Gene Encoding Translation Initiation Factor Research Articles

EIF4E-mediated resistance to potyviruses in plants: from natural alleles to edited genes

Resistance to viruses is an important aspect of plant breeding. One way to achieve it is to select genetic resistances based on the susceptibility factors hijacked by the virus to infect the plants. Here, we recount work done on genes encoding translation initiation factors eIF4E, some of the most successful targets for obtaining resistance to potyviruses, starting from their characterization 20 years ago. With examples from different plant species, pepper, tomato, tobacco and arabidopsis, we present the basis of this type of resistances and their characteristics, highlighting the role of gene redundancy among 4E factors, their specificity for the virus and the need for the plant of a trade-off between resistance and development. Finally, we show how the new genome editing techniques could be used in plant breeding to develop eIF4E-based resistances in crops, mimicking the functional alleles that have been selected during evolution in many crops.

The Efficiency of Various DNA Polymerases for Amplification of Long Sequences from Genomic DNA and cDNA of Cultivated Potatoes

Amplification of long fragments from complex templates, such as eukaryotic genomic DNA, is considered a difficult task for most DNA polymerases. In this research, six DNA polymerases were used to amplify full-length sequences from the genomic DNA of Solanum tuberosum genes encoding translation initiation factors of the eIF4E family, as well as for the synthesis of fragments of the potato Y virus genome from cDNA of potato plants infected by this virus. It was found that the efficiency of amplification by various DNA polymerases generally decreased with the increasing length of the amplicons. LongAmp and Platinum SuperFi II polymerases demonstrated the highest efficiency in the synthesis of long fragments, which made it possible to synthesize PCR products with a length of more than 10 000 base pairs with high efficiency. The lowest efficiency was demonstrated by Encyclo polymerase. None of the DNA polymerases provided efficient amplification of all the studied DNA fragments. At the same time, any of the studied DNA fragments could be effectively amplified using at least one DNA polymerase variant. Thus, the choice of DNA polymerase was of key importance for the efficiency of the synthesis of a desired PCR product.

The Efficiency of Various DNA Polymerases for Amplification of Long Sequences from Genomic DNA and cDNA of Cultivated Potato

Amplification of long fragments from complex templates, such as eukaryotic genomic DNA, is considered a difficult task for most DNA polymerases. In this research, 6 variants of DNA polymerases were used to amplify full-length sequences from the genomic DNA of Solanum tuberosum genes encoding translation initiation factors of the eIF4E family, as well as for the synthesis of fragments of the potato Y virus genome from cDNA of potato plants infected by this virus. It was found that the efficiency of amplification by various DNA polymerases generally decreased with increasing length of the amplicons. LongAmp and Platinum SuperFi II polymerases demonstrated the highest efficiency in the synthesis of long fragments, which made it possible to synthesize PCR products with a length of more than 10,000 base pairs with high efficiency. The lowest efficiency was demonstrated by Encyclo polymerase. None of the DNA polymerases provided efficient amplification of all the studied DNA fragments. At the same time, any of the studied DNA fragments could be effectively amplified using at least one DNA polymerase variant. Thus, the choice of DNA polymerase was of key importance for the efficiency of the synthesis of a desired PCR product.

Comparative transcriptomic analyses reveal differences in the responses of diploid and triploid Pacific oysters (Crassostrea gigas) to thermal stress

