Conchita Ferraz

Life-Cycle and Genome of OtV5, a Large DNA Virus of the Pelagic Marine Unicellular Green Alga Ostreococcus tauri

Large DNA viruses are ubiquitous, infecting diverse organisms ranging from algae to man, and have probably evolved from an ancient common ancestor. In aquatic environments, such algal viruses control blooms and shape the evolution of biodiversity in phytoplankton, but little is known about their biological functions. We show that Ostreococcus tauri, the smallest known marine photosynthetic eukaryote, whose genome is completely characterized, is a host for large DNA viruses, and present an analysis of the life-cycle and 186,234 bp long linear genome of OtV5. OtV5 is a lytic phycodnavirus which unexpectedly does not degrade its host chromosomes before the host cell bursts. Analysis of its complete genome sequence confirmed that it lacks expected site-specific endonucleases, and revealed the presence of 16 genes whose predicted functions are novel to this group of viruses. OtV5 carries at least one predicted gene whose protein closely resembles its host counterpart and several other host-like sequences, suggesting that horizontal gene transfers between host and viral genomes may occur frequently on an evolutionary scale. Fifty seven percent of the 268 predicted proteins present no similarities with any known protein in Genbank, underlining the wealth of undiscovered biological diversity present in oceanic viruses, which are estimated to harbour 200Mt of carbon.

Comparative Genomic Analysis of Three Strains of Ehrlichia ruminantium Reveals an Active Process of Genome Size Plasticity

Ehrlichia ruminantium is the causative agent of heartwater, a major tick-borne disease of livestock in Africa that has been introduced in the Caribbean and is threatening to emerge and spread on the American mainland. We sequenced the complete genomes of two strains of E. ruminantium of differing phenotypes, strains Gardel (Erga; 1,499,920 bp), from the island of Guadeloupe, and Welgevonden (Erwe; 1,512,977 bp), originating in South Africa and maintained in Guadeloupe in a different cell environment. Comparative genomic analysis of these two strains was performed with the recently published parent strain of Erwe (Erwo) and other Rickettsiales (Anaplasma, Wolbachia, and Rickettsia spp.). Gene order is highly conserved between the E. ruminantium strains and with A. marginale. In contrast, there is very little conservation of gene order with members of the Rickettsiaceae. However, gene order may be locally conserved, as illustrated by the tuf operons. Eighteen truncated protein-encoding sequences (CDSs) differentiate Erga from Erwe/Erwo, whereas four other truncated CDSs differentiate Erwe from Erwo. Moreover, E. ruminantium displays the lowest coding ratio observed among bacteria due to unusually long intergenic regions. This is related to an active process of genome expansion/contraction targeted at tandem repeats in noncoding regions and based on the addition or removal of ca. 150-bp tandem units. This process seems to be specific to E. ruminantium and is not observed in the other Rickettsiales.

Starch Division and Partitioning. A Mechanism for Granule Propagation and Maintenance in the Picophytoplanktonic Green Alga Ostreococcus tauri

Whereas Glc is stored in small-sized hydrosoluble glycogen particles in archaea, eubacteria, fungi, and animal cells, photosynthetic eukaryotes have resorted to building starch, which is composed of several distinct polysaccharide fractions packed into a highly organized semicrystalline granule. In plants, both the initiation of polysaccharide synthesis and the nucleation mechanism leading to formation of new starch granules are currently not understood. Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, defines the tiniest eukaryote with one of the smallest genomes. We show that it accumulates a single starch granule at the chloroplast center by using the same pathway as higher plants. At the time of plastid division, we observe elongation of the starch and division into two daughter structures that are partitioned in each newly formed chloroplast. These observations suggest that in this system the information required to initiate crystalline polysaccharide growth of a new granule is contained within the preexisting polysaccharide structure and the design of the plastid division machinery.

Genome-wide identification of in vivo Drosophila Engrailed-binding DNA fragments and related target genes.

Chromatin immunoprecipitation after UV crosslinking of DNA/protein interactions was used to construct a library enriched in genomic sequences that bind to the Engrailed transcription factor in Drosophila embryos. Sequencing of the clones led to the identification of 203 Engrailed-binding fragments localized in intergenic or intronic regions. Genes lying near these fragments, which are considered as potential Engrailed target genes, are involved in different developmental pathways, such as anteroposterior patterning, muscle development, tracheal pathfinding or axon guidance. We validated this approach by in vitro and in vivo tests performed on a subset of Engrailed potential targets involved in these various pathways. Finally, we present strong evidence showing that an immunoprecipitated genomic DNA fragment corresponds to a promoter region involved in the direct regulation of frizzled2 expression by engrailed in vivo.

