angaGEDUCI: Anopheles gambiae gene expression database with integrated comparative algorithms for identifying conserved DNA motifs in promoter sequences
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The completed sequence of the Anopheles gambiae genome has enabled genome-wide analyses of gene expression and regulation in this principal vector of human malaria. These investigations have created a demand for efficient methods of cataloguing and analyzing the large quantities of data that have been produced. The organization of genome-wide data into one unified database makes possible the efficient identification of spatial and temporal patterns of gene expression, and by pairing these findings with comparative algorithms, may offer a tool to gain insight into the molecular mechanisms that regulate these expression patterns.
We provide a publicly-accessible database and integrated data-mining tool, angaGEDUCI, that unifies 1) stage- and tissue-specific microarray analyses of gene expression in An. gambiae at different developmental stages and temporal separations following a bloodmeal, 2) functional gene annotation, 3) genomic sequence data, and 4) promoter sequence comparison algorithms. The database can be used to study genes expressed in particular stages, tissues, and patterns of interest, and to identify conserved promoter sequence motifs that may play a role in the regulation of such expression. The database is accessible from the address http://www.angaged.bio.uci.edu.
By combining gene expression, function, and sequence data with integrated sequence comparison algorithms, angaGEDUCI streamlines spatial and temporal pattern-finding and produces a straightforward means of developing predictions and designing experiments to assess how gene expression may be controlled at the molecular level.
KeywordsTranscription Factor Binding Site Transcription Factor Database Functional Gene Annotation Gambiae Genome Specific Transcription Factor Binding Site
The sequenced genome of the principal vector of human malaria parasites in subSaharan Africa, Anopheles gambiae , has raised expectations for the development of new and unexpected ways to manage or manipulate vector populations to control disease transmission . As part of efforts to meet these expectations, we generated and organized large data sets using gene expression microarrays to quantify genome-wide transcription in different developmental stages and tissues of this mosquito [3, 4]. Arrangement of these data into a searchable format has streamlined the elucidation of genes expressed with stage-, tissue-, and sex-specificity. In addition, by juxtaposing these microarray findings with DNA comparative algorithms, the regulation of genes co-ordinately expressed in specific spatial and temporal patterns can be studied at a mechanistic level. We provide here a public database and web-based data-mining tool that combine stage and tissue expression microarray data, functional annotation, and regulatory DNA sequence comparison algorithms to provide insight into gene expression and regulation in An. gambiae.
Construction and content
Stage-specific transcriptional signal values were imported from genome-wide microarray analyses of An. gambiae larvae, male sugar-fed adults, female sugar-fed adults, and female blood-fed adults 3, 24, 48, 72, 96 hours and 15 days after a bloodmeal using Affymetrix GCOS software. Values from tissue-specific microarray analyses also were imported using GCOS to quantify genome-wide transcription in fat bodies, midgut, and ovaries at 24 hours after bloodfeeding [3, 4]. Functional gene annotation was imported from the Ano-Xcel database  to populate angaGEDUCI with keywords and annotation from the ENSEMBL, NCBI non-redundant, GO, PFAM, and SMART databases. Promoter sequences were selected as regions 1.5 kilobases (kb) in length adjacent to the 5'-ends of transcription start sites of genes using genomic data from ENSEMBL (Assembly: AgamP3, Feb 2006; Genebuild: VectorBase, Feb 2006; Database version: 37.3). Transcription factor binding sites from several classes of organisms were imported from the Transcription Factors Database (TFD) available publicly at ftp://ftp.ncbi.nih.gov/repository/TFD/datasets/. Of the 7,066 sites listed in TFD, 6639 (94.0%) are eight nucleotides or longer and 623 (8.82%) contain degenerate notation. Five-hundred and eleven sites in the database were identified in insects (7.23%), of which 499 (97.7%) are eight nucleotides or longer, and 34 (6.65%) contain degeneracy.
Description of a gene entry
Comparing promoters to identify conserved DNA sequence motifs
After clustering genes into gene sets that show similar patterns of expression, the data-mining interface analysis menu can be used to search for common DNA motifs that may act as regulatory sequences in coordinating these expression patterns. Two parameters must be selected to begin the analysis: 1) motif match length: the desired conserved sequence motif length to search for in the analysis, 2) mismatches: the number of base mismatches allowed between two nearly-conserved sequence motifs without disqualification.
Visualization of transcription profiles
Import gene set
Submit a microarray study
The angaGEDUCI database has the capacity to store and integrate additional Affymetrix microarray studies that examine gene expression in An. gambiae. The Submit Study link provides a short form for uploading microarray data and specifications.
Utility and Discussion
The angaGEDUCI database identifies genes that meet stage- and tissue-specific expression criteria, and incorporates keyword searching and promoter sequence analysis into one unified data-mining tool. A case study best illustrates the utility of this integration. In this example, we will identify genes linked to the complex regulation of phenoloxidase, an enzyme involved in the melanization of invading parasites and micro-organisms as part of invertebrate innate immunity [7, 8]. Specifically, we will search for pro-phenoloxidase genes that are preferentially found in fat bodies and expressed highly three hours after bloodfeeding. Three filters will be used to complete this inquiry (Figure 1). First, a filter selects genes that contain the keyword "prophenoloxidase" in their functional annotation. Eighty-eight of the 13,639 transcripts in the An. gambiae genome contain this keyword. Second, a stage-specific filter identifies 14 of these 88 transcripts that show 5-fold up-regulated expression three hours after bloodfeeding (BF3h) as compared to sugarfed mosquitoes (NBF). Third, a tissue-specific filter isolates six of these 14 transcripts that are expressed 5-fold higher in fat bodies as compared to their corresponding expression in the midgut and ovaries (Figure 2).
While existing databases may allow individualized searching by expression, keyword, or sequence criteria, it is the unification of these fields that makes angaGEDUCI a unique facilitator of experimental design. The database may be used in many different ways, but perhaps most useful is the ability to use the stage- and tissue-specific expression microarray data to identify genes that are expressed in spatial and temporal patterns of interest and then compare the promoter regions of such genes to investigate putative means of facilitating such expression. The experimentally validated utility of such applications may pave the way for similar investigations into the regulatory role of conserved DNA sequence motifs in other control regions within the genome, such as putative microRNA target sites that may be found in 3' UTRs.
In addition to its current microarray data based on genome-wide tissue- and stage-specific gene expression, angaGEDUCI has been built with the goal of expanding its scope to house, integrate, and display additional microarray studies of An. gambiae. For example, Affymetrix microarray data from a study investigating gene expression in An. gambiae following infection with Plasmodium falciparum can be integrated with the existing data in the database to produce a clearer picture of how the mosquito responds to parasite challenge at the transcriptional level. This flexibility assures that angaGEDUCI is capable of growing alongside the increasing quantity of data being produced from other studies. By working closely with Vectorbase and other laboratories in this way, it is hoped that angaGEDUCI will act as a catalyst in accelerating the study and understanding of gene expression and regulation in this important and devastating vector of disease.
Availability and requirements
The Anopheles gambiae Gene Expression Database at UCI is publicly accessible from the URL: http://www.angaged.bio.uci.edu. Questions and comments are welcomed through the site.
The authors thank Dr. Norman Jacobson for his advice and Lynn Olson for help in preparing the manuscript. This work was supported by a grant from the National Institutes of Health (AI29746 to AAJ).
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