Coral life history and symbiosis: functional genomic resources for two reef building Caribbean corals, Acropora palmata and Montastraea faveolata.

BACKGROUND: Scleractinian corals are the foundation of reef ecosystems in tropical marine environments. Their great success is due to interactions with endosymbiotic dinoflagellates (Symbiodinium spp.), with which they are obligately symbiotic. To develop a foundation for studying coral biology and coral symbiosis, we have constructed a set of cDNA libraries and generated and annotated ESTs from two species of corals, Acropora palmata and Montastraea faveolata. RESULTS: We generated 14,588 (Ap) and 3,854 (Mf) high quality ESTs from five life history/symbiosis stages (spawned eggs, early-stage planula larvae, late-stage planula larvae either infected with symbionts or uninfected, and adult coral). The ESTs assembled into a set of primarily stage-specific clusters, producing 4,980 (Ap), and 1,732 (Mf) unigenes. The egg stage library, relative to the other developmental stages, was enriched in genes functioning in cell division and proliferation, transcription, signal transduction, and regulation of protein function. Fifteen unigenes were identified as candidate symbiosis-related genes as they were expressed in all libraries constructed from the symbiotic stages and were absent from all of the non symbiotic stages. These include several DNA interacting proteins, and one highly expressed unigene (containing 17 cDNAs) with no significant protein-coding region. A significant number of unigenes (25) encode potential pattern recognition receptors (lectins, scavenger receptors, and others), as well as genes that may function in signaling pathways involved in innate immune responses (toll-like signaling, NFkB p105, and MAP kinases). Comparison between the A. palmata and an A. millepora EST dataset identified ferritin as a highly expressed gene in both datasets that appears to be undergoing adaptive evolution. Five unigenes appear to be restricted to the Scleractinia, as they had no homology to any sequences in the nr databases nor to the non-scleractinian cnidarians Nematostella vectensis and Hydra magnipapillata. CONCLUSION: Partial sequencing of 5 cDNA libraries each for A. palmata and M. faveolata has produced a rich set of candidate genes (4,980 genes from A. palmata, and 1,732 genes from M. faveolata) that we can use as a starting point for examining the life history and symbiosis of these two species, as well as to further expand the dataset of cnidarian genes for comparative genomics and evolutionary studies.

Construction and characterization of EST libraries from the porcelain crab, Petrolisthes cinctipes.

The thermal phenotype of an organism (heat and cold tolerance, thermal range, and thermal plasticity) is an essential feature of how the organism performs across thermal environments and in response to thermal stress. Porcelain crabs are of interest in addressing questions of thermal phenotype because of their high species diversity and the large variation in thermal phenotype among species, as well as the biogeographic patterning of these crabs along environmental stress gradients. We are studying the cellular bases of thermal phenotype and physiological responses to environmental stress using a functional genomics cDNA microarray approach. To do this, we have isolated total RNA from a range of tissues from 1 species of porcelain crab (Petrolisthes cinctipes) exposed to a suite of thermal conditions, and have used this RNA to construct a 13 824-clone EST library. Here, we describe construction, EST sequencing, assembly and clustering, and results of BLASTx homology search for our initial 13 824-clone library. From 12 060 usable ESTs, 6717 consensus sequences were identified, and roughly 50% of these have homology to known proteins. At present, an additional 50 000-75 000-clone library of P. cinctipes ESTs is being generated, with the aim of developing a library with near-complete coverage of the transcriptome. The libraries and sequence information that will be generated as a result of this project should be of value for crustacean biologists working across a broad range of scientific disciplines (for example, physiology, developmental biology, biological rhythms, ecology, fisheries biology), as well as in studies of molecular evolution and phylogeography.

The Drosophila gene collection: identification of putative full-length cDNAs for 70% of D. melanogaster genes.

Collections of full-length nonredundant cDNA clones are critical reagents for functional genomics. The first step toward these resources is the generation and single-pass sequencing of cDNA libraries that contain a high proportion of full-length clones. The first release of the Drosophila Gene Collection Release 1 (DGCr1) was produced from six libraries representing various tissues, developmental stages, and the cultured S2 cell line. Nearly 80,000 random 5' expressed sequence tags (5' expressed sequence tags [ESTs]from these libraries were collapsed into a nonredundant set of 5849 cDNAs, corresponding to ~40% of the 13,474 predicted genes in Drosophila. To obtain cDNA clones representing the remaining genes, we have generated an additional 157,835 5' ESTs from two previously existing and three new libraries. One new library is derived from adult testis, a tissue we previously did not exploit for gene discovery; two new cap-trapped normalized libraries are derived from 0-22-h embryos and adult heads. Taking advantage of the annotated D. melanogaster genome sequence, we clustered the ESTs by aligning them to the genome. Clusters that overlap genes not already represented by cDNA clones in the DGCr1 were analyzed further, and putative full-length clones were selected for inclusion in the new DGC. This second release of the DGC (DGCr2) contains 5061 additional clones, extending the collection to 10,910 cDNAs representing >70% of the predicted genes in Drosophila.

A Drosophila full-length cDNA resource.

BACKGROUND: A collection of sequenced full-length cDNAs is an important resource both for functional genomics studies and for the determination of the intron-exon structure of genes. Providing this resource to the Drosophila melanogaster research community has been a long-term goal of the Berkeley Drosophila Genome Project. We have previously described the Drosophila Gene Collection (DGC), a set of putative full-length cDNAs that was produced by generating and analyzing over 250,000 expressed sequence tags (ESTs) derived from a variety of tissues and developmental stages. RESULTS: We have generated high-quality full-insert sequence for 8,921 clones in the DGC. We compared the sequence of these clones to the annotated Release 3 genomic sequence, and identified more than 5,300 cDNAs that contain a complete and accurate protein-coding sequence. This corresponds to at least one splice form for 40% of the predicted D. melanogaster genes. We also identified potential new cases of RNA editing. CONCLUSIONS: We show that comparison of cDNA sequences to a high-quality annotated genomic sequence is an effective approach to identifying and eliminating defective clones from a cDNA collection and ensure its utility for experimentation. Clones were eliminated either because they carry single nucleotide discrepancies, which most probably result from reverse transcriptase errors, or because they are truncated and contain only part of the protein-coding sequence.