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The cultivated potato (Solanum tuberosum) shares similar biology with other members of the Solanaceae, yet has features unique within the family, such as modified stems (stolons) that develop into edible tubers. To better understand potato biology, we have undertaken a survey of the potato transcriptome using expressed sequence tags (ESTs) from diverse tissues. A total of 61,940 ESTs were generated from aerial tissues, below-ground tissues, and tissues challenged with the late-blight pathogen (Phytophthora infestans). Clustering and assembly of these ESTs resulted in a total of 19,892 unique sequences with 8,741 tentative consensus sequences and 11,151 singleton ESTs. We were able to identify a putative function for 43.7% of these sequences. A number of sequences (48) were expressed throughout the libraries sampled, representing constitutively expressed sequences. Other sequences (13,068, 21%) were uniquely expressed and were detected only in a single library. Using hierarchal and k means clustering of the EST sequences, we were able to correlate changes in gene expression with major physiological events in potato biology. Using pair-wise comparisons of tuber-related tissues, we were able to associate genes with tuber initiation, dormancy, and sprouting. We also were able to identify a number of characterized as well as novel sequences that were unique to the incompatible interaction of late-blight pathogen, thereby providing a foundation for further understanding the mechanism of resistance.The Solanaceae family contains several species of agronomic importance such as tomato (Lycopersicon esculentum), potato (Solanum tuberosum), pepper (Capsicum annuum), eggplant (Solanum melongena), petunia (Petunia ϫ hybrida), and tobacco (Nicotiana tabacum). Species within the Solanaceae are highly related as evidenced by conserved sequence identity at the gene level and synteny among the homologous chromosomes (Bonierbale et al., 1988;Tanksley et al., 1992;Livingstone et al., 1999). Although members of the Solanaceae family share a number of features at the genome level, potato has a number of features that makes it unique among the Solanaceae. The most important physiological feature is the development of an edible tuber from stolons and consequently, on a global scale, potato is the fourth largest crop species grown as a food source with 300 million metric tons grown annually (http://www.cipotato.org/potato/ potato.htm). However, despite its significance as a major food source, the process of tuber development is not well understood at the molecular level. In addition, potato is susceptible to the late-blight pathogen (Phytophthora infestans), which is not only a historically significant disease that resulted in the deaths of millions of people (for review, see Schumann, 1991), but it also has recently reemerged as a significant pathogen on potato (Fry and Goodwin, 1997).The development of high-throughput sequencing technology has provided a mechanism to gain insight into genomes at the DNA and the RNA level. For assessme...

Most studies of gene expression in Plasmodium have been concerned with asexual and͞or sexual erythrocytic stages. Identification and cloning of genes expressed in the preerythrocytic stages lag far behind. We have constructed a high quality cDNA library of the Plasmodium sporozoite stage by using the rodent malaria parasite P. yoelii, an important model for malaria vaccine development. The technical obstacles associated with limited amounts of RNA material were overcome by PCR-amplifying the transcriptome before cloning. Contamination with mosquito RNA was negligible. Generation of 1,972 expressed sequence tags (EST) resulted in a total of 1,547 unique sequences, allowing insight into sporozoite gene expression. The circumsporozoite protein (CS) and the sporozoite surface protein 2 (SSP2) are well represented in the data set. A BLASTX search with all tags of the nonredundant protein database gave only 161 unique significant matches (P(N) < 10 ؊4 ), whereas 1,386 of the unique sequences represented novel sporozoite-expressed genes. We identified ESTs for three proteins that may be involved in host cell invasion and documented their expression in sporozoites. These data should facilitate our understanding of the preerythrocytic Plasmodium life cycle stages and the development of preerythrocytic vaccines.Plasmodium yoelii yoelii ͉ expressed sequence tag P rotozoan parasites of the genus Plasmodium are the causative agents of malaria, the most devastating parasitic disease in humans. The parasites occur in distinct morphological and antigenic stages as they progress through a complex life cycle, thwarting decades of efforts to develop an effective malaria vaccine. Plasmodium is transmitted via the bite of an infected Anopheles mosquito, which releases the sporozoite stage into the skin. Sporozoites enter the bloodstream and, on reaching the liver, invade hepatocytes and develop into exo-erythrocytic forms (EEF). After multiple cycles of DNA replication, the EEF contains thousands of merozoites (liver schizont) that are released into the blood stream and initiate the erythrocytic cycle (asexual blood stage) that causes the disease malaria. Changes in life cycle stages are accompanied by major changes in gene expression and therefore by major changes in antigenic composition. The form of the parasite best studied is the asexual blood stage, mainly because of its comparatively easy experimental accessibility. Therefore, most Plasmodium proteins that have been well characterized are expressed during the erythrocytic cycle, among them some major erythrocytic-stage vaccine candidates such as merozoite surface protein-1 (MSP-1) and apical membrane antigen-1 (AMA-1; ref. 1). Erythrocytic-stage vaccines are aimed at inducing an immune response that suppresses or eradicates parasite load in the blood. In contrast, preerythrocytic vaccines are aimed at eliciting an immune response that destroys the sporozoites and the EEF, thereby preventing progression of the parasite to the blood stage. The feasibility of a preerythrocytic v...

