We sequenced seven peptidoglycan identification protein genes (and one line of and used a variety of tests to determine whether the observed mutations were selectively neutral
We sequenced seven peptidoglycan identification protein genes (and one line of and used a variety of tests to determine whether the observed mutations were selectively neutral. 2001). Studies of have similarly Verubulin indicated genetic variation for susceptibility to within mosquito populations (Niare et al. 2002). This led the authors to suggest, by analogy with plants, that this variation may be associated with differences between pattern recognition receptors. Further, comparison between the genome sequence of and that of indicates that while core signaling processes are conserved within immune responses, there is a diversification in receptor and effector molecules between the taxa, with a deficit of orthologues and an excess of gene family expansions (Christophides et al. 2002). This further suggests that coevolutionary interactions may be important in the evolution of the innate immune system. Our aim is to test whether innate immune system parasite recognition molecules coevolve with parasites. The null hypothesis of coevolutionary interaction predicts that, unlike their counterparts in the acquired immune system, these molecules will be subject to neither directional nor balancing selection. To this end, we have investigated the peptidoglycan recognition proteins (PGRPs) of due to its ability to bind bacterial peptidoglycan and activate an immune response (Yoshida et al. 1996). Subsequent analyses found that the genome contains at least 12 genes (Werner et al. 2000), at least 5 of which are up regulated following immune challenge (PGRP-SA, -SB1, -SC2, -SD, and -LB [De Gregorio et al. 2001; Irving et al. 2001; Werner et al. 2000]). Seven of the genes produce short transcripts (is required to activate the immune response to grampositive bacteria, and elicits a response to gram-negative bacteria (Choe et al. 2002; Gottar et al. 2002; Michel et al. 2001; Ramet et al. 2002). Although it is clear that several of the PGRP genes play a role in pattern recognition, some may have other functions. For example, PGRP-SC1B, which is expressed in the gut, has enzymatic activity Verubulin and can degrade peptidoglycan (Mellroth et al. 2003). It is possible that this protein is acting as a scavenger, digesting immunogenic molecules from the gut to prevent them inducing an unnecessary immune response. The five amino acid residues necessary for enzymatic activity are conserved in PGRP-SC1A, -SB1, -SB2, -SC2, and -LB, suggesting that these genes may play a similar role (Mellroth et al. 2003). We have Verubulin sequenced seven genes from 12 lines and a single line. We then inferred the selection pressures acting on the genes from patterns of intraspecific polymorphism and interspecific divergence. We found that there was strong purifying selection acting to conserve the protein sequence, and no evidence of either directional selection or balancing selection. Methods The genes were sequenced from a worldwide Rabbit Polyclonal to AOS1 collection of isofemale lines (Table 1) and line C167.4. In two cases we were unable to amplify a particular gene from certain lines, and another line was substituted (SC1B was sequenced from the Seattle S14 rather than Monty5, and SC2 was sequenced from S14 rather than S30). The X chromosome genes were sequenced from a DNA extracted from a single male individual. The genes on chromosomes 2 and 3 were sequenced from fly lines that were first made isogenic for these chromosomes via balancer chromosome-mediated chromosome substitution. Table 1 lines genes were sequenced, all of which encode extracellular proteins (Table 2). They were chosen as they have the shortest genomic sequences (both the Verubulin fewest introns and the shortest exons), which allowed us to maximize the number of genes sequenced. The PCR primers were designed in the noncoding flanking sequence from the genome sequence (Table 2) (Adams et al. 2000). In two cases where these primers did not amplify the gene in in was amplified using the forward primer in Table 2 and the reverse primer 5-GCCATTACTTGTAGTTGATTTAGGGAT. in was amplified using the forward primer 5-GGTATTTAACGCTTCGTTTCCCGTA and the reverse primer 5-TTGCTTTCTAGTTTTGCCTCCAGT. Both of these reverse primers overlap with the coding sequence, so several codons were missing from the final sequences. All the other sequences included the entire coding sequence and any introns. They have been submitted to the EMBL database under the accession numbers “type”:”entrez-nucleotide-range”,”attrs”:”text”:”AJ556551-AJ556634″,”start_term”:”AJ556551″,”end_term”:”AJ556634″,”start_term_id”:”37665198″,”end_term_id”:”37665364″AJ556551-AJ556634. Table 2 genes, their chromosomal location, function, and PCR primers (Adams et al. 2000; Michel et al. 2001; Werner et al. 2000) DNA polymerase and 35 thermal cycles (95C for 30 s, 56C for 1 min, and 72C for 1 min)..