DNA libraries for sequencing the genome of Ostreococcus tauri (Chlorophyta, prasinophyceae): The smallest free-living eukaryotic cell

Ostreococcus tauri is a marine photosynthetic picoeukaryote presenting a minimal cellular organization with one nucleus, one chloroplast, and one mitochondrion. It has the smallest genome described among free‐living eukaryotic cells, and we showed by pulsed‐field gel electrophoresis (PFGE) that it is divided between 15 bands ranging from 1.2 to 0.15 Mb, giving a total size of 9.7 Mb. A Bacterial Artificial Chromosome (BAC) library was prepared from genomic DNA extracted from a culture of O. tauri. A total of 2457 clones was obtained with an average insert size of around 70 kb, representing an 18‐fold coverage of the genome. The library was spotted on high density filters, and several probes of coding sequences were hybridized to both the high density BAC library filters and directly to the dried PFGE gels of the O. tauri genomic DNA. These hybridizations allowed a preliminary organization of the library and the localization of several markers on the chromosomes. Randomly selected fragments were also sequenced, representing 12% of the O. tauri genome. Many sequences showed significant similarities in data banks, mainly with plant and algae sequences. About 1000 coding sequences could be identified. These data confirmed the position of O. tauri in the green lineage and the hypothesis of a very compact organization of its genome.

Transcription Analysis of the Major Antigenic Protein 1 Multigene Family of Three In Vitro-Cultured Ehrlichia ruminantium Isolates

Ehrlichia ruminantium, an obligate intracellular bacterium transmitted by ticks of the genus Amblyomma, causes heartwater disease in ruminants. The gene coding for the major antigenic protein MAP1 is part of a multigene family consisting of a cluster containing 16 paralogs. In the search for differentially regulated genes between E. ruminantium grown in endothelial and tick cell lines that could be used in vaccine development and to determine if differences in the map1 gene cluster exist between different isolates of E. ruminantium, we analyzed the map1 gene cluster of the Senegal and Gardel isolates of E. ruminantium. Both isolates contained the same number of genes, and the same organization as found in the genome sequence of the Welgevonden isolate (H. Van Heerden, N. E. Collins, K. A. Brayton, C. Rademeyer, and B. A. Allsopp, Gene 330:159-168, 2004). However, comparison of two subpopulations of the Gardel isolate maintained in different laboratories demonstrated that recombination between map1-3 and map1-2 had occurred in one subpopulation with deletion of one entire gene. Reverse transcription-PCR on E. ruminantium derived mRNA from infected cells using gene-specific primers revealed that all 16 map1 paralogs were transcribed in endothelial cells. In one vector (Amblyomma variegatum) and several nonvector tick cell lines infected with E. ruminantium, transcripts were found for between 4 and 11 paralogs. In all these cases the transcript for the map1-1 gene was detected and was predominant. Our results indicate that the map1 gene cluster is relatively conserved but can be subject to recombination, and differences in the transcription of map1 multigenes in host and vector cell environments exist.

The first green lineage cdc25 dual-specificity phosphatase

The Cdc25 protein phosphatase is a key enzyme involved in the regulation of the G2/M transition in metazoans and yeast. However, no Cdc25 ortholog has so far been identified in plants, although functional studies have shown that an activating dephosphorylation of the CDK-cyclin complex regulates the G2/M transition. In this paper, the first green lineage Cdc25 ortholog is described in the unicellular alga Ostreococcus tauri. It encodes a protein which is able to rescue the yeast S. pombe cdc25-22 conditional mutant. Furthermore, microinjection of GST-tagged O. tauri Cdc25 specifically activates prophase-arrested starfish oocytes. In vitro histone H1 kinase assays and anti-phosphotyrosine Western Blotting confirmed the in vivo activating dephosphorylation of starfish CDK1-cyclinB by recombinant O. tauri Cdc25. We propose that there has been co-evolution of the regulatory proteins involved in the control of M-phase entry in the metazoan, yeast and green lineages. Link to supplemental material: http://www.landesbioscience.com/journals/cc/khadarooCC3-4-sup.pdf

Comparative genomics of three strains of Ehrlichia ruminantium: a review.

Abstract:  The tick‐borne Rickettsiale Ehrlichia ruminantium (E. ruminantium) is the causative agent of heartwater in Africa and the Caribbean. Heartwater, responsible for major losses on livestock in Africa represents also a threat for the American mainland. Three complete genomes corresponding to two different groups of differing phenotypes, Gardel and Welgevonden, have been recently described. One genome (Erga) represents the Gardel group from Guadeloupe Island and two genomes (Erwo and Erwe) belong to the Welgevonden group. Erwo, isolated in South Africa, is the parental strain of Erwe, which was maintained for 18 years in Guadeloupe under different culture conditions than Erwo. The three strains display genomes of differing sizes with 1,499,920 bp, 1,512,977 bp, and 1,516,355 bp for Erga, Erwe, and Erwo, respectively. Gene sequences and order are highly conserved between the three strains, although several gene truncations could be pinpointed, most of them occurring within three regions of accumulated differences (RAD). E. ruminantium displays a strong leading/lagging compositional bias inducing a strand‐specific codon usage. Finally, a striking feature of E. ruminantium is the presence of long intergenic regions containing tandem repeats. These repeats are at the origin of an active process, specific to E. ruminantium, of genome expansion/contraction based on the addition or removal of tandem units.