Stress activation and genomic impact of Tnt1 retrotransposons in Solanaceae.

Tnt1 elements are a superfamily of LTR-retrotransposons distributed in the Solanaceae plant family and represent good model systems for studying regulatory and evolutionary controls established between hosts and transposable elements. Tnt1 retrotransposons tightly control their activation, by restricting expression to specific conditions. The Tnt1A element, originally discovered in tobacco, is expressed in response to stress, and its activation by microbial factors is followed by amplification, demonstrating that factors of pathogen origin can generate genetic diversity in plants. The Tnt1A promoter has the potential to be activated by various biotic and abiotic stimuli but a number of these are specifically repressed in tobacco and are revealed only when the LTR promoter is placed in a heterologous context. We propose that a tobacco- and stimulus-specific repression has been established in order to minimize activation in conditions that might generate germinal transposition. In addition to tight transcriptional controls, Tnt1A retrotransposons self-regulate their activity through gradual generation of defective copies that have reduced transcriptional activity. Tnt1 retrotransposons found in various Solanaceae species are characterized by a high level of variability in the LTR sequences involved in transcription, and have evolved by gaining new expression patterns, mostly associated with responses to diverse stress conditions. Tnt1A insertions associated with genic regions are initially favored but seem subsequently counter-selected, while insertions in repetitive DNA are maintained. On the other hand, amplification and loss of insertions may result from more brutal occurrences, as suggested by the large restructuring of Tnt1 populations observed in tobacco compared to each of its parental species. The distribution of Tnt1 elements thus appears as a dynamic flux, with amplification counterbalanced by loss of insertions. Tnt1 insertion polymorphisms are too high to reveal species relationships in the Nicotiana genus, but can be used to evaluate species relationships in the Lycopersicon and Capsicum genera. This also demonstrates that the behavior of Tnt1 retrotransposons differs between host species, most probably in correlation to differences in expression conditions and in the evolutionary and environmental history of each host.

Benzyl derivatives of 2,1,3-benzo- and benzothieno[3,2-a]thiadiazine 2,2-dioxides: first phosphodiesterase 7 inhibitors.

The synthesis of a new family of benzyl derivatives of 2,1,3-benzo- and benzothieno[3,2-a]thiadiazine 2,2-dioxides was achieved. The biological data revealed the first heterocyclic family of compounds with PDE 7 inhibitory properties appearing to be a new objective for the treatment of T-cell-dependent disorders. The IC(50) values or percent inhibition values of the compounds against PDE 7 were calculated by testing them against human recombinant PDE 7 expressed in S. cerevisiae. In this expression system the only cyclic nucleotide hydrolyzing activity present in cell extracts corresponded to human PDE 7. Isoenzyme selectivity PDE 7 versus PDE 4 and PDE 3 was also measured. Considering simultaneously inhibition of the three different isoenzymes, monobenzyl derivatives 15 and 23 showed interesting PDE 7 potency (around 10 microM); although not statistically significant, a trend toward selectivity with respect to PDE 3 and PDE 4 was obtained. Benzothiadiazine 16, although less potent at PDE 7 (IC(50) = 25 microM), also showed a trend of selectivity toward PDE 3 and PDE 4. These compounds are considered the best leads for further optimization.

Plant transposable elements.

Genomic programs are yielding tremendous amounts of data about plant genomes and their expression. In order to exploit and understand this data it will be necessary to determine the mechanisms leading to natural variation of patterns of gene expression. The ability to understand how gene expression varies among populations (and not only within the population used in the genomics program) and following the exposure of plants to various stress conditions will be fundamental to progress in the post-genomics phase. Transposable elements (TEs) make up nearly half of the total amount of DNA in many plant genomes, so definition of their influence on genome structure and gene expression is of clear significance to the understanding of global genome regulation and phenotype variations. We describe here the different types of plant TEs and recent examples on how they contribute to structure, evolution and genetic control architecture of plant genomes.

Characterisation of LTR sequences involved in the protoplast specific expression of the tobacco Tnt1 retrotransposon.

