RNA-based gene duplication: mechanistic and evolutionary insights.

Gene copies that stem from the mRNAs of parental source genes have long been viewed as evolutionary dead-ends with little biological relevance. Here we review a range of recent studies that have unveiled a significant number of functional retroposed gene copies in both mammalian and some non-mammalian genomes. These studies have not only revealed previously unknown mechanisms for the emergence of new genes and their functions but have also provided fascinating general insights into molecular and evolutionary processes that have shaped genomes. For example, analyses of chromosomal gene movement patterns via RNA-based gene duplication have shed fresh light on the evolutionary origin and biology of our sex chromosomes.

Gene transfer into the airway epithelium of animals by targeting the polymeric immunoglobulin receptor.

Genes of interest can be targeted specifically to respiratory epithelial cells in intact animals with high efficiency by exploiting the receptor-mediated endocytosis of the polymeric immunoglobulin receptor. A DNA carrier, consisting of the Fab portion of polyclonal antibodies raised against rat secretory component covalently linked to poly-L-lysine, was used to introduce plasmids containing different reporter genes into airway epithelial cells in vivo. We observed significant levels of luciferase enzyme activity in protein extracts from the liver and lung, achieving maximum values of 13,795 +/- 4,431 and 346,954 +/- 199,120 integrated light units (ILU) per milligram of protein extract, respectively. No luciferase activity was detected in spleen or heart, which do not express the receptor. Transfections using complexes consisting of an irrelevant plasmid (pCMV lacZ) bound to the bona fide carrier or the expression plasmid (pGEMluc) bound to a carrier based on an irrelevant Fab fragment resulted in background levels of luciferase activity in all tissues examined. Thus, only tissues that contain cells bearing the polymeric immunoglobulin receptor are transfected, and transfection cannot be attributed to the nonspecific uptake of an irrelevant carrier-DNA complex. Specific mRNA from the luciferase gene was also detected in the lungs of transfected animals. To determine which cells in the lungs are transfected by this method, DNA complexes were prepared containing expression plasmids with genes encoding the bacterial beta-galactosidase or the human interleukin 2 receptor. Expression of these genes was localized to the surface epithelium of the airways and the submucosal glands, and not the bronchioles and alveoli. Receptor-mediated endocytosis can be used to introduce functional genes into the respiratory epithelium of rats, and may be a useful technique for gene therapy targeting the lung.

The interplay between relatedness and horizontal gene transfer drives the evolution of plasmid-carried public goods.

Plasmids carry a wide range of genes that are often involved in bacterial social behaviour. The question of why such genes are frequently mobile has received increasing attention. Here, we use an explicit population genetic approach to model the evolution of plasmid-borne bacterial public goods production. Our findings highlight the importance of both transmission and relatedness as factors driving the evolution of plasmid-borne public goods production. We partition the effects of plasmid transfer of social traits into those of infectivity and the effect of increased relatedness. Our results demonstrate that, owing to its effect on relatedness, plasmid mobility increases the invasion and stability of public goods, in a way not seen in individually beneficial traits. In addition, we show that plasmid transfer increases relatedness when public goods production is rare but this effect declines when production is common, with both scenarios leading to an increase in the frequency of plasmid-borne public goods. Plasmids remain important vectors for the spread of social genes involved in bacterial virulence thus an understanding of their dynamics is highly relevant from a public health perspective.

