Developmental gene regulation in Giardia lamblia: first evidence for an encystation-specific promoter and differential 5' mRNA processing.
ABSTRACT Giardia lamblia must encyst to survive in the environment and subsequently infect new hosts. We investigated the expression of glucosamine-6-phosphate isomerase (Gln6PI), the first enzyme required for biosynthesis of N-acetylgalactosamine, for the major cyst wall polysaccharide. We isolated two Gln6PI genes that encode proteins with large areas of identity, but distinctive central and terminal regions. Both recombinant enzymes have comparable kinetics. Interestingly, these genes have distinct patterns of expression. Gln6PI-A has a conventional, short 5' untranslated region (UTR), and is expressed at a low level during vegetative growth and encystation. The Gln6PI-B gene has two transcripts - one is expressed constitutively and the second species is highly upregulated during encystation. The non-regulated Gln6PI-B transcript has the longest 5'-UTR known for Giardia and is 5' capped or blocked. In contrast, the Gln6PI-B upregulated transcript has a short, non-capped 5'-UTR. A small promoter region (< 56 bp upstream from the start codon) is sufficient for the regulated expression of Gln6PI-B. Gln6PI-B also has an antisense overlapping transcript that is expressed constitutively. A shorter antisense transcript is detected during encystation. This is the first report of a developmentally regulated promoter in Giardia, as well as evidence for a potential role of 5' RNA processing and antisense RNA in differential gene regulation.
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ABSTRACT: Galactosamine, a Giardia filamentous cyst wall specific-sugar, is below the limits of detection in non-encysting trophozoites. Radiolabeling studies suggest that Giardia synthesize galactosamine primarily from endogenous glucose rather than salvage it from the environment. Enzymes responsible for galactosamine synthesis from glucose are induced during encystment and have been characterized in crude homogenates and in supernatant (soluble) fractions. These enzymes (specific activity; time after encystment is induced for maximal activity; x-fold increase) include glucosamine 6-phosphate isomerase (in the deaminating direction, 167 mU mg protein-1; 20 h; x 182-fold; in the aminating direction, 258 mU mg protein-1; 20 h; x 13-fold), glucosamine 6-phosphate N-acetylase (11 mU mg protein-1; 20 h; x 20-fold), phosphoacetylglucosamine mutase (160 mU mg protein-1; 20 h; x 12-fold), UDP-N-acetylglucosamine pyrophosphorylase (22 mU mg protein-1; 48 h; x 8-fold), and UDP-N-acetylglucosamine 4'-epimerase (13 mU mg protein-1; 48 h; x 4000-fold). This represents the first report of these enzymes and of an inducible carbohydrate-synthesizing pathway in any protozoan.Molecular and Biochemical Parasitology 01/1993; 56(2):301-9. · 2.73 Impact Factor
- Transactions of the Royal Society of Tropical Medicine and Hygiene 02/1983; 77(4):487-8. · 1.82 Impact Factor
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ABSTRACT: Giardiavirus (GLV) is a 6,277-bp double-stranded RNA virus of Giardia lamblia, one of the earliest eukaryotic divergents from the prokaryotes. Our previous success in GLV-mediated transfection of G. lamblia has provided an effective way of monitoring the mechanisms underlining GLV gene replication and mRNA translation in this organism. Here we have investigated the cis-acting signals in the GLV genome that regulate replication, transcription, and translation of an inserted firefly luciferase gene in GLV-infected G. lamblia. By modifying the two terminal regions of a full-length GLV cDNA clone used to flank a luciferase gene, various in vitro chimeric transcripts were generated and introduced into GLV-infected G. lamblia via electroporation. Expression of luciferase (+) strand and (-) strand RNAs in the transfected cells was monitored and the luciferase activity assayed. The results indicated that the 5'-untranslated region (UTR) of 366 nt and the 3'-terminal 2,022 nt of the viral transcript are both needed for optimal expression of the two RNA strands. Although the entire 5'-UTR is needed for the chimeric mRNA synthesis, both the primary sequence and the secondary structure at the 3' end of GLV transcript are essential for the synthesis of (-) strand RNA. When the 5' end of GLV transcript was extended 265 nt into the capsid protein open reading frame and fused with that of luciferase, there was no change in the level of luciferase chimeric RNA, but a 5,000-fold increase of luciferase activity was observed that may be attributed to an enhanced translational efficiency of the chimeric mRNA in G. lamblia.RNA 09/1996; 2(8):824-34. · 5.09 Impact Factor