Comparative genomics promises to rapidly accelerate the identification and functional classification of biologically important human genes. We developed the TIGR Orthologous Gene Alignment (TOGA; 〈http://www.tigr.org/tdb/toga/toga.shtml〉) database to provide a cross-reference between fully and partially sequenced eukaryotic transcribed sequences. Starting with the assembled expressed sequence tag (EST) and gene sequences that comprise the 28 TIGR Gene Indices, we used high-stringency pair-wise sequence searches and a reflexive, transitive closure process to associate sequence-specific best hits, generating 32,652 tentative ortholog groups (TOGs). This has allowed us to identify putative orthologs and paralogs for known genes, as well as those that exist only as uncharacterized ESTs and to provide links to additional information including genome sequence and mapping data. TOGA provides an important new resource for the analysis of gene function in eukaryotes. In addition, an analysis of the most widely represented sequences can begin to provide insight into eukaryotic biological processes

An essential component of functional genomics studies is the sequence of DNA expressed in tissues of interest. To provide a resource of bovine-specific expressed sequence data and facilitate this powerful approach in cattle research, four normalized cDNA libraries were produced and arrayed for high-throughput sequencing. The libraries were made with RNA pooled from multiple tissues to increase efficiency of normalization and maximize the number of independent genes for which sequence data were obtained. Target tissues included those with highest likelihood to have impact on production parameters of animal health, growth, reproductive efficiency, and carcass merit. Success of normalization and inter-and intralibrary redundancy were assessed by collecting 6000-23,000 sequences from each of the libraries (68,520 total sequences deposited in GenBank). Sequence comparison and assembly of these sequences was performed in combination with 56,500 other bovine EST sequences present in the GenBank dbEST database to construct a cattle Gene Index (available from The Institute for Genomic Research at http://www.tigr.org/tdb/tgi.shtml). The 124,381 bovine ESTs present in GenBank at the time of the analysis form 16,740 assemblies that are listed and annotated on the Web site. Analysis of individual library sequence data indicates that the pooled-tissue approach was highly effective in preparing libraries for efficient deep sequencing.The rapid progress of genomic research in diverse organisms such as yeast, fruit flies, nematodes, mice, and humans has been driven by a combination of mapping, sequencing, and identification of expressed portions of each genome. Progress in these areas has lagged in the livestock species, limiting the use of functional genomics approaches to current problems in production-animal agriculture. Resources for a public effort to sequence the genomes of livestock species are not currently available. However, more modest sequencing efforts aimed at cDNA libraries have substantial value to the research community, especially when combined with mapping efforts to produce comparative maps with other mammalian species. Comparative maps make use of the general conservation of synteny between mammals and allow the livestock community to tap into the wealth of information generated in the human genome effort.To provide a resource of livestock-specific expressed sequence data to facilitate proteomics and functional genomics approaches in animal science, an EST-sequencing program was initiated with the aim to maximize the efficiency of obtaining sequence from the highest possible number of independent genes. Four normalized bovine cDNA libraries specifically designed for this task were produced and arrayed for high-throughput sequencing. To make the data more accessible and useful, assembly and annotation analyses were performed and an interactive Web site constructed by The Institute for Genomic Research (TIGR) to view and analyze bovine genes. We report the construction of this Cattle Gene Index and assessment ...