CHARACTERIZATION OF NrsZ, A NOVEL SMALL REGULATORY NON-CODING RNA INVOLVED IN THE ADAPTATION OF PSEUDOMONAS AERUGINOSA PAO1

The opportunistic ubiquitous pathogen Pseudomonas aeruginosa strain PAOl is a versatile Gram-negative bacterium that has the extraordinary capacity to colonize a wide diversity of ecological niches and to cause severe and persistent infections in humans. To ensure an optimal coordination of the genes involved in nutrient utilization, this bacterium uses the NtrB/C and/or the CbrA/B two-component systems, to sense nutrients availability and to regulate in consequence the expression of genes involved in their uptake and catabolism. NtrB/C is specialized in nitrogen utilization, while the CbrA/B system is involved in both carbon and nitrogen utilization and both systems activate their target genes expression in concert with the alternative sigma factor RpoN. Moreover, the NtrB/C and CbrA/B two- component systems regulate the secondary metabolism of the bacterium, such as the production of virulence factors. In addition to the fine-tuning transcriptional regulation, P. aeruginosa can rapidly modulate its metabolism using small non-coding regulatory RNAs (sRNAs), which regulate gene expression at the post-transcriptional level by diverse and sophisticated mechanisms and contribute to the fast physiological adaptability of this bacterium. In our search for novel RpoN-dependent sRNAs modulating the nutritional adaptation of P. aeruginosa PAOl, we discovered NrsZ (Nitrogen regulated sRNA), a novel RpoN-dependent sRNA that is induced under nitrogen starvation by the NtrB/C two-component system. NrsZ has a unique architecture, formed of three similar stem-loop structures (SL I, II and II) separated by variant spacer sequences. Moreover, this sRNA is processed in short individual stem-loop molecules, by internal cleavage involving the endoribonuclease RNAse E. Concerning NrsZ functions in P. aeruginosa PAOl, this sRNA was shown to trigger the swarming motility and the rhamnolipid biosurfactants production. This regulation is due to the NrsZ-mediated activation of rhlA expression, a gene encoding for an enzyme essential for swarming motility and rhamnolipids production. Interestingly, the SL I structure of NrsZ ensures its regulatory function on rhlA expression, suggesting that the similar SLs are the functional units of this modular sRNA. However, the regulatory mechanism of action of NrsZ on rhlA expression activation remains unclear and is currently being investigated. Additionally, the NrsZ regulatory network was investigated by a transcriptome analysis, suggesting that numerous genes involved in both primary and secondary metabolism are regulated by this sRNA. To emphasize the importance of NrsZ, we investigated its conservation in other Pseudomonas species and demonstrated that NrsZ is conserved and expressed under nitrogen limitation in Pseudomonas protegens Pf-5, Pseudomonas putida KT2442, Pseudomonas entomophila L48 and Pseudomonas syringae pv. tomato DC3000, strains having different ecological features, suggesting an important role of NrsZ in the adaptation of Pseudomonads to nitrogen starvation. Interestingly the architecture of the different NrsZ homologs is similarly composed by SL structures and variant spacer sequences. However, the number of SL repetitions is not identical, and one to six SLs were predicted on the different NrsZ homologs. Moreover, NrsZ is processed in short molecules in all the strains, similarly to what was previously observed in P. aeruginosa PAOl, and the heterologous expression of the NrsZ homologs restored rhlA expression, swarming motility and rhamnolipids production in the P. aeruginosa NrsZ mutant. In many aspects, NrsZ is an atypical sRNA in the bacterial panorama. To our knowledge, NrsZ is the first described sRNA induced by the NtrB/C. Moreover, its unique modular architecture and its processing in similar short SL molecules suggest that NrsZ belongs to a novel family of bacterial sRNAs. -- L'agent pathogène opportuniste et ubiquitaire Pseudomonas aeruginosa souche PAOl est une bactérie Gram négative versatile ayant l'extraordinaire capacité de coloniser différentes niches écologiques et de causer des infections