BMC Genomics 2006, 7:116 http://www.biomedcentral.com/1471-2164/7/116 FCiognusreerv6ed DNA sequence motifs in putative promoter regions Conserved DNA sequence motifs in putative promoter regions. Analysis output from comparing putative promoter regions of the six prophenoloxidase transcripts identified in the case study, searching for conserved DNA sequence motifs that are 12 nucleotides in length with no mismatches allowed. Each conserved DNA sequence (motif) is followed by the number of genes (#genes) within the gene set where this motif was found, the total occurrences of the motif (count), taking into account that some genes may contain multiple instances of a motif, the corresponding frequency (%set) of occurrence of this motif within the current gene set, the frequency of occurrence of the motif within: all cDNAs (%cdna), all genes [including introns] (%gene), and all promoters (%prom), in the An. gambiae genome, and the fold difference between the frequency of occurrence of the motif in this gene set as compared to its frequency in all cDNAs (cdna-fold), all genes (gene-fold), and all promoter regions (prom-fold), in the An. gambiae genome. Each transcription factor binding site that matches or occurs within a conserved motif is indicated (factors), along with the class of organism in which the binding site was described origi- nally. Motifs that do not match or contain a known transcription factor binding site are highlighted in orange. The gene identifi- ers containing each sequence motif are shown in the last column (genes). 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 mosqui- toes (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). The analysis menu can be used with this gene set of inter- est to search for common DNA sequence motifs that occur within the promoter regions of the genes corresponding to these transcripts. Analysis of the promoter regions of the six prophenoloxidase-related genes shows the occur- rence of 14 conserved 12-basepair DNA sequence motifs (Figure 6). Of these 14 motifs, 10 match known transcrip- tion factor binding sites while the other four do not. Addi- tional motifs of interest can be found by executing the promoter analysis as a search for a conserved motif length less than 12 nucleotides in length or by specifying a number of mismatches that may be allowed within a nearly-conserved but imperfectly-matching motif. Depending on how these parameters are adjusted, the output from the promoter analysis of a gene set may gen- erate more or less conserved motifs, as well as a different number of motifs that are or are not matched to known transcription factor binding sites. A survey of the data pro- duced with different specifications of these parameters in the analysis of the prophenoloxidase gene set is included Page 6 of 11 (page number not for citation purposes)