Recombination is suppressed and variability reduced in a nascent Y chromosome.

Several hypotheses have been elaborated to account for the evolutionary decay commonly observed in full-fledged Y chromosomes. Enhanced drift, background selection and selective sweeps, which are expected to result from reduced recombination, may all share responsibilities in the initial decay of proto-Y chromosomes, but little empirical information has been gathered so far. Here we take advantage of three markers that amplify on both of the morphologically undifferentiated sex chromosomes of the European tree frog (Hyla arborea) to show that recombination is suppressed in males (the heterogametic sex) but not in females. Accordingly, genetic variability is reduced on the Y, but in a way that can be accounted for by merely the number of chromosome copies per breeding pair, without the need to invoke background selection or selective sweeps.

Identification of cancer/testis-antigen genes by massively parallel signature sequencing.

Massively parallel signature sequencing (MPSS) generates millions of short sequence tags corresponding to transcripts from a single RNA preparation. Most MPSS tags can be unambiguously assigned to genes, thereby generating a comprehensive expression profile of the tissue of origin. From the comparison of MPSS data from 32 normal human tissues, we identified 1,056 genes that are predominantly expressed in the testis. Further evaluation by using MPSS tags from cancer cell lines and EST data from a wide variety of tumors identified 202 of these genes as candidates for encoding cancer/testis (CT) antigens. Of these genes, the expression in normal tissues was assessed by RT-PCR in a subset of 166 intron-containing genes, and those with confirmed testis-predominant expression were further evaluated for their expression in 21 cancer cell lines. Thus, 20 CT or CT-like genes were identified, with several exhibiting expression in five or more of the cancer cell lines examined. One of these genes is a member of a CT gene family that we designated as CT45. The CT45 family comprises six highly similar (>98% cDNA identity) genes that are clustered in tandem within a 125-kb region on Xq26.3. CT45 was found to be frequently expressed in both cancer cell lines and lung cancer specimens. Thus, MPSS analysis has resulted in a significant extension of our knowledge of CT antigens, leading to the discovery of a distinctive X-linked CT-antigen gene family.

Natural gene-expression variation in Down syndrome modulates the outcome of gene-dosage imbalance.

Down syndrome (DS) is characterized by extensive phenotypic variability, with most traits occurring in only a fraction of affected individuals. Substantial gene-expression variation is present among unaffected individuals, and this variation has a strong genetic component. Since DS is caused by genomic-dosage imbalance, we hypothesize that gene-expression variation of human chromosome 21 (HSA21) genes in individuals with DS has an impact on the phenotypic variability among affected individuals. We studied gene-expression variation in 14 lymphoblastoid and 17 fibroblast cell lines from individuals with DS and an equal number of controls. Gene expression was assayed using quantitative real-time polymerase chain reaction on 100 and 106 HSA21 genes and 23 and 26 non-HSA21 genes in lymphoblastoid and fibroblast cell lines, respectively. Surprisingly, only 39% and 62% of HSA21 genes in lymphoblastoid and fibroblast cells, respectively, showed a statistically significant difference between DS and normal samples, although the average up-regulation of HSA21 genes was close to the expected 1.5-fold in both cell types. Gene-expression variation in DS and normal samples was evaluated using the Kolmogorov-Smirnov test. According to the degree of overlap in expression levels, we classified all genes into 3 groups: (A) nonoverlapping, (B) partially overlapping, and (C) extensively overlapping expression distributions between normal and DS samples. We hypothesize that, in each cell type, group A genes are the most dosage sensitive and are most likely involved in the constant DS traits, group B genes might be involved in variable DS traits, and group C genes are not dosage sensitive and are least likely to participate in DS pathological phenotypes. This study provides the first extensive data set on HSA21 gene-expression variation in DS and underscores its role in modulating the outcome of gene-dosage imbalance.

A characteristic EEG pattern in 4p-syndrome: case report and review of the literature.