The tobacco Tnt1 retrotransposon is the only plant retrotransposon that has been shown to be transcriptionally active, and its transcription is strongly induced when preparing leaf-derived protoplasts. We have analysed in this paper the LTR sequences important for Tnt1 expression in tobacco protoplasts. We show that LTR sequences upstream of the TATA box are sufficient to confer protoplast-dependent induction to a heterologous promoter. We also show that this region contains two short activator elements, and that one of these sequences, BII, interacts with protoplast-specific nuclear factors.

Sequential expression and differential hormonal regulation of proteolytic activities during germination in Zea mays L.

We have characterized the proteolytic activities (proteases in cooperation with carboxypeptidases) involved in the different stages of germination of maize (Zea mays L.) grains. A sequential expression of different groups of proteases (aspartic, cysteine, serine and metallo-proteases) with pH optima in the acidic range has been found by using specific protease inhibitors. Pepstatin-sensitive proteolytic activity (aspartic-protease activity) is dominant in resting grains. Germination is accompanied by the appearance of a proteolytic activity which can be enhanced by low-molecular-weight thiol compounds and inhibited by thiol-protease inhibitors, which is indicative of the involvement of cysteine protease(s). This burst of cysteine-protease activity is coincident with the disappearance of the main storage-protein fractions. We conclude from this that cysteine protease(s), with an acid pH optimum, are good candidate(s) for the proteolytic attack of stored protein reserves in maize. After this stage, where cysteine-protease activity is dominant, a period with larger total proteolytic activity starts, coincidentally with the expression of the different types of other proteolytic activities (serine, aspartic and metallo proteases), in addition to the cysteine-protease activity above mentioned. When the development of carboxypeptidase activity during germination was analyzed, the highest activities were found during the earlier and later stages. This result is indicative of a cooperative interaction between carboxypeptidase and endoproteolytic systems in order to obtain a more effective mobilization of storage proteins in germinating maize grains. The phytohormones, gibberellic acid (GA3) and abscisic acid (ABA) which can stimulate or inhibit, respectively, the total proteolytic activity in extracts from germinating grains, exert a differential effect on the different proteolytic activities here detected.

Sequence variability within the tobacco retrotransposon Tnt1 population.

Retroviruses consist of populations of different but closely related genomes referred to as quasispecies. A high mutation rate coupled with extremely rapid replication cycles allows these sequences to be highly interconnected in a rapid equilibrium. It is not known if other retroelements can show a similar population structure. We show here that when the tobacco Tnt1 retrotransposon is expressed, its RNA is not a unique sequence but a population of different but closely related sequences. Nevertheless, this highly variable population is not in a rapid equilibrium and could not be considered as a quasispecies. We have thus named the structure presented by Tnt1 RNA quasispecies-like. We show that the expression of Tnt1 in different situations gives rise to different populations of Tnt1 RNA sequences, suggesting an adaptive capacity for this element. The analysis of the variability within the total genomic population of Tnt1 elements shows that mutations frequently occur in important regulatory elements and that defective elements are often produced. We discuss the implications that this population structure could have for Tnt1 regulation and evolution.

In vivo characterization of transcriptional regulatory sequences involved in the defence-associated expression of the tobacco retrotransposon Tnt1.

The expression of the tobacco retrotransposon Tnt1 is induced by wounding, pathogen infections as well as microbial elicitors and abiotic factors known to induce the plant defence response. We report here that the LTR U3 region is sufficient to mediate transcriptional activation by biotic and abiotic elicitors in stable transgenic conditions. We have used in vivo footprinting techniques in order to analyse the cis-regulatory elements of the LTR U3 region that mediate the induction of Tnt1 expression. Our results indicate that a tandemly repeated short element, named BII box, is involved in the transcriptional activation of the tobacco retrotransposon Tnt1 in association with the plant defence signaling cascade.

Presence of miniature inverted-repeat transposable elements (MITEs) in the genome of Arabidopsis thaliana: characterisation of the Emigrant family of elements.

Although the genome of Arabidopsis thaliana has a small amount of repetitive DNA, it contains representatives of most classes of mobile elements. However, to date, no miniature inverted-repeat transposable element (MITE) has been described in this plant. Here, we describe a new family of repeated sequences that we have named Emigrant, which are dispersed in the genome of Arabidopsis and fulfil all the requirements of MITEs. These sequences are short, AT-rich, have terminal inverted repeats (TIRs), and do not seem to have any coding capacity. Evidence for the mobility of Emigrant elements has been obtained from the absence of one of these elements in a specific Arabidopsis ecotype. Emigrant is also present in the genome of different Brassicae and its TIRs are 74% identical to those of Wujin elements, a recently described family of MITEs from the yellow fever mosquito Aedes aegypti.