sévères et persistantes chez l'être humain. Afin d'assurer une coordination optimale des gènes impliqués dans l'utilisation de différents nutriments, cette bactérie se sert de systèmes à deux composants tel que NtrB/C et CbrA/B afin de détecter la disponibilité des ressources nutritives, puis de réguler en conséquence l'expression des gènes impliqués dans leur importation et leur catabolisme. Le système NtrB/C régule l'utilisation des sources d'azote alors que le système CbrA/B est impliqué à la fois dans l'utilisation des sources de carbone et d'azote. Ces deux systèmes activent l'expression de leurs gènes-cibles de concert avec le facteur sigma alternatif RpoN. En outre, NtrB/C et CbrA/B régulent aussi le métabolisme secondaire, contrôlant notamment la production d'importants facteurs de virulence. En plus de toutes ces régulations génétiques fines ayant lieu au niveau transcriptionnel, P. aeruginosa est aussi capable de moduler son métabolisme en se servant de petits ARNs régulateurs non-codants (ARNncs), qui régulent l'expression génétique à un niveau post- transcriptionnel par divers mécanismes sophistiqués et contribuent à rendre particulièrement rapide l'adaptation physiologique de cette bactérie. Au cours de nos recherches sur de nouveaux ARNncs dépendant du facteur sigma RpoN et impliqués dans l'adaptation nutritionnelle de P. aeruginosa PAOl, nous avons découvert NrsZ (Nitrogen regulated sRNA), un ARNnc induit par la cascade NtrB/C-RpoN en condition de carence en azote. NrsZ a une architecture unique, composée de trois structures en tige- boucle (TB I, II et III) hautement similaires et séparées par des « espaceurs » ayant des séquences variables. De plus, cet ARNnc est clivé en petits fragments correspondant au trois molécules en tige-boucle, par un processus de clivage interne impliquant l'endoribonucléase RNase E. Concernant les fonctions de NrsZ chez P. aeruginosa PAOl, cet ARNnc est capable d'induire la motilité de type « swarming » et la production de biosurfactants, nommés rhamnolipides. Cette régulation est due à l'activation par NrsZ de l'expression de rhlA, un gène essentiel pour la motilité de type swarming et pour la production de rhamnolipides. Étonnamment, la structure TB I est capable d'assurer à elle seule la fonction régulatrice de NrsZ sur l'expression de rhlA, suggérant que ces molécules TBs sont les unités fonctionnelles de cet ARNnc modulaire. Cependant, le mécanisme moléculaire par lequel NrsZ active l'expression de rhlA demeure à ce jour incertain et est actuellement à l'étude. En plus, le réseau de régulations médiées par NrsZ a été étudié par une analyse de transcriptome qui a indiqué que de nombreux gènes impliqués dans le métabolisme primaire ou secondaire seraient régulés par NrsZ. Pour accentuer l'importance de NrsZ, nous avons étudié sa conservation dans d'autres espèces de Pseudomonas. Ainsi, nous avons démontré que NrsZ est conservé et exprimé en situation de carence d'azote par les souches Pseudomonas protegens Pf-5, Pseudomonas putida KT2442, Pseudomonas entomophila L48, Pseudomonas syringae pv. tomato DC3000, quatre espèces ayant des caractéristiques écologiques très différentes, suggérant que NrsZ joue un rôle important dans l'adaptation du genre Pseudomonas envers la carence en azote. Chez toutes les souches étudiées, les différents homologues de NrsZ présentent une architecture similaire faite de TBs conservées et d'espaceurs. Cependant, le nombre de TBs n'est pas identique et peut varier de une à six copies selon la souche. Les différentes versions de NrsZ sont clivées en petites molécules dans ces quatre souches, comme il a été observé chez P. aeruginosa PAOl. De plus, l'expression hétérologue des différentes variantes de NrsZ est capable de restaurer l'expression de rhlA, la motilité swarming et la production de rhamnolipides dans une souche de P. aeruginosa dont nrsZ a été inactivé. Par bien des aspects, NrsZ est un ARNnc atypique dans le monde bactérien. À notre connaissance, NrsZ est le premier ARNnc décrit comme étant régulé par le système NtrB/C. De plus, son unique architecture modulaire et son clivage en petites molécules similaires suggèrent que NrsZ appartient à une nouvelle famille d'ARNncs bactériens.

