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Background Internal ribosome entry sites (IRESs) allow the translation of a transcript independent of its cap structure. They are distributed in some viruses and cellular RNA. The element is applied in dual gene expression in a single vector. Although it appears the lower efficiency of IRES-mediated translation than that of cap-dependent translation, it is with the crucial needs to know the precise differences in translational efficacy between upstream cistrons (cap-dependent) and downstream cistrons (IRES-mediate, cap-independent) before applying the bicistronic vector in biomedical applications. Material and methods This study aimed to provide real examples and showed the precise differences for translational efficiency dependent upon target gene locations. We generated various bicistronic constructs with quantifiable reporter genes as upstream and downstream cistrons of the encephalomyocarditis virus (EMCV) IRES to precisely evaluate the efficacy of IRES-mediated translation in mammalian cells. Results There was no significant difference in protein production when the reporter gene was cloned as an upstream cistron. However, lower levels of protein production were obtained when the reporter gene was located downstream of the IRES. Moreover, in the presence of an upstream cistron, a markedly reduced level of protein production was observed. Conclusion Our findings demonstrate the version of the EMCV IRES that is provided in many commercial vectors is relatively less efficient than cap-dependent translation and provide valuable information regarding the utilization of IRES to facilitate the expression of more than one protein from a transcript.

During implantation, cytotrophoblasts undergo epithelial-to-mesenchymal transition (EMT) as they differentiate into invasive extravillous trophoblasts (EVTs). The primate-specific microRNA cluster on chromosome 19 (C19MC) is exclusively expressed in the placenta, embryonic stem cells and certain cancers however, its role in EMT gene regulation is unknown. In situ hybridization for miR-517a/c, a C19MC cistron microRNA, in first trimester human placentas displayed strong expression in villous trophoblasts and a gradual decrease from proximal to distal cell columns as cytotrophoblasts differentiate into invasive EVTs. To investigate the role of C19MC in the regulation of EMT genes, we employed the CRISPR/dCas9 Synergistic Activation Mediator (SAM) system, which induced robust transcriptional activation of the entire C19MC cistron and resulted in suppression of EMT associated genes. Exposure of human iPSCs to hypoxia or differentiation of iPSCs into either cytotrophoblast-stem-like cells or EVT-like cells under hypoxia reduced C19MC expression and increased EMT genes. Furthermore, transcriptional activation of the C19MC cistron induced the expression of OCT4 and FGF4 and accelerated cellular reprogramming. This study establishes the CRISPR/dCas9 SAM as a powerful tool that enables activation of the entire C19MC cistron and uncovers its novel role in suppressing EMT genes critical for maintaining the epithelial cytotrophoblasts stem cell phenotype.

Soybean mosaic virus and Clover yellow vein virus are two definite species of the genus Potyvirus within the family Potyviridae. Soybean mosaic virus-N (SMV-N) is well adapted to cultivated soybean (Glycine max) genotypes and wild soybean (G. soja), whereas it remains undetectable in inoculated broad bean (Vicia faba). In contrast, clover yellow vein virus No. 30 (ClYVV-No. 30) is capable of systemic infection in broad bean and wild soybean; however, it infects cultivated soybean genotypes only locally. In this study, SMV-N was shown to also infect broad bean locally; hence, broad bean is a host for SMV-N. Based on these observations, it was hypothesized that lack of systemic infection by SMV-N in broad bean and by ClYVV-No. 30 in cultivated soybean is attributable to the incompatibility of multifunctional helper-component proteinase (HC-Pro) in these hosts. The logic of selecting the HC-Pro cistron as a target is based on its established function in systemic movement and being a relevant factor in host range specificity of potyviruses. To test this hypothesis, chimeras were constructed with precise exchanges of HC-Pro cistrons between SMV-N and ClYVV-No. 30. Upon inoculation, both chimeras were viable in infection, but host range specificity of the recombinant viruses did not differ from those of the parental viruses. These observations suggest that (i) HC-Pro cistrons from SMV-N and ClYVV-No. 30 are functionally compatible in infection despite 55.6 and 48.9% nucleotide and amino acid sequence identity, respectively, and (ii) HC-Pro cistrons from SMV-N and ClYVV-No. 30 are not the determinants of host specificity on cultivated soybean or broad beans, respectively.

Cucumber vein yellowing virus (CVYV) is an emerging virus on cucurbits in the Mediterranean Basin, against which few resistance sources are available, particularly in melon. The melon accession PI 164323 displays complete resistance to isolate CVYV-Esp, and accession HSD 2458 presents a tolerance, i.e., very mild symptoms despite virus accumulation in inoculated plants. The resistance is controlled by a dominant allele Cvy-11, while the tolerance is controlled by a recessive allele cvy-2, independent from Cvy-11. Before introducing the resistance or tolerance in commercial cultivars through a long breeding process, it is important to estimate their specificity and durability. Upon inoculation with eight molecularly diverse CVYV isolates, the resistance was found to be isolate-specific because many CVYV isolates induced necrosis on PI 164323, whereas the tolerance presented a broader range. A resistance-breaking isolate inducing severe mosaic on PI 164323 was obtained. This isolate differed from the parental strain by a single amino acid change in the VPg coding region. An infectious CVYV cDNA clone was obtained, and the effect of the mutation in the VPg cistron on resistance to PI 164323 was confirmed by reverse genetics. This represents the first determinant for resistance-breaking in an ipomovirus. Our results indicate that the use of the Cvy-11 allele alone will not provide durable resistance to CVYV and that, if used in the field, it should be combined with other control methods such as cultural practices and pyramiding of resistance genes to achieve long-lasting resistance against CVYV.

In bacteria, small non-coding RNAs (sRNAs) bind to target mRNAs and regulate their translation and/or stability. In the polycistronic galETKM operon of Escherichia coli, binding of the Spot 42 sRNA to the operon transcript leads to the generation of galET mRNA. The mechanism of this regulation has remained unclear. We show that sRNA-mRNA base pairing at the beginning of the galK gene leads to both transcription termination and transcript cleavage within galK, and generates galET mRNAs with two different 3’-OH ends. Transcription termination requires Rho, and transcript cleavage requires the endonuclease RNase E. The sRNA-mRNA base-paired segments required for generating the two galET species are different, indicating different sequence requirements for the two events. The use of two targets in an mRNA, each of which causes a different outcome, appears to be a novel mode of action for a sRNA. Considering the prevalence of potential sRNA targets at cistron junctions, the generation of new mRNA species by the mechanisms reported here might be a widespread mode of bacterial gene regulation., Author summary sRNAs are regulators of gene expression in all forms of life. In bacteria such as E. coli, sRNAs base-pair with mRNA, which can have many consequences such as premature transcription termination by Rho and degradation of mRNA by the endoribonuclease RNase E. Here we show that the two processes can occur on the same sRNA-mRNA pair and produce shorter but stable mRNA that can be of functional significance. Thus, in sRNA regulation of mRNA, transcript cleavage can be an additional mechanism to well-known transcription termination to generate new mRNA species. The choice between the mechanisms is dictated by where on the mRNA the base pairing with sRNA takes place.

Cervical cancer is the fourth most common cause of cancer in women worldwide in terms of both incidence and mortality. Persistent infection with high-risk types of human papillomavirus (HPV), namely 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, constitute a necessary cause for the development of cervical cancer. Viral oncoproteins E6 and E7 play central roles in the carcinogenic process by virtue of their interactions with cell master proteins such as p53, retinoblastoma (Rb), mammalian target of rapamycin (mTOR), and c-MYC. For the synthesis of E6 and E7, HPVs use a bicistronic messenger RNA (mRNA) that has been studied in cultured cells. Here, we report that in cervical tumors, HPV-18, -39, and -45 transcribe E6/E7 mRNAs with extremely short 5' untranslated regions (UTRs) or even lacking a 5' UTR (i.e., zero to three nucleotides long) to express E6. We show that the translation of HPV-18 E6 cistron is regulated by the motif ACCaugGCGCG(C/A)UUU surrounding the AUG start codon, which we term Translation Initiation of Leaderless mRNAs (TILM). This motif is conserved in all HPV types of the phylogenetically coherent group forming genus alpha, species 7, which infect mucosal epithelia. We further show that the translation of HPV-18 E6 largely relies on the cap structure and eIF4E and eIF4AI, two key translation initiation factors linking translation and cancer but does not involve scanning. Our results support the notion that E6 forms the center of the positive oncogenic feedback loop node involving eIF4E, the mTOR cascade, and p53.

