Improved Screening of cDNAs Generated by mRNA Differential Display Enables the Selection of True Positives and the Isolation of Weakly Expressed Messages

The high percentage of false positives generated by differential display (as high as 85%) has previously limited the potential of the method. This report describes an efficient methodology that enables false positives to be discarded prior to cloning, via reverse Northern analysis. This first step of the screening also allows the detection of putative lowabundance differential clones. Following cloning, a second reverseNorthern combined with partial DNA sequencing and RT-PCR detection allows isolation of all differential cDNAs including very lowabundance clones. Use of the sequential screening procedure described here led to the isolation of novel tomato genes responding to the plant hormone ethylene while minimising labor and materials input

ER5, a tomato cDNA encoding an ethylene-responsive LEA-like protein: characterization and expression in response to drought, ABA and wounding

We report the isolation by differential display of a novel tomato ethylene-responsive cDNA, designated ER5. RT-PCR analysis of ER5 expression revealed an early (15 min) and transient induction by ethylene in tomato fruit, leaves and roots. ER5 mRNA accumulated during 2 h of ethylene treatment and thereafter underwent a dramatic decline leading to undetectable expression after 5 h of treatment. The full-length cDNA clone of 748 bp was obtained and DNA sequence analysis showed strong homologies to members of the atypical hydrophobic group of the LEA protein family. The predicted amino acid sequence shows 67%, 64%, 64%, and 61%sequence identity with the tomato Lemmi9, soybean D95-4, cotton Lea14-A, and resurrection plant pcC27-45 gene products, respectively. As with the other members of this group, ER5 encodes a predominantly hydrophobic protein. Prolonged drought stress stimulates ER5 expression in leaves and roots, while ABA induction of this ethylene-responsive clone is confined to the leaves. The use of 1-MCP, an inhibitor of ethylene action, indicates that the drought induction of ER5 is ethylene-mediated in tomato roots. Finally, wounding stimulates ER5 mRNA accumulation in leaves and roots. Among the Lea gene family this novel clone is the first to display an ethylene-regulated expression

Recent Developments on the Role of Ethylene in the Ripening of Climacteric Fruit

It has long been recognised that ethylene plays a major role in the ripening process of climacteric fruit. A more thorough analysis, however, has revealed that a number of biochemical and molecular processes associated with climacteric fruit ripening are ethylene-independent. One of the crucial steps of the onset of ripening is the induction of autocatalytic ethylene production. In ethylene-suppressed melons, ACC synthase activity is induced at the same time as in control melons, indicating that ACC biosynthesis during the early stages of ripening seems to be a developmentally-regulated (ethylene-independent) process. The various ripening events exhibit differential sensitivity to ethylene. For instance, the threshold level for degreening of the rind is 1ppm, while 2.5 ppm are required to trigger some components of the softening process. The saturating level of ethylene producing maximum effects is less than 5 ppm, which is by far lower than the internal ethylene concentrations found in the fruit at the climacteric peak (over 100 ppm). In many fruit chilling temperatures hasten ethylene production and ripening and in some late season pear varieties, exposure to chilling temperatures is even absolutely required for the attainment of the capacity to synthesize autocatalytic ethylene. This is correlated with the stimulation of expression of ACC oxidase and of members of the ACC synthase gene family. Ethylene operates via a perception and transduction pathway to induce the expression of genes responsible for the biochemical and physiological changes observed during ripening. However, only a few genes induced via the ethylene transduction pathway have been described so far. We have used a differential display method to isolate novel ethylene-reponsive (ER) cDNA clones of tomato that potentially play a role in propagating the ethylene response and in regulating fruit ripening. Collectively, these data permit a general scheme of the molecular mechanisms of fruit ripening to be proposed

Tomato EF-Tsmt, a functional mitochondrial translation elongation factor from higher plants

Ethylene-induced ripening in tomato (Lycopersicon esculentum) resulted in the accumulation of a transcript designated LeEF-Tsmt that encodes a protein with significant homology to bacterial Ts translational elongation factor (EF-Ts). Transient expression in tobacco and sunflower protoplasts of full-length and truncated LeEF-Tsmt- GFP fusion constructs and confocal microscopy observations clearly demonstrated the targeting of LeEF-Tsmt to mitochondria and not to chloroplasts and the requirement for a signal peptide for the proper sorting of the protein. Escherichia coli recombinant LeEF-Tsmt co-eluted from Ni-NTA resins with a protein corresponding to the molecular weight of the elongation factor EF-Tu of E. coli, indicating an interaction with bacterial EF-Tu. Increasing the GDP concentration in the extraction buffer reduced the amount of EF-Tu in the purified LeEF-Tsmt fraction. The purified LeEF-Tsmt stimulated the poly(U)-directed polymerization of phenylalanine 10-fold in the presence of EF-Tu. Furthermore, LeEF-Tsmt was capable of catalysing the nucleotide exchange reaction with E. coli EF-Tu. Altogether, these data demonstrate that LeEF-Tsmt encodes a functional mitochondrial EF-Ts. LeEFTsmt represents the first mitochondrial elongation factor to be isolated and functionally characterized in higher plants

SARS-CoV-2 nsp10/nsp16 MTase kinetic parameters and substrate specificity.

