Methods to Prepare RNA and to Isolate Developmentally Regulated Genes fromEimeria

White, Michael; Radke, Jay R.

doi:10.1006/meth.1997.0508

Cited by 10 publications

(4 citation statements)

References 37 publications

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“…The DD RT‐PCR results presented in this paper have shown that the overall profiles were reproducible but of lower complexity (no more than 30 major bands per primer pair) compared to what has previously been published in other eukaryotic systems (between 50–100 bands per primer pair) (Liang et al 1995; White and Radke 1997). The low complexity is an advantage that could minimize the analysis of false positive bands, one of the limitations of the DD RT‐PCR (Liang et al 1995).…”

Section: Discussionsupporting

confidence: 58%

Searching for Excystment‐Regulated Genes in Sterkiella histriomuscorum (Ciliophora, Oxytrichidae): A mRNA Differential Display Analysis of Gene Expression in Excysting Cells

Villalobo

Moch

Perasso

et al. 2001

J Eukaryotic Microbiology

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In the absence of food, the oxytrichid Sterkiella histriomuscorum transforms like many ciliates into resting cysts. When transferred back into feeding medium, the cyst re-transforms into a vegetative cell. The entry into and exit from the dormant cyst stage are complex developmental processes still poorly investigated at the molecular level. Assuming that these changes in state could involve changes in gene expression, we have used the technique of mRNA differential display to detect differentially expressed genes in cysts and two different stages of excysting cell. Variation in the temporal expression pattern of transcripts could be detected and, in using an inverse-PCR strategy on circularized macronuclear DNA, we have sequenced the macronuclear genes of three of the isolated cDNAs. which correspond to 1) a nucleotide-binding domain-encoding gene, 2) a DHHC-domain-carrying gene, and 3) a phosphatase type 2C-encoding gene. For the first two genes, Northern blot analyses supported an excystment-associated regulated gene expression. We discuss their possible role during excystment and we show that the combination of differential display and inverse PCR constitutes a powerful approach to isolate excystment-regulated genes in hypotrichs.

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Section: Discussionsupporting

confidence: 58%

Searching for Excystment‐Regulated Genes in Sterkiella histriomuscorum (Ciliophora, Oxytrichidae): A mRNA Differential Display Analysis of Gene Expression in Excysting Cells

Villalobo

Moch

Perasso

et al. 2001

J Eukaryotic Microbiology

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“…Total RNA from the oocysts was extracted, ethanol precipitated twice, and resuspended in RNasefree water (15). Total RNA from Me49 tachyzoites and bradyzoites were obtained after incubation in TRIzol for 5 min at room temperature, extraction with 20% chloroform, and precipitation with 50% isopropanol.…”

Section: Methodsmentioning

confidence: 99%

A spatially localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma

Brossier

Jewett

Sibley

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

188

249

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Apicomplexan parasites cause serious human and animal diseases, the treatment of which requires identification of new therapeutic targets. Host-cell invasion culminates in the essential cleavage of parasite adhesins, and although the cleavage site for several adhesins maps within their transmembrane domains, the protease responsible for this processing has not been discovered. We have identified, cloned, and characterized the five nonmitochondrial rhomboid intramembrane proteases encoded in the recently completed genome of Toxoplasma gondii. Four T. gondii rhomboids (TgROMs) were active proteases with similar substrate specificity. TgROM1, TgROM4, and TgROM5 were expressed in the tachyzoite stage responsible for the disease, whereas TgROM2 and TgROM3 were expressed in the oocyst stage involved in transmission. Although both TgROM5 and TgROM4 localized to the cell surface in tachyzoites, TgROM5 was primarily at the posterior of the parasite, whereas adhesins were sequestered in internal micronemes. Upon microneme secretion, as occurs during invasion, the MIC2 adhesin was secreted to the apical end and translocated to the posterior, the site of cleavage, where it colocalized only with TgROM5. Moreover, only TgROM5 was able to cleave MIC adhesins in a cell-based assay, indicating that it likely provides the key protease activity necessary for invasion. T. gondii rhomboids have clear homologues in other apicomplexans including malaria; thus, our findings provide a model for studying invasion by this deadly pathogen and offer a target for therapeutic intervention.microneme ͉ microneme protein protease 1 ͉ regulated intramembrane proteolysis ͉ MIC2 ͉ protease

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“…Total RNA from VEG oocysts (partially sporulated for 48 h and representing a mixture of VEG oocysts at different stages of sporulation) was purified according to published protocols (White and Radke, 1997) with the following modifications. Before lysis, oocysts (30–60 million oocysts) were resuspended in 4 mls of guanidine thiocyanate solution(White and Radke, 1997), transferred to a 5 ml French pressure cell (Sim‐Amico, Urbana, IL), and the material frozen in the cell at − 80°C for 30 min. The frozen solution was pressed under 20 000 p.s.i.…”

Section: Methodsmentioning

confidence: 99%

“…which was sufficient to break > 90% of the oocysts in a single pass. The RNA was extracted and ethanol precipitated twice (White and Radke, 1997) and resuspended in RNase‐free water. Total RNA was converted to cDNA fragments by a directional strategy (Zhu et al ., 2001), the fragments were digested with SfiI , sized on Sepharose CL‐2B‐300 columns (Sigma‐Aldrich) to exclude < 500 nt fragments, and cloned into the pDHFR*‐TscABP‐P30 expression vector (Striepen et al ., 2002).…”

Section: Methodsmentioning

confidence: 99%

Identification of a sporozoite‐specific member of the Toxoplasma SAG superfamily via genetic complementation

Radke

Gubbels

Jerome

et al. 2004

Molecular Microbiology

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SummaryToxoplasma gondii sporozoites possess an array of stage-specific antigens that are localized to the membrane and internal cellular space, as well as secreted into the primary parasitophorous vacuole. Specific labelling of viable sporozoites excysted from oocysts reveals a complex admixture of surface proteins partially shared with tachyzoites. SAG1, SRS3 and SAG3 were detected on sporozoites as well as numerous minor antigens. In contrast, tachyzoite SAG2A and B were completely absent whereas a dominant 25 kDa protein was unique to the sporozoite surface. The sporozoite gene encoding this protein was identified in tachyzoites genetically complemented with a sporozoite cDNA library and cloned via site-specific recombination into a bacterial shuttle vector. The sporozoite cDNA identified in these experiments encoded a protein with conserved structural features of the prototypical T. gondii SAG1 (P30) and shared sequence identity with surface proteins from Sarcocystis spp. This new member of the SAG superfamily was designated SporoSAG. Expression of SporoSAG in tachyzoites conferred enhanced invasion on transgenic parasites suggesting a role for this protein in oocyst/sporozoite transmission to susceptible hosts.

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Methods to Prepare RNA and to Isolate Developmentally Regulated Genes fromEimeria

Cited by 10 publications

References 37 publications

Searching for Excystment‐Regulated Genes in Sterkiella histriomuscorum (Ciliophora, Oxytrichidae): A mRNA Differential Display Analysis of Gene Expression in Excysting Cells

Searching for Excystment‐Regulated Genes in Sterkiella histriomuscorum (Ciliophora, Oxytrichidae): A mRNA Differential Display Analysis of Gene Expression in Excysting Cells

A spatially localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma

Identification of a sporozoite‐specific member of the Toxoplasma SAG superfamily via genetic complementation

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