scholarly journals Investigating the coding capacity of rotaviruses using a newly developed reverse genetics system

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Ola Lee ◽  
Hui Min Lee ◽  
Rute Maria Pinto ◽  
Paul Digard ◽  
Eleanor Gaunt
Keyword(s):  
Reverse Genetics ◽  
Site Directed Mutagenesis ◽  
Dairy Calves ◽  
Start Codon ◽  
High Morbidity ◽  
Severe Diarrhoea ◽  
Bovine Rotavirus ◽  
Translation Start ◽  
Kozak Context ◽  
Coding Capacity

Bovine rotavirus (RV) infection causes severe diarrhoea in young dairy calves and has a significant economic impact on livestock production as a result of high morbidity and mortality caused. Development of technologies to engineer infectious RV using an entirely plasmid-based reverse genetics (RG) system has proven challenging. A breakthrough was made when Kanaiand co-authors (PNAS, 2017)developed a plasmid-only-based RG system for the simian RV strain SA11.We are currently developing an analogous RG system for the bovine RF RV strain. Having parallel systems for different RV strains will help to validate phenotypic changes induced by site-directed mutagenesis (SDM) within the RV genome. The coding capacity of the 11-segmented dsRNA RV genome has been largely unexplored. Using bioinformatic analyses, we have identified four segments with up to five putative alternative initiation codons which are in moderate or strong Kozak context. Furthermore, some occur in segments for which the canonical start codon occurs within 15 nucleotides of the start of the segment, further suggesting the possibility of alternative translation start sites to generate coding diversity. We are now applying our RG systems to investigate RV coding capacity using TnT transcription assays, radiolabelling and SDM.

scholarly journals Translationskopplung via Termination-Reinitiation in Archaea und Bacteria

2021 ◽  
Author(s):  
◽  
Madeleine Huber
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Stop Codon ◽  
Haloferax Volcanii ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Directed Mutagenesis ◽  
Ribosome Display ◽  
E Coli