Three new species ofLachemilla(Rosaceae), two from Colombia and one from Peru, are described and illustrated.Lachemillarothmalerianais characterized by its stout stems, sericeous-villous indumentum, and wide ascending sheaths with trilobate lateral lobes.Lachemillaargenteapresents a unique combination of tripartite basal leaves with an adaxial silvery villous indumentum, and decumbent branches with verticillate lobed sheaths. Finally,Lachemillacyaneahas distinctly basal reniform leaves with a blue-green color and hirsute pubescence. Phylogenetic analyses of the nuclear ribosomal cistron and multiple regions of the plastid genome revealed the allopolyploid origin of the three new taxa.

The complete genome sequence of a wild tomato mosaic virus (WTMV) isolate (named WTMV-Sn) was determined and identified in Solanum nigrum in China. The complete genome of WTMV-Sn is 9,659 nucleotides in length, excluding the poly(A) tail and encodes a polyprotein of 3,074 amino acids. This is the first report of WTMV infecting S. nigrum. Despite the high degree of sequence similarity between the WTMV-Sn and WTMV-XC-1 isolates, the 349 nucleotides at the 5' terminus of WTMV-Sn appear to have originated by recombination with another isolate. The recombination parent remains unknown, but the recombination region shares 74.57% sequence identity with isolate WTMV-Laichau, which is below the species demarcation threshold for the genus Potyvirus. A pathogenicity test showed that WTMV-Sn can infect tobacco. This suggests that variation in the P1 cistron of WTMV-Sn may contribute to its ability to infect S. nigrum.

The present study demonstrates how different potato virus Y (PVY) strains affect the miRNA balance in tobacco cv. Samsun. The two prevalent strains PVYNTN and PVYN−Wi caused severe and mild veinal necrosis (VN) respectively, and the unique PVYZ-NTN strain induced milder vein clearing (VCl) in the upper non-inoculated leaves. A single amino acid polymorphisms (SAPs) I252V and a Q412 to R412 substitution in the HC-Pro cistron of the PVYZ-NTN strain might relate to the loss of VN in tobacco. The abundance of 18 out of the 26 tested miRNAs was increased upon infection by the severe strains PVYNTN and PVYN−Wi. Expression of a group of defense related transcripts were increased accordingly. Two miRNAs, nta-miR6020a-5p and nta-miR6164a/b, which target the TIR-NBS-LRR type resistant TMV N genes involving in signal transduction, might correlate with the PVYNTN and PVYN-Wi induced VN. The down-regulated mRNAs, e.g., RAP2-7 and TOE3, PXC3, LRR-RLK, ATHB-14 and TCP4 targeted by nta-miR172, nta-miR390, nta-miR482, nta-miR166 and nta-miR319/159 respectively, were related to regulation of transcription, protein phosphorylation and cell differentiation. The observed strain-specific alteration of miRNAs and their targets are host dependent and corresponds to the symptom severity and the viral HC-Pro RNA levels.

The ability of Escherichia coli to grow on l-lactate as a sole carbon source depends on the expression of the lldPRD operon. A striking feature of this operon is that the gene encoding the transcriptional regulator (LldR) is located between the genes encoding the permease (LldP) and the dehydrogenase (LldD). In this study, we report that the dosages of the LldP, LldR, and LldD proteins are not modulated on the transcriptional level. Instead, modulation of the protein dosage is correlated primarily with RNase E-dependent mRNA-processing events that take place within the lldR mRNA, leading to the immediate inactivation of lldR, to differential segmental stabilities of the resulting cleavage products, and to differences in the translation efficiencies of the three cistrons. A model for the processing events controlling the molar quantities of the proteins in the lldPRD operon is presented and discussed. IMPORTANCE Adjustment of gene expression is critical for proper cell function. In the case of polycistronic transcripts, posttranscriptional regulatory mechanisms can be used to fine-tune the expression of individual cistrons. Here, we elucidate how the protein dosage of the Escherichia coli lldPRD operon, which presents the paradox of having the gene encoding a regulator protein located between genes that code for a permease and an enzyme, is regulated. Our results demonstrate that the key event in this regulatory mechanism involves the RNase E-dependent cleavage of the primary lldPRD transcript at an internal site(s) located within the lldR cistron, resulting in a drastic decrease in the amount of intact lldR mRNA, in differential segmental stabilities of the resulting cleavage products, and in differences in the translation efficiencies of the three cistrons.

The circular extrachromosomal ribosomal DNA (rDNA) element of Naegleria fowleri strain LEE was molecularly cloned and fully sequenced. The element comprises 15,786 bp and encodes a single copy of the organism’s rDNA cistron. The nonribosomal sequence contains five potential open reading frames, two large direct repeat sequences, and numerous smaller repeated-sequence regions., The circular extrachromosomal ribosomal DNA (rDNA) element of Naegleria fowleri strain LEE was molecularly cloned and fully sequenced. The element comprises 15,786 bp and contains a single copy of the organism’s rDNA cistron. The nonribosomal sequence contains five potential open reading frames, two large direct repeat sequences, and numerous smaller repeated-sequence regions.

In bacteria, mRNA decay is a major mechanism for regulating gene expression. In Escherichia coli, mRNA decay initiates with endonucleolytic cleavage by RNase E. Translating ribosomes impede RNase E cleavage, thus providing stability to mRNA. In transcripts containing multiple cistrons, the translation of each cistron initiates separately. The effect of internal translation initiations on the decay of polycistronic transcripts remains unknown, which we have investigated here using the four-cistron galETKM transcript. We find that RNase E cleaves a few nucleotides (14–36) upstream of the translation initiation site of each cistron, generating decay intermediates galTKM, galKM, and galM mRNA with fewer but full cistrons. Blocking translation initiation reduced stability, particularly of the mutated cistrons and when they were the 5′-most cistrons. This indicates that, together with translation failure, the location of the cistron is important for its elimination. The instability of the 5′-most cistron did not propagate to the downstream cistrons, possibly due to translation initiation there. Cistron elimination from the 5′ end was not always sequential, indicating that RNase E can also directly access a ribosome-free internal cistron. The finding in gal operon of mRNA decay by cistron elimination appears common in E. coli and Salmonella.

Temperature plays an important role in bacteria-host interactions and can be a determining factor for host switching. In this study we sought to investigate the reasons behind growth temperature restriction in the entomopathogenic enterobacterium Photorhabdus. Photorhabdus has a complex dual symbiotic and pathogenic life cycle. The genus consists of 19 species but only one subgroup, previously all classed together as P. asymbiotica, have been shown to cause human disease. These clinical isolates necessarily need to be able to grow at 37 °C, whilst the remaining species are largely restricted to growth temperatures below 34 °C and are therefore unable to infect mammalian hosts. Here, we have isolated spontaneous mutant lines of P. laumondii DJC that were able to grow up to 36 °C-37 °C. Following whole genome sequencing of 29 of these mutants we identified a single gene, encoding a protein with a RecG-like helicase domain, that for the majority of isolates contained single nucleotide polymorphisms. Importantly, provision of the wild-type allele of this gene in trans restored the temperature restriction, confirming the mutations are recessive, and the dominant effect of the protein product of this gene. The gene appears to be part of a short three cistron operon, which we have termed the Temperature Restricting Locus (TRL). Transcription reporter strains revealed that this operon is induced upon the switch from 30 °C to 37 °C, leading to replication arrest of the bacteria. TRL is absent from all of the human pathogenic species so far examined, although its presence is not uniform in different strains of the P. luminescens subgroup. In a wider context, the presence of this gene is not limited to Photorhabdus, being found in phylogenetically diverse proteobacteria. We therefore suggest that this system may play a more fundamental role in temperature restriction in diverse species, relating to as yet cryptic aspects of their ecological niches and life cycle requirements.

