Comparative analysis of antisense RNA,double-stranded RNA,and delta ribozyme-mediated gene regulation in Toxoplasma gondii

RNA tools, namely, antisense RNA, double-stranded RNA (dsRNA), and delta ribozyme, were comparatively analyzed for the development of effective RNA-based gene modulators. The gene encoding uracil phosphoribosyltransferase (UPRT) of Toxoplasma gondii was used as a target and a negative selectable marker. Using plasmid transformation and drug selection assays, we obtained T. gondii transformants resistant to 5-fluoro-2'-deoxyuridine (FDUR), the cytotoxic prodrug and substrate of UPRT, when the plasmids expressing dsRNA and active delta ribozyme were used. No resistant transformants were detected when the plasmids carrying the antisense RNA, the inactive delta ribozyme, or the chloramphenicol acetyltransferase (CAT) genes were used. Parasites generated using the plasmids expressing dsRNA and the delta ribozyme become resistant to FDUR with an LD50 of 50 +/- 5 microM and 25 +/- 8 microM, respectively. These values are approximately 25-fold and 12-fold higher than that of the RH parental parasite strain, indicating that UPRT activity of the transformed parasites was drastically inhibited. Using Northern and Southern blot analysis, we demonstrated that dsRNA and the delta ribozyme interrupt the expression of UPRT. These two RNA tools should, thus, be very useful for the study of gene expression.     

Biosynthetic labeling of RNA with uracil phosphoribosyltransferase allows cell-specific microarray analysis of mRNA synthesis and decay

Standard microarrays measure mRNA abundance, not mRNA synthesis, and therefore cannot identify the mechanisms that regulate gene expression. We have developed a method to overcome this limitation by using the salvage enzyme uracil phosphoribosyltransferase (UPRT) from the protozoan Toxoplasma gondii. T. gondii UPRT has been well characterized because of its application in monitoring parasite growth: mammals lack this enzyme activity and thus only the parasite incorporates (3)H-uracil into its nucleic acids. In this study we used RNA labeling by UPRT to determine the roles of mRNA synthesis and decay in the control of gene expression during T. gondii asexual development. We also used this approach to specifically label parasite RNA during a mouse infection and to incorporate thio-substituted uridines into the RNA of human cells engineered to express T. gondii UPRT, indicating that engineered UPRT expression will allow cell-specific analysis of gene expression in organisms other than T. gondii.     

美洲棉铃虫细胞中dsRNA介导的egfp基因沉默分析

为了分析在美洲棉铃虫细胞( HzAM1)内RNAi的效果,将egfp基因克隆到含有双向T7启动子/终止子的质粒载体中,在体外合成全长的增强型绿色荧光蛋白(egfp)基因dsRNA,将dsRNA和含有能在昆虫细胞内表达eGFP的质粒一起转染HzAM1细胞,分析dsRNA对eGFP表达的抑制作用.结果显示,由egfp基因转录的长dsRNA能有效抑制HzAM1细胞内eGFP的表达,而且该抑制作用表现为剂量依赖效应.但是抑制作用并不彻底,在高剂量的dsRNA处理下,仍有部分细胞内能观察到eGFP的表达.     

Inhibition of gene expression by administration of homologous double-stranded RNA in Drosophila melanogaster cell culture

Specific inhibition of gene expression by exogenous homologous double-stranded RNA (dsRNA) in invertebrates and in the early development of vertebrates is termed RNA interference. Cultured cells were cotransfected with reporter plasmids and dsRNA. The inhibitor effect on reporter gene expression depended on the extent of homology between dsRNA and the target gene. RNA interference was also studied in cells cotransfected with plasmids directing synthesis of sense and antisense RNAs. Production of antisense RNA only slightly inhibited expression of the reporter gene. Simultaneous expression of both sense and antisense RNAs from a special plasmid did not inhibit expression of the reporter construct.     

细菌表达dsRNA介导的家蚕FTZ-F1基因的RNA干扰

为探索细菌表达目标基因dsRNA介导的RNAi技术是否在家蚕Bombyx mori可行, 本研究引入了在其他物种中广泛应用的细菌表达dsRNA的RNAi系统: HT115细菌株和L4440质粒。利用L4440载体两端含有T7启动子的特点, 设计并构建了针对家蚕核受体FTZ-F1基因的RNA干扰（RNA interference）载体, 将构建好的质粒转入大肠杆菌Escherichia coli HT115, 在IPTG诱导下成功获得目标基因对应双链RNA（dsRNA）。 结果显示: 通过对5龄第7天家蚕幼虫注射IPTG诱导后提取的FTZ F1基因对应的dsRNA 25 μg, 85%的蛹变态发育过程明显延迟, 不能实现幼虫到蛹的形态完全转变。荧光定量PCR分析显示目标基因的表达得到了特异的抑制。实验结果初步表明, 通过细菌表达目标基因dsRNA介导的RNAi策略, 以其经济、高效的特点, 具有广泛应用于家蚕基因功能研究中的潜力。     