Operons wurden zuerst im Jahre 1961 beschrieben. Bis heute ist bekannt, dass die prokaryotischen Domänen Bacteria und Archaea Gene sowohl in monocistronischen als auch in bi- oder polycistronischen Transkripten exprimieren können. Häufig überlappen Gene sogar in ihren Sequenzen. Diese überlappenden Genpaare stehen nicht in Korrelation mit der Kompaktheit ihres Genoms. Das führt zu der Annahme, dass eine Art der Regulation vorliegt, welche weitere Proteine oder Gene nicht benötigt. Diese könnte eine gekoppelte Translation sein. Das bedeutet die Translation des stromabwärts-liegenden Gens ist abhängig von der Translation eines stromaufwärts-liegenden Gens. Diese Abhängigkeit kann zum Beispiel durch lang reichende Sekundärstrukturen entstehen, bei welchen Ribosomenbindestellen (RBS) des stromabwärts-liegenden Gens blockiert sind. Die de novo-Initiation am stromabwärts-liegenden Gen kann nur stattfinden, wenn das erste Gen translatiert wird und dabei die Sekundärstruktur an der RBS aufgeschmolzen wird. Für Genpaare in E. coli ist dieser Mechanismus gut untersucht. Ein anderes Beispiel für die Translationskopplung ist die Termination-Reinitiation, bei welcher ein Ribosom das erste Gen translatiert bis zum Stop-Codon, dort terminiert und direkt am stromabwärts-liegenden Start-Codon reinitiiert. Der Mechanismus via Termination-Reinitiation ist bis jetzt nur für eukaryontische Viren beschrieben worden. Im Gegensatz zu einer Kopplung über Sekundärstrukturen kommt es bei der Termination-Reinitiation am stromabwärts-liegenden Gen nicht zu einer de novo-Initiation sondern eine Reinitiation des Ribosoms findet statt. Diese Arbeit analysiert jene Art der Translationskopplung an Genen polycistronischer mRNAs in jeweils einem Modellorganismus als Vertreter der Archaea (Haloferax volcanii) und Bacteria (Escherichia coli). Hierfür wurden Reportergenvektoren erstellt, welche die überlappenden Genpaare an Reportergene fusionierten. Für diese Reportergene ist es möglich die Transkriptmenge zu quantifizieren sowie für die exprimierten Proteine Enzymassays durchgeführt werden können. Aus beiden Werten können Translationseffizienzen berechnet werden indem jeweils die Enzymaktivität pro Transkriptmenge ermittelt wird. Durch ein prämatures Stop-Codon in diesen Konstrukten ist es möglich zu unterscheiden ob es für die Translation des zweiten Gens essentiell ist, dass das Ribosom den Überlapp erreicht. Hiermit konnte für neun Genpaare in H. volcanii und vier Genpaare in E. coli gezeigt werden, dass eine Art der Kopplung stattfindet bei der es sich um eine Termination-Reinitiation handelt. Des Weiteren wurde analysiert, welche Auswirkungen intragene Shine-Dalgarno Sequenzen bei dem Event der Translationskopplung besitzen. Durch die Mutation solcher Motive und dem Vergleich der Translationseffizienzen der Konstrukte, mit und ohne einer SD Sequenz, wird für alle analysierten Genpaare beider Modellorganismen gezeigt, dass die SD Sequenz einen Einfluss auf diese Art der Kopplung hat. Zwischen den Genpaaren ist dieser Einfluss jedoch stark variabel. Weiterhin wurde der maximale Abstand zwischen zwei bicistronischen Genen untersucht, für welchen Translationskopplung via Termination-Reinitiation noch stattfinden kann. Hierfür wird durch site-directed mutagenesis jeweils ein prämatures Stop-Codon im stromaufwärts-liegenden Gen eingebracht, welches den intergenen Abstand zwischen den Genen in den jeweiligen Konstrukten vergrößert. Der Vergleich aller Konstrukte eines Genpaars zeigt in beiden Modellorganismen, dass die Termination-Reinitiation vom intergenen Abstand abhängig ist und die Translationseffizienz des stromabwärts-liegenden Reporters bereits ab 15 Nukleotiden Abstand abnimmt. Eine weitere Fragestellung dieser Arbeit war es, den genauen Mechanismus der Termination-Reinitiation zu analysieren. Für Ribosomen gibt es an der mRNA nach der Termination der Translation zwei Möglichkeiten: Entweder als 70S Ribosom bestehen zu bleiben und ein weiteres Start-Codon auf der mRNA zu suchen oder in seine beiden Untereinheiten zu dissoziieren, während die 50S Untereinheit die mRNA verlässt und die 30S Untereinheit über Wechselwirkungen an der mRNA verbleiben kann. Um diesen Mechanismus auf molekularer Ebene zu untersuchen, wird ein Versuchsablauf vorgestellt. Dieser ermöglicht das Event bei der Termination-Reinitiation in vitro zu analysieren. Eine Unterscheidung von 30S oder 70S Ribosomen bei der Reinitiation der Translation des stromabwärts-liegenden Gens wird ermöglicht. Die Idee dabei basiert auf einem ribosome display, bei welchem Translationskomplexe am Ende der Translation nicht in ihre Bestandteile zerfallen können, da die eingesetzte mRNA kein Stop-Codon enthält Der genaue Versuchsablauf, die benötigten Bestandteile sowie proof-of-principal Versuche sind in der Arbeit dargestellt und mögliche Optimierungen werden diskutiert.

The metabolite repair enzyme Nit1 is a dual-targeted amidase that disposes of damaged glutathione in Arabidopsis

2019 ◽  
Vol 476 (4) ◽  
pp. 683-697 ◽  
Author(s):  
Thomas D. Niehaus ◽  
Jenelle A. Patterson ◽  
Danny C. Alexander ◽  
Jakob S. Folz ◽  
Michal Pyc ◽  
...  
Keyword(s):  
In Vitro Transcription ◽  
Start Codon ◽  
Repair System ◽  
Leaf Extracts ◽  
Physiological Processes ◽  
Translation Start ◽  
Targeting Peptide ◽  
Cellular Compartments ◽  
Metabolite Repair

Abstract The tripeptide glutathione (GSH) is implicated in various crucial physiological processes including redox buffering and protection against heavy metal toxicity. GSH is abundant in plants, with reported intracellular concentrations typically in the 1–10 mM range. Various aminotransferases can inadvertently transaminate the amino group of the γ-glutamyl moiety of GSH to produce deaminated glutathione (dGSH), a metabolite damage product. It was recently reported that an amidase known as Nit1 participates in dGSH breakdown in mammals and yeast. Plants have a hitherto uncharacterized homolog of the Nit1 amidase. We show that recombinant Arabidopsis Nit1 (At4g08790) has high and specific amidase activity towards dGSH. Ablating the Arabidopsis Nit1 gene causes a massive accumulation of dGSH and other marked changes to the metabolome. All plant Nit1 sequences examined had predicted plastidial targeting peptides with a potential second start codon whose use would eliminate the targeting peptide. In vitro transcription/translation assays show that both potential translation start codons in Arabidopsis Nit1 were used and confocal microscopy of Nit1–GFP fusions in plant cells confirmed both cytoplasmic and plastidial localization. Furthermore, we show that Arabidopsis enzymes present in leaf extracts convert GSH to dGSH at a rate of 2.8 pmol min−1 mg−1 in the presence of glyoxalate as an amino acceptor. Our data demonstrate that plants have a dGSH repair system that is directed to at least two cellular compartments via the use of alternative translation start sites.