Deletions on the short arm of chromosome 4 cause Wolf-Hirschhorn syndrome (WHS) and Pitt-Rogers-Danks syndrome (PRDS). WHS is associated with severe growth and mental retardation, microcephaly, a characteristic facies and congenital malformations. The PRDS phenotype is similar to WHS but generally less severe. Seizures occur in the majority of WHS and PRDS patients. Sgrò et al. [17] described a stereotypic electroclinical pattern in four unrelated WHS patients, consisting of intermittent bursts of 2-3 Hz high voltage slow waves with spike wave activity in the parietal areas during drowsiness and sleep associated with myoclonic jerks. We report a patient with PRDS and the typical EEG pattern and review 14 WHS patients with similar EEG findings reported in the literature. CONCLUSION: Awareness and recognition of the characteristic electroclinical findings in Wolf-Hirschhorn syndrome and Pitt-Rogers-Danks syndrome might help in the early diagnosis of such patients.

Chromosomal evolution and zoogeographic origin of southeast Asian shrews (genus Crocidura).

To the origins and evolution of Indomalayan shrews, we investigated the chromosomal variations of 14 species of Crocidura from SE Asia. Intraspecific polymorphism was mainly due to variation in the number of short chromosomal arms but C. lepidura and C. hutanis showed a polymorphism due to a centric fusion. The undifferentially stained karyotypes were similar in 9 species, all possessing 2n = 38 and FN = 54-56 (68); C. fuliginosa had 2n = 40 and FN = 54-58. These karyotypes are close to the presumed ancestral state for the genus Crocidura. Four species from Sulawesi had a reduced diploid number (2n = 30-34), a trend not observed among other SE Asian species but present in few Palaearctic taxa. Compared to the apparent stasis of karyotypic evolution observed among other SE Asian species, the high degree of interspecific differences reported among Sulawesian shrews is unusual and needs further investigation. Stasis and reduction in diploid number found in both Indomalayan and Palaeractic species suggest that these two groups share a common ancestry. This is in sharp contrast to most Afrotropical species which evolved towards higher diploid and fundamental numbers. The zoogeographical implications of these results are discussed.

Regulation of the activity of DnaA and its role during the cell cycle of caulobacter crescentus

