading Frames ◽  
Reading Frames

Translational activation of GCN4 mRNA in a cell-free system is triggered by uncharged tRNAs

1990 ◽  
Vol 10 (8) ◽  
pp. 4375-4378
Author(s):  
G Krupitza ◽  
G Thireos
Keyword(s):  
Open Reading Frames ◽  
Yeast Cells ◽  
Free System ◽  
In Vitro System ◽  
A Cell ◽  
Translational Activation ◽  
Reading Frames ◽  
Small Open Reading Frames

Translation of GCN4 mRNA is activated when yeast cells are grown under conditions of amino acid limitation. In this study, we established the conditions through which translation of the GCN4 mRNA could be activated in a homologous in vitro system. This activation paralleled the in vivo situation: it required the small open reading frames located in the 5' untranslated region of the GCN4 mRNA, and it was coupled with reduced rates of 43S preinitiation complex formation. Translational derepression in vitro was triggered by uncharged tRNA molecules, demonstrating that deacylated tRNAs are more proximal signals for translational activation of the GCN4 mRNA.

scholarly journals Translational activation of GCN4 mRNA in a cell-free system is triggered by uncharged tRNAs.

1990 ◽  
Vol 10 (8) ◽  
pp. 4375-4378 ◽  
Author(s):  
G Krupitza ◽  
G Thireos
Keyword(s):  
Open Reading Frames ◽  
Yeast Cells ◽  
Free System ◽  
In Vitro System ◽  
A Cell ◽  
Translational Activation ◽  
Reading Frames ◽  
Small Open Reading Frames

Translation of GCN4 mRNA is activated when yeast cells are grown under conditions of amino acid limitation. In this study, we established the conditions through which translation of the GCN4 mRNA could be activated in a homologous in vitro system. This activation paralleled the in vivo situation: it required the small open reading frames located in the 5' untranslated region of the GCN4 mRNA, and it was coupled with reduced rates of 43S preinitiation complex formation. Translational derepression in vitro was triggered by uncharged tRNA molecules, demonstrating that deacylated tRNAs are more proximal signals for translational activation of the GCN4 mRNA.

Prediction and functional analysis of the replication origin of the linear plasmid pSCL2 inStreptomyces clavuligerus

2006 ◽  
Vol 52 (4) ◽  
pp. 293-300 ◽  
Author(s):  
Wei Wu ◽  
Shannon K. D Leblanc ◽  
James Piktel ◽  
Susan E Jensen ◽  
Kenneth L Roy
Keyword(s):  
Amino Acids ◽  
Functional Analysis ◽  
Replication Origin ◽  
Streptomyces Clavuligerus ◽  
Replication Initiation ◽  
Open Reading Frames ◽  
Linear Plasmid ◽  
Replication Proteins ◽  
Bacterial Plasmid ◽  
Reading Frames

pSCL2 (120 kb), one of the linear plasmids found in Streptomyces clavuligerus NRRL3585, was isolated and partially sequenced. Computational analysis of the central region of pSCL2 revealed the presence of two open reading frames that appear to encode proteins highly homologous to RepL1 and RepL2, replication proteins from pSLA2-L, the large linear plasmid in Streptomyces rochei. The S. clavuligerus open reading frames were designated repC1 and repC2, encoding the proteins RepC1 (150 amino acids) and RepC2 (102 amino acids), respectively. The RepC and RepL proteins have identical translation features and very similar predicted secondary and tertiary structures. Functional analysis confirmed that RepC1 is essential for replication initiation of pSCL2, whereas RepC2 is dispensable but may play a role in copy number control. The RepC and RepL proteins do not show similarity to any other bacterial plasmid replication proteins. Three regions of DNA sequence, Box 1 (1050–850 bp), Box 2 (723–606 bp), and Box 3 (224–168 bp), located upstream of repC1, were also shown to be essential or very important for replication of pSCL2.Key words: pSCL2, Streptomyces clavuligerus, replication origin.

scholarly journals Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica.