Internal ribosome entry site (IRES) sequences have become a valuable tool in the construction of gene transfer and therapeutic vectors for multi-cistronic gene expression from a single mRNA transcript. The optimal conditions for effective use of this sequence to construct a functional expression vector are not precisely defined but it is generally assumed that the internal ribosome entry site dependent expression of the second gene in such as cassette is less efficient than the cap-dependent expression of the first gene. Mainly tailoring inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector further in construction of optimised cassette for gene therapy of familial hypercholesterolemia. We tailored the size of the inter-cistronic spacer sequence at the 5′ region of the internal ribosome entry site sequence using sequential deletions and demonstrated that the expression of the 3′ gene can be significantly increased to similar levels as the cap-dependent expression of the 5’ gene. Maximum expression efficiency of the downstream gene was obtained when the spacer is composed of 18–141 base pairs. In this case a single mRNA transcriptional unit containing both the first and the second Cistron was detected. Whilst constructs with spacer sequences of 216 bp or longer generate a single transcriptional unit containing only the first Cistron. This suggests that long spacers may affect transcription termination. When the spacer is 188 bp, both transcripts were produced simultaneously in most transfected cells, while a fraction of them expressed only the first but not the second gene. Expression analyses of vectors containing optimised cassettes clearly confirm that efficiency of gene transfer and biological activity of the expressed transgenic proteins in the transduced cells can be achieved. Furthermore, Computational analysis was carried out by molecular dynamics (MD) simulation to determine the most emerges as viable containing specific binding site and bridging of 5′ and 3′ ends involving direct RNA-RNA contacts and RNA-protein interactions. These results provide a mechanistic basis for translation stimulation and RNA resembling for the synergistic stimulation of cap-dependent translation. Keywords: Internal ribosome entry site, IRES, Gene therapy, Inter-cistronic sequences, MD simulation, Protein structure modeling, Familial hypercholesterolemia

Two intercistronic regions were identified as functional intercistronic expression elements (IEE) for the simultaneous expression of aphA-6 and gfp in a synthetic operon in the chloroplast of C. reinhardtii. Chlamydomonas reinhardtii, a biflagellate photosynthetic microalga, has been widely used in basic and applied science. Already three decades ago, Chlamydomonas had its chloroplast genome transformed and to this day constitutes the only alga routinely used in transplastomic technology. Despite the fact that over a 100 foreign genes have been expressed from the chloroplast genome, little has been done to address the challenge of expressing multiple genes in the form of operons, a development that is needed and crucial to push forward metabolic engineering and synthetic biology in this organism. Here, we studied five intercistronic regions and investigated if they can be used as intercistronic expression elements (IEE) in synthetic operons to drive the expression of foreign genes in the chloroplast of C. reinhardtii. The intercistronic regions were those from the psbB-psbT, psbN-psbH, psaC-petL, petL-trnN and tscA-chlN chloroplast operons, and the foreign genes were the aminoglycoside 3'-phosphotransferase (aphA-6), which confers resistance to kanamycin, and the green fluorescent protein gene (gfp). While all the intercistronic regions yielded lines that were resistant to kanamycin, only two (obtained with intercistronic regions from psbN-psbH and tscA-chlN) were identified as functional IEEs, yielding lines in which the second cistron (gfp) was translated and generated GFP. The IEEs we have identified could be useful for the stacking of genes for metabolic engineering or synthetic biology circuits in the chloroplast of C. reinhardtii.

Small noncoding RNAs (sRNAs) from mRNA 3′ UTRs seem to present a previously unrecognized layer of bacterial post-transcriptional control whereby mRNAs influence each other's expression, independently of transcriptional control. Studies in Escherichia coli and Salmonella enterica showed that such sRNAs are natural products of RNase E-mediated mRNA decay and associate with major RNA-binding proteins (RBPs) such as Hfq and ProQ. If so, there must be additional sRNAs from mRNAs that accumulate only under specific physiological conditions. We test this prediction by characterizing candidate NarS that represents the 3′ UTR of nitrate transporter NarK whose gene is silent during standard aerobic growth. We find that NarS acts by Hfq-dependent base pairing to repress the synthesis of the nitrite transporter, NirC, resulting in mRNA cross-regulation of nitrate and nitrite transporter genes. Interestingly, the NarS-mediated repression selectively targets the nirC cistron of the long nirBDC-cysG operon, an observation that we rationalize as a mechanism to protect the bacterial cytoplasm from excessive nitrite toxicity during anaerobic respiration with abundant nitrate. Our successful functional assignment of a 3′ UTR sRNA from a non-standard growth condition supports the notion that mRNA crossregulation is more pervasive than currently appreciated.

UMR BFP - Equipe Virologie; International audience; We developed an innovative RNAi concept based on two gene constructs built from the capsid gene (CP) cistron of the Plum pox virus (PPV) genome. First, designated as amiCPRNA, a potential molecule interfering with PPV genome translation and the second one is the ami-siCPRNA to target viral genome translation and PPV RNA replication. Following the previous engineering of these constructs in an experimental herbaceous host, they were introduced into Prunus domestica (plum tree) genome. Previously propagated onto a susceptible rootstock, these clones were graft-inoculated with PPV. After four dormancy cycles, and consistent with our experience of PPV infection, some clones showed a common phenomenon of silencing that can differ between the detailed plant phenotypes. Three different phenotypes were developed by the amisiCPRNA clones. First, the high resistance character shown by the amisiCPRNA plum-7 that was similar to the resistance expressed by HoneySweet plum. Secondly, a recovery reaction was developed by the two other amisiCPRNA plum-3 and plum-4 that differed from the rest, characterized as susceptible clones, among these were the amiCPRNA plums. Having assessed the behavior of these plums versus the herbaceous host accumulating the similar form of RNAi: ami-, si-, and ami-siRNA, challenging assays in perennials consistently reflect the natural context of viral genome targeting

Methylation of lysine 4 in histone 3 (H3K4) is a post-translational modification that promotes gene expression. H3K4 methylation can be reversed by specific demethylases with an enzymatic Jumonji C domain. In Arabidopsis thaliana, H3K4-specific JUMONJI (JMJ) proteins distinguish themselves by the association with an F/Y-rich (FYR) domain. Here, we report that jmj14 mutations partially suppress reduced root meristem size and growth vigor of brevis radix (brx) mutants. Similar to its close homologs, JMJ15, JMJ16 and JMJ18, the JMJ14 promoter confers expression in mature root vasculature. Yet, unlike jmj14, neither jmj16 nor jmj18 mutation markedly suppresses brx phenotypes. Domain-swapping experiments suggest that the specificity of JMJ14 function resides in the FYR domain. Despite JMJ14 promoter activity in the mature vasculature, jmj14 mutation affects root meristem size. However, JMJ14 protein is observed throughout the meristem, suggesting that the JMJ14 transcript region contributes substantially to the spatial aspect of JMJ14 expression. In summary, our data reveal a role for JMJ14 in root growth in sensitized genetic backgrounds that depends on its FYR domain and regulatory input from the JMJ14 cistron.