Identification and characterisation of a regulatory region in the Toxoplasma gondii hsp70 genomic locus

Toxoplasma gondii is an important human and veterinary pathogen. The induction of bradyzoite development in vitro has been linked to temperature, pH, mitochondrial inhibitors, sodium arsenite and many of the other stressors associated with heat shock protein induction. Heat shock or stress induced activation of a set of heat shock protein genes, is characteristic of almost all eukaryotic and prokaryotic cells. Studies in other organisms indicate that heat shock proteins are developmentally regulated. We have established that increases in the expression of bag1/hsp30 and hsp70 are associated with bradyzoite development. The T. gondii hsp70 gene locus was cloned and sequenced. The regulatory regions of this gene were analysed by deletion analysis using beta-galactosidase expression vectors transiently transfected into RH strain T. gondii. Expression was measured at pH 7.1 and 8.1 (i.e. pH shock) and compared to the expression obtained with similar constructs using BAG1 and SAG1 promoters. A pH-regulated region of the Tg-hsp70 gene locus was identified which has some similarities to heat shock elements described in other eukaryotic systems. Green fluorescent protein expression vectors driven by the Tg-hsp70 regulatory region were constructed and stably transfected into T. gondii. Expression of green fluorescent protein in these parasites was induced by pH shock in those lines carrying the Tg-hsp70 regulatory constructs. Gel shift analysis was carried out using oligomers corresponding to the pH-regulated region and a putative DNA binding protein was identified. These data support the identification of a pH responsive cis-regulatory element in the T. gondii hsp70 gene locus. A model of the interaction of hsp70 and small heat shock proteins (e.g. BAG1) in development is presented.     

Improved techniques for endogenous epitope tagging and gene deletion in Toxoplasma gondii

Toxoplasma gondii is an excellent model organism for studies on the biology of the Apicomplexa due to its ease of in vitro cultivation and genetic manipulation. Large-scale reverse genetic studies in T. gondii have, however, been difficult due to the low frequency of homologous recombination. Efforts to ensure homologous recombination have necessitated engineering long flanking regions in the targeting construct. This requirement makes it difficult to engineer chromosomally targeted epitope tags or gene knock out constructs only by restriction enzyme mediated cloning steps. To address this issue we employed multisite Gateway® recombination techniques to generate chromosomal gene manipulation targeting constructs. Incorporation of 1.5 to 2.0 kb flanking homologous sequences in PCR generated targeting constructs resulted in 90% homologous recombination events in wild type T. gondii (RH strain) as determined by epitope tagging and target gene deletion experiments. Furthermore, we report that split marker constructs were equally efficient for targeted gene disruptions using the T. gondii UPRT gene locus as a test case. The methods described in this paper represent an improved strategy for efficient epitope tagging and gene disruptions in T. gondii.     

Evidence that processed small dsRNAs may mediate sequence-specific mRNA degradation during RNAi in Drosophila embryos

BACKGROUND: RNA interference (RNAi) is a phenomenon in which introduced double-stranded RNAs (dsRNAs) silence gene expression through specific degradation of their cognate mRNAs. Recent analyses in vitro suggest that dsRNAs may be copied, or converted, into 21-23 nucleotide (nt) guide RNAs that direct the nucleases responsible for RNAi to their homologous mRNA targets. Such small RNAs are also associated with gene silencing in plants. RESULTS: We developed a quantitative single-embryo assay to examine the mechanism of RNAi in vivo. We found that dsRNA rapidly induced mRNA degradation. A fraction of dsRNAs were converted into 21-23 nt RNAs, and their time of appearance and persistence correlated precisely with inhibition of expression. The strength of RNAi increased disproportionately with increasing dsRNA length, but an 80bp dsRNA was capable of effective gene silencing. RNAi was saturated at low dsRNA concentration and inhibited by excess unrelated dsRNA. The antisense strand of the dsRNA determined target specificity, and excess complementary sense or antisense single-stranded RNAs (ssRNAs) competed with the RNAi reaction. CONCLUSIONS: Processed dsRNAs can act directly to mediate RNAi, with the antisense strand determining mRNA target specificity. The involvement of 21-23 nt RNAs is supported by the kinetics of the processing reaction and the observed size dependence. RNAi depends on a limiting factor, possibly the nuclease that generates the 21-23 mer species. The active moiety appears to contain both sense and antisense RNA strands.     

