Transcriptional regulation of two stage-specifically expressed genes in the protozoan parasite Toxoplasma gondii

The protozoan parasite Toxoplasma gondii differentially expresses two distinct enolase isoenzymes known as ENO1 and ENO2, respectively. To understand differential gene expression during tachyzoite to bradyzoite conversion, we have characterized the two T.gondii enolase promoters. No homology could be found between these sequences and no TATA or CCAAT boxes were evident. The differential activation of the ENO1 and ENO2 promoters during tachyzoite to bradyzoite differentiation was investigated by deletion analysis of 5′-flanking regions fused to the chloramphenicol acetyltransferase reporter followed by transient transfection. Our data indicate that in proliferating tachyzoites, the repression of ENO1 involves a negative distal regulatory region (nucleotides −1245 to −625) in the promoter whereas a proximal regulatory region in the ENO2 promoter directs expression at a low level. In contrast, the promoter activity of ENO1 is highly induced following the conversion of tachyzoites into resting bradyzoites. The ENO2 promoter analysis in bradyzoites showed that there are two upstream repression sites (nucleotides −1929 to −1067 and −456 to −222). Furthermore, electrophoresis mobility shift assays demonstrated the presence of DNA-binding proteins in tachyzoite and bradyzoite nuclear lysates that bound to stress response elements (STRE), heat shock-like elements (HSE) and other cis-regulatory elements in the upstream regulatory regions of ENO1 and ENO2. Mutation of the consensus AGGGG sequence, completely abolished protein binding to an oligonucleotide containing this element. This study defines the first characterization of cis-regulatory elements and putative transcription factors involved in gene regulation of the important pathogen T.gondii

Identification et caractérisation d'une enzyme de réparation de l'ADN chez Toxoplasma Gondii

LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Disruption of Murine Mus81 Increases Genomic Instability and DNA Damage Sensitivity but Does Not Promote Tumorigenesis

The Mus81-Eme1 endonuclease is implicated in the efficient rescue of broken replication forks in Saccharomyces cerevisiae and Schizosaccharomyces pombe. We have used gene targeting to study the function of the Mus81-Eme1 endonuclease in mammalian cells. Mus81-deficient mice develop normally and are fertile. Surprisingly, embryonic fibroblasts from Mus81(−/−) animals fail to proliferate in vitro. This proliferation defect can be rescued by expression of the papillomavirus E6 protein that promotes degradation of p53. When grown in culture, Mus81(−/−) cells have elevated levels of DNA damage, acquire chromosomal aberrations, and are hypersensitive to agents that generate DNA cross-links. In contrast to the situation in yeast, murine Mus81 is not required for replication restart following camptothecin treatment. Mus81(−/−) mice and cells are hypersensitive to DNA cross-linking agents. Cross-link-induced double-strand break formation is normal in Mus81(−/−) cells, but the resolution of repair intermediates is not. The persistence of Rad51 foci in Mus81(−/−) cells suggests that Mus81 acts at a late step in the repair of cross-link-induced lesions. Despite these defects, Mus81(−/−) mice do not show increased predisposition to lymphoma or any other malignancy in the first year of life

Structural and functional characterization of the TgDRE multidomain protein, a DNA repair enzyme from Toxoplasma gondii.

The parasite Toxoplasma gondii expresses a 55 kDa protein or TgDRE that belongs to a novel family of proteins characterized by the presence of three domains, a human splicing factor 45-like motif (SF), a glycine-rich motif (G-patch), and a RNA recognition motif (RRM). The two latter domains are mainly known as RNA-binding domains, and their presence in TgDRE, whose partial DNA repair function was demonstrated, suggests that the protein could also be involved in the RNA metabolism. In this work, we characterized the structure and function of the different domains by using single or multidomain proteins to define their putative role. The SF45-like domain has a helical conformation and is involved in the oligomerization of the protein. The G-patch domain, mainly unstructured on its own as well as in the presence of the SF upstream and RRM downstream domains, is able to bind small RNA oligonucleotides. We also report the structure determination of the RRM domain from the NMR data. It adopts a classical betaalphabetabetaalphabeta topology consisting of a four-stranded beta sheet packed against two alpha helices but does not present the key residues for the RNA interaction. In contrast, our analysis shows that the RRM of TgDRE is not only unable to bind small RNA oligonucleotides but it also shares the protein-protein interaction characteristics with two unusual RRMs of the U2AF heterodimeric splicing factor. The presence of both RNA- and protein-binding domains seems to indicate that TgDRE could also be involved in RNA metabolism

Identification of transcriptional regulatory elements in the and genes by transient CAT assays

