Identification of an Alternative Splicing Product of the Otx2 Gene Expressed in the Neural Retina and Retinal Pigmented Epithelial Cells.

To investigate the complexity of alternative splicing in the retina, we sequenced and analyzed a total of 115,706 clones from normalized cDNA libraries from mouse neural retina (66,217) and rat retinal pigmented epithelium (49,489). Based upon clustering the cDNAs and mapping them with their respective genomes, the estimated numbers of genes were 9,134 for the mouse neural retina and 12,050 for the rat retinal pigmented epithelium libraries. This unique collection of retinal of messenger RNAs is maintained and accessible through a web-base server to the whole community of retinal biologists for further functional characterization. The analysis revealed 3,248 and 3,202 alternative splice events for mouse neural retina and rat retinal pigmented epithelium, respectively. We focused on transcription factors involved in vision. Among the six candidates suitable for functional analysis, we selected Otx2S, a novel variant of the Otx2 gene with a deletion within the homeodomain sequence. Otx2S is expressed in both the neural retina and retinal pigmented epithelium, and encodes a protein that is targeted to the nucleus. OTX2S exerts transdominant activity on the tyrosinase promoter when tested in the physiological environment of primary RPE cells. By overexpressing OTX2S in primary RPE cells using an adeno associated viral vector, we identified 10 genes whose expression is positively regulated by OTX2S. We find that OTX2S is able to bind to the chromatin at the promoter of the retinal dehydrogenase 10 (RDH10) gene.

The streamlined genome of Phytomonas spp. relative to human pathogenic kinetoplastids reveals a parasite tailored for plants.

Members of the family Trypanosomatidae infect many organisms, including animals, plants and humans. Plant-infecting trypanosomes are grouped under the single genus Phytomonas, failing to reflect the wide biological and pathological diversity of these protists. While some Phytomonas spp. multiply in the latex of plants, or in fruit or seeds without apparent pathogenicity, others colonize the phloem sap and afflict plants of substantial economic value, including the coffee tree, coconut and oil palms. Plant trypanosomes have not been studied extensively at the genome level, a major gap in understanding and controlling pathogenesis. We describe the genome sequences of two plant trypanosomatids, one pathogenic isolate from a Guianan coconut and one non-symptomatic isolate from Euphorbia collected in France. Although these parasites have extremely distinct pathogenic impacts, very few genes are unique to either, with the vast majority of genes shared by both isolates. Significantly, both Phytomonas spp. genomes consist essentially of single copy genes for the bulk of their metabolic enzymes, whereas other trypanosomatids e.g. Leishmania and Trypanosoma possess multiple paralogous genes or families. Indeed, comparison with other trypanosomatid genomes revealed a highly streamlined genome, encoding for a minimized metabolic system while conserving the major pathways, and with retention of a full complement of endomembrane organelles, but with no evidence for functional complexity. Identification of the metabolic genes of Phytomonas provides opportunities for establishing in vitro culturing of these fastidious parasites and new tools for the control of agricultural plant disease.

Insights into metazoan evolution from Alvinella pompejana cDNAs.

BACKGROUND: Alvinella pompejana is a representative of Annelids, a key phylum for evo-devo studies that is still poorly studied at the sequence level. A. pompejana inhabits deep-sea hydrothermal vents and is currently known as one of the most thermotolerant Eukaryotes in marine environments, withstanding the largest known chemical and thermal ranges (from 5 to 105°C). This tube-dwelling worm forms dense colonies on the surface of hydrothermal chimneys and can withstand long periods of hypo/anoxia and long phases of exposure to hydrogen sulphides. A. pompejana specifically inhabits chimney walls of hydrothermal vents on the East Pacific Rise. To survive, Alvinella has developed numerous adaptations at the physiological and molecular levels, such as an increase in the thermostability of proteins and protein complexes. It represents an outstanding model organism for studying adaptation to harsh physicochemical conditions and for isolating stable macromolecules resistant to high temperatures. RESULTS: We have constructed four full length enriched cDNA libraries to investigate the biology and evolution of this intriguing animal. Analysis of more than 75,000 high quality reads led to the identification of 15,858 transcripts and 9,221 putative protein sequences. Our annotation reveals a good coverage of most animal pathways and networks with a prevalence of transcripts involved in oxidative stress resistance, detoxification, anti-bacterial defence, and heat shock protection. Alvinella proteins seem to show a slow evolutionary rate and a higher similarity with proteins from Vertebrates compared to proteins from Arthropods or Nematodes. Their composition shows enrichment in positively charged amino acids that might contribute to their thermostability. The gene content of Alvinella reveals that an important pool of genes previously considered to be specific to Deuterostomes were in fact already present in the last common ancestor of the Bilaterian animals, but have been secondarily lost in model invertebrates. This pool is enriched in glycoproteins that play a key role in intercellular communication, hormonal regulation and immunity. CONCLUSIONS: Our study starts to unravel the gene content and sequence evolution of a deep-sea annelid, revealing key features in eukaryote adaptation to extreme environmental conditions and highlighting the proximity of Annelids and Vertebrates.