Genetic and environmental factors affect the efficiency of pork production by influencing gene expression during porcine reproduction, tissue development, and growth. The identification and functional analysis of gene products important to these processes would be greatly enhanced by the development of a database of expressed porcine gene sequence. Two normalized porcine cDNA libraries (MARC 1PIG and MARC 2PIG), derived respectively from embryonic and reproductive tissues, were constructed, sequenced, and analyzed. A total of 66,245 clones from these two libraries were 5?-end sequenced and deposited in GenBank. Cluster analysis revealed that within-library redundancy is low, and comparison of all porcine ESTs with the human database suggests that the sequences from these two libraries represent portions of a significant number of independent pig genes. A Porcine Gene Index (PGI), comprising 15,616 tentative consensus sequences and 31,466 singletons, includes all sequences in public repositories and has been developed to facilitate further comparative map development and characterization of porcine genes (http://www.tigr.org/tdb/ssgi/). The clones and sequences from these libraries provide a catalog of expressed porcine genes and a resource for development of high-density hybridization arrays for transcriptional profiling of porcine tissues. In addition, comparison of porcine ESTs with sequences from other species serves as a valuable resource for comparative map development. Both arrayed cDNA libraries are available for unrestricted public use.

Functional genomic studies of the mammary gland require an appropriate collection of cDNA sequences to assess gene expression patterns from the different developmental and operational states of underlying cell types. To better capture the range of gene expression, a normalized cDNA library was constructed from pooled bovine mammary tissues, and 23,202 expressed sequence tags (EST) were produced and deposited into GenBank. Assembly of these EST with sequences in the Bos taurus Gene Index (BtGI) helped to form 5751 of the current 23,883 tentative consensus (TC) sequences. The majority (87%) of these 5751 assemblies contained only one to three mammary-derived EST. In contrast, 18% of the mammary EST assembled with TC sequences corresponding to 12 genes. These results suggest library normalization was only partially effective, because the reduction in EST for genes abundantly transcribed during lactation could be attributed to pooling. For better assessment of novel content in the mammary library and to add to existing annotation of all bovine sequence elements, gene ontology assignments, and comparative sequence analyses against human genome sequence, human and rodent gene indices, and an index of orthologous alignments of genes across eukaryotes (TOGA) were performed, and results were added to existing BtGI annotation. Over 35,000 of the bovine elements significantly matched human genome sequence, and the positions of some alignments (3%) were unique relative to those using human expressed sequences. Because 3445 TC sequences had no significant match with any data set, mammary-derived cDNA clones representing 23 of these elements were analyzed further for expression and novelty. Only one clone met criteria suggesting the corresponding gene was a divergent ortholog or expressed sequence unique to cattle. These results demonstrate that bovine sequence expression data serve as a resource for characterizing mammalian transcriptomes and identifying those genes potentially unique to ruminants.

Microarray expression analysis is providing unprecedented data on gene expression in humans and mammalian model systems. Although such studies provide a tremendous resource for understanding human disease states, one of the significant challenges is cross-referencing the data derived from different species, across diverse expression analysis platforms, in order to properly derive inferences regarding gene expression and disease state. To address this problem, we have developed RESOURCERER, a microarray-resource annotation and cross-reference database built using the analysis of expressed sequence tags (ESTs) and gene sequences provided by the TIGR Gene Index (TGI) and TIGR Orthologous Gene Alignment (TOGA) databases [now called Eukaryotic Gene Orthologs (EGO)].