The initiation of chromosomal replication must be tightly regulated so that the genome is replicated only once per cell cycle. In most bacteria, DnaA binds to the origin of replication and initiates chromosomal replication. DnaA is a dual-function protein that also acts as an important transcription factor that regulates the expression of many genes in bacteria. Thus, understanding how this protein is regulated during the bacterial cell cycle is of major importance. The α-proteobacterium Caulobacter crescentus is an excellent model to study the bacterial cell cycle, mainly because it is possible to isolate synchronized cell cultures and because it initiates the replication of its chromosome once per cell cycle and at a specific time of the cell cycle. This latest feature is of special interest for the major aim of my thesis work, which focused on the temporal and spatial regulation of the activity of the essential DnaA protein in C. crescentus. In Escherichia coli, the Hda protein converts ATP-DnaA into ADP- DnaA by stimulating the ATPase activity of DnaA, to prevent over-initiation of chromosome replication. We propose that there exists a similar mechanism in C. crescentus, which is not only involved in the temporal control of chromosome replication, but also in the control of gene expression. First, we provided evidences indicating that the hydrolysis of the ATP bound to DnaA is essential for the viability of C. crescentus. Our results suggest that ATP-DnaA promotes the initiation of chromosome replication, since we found that cells over-expressing a DnaA protein with a mutated ATPase domain, DnaA(R357A), over-initiated chromosome replication, unlike cells expressing the wild-type DnaA protein at similar levels. By contrast, the DnaA(R357A) protein was less active than DnaA in promoting the transcription of three essential genes, suggesting that these may be more efficiently activated by ADP-DnaA than ATP-DnaA. We propose that the ATP-DnaA to ADP-DnaA switch down-regulates the initiation of DNA replication while activating the transcription of several essential genes involved in subsequent cell cycle events. Second, we studied the role of the HdaA protein, homologous to Hda, in promoting the ATP- DnaA to ADP-DnaA switch in C. crescentus. HdaA is essential for viability and its depletion in the cell leads to an over-replication of the chromosome, indicating that HdaA is a negative regulator of DNA replication. HdaA dynamically co-localizes with the replisome. In this work, we identified DnaN, the β-clamp of the DNA polymerase, as the replisome component that interacts directly with HdaA and that recruits HdaA to the replisome in live C. crescentus cells. We also showed that a mutant HdaA protein that cannot interact or co-localize with DnaN is not functional, indicating that HdaA is probably activated by DnaN. However, we found that another non-functional HdaA protein, mutated in the conserved Arginine finger of its AAA+ domain, was able to localize at the replisome, suggesting that the AAA+ domain of HdaA exerts its essential function after the recruitment of HdaA to the replisome. We propose that HdaA stimulates the ATPase activity of DnaA once DNA replication is ongoing, via its interaction with DnaN and the activity of the two conserved R fingers of DnaA and HdaA. Finally, we created different strains in which HdaA, DnaN or DnaA were over-produced. We observed that the over-production of HdaA seems to lead to a delay in chromosome replication, while the over-production of DnaN had an opposite effect. Our results also indicate that the over-production of DnaA may intensify the over-initiation phenotype of cells depleted for HdaA. We conclude that the dynamic interplay of HdaA and DnaN in the cell contributes to regulating the ATP-DnaA/ADP-DnaA ratio in the cell, to ensure once per cell cycle initiation of chromosomal replication in C. crescentus. Altogether, our work provided important information on the regulation of the activity of DnaA in C. crescentus. Since DnaA, HdaA and DnaN are well-conserved proteins, most of our findings are useful to understand how chromosome replication and gene expression are controlled by DnaA in many other bacterial species. - L'initiation de la réplication des chromosomes doit être précisément régulée de telle sorte que le génome ne soit répliqué qu'une seule fois par cycle cellulaire. Chez la plupart des bactéries, DnaA se lie à l'origine de réplication du