Florence Rouleux-Bonnin

Genomic Sequence of Spodoptera frugiperda Ascovirus 1a, an Enveloped, Double-Stranded DNA Insect Virus That Manipulates Apoptosis for Viral Reproduction

ABSTRACT Ascoviruses (family Ascoviridae) are double-stranded DNA viruses with circular genomes that attack lepidopterans, where they produce large, enveloped virions, 150 by 400 nm, and cause a chronic, fatal disease with a cytopathology resembling that of apoptosis. After infection, host cell DNA is degraded, the nucleus fragments, and the cell then cleaves into large virion-containing vesicles. These vesicles and virions circulate in the hemolymph, where they are acquired by parasitic wasps during oviposition and subsequently transmitted to new hosts. To develop a better understanding of ascovirus biology, we sequenced the genome of the type species Spodoptera frugiperda ascovirus 1a (SfAV-1a). The genome consisted of 156,922 bp, with a G+C ratio of 49.2%, and contained 123 putative open reading frames coding for a variety of enzymes and virion structural proteins, of which tentative functions were assigned to 44. Among the most interesting enzymes, due to their potential role in apoptosis and viral vesicle formation, were a caspase, a cathepsin B, several kinases, E3 ubiquitin ligases, and especially several enzymes involved in lipid metabolism, including a fatty acid elongase, a sphingomyelinase, a phosphate acyltransferase, and a patatin-like phospholipase. Comparison of SfAV-1a proteins with those of other viruses showed that 10% were orthologs of Chilo iridescent virus proteins, the highest correspondence with any virus, providing further evidence that ascoviruses evolved from a lepidopteran iridovirus. The SfAV-1a genome sequence will facilitate the determination of how ascoviruses manipulate apoptosis to generate the novel virion-containing vesicles characteristic of these viruses and enable study of their origin and evolution.

Evolution of Full-Length and Deleted Forms of the Mariner-Like Element, Botmar1, in the Genome of the Bumble Bee, Bombus terrestris (Hymenoptera: Apidae)

Mariner-like elements (MLE) are Class II transposable elements that are very widespread among eukaryotic genomes. One MLE belonging to the mauritiana subfamily, named Botmar1, has been identified in the genome of the bumble bee, Bombus terrestris. gDNA hybridization with the Botmar1 transposase ORF revealed that about 230 elements are present in each haploid genome of B. terrestris that consist entirely of 1.3- and 0.85-kbp elements. The analysis of their sequences revealed that there are two Botmar1 subfamilies of similar ages in the Bombus terrestris genome: one is composed entirely of 1.3-kpb elements, whereas the second comprises both completed and deleted elements. Our previous data indicated that the internally deleted form, which correspond to the 0.85-kbp Botmar1-related elements occur in other distantly related hymenopteran genomes. Because the presence of similar 1.3- and 0.85-kbp Botmar1-related elements in some distantly related hymenopteran species cannot be explained by horizontal transfers, the nucleic acid sequence properties of these elements were further investigated. We found that certain structural properties in their nucleic acid sequence might explain the occurrence of 0.85-kbp Botmar1-related elements presenting similarly located internal deletions in hymenopteran genomes.

Factors acting on Mos1 transposition efficiency

BackgroundMariner- like elements (MLEs) are widespread DNA transposons in animal genomes. Although in vitro transposition reactions require only the transposase, various factors depending on the host, the physico-chemical environment and the transposon sequence can interfere with the MLEs transposition in vivo.ResultsThe transposition of Mos1, first isolated from drosophila mauritiana, depends of both the nucleic acid sequence of the DNA stuffer (in terms of GC content), and its length. We provide the first in vitro experimental demonstration that MITEs of MLE origin, as small as 80 to 120-bp, are able to transpose. Excessive temperature down-regulates Mos1 transposition, yielding excision products unable to re-integrate. Finally, the super-helicity of the DNA transposon donor has a dramatic impact on the transposition efficiency.ConclusionThe study highlights how experimental conditions can bias interpretation of mariner excision frequency and quality. In vitro, the auto-integration pathway markedly limits transposition efficiency to new target sites, and this phenomenon may also limit events in the natural host. We propose a model for small transposons transposition that bypasses DNA bending constraints.

Free serosal cells originating from the embryo of the wasp Diadromus pulchellus in the pupal body of parasitized leek-moth, Acrolepiosis assectella. Are these cells teratocyte-like?

In braconid species, teratocytes are derived from a serosal cell membrane which envelops the developing parasitoid embryo. On hatching, this membrane dissociates into individual cells, the teratocytes, which then circulate in the haemolymph of the host. We describe herein such a membrane, surrounding the embryo in eggs of the ichneumonid parasitoid wasp, Diadromus pulchellus. This membrane consisted of a single sheet of tightly packed cells with large 12+/-1.4 &mgr;m nuclei. These cells were released after hatching in vitro and cells of the same size were detected in vivo, in the vicinity of the D. pulchellus embryo. The number of nuclei detected suggests that the serosal membrane consists of about 450+/-150 cells. These cells did not grow after hatching of the parasitoid egg in the parasitized host, Acrolepiosis assectella, during the development of the parasitoid wasp larva. Southern blot experiments, using D. pulchellus satellite DNA or the ribosomal genes as probes, showed that free-living floating cells of wasp origin were present in the body of the parasitized host. This is the first time that free-floating teratocyte-like cells have been described in species of the Ichneumonidae.

Transposable elements as tools for reshaping the genome: it is a huge world after all!

