Approaches to understanding the regulation of trypsin gene expression in mosquitoes.

摘要

In order to identify potential cis-acting elements responsible for the correct expression of the mosquito trypsin genes we have resorted to an evolutionary approach. This approach is based on the identification of DNA footprints that are conserved in homologous genes isolated from different species. Several trypsin clones have been isolated from Aedes species specific genomic DNA libraries, and by sequencing been shown to contain an ORF coding for the late trypsin gene. Analysis of the 5′ FLR's of the species specific late trypsin genes, reveals the presence of a conserved TATA-box and transcriptional initiator. A phylogenetic footprinting analysis detected some evolutionarily conserved sequence elements in the 5′ regulatory regions of the late trypsin gene. A cis-element that bears sequence similarity with the target of the transcription factor Tinman has been identified.; Analysis of the trypsin coding region shows that the late trypsin from the most distant species retains approximately 83% amino acid identity with late trypsin from Ae aegypti. Furthermore, unique features of the late trypsin specificity pocket from Ae aegypti are retained in all species examined making this a unique evolutionary molecular tag for this serine protease. The dicotomy of a positive/negative charge observed in the specificity pocket of trypsin members of the chymotrypsin family of serine proteases is retained; however, the conserved aspartate is replaced by a glutamate. The position of this glutamate is displaced towards the N-terminus by a serine, which is characteristic of chymotrypsin-like enzymes from the same family. Moreover, there is an insertion of a proline after the serine amino acid at the C-terminal end of the pocket. Alignment of this trypsin to other members of the same protease family strongly suggests that it is related to a unique group of proteases called serine collagenases. The most important enzymatic characteristic of enzymes belonging to this group is there lack of substrate specificity. Outside of the typical metalloproteases, these are the only proteases known to cleave collagen. What these observations mean in terms of the evolution of enzyme specificity aid structural fold remain to be elucidated.