Triploid oysters often have higher growth rates and survival rates than diploid oysters. However, some studies have observed the opposite patterns. Triploid oysters have also been observed to show reduced resistance to stress and disease compared with diploid oysters. We compared the transcriptional patterns of diploid and triploid Pacific Oysters (Crassostrea gigas) in response to acute and prolonged thermal stress. The survival rate of triploid C. gigas was lower than that of diploid oysters after prolonged heat stress at 28 °C. A total of 779 and 396 differentially expressed genes (DEGs) were detected in diploid and triploid oysters exposed to acute heat stress, respectively, and the number of DEGs was 418 in diploids and 483 in triploids after prolonged heat stress. Functional enrichment analysis revealed that the DEGs were significantly enriched in protein processing in the endoplasmic reticulum pathway, NF-kappa B signaling pathway, NOD-like receptor signaling pathway, and TNF signaling pathway in both diploid and triploid C. gigas under heat stress. Genes encoding translation initiation factors, including MKNK1, eIF4G3, eIF2a kinase 1, and eIF2a kinase 3, were differentially expressed under acute heat stress in diploid oysters. The number of differentially expressed HSP and IAP genes was greater in diploids than in triploids under acute heat stress, and the expression levels of most of these HSP and IAP genes were higher in diploid oysters than in triploid oysters. Overall, our findings revealed differences in the response of diploid and triploid C. gigas to thermal stress. Triploid C. gigas might be less capable of rapidly modifying the inflammatory response and apoptosis in response to heat stress compared with diploid C. gigas. Different genetic lines of diploid and tetraploid oysters should be used to produce triploid oysters with increased stress resistance.

Adaptive laboratory evolution of the fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for improved solvent tolerance

Cyanobacteria hold promise as cell factories for the photoautotrophic conversion of carbon dioxide to useful chemicals. For the eventual commercial viability of such processes, cyanobacteria need to be engineered for (i) efficient channeling of carbon flux toward the product of interest and (ii) improved product tolerance, the latter being the focus of this study. We chose the recently reported, fast-growing, high light and CO2 tolerant cyanobacterium Synechococcus elongatus PCC 11801 for adaptive laboratory evolution. In two parallel experiments that lasted over 8400h of culturing and 100 serial passages, S.elongatus PCC 11801 was evolved to tolerate 5g/L n-butanol or 30g/L 2,3-butanediol representing a 100% improvement in concentrations tolerated. The evolved strains retained alcohol tolerance even after being passaged several times without the alcohol stress suggesting that the changes were permanent. Whole genome sequencing of the n-butanol evolved strains revealed mutations in a number of stress responsive genes encoding translation initiation factors, RpoB and an ABC transporter. In 2,3-butanediol evolved strains, genes for ClpC, a different ABC transporter, glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase were found to be mutated. Furthermore, the evolved strains showed significant improvement in tolerance toward several other alcohols. Notably, the n-butanol evolved strain could tolerate up to 32g/L ethanol, thereby making it a promising host for photosynthetic production of biofuels via metabolic engineering.

A new eIF4E1 allele characterized by RNAseq data mining is associated with resistance to potato virus Y in tomato albeit with a low durability.

Allele mining on susceptibility factors offers opportunities to find new sources of resistance among crop wild relatives for breeding purposes. As a proof of concept, we used available RNAseq data to investigate polymorphisms among the four tomato genes encoding translation initiation factors [eIF4E1 and eIF4E2, eIFiso4E and the related gene new cap-binding protein(nCBP)] to look for new potential resistance alleles to potyviruses. By analysing polymorphism among RNAseq data obtained for 20 tomato accessions, 10 belonging to the cultivated type Solanum lycopersicum and 10 belonging to the closest related wild species Solanum pimpinellifolium, we isolated one new eIF4E1 allele, in the S. pimpinellifolium LA0411 accession, which encodes a potential new resistance allele, mainly due to a polymorphism associated with an amino acid change within eIF4E1 region II. We confirmed that this new allele, pot12, is indeed associated with resistance to potato virus Y, although with a restricted resistance spectrum and a very low durability potential. This suggests that mutations occurring in eIF4E region II only may not be sufficient to provide efficient and durable resistance in plants. However, our study emphasizes the opportunity brought by RNAseq data to mine for new resistance alleles. Moreover, this approach could be extended to seek for putative new resistance alleles by screening for variant forms of susceptibility genes encoding plant host proteins known to interact with viral proteins.