Vitellogenin B2 gene in Xenopus laevis: isolation, in vitro transcription and relation to other vitellogenin genes.

The isolation of the four Xenopus laevis vitellogenin genes has been completed by the purification from a DNA library of the B2 gene together with its flanking sequences. The overlapping DNA fragments analyzed cover 34 kilobases. The B2 gene which has a length of 17.5 kilobases was characterized by heteroduplex and R-loop mapping in the electron microscope and by in vitro transcription in a HeLa whole-cell extract. Its structural organization is compared with that of the closely related B1 gene. The mRNA-coding sequence of about 6 kilobases is interrupted 34 times in the B1 gene and 33 times in the B2 gene. Sequence homology between the two genes was not only found in exons. In addition, 54% of the intron sequences as well as 63% and 48.5% respectively of the 5' and 3' flanking sequences, show enough homology to form stable duplexes. These findings are compared with earlier results obtained with the two other closely related members of the vitellogenin gene family, the A1 and the A2 genes.

Monophyly of beta-tubulin and H+-ATPase gene variants in Glomus intraradices: consequences for molecular evolutionary studies of AM fungal genes.

Arbuscular mycorrhizal fungi (AMF) are an ecologically important group of fungi. Previous studies showed the presence of divergent copies of beta-tubulin and V-type vacuolar H+-ATPase genes in AMF genomes and suggested horizontal gene transfer from host plants or mycoparasites to AMF. We sequenced these genes from DNA isolated from an in vitro cultured isolate of Glomus intraradices that was free of any obvious contaminants. We found two highly variable beta-tubulin sequences and variable H+-ATPase sequences. Despite this high variation, comparison of the sequences with those in gene banks supported a glomeromycotan origin of G. intraradices beta-tubulin and H+-ATPase sequences. Thus, our results are in sharp contrast with the previously reported polyphyletic origin of those genes. We present evidence that some highly divergent sequences of beta-tubulin and H+-ATPase deposited in the databases are likely to be contaminants. We therefore reject the prediction of horizontal transfer to AMF genomes. High differences in GC content between glomeromycotan sequences and sequences grouping in other lineages are shown and we suggest they can be used as an indicator to detect such contaminants. H+-ATPase phylogeny gave unexpected results and failed to resolve fungi as a natural group. beta-Tubulin phylogeny supported Glomeromeromycota as sister group of the Chytridiomycota. Contrasts between our results and trees previously generated using rDNA sequences are discussed.

Evolution of mRNA expression levels of autosomal and sex-linked genes in amniotes

