A 22-nucleotide spliced leader sequence in the human parasitic nematode Brugia malayi is identical to the trans-spliced leader exon in Caenorhabditis elegans ( Communicated by Lester 0. Krampitz, July 25, 1988 (received for review June 6, 1988 ABSTRACTThe mRNAs encoding a 63-kDa antigen in the human parasitic nematode Brugia Malayi contain a spliced leader sequence of 22 nucleotides (nt) that is identical to the trans-spliced leader found on certain actin mRNAs in the distantly related nematode Caenorhabditis elegans. The 22-nt sequence does not appear to be encoded near the 63-kDa genes but is present in multiple copies in several locations within the parasite genome, including the 5S rRNA gene repeat. The 5S-linked copies of the 22-nt sequence are transcribed to yield a 109-nt nonpolyadenylylated RNA with the 22-nt leader sequence at its 5' end. We suggest that the 22-nt leader is acquired by 63-kDa antigen mRNAs through trans-splicing. These results indicate that trans-splicing is widespread in nematodes and argue for the functional significance of the 22-nt spliced leader exon in nematode mRNA metabolism.Evidence suggests that intermolecular (trans) splicing is used in a variety of organisms during the maturation of some mRNAs. This is particularly clear for trypanosomatid protozoans, where all mRNAs contain a common leader derived from a small nonpolyadenylylated miniexon transcript (for review, see ref. 1). A trans-splicing mechanism of leader addition is supported by the primary structure of the miniexon transcript and the existence of appropriate branched intermediates (2, 3). Recent observations indicate that transsplicing might also be used in the formation of mRNA for chloroplast ribosomal protein S12 (4) and in the maturation of certain actin mRNAs in Caenorhabditis elegans (5).In C. elegans, mRNAs derived from three of four actin genes contain a 22-nucleotide (nt) leader sequence that is not encoded within 15 kilobases (kb) of the actin genes. This leader sequence is found as the first 22 nt of an abundant 100-base RNA transcribed from within the 5S rRNA gene cluster (5). Several lines of evidence, including the demonstration of branched intermediates containing a portion of the 100-nt RNA, suggest that the 22-nt leader is acquired by trans-splicing (5, 16). In contrast to the situation in trypanosomes, only a subset of C. elegans mRNAs appear to contain the trans-spliced leader. Furthermore, because C. elegans actin genes contain multiple introns, trans-splicing apparently occurs in conjunction with conventional cis-splicing. As discussed by Krause and Hirsh (5) the use of trans-splicing in C. elegans raises the possibility that this mechanism could be widespread in eukaryotes and may be a regulatory mechanism in gene expression.We have recently described the isolation and characterization of cDNA and genomic clones encoding a 63-kDa protective antigen in the human parasitic nematode Brugia malayi, the causative agent of lymphatic filariasis (6, 7).Nuclease protection and primer-extension experime...
To facilitate biochemical studies of protective filarial antigens, a Agtl1 cDNA library was constructed from Brugia malayi adult mRNA and screened with rabbit sera that recognizes a limited set of filarial antigens of approximately 25, 42, 60, and 112 kDa. Antigens of z25 and ==60 kDa have been shown previously to induce enhanced clearance of microrflaremia in mice. A 154-base pair clone detected by immunological reactivity was used to isolate by hybridization a nearly fulllength cDNA clone of 1.8 kilobases. Nucleotide-sequence analysis indicated that this clone was derived from a mRNA encoding a 63-kDa antigen. A fusion polypeptide containing 37 kDa of the Escherichia coli TrpE protein (anthranilate synthase) and 55 kDa of the cloned protein was recognized in immunoblot experiments with antisera raised against a partially purified preparation of the 460-kDa protective filarial antigen. These data relate the cloned antigen to a potentially protective antigen in lymphatic fiariasis.Lymphatic filariasis caused by the nematodes Wuchereria bancrofti, Brugia malayi, and B. timori affects >90 million people worldwide and is a significant cause of morbidity in endemic areas (1). Infection of humans is initiated by deposition of third-stage larvae in the skin during bloodletting by several species of mosquitoes. These larvae develop into lymphatic-dwelling adult worms over several months. Mature female worms subsequently produce microfilariae, which are released into the bloodstream where they may be ingested by the obligate mosquito vector. Reduction or elimination ofthese blood-borne microfilariae could interrupt transmission of infection in endemic areas (2).Experiments using animal models of filarial infections suggest that vaccination is a feasible strategy to reduce microfilaremia. Resistance manifested by 50-80% reduction in adult worm burdens and 90% decrease in the level of microfilaremia has been elicited by active immunization of jirds (Meriones unguiculatus; Mongolian gerbil) (3) and other mammals (4, 5) with irradiated third-stage larvae of Brugia species. In addition, Canlas et al. (6) have shown that a monoclonal antibody to 70-and 75-kDa microfilarial molecules induces a transient depression of microfilaremia in B. malayi-infected jirds.We reported that immunization of jirds and mice with a soluble extract of B. malayi microfilariae induces 50% reduction in adult worm load and 80-100% decrease in parasitemia (7,8
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