chromosome et en initie sa réplication. DnaA est aussi un facteur de transcription qui régule l'expression de nombreux gènes bactériens. De ce fait, il est très important de comprendre comment DnaA est régulée au cours du cycle cellulaire bactérien. L'a-protéobactérie Caulobacter crescentus est un excellent modèle pour étudier le cycle cellulaire bactérien, essentiellement parce qu'il est aisé d'isoler des populations de cellules synchronisées à la même étape du cycle cellulaire et parce que cette bactérie n'initie la réplication de son chromosome qu'une seule fois et à un moment précis de son cycle. Cette dernière caractéristique est particulièrement pertinente pour l'objectif de mon travail doctoral, qui consistait à comprendre comment l'activité de la protéine essentielle DnaA est régulée dans l'espace et dans le temps chez C. crescentus. Chez Escherichia coli, la protéine Hda convertie DnaA-ATP en DnaA-ADP en stimulant l'activité ATPasique de DnaA, ce qui empêche la sur-initiation de la réplication du chromosome. Nous proposons qu'un mécanisme similaire existe chez C. crescentus. Il serait non seulement nécessaire au contrôle de la réplication du chromosome, mais aussi au contrôle de l'expression de certains gènes. Dans un premier temps, nous avons mis en évidence le fait que l'hydrolyse de l'ATP lié à DnaA est un processus essentiel à la viabilité de C. crescentus. Nos résultats suggèrent que DnaA-ATP initie la réplication du chromosome, comme nous avons observé que des cellules qui sur-expriment une protéine DnaA(R357A) mutée sans domaine ATPasique fonctionnel, sur-initie la réplication de leur chromosome, contrairement aux cellules qui sur-expriment la protéine DnaA sauvage à des niveaux équivalents. Au contraire, la protéine DnaA(R357A) était moins active que la protéine DnaA sauvage pour promouvoir la transcription de trois gènes essentiels, ce qui suggère que ces derniers sont peut-être plus efficacement activés par DnaA-ADP que DnaA-ATP. Nous proposons que la conversion de DnaA-ATP en DnaA-ADP réprime l'initiation de la réplication, tandis qu'elle active la transcription de plusieurs gènes impliqués dans des étapes plus tardives du cycle cellulaire. Dans un deuxième temps, nous avons étudié le rôle de la protéine HdaA, homologue à Hda, dans la conversion de DnaA-ATP en DnaA-ADP chez C. crescentus. Cette protéine est essentielle à la viabilité de C. crescentus et sa déplétion donne des cellules qui sur-initient la réplication de leur chromosome, suggérant que HdaA est un répresseur de la réplication du chromosome. HdaA co-localise de manière dynamique avec le réplisome. Lors de mon travail doctoral, nous avons démontré que DnaN, le β-clamp de l'ADN polymérase, est l'élément qui recrute HdaA au réplisome in vivo. Nous avons aussi montré qu'une protéine HdaA mutante qui ne peut pas interagir ou co-localiser avec DnaN, n'est pas fonctionnelle, ce qui suggère que HdaA est activée par DnaN. Nous avons néanmoins aussi isolé une autre protéine HdaA non fonctionnelle, dont une arginine conservée de son domaine AAA+ était mutée, mais qui pouvait toujours co-localiser avec le réplisome, ce qui suggère que le domaine AAA+ de HdaA est nécessaire après le recrutement de HdaA au réplisome. Nous proposons que HdaA stimule l'activité ATPasique de DnaA qu'une fois que la réplication a commencé, grâce à son interaction avec DnaN et aux deux arginines conservées des protéines HdaA et DnaA. Finalement, nous avons construit différentes souches sur-exprimant HdaA, DnaN ou DnaA. Nous avons observé que la sur-production de HdaA retarde la réplication du chromosome, tandis que la sur-production de DnaN a un effet opposé. Nos observations suggèrent aussi que la sur-expression de DnaA dans des cellules déplétées pour HdaA aggrave leur phénotype de sur-initiation. Nous en concluons que HdaA et DnaN collaborent étroitement et de manière dynamique pour réguler le rapport DnaA-ATP/DnaA-ADP dans la cellule, pour s'assurer que la réplication du chromosome ne soit initiée qu'une seule fois par cycle cellulaire chez C. crescentus. Globalement, notre travail a mis en évidence des informations importantes sur la régulation de l'activité de DnaA chez C. crescentus. Comme DnaA, HdaA et DnaN sont des protéines très conservées, la plupart de nos découvertes sont utiles pour mieux comprendre comment la réplication du chromosome bactérien et l'expression des gènes sont contrôlées par DnaA chez de nombreuses autres espèces bactériennes.