Construction of a medicinal leech transcriptome database and its application to the identification of leech homologs of neural and innate immune genes.

BACKGROUND: The medicinal leech, Hirudo medicinalis, is an important model system for the study of nervous system structure, function, development, regeneration and repair. It is also a unique species in being presently approved for use in medical procedures, such as clearing of pooled blood following certain surgical procedures. It is a current, and potentially also future, source of medically useful molecular factors, such as anticoagulants and antibacterial peptides, which may have evolved as a result of its parasitizing large mammals, including humans. Despite the broad focus of research on this system, little has been done at the genomic or transcriptomic levels and there is a paucity of openly available sequence data. To begin to address this problem, we constructed whole embryo and adult central nervous system (CNS) EST libraries and created a clustered sequence database of the Hirudo transcriptome that is available to the scientific community. RESULTS: A total of approximately 133,000 EST clones from two directionally-cloned cDNA libraries, one constructed from mRNA derived from whole embryos at several developmental stages and the other from adult CNS cords, were sequenced in one or both directions by three different groups: Genoscope (French National Sequencing Center), the University of Iowa Sequencing Facility and the DOE Joint Genome Institute. These were assembled using the phrap software package into 31,232 unique contigs and singletons, with an average length of 827 nt. The assembled transcripts were then translated in all six frames and compared to proteins in NCBI's non-redundant (NR) and to the Gene Ontology (GO) protein sequence databases, resulting in 15,565 matches to 11,236 proteins in NR and 13,935 matches to 8,073 proteins in GO. Searching the database for transcripts of genes homologous to those thought to be involved in the innate immune responses of vertebrates and other invertebrates yielded a set of nearly one hundred evolutionarily conserved sequences, representing all known pathways involved in these important functions. CONCLUSIONS: The sequences obtained for Hirudo transcripts represent the first major database of genes expressed in this important model system. Comparison of translated open reading frames (ORFs) with the other openly available leech datasets, the genome and transcriptome of Helobdella robusta, shows an average identity at the amino acid level of 58% in matched sequences. Interestingly, comparison with other available Lophotrochozoans shows similar high levels of amino acid identity, where sequences match, for example, 64% with Capitella capitata (a polychaete) and 56% with Aplysia californica (a mollusk), as well as 58% with Schistosoma mansoni (a platyhelminth). Phylogenetic comparisons of putative Hirudo innate immune response genes present within the Hirudo transcriptome database herein described show a strong resemblance to the corresponding mammalian genes, indicating that this important physiological response may have older origins than what has been previously proposed.

Stage-specific gene expression during spermatogenesis in the dogfish (Scyliorhinus canicula).

In the dogfish testis, the cystic arrangement and polarization of germ cell stages make it possible to observe all stages of spermatogenesis in a single transverse section. By taking advantage of the zonation of this organ, we have used suppressive subtractive libraries construction, real-time PCR, and in situ hybridization to identify 32 dogfish genes showing differential expressions during spermatogenesis. These include homologs of genes already known to be expressed in the vertebrate testis, but found here to be specifically expressed either in pre-meiotic and/or meiotic zones (ribosomal protein S8, high-mobility group box 3, ubiquitin carboxyl-terminal esterase L3, 20beta-hydroxysteroid dehydrogenase, or cyclophilin B) or in post-meiotic zone (speriolin, Soggy, zinc finger protein 474, calreticulin, or phospholipase c-zeta). We also report, for the first time, testis-specific expression patterns for dogfish genes coding for A-kinase anchor protein 5, ring finger protein 152, or F-box only protein 7. Finally, the study highlights the differential expression of new sequences whose identity remains to be assessed. This study provides the first molecular characterization of spermatogenesis in a chondrichthyan, a key species to gain insight into the evolution of this process in gnathostomes.