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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.

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.

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.

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

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.

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.

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.

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.

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.

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.

Luciferases are prominent reporters in molecular and cellular biology investigations including miRNA target studies and the determination of Internal Ribosome Entry Site (IRES) activities in bicistronic assays. A majority of the current bicistronic vectors contain a firefly luciferase reporter as the second cistron. One reason for this is the presence of cryptic transcription start sites inside the luciferase gene. We present here an experimental evaluation of the cryptic transcription within the latest version of the firefly luciferase gene, luc2. Using flow cytometric analysis, we observed a negligible amount of cryptic transcriptional activity that was only slightly above the background of untransfected cells. Nevertheless, quantitative reverse transcription PCR experiments revealed a six-to-nine-fold gradual increase of transcription along the coding region of the gene. The level of cryptic transcription from the coding region of the improved luc2 firefly luciferase gene is significantly lower when compared to the luc+ gene. In summary, the luc2 better fulfills the requirements of bicistronic assays than the previous luc+ version. The observed low cryptic transcription activity in luc2 could be limiting only in cases where weak IRESs are studied.

Tacrolimus (FK506) is a 23-membered macrolide immunosuppressant used in current clinics. Understanding how the tacrolimus biosynthetic gene cluster is regulated is important to increase its industrial production. Here, we analysed the effect of the disruption of fkbN (encoding a LAL-type positive transcriptional regulator) on the whole transcriptome of the tacrolimus producer Streptomyces tsukubaensis using microarray technology. Transcription of fkbN in the wild type strain increases from 70 h of cultivation reaching a maximum at 89 h, prior to the onset of tacrolimus biosynthesis. Disruption of fkbN in S. tsukubaensis does not affect growth but prevents tacrolimus biosynthesis. Inactivation of fkbN reduces the transcription of most of the fkb cluster genes, including some all (for allylmalonyl-CoA biosynthesis) genes but does not affect expression of allMNPOS or fkbR (encoding a LysR-type regulator). Disruption of fkbN does not suppress transcription of the cistron tcs6-fkbQ-fkbN; thus, FkbN self-regulates only weakly its own expression. Interestingly, inactivation of FkbN downregulates the transcription of a 4′-phosphopantetheinyl transferase coding gene, which product is involved in tacrolimus biosynthesis, and upregulates the transcription of a gene cluster containing a cpkA orthologous gene, which encodes a PKS involved in coelimycin P1 biosynthesis in Streptomyces coelicolor. We propose an information theory-based model for FkbN binding sequences. The consensus FkbN binding sequence consists of 14 nucleotides with dyad symmetry containing two conserved inverted repeats of 7 nt each. This FkbN target sequence is present in the promoters of FkbN-regulated genes.

RNA viruses use various strategies to condense their genetic information into small genomes. Potyviruses not only use the polyprotein strategy, but also embed an open reading frame, pipo, in the P3 cistron in the –1 reading frame. PIPO is expressed as a fusion protein with the N-terminal half of P3 (P3N-PIPO) via transcriptional slippage of viral RNA-dependent RNA polymerase (RdRp). We herein show that clover yellow vein virus (ClYVV) produces a previously unidentified factor, P3N-ALT, in the +1 reading frame via transcriptional slippage at a conserved G1–2A6–7 motif, as is the case for P3N-PIPO. The translation of P3N-ALT terminates soon, and it is considered to be a C-terminal truncated form of P3. In planta experiments indicate that P3N-ALT functions in cell-to-cell movement along with P3N-PIPO. Hence, all three reading frames are used to produce functional proteins. Deep sequencing of ClYVV RNA from infected plants endorses the slippage by viral RdRp. Our findings unveil a virus strategy that optimizes the coding capacity.