The evolutionary analysis of the Tnt1 retrotransposon in Nicotiana species reveals the high variability of its regulatory sequences.

We studied the evolution of the tobacco Tnt1 retrotransposon by analyzing Tnt1 partial sequences containing both coding domains and U3 regulatory sequences obtained from a number of Nicotiana species. We detected three different subfamilies of Tnt1 elements, Tnt1A, Tnt1B, and Tnt1C, that differ completely in their U3 regions but share conserved flanking coding and LTR regions. U3 divergence between the three subfamilies is found in the region that contains the regulatory sequences that control the expression of the well-characterized Tnt1-94 element. This suggests that expression of the three Tnt1 subfamilies might be differently regulated. The three Tnt1 subfamilies were present in the Nicotiana genome at the time of species divergence, but have evolved independently since then in the different genomes. Each Tnt1 subfamily seems to have conserved its ability to transpose in a limited and different number of Nicotiana species. Our results illustrate the high variability of Tnt1 regulatory sequences. We propose that this high sequence variability could allow these elements to evolve regulatory mechanisms in order to optimize their coexistence with their host genome.

A gene coding for a basic pathogenesis-related (PR-like) protein from Zea mays. Molecular cloning and induction by a fungus (Fusarium moniliforme) in germinating maize seeds.

Pathogenesis-related proteins (PRs) are plant proteins produced in leaves in response to infection by pathogens including viruses, viroids, fungi and bacteria. Information on the presence and/or expression of PRs in monocotyledonous plants is scare. Here we report the identification of cDNA and genomic clones coding for a basic form of a protein from germinating maize seeds having a high homology with the group of PR-1 from tobacco. A cDNA library enriched in aleurone-specific sequences was prepared from maize seeds two days after germination. One clone was found to contain an open reading frame encoding a protein homologous to PR proteins from tomato (p14) and tobacco (PR-1 group). Sequence analysis of the corresponding genomic clone revealed that it was encoded by a single exon. Besides, DNA blot hybridization indicates that this PR-like protein is encoded by a single-copy gene in maize. The accumulation of its mRNA increases after rehydration of desiccated seeds. Furthermore, a relationship was found between its expression and infection by a natural pathogen of maize, the fungus Fusarium moniliforme. The possible role of this protein as a response mechanism following fungal infection in cereal seeds is discussed.

Mrs, a new subfamily of Tourist transposable elements.

We have characterised a new family of repetitive sequences that we have named Mrs (maize repetitive sequences). Mrs elements are associated with different maize genes and seem to be specific for the genome of Zea species. Mrs elements are short, AT-rich and contain terminal inverted repeats (TIRs). The sequence of their TIRs, as well as the fact that they are flanked by short repetitions that tend to be TAA, allows us to propose Mrs as a new subfamily of Tourist transposable elements.

Expression of the gene encoding the PR-like protein PRms in germinating maize embryos.

The PRms protein is a pathogenesis-related (PR)-like protein whose mRNA accumulates during germination of maize seeds. Expression of the PRms gene is induced after infection of maize seeds with the fungus Fusarium moniliforme. To further our investigations on the expression of the PRms gene we examined the accumulation of PRms mRNA in different tissues of maize seedlings infected with F. moniliforme and studied the effect of fungal elicitors, the mycotoxin moniliformin, the hormone gibberellic acid, and specific chemical agents. Our results indicate that fungal infection, and treatment either with fungal elicitors or with moniliformin, a mycotoxin produced by F. moniliforme, increase the steady-state level of PRms mRNA. PRms mRNA accumulation is also stimulated by the application of the hormone gibberellic acid or by treatment with silver nitrate, whereas acetylsalicylic acid has no effect. In situ RNA hybridization in isolated germinating embryo sections demonstrates that the PRms gene is expressed in the scutellum, particularly in a group of inner cells, and in the epithelium lying at the interface of the scutellum and the endosperm. The pattern of expression of the PRms gene closely resembles that found for hydrolytic enzymes, being confined to the scutellum and the aleurone layer of the germinating maize seed. Our results suggest that the PRms protein has a function during the normal process of seed germination that has become adapted to serve among the defence mechanisms induced in response to pathogens during maize seed germination.

