RESUMO
The genomic organisation of two abundant larval transcript (alt) genes from the filarial nematode Brugia malayi has been defined. The products of these genes are 78% identical in amino acid sequence, and are highly expressed in a stage-specific manner by mosquito-borne infective larvae. alt-1 is present as two near-identical copies organised in an inverted repeat of approximately 7.6 kb, occupying a total of 16 kb of the genome. alt-2 is a single-copy gene at a different locus to alt-1. The two alt-1 genes (alt-1.1 and -1.2) are 99.7% identical in coding sequence and 99.5% in intronic sequences. Both alt-1 and -2 contain 3 introns, and the third intron of alt-2 exhibits a size polymorphism evident in different individual parasites from the laboratory-maintained strain. Genomic sequence up- and down-stream from alt-1.1/1.2 (26 and 6 kb, respectively) and alt-2 (6 and 4 kb, respectively) show that neither gene is in a multiple array or an operon. Most notably, the neighbouring genes of alt-1 and -2 show no similarity to each other, or to the genes flanking the distant alt homologue in Caenorhabditis elegans. Despite this diversity in flanking genes, the 5' UTR tracts extending some 800 bp upstream of each B. malayi alt gene show a high degree of similarity (overall 59% identity with tracts of 77-86% identity). Surmising that this region may contain conserved promoter elements, constructs containing the B. malayi alt 5' UTR with or without coding sequence were made fused to beta-galactosidase reporter protein. These constructs were injected into the syncytical gonad of C. elegans and progeny stained for beta-gal expression. Our results show relatively strong expression in the gut cells of C. elegans for both alt-1 and -2 constructs, commencing in larval worms and continuing into adulthood. Moreover, expression was enhanced when constructs contained segments of alt-1 coding and intronic sequence in addition to the 5' UTR. We conclude that the high level of alt transcription in filarial L3s is not due to expression from a multi-copy gene family but to a set of strong promoter elements shared between the two alt genes.