[EN] Plant viruses are obligate intracellular parasites which were used to develop recombinant plant virus vectors to express heterologous proteins and to modify endogenous metabolic pathways of natural products in plants. The main limitation of many plant virus-based systems is the difficulty to co-express various heterologous proteins in the same cell with proper subcellular localization, which is a crucial question in metabolic engineering. This work provides a solution to overcome this problem by using a potyvirus-based vector system. Potyviruses (genus Potyvirus, family Potyviridae) are plus-strand single-stranded RNA viruses, which have a genome expression strategy that allows the equimolar production of most viral proteins. On the basis of an infectious clone of Tobacco etch virus (TEV), Bedoya et al. (2010) developed an expression system in which the RNA-dependent RNA polymerase (NIb) gene was replaced by an expression cassette, harboring several heterologous proteins. This viral vector was able to express three fluorescent proteins with nucleocytoplasmic localization in equimolar amounts in transgenic tobacco plants in which NIb was supplemented in trans. Despite of the apparent simplicity of potyvirus genome expression strategy, foreign cDNA insertion is a complicated task. Thus, our first goal was to analyze the effect of gene insertion on TEV genome stability. As a result of this work, a novel insertion position was discovered at the amino-terminal end of the potyvirus polyprotein, which opened the possibility to explore new questions of recombinant protein expression. Since metabolic pathways are highly compartmentalized, proper subcellular targeting of enzymes is an essential task. Thus, our second objective centralized on the subcellular targeting of expressed proteins from the TEV-based viral vector. cDNAs coding for the green fluorescent protein (GFP) fused to chloroplast, nucleus and mitochondria targeting signal sequences were inserted into the newly described amino-terminal insertion position or into an internal site, replacing the NIb cistron. Our results showed that for protein delivery to chloroplasts and mitochondria, foreign genes have to be inserted at the amino-terminal site of the viral vector, but for nuclear delivery, both insertion positions are suitable. The last objective of this work was to investigate whether the potyvirus-based vector was able to express an entire heterologous multistep biosynthetic pathway in plant cells. For this aim we purposed to produce lycopene, a plant pigment with health promoting properties. To do so, we inserted cDNAs coding for the enzymes of a three-step metabolic pathway of bacterial origin into the potyvirus-based vector. Infected tobacco plants developed orange symptoms indicating lycopene accumulation, which was confirmed by high-performance liquid chromatography analysis and microscopy observations. Our results also illustrated that the sole expression of Pantoea ananatis phytoene synthase, crtB, is enough to induce carotenoid accumulation, conferring yellow coloration to the infected tissue and serves as reporter system to visually track viral infection in several plant species., [ES] Los virus de plantas son parásitos intracelulares obligados que han sido utilizados para desarrollar vectores virales y expresar proteínas heterólogas y modificar rutas metabólicas endógenas de productos naturales. La principal limitación de muchos sistemas basados en virus de plantas es la dificultad de coexpresar diversas proteínas heterólogas en la misma célula con la localización subcelular apropiada, lo cual es una cuestión crucial en ingeniería metabólica. Este trabajo presenta una solución para superar este problema mediante el uso de un vector viral basado en un potyvirus. Los potyvirus (género Potyvirus, familia Potyviridae) son virus de RNA de cadena positiva simple que tienen una estrategia de expresión génica que permite la producción de la mayoría de las proteínas virales en cantidades equimolares. Basado en un clon infeccioso del virus del grabado del tabaco (Tobacco etch virus, TEV) Bedoya et al. (2010) desarrollaron un sistema de expresión en el que el gen de la RNA polimerasa dependiente de RNA (NIb) fue sustituido por un casete de expresión, que albergaba varias proteínas heterólogas. Este vector viral fue capaz de expresar tres proteínas fluorescentes con localización nucleocitoplásmica en cantidades equimolares en plantas de tabaco transgénicas que complementaban el cistron NIb en trans. A pesar de la aparente simplicidad de la estrategia de expresión génica de los potyvirus, la inserción de un cDNA foráneo es una tarea complicada. Por lo tanto, nuestro primer objetivo fue analizar el efecto de la inserción en la estabilidad del genoma de TEV. Como resultado de este trabajo, descubrimos una nueva posición de inserción en el extremo amino-terminal de la poliproteína viral que nos permitió explorar otras cuestiones sobre la expresión de proteínas recombinantes. Dado que las vías metabólicas son muy compartimentalizadas, la adecuada localización subcelular de enzimas es una tarea esencial en ingeniería metabólica. Por eso, nuestro segundo objetivo se centró en la distribución de las proteínas heterológas expresadas con el vector viral a diferentes orgánulos subcelulares. cDNAs que codificaban la proteína fluorescente verde (green fluorescent protein, GFP) fusionada a péptidos señal se insertaron en la nueva posición amino-terminal y en un sitio interno, sustituyendo el cistrón NIb, para enviarla al cloroplasto, núcleo y a la mitocondria. Nuestros resultados mostraron que para la distribución de proteínas al cloroplasto y mitocondria, los genes foráneos deben ser insertados en el sitio amino-terminal del vector viral, pero para la distribución nuclear, ambas posiciones son adecuadas. El último objetivo de este trabajo fue estudiar si el vector viral basado en potyvirus es capaz de expresar una ruta biosíntética de múltiples pasos en células vegetales. Para ello nos propusimos producir licopeno, un pigmento vegetal con propiedades beneficiosas para la salud humana. Para ello, insertamos un cDNA que codificaba las enzimas de una ruta metabólica de tres pasos de origen bacteriano en el vector viral. Las plantas de tabaco infectadas con el vector viral desarrollaron síntomas de color naranja indicando la acumulación de licopeno, que fue confirmado por análisis de cromatografía líquida de alta eficacia y observaciones de microscopía. Nuestros resultados también ilustraron que la sola expresión de la fitoeno sintasa de Pantonea ananatis, crtB, es suficiente para inducir la acumulación de carotenoides que confieren una coloración amarilla al tejido infectado y sirve como sistema reportero visual en varias especies de plantas., [CA] Els virus de plantes són paràsits intracel·lulars obligats que han estat utilitzats per a desenvolupar vectors virals i expressar proteïnes heteròlogues y modificar rutes metabòliques endògenes de productes naturals silenciant certs gens o expressant factors de transcripció i enzims metabòlics. La principal limitació de molts sistemes basats en virus de plantes és la dificultat de coexpressar diverses proteïnes heteròlogues en la mateixa cèl·lula amb la localització subcel·lular apropiada, cosa que és una qüestió crucial en enginyeria metabòlica. Aquest treball presenta una solució per a superar aquest problema mitjançant l'ús d'un vector viral basat en un potyvirus. Els potyvirus (gènere Potyvirus, família Potyviridae) són virus d'RNA de cadena positiva simple que tenen una estratègia d'expressió gènica que permet la producció de la majoria de les proteïnes virals en quantitats equimolars. Basat en un clon infecciós del virus del gravat del tabac (Tobacco etch virus, TEV) Bedoya et al. (2010) van desenvolupar un sistema d'expressió en el qual el gen de l'RNA polimerasa depenent d'RNA (NIb) va ser substituït per un casset d'expressió, que albergava diverses proteïnes heteròlogues. Aquest vector viral va ser capaç d'expressar tres proteïnes fluorescents amb localització nucleocitoplàsmica en quantitats equimolars en plantes de tabac transgèniques que complementaven el cistró NIb en trans. Malgrat l'aparent simplicitat de l'estratègia d'expressió gènica dels potyvirus, la inserció d'un cDNA forà és una tasca complicada. Per tant, el nostre primer objectiu va ser analitzar l'efecte de la inserció en l'estabilitat del genoma de TEV. Com a resultat d'aquest treball, hem descobert una nova posició d'inserció en l'extrem amino terminal de la poliproteïna viral que ens va permetre explorar altres qüestions sobre l'expressió de proteïnes recombinants. Atès que les vies metabòliques són molt compartimentalitzades, l'adequada localització subcel·lular d'enzims és una tasca essencial en enginyeria metabòlica. Per açò, el nostre segon objectiu es va centrar en la distribució de les proteïnes heteròlogues expressades amb el vector viral a diferents orgànuls subcelul·lars. cDNAs que codificaven la proteïna fluorescent verda (green fluorescent protein, GFP) fusionada a pèptids senyal es van inserir en la nova posició amino terminal i en un lloc intern, substituint el cistró NIb, per a enviar-la al cloroplast, nucli i al mitocondri. Els nostres resultats van mostrar que per a la distribució de proteïnes al cloroplast i mitocondri, els gens forans han de ser inserits en el lloc amino terminal del vector viral, però per a la distribució nuclear, ambdues posicions són adequades. El lloc amino terminal va resultar ser més adequat per a produir quantitats més grans de proteïnes recombinants, però el lloc d'inserció intern va demostrar ser més estable. Sobre la base d'aquests resultats, hem sigut capaços de distribuir dues proteïnes fluorescents diferents als cloroplasts i nuclis des d'un únic vector viral. L'últim objectiu d'aquest treball va ser estudiar si el vector viral basat en potyvirus és capaç d'expressar una ruta biosintètica de múltiples passos en cèl·lules vegetals. Per açò ens vam proposar produir licopè, un pigment vegetal amb propietats beneficioses per a la salut humana. Per això inserírem un cDNA que codificaba els tres enzims de una ruta metabòlica de tres passos d'origen bacterià en el vector viral. Les plantes de tabac infectades amb el vector viral van desenvolupar símptomes de color taronja indicant l'acumulació de licopè, que va ser confirmat per anàlisi de cromatografia líquida d'alta eficàcia i observacions de microscòpia. Els nostres resultats també van il·lustrar que la sola expressió de fitoè sintasa de Pantonea ananatis, crtB, és suficient per a induir l'acumulació de carotenoides que confereixen una colora