Molecular characterization of a human matrix attachment region epigenetic regulator.

Matrix attachment regions (MAR) generally act as epigenetic regulatory sequences that increase gene expression, and they were proposed to partition chromosomes into loop-forming domains. However, their molecular mode of action remains poorly understood. Here, we assessed the possible contribution of the AT-rich core and adjacent transcription factor binding motifs to the transcription augmenting and anti-silencing effects of human MAR 1-68. Either flanking sequences together with the AT-rich core were required to obtain the full MAR effects. Shortened MAR derivatives retaining full MAR activity were constructed from combinations of the AT-rich sequence and multimerized transcription factor binding motifs, implying that both transcription factors and the AT-rich microsatellite sequence are required to mediate the MAR effect. Genomic analysis indicated that MAR AT-rich cores may be depleted of histones and enriched in RNA polymerase II, providing a molecular interpretation of their chromatin domain insulator and transcriptional augmentation activities.

Cervical nerve root synovial sarcoma in a child with chromosomal (X;18) translocation. Case report and review of the literature.

We report on an 11-year-old female with a history of cervicobrachialgia and progressive weakness of the right arm. Cervical spine MRI showed an enhancing heterogeneous intradural mass occupying the right C6-C7 foramen. She underwent a right C6-C7 foraminotomy with a complete macroscopic removal of the lesion. Pathological examination revealed a synovial sarcoma. Treatment was completed by chemotherapy and proton radiotherapy, and the girl remained free of symptoms for 3 years. After appearance of new symptoms, a local recurrence was confirmed, and despite aggressive treatment with salvage chemotherapy and radiotherapy, the disease progressed beyond medical control, and the child died, 6 years after diagnosis. Early recognition of this rare entity compared to its more benign differential diagnosis is crucial, as an aggressive management is needed.

Studying the molecular causes of ageing in ants

RÉSUMÉ DE THÈSE Au cours de ma thèse, je me suis intéressée aux causes physiologiques du vieillissement en utilisant les fourmis comme modèle. Les trois castes de fourmis - les mâles, les ouvrières et les reines - présentent des longévités très différentes, tout en étant génétiquement identiques. Ceci implique que les différences de longévité sont dues à des variations entre castes dans le pattern d'expression de gènes. Mon travail chez la fourmi a consisté d'une part à mettre en place les outils pour identifier de tels gènes à grande échelle, de l'autre à étudier le rôle de gènes et de mécanismes qui affectent la longévité chez d'autres espèces. Pour identifier de nouveaux gènes potentiellement impliqués dans le vieillissement, nous avons développé des puces à ADN. Cette technique permet la comparaison du niveau d'expression de milliers de gènes entre deux échantillons. L'application de cette méthode aux reines et ouvrières adultes nous a jusqu'à présent permis d'identifier neuf gènes surexprimés chez les reines. Trois d'entre eux sont potentiellement impliqués dans le maintien et la réparation du soma, deux processus qui sont supposés avoir un impact crucial sur la longévité. Parmi les mécanismes impliqués dans le vieillissement chez d'autres espèces, nous nous sommes principalement intéressés aux télomères, qui sont les extrémités des chromosomes. Chez les vertébrés, les télomères se raccourcissent à chaque division cellulaire, entre autres parce que l'ADN polymérase ne peut répliquer cette partie des chromosomes en entier. Or des télomères courts entravent la prolifération des cellules et peuvent même induire l'apoptose, ce qui pourrait se répercuter sur la capacité des organismes à régénérer des tissus. J'ai pu montrer que chez les fourmis mâles (la caste qui vit le moins longtemps) les télomères se raccourcissent beaucoup plus vite que chez les reines et les ouvrières. L'explication la plus plausible pour cette différence est que les mâles, étant adapté à une vie très éphémère, n'investissent qu'un minimum d'énergie dans la machinerie de maintenance qui assure le bon fonctionnement des cellules. Ces résultats sont intéressants car ils permettent pour la première fois de faire le lien entre les théories évolutives du vieillissement et la biologie des télomères. THESIS ABSTRACT During my thesis I used ants as a model to study the proximate (i.e., molecular) causes of ageing and lifespan determination. Ant queens, workers and males differ tremendously in lifespan, although all three castes are genetically identical. Importantly, this implies that genes and molecular pathways responsible for modulating lifespan are regulated in a caste-specific manner. To find new genes potentially involved in ageing, we first constructed 371-gene-cDNA microarrays for the ant L. niger. This molecular tool can be used to survey the relative gene expression levels of two samples for thousands of genes simultaneously. By applying this method to adult queens and workers we identified nine genes that are overexpressed in queens. Three of them are putatively involved in somatic maintenance and repair, two processes that have been previously suggested as important for ageing and lifespan determination. We expect to identify many more candidate genes in the near future by using the 9000-gene fire ant microarrays we have recently developed. We also investigated whether factors linked to ageing in other organisms could affect lifespan determination in ants. One project was on telomeres, the ends of linear chromosomes. For various reasons telomeres shorten with every cell division. Since short telomeres can lead to cellular defects such as impaired cell division, telomeres have been hypothesized as playing a role in ageing. We tested whether telomere length in ant somatic tissues correlates with caste-specific lifespan in young adults. The short-lived L. niger mates did indeed have significantly shorter telomeres than the longer-lived queens and workers, probably because telomere attrition is faster in males than in queens and workers. Queens did not, however, have longer telomeres than the shorter-lived workers. These findings are consistent with the idea that telomere length may play a role in ageing under some circumstances, but they also clearly demonstrate that other factors must be involved. We argue that sex-specific telomere length patterns in ants ultimately reflect adaptive differences in the level of somatic maintenance between males and females, and thus create a link between telomere biology and the evolutionary theory of ageing.