Three Tnt1 subfamilies show different stress-associated patterns of expression in tobacco. Consequences for retrotransposon control and evolution in plants.

The genomes of most Nicotiana species contain three different subfamilies of the Tnt1 retrotransposon, which differ completely in their U3 sequence, whereas the rest of the sequence is relatively constant. The results presented here show that all three Tnt1 subfamilies are expressed in tobacco (Nicotiana tabacum) and that the U3 sequence variability correlates with differences in the pattern of expression of the Tnt1 elements. Each of the three Tnt1 subfamilies is induced by stress, but their promoters have a different response to different stress-associated signaling molecules. The Tnt1A subfamily is particularly strongly induced by elicitors and methyl jasmonate, whereas expression of the Tnt1C subfamily is more sensitive to salicylic acid and auxins. The direct relationship between U3 sequence variability and differences in the stress-associated expression of the Tnt1 elements present in a single host species gives support to our model that postulates that retrotransposons have adapted to their host genomes through the evolution of highly regulated promoters that mimic those of the stress-induced plant genes. Moreover, here we show that the analysis of the transcriptional control of a retrotransposon population such as Tnt1 provides new insights into the study of the complex and still poorly understood network of defense- and stress-induced plant signal transduction pathways.

Quasispecies in retrotransposons: a role for sequence variability in Tnt1 evolution.

Retroviral replication is a very error-prone process. Replication of retroviruses gives rise to populations of closely related but different genomes referred to as 'quasispecies'. This huge swarm of different sequences constitutes a reservoir of potentially useful genomes in case of an environmental change, endowing retroviruses with extreme adaptability. Retrotransposons are mobile genetic elements closely related to retroviruses, and retrotransposition is as error prone as retroviral replication. The Tnt1 retrotransposon is present in hundreds of copies in the genome of tobacco that show a high level of sequence heterogeneity. When Tnt1 is expressed, its RNA is not a single sequence but a population of sequences displaying a quasispecies-like structure. This population structure gives to Tnt1, as in the case of retroviruses, a high sequence plasticity and an adaptive capacity. We propose this adaptivity as the major reason for Tnt1 maintenance in Nicotiana genomes and we discuss in this paper the importance of sequence variability for Tnt1 evolution.

Functional analysis of the tobacco Tnt1 retrotransposon.

Retroelements represent by far the largest and most widespread class of mobile genetic elements. Representative of several classes of retrotransposons have been characterized in a broad range of plant species, but only a few of them have been shown to be active. Among these, the tobacco Tnt1 retrotransposon has been isolated after insertion mutagenesis and is one of the very few to be transcriptionally active. Tnt1 expression is strongly regulated in a tissue-specific and developmental manner. Moreover, Tnt1 expression is induced by a range of biotic or abiotic elicitors, which all have in common the ability to induce the plant defense response. Regulatory sequences involved in this elicitor-mediated induction have been located in the LTR U3 region. The link between Tnt1 activation and the plant defense response might represent an example of the involvement of transposable elements in genome restructurations needed in response to environmental fluctuations such as pathogen attacks.

A 20 bp cis-acting element is both necessary and sufficient to mediate elicitor response of a maize PRms gene.

Transient gene expression assays in barley aleurone protoplasts were used to identify a cis-regulatory element involved in the elicitor-responsive expression of the maize PRms gene. Analysis of transcriptional fusions between PRms 5' upstream sequences and a chloramphenicol acetyltransferase reporter gene, as well as chimeric promoters containing PRms promoter fragments or repeated oligonucleotides fused to a minimal promoter, delineated a 20 bp sequence which functioned as an elicitor-response element (ERE). This sequence contains a motif (-246 AATTGACC) similar to sequences found in promoters of other pathogen-responsive genes. The analysis also indicated that an enhancing sequence(s) between -397 and -296 is required for full PRms activation by elicitors. The protein kinase inhibitor staurosporine was found to completely block the transcriptional activation induced by elicitors. These data indicate that protein phosphorylation is involved in the signal transduction pathway leading to PRms expression.