Summary Resistance to Soybean mosaic virus (SMV) in soybean is conferred by three dominant genes: Rsv1, Rsv3 and Rsv4. Over the years, scientists in the USA have utilized a set of standard pathotypes, SMV-G1 to SMV-G7, to study interaction with Rsv-genotype soybeans. However, these pathotypes were isolated from a collection of imported soybean germplasm over 30 years ago. In this study, 35 SMV field isolates collected in recent years from 11 states were evaluated for gain of virulence on soybean genotypes containing individual Rsv genes. All isolates were avirulent on L78-379 (Rsv1), whereas 19 were virulent on L29 (Rsv3). On PI88788 (Rsv4), 14 of 15 isolates tested were virulent; however, only one was capable of systemically infecting all of the inoculated V94-5152 (Rsv4). Nevertheless, virulent variants from 11 other field isolates were rapidly selected on initial inoculation onto V94-5152 (Rsv4). The P3 cistrons of the original isolates and their variants on Rsv4-genotype soybeans were sequenced. Analysis showed that virulence on PI88788 (Rsv4) was not associated, in general, with selection of any new amino acid, whereas Q1033K and G1054R substitutions were consistently selected on V94-5152 (Rsv4). The role of Q1033K and G1054R substitutions, individually or in combination, in virulence on V94-5152 (Rsv4) was confirmed on reconstruction in the P3 cistron of avirulent SMV-N, followed by biolistic inoculation. Collectively, our data demonstrate that SMV has evolved virulence towards Rsv3 and Rsv4, but not Rsv1, in the USA. Furthermore, they confirm that SMV virulence determinants on V94-5152 (Rsv4) reside on P3.

A vast amount of research on the regulation of gene expression has relied on plasmid reporter assays. In this study, we show that plasmids widely used for this purpose constitutively produce substantial amounts of RNA from a TATA-containing cryptic promoter within the origin of replication. Readthrough of these RNAs into the intended transcriptional unit potently stimulated reporter activity when the inserted test sequence contained a 3' splice site (ss). We show that two human sequences, originally reported to be internal ribosome entry sites and later to instead be promoters, mimic both types of element in dicistronic reporter assays by causing these cryptic readthrough transcripts to splice in patterns that allow efficient translation of the downstream cistron. Introduction of test sequences containing 3' ss into monocistronic luciferase reporter vectors widely used in the study of transcriptional regulation also created the false appearance of promoter function via the same mechanism. Across a large number of variants of these plasmids, we found a very highly significant correlation between reporter activity and levels of such spliced readthrough transcripts. Computational estimation of the frequency of cryptic 3' ss in genomic sequences suggests that misattribution of cis-regulatory function may be a common occurrence.

Soybean mosaic virus (SMV; Potyvirus, Potyviridae) is a plant pathogenic virus that infects many commercially important plants worldwide, including soybean, and impairs their production and quality. SMV can be classified into strains based on differences in virulence. Many of these strains have been sequenced; however, only a few complete genomic sequences of virus strains and isolates from soybean grown in China have been reported. In this study, the complete genomic sequences of three SMV isolates of different virulence from plants in China were determined and compared with the sequences of 54 other SMV strains and isolates. Genomic nucleotide diversity analysis and the Neutrality Test showed that the sequence of the 5′ untranslated regions (5′UTR) of the virus genome-linked protein (VPg) and first protein (P1) genes had been altered by negative selection pressure. Phylogenetic analyses indicated that sequence variation in the cytoplasmic inclusion (CI) cistron of the virus was correlated close...