Simple construction of chimeric hairpin RNA for virus resistance in plants

RNA silencing has been adopted to develop virus-resistant plants through expression of virus-derived hairpin RNAs. Due to the high sequence specificity of RNA silencing, this technology has been limited to the targeting of single viruses. Simultaneous targeting of multiple viruses or plant genes can be achieved by using a chimeric cassette. In this study, a simple method was developed to construct chimeric hairpin RNA rapidly and efficiently. This method splices two DNA fragments from viruses or plant genes to be a chimeric sequence using Overlap Extension PCR (OE-PCR); then this chimeric sequence was assembled with an intron sequence to generate an intron-containing hairpin RNA construct in one step mediated by OE-PCR. This method is neither dependent on restriction enzymes nor requires expensive consumables, so a chimeric hairpin RNA can be constructed rapidly and costlessly. Two chimeric hairpin RNA constructs were amplified successfully using this method, with the targeting sequences from both papaya ringspot virus (PRSV) and two plant genes encoding translation initiation factors eIF4E and eIFiso4E. This novel method is a useful strategy to construct chimeric hairpin RNA for RNA silencing in plants.

Clonal origin of aminoglycoside-resistant Citrobacter freundii isolates in a Danish county.

During 1997, attention was drawn to an increased frequency of aminoglycoside-resistant Citrobacterfreundii in a Danish county, when a total of 24 resistant C. freundii isolates was detected. In this study, 15 such isolates were typed by pulsed-field gel electrophoresis, riboprinting and partial sequencing of the gene encoding translation initiation factor 2. Fourteen of the 15 isolates were identical, as evaluated by their antibiograms and by all these typing methods. This epidemic strain harboured the aminoglycoside resistance genes aac(3)-II and ant(3")-I, with the latter located in tandem with a dihydrofolate reductase gene in a class I integron. The source of the strain remains unresolved. Representative isolates were obtained from various specimens from hospitals and general practice throughout the county, with no evidence of patient-to-patient transmission.

Identification of Enterobacteriaceae by partial sequencing of the gene encoding translation initiation factor 2.

Nucleotide sequence analysis is increasingly being used to identify bacteria. In this work, a PCR assay based on degenerate primers was used to obtain the partial sequence of infB, the gene encoding translation initiation factor 2 (IF2), in 39 clinical isolates of different Enterobacteriaceae. The partial sequence encodes the GTP-binding domain of IF2. Together with sequences from the literature, a total of 15 species, each represented by one to seven strains, was investigated. Phylogenetic analysis yielded an evolutionary tree which had a topology similar to a tree constructed using available 16S rRNA sequences. It is concluded that the inter-species variation of the infB gene fragment is sufficient for its use in the characterization of strains that have aberrant phenotypic reactions.

A systematic nomenclature for new translation initiation factor genes from S. pombe and other fungi.

Eukaryotic translation initiation factors and their corresponding genes have been characterized using biochemical and genetic methods from a variety of different organisms. The designations of the factors relate to their apparent roles in the biochemical process. Many gene names indicate genetic interactions with other genes or the functional attributes used to identify them. On the other hand, progress in systematic sequencing of the genomes of organisms like Saccharomyces cerevisiae and Schizosaccharomyces pombe has revealed many genes homologous to known translation initiation factor genes. The genes defined by the systematic sequencing approach are assigned numerical designations completely unrelated to their biological function. So far there have been publications on only three genes encoding translation initiation factors from Schizosaccharomyces pombe. We therefore see this an an ideal opportunity to propose a systematic and logical nomenclature for genes encoding translation initiation factor genes that can be applied to all further genes of this type that are characterized in this fission yeast.

The role of the AUU initiation codon in the negative feedback regulation of the gene for translation initiation factor IF3 in Escherichia coli.