Expansion of a hexanucleotide repeat (HRE), GGGGCC, in the C9ORF72 gene is recognized as the most common cause of familial amyotrophic lateral sclerosis (FALS), frontotemporal dementia (FTD) and ALS-FTD, as well as 5–10% of sporadic ALS. Despite the location of the HRE in the non-coding region (with respect to the main C9ORF72 gene product), dipeptide repeat proteins (DPRs) that are thought to be toxic are translated from the HRE in all three reading frames from both the sense and antisense transcript. Here, we identified a CUG that has a good Kozak consensus sequence as the translation initiation codon. Mutation of this CTG significantly suppressed polyglycine-alanine (GA) translation. GA was translated when the G(4)C(2) construct was placed as the second cistron in a bicistronic construct. CRISPR/Cas9-induced knockout of a non-canonical translation initiation factor, eIF2A, impaired GA translation. Transfection of G(4)C(2) constructs induced an integrated stress response (ISR), while triggering the ISR led to a continuation of translation of GA with a decline in conventional cap-dependent translation. These in vitro observations were confirmed in chick embryo neural cells. The findings suggest that DPRs translated from an HRE in C9ORF72 aggregate and lead to an ISR that then leads to continuing DPR production and aggregation, thereby creating a continuing pathogenic cycle.

The lactic acid bacteria (LAB) Leuconostoc citreum are non-sporulating hetero-fermentative bacteria that play an important role in the fermented food industry. In this study, for the enhanced and reliable production of recombinant proteins in L. citreum, we developed a bicistronic design (BCD) expression system which includes a short leader peptide (1st cistron) followed by target genes (2nd cistron) under the control of a single promoter. Using superfolder green fluorescent protein (sfGFP) as a reporter, the functionality of BCD in L. citreum was verified. Further, to improve the expression in BCD, we tried to engineer a Shine-Dalgarno sequence (SD2) for the 2nd cistron and a promoter by FACS screening of random libraries, and both strong SD2 (eSD2) and promoter (P710V4) were successfully isolated. The usefulness of the engineered BCD with P710V4 and eSD2 was further validated using three model proteins—glutathione-s-transferase, human growth hormone, and α-amylase. All examined proteins were successfully produced with levels highly increased compared with those in the original BCD as well as the monocistronic design (MCD) expression system.

SUMMARY Taxonomy. Soybean mosaic virus (SMV) is a species within the genus Potyvirus, family Potyviridae that includes almost a quarter of all known plant RNA viruses affecting agriculturally important plants. The Potyvirus genus is the largest of all genera of plant RNA viruses with 160 species. Particle. The filamentous particles of SMV, typical of potyviruses, are about 7,500 A long and 120 A in diameter with a central hole of about 15 A in diameter. Coat protein residues are arranged in helice of about 34 A pitch having slightly less than 9 subunits per turn. Genome. The SMV genome consists of a single-stranded positive-sense polyadenylated RNA of approximately 9.6 kb with a virus-encoded protein (VPg) linked at the 5' terminus. The genomic RNA contains a single large open reading frame (ORF). The polypeptide produced from the large ORF is processed proteolytically by three viral-encoded proteinases to yield about 10 functional proteins. A small ORF, partially overlapping the P3 cistron, pipo, is encoded as a fusion protein in the N-terminus of P3 (P3N+PIPO). Biological properties. SMV's host range is restricted mostly to two plant species of a single genus; Glycine max (cultivated soybean) and G. soja (wild soybean). SMV is transmitted by aphids non-persistently and by seeds. Variability of SMV is recognized by reactions on cultivars with dominant resistance (R) genes. Recessive resistance genes are not known. Geographical distribution and economic importance. As a consequence of its seed transmissibility, SMV is present in all soybean growing areas of the world. SMV infections can reduce significantly seed quantity and quality (e.g., mottled seed coats, reduced seed size and viability, and altered chemical composition). Control. The most effective means of managing losses from SMV are planting virus-free seeds and cultivars containing single or multiple R genes. Key attractions. The interactions of SMV with soybean genotypes containing different dominant R genes and understanding functional role(s) of SMV-encoded proteins in virulence, transmission and pathogenicity have been intensively investigated. The SMV-soybean pathosystem has become an excellent model for examining the genetics and genomics of uniquely complex gene-for-gene resistance model in a crop of worldwide importance. This article is protected by copyright. All rights reserved.

Molecular biological experiments sometimes require expression of two or more genes in a single cell with an accurate ratio between their expression levels. One of the methods to provide this control is the use of the internal ribosome entry site (IRES) from the encephalomyocarditis virus as a separating insert between two target genes in the expression vector. Previously, it was shown that the efficacy of translation of the gene after IRES varies considerably in a range from 6 to 100% depending on the cell type. In neurons, the exact ratio between the expression levels of genes that are located before and after the IRES in the expression vector is unknown. Here, we analyzed the ratio between the amounts of products of the first and second genes located before and after the IRES in a plasmid that was used to transfect neurons in a primary hippocampal culture. We created two plasmid vectors that contain genes of the yellow (Venus) and red (mCherry) fluorescent proteins in different orders, which are separated by the IRES. We found that the unmodified IRES sequence of the encephalomyocarditis virus decreases the expression in the second cistron by a factor of 2.7 in a primary culture of hippocampal neurons. These data will help us to use currently available libraries of mutant IRES sequences for accurate control of the relationships between the expression of different target genes in neurons.

Human neurturin (NTN) is a cystine knot growth factor with potential therapeutic use in diseases such as Parkinson's and diabetes. Scalable high titer production of native NTN is particularly challenging because of the cystine knot structure which consists of an embedded ring comprised of at least three disulfide bonds. We sought to pursue enhanced scalable production of NTN in Escherichia coli. Our initial efforts focused on codon optimization of the first two codons following AUG but these studies resulted in only a marginal increase in NTN expression. Therefore, we pursued an alternative strategy of using a bicistronic vector for NTN expression designed to reduce mRNA secondary structure to achieve increased ribosome binding and re-initiation. The first cistron was designed to prevent sequestration of the translation initiation region in a secondary conformation. The second cistron, which contained the NTN coding sequence itself, was engineered to disrupt double bonded base pairs and destabilize the secondary structure for ribosome re-initiation. The ensemble approach of reducing NTN's mRNA secondary structure and using the bicistronic vector had an additive effect resulting in significantly increased NTN expression. Our stain selection studies were conducted in a miniaturized bioreactor. An optimized strain was selected and scaled up to a 100 liter fermentor which yielded an inclusion body titer of 2 g/L. The inclusion bodies were refolded to yield active NTN. We believe that our strategy is applicable to other candidate proteins that are difficult-to-express due to stable mRNA secondary structures. This article is protected by copyright. All rights reserved

Potyviruses are plus-strand RNA viruses that can be easily transformed into expression vectors to quickly express one carotenogenic enzyme or transcription factor, or more, in plant tissues. Unlike the technically challenging and time-consuming process of plant transformation, manipulation of a roughly 10,000 nt-long viral genome is rather straightforward via common molecular biology techniques. Here I describe how to insert the cDNAs of the proteins of interest into two particular positions of the cDNA of a Tobacco etch virus (TEV) mutant that lacks the viral NIb cistron and only infects the plants in which this protein is expressed. This deletion increases the space to harbor foreign sequences. The selection of the expression site must be made according to subcellular localization requirements. The recombinant virus is then inoculated into Nicotiana benthamiana plants by means of Agrobacterium tumefaciens. The expression of the viral genome entails the production of carotenogenic proteins in the plant tissues with a consequent effect on the plant carotenoid pathway.

