The long term goal of this research is to understand, in detail, the molecular mechanisms of transcriptional and post-transcriptional (RNA-processing) gene regulation in the human parasitic nematodes, Brugia malayi, a causative agent of lymphatic filariasis, and Ascaris lumbricoides, the most common human intestinal parasite. In preliminary experiments it has been shown that a subset of mRNAs in B. malayi and Ascaris contain a common trans-spliced leader exon. In both organisms, the leader exon is derived from a short non-polyadenylated RNA (SL-RNA) transcribed from within the 5S rRNA gene locus. The SL-RNAs are transcribed by RNA polymerase II and possess the trimethyl-guanosine cap structure characteristic of snRNAs. The SL-RNA genes of both B. malayi and Ascaris are accurately transcribed in a cell-free extract derived from developing Ascaris embryos. In vitro transcripts of SL-RNA genes are post-transcriptionally modified by trimethylation of their cap structures. To define the genes in B. malayi and Ascaris which give rise to transcripts which are trans-spliced, cDNA libraries will be constructed where second strand synthesis is primed by an oligonucleotide corresponding to the spliced leader. Characterization of cDNA clones derived from trans-spliced mRNAs will indicate whether a similar spectrum of specific mRNAs are trans-spliced in B. malayi and Ascaris. Using deletional analysis and site-directed mutagenesis, the sequence elements which direct initiation and termination of SL-RNA synthesis in B. malayi and Ascaris will be defined. Mutational analysis will also be used to determine which features of the SL-RNAs serve to direct trimethylation of SL-RNA caps. Constructs containing the 5' flanking regions of protein coding genes will be used also as substrates for in vitro transcription. If these constructs are transcribed, appropriate mutational techniques will be used to define transcriptional control elements of genes encoding trans-spliced mRNAs and genes encoding mRNAs which are not trans-spliced. Finally, direct RNA sequence analysis will be used to completely characterize the snRNAs of B. malayi and Ascaris. This research program may provide information relevant to the basic mechanisms of gene expression in parasitic nematodes. Such information is a necessary prerequisite to the understanding of complex questions regarding the molecular basis of parasitism and may suggest novel therapeutic strategies for control of parasite nematode infection.
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