Transposable elements (TEs) are discrete pieces of DNA that can move from one site to another within genomes and sometime between genomes. They are found in all major branches of life. Because of their wide distribution and considerable diversity, they are a considerable source of genomic variation and as such, they constitute powerful drivers of genome evolution. Moreover, it is becoming clear that the epigenetic regulation of certain genes is derived from defense mechanisms against the activity of ancestral transposable elements. TEs now tend to be viewed as natural molecular tools that can reshape the genome, which challenges the idea that TEs are natural tools used to answer biological questions. In the first part of this chapter, we review the classification and distribution of TEs, and look at how they have contributed to the structural and transcriptional reshaping of genomes. In the second part, we describe methodological innovations that have modified their contribution as molecular tools.

Physical properties of DNA components affecting the transposition efficiency of the mariner Mos1 element

Previous studies have shown that the transposase and the inverted terminal repeat (ITR) of the Mos1mariner elements are suboptimal for transposition; and that hyperactive transposases and transposon with more efficient ITR configurations can be obtained by rational molecular engineering. In an attempt to determine the extent to which this element is suboptimal for transposition, we investigate here the impact of the three main DNA components on its transposition efficiency in bacteria and in vitro. We found that combinations of natural and synthetic ITRs obtained by systematic evolution of ligands by exponential enrichment did increase the transposition rate. We observed that when untranslated terminal regions were associated with their respective natural ITRs, they acted as transposition enhancers, probably via the early transposition steps. Finally, we demonstrated that the integrity of the Mos1 inner region was essential for transposition. These findings allowed us to propose prototypes of optimized Mos1 vectors, and to define the best sequence features of their associated marker cassettes. These vector prototypes were assayed in HeLa cells, in which Mos1 vectors had so far been found to be inactive. The results obtained revealed that using these prototypes does not circumvent this problem. However, such vectors can be expected to provide new tools for the use in genome engineering in systems such as Caenorhabditis elegans in which Mos1 is very active.

Optimization of the piggyBac Transposon Using mRNA and Insulators: Toward a More Reliable Gene Delivery System

Integrating and expressing stably a transgene into the cellular genome remain major challenges for gene-based therapies and for bioproduction purposes. While transposon vectors mediate efficient transgene integration, expression may be limited by epigenetic silencing, and persistent transposase expression may mediate multiple transposition cycles. Here, we evaluated the delivery of the piggyBac transposase messenger RNA combined with genetically insulated transposons to isolate the transgene from neighboring regulatory elements and stabilize expression. A comparison of piggyBac transposase expression from messenger RNA and DNA vectors was carried out in terms of expression levels, transposition efficiency, transgene expression and genotoxic effects, in order to calibrate and secure the transposition-based delivery system. Messenger RNA reduced the persistence of the transposase to a narrow window, thus decreasing side effects such as superfluous genomic DNA cleavage. Both the CTF/NF1 and the D4Z4 insulators were found to mediate more efficient expression from a few transposition events. We conclude that the use of engineered piggyBac transposase mRNA and insulated transposons offer promising ways of improving the quality of the integration process and sustaining the expression of transposon vectors.

Transgene Site-Specific Integration: Problems and Solutions

Integrative gene transfer performed by viral and non-viral vectors have demonstrated their effectiveness, but have been linked to some adverse events, such as clonal expansion and tumorigenesis. These observations have raised serious concerns about the safety of gene transfer methods, and have led to many attempts to find new solutions. In this chapter, we summarize the major problems encountered with viral and non-viral-vectors and various ways of avoiding insertional mutagenesis, the induction of innate immunity and transgene silencing are described. We also list the main strategies for optimizing vector architecture so as to ensure safe and long-term expression of the transgene. Several new approaches have succeeded in targeting transgene integration to a specific locus using recombinases, homing endonucleases, zinc finger nucleases, integrases and transposases or a combination of them. Here, we report various criteria that can be used to define what is a good insertion site in the human genome.

Alteration Analysis of Bone Marrow Mesenchymal Stromal Cells from De Novo Acute Myeloid Leukemia Patients at Diagnosis

Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) frequently display alterations in several hematologic disorders, such as acute lymphoid leukemia, acute myeloid leukemia (AML), and myelodysplastic syndromes. In acute leukemias, it is not clear whether MSC alterations contribute to the development of the malignant clone or whether they are simply the effect of tumor expansion on the microenvironment. We extensively investigated the characteristics of MSCs isolated from the BM of patients with de novo AML at diagnosis (L-MSCs) in terms of phenotype (gene and protein expression, apoptosis and senescence levels, DNA double-strand break formation) and functions (proliferation and clonogenic potentials, normal and leukemic hematopoiesis-supporting activity). We found that L-MSCs show reduced proliferation capacity and increased apoptosis levels compared with MSCs from healthy controls. Longer population doubling time in L-MSCs was not related to the AML characteristics at diagnosis (French-American-British type, cytogenetics, or tumor burden), but was related to patient age and independently associated with poorer patient outcome, as was cytogenetic prognostic feature. Analyzing, among others, the expression of 93 genes, we found that proliferative deficiency of L-MSCs was associated with a perivascular feature at the expense of the osteo-chondroblastic lineage with lower expression of several niche factors, such as KITLG, THPO, and ANGPT1 genes, the cell adhesion molecule VCAM1, and the developmental/embryonic genes, BMI1 and DICER1. L-MSC proliferative capacity was correlated positively with CXCL12, THPO, and ANGPT1 expression and negatively with JAG1 expression. Anyway, these changes did not affect their in vitro capacity to support normal hematopoiesis and to modify leukemic cell behavior (protection from apoptosis and quiescence induction). Our findings indicate that BM-derived MSCs from patients with newly diagnosed AML display phenotypic and functional alterations such as proliferative deficiency that could be attributed to tumor progression, but does not seem to play a special role in the leukemic process.