Concerted evolution of the ribosomal DNA array has been studied in numerous eukaryotic taxa, yet is still poorly understood. rDNA genes are repeated dozens to hundreds of times in the eukaryotic genome (Eickbush and Eickbush, 2007) and it is believed that these arrays are homogenized through concerted evolution (Zimmer et al., 1980; Dover, 1993) preventing the accumulation of intragenomic, and intraspecific, variation. However, numerous studies have reported rampant intragenomic and intraspecific variation in the rDNA array (Ganley and Kobayashi, 2011; Naidoo et al., 2013; Hughes and Petersen, 2001; Lindner and Banik, 2011; Li et al., 2013; Lindner et al., 2013; Hughes et al., 2018), contradicting our current understanding of concerted evolution. The internal transcribed spacers (ITS) of the rDNA cistron are the most commonly used DNA barcoding region in Fungi (Schoch et al., 2012), and rely on concerted evolution to homogenize the rDNA array leading to a "barcode gap" (Puillandre et al., 2012). Here we show that in Boletus edulis Bull., ITS intragenomic variation persists at low allele frequencies throughout the rDNA array, this variation does not correlate with genomic relatedness between populations, and rDNA genes may not evolve in a strictly concerted fashion despite the presence of unequal recombination and gene conversion. Under normal assumptions, heterozygous positions found in ITS sequences represent hybridization between populations, yet through allelic mapping of the rDNA array we found numerous heterozygous alleles to be stochastically introgressed throughout, presenting a dishonest signal of gene flow. Moreover, despite the signal of gene flow in ITS, our organisms were highly inbred, indicating a disconnect between true gene flow and barcoding signals. In addition, we show that while the mechanisms of concerted evolution are ongoing in pseudo-heterozygous individuals, they are not fully homogenizing the ITS array. Concerted evolution of the rDNA array may insufficiently homogenize the ITS gene, allowing for misleading signals of gene flow to persist, vastly complicating the use of the ITS locus for DNA barcoding in Fungi.

The Clostridium perfringens alpha toxin (CPA), encoded by the plc gene, is the causative pathogen of gas gangrene, which is a lethal infection. In this study, we used an E. coli system for the efficient production of recombinant proteins and developed a bicistronic design (BCD) expression construct consisting of two copies of the C-terminal (247–370) domain of the alpha toxin (CPA-C) in the first cistron, followed by Cholera Toxin B (CTB) linked with another two copies of CPA-C in the second cistron that is controlled by a single promoter. Rabbits were immunized twice with purified proteins (rCPA-C rCTB-CPA-C) produced in the BCD expression system, with an inactivated recombinant E. coli vaccine (RE), C. perfringens formaldehyde-inactivated alpha toxoid (FA-CPA) and C. perfringens l -lysine/formaldehyde alpha toxoid (LF-CPA) vaccines. Following the second vaccination, 0.1 mL of pooled sera of the RE-vaccinated rabbits could neutralize 12× mouse LD100 (100% lethal dose) of CPA, while that of the rCPA-C rCTB-CPA-C-vaccinated rabbits could neutralize 6× mouse LD100 of CPA. Antibody titers against CPA were also assessed by ELISA, reaching titers as high as 1:2048000 in the RE group; this was significantly higher compared to the C. perfringens alpha toxoid vaccinated groups (FA-CPA and LF-CPA). Rabbits from all vaccinated groups were completely protected from a 2× rabbit LD100 of CPA challenge. These results demonstrate that the recombinant proteins are able to induce a strong immune responses, indicating that they may be potentially utilized as targets for novel vaccines specifically against the C. perfringens alpha toxin.

To better understand the structure and variability of the 45S rDNA cistron and its evolutionary dynamics in grasshoppers, we performed a detailed analysis combining classical and molecular cytogenetic data with whole-genome sequencing in Abracris flavolienata, which shows extraordinary variability in the chromosomal distribution for this element. We found astonishing variability in the number and size of rDNA clusters at intra- and inter-population levels. Interestingly, FISH using distinct parts of 45S rDNA cistron (18S rDNA, 28S rDNA, and ITS1) as probes revealed a distinct number of clusters, suggesting independent mobility and amplification of the 45S rDNA components. This hypothesis is consistent with the higher genomic coverage of almost the entire cistron of 45S rDNA observed in A. flavolineata compared to other grasshoppers, besides coverage variability along the 45S rDNA cistron in the species. In addition, these differences in coverage for distinct components of the 45S rDNA cistron indicate emergence of pseudogenes evidenced by existence of truncated sequences, demonstrating the rDNA dynamics in the species. Although the chromosomal distribution of 18S rDNA was highly variable, the chromosomes 1, 3, 6, and 9 harbored rDNA clusters in all individuals with the occurrence of NOR activity in pair 9, suggesting ancestry or selective pressures to prevent pseudogenization of rDNA sequences in this chromosome pair. Additionally, small NORs and cryptic rDNA loci were observed. Finally, there was no evidence of enrichment and association of transposable elements, at least, inside or nearby rDNA cistron. These findings broaden our knowledge of rDNA dynamics, revealing an independent movement and amplification of segments of 45S rDNA cistron, which in A. flavolineata could be attributed to ectopic recombination. Fil: Ferretti, Ana B. S. M.. Universidade Estadual Paulista Julio de Mesquita Filho. Faculdade de Engenharia.; Brasil Fil: Ruiz Ruano, Francisco J.. Uppsala University; Suecia. Universidad de Granada; España Fil: Milani, Diogo. Universidade Estadual Paulista Julio de Mesquita Filho. Faculdade de Engenharia.; Brasil Fil: Loreto, Vilma. Universidade Federal de Pernambuco; Brasil Fil: Marti, Dardo Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina Fil: Ramos, Érica Daniela. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil Fil: Martins, Cesar. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil Fil: Cabral de Mello, Diogo C.. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil

Background Recombinant human Fibroblast growth factor 21 (rhFGF21) is an endocrine hormone that has profound effects on treatment of metabolic diseases. However, rhFGF21 is prone to form inclusion body when expressed in bacteria, which results in, the downstream process of purification of bioactive rhFGF21 is time-consuming and labor intensive. The aim of this work is to explore a new method for improving the soluble expression and secretion level of rhFGF21 in B. subtilis. Results A codon optimized rhFGF21 gene was expressed under the control of a strong inducible promoter PmalA in B. subtilis. A mini-cistron cassette (from gsiB) was located upstream of rhFGF21 in expression vector (pMATEFc5), which could reduce the locally stabilized mRNA secondary structure of transcripts and enhance the efficiency of translation initiation. Then various chaperones were further overexpressed to improve the expression efficiency of rhFGF21. Results showed that overexpression of the chaperone DnaK contributed to the increase of solubility of rhFGF21. Moreover, an extracellular proteases deficient strain B. subtilis Kno6cf was used to accumulate the secreted rhFGF21 solidly. In addition, eleven signal peptides from B. subtilis were evaluated and the SPdacB appeared the highest secretion yield of rhFGF21 in B. subtilis. Finally, the combinatorial optimized strain achieved an about ninefold increase of the soluble rhFGF21 production after 24 h of flask fermentation in comparison with the initial production strain. Conclusion This work provided a comprehensive strategy for secretory expressing the heterologous protein rhFGF21 in B. subtilis. To our knowledge, this is the first report of the highly efficient production of rhFGF21 in B. subtilis and this approach may provide some suggestions for heterologous proteins production in B. subtilis. Electronic supplementary material The online version of this article (10.1186/s12934-019-1066-4) contains supplementary material, which is available to authorized users.

We discovered a soybean mosaic virus (SMV) variant (4278-1) that caused systemic infections in Nicotiana benthamiana plants, resulting in stem stunting and leaf shriveling. The virus had a particle morphology and incubation period similar to those of other SMV isolates but differed from them in the leaf symptoms it caused when infecting soybean and N. benthamiana. The genome of this variant consisted of a 9994-nt single-stranded RNA, which was different from most of the other known SMV isolates (approximately 9600 nt). Interestingly, we found evidence that two recombination events (nt 1-476 and nt 1145-1349) had occurred between 4278-1 and a watermelon mosaic virus analogue (WMV analogue), in the 5' untranslated region and the P1 cistron.