scholarly journals Hepatocyte nuclear factor-4α regulates expression of the serotonin transporter in intestinal epithelial cells

2020 ◽  
Vol 318 (6) ◽  
pp. C1294-C1304
Author(s):  
Nathaniel W. Holton ◽  
Megha Singhal ◽  
Anoop Kumar ◽  
Alexander L. Ticho ◽  
Christopher R. Manzella ◽  
...  
Keyword(s):  
Serotonin Transporter ◽  
Site Directed Mutagenesis ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Intestinal Epithelial ◽  
Start Site ◽  
Cellular Functions ◽  
Mrna Variant ◽  
Translation Start ◽  
Bowel Diseases

The serotonin transporter (SERT) functions to regulate the availability of serotonin (5-HT) in the brain and intestine. An intestine-specific mRNA variant arising from a unique transcription start site and alternative promoter in the SERT gene has been identified (iSERT; spanning exon 1C). A decrease in SERT is implicated in several gut disorders, including inflammatory bowel diseases (IBD). However, little is known about mechanisms regulating the iSERT variant, and a clearer understanding is warranted for targeting SERT for the treatment of gut disorders. The current studies examined the expression of iSERT across different human intestinal regions and investigated its regulation by HNF4α (hepatic nuclear factor-4α), a transcription factor important for diverse cellular functions. iSERT mRNA abundance was highest in the human ileum and Caco-2 cell line. iSERT mRNA expression was downregulated by loss of HNF4α (but not HNF1α, HNF1β, or FOXA1) in Caco-2 cells. Overexpression of HNF4α increased iSERT mRNA concomitant with an increase in SERT protein. Progressive promoter deletion and site-directed mutagenesis revealed that the HNF4α response element spans nucleotides −1,163 to −1150 relative to the translation start site. SERT mRNA levels in the intestine were drastically reduced in the intestine-specific HNF4α-knockout mice relative to HNF4αFL/FL mice. Both HNF4α and SERT mRNA levels were also downregulated in mouse model of ileitis (SAMP) compared with AKR control mice. These results establish the transcriptional regulation of iSERT at the gut-specific internal promoter (hSERTp2) and have identified HNF4α as a critical modulator of basal SERT expression in the intestine.

scholarly journals eIF2α interactions with mRNA control accurate start codon selection by the translation preinitiation complex

2020 ◽  
Vol 48 (18) ◽  
pp. 10280-10296
Author(s):  
Anil Thakur ◽  
Swati Gaikwad ◽  
Anil K Vijjamarri ◽  
Alan G Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Ternary Complex ◽  
Start Codon ◽  
Preinitiation Complex ◽  
Closed Conformation ◽  
Open Conformation ◽  
Kozak Context ◽  
Codon Selection

Abstract In translation initiation, AUG recognition triggers rearrangement of the 48S preinitiation complex (PIC) from an open conformation to a closed state with more tightly-bound Met-tRNAi. Cryo-EM structures have revealed interactions unique to the closed complex between arginines R55/R57 of eIF2α with mRNA, including the −3 nucleotide of the ‘Kozak’ context. We found that R55/R57 substitutions reduced recognition of a UUG start codon at HIS4 in Sui− cells (Ssu− phenotype); and in vitro, R55G-R57E accelerated dissociation of the eIF2·GTP·Met-tRNAi ternary complex (TC) from reconstituted PICs with a UUG start codon, indicating destabilization of the closed complex. R55/R57 substitutions also decreased usage of poor-context AUGs in SUI1 and GCN4 mRNAs in vivo. In contrast, eIF2α-R53 interacts with the rRNA backbone only in the open complex, and the R53E substitution enhanced initiation at a UUG codon (Sui− phenotype) and poor-context AUGs, while reducing the rate of TC loading (Gcd− phenotype) in vivo. Consistently, R53E slowed TC binding to the PIC while decreasing TC dissociation at UUG codons in vitro, indicating destabilization of the open complex. Thus, distinct interactions of eIF2α with rRNA or mRNA stabilize first the open, and then closed, conformation of the PIC to influence the accuracy of initiation in vivo.

