Abundant larval transcript-1 and -2 genes from Brugia malayi : diversity of genomic environments but conservation of 5ı promoter sequences functional in Caenorhabditis elegansı
Gregory, William F
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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 ı /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. T he 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 5ı U T R tracts extending some 800 bp upstream of each B. malayi alt gene show a high degree of similarity (overall 59% identity with tracts of 77ˇ /86% identity). Surmising that this region may contain conserved promoter elements, constructs containing the B. malayi alt 5ı U T R with or without coding sequence were made fused to ı -galactosidase reporter protein. T hese constructs were injected into the syncytical gonad of C. elegans and progeny stained for ı -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 5ı U T R . We conclude that the high level of alt transcription in filarial L 3s 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.