It is general wisdom that termination of bacterial protein synthesis is obligatorily followed by recycling governed by the factors ribosomal recycling factor (RRF), EF-G, and IF3, where the ribosome dissociates into its subunits. In contrast, a recently described 70S-scanning mode of initiation holds that after termination, scanning of 70S can be triggered by fMet-tRNA to the initiation site of a downstream cistron. Here, we analyze the apparent conflict. We constructed a bicistronic mRNA coding for luciferases and showed with a highly resolved in vitro system that the expression of the second cistron did not at all depend on the presence of active RRF. An in vivo analysis cannot be performed in a straightforward way, since RRF is essential for viability and therefore, the RRF gene cannot be knocked out. However, we found an experimental window, where the RRF amount could be reduced to below 2.5%, and in this situation, the expression of the second cistron of a bicistronic luciferase mRNA was only moderately reduced. Both in vitro and in vivo results suggested that RRF-dependent recycling is not an obligatory step after termination, in agreement with the previous findings concerning 70S-scanning initiation. In this view, recycling after termination is a special case of the general RRF function, which happens whenever fMet-tRNA is not available for triggering 70S scanning. (C) 2016 Elsevier Ltd. All rights reserved.

In this study, the full-length nucleotide sequences of four Iranian PVY isolates belonging to PVY(N) strain were determined. The genome of Iranian PVY isolates were 9,703-9,707 nucleotides long encoding all potyviral cistrons including P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb and CP with coding regions of 825, 1,395, 1,095, 156, 1,902, 156, 564, 732, 1,557 and 801 nucleotides in length, respectively. The length of pipo, embedded in the P3 cistron, was 231 nucleotides. Phylogenetic analysis showed that the Iranian isolates clustered with European recombinant NTN isolates in the N lineage. Recombination analysis demonstrated that Iranian PVY(N) isolates had a typical European PVY(NTN) genome having three recombinant junctions while PVY(N) and PVY(O) were identified as the parents. We used dN/dS methods to detect candidate amino acid positions for positive selection in viral proteins. The mean ω ratio differed among different genes. Using model M0, ω values were 0.267 (P1), 0.085 (HC-Pro), 0.153 (P3), 0.050 (CI), 0.078 (VPg), 0.087 (NIa-pro), 0.079 (NIb) and 0.165 (CP). The analysis showed different sites within P1, P3 and CP were under positive selection pressure, however, the sites varied among PVY populations. To the best of our knowledge, our analysis provides the first demonstration of population structure of PVY(N) strain in mid-Eurasia Iran using complete genome sequences and highlights the importance of recombination and selection pressure in the evolution of PVY.

Turnip mosaic virus (TuMV) infections affect many Arabidopsis developmental traits. This paper analyzes, at different levels, the development-related differential alterations induced by different strains of TuMV, represented by isolates UK 1 and JPN 1. The genomic sequence of JPN 1 TuMV isolate revealed highest divergence in the P1 and P3 viral cistrons, upon comparison with the UK 1 sequence. Infectious viral chimeras covering the whole viral genome uncovered the P3 cistron as a major viral determinant of development alterations, excluding the involvement of the PIPO open reading frame. However, constitutive transgenic expression of P3 in Arabidopsis did not induce developmental alterations nor modulate the strong effects induced by the transgenic RNA silencing suppressor HC-Pro from either strain. This highlights the importance of studying viral determinants within the context of actual viral infections. Transcriptomic and interactomic analyses at different stages of plant development revealed large differences in the number of genes affected by the different infections at medium infection times but no significant differences at very early times. Biological functions affected by UK 1 (the most severe strain) included mainly stress response and transport. Most cellular components affected cell-wall transport or metabolism. Hubs in the interactome were affected upon infection. © 2015 The American Phytopathological Society.

The complete 9,556-nt genomic sequence of a putative new potyvirus, tentatively named “platycodon mild mottle virus” (PlaMMV), infecting Platycodon grandiflorum was determined (GenBank accession no. MH779625). A single large open reading frame (ORF; nt 121-9360, 3079 aa) encoding a polyprotein that was predicted to be cleaved into 10 mature proteins was detected. By comparison with other potyviruses, nine cleavage sites and conserved domains/motifs were identified. PlaMMV also has a highly conserved GA6 motif (nt 2857-2863) that could enable a small overlapping ORF (PIPO) embedded within the P3 cistron to be translated by a viral polymerase slippage mechanism. A BLAST analysis showed that the complete PlaMMV nucleotide sequence shared 70% identity (40% query coverage) with carrot thin leaf virus (JX156434) and that the complete amino acid sequence of the PlaMMV polyprotein shared 54% identity (98% query coverage) with Thunberg fritillary mosaic virus (CAI59123). These results suggest that PlaMMV is a new member of the genus Potyvirus in the family Potyviridae.

A yeast strain was isolated during a study on vineyard-associated yeast strains from Apulia in Southern Italy. ITS and LSU D1/D2 rDNA sequences showed this strain not to belong to any known species and was described as the type strain of Ogataea uvarum sp. nov., a close relative of O. philodendri. Several secondary peaks appeared in the sequences, suggesting internal heterogeneity among the copies of the rDNA. This hypothesis was tested by sequencing single clones of the marker region. The analyses showed different levels of variability throughout the operon with differences between the rRNA encoding genes and the internally transcribed regions. O. uvarum and O. philodendri share high frequency variants, i.e., variants frequently found in many clones, whereas there is a large variability of the low frequency polymorphisms, suggesting that the mechanism of homogenization is more active with the former than with the latter type of variation. These findings indicate that low frequency variants are detected in Sanger sequencing as secondary peaks whereas in Next Generation Sequencing (NGS) of metagenomics DNA would lead to an overestimate of the alpha diversity. For the first time in our knowledge, this investigation shed light on the variation of the copy number of the rDNA cistron during the yeast speciation process. These polymorphisms can be used to investigate on the processes occurring in these taxonomic markers during the separation of fungal species, it being a genetic process highly frequent in the complex microbial ecosystem existing in grape, must and wine.

Genetic coding in bacteria largely operates via the “one gene-one protein” paradigm. However, the peculiarities of the mRNA structure, the versatility of the genetic code, and the dynamic nature of translation sometimes allow organisms to deviate from the standard rules of protein encoding. Bacteria can use several unorthodox modes of translation to express more than one protein from a single mRNA cistron. One such alternative path is the use of additional translation initiation sites within the gene. Proteins whose translation is initiated at different start sites within the same reading frame will differ in their N termini but will have identical C-terminal segments. On the other hand, alternative initiation of translation in a register different from the frame dictated by the primary start codon will yield a protein whose sequence is entirely different from the one encoded in the main frame. The use of internal mRNA codons as translation start sites is controlled by the nucleotide sequence and the mRNA folding. The proteins of the alternative proteome generated via the “genes-within-genes” strategy may carry important functions. In this review, we summarize the currently known examples of bacterial genes encoding more than one protein due to the utilization of additional translation start sites and discuss the known or proposed functions of the alternative polypeptides in relation to the main protein product of the gene. We also discuss recent proteome- and genome-wide approaches that will allow the discovery of novel translation initiation sites in a systematic fashion.