Fibrosarcoma-like lipomatous neoplasm: a reappraisal of so-called spindle cell liposarcoma defining a unique lipomatous tumor unrelated to other liposarcomas

The term "spindle cell liposarcoma" has been applied to liposarcomas (LPSs) composed predominantly or exclusively of spindled cells. These tumors have been considered variants of well-differentiated LPS (WDL), myxoid LPS, and spindle cell lipoma, suggesting that this is a heterogenous group of lesions. Using strict morphologic criteria and molecular and immunohistochemical analyses, we have identified a homogenous group of spindle cell lipomatous tumors, histologically and genetically distinct from other forms of LPS, which we have called "fibrosarcoma-like lipomatous neoplasm." Cases classified as "spindle cell LPS" or "low-grade LPS with spindle cell features" were reviewed. Final selection criteria included: (1) an exclusive low-grade spindle cell component resembling fibrosarcoma; (2) a mixture of bland fibroblastic cells resembling the preadipocyte and early-adipocyte stage of embryonic fat; and (3) molecular-genetic analysis that excluded other forms of lipomatous tumors. Of the initial 25 cases identified, comparative genomic hybridization (CGH) was uninformative in 2 cases; 5 were reclassified as WDL on the basis of molecular data (MDM2 amplification) and 6 as spindle cell lipoma (CGH profiles with a few gains and losses including a constant loss of chromosome 13 and frequent losses of chromosomes 16 and 6). The 12 remaining cases showed flat CGH profiles; of these cases, 11 were negative for DDIT3 gene rearrangements, and 1 result was uninterpretable. Patients ranged in age from 15 to 82 years (mean 50 y); male patients were affected slightly more often (7:5). Tumors arose in the deep (6) and superficial (3) soft tissue of the groin (4), buttock (3), thigh (2), flank (1), shoulder (1), and paratesticular tissue (1) and ranged in size from 2 to 20 cm (mean 7.5 cm). Clinical follow-up in 11 patients (9 mo to 20 y; mean 68 mo) showed no recurrences or metastases. As defined above, "fibrosarcoma-like lipomatous neoplasm" is a unique lipomatous tumor that should be distinguished from WDL/(low-grade) dedifferentiated LPS and myxoid LPS on combined histologic/molecular features because of its better prognosis.

Variability of the nucleolar organizer activity in human lymphocytes via Ag-staining.

Chromosomes with Ag staining that varies from one metaphase to the other can be distinguished from those with an Ag-staining that is the same in all metaphases. The intercellular variation of an Ag-NOR can be attributed to many different factors. Whatever the importance of technical factors, they do not seem to account for the large variations in Ag-staining which were observed for each ac. This suggests the existence of a natural intercellular variability of the NOR's activity. The variation of the Ag-stainability of a given NOR, the diversity of Ag-stainings observed on the ten ac of one individual and the differences that exist between individuals raise the question of the existence of a compensation of activity between nucleolar organizers. The study, for each individual, of the mean sum of staining per metaphase reveals that this value is not absolutely constant from one individual to another; in the carriers of Robertsonian fusions it is smaller than in chromosomally normal individuals. The analysis of the transmission shows that inactive NORs remain inactive and that active NORs present a variation in the activity from one generation to the next.

Role of trabectedin in the treatment of soft tissue sarcoma.