In addition to differences in protein-coding gene sequences, changes in expression resulting from mutations in regulatory sequences have long been hypothesized to be responsible for phenotypic differences between species. However, unlike comparison of genome sequences, few studies, generally restricted to pairwise comparisons of closely related mammalian species, have assessed between-species differences at the transcriptome level. They reported that gene expression evolves at different rates in various organs and in a pattern that is overall consistent with neutral models of evolution. In the first part of my thesis, I investigated the evolution of gene expression in therian mammals (i.e.7 placental and marsupials), based on microarray data from human, mouse and the gray short-tailed opossum (Monodelphis domestica). In addition to autosomal genes, a special focus was given to the evolution of X-linked genes. The therian X chromosome was recently shown to be younger than previously thought and to harbor a specific gene content (e.g., genes involved in brain or reproductive functions) that is thought to have been shaped by specific sex-related evolutionary forces. Sex chromosomes derive from ordinary autosomes and their differentiation led to the degeneration of the Y chromosome (in mammals) or W chromosome (in birds). Consequently, X- or Z-linked genes differ in gene dose between males and females such that the heterogametic sex has half the X/Z gene dose compared to the ancestral state. To cope with this dosage imbalance, mammals have been reported to have evolved mechanisms of dosage compensation.¦In the first project, I could first show that transcriptomes evolve at different rates in different organs. Out of the five tissues I investigated, the testis is the most rapidly evolving organ at the gene expression level while the brain has the most conserved transcriptome. Second, my analyses revealed that mammalian gene expression evolution is compatible with a neutral model, where the rates of change in gene expression levels is linked to the efficiency of purifying selection in a given lineage, which, in turn, is determined by the long-term effective population size in that lineage. Thus, the rate of DNA sequence evolution, which could be expected to determine the rate of regulatory sequence change, does not seem to be a major determinant of the rate of gene expression evolution. Thus, most gene expression changes seem to be (slightly) deleterious. Finally, X-linked genes seem to have experienced elevated rates of gene expression change during the early stage of X evolution. To further investigate the evolution of mammalian gene expression, we generated an extensive RNA-Seq gene expression dataset for nine mammalian species and a bird. The analyses of this dataset confirmed the patterns previously observed with microarrays and helped to significantly deepen our view on gene expression evolution.¦In a specific project based on these data, I sought to assess in detail patterns of evolution of dosage compensation in amniotes. My analyses revealed the absence of male to female dosage compensation in monotremes and its presence in marsupials and, in addition, confirmed patterns previously described for placental mammals and birds. I then assessed the global level of expression of X/Z chromosomes and contrasted this with its ancestral gene expression levels estimated from orthologous autosomal genes in species with non-homologous sex chromosomes. This analysis revealed a lack of up-regulation for placental mammals, the level of expression of X-linked genes being proportional to gene dose. Interestingly, the ancestral gene expression level was at least partially restored in marsupials as well as in the heterogametic sex of monotremes and birds. Finally, I investigated alternative mechanisms of dosage compensation and found that gene duplication did not seem to be a widespread mechanism to restore the ancestral gene dose. However, I could show that placental mammals have preferentially down-regulated autosomal genes interacting with X-linked genes which underwent gene expression decrease, and thus identified a novel alternative mechanism of dosage compensation.

Visualization of RNA polymerase II ternary transcription complexes formed in vitro on a Xenopus laevis vitellogenin gene.

Stable ternary transcription complexes assembled in vitro, using a HeLa whole-cell extract, have been isolated and visualized by electron microscopy. The formation of these stable complexes on the DNA fragment used as template, the 5' end region of the Xenopus laevis vitellogenin gene B2, depends on factors present in the whole-cell extract, RNA polymerase II and at least two nucleotides. Interestingly, bending in the DNA fragment was frequently observed at the binding site of RNA polymerase II. Dinucleotides that can prime initiation within a short sequence of approximately 10 contiguous nucleotides centered around the initiation site used in vivo, also favour the formation of stable complexes. In addition, pre-initiation complexes were isolated and it was shown that factors in the extract involved in their formation are more abundant than the RNA polymerase II molecules available for binding. The possible implication of this observation relative to the in vivo situation is discussed.

Gene transfer into Xenopus hepatocytes: transcriptional regulation by members of the nuclear receptor superfamily.

A procedure to culture Xenopus laevis hepatocytes that allows the cells in primary culture to be subjected to gene transfer experiments has been developed. The cultured cells continue to present tissue-specific markers such as expression of the albumin gene or estrogen-controlled vitellogenin gene expression, which are both restricted to liver. Two efficient and reproducible gene transfer procedures have been adapted to the Xenopus hepatocytes, namely lipofection and calcium phosphate-mediated precipitation. The transcription of transfected reporter genes controlled by estrogen-, glucocorticoid- or peroxisome proliferator-response elements was stimulated by endogenous or co-transfected receptor in a ligand-dependent manner. Furthermore, the expression of a reporter gene under the control of the entire promoter of the vitellogenin B1 gene mimicked the expression of the chromosomal vitellogenin gene with respect to basal and estrogen-induced activity. Thus, this culture-transfection system will prove very useful to study the regulation of genes expressed in the liver under the control of various hormones or xenobiotics.

Triiodothyronine has diverse and multiple stimulating effects on expression of the major myelin protein genes.