Oligopeptide "2A" and "2A-like" sequences ("2As"; 18-25aa) are found in a range of RNA virus genomes controlling protein biogenesis through "recoding" of the host-cell translational apparatus. Insertion of multiple 2As within a single open reading frame (ORF) produces multiple proteins; hence, 2As have been used in a very wide range of biotechnological and biomedical applications. During translation, these 2A peptide sequences mediate a eukaryote-specific, self-"cleaving" event, termed "ribosome skipping" with very high efficiency. A particular advantage of using 2As is the ability to simultaneously translate a number of proteins at an equal level in all eukaryotic systems although, naturally, final steady-state levels depend upon other factors-notably protein stability. By contrast, the use of internal ribosome entry site elements for co-expression results in an unbalanced expression due to the relative inefficiency of internal initiation. For example, a 1:1 ratio is of particular importance for the biosynthesis of the heavy-chain and light-chain components of antibodies: highly valuable as therapeutic proteins. Furthermore, each component of these "artificial polyprotein" systems can be independently targeted to different sub-cellular sites. The potential of this system was vividly demonstrated by concatenating multiple gene sequences, linked via 2A sequences, into a single, long, ORF-a polycistronic construct. Here, ORFs comprising the biosynthetic pathways for violacein (five gene sequences) and β-carotene (four gene sequences) were concatenated into a single cistron such that all components were co-expressed in the yeast Pichia pastoris. In this review, we provide useful information on 2As to serve as a guide for future utilities of this co-expression technology in basic research, biotechnology, and clinical applications.

Trunk and branch cankers are among the most important diseases compromising avocado production worldwide. A novel species, Neocosmospora perseae sp. nov. is described isolated from trunk lesions on Persea americana in the main avocado producing area of Sicily, Italy. The new species is characterised using a polyphasic approach including morphological characters and a multilocus molecular phylogenetic analysis based on partial sequences of the translation elongation factor-1α, the internal transcribed spacer regions plus the large subunit of the rDNA cistron, and the RNA polymerase II second largest subunit. Pathogenicity tests and the fulfilment of Koch’s postulates confirm N. perseae as a novel canker pathogen of Persea americana.

The generation of stable, high-level monoclonal antibody (mAb) producing cell lines remains a major challenge in biopharmaceutical industry. The commonly used plasmid vectors for mAb expression, which express light chain (LC), heavy chain (HC), and selection marker genes on separate vectors or via multiple promoters on a single vector, are not able to accurately control the ratio of LC over HC expression and tend to result in non-expressing clones. To overcome these issues, we have developed a tricistronic vector using two internal ribosome entry sites (IRES) to express the LC, HC, and dihydrofolate reductase (DHFR) selection marker genes in one transcript. In this tricistronic vector, the three genes are under the control of a hapten-modified human cytomegalovirus (hCMV) promoter containing a core CpG island element (IE) to enhance the production stability. The LC gene is arranged as the first cistron followed by a wild-type IRES to control the HC expression. Such design expresses excess LC polypeptides which enhance mAb expression level and reduce aggregate. A mutated IRES with attenuated strength is applied on DHFR to reduce its expression for enhancing the stringency of selection for high producers. This vector allows easy generation of stable, high mAb producing CHO DG44 pools and clones for antibody development and manufacturing.

International audience; In spite of their widespread occurrence, only few host jumps by plant viruses have been evidenced and the molecular bases of even fewer have been determined. A combination of three independent approaches, (i) experimental evolution followed by reverse genetics analysis, (ii) positive selection analysis and (iii) locus-by-locus analysis of molecular variance (AMOVA) allowed reconstructing the Potato virus Y (PVY; genus Potyvirus, family Potyviridae) jump to pepper (Capsicum annuum), probably from other solanaceous plants. Synthetic chimeras between infectious cDNA clones of two PVY isolates with contrasted levels of adaptation to C. annuum showed that the P3 and, to a lower extent, the CI cistron played important roles in infectivity towards C. annuum. The three analytical approaches pinpointed a single nonsynonymous substitution in the P3 and P3N-PIPO cistrons that evolved several times independently and conferred adaptation to C. annuum. In addition to increasing our knowledge of host jumps in plant viruses, this study illustrates also the efficiency of locus-by-locus AMOVA and combined approaches to reconstruct adaptive mutations in the genome of RNA viruses.

The complete nuclear ribosomal DNA (nrDNA) cistron sequence of Pyropia yezoensis has been sequenced, of which each unit is composed of the intergenic spacer (IGS), the small-subunit (SSU) ribosomal RNA (rRNA) gene, the internal transcribed spacer 1 (ITS1), the 5.8S rRNA gene, the internal transcribed spacer 2 (ITS2), and the large-subunit (LSU) rRNA gene. This cistron occurs in tandem repeats that can number in 100s to 1000s. Furthermore, in the analysis of the sequence data, we determined that introns were present in the small-subunit rRNA genes, for which the exon sequences (1834 bp) are almost identical. The intron numbers ranging from zero to two in the small-subunit rRNA genes are different between multiple individuals. However, the full length of the large-subunit rRNA genes is 4770 bp without any introns, and the 5.8S rRNA gene is 159 bp. Moreover, the 5′ and 3′ terminal sequences of the genic regions of the ribosomal cistron are extremely conservative even among various species in red alga. In addition, the range for ITS1 is 371–372 bp and for ITS2 is 532 to 535 bp. The IGS is 5984 bp in length and possesses upstream direct repeats, downstream repeats, and middle inverted repeats that make sequencing untoward. This is the first time that the complete nucleotide of the IGS region of P. yezoensis has been sequenced. The provision of the full length of nrDNA cistron will offer useful information to be widely utilized for identification, taxonomic relationships, and phylogenies in red algae.

Molecular analyses employing sequencing of the complete ribosomal RNA cistron (18S rDNA, ITS1, 5.8S rDNA, ITS2, and 28S rDNA) and transcriptome analysis of the RuBisCO gene (rbcL) were done on Chlorella vulgaris UTEX 2714. The constructed phylogenetic trees showed that C. vulgaris UTEX 2714 is Chlorella sorokiniana. Growth analysis and production of chlorophyll a over a range of increasing cultivation temperatures (27–40 °C) showed that this strain is far less thermotolerant in comparison to a common C. sorokiniana strain. A change in the taxonomic designation of strain UTEX 2714 is proposed.

Sugarcane streak mosaic virus (SCSMV) is a distinct member of Poacevirus in Potyviridae family causing mosaic disease in sugarcane. In this study, we computationally predicted potyvirus specific overlapping ORF, PIPO (~139 amino acid) in P3 cistron of known SCSMV genome in +1 reading frame using MLOGD. The nucleotide variations in P3 and the predicted PIPO were experimentally validated using SCSMV isolates from India. Phylogenetic relatedness of identified sequences (P3, PIPO) reflects strong selection and at least three subpopulations within the strains of SCSMV in India. These finding considerably broaden future investigation of the SCSMV pathogenesis in sugarcane.

The present study aimed to investigate the regulatory mechanism of the AmpC enzyme by analyzing the construction and function of AmpCR, AmpE and AmpG genes in the Dhahran (DHA)‑1 plasmid of Klebsiella pneumoniae (K. pneumoniae). The production of AmpC and extended‑spectrum β‑lactamase (ESBL) were determined following the cefoxitin (FOX) inducing test for AmpC, preliminary screening and confirmation tests for ESBL in 10 DHA‑1 plasmid AmpC enzymes of K. pneumoniae strains. AmpCR, AmpD, AmpE and AmpG sequences were analyzed by polymerase chain reaction. The pACYC184‑X plasmid analysis system was established and examined by regulating the pAmpC enzyme expression. The electrophoretic bands of AmpCR, AmpD, AmpE and AmpG were expressed. Numerous mutations in AmpC + AmpR (AmpCR) and in the intergenic region cistron of AmpC‑AmpR, AmpD, AmpE and AmpG were observed. The homology of AmpC and AmpR, in relation to the Morganella morganii strain, was 99%, which was determined by comparing the gene sequences of Kp1 with those of Kp17 AmpCR. The specific combination of AmpR and labeled probe demonstrated a band retarded phenomenon and established a spatial model of AmpR. All the enzyme production strains demonstrated Val93→Ala in AmpG; six transmembrane domains were found in AmpE in all strains, with the exception of Kp1 and Kp4, which had only three transmembrane segments that were caused by mutation. The DHA‑1 plasmid AmpC enzymes encoded by plasmid are similar to the inducible chromosomal AmpC enzymes, which are also regulated by AmpD, AmpE, AmpR and AmpG.