Interest in marine natural products has allowed the discovery of new drugs and trabectedin (ET-743, Yondelis), derived from the marine tunicate Ecteinascidia turbinata, was approved for clinical use in 2007. It binds to the DNA minor groove leading to interferences with the intracellular transcription pathways and DNA-repair proteins. In vitro antitumor activity was demonstrated against various cancer cell lines and soft tissue sarcoma cell lines. In phase I studies tumor responses were observed also in osteosarcomas and different soft tissue sarcoma subtypes. The most common toxicities were myelosuppression and transient elevation of liver function tests, which could be reduced by dexamethasone premedication. The efficacy of trabectedin was established in three phase II studies where it was administered at 1.5 mg/m2 as a 24 h intravenous infusion repeated every three weeks, in previously treated patients. The objective response rate was 3.7%-8.3% and the tumor control rate (which included complete response, partial response and stable disease) was obtained in half of patients for a median overall survival reaching 12 months. In nonpretreated patients the overall response rate was 17%. Twenty-four percent of patients were without progression at six months. The median overall survival was almost 16 months with 72% surviving at one year. Predictive factors of response are being explored to identify patients who are most likely to respond to trabectedin. Combination with other agents are currently studied with promising results. In summary trabectedin is an active new chemotherapeutic agents that has demonstrated its role in the armamentarium of treatments for patients with sarcomas.

Molecular analysis of a novel gene cluster encoding an insect toxin in plant-associated strains of Pseudomonas fluorescens

Pseudomonas fluorescens CHA0 and the related strain Pf-5 are well-characterized representatives of rhizosphere bacteria that have the capacity to protect crop plants from fungal root diseases, mainly by releasing a variety of exoproducts that are toxic to plant pathogenic fungi. Here, we report that the two plant-beneficial pseudomonads also exhibit potent insecticidal activity. Anti-insect activity is linked to a novel genomic locus encoding a large protein toxin termed Fit (for P. fluorescensinsecticidal toxin) that is related to the insect toxin Mcf (Makes caterpillars floppy) of the entomopathogen Photorhabdus luminescens, a mutualist of insect-invading nematodes. When injected into the haemocoel, even low doses of P. fluorescens CHA0 or Pf-5 killed larvae of the tobacco hornworm Manduca sexta and the greater wax moth Galleria mellonella. In contrast, mutants of CHA0 or Pf-5 with deletions in the Fit toxin gene were significantly less virulent to the larvae. When expressed from an inducible promoter in a non-toxic Escherichia coli host, the Fit toxin gene was sufficient to render the bacterium toxic to both insect hosts. Our findings establish the Fit gene products of P. fluorescens CHA0 and Pf-5 as potent insect toxins that define previously unappreciated anti-insect properties of these plant-colonizing bacteria

Origin and genome evolution of polyploid green toads in Central Asia: evidence from microsatellite markers.

Polyploidization, which is expected to trigger major genomic reorganizations, occurs much less commonly in animals than in plants, possibly because of constraints imposed by sex-determination systems. We investigated the origins and consequences of allopolyploidization in Palearctic green toads (Bufo viridis subgroup) from Central Asia, with three ploidy levels and different modes of genome transmission (sexual versus clonal), to (i) establish a topology for the reticulate phylogeny in a species-rich radiation involving several closely related lineages and (ii) explore processes of genomic reorganization that may follow polyploidization. Sibship analyses based on 30 cross-amplifying microsatellite markers substantiated the maternal origins and revealed the paternal origins and relationships of subgenomes in allopolyploids. Analyses of the synteny of linkage groups identified three markers affected by translocation events, which occurred only within the paternally inherited subgenomes of allopolyploid toads and exclusively affected the linkage group that determines sex in several diploid species of the green toad radiation. Recombination rates did not differ between diploid and polyploid toad species, and were overall much reduced in males, independent of linkage group and ploidy levels. Clonally transmitted subgenomes in allotriploid toads provided support for strong genetic drift, presumably resulting from recombination arrest. The Palearctic green toad radiation seems to offer unique opportunities to investigate the consequences of polyploidization and clonal transmission on the dynamics of genomes in vertebrates.