<p><b>Copyright information:</b></p><p>Taken from "Transcriptional regulation of two stage-specifically expressed genes in the protozoan parasite "</p><p>Nucleic Acids Research 2005;33(5):1722-1736.</p><p>Published online 22 Mar 2005</p><p>PMCID:PMC1903550.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Transfections of tachyzoites were performed with () and () promoter–reporter constructs that are schematically depicted on the left of the panels. The transfected tachyzoites were also subjected to stress thereby converting to bradyzoites (see Materials and Methods). () Activities of the and promoters during tachyzoite to bradyzoite conversion. The plasmids E2F or E1F contain the 3′-UTR of gene while E2F10 and E1F9 contain the 3′-UTR of and , respectively. The CAT-constructs depicted on the left-hand side (E2F versus E2F10 and E1F versus E1F9) were transfected into tachyzoites and these organisms were divided equally into two flasks of human foreskin fibroblasts. After 6 h of invasion, one flask was subjected to experimental stress conditions to induce bradyzoite conversion and the second flask was kept in tachyzoite growth conditions (see Materials and Methods). Levels of CAT signal in tachyzoites and bradyzoites were determined after removing the promoterless CAT vector activities and adjustments for β-galactosidase activity used as internal control. The data are averages of four independent experiments. Error bars represent the mean and SD values of four independent experiments

( and ) Overview of the results obtained by promoter analysis and by EMSAs

<p><b>Copyright information:</b></p><p>Taken from "Transcriptional regulation of two stage-specifically expressed genes in the protozoan parasite "</p><p>Nucleic Acids Research 2005;33(5):1722-1736.</p><p>Published online 22 Mar 2005</p><p>PMCID:PMC1903550.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The promoter regions of and are shown. The forward primers used for CAT expressing constructs are indicated in red while the common reverse primers are shown in blue. The name of these constructs are displayed in red on top of the forward primers. The DNA fragments used in EMSAs are indicated by bold bars and underlined. The transcription start sites are shown by black arrows. The -regulatory elements identified are displayed as boldface, black, underlined sequences on top and at the beginning of the EMSA fragments. () The nature of -regulatory elements and the corresponding putative -acting or transcription factors are indicated in the table. The oligonucleotide sequences were checked for homology to sequences in the TRANSFAC and TFSEARCH databases

Localization of protein binding within the 35 and 42 bp DNA of oligonucleotides 3b and 2p2

<p><b>Copyright information:</b></p><p>Taken from "Transcriptional regulation of two stage-specifically expressed genes in the protozoan parasite "</p><p>Nucleic Acids Research 2005;33(5):1722-1736.</p><p>Published online 22 Mar 2005</p><p>PMCID:PMC1903550.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () The sequence of oligonucleotide 3b and the extent of the oligonucleotides 3b1 and 3b2 used in the competition experiments using the tachyzoite nuclear extracts are shown. () Right panel: lane 1, free probe; lane 2, no competitor; lane 3, specific competitor (100-fold excess); and lanes 5 and 6 show competition with oligonucleotides 3b1 and 3b2, respectively (100-fold excess each). Arrowheads show the oligonucleotide–protein complexes. Band shift assay with oligonucleotides 2p1 and 2p2. The sequence of oligonucleotide 2p2 and the extent of the oligonucleotides 2p2a, 2p2b and 2p2c used in the competition experiments are shown in (A). Lane 1: free probe; lane 2, no competitor; and lane 3, specific competitor (100-fold excess). Lanes 4–6 show competition of labelled oligonucleotide 2p2 with the short overlapping self oligonucleotides shown in (A). Note the disappearance of the oligonucleotide protein complexes in lanes 5 and 6 (arrowed)

Increased DNA–protein interactions with nuclear extract from stress-induced bradyzoites

<p><b>Copyright information:</b></p><p>Taken from "Transcriptional regulation of two stage-specifically expressed genes in the protozoan parasite "</p><p>Nucleic Acids Research 2005;33(5):1722-1736.</p><p>Published online 22 Mar 2005</p><p>PMCID:PMC1903550.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Electrophoretic band shift assays showing DNA–protein complexes with promoter DNA oligonucleotides containing STRE motifs (5b1 and 5b2) or HSE-like motifs (7b+7c) and nuclear extract from non-stressed tachyzoites (lanes 1–3), nuclear extract from stress-induced bradyzoites (lanes 4–6) or nuclear extract from heat shock treated tachyzoites (oligo 7b+7c, lanes 7–9). Equal amount of nuclear extracts have been used. Lane 1, free probe; lane 2, probe with tachyzoite nuclear extract and no competitor; lane 3, probe with tachyzoite nuclear extract and specific competitor (200-fold excess of the homologous cold fragment); lane 4, free probe; lane 5, probe with stress-induced bradyzoite nuclear extract and specific competitor (200-fold excess); lane 6, probe with stress-induced bradyzoite nuclear extract and specific competitor (200-fold excess); lane 7, free probe; lane 8, probe with nuclear extract from tachyzoite heat shock treated and no competitor; and lane 9, probe with tachyzoite nuclear extract and specific competitor (200-fold excess). Arrowheads show the DNA–protein complexes