The expression of the infC gene encoding translation initiation factor IF3 is negatively autoregulated at the level of translation, i.e. the expression of the gene is derepressed in a mutant infC background where the IF3 activity is lower than that of the wild type. The special initiation codon of infC, AUU, has previously been shown to be essential for derepression in vivo. In the present work, we provide evidence that the AUU initiation codon causes derepression by itself, because if the initiation codon of the thrS gene, encoding threonyl-tRNA synthetase, is changed from AUG to AUU, its expression is also derepressed in an infC mutant background. The same result was obtained with the rpsO gene encoding ribosomal protein S15. We also show that derepression of infC, thrS, and rpsO is obtained with other 'abnormal' initiation codons such as AUA, AUC, and CUG which initiate with the same low efficiency as AUU, and also with ACG which initiates with an even lower efficiency. Under conditions of IF3 excess, the expression of infC is repressed in the presence of the AUU or other 'abnormal' initiation codons. Under the same conditions and with the same set of 'abnormal' initiation codons, the repression of thrS and rpsO expression is weaker. This result suggests that the infC message has specific features that render its expression particularly sensitive to excess of IF3. We also studied another peculiarity of the infC message, namely the role of a GC-rich sequence located immediately downstream of the initiation codon and conserved through evolution. This sequence was proposed to interact with a conserved region in 16S RNA and enhance translation initiation. Unexpectedly, mutating this GC-rich sequence increases infC expression, indicating that this sequence has no enhancing role. Chemical and enzymatic probing of infC RNA synthesized in vitro indicates that this GC-rich sequence might pair with another region of the mRNA. On the basis of our in vivo results we propose, as suspected from earlier in vitro results, that IF3 regulates the expression of its own gene by using its ability to differentiate between 'normal' and 'abnormal' initiation codons.

Growth factor-independent expression of the gene encoding eukaryotic translation initiation factor 4E in transformed cell lines

Activation of protein synthesis is necessary for the transition of cells from quiescence to proliferation, while withdrawal of growth factors leads to decrease in protein synthesis and transition of normal cells into the resting period. It is shown in this paper that serum growth factors are required for activation of expression of gene encoding translation initiation factor 4E (eIF-4E) in non-transformed NIH 3T3 and Rat-1 fibroblasts but this requirement is lost in C6 glioblastoma, A431 carcinoma and N-myc transformed Rat-1 cells. These data raise the possibility that neoplastic transformation leads to growth factor-independent expression of eIF-4E, thus facilitating continuous growth and replication of transformed cells.

Growth factor-independent expression of the gene encoding eukaryotic translation initiation factor 4E in transformed cell lines

Activation of protein synthesis is necessary for the transition of cells from quiescence to proliferation, while withdrawal of growth factors leads to decrease in protein synthesis and transition of normal cells into the resting period. It is shown in this paper that serum growth factors are required for activation of expression of gene encoding translation initiation factor 4E (eIF-4E) in non-transformed NIH 3T3 and Rat-1 fibroblasts but this requirement is lost in C6 glioblastoma, A431 carcinoma and N-myc transformed Rat-1 cells. These data raise the possibility that neoplastic transformation leads to growth factor-independent expression of eIF-4E, thus facilitating continuous growth and replication of transformed cells.

Suppression of recJ mutations of Escherichia coli by mutations in translation initiation factor IF3.

We have isolated genetic suppressors of mutations in the recJ gene of Escherichia coli in a locus we term srjA. These srjA mutations cause partial to complete alleviation of the recombination and UV repair defects conferred by recJ153 and recJ154 mutations in a recBC sbcA genetic background. The srjA gene was mapped to 37.5 min on the E. coli chromosome. This chromosomal region from the srjA5 strain was cloned into a plasmid vector and was shown to confer recJ suppression in a dominant fashion. Mutational analysis of this plasmid mapped srjA to the infC gene encoding translation initiation factor 3 (IF3). Sequence analysis revealed that all three srjA alleles cause amino acid substitutions of IF3. Suppression of recJ was shown to be allele specific: recJ153 and recJ154 mutations were suppressible, but recJ77 and the insertion allele recJ284::Tn10 were not. In addition, growth medium-conditional lethality was observed for strains carrying srjA mutations with the nonsuppressible recJ alleles. When introduced into recJ+ strains, srjA mutations conferred hyperrecombinational and hyper-UVr phenotypes. An interesting implication of these genetic properties of srjA suppression is that IF3 may regulate the expression of recJ and perhaps other recombination genes and hence may regulate the recombinational capacity of the cell.