scholarly journals Regulatory elements of the erythropoietin gene

Blood ◽  
1991 ◽  
Vol 77 (2) ◽  
pp. 278-285 ◽  
Author(s):  
S Imagawa ◽  
MA Goldberg ◽  
J Doweiko ◽  
HF Bunn
Keyword(s):  
Transfection Efficiency ◽  
Hepatoma Cell ◽  
Internal Standard ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Hepatoma Cell Line ◽  
First Intron ◽  
Initiation Of Translation ◽  
Flanking Region

Abstract Because the human hepatoma cell line Hep3B produces erythropoietin (Epo) in a regulated fashion, it can be used to investigate the cis- acting regulatory elements of the Epo gene. Comparison of primate and mouse sequences shows strong homology not only in the coding sequence but also within the 5′ flanking region, the first intron, and the 3′ flanking region. These portions of the Epo gene were inserted 5′ and 3′ to a reporter gene, human growth hormone (GH). 5A is a 1,192-base pair (bp) HindIII-Xbal fragment that extends from 378 bp 5′ to the cap site through the first intron. To obviate the problem of false initiation of translation from the Epo ATG start codon, this site was changed to TAG by site-directed mutagenesis. 3A is a 255-bp Accl-BglII fragment that extends 67 bp upstream from the Epo termination codon and covers most of the 3′ noncoding region of homology. The plasmid DNAs were transfected by electroporation into Hep3B cells with RSVCAT as an internal standard to correct for transfection efficiency. One aliquot of cells was exposed to 50 mumol/L CoCl2 or to 1% O2. At the end of the incubations, GH and Epo were measured in the cell media and the cell pellet was assayed for CAT. Production of GH was stimulated 1.7-fold by cobalt or hypoxia. Furthermore, addition of 3A to the GH gene, irrespective of orientation, stimulated GH production 2.6-fold with CoCl2 and 2.3-fold with hypoxia. Stable cell lines were produced by cotransfection of the above constructions, along with the selectable marker pSV-Neo. In two clones, exposure to hypoxia resulted in much more marked (16-fold) induction of GH. Stimulus of both GH and Epo production by hypoxia was partially abrogated by carbon monoxide. These results demonstrate the presence of promoter and enhancer elements within the human Epo gene that are appropriately responsive to hypoxia and cobalt.

Matrix metalloproteinase-3 induction in rat brain astrocytes: focus on the role of two AP-1 elements

2008 ◽  
Vol 410 (3) ◽  
pp. 605-611 ◽  
Author(s):  
Kwang Soo Kim ◽  
Hee Young Kim ◽  
Eun-hye Joe ◽  
Ilo Jou
Keyword(s):  
Rat Brain ◽  
Interferon Γ ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Activator Protein 1 ◽  
Mobility Shift ◽  
Matrix Metalloproteinase 3 ◽  
Mitogen Activated Protein ◽  
Electrophoretic Mobility Shift Assays

Many brain cells secrete MMPs (matrix metalloproteinases), and increased or misregulated MMP levels are found in neurodegenerative disorders. Here we report that MMP-3 transcription and protein secretion were increased in rat brain astrocytes stimulated with lipopolysaccharide, gangliosides or interferon-γ. Sequential deletion of the MMP-3 promoter revealed that sequences between −0.5 kb and the start codon were crucial for the transcriptional induction of MMP-3. In addition, experiments using pharmacological inhibitors of individual mitogen-activated protein kinases revealed that MMP-3 induction and promoter activity involved Jun N-terminal kinase, a representative upstream signal of AP-1 (activator protein-1). Sequence analyses of the region of the MMP-3 promoter 500 bp from the start codon indicated the presence of three AP-1 binding sequences. Among them, electrophoretic-mobility-shift assays as well as site-directed mutagenesis of individual AP-1 sequences revealed that distal and middle, but not proximal, sequences largely mediated its induction. Together, these results indicate that AP-1 could control MMP-3 induction in brain astrocytes and that its regulation through specific AP-1 elements could be exploited in the treatment of brain pathologies in which increased expression of MMP-3 plays crucial roles.

