Developmental gene regulation in Giardia lamblia: First evidence for an encystation-specific promoter and differential 5' mRNA processing

Division of Infectious Diseases, Department of Pathology, University of California at San Diego, 214 Dickinson St., San Diego, CA 92103-8416, USA.
Molecular Microbiology (Impact Factor: 4.42). 10/1999; 34(2):327-40. DOI: 10.1046/j.1365-2958.1999.01602.x
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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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Available from: Jeffrey D Silberman, Oct 13, 2014
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    • "In Giardia lamblia, also called G. intestinalis or G. duodenalis, the protein and/or gene expression level of cyst wall protein (CWP) 1, CWP2 (Lujan et al. 1995; Mowatt et al. 1995), CWP3 (Sun et al. 2003), and many enzymes (Knodler et al. 1999; Lopez et al. 2003) were reported to be upregulated (reviewed in Carranza and Lujan 2010; Lauwaet et al. 2007; Lujan 2006, 2011). The functions of the enzymes whose expression level is enhanced in the G. lamblia encystment process and the mechanisms of encystment-specific gene regulation (Knodler et al. 1999; Pan et al. 2009; Sun et al. 2002, 2006; Wang et al. 2007; Yang et al. 2003) have been revealed (reviewed in Carranza and Lujan 2010; Lauwaet et al. 2007; Lujan 2006, 2011). For example, gMyb2 was identified as a transcription factor (Sun et al. 2002; Yang et al. 2003) and reported to play an important role in the transcriptional regulation of encystation genes, and may help coordinate the synthesis of CWPs polysaccharide (Sun et al. 2002). "
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    ABSTRACT: In the encystment process of the ciliate protist Colpoda cucullus, we observed that the cell total protein abundance was reduced at 12 h - 1 d after the onset of encystment induction subsequent to the reduction of mRNA abundance. We analyzed the alteration of the expression levels of water-insoluble proteins by two-dimensional polyacrylamide gel electrophoresis using polyoxyethylene (20) sorbitan monooleate (Tween-80), and we identified proteins whose expression levels were altered in the encystment process by a liquid chromatography tandem mass spectrometry analysis. The expression level of a 60-kDa protein (p60; heat shock protein 60) was temporarily enhanced and that of a 55-kDa protein (p55; actin) and a 49-kDa protein (p49; actin) was enhanced in the Colpoda encystment process. In mature cysts, the expression level of p55 and p49 tended to be reduced, whereas the expression level of a 50-kDa protein (p50d; α-tubulin), a 25-kDa protein (p25; α-tubulin) and a 52-kDa protein (p52c; β-tubulin) were enhanced. This article is protected by copyright. All rights reserved.
    Journal of Eukaryotic Microbiology 10/2013; 61(1). DOI:10.1111/jeu.12086 · 3.22 Impact Factor
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    • "Most of the 5′-UTRs are less than 20 nt [14]. However, Knodler et al. [15] reported two long 5′-UTRs of 146 nt and 280 nt in an investigation of glucosamine-6-phosphate isomerase expression. The promoter system is thought to be a simple TATA box-like system. "
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    ABSTRACT: Giardia lamblia is a protozoan parasite that is found worldwide and has both medical and veterinary importance. We applied the transcription start sequence (TSS-seq) and RNA sequence (RNA-seq) techniques to study the transcriptome of the assemblage A WB strain trophozoite. We identified 8000 transcription regions (TR) with significant transcription. Of these regions, 1881 TRs were more than 500 nucleotides upstream of an annotated ORF. Combining both techniques helped us to identify 24 ORFs that should be re-annotated and 60 new ORFs. From the 8000 TRs, we were able to identify an AT-rich consensus that includes the transcription initiation site. It is possible that transcription that was previously thought to be bidirectional is actually unidirectional.
    PLoS ONE 10/2013; 8(10):e76184. DOI:10.1371/journal.pone.0076184 · 3.23 Impact Factor
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    • "Giardial RNA polymerase II has no regular heptad repeats in the carboxyl-terminal domain and transcription by RNA polymerase II is highly resistant to α-amanitin [22], [24]. The giardial promoter regulatory mechanism may be unusual because unusually short 5′-flanking regions (<65 bp) are sufficient for the expression of many giardial protein-coding genes [12], [13], [15], [25], [26], [27]. Within the short promoter regions, no consensus TATA boxes or other cis-acting elements characteristic of higher eukaryotic promoters have been observed [12], [13], [15], [25], [26], [27], [28]. "
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    ABSTRACT: The protozoan Giardia lamblia differentiates from a pathogenic trophozoite into an infectious cyst to survive outside of the host. During encystation, genes encoding cyst wall proteins (CWPs) are coordinately induced. Pax family transcription factors are involved in a variety of developmental processes in animals. Nine Pax proteins have been found to play an important role in tissue and organ development in humans. To understand the progression from primitive to more complex eukaryotic cells, we tried to identify putative pax genes in the G. lamblia genome and found two genes, pax1 and pax2, with limited similarity. We found that Pax1 may transactivate the encystation-induced cwp genes and interact with AT-rich initiatior elements that are essential for promoter activity and transcription start site selection. In this study, we further characterized Pax2 and found that, like Pax1, Pax2 was present in Giardia nuclei and it may specifically bind to the AT-rich initiator elements of the encystation-induced cwp1-3 and myb2 genes. Interestingly, overexpression of Pax2 increased the cwp1-3 and myb2 gene expression and cyst formation. Deletion of the C-terminal paired domain or mutation of the basic amino acids of the paired domain resulted in a decrease of nuclear localization, DNA-binding activity, and transactivation activity of Pax2. These results are similar to those found in the previous Pax1 study. In addition, the profiles of gene expression in the Pax2 and Pax1 overexpressing cells significantly overlap in the same direction and ERK1 associated complexes may phosphorylate Pax2 and Pax1, suggesting that Pax2 and Pax1 may be downstream components of a MAPK/ERK1 signaling pathway. Our results reveal functional redundancy between Pax2 and Pax1 in up-regulation of the key encystation-induced genes. These results illustrate functional redundancy of a gene family can occur in order to increase maintenance of important gene function in the protozoan organism G. lamblia.
    PLoS ONE 02/2012; 7(2):e30614. DOI:10.1371/journal.pone.0030614 · 3.23 Impact Factor
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