The chloroplast atpB and atpE genes encode subunits b and e of the ATP synthase, respectively. They are co-transcribed as dicistronic mRNAs in flowering plants. An unusual feature is an overlap (AUGA) of the atpB stop codon (UGA) with the atpE start codon (AUG). Hence, atpE translation has been believed to depend on atpB translation (i.e. translational coupling). Using an in vitro translation system from tobacco chloroplasts, we showed that both atpB and atpE cistrons are translated from the tobacco dicistronic mRNA, and that the efficiency of atpB translation is higher than that of atpE translation. When the atpB 5 0 -UTR was replaced with lower efficiency 5 0 -UTRs, atpE translation was higher than atpB translation. Removal of the entire atpB 5 0 -UTR arrested atpB translation but atpE translation still proceeded. Introduction of a premature stop codon in the atpB cistron did not abolish atpE translation. These results indicate that atpE translation is independent of atpB translation. Mutation analysis showed that the atpE cistron possesses its own cis-element(s) for translation, located � 25nt upstream from the start codon.

This study examined the morphological and molecular characteristics of the microsporidium Endoreticulatus sp. Zhenjiang, isolated from the silkworm (Bombyx mori). The fresh spores were oval, 2.9 ± 0.2 μm in length and 1.2 ± 0.2 μm in width. The complete rRNA cistron has a length of 4,432 bp (GenBank accession no. FJ772431), including the large subunit rRNA (2,460 bp), the internal transcribed spacer (187 bp), the small subunit rRNA (1,254 bp), the intergenic spacer (276 bp), and the 5S region (115 bp). The organization of the rRNA gene is 5'-LSU-ITS-SSU-IGS-5S-3', which is reverse compared to the organization of most microsporidian rRNA regions. Phylogenetic analysis based on small subunit rRNA sequences showed that this isolate belongs to the genus Endoreticulatus, and is closely related to Glugoides intestinalis. Furthermore, both had a similar reverse arrangement of the rRNA gene. Our study provides another example of a microsporidian species with a novel organization of rRNA genes, demonstrating that the reverse arrangement is exhibited not only by the microsporidian genus Nosema but may also occur in a clade that contains the genera Endoreticulatus and Glugoides.

A small open reading frame, termed 'pipo', is embedded in the P3 cistron of potyviruses. Currently, knowledge on pipo and its role(s) in the life cycle of potyviruses is limited. The P3 and helper-component proteinase (HC-Pro) cistrons of Soybean mosaic virus (SMV) harbour determinants affecting virulence on functionally immune Rsv1-genotype soybeans. Interestingly, a key virulence determinant of SMV on Rsv1-genotype soybeans (i.e. soybeans containing the Rsv1 resistance gene) that resides at polyprotein codon 947 overlaps both P3 and a pipo-encoded codon. This raises the question of whether PIPO or P3 is the virulence factor. To answer this question, the corresponding pipo of an avirulent and two virulent strains of SMV were studied by comparative genomics, followed by syntheses and analyses of site-directed mutants. Our data demonstrate that the virulence of SMV on Rsv1-genotype soybeans is affected by P3 and not the overlapping pipo-encoded protein.

Two recessive genes (cyv1 and cyv2) are known to confer resistance against Clover yellow vein virus (ClYVV) in pea. cyv2 has recently been revealed to encode eukaryotic translation initiation factor 4E (eIF4E) and is the same allele as sbm1 and wlm against other potyviruses. Although mechanical inoculation with crude sap is rarely able to cause infection of a cyv2 pea, biolistic inoculation of the infectious ClYVV cDNA clone does. At the infection foci, the breaking virus frequently emerges, resulting in systemic infection. Here, a derived cleaved-amplified polymorphic sequence analysis showed that the breakings were associated with a single nonsynonymous mutation on the ClYVV genome, corresponding to an amino-acid substitution at position 24 (isoleucine to valine) on the P1 cistron. ClYVV with the point mutation was able to break the resistance. This is a first report demonstrating that P1 is involved in eIF4E-mediated recessive resistance.

Tobacco rattle virus (TRV) causes stem mottle on potato leaves and necrotic arcs and rings in potato tubers, known as corky ringspot disease. Recently, TRV was reported in Michigan potato tubers cv. FL1879 exhibiting corky ringspot disease. Sequence analysis of the RNA-1-encoded 16-kDa gene of the Michigan isolate, designated MI-1, revealed homology to TRV isolates from Florida and Washington. Here, we report the complete genomic sequence of RNA-1 (6,791 nt) and RNA-2 (3,685 nt) of TRV MI-1. RNA-1 is predicted to contain four open reading frames, and the genome structure and phylogenetic analyses of the RNA-1 nucleotide sequence revealed significant homologies to the known sequences of other TRV-1 isolates. The relationships based on the full-length nucleotide sequence were different from than those based on the 16-kDa gene encoded on genomic RNA-1 and reflect sequence variation within a 20-25-aa residue region of the 16-kDa protein. MI-1 RNA-2 is predicted to contain three ORFs, encoding the coat protein (CP), a 37.6-kDa protein (ORF 2b), and a 33.6-kDa protein (ORF 2c). In addition, it contains a region of similarity to the 3' terminus of RNA-1, including a truncated portion of the 16-kDa cistron. Phylogenetic analysis of RNA-2, based on a comparison of nucleotide sequences with other members of the genus Tobravirus, indicates that TRV MI-1 and other North American isolates cluster as a distinct group. TRV M1-1 is only the second North American isolate for which there is a complete sequence of the genome, and it is distinct from the North American isolate TRV ORY. The relationship of the TRV MI-1 isolate to other tobravirus isolates is discussed.