The promoter of the tobacco Tnt1 retrotransposon is induced by wounding and by abiotic stress.

The transcription of the tobacco Tnt1 retrotransposon was previously shown to be induced, in tobacco and in heterologous species, by microbial elicitors and by pathogen infections. We report here that the expression of the Tnt1 promoter is also activated in heterologous species such as tomato and Arabidopsis by wounding, freezing and by other abiotic factors known to induce the plant defence response, such as salicylic acid, CuCl2, or oxidative stress. A similar regulation is observed in tobacco for most treatments. The induction of the Tnt1 promoter expression by wounding remains localized around injury points. In CuCl2-treated Arabidopsis plants, the transcription of Tnt1 is correlated with accumulation of the phytoalexin camalexin and with the expression of the EL13 defence gene. The interest of the Tnt1 promoter as a sensitive indicator of the plant defence responses is discussed.

Phosphodiesterases 4D and 7A splice variants in the response of HUVEC cells to TNF-alpha(1).

The mRNA accumulation of phosphodiesterases PDE4D and PDE7A was studied by RNA blot analysis in human umbilical vein endothelial cells (HUVEC) incubated with TNFalpha for different periods. A contrasting behaviour was observed in the mRNA accumulation of the two genes. Further analysis by RT-PCR of the PDE4D and PDE7A splice variants gave different accumulation patterns which may indicate that differential splicing has a role in the regulation of these enzymes. Three previously undescribed PDE4D isoforms, with different accumulation patterns, were also detected. They code for truncated PDE4D isoforms, which could participate in the regulation of PDE4D activity.

The expression of the tobacco Tnt1 retrotransposon is linked to plant defense responses.

Activation of retrotransposons by stresses and external changes is common in all eukaryotic systems, including plants. The transcription of the tobacco Tnt1 retrotransposon was studied in its natural host as well as in Arabidopsis and tomato. It is activated by factors of microbial origin, by external stresses, and by viral, bacterial, and fungal attacks. Tnt1 expression is linked with the biological responses of the plant to the elicitor or to the pathogen attack and in particular with the early steps of the metabolic pathways leading to the activation of plant defense genes. In most cases, the basic features of Tnt1 regulation in tobacco are maintained in tomato and Arabidopsis, but some host-specific regulations were shown. The U3 region of the Tnt1 LTR contains the major cis-acting components of Tnt1 transcriptional activation in association with the plant defense responses. Furthermore, the Tnt1 U3 region, and especially the tandemly repeated BII boxes, contains several sequences similar to well-characterized motifs involved in the activation of several plant defense genes. The possible origin of Tnt1 regulatory sequences as well as the biological implications of Tnt1 activation by pathogen attacks are discussed.

Serological profile of tissue autoantibodies during acute and chronic delta hepatitis.

In order to assess the serological profile in relation to other serological and histological markers of hepatitis delta virus (HDV) infection we have investigated the presence of autoantibodies during acute and chronic delta infection in 353 serum samples from different patients with acute and chronic hepatitis and autoimmune diseases. Basal cell layer antibodies (BCLA) were found in 58% acute hepatitis B, in 73% chronic hepatitis D and in 4% primary biliary cirrhosis. Stellate thymic epithelial cell antibodies (SECA) were detected in 40% acute D hepatitis and in 49% chronic D hepatitis. No tissue autoantibodies were detected in 50 acute B hepatitis, 35 autoimmune chronic liver diseases, 24 connective tissue diseases or 25 controls. In addition, two previously unreported specificities of anti-thymic antibodies reacting with reticular (TRA) and perithymocytic cells (PTA) were identified in 33% and 9% acute D hepatitis, respectively, and in 13% and 6% chronic D hepatitis cases. Among patients with acute HBV-HDV coinfection these antibodies were detected transiently (less than 4 weeks) and at low titer, whereas in those with chronic infection autoantibodies levels tend to be high and persistent throughout the follow-up. Among patients with chronic HDV infection no correlation was found between the presence of tissue autoantibodies and hepatic delta antigen expression and serum HDV-RNA which suggest that autoimmune phenomena observed during chronic delta infection are not related to the level of viral replication.