1990 ◽  
Vol 10 (9) ◽  
pp. 4795-4806 ◽  
Author(s):  
J W Xuan ◽  
P Fournier ◽  
N Declerck ◽  
M Chasles ◽  
C Gaillardin
Keyword(s):  
Yarrowia Lipolytica ◽  
Stop Codon ◽  
Open Reading Frames ◽  
Yeast Cells ◽  
Lacz Gene ◽  
Galactosidase Activity ◽  
Phenotypic Effect ◽  
Frame Fusion ◽  
Beta Galactosidase ◽  
Reading Frames

Mutants affected at the LYS5 locus of Yarrowia lipolytica lack detectable dehydrogenase (SDH) activity. The LYS5 gene has previously been cloned, and we present here the sequence of the 2.5-kilobase-pair (kb) DNA fragment complementing the lys5 mutation. Two large antiparallel open reading frames (ORF1 and ORF2) were observed, flanked by potential transcription signals. Both ORFs appear to be transcribed, but several lines of evidence suggest that only ORF2 is translated and encodes SDH. (i) The global amino acid compositions of Saccharomyces cerevisiae SDH and of the putative ORF2 product are similar and that of ORF1 is dissimilar. (ii) An in-frame translational fusion of ORF2 with the Escherichia coli lacZ gene was introduced into yeast cells and resulted in a beta-galactosidase activity regulated similarly to SDH; no beta-galactosidase activity was obtained with an in-frame fusion of ORF1 with lacZ. (iii) The introduction of a stop codon at the beginning of ORF2 prevented SDH expression in yeast cells, whereas no phenotypic effect was observed when ORF1 translation was blocked.

scholarly journals Glycosylation Defects and Virulence Phenotypes ofLeishmania mexicana Phosphomannomutase and Dolicholphosphate-Mannose Synthase Gene Deletion Mutants

2001 ◽  
Vol 21 (23) ◽  
pp. 8168-8183 ◽  
Author(s):  
Attila Garami ◽  
Angela Mehlert ◽  
Thomas Ilg
Keyword(s):  
Gene Deletion ◽  
Open Reading Frames ◽  
Mammalian Host ◽  
Deletion Mutants ◽  
Leishmania Mexicana ◽  
Gpi Anchor ◽  
Gene Encoding ◽  
Mouse Macrophages ◽  
Ample Supply ◽  
Reading Frames

ABSTRACT Leishmania parasites synthesize an abundance of mannose (Man)-containing glycoconjugates thought to be essential for virulence to the mammalian host and for viability. These glycoconjugates include lipophosphoglycan (LPG), proteophosphoglycans (PPGs), glycosylphosphatidylinositol (GPI)-anchored proteins, glycoinositolphospholipids (GIPLs), and N-glycans. A prerequisite for their biosynthesis is an ample supply of the Man donors GDP-Man and dolicholphosphate-Man. We have cloned from Leishmania mexicana the gene encoding the enzyme phosphomannomutase (PMM) and the previously described dolicholphosphate-Man synthase gene (DPMS) that are involved in Man activation. Surprisingly, gene deletion experiments resulted in viable parasite lines lacking the respective open reading frames (ΔPMM and ΔDPMS), a result against expectation and in contrast to the lethal phenotype observed in gene deletion experiments with fungi. L. mexicanaΔDPMS exhibits a selective defect in LPG, protein GPI anchor, and GIPL biosynthesis, but despite the absence of these structures, which have been implicated in parasite virulence and viability, the mutant remains infectious to macrophages and mice. By contrast, L. mexicana ΔPMM are largely devoid of all known Man-containing glycoconjugates and are unable to establish an infection in mouse macrophages or the living animal. Our results define Man activation leading to GDP-Man as a virulence pathway in Leishmania.

Efficient long fragment editing technique enables large-scale and scarless bacterial genome engineering

2020 ◽  
Author(s):  
Chaoyong Huang ◽  
Liwei Guo ◽  
Jingge Wang ◽  
Ning Wang ◽  
Yi-Xin Huo
Keyword(s):  
Metabolic Engineering ◽  
Large Scale ◽  
Genome Engineering ◽  
Bacterial Genome ◽  
Open Reading Frames ◽  
Deletion Mutants ◽  
Dna Fragments ◽  
E Coli ◽  
Stable Plasmid ◽  
Reading Frames

ABSTRACTBacteria are versatile living systems that enhance our understanding of nature and enable biosynthesis of valuable chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable strains. However, the existing genome editing methods cannot meet the needs of engineers. We herein report an efficient long fragment editing method for large-scale and scarless genome engineering in Escherichia coli. The method enabled us to insert DNA fragments up to 12 kb into the genome and to delete DNA fragments up to 186.7 kb from the genome, with positive rates over 95%. We applied this method for E. coli genome simplification, resulting in 12 individual deletion mutants and four cumulative deletion mutants. The simplest genome lost a total of 370.6 kb of DNA sequence containing 364 open reading frames. Additionally, we applied this technique to metabolic engineering and obtained a genetically stable plasmid-independent isobutanol production strain that produced 1.3 g/L isobutanol via shake-flask fermentation. These results suggest that the method is a powerful genome engineering tool, highlighting its potential to be applied in synthetic biology and metabolic engineering.

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