scholarly journals Modulation of covR Expression in Streptococcus mutans UA159

2008 ◽  
Vol 190 (13) ◽  
pp. 4478-4488 ◽  
Author(s):  
Patrick Chong ◽  
Laura Drake ◽  
Indranil Biswas
Keyword(s):  
Dental Caries ◽  
Streptococcus Mutans ◽  
Growth Phase ◽  
Response Regulator ◽  
Transcriptional Start Site ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Pcr Analysis ◽  
Exponential Growth Phase ◽  
Mobility Shift

ABSTRACT The biofilm-forming Streptococcus mutans is a gram-positive bacterium that resides in the human oral cavity and is considered to be the primary etiological agent in the formation of dental caries. The global response regulator CovR, which lacks a cognate sensor kinase, is essential for the pathogenesis and biofilm formation of this bacterium, but it is not clear how covR expression is regulated in S. mutans. In this communication, we present the results of our studies examining various factors that regulate the expression of covR in S. mutans UA159. The results of Southern hybridization and PCR analysis indicated that CovR is an orphan response regulator in various isolates of S. mutans. The transcriptional start site for covR was found to be 221 base pairs upstream of the ATG start codon, and site-directed mutagenesis of the upstream TATAAT box confirmed our findings. The expression of covR is growth phase dependent, with maximal expression observed during exponential-growth phase. While changes to the growth temperature did not significantly affect the expression of covR, increasing the pH or the concentration of Mg2+ in the growth medium leads to an increase in covR expression. The results of semiquantitative reverse transcriptase PCR analysis and in vivo transcriptional-fusion reporter assays indicated that CovR autoregulates its own expression; this was verified by the results of electrophoretic mobility shift assays and DNase I protection assays, which demonstrated direct binding of CovR to the promoter region. Apparently, regulation by Mg2+ and the autoregulation of covR are not linked. A detailed analysis of the regulation of CovR may lead to a better understanding of the pathogenesis of S. mutans, as well as providing further insight into the prevention of dental caries.

The Translation Start Codon Region Is Sensitive to Antisense PNA Inhibition inEscherichia coli

2003 ◽  
Vol 13 (6) ◽  
pp. 427-433 ◽  
Author(s):  
Rikard Dryselius ◽  
Satish Kumar Aswasti ◽  
Gunaratna K. Rajarao ◽  
Peter E. Nielsen ◽  
Liam Good
Keyword(s):  
Start Codon ◽  
Inescherichia Coli ◽  
Translation Start Codon ◽  
Translation Start

scholarly journals Identification of a nisI Promoter within the nisABCTIP Operon That May Enable Establishment of Nisin Immunity Prior to Induction of the Operon via Signal Transduction

2006 ◽  
Vol 188 (24) ◽  
pp. 8496-8503 ◽  
Author(s):  
Haiping Li ◽  
Daniel J. O'Sullivan
Keyword(s):  
Transcription Initiation ◽  
Start Codon ◽  
Northern Analysis ◽  
Pcr Analysis ◽  
Translation Start ◽  
Promoter Probe ◽  
Genes Encoding ◽  
Promoter Probe Vector ◽  
Successful Establishment ◽  
High Degree

ABSTRACT Certain strains of Lactococcus lactis produce the broad-spectrum bacteriocin nisin, which belongs to the lantibiotic class of antimicrobial peptides. The genes encoding nisin are organized in three contiguous operons: nisABTCIP, encoding production and immunity (nisI); nisRK, encoding regulation; and nisFEG, also involved in immunity. Transcription of nisABTCIP and nisFEG requires autoinduction by external nisin via signal transducing by NisRK. This organization poses the intriguing question of how sufficient immunity (NisI) can be expressed when the nisin cluster enters a new cell, before it encounters external nisin. In this study, Northern analysis in both Lactococcus and Enterococcus backgrounds revealed that nisI mRNA was present under conditions when no nisA transcription was occurring, suggesting an internal promoter within the operon. The nisA transcript was significantly more stable than nisI, further substantiating this. Reverse transcriptase PCR analysis revealed that the transcription initiated just upstream from nisI. Fusing this region to a lacZ gene in a promoter probe vector demonstrated that a promoter was present. The transcription start site (TSS) of the nisI promoter was mapped at bp 123 upstream of the nisI translation start codon. Ordered 5′ deletions revealed that transcription activation depended on sequences located up to bp −234 from the TSS. The presence of poly(A) tracts and computerized predictions for this region suggested that a high degree of curvature may be required for transcription initiation. The existence of this nisI promoter is likely an evolutionary adaptation of the nisin gene cluster to enable its successful establishment in other cells following horizontal transfer.

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