If the importance of triiodothyronine (T3) on brain development including myelinogenesis has long been recognized, its mechanism of action at the gene level is still not fully elucidated. We studied the effect of T3 on the expression of myelin protein genes in aggregating brain cell cultures. T3 increases the concentrations of mRNA transcribed from the following four myelin protein genes: myelin basic protein (Mbp), myelin-associated glycoprotein (Mag), proteolipid protein (Plp), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (Cnp). T3 is not only a triggering signal for oligodendrocyte differentiation, but it has continuous stimulatory effects on myelin gene expression. Transcription in isolated nuclei experiments shows that T3 increases Mag and Cnp transcription rates. After inhibiting transcription with actinomycin D, we measured the half-lives of specific mRNAs. Our results show that T3 increases the stability of mRNA for myelin basic protein, and probably proteolipid protein. In vitro translation followed by myelin basic protein-specific immunoprecipitation showed a direct stimulatory effect of T3 on myelin basic protein mRNA translation. Moreover, this stimulation was higher when the mRNA was already stabilized in culture, indicating that stabilization is achieved through mRNA structural modifications. These results demonstrate the diverse and multiple mechanisms of T3 stimulation of myelin protein genes.

Genome-wide search reveals a novel GacA-regulated small RNA in Pseudomonas species.

BACKGROUND: Small RNAs (sRNAs) are widespread among bacteria and have diverse regulatory roles. Most of these sRNAs have been discovered by a combination of computational and experimental methods. In Pseudomonas aeruginosa, a ubiquitous Gram-negative bacterium and opportunistic human pathogen, the GacS/GacA two-component system positively controls the transcription of two sRNAs (RsmY, RsmZ), which are crucial for the expression of genes involved in virulence. In the biocontrol bacterium Pseudomonas fluorescens CHA0, three GacA-controlled sRNAs (RsmX, RsmY, RsmZ) regulate the response to oxidative stress and the expression of extracellular products including biocontrol factors. RsmX, RsmY and RsmZ contain multiple unpaired GGA motifs and control the expression of target mRNAs at the translational level, by sequestration of translational repressor proteins of the RsmA family. RESULTS: A combined computational and experimental approach enabled us to identify 14 intergenic regions encoding sRNAs in P. aeruginosa. Eight of these regions encode newly identified sRNAs. The intergenic region 1698 was found to specify a novel GacA-controlled sRNA termed RgsA. GacA regulation appeared to be indirect. In P. fluorescens CHA0, an RgsA homolog was also expressed under positive GacA control. This 120-nt sRNA contained a single GGA motif and, unlike RsmX, RsmY and RsmZ, was unable to derepress translation of the hcnA gene (involved in the biosynthesis of the biocontrol factor hydrogen cyanide), but contributed to the bacterium's resistance to hydrogen peroxide. In both P. aeruginosa and P. fluorescens the stress sigma factor RpoS was essential for RgsA expression. CONCLUSION: The discovery of an additional sRNA expressed under GacA control in two Pseudomonas species highlights the complexity of this global regulatory system and suggests that the mode of action of GacA control may be more elaborate than previously suspected. Our results also confirm that several GGA motifs are required in an sRNA for sequestration of the RsmA protein.

Genomic Study of RNA Polymerase II and III SNAP(c)-Bound Promoters Reveals a Gene Transcribed by Both Enzymes and a Broad Use of Common Activators.

SNAP(c) is one of a few basal transcription factors used by both RNA polymerase (pol) II and pol III. To define the set of active SNAP(c)-dependent promoters in human cells, we have localized genome-wide four SNAP(c) subunits, GTF2B (TFIIB), BRF2, pol II, and pol III. Among some seventy loci occupied by SNAP(c) and other factors, including pol II snRNA genes, pol III genes with type 3 promoters, and a few un-annotated loci, most are primarily occupied by either pol II and GTF2B, or pol III and BRF2. A notable exception is the RPPH1 gene, which is occupied by significant amounts of both polymerases. We show that the large majority of SNAP(c)-dependent promoters recruit POU2F1 and/or ZNF143 on their enhancer region, and a subset also recruits GABP, a factor newly implicated in SNAP(c)-dependent transcription. These activators associate with pol II and III promoters in G1 slightly before the polymerase, and ZNF143 is required for efficient transcription initiation complex assembly. The results characterize a set of genes with unique properties and establish that polymerase specificity is not absolute in vivo.