Two-cistronic expression plasmids are useful for high-level expression of heterologous genes in Escherichia coli cells by preventing the inhibition of translational initiation. In the process of constructing a two-cistronic expression plasmid pCbSTCR-4 containing the fragments of the porcine cytochrome b(5) (Psb5) and NADPH-cytochrome P450 reductase (PsCPR) genes as the first and second cistrons, respectively, the presence of a specific region in the first cistron that lowered the accumulation level of the PsCPR was suggested [Kimura, S., et al. (2005) J. Biochem. 137, 523-533]. In this study, a disturbing nucleotide sequence similar to a Shine-Dalgarno (SD) sequence (SD-like sequence), AGGAG, was identified at the 5'-upstream region near the SD sequence for the second cistron. Silent mutations in the SD-like sequence that lowered the similarity to a typical SD sequence increased the accumulation level of PsCPR. SD-like sequences introduced into mono-cistronic expression plasmids for the Psb5 and PsCPR genes also decreased the accumulation level of these proteins. The SD-like sequence also decreased the accumulation level of the insoluble PsCPR protein. This type of ribosome-binding site interference is useful not only for precise control of protein accumulation but also for increasing the soluble form of recombinant proteins in E. coli cells.

Potato tubers (Solanum tuberosum L. cv. Monalisa) showing superficial necrotic rings typical of Potato virus Y NTN (PVYNTN) infection were collected from commercial potato fields in the State of Sao Paulo (Brazil). An isolate named IAC-PVYNTN was characterized by sequencing 822 and 836 nucleotides from the coat protein and P1 protein coding genes, respectively. The IAC-PVYNTN was observed as a European/North American recombinant isolate closely related to three Eu-PVYNTN isolates that share an alternative recombination site in the coat protein cistron near position 144. Therefore, the IAC-PVYNTN isolate may represent a novel recombinant variant between PVYN N-605 Swiss (European) and PVY O-139 Canadian (North American). Cluster analysis of the P1 sequences found IAC-PVYNTN in the sub-cluster of Eu-PVYN/NTN/N:O. This study supports previous evidence of a common origin for PVYN/NTN in Europe and Brazil.

Initial progress in the science of 'genetics' in the first half of the 20th century was mainly driven by studies utilizing mutations and consequent changes in phenotype. The structural and functional nature of the gene was far from being understood. That state of understanding started changing from the 1940s. In the following decades, with the discovery of the double helix followed by the discoveries on gene structure and expression, there was a period when the structural and functional aspects of the gene could be conceived in terms of one entity, the cistron. However, the discovery of intervening sequences caused this unified concept to fall apart, making the gene concept a subject of philosophical debate again. Meanwhile, over time, technological progress in molecular biology had the field forge ahead rapidly, ultimately leading to the sequencing of the human genome and genomes of other species, and giving birth to the science of genomics. Developments in genomics have given rise to certain applied sub-disciplines like pharmacogenomics and toxicogenomics. While the full potential of pharmaco- and toxicogenomics is yet to be harnessed, they nevertheless have had an impact in drug development and safety assessment, such that the future promise of genomics seems achievable. At present, it is a good opportunity to revisit the path from the development of the initial gene concept and the philosophical debate surrounding the meaning of the term 'gene' to the current state of understanding of genes and genomes and their role in health and disease.

Interaction of recessive visible alleles (fag, fano, spl, nd, nd2) and different Abruptex (Ax) alleles of the Notch locus of Drosophila melanogaster was analysed at 18°C. 25°C and 29°C in transheterozygotes. The homozygous viable Abruptex alleles complement most of the recessives at all temperatures. The expression of hypomorphic Abruptex alleles (AxE2, Ax16172, Ax71d) remains constant at all temperatures, whereas the expression of hypermorphic alleles (Ax28, Ax9B2) is enhanced at elevated temperatures. The homozygous lethal Abruptex alleles (Ax59b8.1, Ax59d5) not only complement all of the recessives at 18°C, but their own phenotypic expression is usually very strong. At 25°C they fail to complement spl and fa8, and their own phenotypic expression is reduced. At 29°C the lethal Abruptex alleles do not complement any of the recessives except nd, and their own expression is reduced or almost absent. At low temperatures the lethal Abruptex alleles compete efficiently with the wild-type allele, but at elevated temperatures their competetive ability is decreased. The results strongly support the view that the Notch complex gene is a single cistron, but that the Abruptex site may possibly have three functionally different mutant forms: hypomorphic, hypermorphic, and antimorphic. The morphism may, however, he different in different functions of the gene which may suggest independence of the pleiotropic effects of the gene.

The chloroplast NAD(P)H dehydrogenase complex, a homologue of mitochondrial complex I, consists of >15 subunits, of which 11 are encoded by the chloroplast genome ( ndhA-K ). The ndhC and ndhK genes are partially overlapped and cotranscribed in many land plants. The downstream ndhK mRNA possesses 4 possible AUG initiation codons in many dicot plants. By using an efficient in vitro translation system from tobacco chloroplasts, we defined that the major initiation site of tobacco ndhK mRNAs is the third AUG that is located 4 nt upstream from the ndhC stop codon. Mutation of the ndhC stop codon (UAG) arrested translation of the ndhK cistron. Frameshift of the ndhC coding strand inhibited also translation of the distal cistron. The results indicated that ndhK translation depends on termination of the preceding cistron, namely translational coupling. Surprisingly, removal of the ndhC 5′-UTR and its AUG still supported substantial translation of the ndhK cistron. This translation was abolished again by removing the ndhC stop codon. Although translation of the downstream cistron of an overlapping mRNA is generally very low, we found that the ndhC / K mRNA produces NdhK and NdhC in similar amounts. Based on subunit compositions of the bacterial complex I, the stoichiometry of NdhK and NdhC is suggested to be 1:1 in chloroplasts. To meet this stoichiometry, the ndhC / K mRNA is translated not only by a translational coupling event but also by a termination codon-dependent pathway.