In this study, we found that the infectivity of zucchini yellow mosaic virus (ZYMV) in watermelon lines H1 and K6 changed from partial to complete after propagation in the susceptible watermelon line ZXG637. When using cucumber infected with strain ZYMV-CH87 as an inoculum (named ZYMV-CH87C), the mean incidences of infection in lines H1 and K6 were 6% and 11%, respectively. However, when these lines were inoculated with ZXG637 infected with ZYMV-CH87C (named ZYMV-637), 100% of the plants became infected. Sequencing of ZYMV from these different inoculums revealed two nucleotide changes in the P3 cistron in ZYMV-637, which resulted in changes in the amino acids at positions 768 and 857 of the P3 protein, compared with the original strain ZYMV-CH87. We named this variant the M768I857-variant. The M768I857-variant was detected at low levels (3.9%) in ZYMV-CH87C. When ZYMV-CH87C was passaged with ZXG637, the M768I857-variant was selected by the host, and the original sequence was replaced entirely after two passages. These results may be explained by host-associated selection due to an unknown host-encoded factor. Using the M768I857-variant as an inoculum, 100% of the H1 and K6 plants showed systemic symptoms. These results suggest that (1) changing the individual amino acids at the end of the P3 N-terminus induces resistance-breaking, and (2) the P3 N-terminus may be involved in host recognition.

The sequences of different plant viral leaders with known translation enhancer ability show partial complementarity to the central region of 18S rRNA. Such complementarity might serve as a means to attract 40S ribosomal subunits and explain in part the translation-enhancing property of these sequences. To verify this notion, we designed beta-glucuronidase (GUS) mRNAs differing only in the nature of 10 nt inserts in the center of their 41 base leaders. These were complementary to consecutive domains of plant 18S rRNA. Sucrose gradient analysis revealed that leaders with inserts complementary to regions 1105-1114 and 1115-1124 ('ARC-1') of plant 18S rRNA bound most efficiently to the 40S ribosomal subunit after dissociation from 80S ribosomes under conditions of high ionic strength, a treatment known to remove translation initiation factors. Using wheat germ cell-free extracts, we could demonstrate that mRNAs with these leaders were translated more than three times more efficiently than a control lacking such a complementarity. Three linked copies of the insert enhanced translation of reporter mRNA to levels comparable with those directed by the natural translation enhancing leaders of tobacco mosaic virus and potato virus Y RNAs. Moreover, inserting the same leaders as intercistronic sequences in dicistronic mRNAs substantially increased translation of the second cistron, thereby revealing internal ribosome entry site activity. Thus, for plant systems, the complementary interaction between mRNA leader and the central region of 18S rRNA allows cap-independent binding of mRNA to the 43S pre-initiation complex without assistance of translation initiation factors.

The chloroplast is a prime target for genetic engineering in plants, offering various advantages over nuclear transformation. For example, chloroplasts allow the expression of polycistronic transcripts and thus to engineer complex metabolic pathways. Each cistron within such a longer transcript needs its own expression elements. Within the 5'-UTR, such expression elements are needed for stabilizing mRNAs and for translation of the downstream reading frame. One of the few effective expression elements used so far in transplastomic approaches is the intercistronic expression element (IEE). The IEE is derived from the psbT-psbH intergenic region and includes a target sequence of the RNA binding protein HCF107. We here show that excessive expression of the IEE can lead to specific defects of endogenous chloroplast mRNA stabilization, likely via depletion of HCF107. Key players in chloroplast transcript stabilization and translation are pentatricopeptide repeat (PPR) proteins, which are structurally related to HCF107. PPR proteins that stabilize mRNAs leave behind short RNA footprints that are indicators of their activity. We identified such sRNAs in tobacco, and demonstrate that they are sufficient to stabilize and stimulate translation of mRNAs from synthetic dicistronic transgenes in chloroplasts. Thus, minimal sequence elements are generally adequate to support key steps in chloroplast gene expression, i.e. RNA stability and translation. Furthermore, our analysis expands the repertoire of available expression elements to facilitate the assembly and expression of multi-gene ensembles in the chloroplast.

Like other positive-strand RNA viruses, plant potyviruses assemble viral replication complexes (VRCs) on modified cellular membranes. Potyviruses encode two membrane proteins, 6K2 and P3. The former is known to play pivotal roles in the formation of membrane-associated VRCs. However, P3 remains to be one of the least characterized potyviral proteins. The P3 cistron codes for P3 as well as P3N-PIPO which results from RNA polymerase slippage. In this study, we show that the P3N-PIPO of Turnip mosaic virus (TuMV) is required for viral cell-to-cell movement but not for viral replication. We demonstrate that the C-terminal region of P3 (P3C) is indispensable for P3 to form cytoplasmic punctate inclusions and target VRCs. We reveal that TuMV mutants that lack P3C are replication-defective. Taken together, these data suggest that the P3 cistron has two distinct functions: P3N-PIPO as a dedicated movement protein and P3 as an essential component of the VRC.

Robust RNAi-mediated resistance to multiple Potyvirus strains and isolates, but not to Secovirus BPMV, was conferred by expressing a short SMV P3 hairpin in soybean plants. Engineering resistance to multiple Potyvirus strains is of great interest because of a wide variability of the virus strains, and mixed infections of multiple viruses or strains commonly associated with field grown soybean. In this study, RNAi-mediated silencing of the soybean mosaic virus (SMV) P3 cistron, which is reported to participate in virus movements and pathogenesis and to be the putative determinant of SMV virulence, was used to induce resistance to multiple Potyvirus strains and isolates in soybean. A 302 bp inverted repeat (IR) of the P3 cistron, isolated from the SMV strain SC3, was introduced into soybean. The transgenic lines exhibited stable and enhanced resistance to SMV SC3 under field conditions over 3 consecutive years. The transgenic lines also showed significantly enhanced resistance to four other SMV strains (SC7, SC15, SC18, and SMV-R, a novel recombinant found in China), the soybean-infecting bean common mosaic virus (BCMV) and watermelon mosaic virus (WMV). Nevertheless, no significant differences were found between transgenic plants and their non-transformed (NT) counterparts in terms of resistance to bean pod mottle virus (BPMV, Secoviridae). Consistent with the results of resistance evaluations, the expression of the respective viral CP cistrons and virus accumulation were significantly lower in seven Potyvirus strains and isolates than in the NT plants, but not in BCMV-inoculated transgenic lines. The results demonstrate the effectiveness of engineering resistance to multiple Potyvirus strains and isolates via RNAi-mediated SMV P3 cistron silencing, and thus provide an effective control strategy against Potyvirus infections in soybean and other crops.

Multigene families correspond to a group of genes tandemly repeated, showing enormous diversity in both number of units and genomic organization. In fishes, unlike rDNAs that have been well explored in cytogenetic studies, U2 small nuclear RNA (snRNA) genes are poorly investigated concerning their chromosomal localization. All Triportheus species (Characiformes, Triportheidae) studied so far carry a ZZ/ZW sex chromosomes system, where the W chromosome contains a huge 18S rDNA cistron. In some species the syntenic organization of rDNAs on autosomes was also verified. To explore this particular organization, we performed three-color-fluorescence in situ hybridization using 5S, 18S rDNA, and U2 snRNA genes as probes in eight Triportheus species. This work represents the first one analyzing the chromosomal distribution of U2 snRNA genes in genomes of Triportheidae. The variability in number of rDNA clusters, and the divergent syntenies for these three multigene families, put in evidence their evolutionary dynamism, revealing a much more complex organization of these genes than previously supposed for closely related species. Our study also provides additional data on the accumulation of repetitive sequences in the sex-specific chromosome. Besides, the chromosomal organization of U2 snDNAs among fish species is also reviewed.

(Bangiales, Rhodophyta)((T.J. Chang