Molecular cloning and sequencing of infC, the gene encoding translation initiation factor IF3, from four enterobacterial species

Molecular cloning and sequencing of infC, the gene encoding translation initiation factor IF3, from four enterobacterial species

Growth rate regulation of translation initiation factor IF3 biosynthesis in Escherichia coli

infC, the gene encoding translation initiation factor IF3 in Escherichia coli, can be transcribed from three promoters. Two of these promoters, PI1 and PI2, are located in the upstream thrS sequence which codes for threonyl-tRNA synthetase. Previous studies had shown that PI2 was the major promoter for infC. In the present study, the extent of transcription from PI1 and/or PI2 at a variety of steady-state growth rates was analyzed by promoter fusion studies. PI2 was the more active promoter (two- to threefold stronger than PI1) at all growth rates tested. A fusion plasmid containing both PI1 and PI2 exhibited a transcription level approximately equal to the sum of those observed with the fusion plasmids containing the individual promoters. The transcriptional activities of PI1 and PI2 did not change as the growth rate was varied from 0.3 to 1.7 doublings per h. In contrast, a fusion plasmid carrying the rrnB P1 promoter displayed the expected growth rate response. The steady-state concentrations of infC mRNA in cells grown at different rates were measured and found not to vary. These results indicate that the previously reported growth rate regulation of IF3 biosynthesis neither is accomplished by transcriptional control nor is a result of differential mRNA stability. In view of these results, the steady-state levels of IF3 in cells grown at a number of different growth rates were determined by quantitative immunoblotting. IF3 levels were found to vary with growth rate in a manner essentially identical to that observed for ribosomes. A model accounting for these results and describing a mechanism for coordinate growth rate-regulated expression of ribosomes and IF3 is presented.

Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system.

The gene encoding translation initiation factor 4E (eIF-4E) from Saccharomyces cerevisiae was randomly mutagenized in vitro. The mutagenized gene was reintroduced on a plasmid into S. cerevisiae cells having their only wild-type eIF-4E gene on a plasmid under the control of the regulatable GAL1 promoter. Transcription from the GAL1 promoter (and consequently the production of wild-type eIF-4E) was then shut off by plating these cells on glucose-containing medium. Under these conditions, the phenotype conferred upon the cells by the mutated eIF-4E gene became apparent. Temperature-sensitive S. cerevisiae strains were identified by replica plating. The properties of one strain, 4-2, were further analyzed. Strain 4-2 has two point mutations in the eIF-4E gene. Upon incubation at 37 degrees C, incorporation of [35S]methionine was reduced to 15% of the wild-type level. Cell-free translation systems derived from strain 4-2 were dependent on exogenous eIF-4E for efficient translation of certain mRNAs, and this dependence was enhanced by preincubation of the extract at 37 degrees C. Not all mRNAs tested required exogenous eIF-4E for translation.

Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system.

The gene encoding translation initiation factor 4E (eIF-4E) from Saccharomyces cerevisiae was randomly mutagenized in vitro. The mutagenized gene was reintroduced on a plasmid into S. cerevisiae cells having their only wild-type eIF-4E gene on a plasmid under the control of the regulatable GAL1 promoter. Transcription from the GAL1 promoter (and consequently the production of wild-type eIF-4E) was then shut off by plating these cells on glucose-containing medium. Under these conditions, the phenotype conferred upon the cells by the mutated eIF-4E gene became apparent. Temperature-sensitive S. cerevisiae strains were identified by replica plating. The properties of one strain, 4-2, were further analyzed. Strain 4-2 has two point mutations in the eIF-4E gene. Upon incubation at 37 degrees C, incorporation of [35S]methionine was reduced to 15% of the wild-type level. Cell-free translation systems derived from strain 4-2 were dependent on exogenous eIF-4E for efficient translation of certain mRNAs, and this dependence was enhanced by preincubation of the extract at 37 degrees C. Not all mRNAs tested required exogenous eIF-4E for translation.

The gene encoding translation initiation factor 3 is highly conserved in gram-negative bacteria

A 1.1-kb HpaI fragment of the Escherichia coli chromosome containing the gene for translation initiation factor 3 was employed as a probe in heterologous hybridization to chromosomal DNA from a variety of other procaryotes. Positive hybridization was observed to DNA derived from all gram-negative bacteria tested. In contrast, no hybridization to DNA from gram-positive bacteria was detected. In addition, homologous sequences were found in Euglena gracilis chloroplast DNA, while this was not the case with Saccharomyces cerevisiae mitochondrial DNA. These results are discussed in light of existing data on the components and mechanism of translation initiation in the various organisms and organelles employed in this study.