The family Potyviridae includes >30% of known plant virus species, many of which are of great agricultural significance. These viruses have a positive sense RNA genome that is ≈10 kb long and contains a single long ORF. The ORF is translated into a large polyprotein, which is cleaved into ≈10 mature proteins. We report the discovery of a short ORF embedded within the P3 cistron of the polyprotein but translated in the +2 reading-frame. The ORF, termed pipo , is conserved and has a strong bioinformatic coding signature throughout the large and diverse Potyviridae family. Mutations that knock out expression of the PIPO protein in Turnip mosaic potyvirus but leave the polyprotein amino acid sequence unaltered are lethal to the virus. Immunoblotting with antisera raised against two nonoverlapping 14-aa antigens, derived from the PIPO amino acid sequence, reveals the expression of an ≈25-kDa PIPO fusion product in planta . This is consistent with expression of PIPO as a P3-PIPO fusion product via ribosomal frameshifting or transcriptional slippage at a highly conserved G 1-2 A 6-7 motif at the 5′ end of pipo . This discovery suggests that other short overlapping genes may remain hidden even in well studied virus genomes (as well as cellular organisms) and demonstrates the utility of the software package MLOGD as a tool for identifying such genes.

Coronaviruses are the largest RNA viruses, and their genomes encode replication machinery capable of efficient replication of both positive- and negative-strand viral RNAs as well as enzymes capable of processing large viral polyproteins into putative replication intermediates and mature proteins. A model described recently by Sawicki et al. (S. G. Sawicki, D. L. Sawicki, D. Younker, Y. Meyer, V. Thiel, H. Stokes, and S. G. Siddell, PLoS Pathog. 1:e39, 2005), based upon complementation studies of known temperature-sensitive (TS) mutants of murine hepatitis virus (MHV) strain A59, proposes that an intermediate comprised of nsp4 to nsp10/11 (∼150 kDa) is involved in negative-strand synthesis. Furthermore, the mature forms of nsp4 to nsp10 are thought to serve as cofactors with other replicase proteins to assemble a larger replication complex specifically formed to transcribe positive-strand RNAs. In this study, we introduced a single-amino-acid change (nsp10:Q65E) associated with the TS-LA6 phenotype into nsp10 of the infectious clone of MHV. Growth kinetic studies demonstrated that this mutation was sufficient to generate the TS phenotype at permissive and nonpermissive temperatures. Our results demonstrate that the TS mutant variant of nsp10 inhibits the main protease, 3CLpro, blocking its function completely at the nonpermissive temperature. These results implicate nsp10 as being a critical factor in the activation of 3CLpro function. We discuss how these findings challenge the current hypothesis that nsp4 to nsp10/11 functions as a single cistron in negative-strand RNA synthesis and analyze recent complementation data in light of these new findings.

Multigene expression vectors are commonly utilized whereby the (over)expression of two or more genes is desired simultaneously and often at supposedly equivalent levels. We have characterized dual-gene and pEE14.4 RlucMFluc expression plasmids in which the second hCMV-MIE promoter is replaced with a c-myc IRES to enable one-step coordinated expression of multiple genes in eukaryotic cells. The efficacy of these plasmids has been tested in Chinese hamster ovary (CHO) cells using renilla and firefly luciferase reporter genes, with the reporter gene in either position 1 or 2 from the 5' to 3' direction, respectively. The dual-gene constructs gave enhanced second position gene expression levels compared to the first gene during transient transfection experiments (4- to 50-fold increase 24 h post-transfection). The pEE14.4 RlucMFluc plasmid gave enhanced first cistron expression compared to the second cistron ( approximately 19-fold increase 24 h post-transfection). More equivalent transient expression levels were obtained by undertaking co-transfection of the appropriate single gene plasmids. Reporter gene mRNA levels in the transfected cells were also evaluated by qRT-PCR and compared to the observed protein expression levels. Analysis of the resulting data showed that transcriptional limitation of the first cistron in the dual-gene expression system and not translational limitation was the major limiting factor. Taken together these data suggest that relative protein expression levels expected from heterologous gene products in a multigene vector cannot be predicted on copy number alone and it is important to characterize multigene or oligocistronic systems prior to use.

SUMMARY A new type of Gm genetic marker combination in an IgG1 immunoglobulin, Gm(z) together with a non-a marker, is here described. This indicates the presence of a new gene, Gmz,non-a, which most likely originated on the basis of an intragenic recombination at the IgG1 cistron. Immunochemical and immunogenetic studies showed that no other IgG1 Gm markers were expressed by the unusual gene, and there was no evidence for deletions or duplications at the IgG1 cistron.

Using the RNA replication machinery of Japanese encephalitis virus (JEV), the authors have established and characterized three strategies for the expression of foreign genes. Initially, approximately 11 kb genomic RNA was engineered to express heterologous genes of various sizes by preferentially inserting a new cistron at the beginning of the 3' nontranslated variable region. RNA transfection yielded recombinant viruses that initiated foreign gene expression after infecting permissive cells. JEV was capable of packaging recombinant genomes as large as approximately 15 kb. However, larger genome size was inversely correlated with RNA replication efficiency and cytopathogenicity, with no significant change in infectivity. Second, a variety of self-replicating propagation-deficient viral replicons were constructed by introducing one to three in-frame deletions into the ectodomains of all the structural proteins of JEV. These replicons displayed a spectrum of RNA replication efficiency upon transfection, suggesting that remnant transmembrane domains play a suppressive role in this process. Third, the authors generated a panel of stable packaging cell lines (PCLs) providing all three JEV structural proteins in trans. These PCLs efficiently packaged viral replicon RNAs into single-round infectious viral replicon particles. These JEV-based virus/vector systems may provide useful tools for a variety of biological applications, including foreign gene expression, antiviral compound screening, and genetic immunization.