(A) SARS-CoV-2 nsp10/nsp16 substrate kinetic studies were carried out using different concentrations of Oligo 3 using 20μM SAM per reaction. (B) Similar studies with the enzyme using 80 μM of Oligo 3 and varying SAM concentration to calculate the Kd value for SAM. The kinetic studies were carried out with 40ng of nsp10/nsp16 complex per reaction at 23°C for 15min. (C) Testing substrate specificities for nsp10/nsp16 enzyme reactions were carried out using 40ng enzyme per reaction with 50μM SAM and 25μM of each Oligo RNAs and 20μM for both Histones. Reactions were carried out at 37°C for 80min in white and solid low-volume 384-well plate, and the MTase-Glo™ Assay was performed as described in Materials and Methods section. Each point represents average of two data points. Data analysis was performed with GraphPad Prism® software, version 9.1.0, for Windows® using a One Site Binding (hyperbola) program (panel A and B) and with Microsoft® Excel 365 program (panel C). All substrate sequences are shown in their specific panel. Data analysis was performed with Microsoft® Excel 2013 program, and GraphPad Prism® software, version 9.1.0, for Windows® using a One Site Binding (hyperbola) program.</p

Determining SARS-CoV-2 nsp14 MTase Activity using RNA substrates.

Monitoring the activity of nsp14 MTase using capped and uncapped RNA substrates as listed under the graphs. (A) Short sequences substrates, and (B) long sequence substrates. Reactions were carried out with 50μM SAM and 40μM of either short RNAs or Oligo RNAs using a white solid 384LV-well plate. SARS-CoV-2 nsp14 reaction was for 90 min at 37°C. The list of RNA substrates and their sequence are shown below the figures. The MTase-Glo™ Assay was performed as described in Materials and Methods section. Each point represents an average of two data points; the error bars represent the standard deviation. Data analysis was performed with GraphPad Prism® software, version 9.1.0, for Windows® using a One Site Binding (hyperbola) program.</p

SARS-CoV-2 NSP14 MTase inhibitors study.

Effect of sinefungin concentrations on nsp14 (1ng) MTase activity at different concentrations of SAM (5 μM, 25 μM, and 50 μM) using 10 μM of RNA 1 (A) or 10 μM of Oligo1 (B). Effect of Aurintricarboxylic acid (ATA) on the activity of nsp14 (1ng) in the presence of varying concentrations of SAM (5 μM,25 μM, and 50 μM) using 10 μM RNA1 (C), or 10μM Oligo1 (D) and varying concentrations of Oligo1 (10 μM,50 μM,100 μM) at 5 μM SAM (E). Reactions were carried out for 90min at 37°C. All reactions were done using a white solid 384LV-well plate, and The MTase-Glo™ Assay was performed as described in Materials and Methods section. Each point represents an average of two data points; the error bars represent the standard deviation. Data analysis was performed with GraphPad Prism® software, version 9.1.0, for Windows® using a Sigmoidal dose response (variable slope) program.</p

Determining optimal stoichiometry of SARS-COV-2 nsp10 & nsp16 MTases.

Monitoring methyltransferase activity of nsp10/nsp16 starting with constant concentration of nsp10 (0.5μM) and increasing ratio of nsp16 (0, 1:1, 1:2, 1:4, and 1:6) in the presence of 5, 25, and 50μM SAM and different concentrations of Oligo 3 (10, 50, and 100μM). Reactions were carried out in white solid low volume 384-well plate for 90min at 37°C. The MTase-Glo™ Assay was performed as described in Method and Materials. Each point represents an average of three data points; the error bars represent the standard deviation Data analysis was performed with Microsoft® Excel 2013 program.</p

Effect of inhibitors on SARS-CoV-2 nsp10/nsp16 MTase activity.

Percent inhibition of sinefungin (0 μM, 50 μM, and 100 μM) on the enzyme activity of SARS-CoV-2 nsp10/nsp16, at three different concentrations of SAM (5 μM, 25 μM, and 50 μM) using 10 μM oligo 3 as substrate (left panel) or different concentrations of Oligo 3 (10 μM, 50 μM, and 100 μM) using 5 μM of SAM. Reactions were carried out with 60ng of NSP10/NSP16 per reaction for 90min at 37°C. All reactions were done using white solid 384LV-well plate, and the MTase-Glo™ Assay was performed as described in Materials and Methods section. Each point represents an average of two data points; the error bars represent the standard deviation was performed with Microsoft® Excel 365 program.</p

SARS-CoV-2 nsp14 MTase kinetic parameters and substrate specificity.

(A) SARS-CoV-2 nsp14 enzyme activity was monitored using a titration of uncapped (RNA1) short sequence and uncapped oligo 1 but methylated at the penultimate nucleotide sugar in the presence of 50μM SAM per reaction. (B) Titration of SAM substrate using saturating concentrations of RNA substrates, 25 μM of oligo 1 and 50 μM of RNA1. The data were generated using 1ng of nsp14 per reaction at 23°C for 30min. (C) Substrate selectivity of nsp14 using different RNA sequences and proteins known to be methylated. Reactions were carried out using 1ng of nsp14 per reaction with 50μM SAM and 25μM of each Oligo RNA and 20μM for both Histones and reactions were carried out at 37°C for 80min in white solid 384LV-well plate. MTase-Glo™ Assay was performed as described in Materials and Methods section. Each point represents an average of two data points; the error bars represent the standard deviation. Data analysis was performed with GraphPad Prism® software, version 9.1.0, for Windows® using a One Site Binding (hyperbola) program (panel A and B) and with Microsoft® Excel 365 program (panel C). All substrate sequences are shown in their specific panel.</p