Development of methods for RNA interference in the sheep gastrointestinal parasite, Trichostrongylus colubriformis

The efficiency of RNA interference (RNAi) delivery to L1 through L3 stage worms of the sheep parasitic nematode Trichostrongylus colubriformis was investigated using several techniques. These were: (i) feeding of Escherichia coli expressing double stranded RNA (dsRNA); (ii) soaking of short interfering (synthetic) RNA oligonucleotides (siRNA) or in vitro transcribed dsRNA molecules; and (iii) electroporation of siRNA or in vitro transcribed dsRNA molecules. Ubiquitin and tropomyosin were used as a target gene because they are well conserved genes whose DNA sequences are available for several nematode parasite species. Ubiquitin siRNA or dsRNA delivered by soaking or electroporation inhibited development in T. colubriformis but with feeding as a delivery method, RNAi of ubiquitin was not successful. Feeding was, however, successful with tropomyosin as a target, suggesting that mode of delivery is an important parameter of RNAi. Electroporation is a particularly efficient means of inducing RNA in nematodes with either short dsRNA oligonucleotides or with long in vitro transcribed dsRNA molecules. These methods permit routine delivery of dsRNA for RNAi in T. colubriformis larval stage parasites and should be applicable to moderate to high-throughput screening.     

High prevalence and genotypes of Toxoplasma gondii isolated from goats, from a retail meat store, destined for human consumption in the USA

Little information is available concerning the presence of viable Toxoplasma gondii in tissues of goats worldwide. In the present study, hearts of 234 goats obtained from a local USA grocery store were examined for T. gondii infection. Blood clot or fluid removed from each heart was tested for antibodies to T. gondii by using the modified agglutination test (MAT). Antibodies to T. gondii were found in 125 (53.4%) of 234 goats, with titers of 1:5 in 20, 1:10 in 44, 1:20 in 16, 1:40 in five, 1:160 in five, 1:320 in five, and 1:640 or higher in 30 goats. Hearts of 112 goats (46 goats <1:5, and 66 goats 1:10 or higher) were used for isolation of viable T. gondii by bioassays in mice. For bioassays, 50 g of the myocardium were digested in an acid pepsin solution and the digest inoculated into mice; the recipient mice were examined for T. gondii infection. Toxoplasma gondii was isolated from 29 goats; from hearts of one of 46 with titers of <1:5, one of nine with titers of 1:10, one of three with titers of 1:40, and 26 of 40 with titers of 1:160 or higher. Two isolates were highly virulent to outbred Swiss Webster mice; all infected mice died of toxoplasmosis, irrespective of the dose. All T. gondii isolates were subsequently grown in cell cultures. Genotyping of the 29 T. gondii isolates using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) from DNA obtained from cell culture grown tachyzoites revealed 12 genotypes. Nine isolates were clonal Type II lineage, four isolates had type II alleles at all loci except a type I allele at the Apico locus, and four isolates were clonal Type III. The remaining 12 strains were divided into nine atypical genotypes, including five new and four previously identified genotypes. DNA sequences of four introns (EF1, HP2, UPRT1 and UPRT7) and two genes (GRA6 and GRA7) were generated for the five new genotypes. Comparing these sequences with previously published data revealed no unique sequences in these goat strains. Taken together, these results indicate high parasite prevalence and moderate genetic diversity of T. gondii in goats, which have important implications in public health. We believe this is the first genetic analysis of T. gondii isolates from goats in the USA.     

The methylerythritol phosphate pathway for isoprenoid biosynthesis in coccidia: presence and sensitivity to fosmidomycin

The apicoplast is a recently discovered, plastid-like organelle present in most apicomplexa. The methylerythritol phosphate (MEP) pathway involved in isoprenoid biosynthesis is one of the metabolic pathways associated with the apicoplast, and is a new promising therapeutic target in Plasmodium falciparum. Here, we check the presence of isoprenoid genes in four coccidian parasites according to genome database searches. Cryptosporidium parvum and C. hominis, which have no plastid genome, lack the MEP pathway. In contrast, gene expression studies suggest that this metabolic pathway is present in several development stages of Eimeria tenella and in tachyzoites of Toxoplasma gondii. We studied the potential of fosmidomycin, an antimalarial drug blocking the MEP pathway, to inhibit E. tenella and T. gondii growth in vitro. The drug was poorly effective even at high concentrations. Thus, both fosmidomycin sensitivity and isoprenoid metabolism differs substantially between apicomplexan species.     

Triplex PCR using new primers for the detection of Toxoplasma gondii

Molecular methods are used increasingly for the detection of Toxoplasma gondii infection. This study developed a rapid, sensitive, and specific conventional triplex PCR for the detection of the B1 gene and ITS1 region of T. gondii using newly designed primers and an internal control based on the Vibrio cholerae HemM gene. The annealing temperature and concentrations of the primers, MgCl(2), and dNTPs were optimized. Two sets of primers (set 1 and 2) were tested, which contained different segments of the T. gondii B1 gene, 529 repeat region and ITS1 region. A series of sensitivity tests were performed using parasite DNA, whole parasites, and spiked human body fluids. Specificity tests were performed using DNA from common protozoa and bacteria. The newly developed assay based on set 2 primers was found to be specific and sensitive. The test was capable of detecting as little as 10 pg T. gondii DNA, 10(4) tachyzoites in spiked body fluids, and T. gondii DNA in the organ tissues of experimentally infected mice. The assay developed in this study will be useful for the laboratory detection of T. gondii infection.