A 4-nucleotide (nt) deletion was made in the 36-nt-long intercistronic region separating the coat and replicase genes of the single-stranded RNA phage MS2. This region is the focus of several RNA structures conferring high fitness. One such element is the operator hairpin, which, in the course of infection, will bind a coat-protein dimer, thereby precluding further replicase synthesis and initiating encapsidation. Another structure is a long-distance base pairing (MJ) controlling replicase expression. The 4-nt deletion does not directly affect the operator hairpin but it disrupts the MJ pairing. Its main effect, however, is a frame shift in the overlapping lysis gene. This gene starts in the upstream coat gene, runs through the 36-nt-long intercistronic region, and ends in the downstream replicase cistron. Here we report and interpret the spectrum of solutions that emerges when the crippled phage is evolved. Four different solutions were obtained by sequencing 40 plaques. Three had cured the frame shift in the lysis gene by inserting one nt in the loop of the operator hairpin causing its inactivation. Yet these low-fitness revertants could further improve themselves when evolved. The inactivated operator was replaced by a substitute and thereafter these revertants found several ways to restore control over the replicase gene. To allow for the evolutionary enrichment of low-probability but high-fitness revertants, we passaged lysate samples before plating. Revertants obtained in this way also restored the frame shift, but not at the expense of the operator. By taking larger and larger lysates samples for such bulk evolution, ever higher-fitness and lower-frequency revertants surfaced. Only one made it back to wild type. As a rule, however, revertants moved further and further away from the wild-type sequence because restorative mutations are, in the majority of cases, selected for their capacity to improve the phenotype by optimizing one of several potential alternative RNA foldings that emerge as a result of the initial deletion. This illustrates the role of structural constraints which limit the path of subsequent restorative mutations.

During an investigation of lignicolous ascomycetes from Luxembourg, we isolated a Phialophora -like species that produced striking red colonies. To confirm the identity of this fungus as Catenulifera rhodogena , we compared it with isolates of Catenulifera rhodogena and Hyphodiscus hymeniophilus on a variety of media. Portions of the β-tubulin gene and the nuclear ribosomal DNA cistron (internal transcribed spacers (ITS) and large subunit (LSU)) were sequenced to examine the relationship of isolates of Catenulifera rhodogena and Hyphodiscus hymeniophilus from different substrates and to test the hypothesis that Cadophora and Catenulifera are congeneric. The phylogenetic position of Catenulifera within the Ascomycota was investigated based on the analysis of the small-subunit (SSU) rDNA sequences. The isolates examined were indistinguishable micromorphologically and closely related phylogenetically. Three strains of Catenulifera rhodogena from bark or wood and one strain from Piptoporus betulinus formed a strongly supported clade in analyses of β-tubulin and ITS sequences. This clade did not encompass the ex-type isolates of Cistella rubescens and Scopulariopsis rhodogena or a second isolate of Catenulifera rhodogena from Piptoporus betulinus. Analysis of partial LSU sequences confirmed the close phylogenetic relationship of Catenulifera rhodogena and Hyphodiscus hymeniophilus but provided no evidence that the isolates could be grouped by substrate or that Catenulifera is synonymous with Cadophora. The position of Catenulifera within the Helotiales was not resolved based on the comparison of LSU and SSU sequences, but the isolate for which we obtained complete SSU sequence grouped with the root endophyte Phialocephala fortinii. Comparison of ITS sequences confirmed the close phylogenetic relationship of Hyphodiscus to members of the Dermateaceae and Hyaloscyphaceae.

Multiple synonymous substitution mutations in the Wheat streak mosaic virus P3 cistron did not affect translation in vitro but rendered the virus incapable of systemic infection. Multiple synonymous substitutions in the cylindrical inclusion cistron did not alter infectivity or in vitro translation. To assess replication and movement phenotypes, P3 mutations were placed in context with a GUS reporter gene. GUS activity measured in barley protoplasts 36 h post-transfection indicated that mutants with synonymous substitutions in P3 retained the ability to replicate at 22–80 % of wild-type levels. Almost no GUS activity was detected in protoplasts transfected with a P3 frame-shift mutant. Histochemical GUS assays conducted 3 days post-inoculation (p.i.) revealed genomes with multiple synonymous substitutions in P3, which were able to establish infection foci limited to small clusters of cells that increased in size only slightly by 5 days p.i. Infection foci produced by wild-type Wheat streak mosaic virus-expressing GUS were much larger at 3 days p.i. and had coalesced by 5 days p.i. No GUS activity was detected in plants inoculated with the frame-shift mutant bearing GUS. Three of four mutants, each with a single synonymous substitution in the 3′-proximal half of the P3 cistron, were wild-type with respect to systemic infectivity. A model RNA secondary structure obtained for the region was disrupted by the debilitating single mutation but not by the other three single mutations. Collectively, these results identify an internal RNA sequence element in the P3 cistron that affects both replication and movement of the viral genome.

Internal ribosome entry sites (IRES) were first identified as sequences in viral genomes that allow cap-independent translation initiation. The IRES sequence from the encephalomyocarditis virus (EMCV) is able to direct internal translation initiation that allows co-expression of multiple genes from one mRNA. Although EMCV-IRES has also been demonstrated to be functional in transgenic plants, the characteristics of the EMCV-IRES in plant cells have not been fully defined. Using a transient expression system by microprojectile bombardment and quantitative reporter gene assay, we have analyzed EMCV-IRES activity in plant cells. We show that the spacing between the IRES sequence and the second cistron is crucial for IRES-dependent translation efficiency. However, the order of cistrons is not an important factor for the EMCV-IRES-dependent translation in plant cells. Studies in different plant species reveal species specificity for EMCV-IRES activity. Analysis of transgenic tobacco plants suggested that the EMCV-IRES-dependent translation is suppressed in roots.

