The Biology of the Glycosylphosphatidylinositol-Specific Phospholipase C of Trypanosoma Brucei

Carrington, Mark; Walters, Dawn; Webb, Helena

doi:10.1016/b978-0-12-159390-2.50021-0

Cited by 10 publications

(18 citation statements)

References 45 publications

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“…First, the EP procyclin 5′UTR and luciferase gene were removed using a complete BamHI and partial Xma1 digest and replaced with a polylinker containing BstBI and HindIII sites to produce p1865. Second, a ClaI HindIII fragment containing all of the GPI-PLC gene (37) plus processing signals for the flanking genes (Figure 4a) was cloned into the BstBI and HindIII sites of p1865 to produce p1885. The plasmid p1885 was linearized with NotI and electroporated into Lister 427 29-13 procyclic forms and integrants selected using zeocin.…”

Section: Methodsmentioning

confidence: 99%

“…The mRNA is moderately abundant in bloodstream forms, but almost undetectable in procyclic forms (37,39), and there is no difference in the transcription rate in the two life cycle stages (39). In this paper, the differential expression of GPI-PLC mRNA in the two life cycle stages is investigated and it is shown that the developmental regulation of steady-state mRNA levels is underpinned by a 10-fold reduced half-life in procyclic forms.…”

Section: Introductionmentioning

confidence: 99%

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Developmentally regulated instability of the GPI-PLC mRNA is dependent on a short-lived protein factor

Webb

Burns

Ellis

et al. 2005

Nucleic Acids Research

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The expression of the vast majority of protein coding genes in trypanosomes is regulated exclusively at the post-transcriptional level. Developmentally regulated mRNAs that vary in levels of expression have provided an insight into one mechanism of regulation; a decrease in abundance is due to a shortened mRNA half-life. The decrease in half-life involves cis-acting elements in the 3′ untranslated region of the mRNA. The trans-acting factors necessary for the increased rate of degradation remain uncharacterized. The GPI-PLC gene in Trypanosoma brucei encodes a phospholipase C expressed in mammalian bloodstream form, but not in the insect procyclic form. Here, it is reported that the differential expression of the GPI-PLC mRNA also results from a 10-fold difference in half-life. Second, the instability of the GPI-PLC mRNA in procyclic forms can be reversed by the inhibition of protein synthesis. Third, specifically blocking the translation of the GPI-PLC mRNA in procyclic forms by the inclusion of a hairpin in the 5′ untranslated region does not result in stabilization of the mRNA. Thus, the effect of protein synthesis inhibitors in stabilizing the GPI-PLC mRNA operates in trans through a short-lived factor dependent on protein synthesis.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Developmentally regulated instability of the GPI-PLC mRNA is dependent on a short-lived protein factor

Webb

Burns

Ellis

et al. 2005

Nucleic Acids Research

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“…There is no detectable GPI-PLC protein in procyclic form trypanosomes (Bülow and Overath 1985;Webb et al 1997), and the mRNA is barely detectable (Carrington et al 1989). The same regulation was conferred on neo R expression by the GPI-PLC 3 0 UTR and enabled a genetic screen for spontaneous revertants to G418 resistance to be performed.…”

Section: Discussionmentioning

confidence: 99%

“…2b). The GPI-PLC 3 0 UTR is 2300 bases long (Carrington et al 1989;Webb et al 2005), whereas the a-tubulin 3 0 UTR is 125 bases long (see EMBL: BX565803). The difference in length between the two 3 0 UTRs means that GPI-PLC mRNAs with one or other of the 3 0 UTRs can be readily distinguished by size on Northern blots.…”

Section: Identification Of Cis-elements In Trypanosome Mrnasmentioning

confidence: 99%

“…The GPI-PLC gene encodes a developmentally regulated phospholipase C expressed in bloodstream form but not procyclic form trypanosomes (Bülow and Overath 1985). The GPI-PLC mRNA is 3600 nt with a 2300-base-long 3 0 UTR (Carrington et al 1989;Webb et al 2005) and is unstable in procyclic forms with a half-life of 3 min (Webb et al 2005). By using the methods outlined above, it was shown that the GPI-PLC 3 0 UTR contains all the information necessary for differential expression, the first (5 0 ) 750 bases of the 3 0 UTR do not confer full instability in procyclic forms, and deletion of the last (3 0 ) 800 bases of the gene results in stability.…”

Section: Introductionmentioning

confidence: 99%

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A novel strategy to identify the location of necessary and sufficient cis-acting regulatory mRNA elements in trypanosomes

Webb

Burns²,

Kimblin³

et al. 2005

RNA

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Expression of nearly all protein coding genes in trypanosomes is regulated post-transcriptionally, predominantly at the level of mRNA half-life. The identification of cis-acting elements involved in mRNA stability has been hindered by a lack of ability to screen for loss-of-regulation mutants. The method described in this article allows the region containing the necessary and sufficient elements within a mRNA to be identified and uses antibiotic resistance genes as both selectable markers and reporters. In the case of unstable mRNAs, the strategy can be extended by performing a screen for spontaneous loss-of-function mutants in regulatory parts of a mRNA. The method was validated by using the GPI-PLC mRNA, which is unstable in procyclic form trypanosomes and showed that the 3 0 UTR of the GPI-PLC mRNA contains all elements required for developmentally regulated instability. Loss-of-instability mutants all contained deletions within the 2300-nucleotide-long 3 0 UTR, and their analysis showed that a deletion including the last 800 nt of the gene stabilized the mRNA. The method is nonpresumptive, allows far more rapid screening for cis-elements than existing procedures, and has the advantage of identifying functional mutants. It is applicable to all eukaryotes using polycistronic transcription.

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Crystal structure of the phosphatidylinositol-specific phospholipase C from the human pathogen Listeria monocytogenes

Moser

Gerstel

Meyer

et al. 1997

Journal of Molecular Biology

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The Biology of the Glycosylphosphatidylinositol-Specific Phospholipase C of Trypanosoma Brucei

Cited by 10 publications

References 45 publications

Developmentally regulated instability of the GPI-PLC mRNA is dependent on a short-lived protein factor

Developmentally regulated instability of the GPI-PLC mRNA is dependent on a short-lived protein factor

A novel strategy to identify the location of necessary and sufficient cis-acting regulatory mRNA elements in trypanosomes

Crystal structure of the phosphatidylinositol-specific phospholipase C from the human pathogen Listeria monocytogenes

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