MAR-mediated integration of plasmid vectors for in vivo gene transfer and regulation.

BACKGROUND: The in vivo transfer of naked plasmid DNA into organs such as muscles is commonly used to assess the expression of prophylactic or therapeutic genes in animal disease models. RESULTS: In this study, we devised vectors allowing a tight regulation of transgene expression in mice from such non-viral vectors using a doxycycline-controlled network of activator and repressor proteins. Using these vectors, we demonstrate proper physiological response as consequence of the induced expression of two therapeutically relevant proteins, namely erythropoietin and utrophin. Kinetic studies showed that the induction of transgene expression was only transient, unless epigenetic regulatory elements termed Matrix Attachment Regions, or MAR, were inserted upstream of the regulated promoters. Using episomal plasmid rescue and quantitative PCR assays, we observed that similar amounts of plasmids remained in muscles after electrotransfer with or without MAR elements, but that a significant portion had integrated into the muscle fiber chromosomes. Interestingly, the MAR elements were found to promote plasmid genomic integration but to oppose silencing effects in vivo, thereby mediating long-term expression. CONCLUSIONS: This study thus elucidates some of the determinants of transient or sustained expression from the use of non-viral regulated vectors in vivo.

Expression of genes encoding cytotoxic cell-associated serine proteases in thymocytes.

A family of homologous serine esterases designated granzyme A-H and the pore-forming protein perforin are present in cytoplasmic granules of mature peripheral cytolytic T lymphocytes and natural killer cells. In vivo, the majority of cytotoxic T cells containing these granule-associated proteins are of the CD4-CD8+ phenotype. It is generally assumed that these cells are derived from immature CD4-CD8- thymocytes. However, the precise intrathymic differentiation steps leading to functionally mature cytotoxic T cells are unclear. Thus we decided to analyze the expression of genes in the thymus which are preferentially expressed in mature cytotoxic cells, i.e. granzyme A, granzyme B, and perforin. In situ hybridization on tissue sections revealed the expression of genes coding for granzyme A and granzyme B in the thymus. No evidence was found, however, for thymocytes expressing the perforin gene. Granzyme A and granzyme B mRNA positive cells in the thymus are almost exclusively CD4-CD8- thymocytes, particularly of the CD3- IL2R- phenotype.

A dual functional origin of transfer in the ICEclc genomic island of Pseudomonas knackmussii B13.

Genomic islands (GEIs) are large DNA segments, present in most bacterial genomes, that are most likely acquired via horizontal gene transfer. Here, we study the self-transfer system of the integrative and conjugative element ICEclc of Pseudomonas knackmussii B13, which stands model for a larger group of ICE/GEI with syntenic core gene organization. Functional screening revealed that unlike conjugative plasmids and other ICEs ICEclc carries two separate origins of transfer, with different sequence context but containing a similar repeat motif. Conjugation experiments with GFP-labelled ICEclc variants showed that both oriTs are used for transfer and with indistinguishable efficiencies, but that having two oriTs results in an estimated fourfold increase of ICEclc transfer rates in a population compared with having a single oriT. A gene for a relaxase essential for ICEclc transfer was also identified, but in vivo strand exchange assays suggested that the relaxase processes both oriTs in a different manner. This unique dual origin of transfer system might have provided an evolutionary advantage for distribution of ICE, a hypothesis that is supported by the fact that both oriT regions are conserved in several GEIs related to ICEclc.