In previous studies, we demonstrated the sufficiency of short nucleotide sequences to facilitate internal initiation of translation in mammalian cells. By using a selection methodology, we have now identified comparable sequences in Saccharomyces cerevisiae . For these studies, a library of constructs expressing dicistronic mRNAs with the HIS3 gene as the second cistron and 18 random nucleotides in the intercistronic region was introduced into a yeast strain in which the endogenous HIS3 gene was deleted. Untransformed cells or those containing the parent construct failed to grow on medium lacking histidine. Intercistronic sequences recovered from cells that did grow were evaluated by using various criteria. Fifty-six of the 18-nt sequences (≈1/400,000) functioned as synthetic internal ribosome entry sites (IRESes). The 14 most active sequences allowed growth in the presence of 0.1–0.6 mM 3-amino-1,2,4-triazole, a competitive inhibitor of the HIS3 gene product. In addition, eight sequences were identified that were not IRESes, but that enhanced HIS3 expression by an alternative mechanism that depended on the 5′ end of the mRNA and appeared to involve either shunting or reinitiation. Comparisons among the 56 selected IRESes identified eight significant sequence matches containing up to 10 nucleotides. Many of the selected sequences also contained extensive complementary matches to yeast 18S rRNA, some at overlapping sites. The identification of cis sequences that facilitate translation initiation in yeast enables detailed biochemical and genetic analyses of underlying mechanisms and may have practical applications for bioengineering.

Summary Glycolysis is one of the best and widely conserved general metabolic pathways. Bacillus subtilis enzymes catalysing the central part of glycolysis, gathering the steps of interconversion of the triose phosphates from dihydroxyacetone-phosphate to phosphoenolpyruvate, are encoded by five genes, gapA , pgk , tpi , pgm and eno . They are transcribed in a hexacistronic operon together with cggR , the first cistron, encoding the repressor of this gapA operon. Using deletion analysis, we have localized the CggR operator between the promoter and the first gene of the operon. CggR was purified and used in gel mobility shift assays and DNase I footprinting experiments to delimit its target sequence. Site-directed mutagenesis and in vivo tests demonstrated that it consists of two direct-repeats (CGGGACN 6 TGTCN 4 CGGGACN 6 TG TC). Sequence analysis and transcriptome comparison of a wild-type and a cggR mutant strain strongly suggested that CggR regulates only the gapA operon. The presence of glycolytic carbon sources induces expression of the gapA operon. Genetic experiments allowed us to identify the metabolic steps required for the formation of the CggR effector. In vitro experiments with the suggested candidates allowed us to demonstrate that fructose-1,6-biphosphate (FBP) acts as an inhibitor of CggR DNA-binding activity (10 mM for full inhibition). FBP is thus the major signal for both CcpA-dependent catabolite repression (or activation) and activation of the central glycolytic genes. Genomic sequence comparisons suggest that these results can apply to numerous low-G + C, Grampositive bacterial species.

Constructs based on a cysteine proteinase inhibitor (cystatin) from sunflower and a protein engineered variant of a rice cystatin (Oc-IΔD86) conferred similar levels of resistance on potato plants to Globodera spp as chicken egg white cystatin (CEWC) under the control of CaMV35S. The level of resistance on challenge by 6.5 viable eggs Globodera spp g–1 soil in a small scale field trial was similar to that provided by the natural partial resistance of cv. Sante. PCR of an internal transcribed spacer region of the ribosomal cistron of the nematode genome established that the challenging population was a mixture of G. pallida and G. rostochiensis with no evidence of a differential effect of the transgenic resistance on the reproductive success of the two species. Transformation of Sante and the South American cultivar Maria Huanca with CaMV35S/CEWC raised the status of both cultivars from partial to full resistance in this study. The results establish the potential of plant cystatins and demonstrate an additive effect of expressing CEWC with natural resistance genes.

Analysis of gene function in primary vascular cells has been particularly limited by low transfection efficiencies. Using internal ribosomal entry site (IRES)-based retroviral vectors, we demonstrate efficient infection (range of 45%–95%) of primary human endothelial and smooth muscle cells with genes varying in size from 1.3 to 4.5 kb. Because IRES vectors are designed to allow the expression of two genes from a single mRNA, we can show excellent correlation between the expression of a reporter gene and an inserted gene of interest. Reporter gene expression allows rapid (24–48 h) and unambiguous identification of transduced cells. Additionally, reporter gene expression can be used to isolate subpopulations of cells that express distinct levels of cistron 1 genes by flow cytometry, and sorted cells maintain relative levels of gene expression over multiple passages in culture. Two examples of the usefulness of these vectors to characterize gene function in primary vascular cells include (i) the inhibition of endothelial cell inflammatory responses in a polyclonal population by the expression of a dominant negative inhibitor of nuclear factor-κB and (ii) monitoring the in vitro evolution of smooth muscle cells provided with a selective growth advantage by transduction with telomerase. Potential applications of retroviral expression strategies in vascular biology are also discussed.

Insertional mutagenesis with mini-Tn10 was performed to identify new genes involved in sporulation of Bacillus subtilis. Here, we report on the characterization of the ybdA locus, which encodes a putative ATP-binding cassette transporter. The ybdA gene is the 6th cistron of the putative ybcOPQST-ybdABDE operon. A deletion mutation in ybdA and an insertional mutation in ybdB exhibited highly oligosporogenous phenotypes and led to a decrease in the transcription controlled by Spo0A, which is a key response regulator required for the initiation of sporulation. We further observed that the transcription of this operon was strongly induced after the end of the exponential growth phase in the wild-type strain, but not in a spo0A null mutant. Our data suggest that the YbdA and YbdB proteins are able to affect incorporation of nutrient signals during initiation of sporulation and may act as components of positive feedback systems of Spo0A activation.