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Var transcription profiling of Plasmodium falciparum 3D7: assignment of cytoadherent phenotypes to dominant transcripts.

Gölnitz U, Albrecht L, Wunderlich G - Malar. J. (2008)

Bottom Line: Each cytoadherence selected parasite line also adhered to untransfected CHO-745 cells and upregulation of the var gene PFD995/PFD1000c was consistently associated with cytoadherence to all but one CHO cell line.In addition, parasites panned on different CHO cell lines revealed candidate var genes which reproducibly associated to the respective cytoadherent phenotype.The transcription profile obtained by RT-PCR/cloning/sequencing differed significantly from that of RT-quantitative PCR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, Avenida Prof, Lineu Prestes 1374, São Paulo - SP, Brazil. uta.goelnitz@gmx.de

ABSTRACT

Background: Cytoadherence of Plasmodium falciparum-infected red blood cells is mediated by var gene-encoded P. falciparum erythrocyte membrane protein-1 and host receptor preference depends in most cases on which of the 50-60 var genes per genome is expressed. Enrichment of phenotypically homogenous parasites by panning on receptor expressing cells is fundamental for the identification of the corresponding var transcript.

Methods: P. falciparum 3D7 parasites were panned on several transfected CHO-cell lines and their var transcripts analysed by i) reverse transcription/PCR/cloning/sequencing using a universal DBLalpha specific oligonucleotide pair and ii) by reverse transcription followed by quantitative PCR using 57 different oligonucleotide pairs.

Results: Each cytoadherence selected parasite line also adhered to untransfected CHO-745 cells and upregulation of the var gene PFD995/PFD1000c was consistently associated with cytoadherence to all but one CHO cell line. In addition, parasites panned on different CHO cell lines revealed candidate var genes which reproducibly associated to the respective cytoadherent phenotype. The transcription profile obtained by RT-PCR/cloning/sequencing differed significantly from that of RT-quantitative PCR.

Conclusion: Transfected CHO cell lines are of limited use for the creation of monophenotypic cytoadherent parasite lines. Nevertheless, 3D7 parasites can be reproducibly selected for the transcription of different determined var genes without genetic manipulation. Most importantly, var transcription analysis by RT-PCR/cloning/sequencing may lead to erroneous interpretation of var transcription profiles.

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var promoter profile of different adhesive parasites phenotypes. The pie charts present relative levels of transcription of each var promoter type from parasites selected on – upsA, upsB/upsBsh, upsC, upsD and upsE. In hatched dark grey, the fraction of transcripts from upsC var genes PFD0995/PFD1000c are highlighted.
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Figure 2: var promoter profile of different adhesive parasites phenotypes. The pie charts present relative levels of transcription of each var promoter type from parasites selected on – upsA, upsB/upsBsh, upsC, upsD and upsE. In hatched dark grey, the fraction of transcripts from upsC var genes PFD0995/PFD1000c are highlighted.

Mentions: The herein used stationary assay of cytoadherence is believed to select for the strongest binding parasite phenotype. In order to elucidate from which promoter type the most abundant transcripts were generated, the relative var transcript copy numbers of each transcript were grouped corresponding to their promoter type upsA, B, C, D and E. As shown in Figure 2, most of the transcribed var genes were from upsC promoters, even when omitting the signal from the PFD0995/PFD1000c locus.


Var transcription profiling of Plasmodium falciparum 3D7: assignment of cytoadherent phenotypes to dominant transcripts.

Gölnitz U, Albrecht L, Wunderlich G - Malar. J. (2008)

var promoter profile of different adhesive parasites phenotypes. The pie charts present relative levels of transcription of each var promoter type from parasites selected on – upsA, upsB/upsBsh, upsC, upsD and upsE. In hatched dark grey, the fraction of transcripts from upsC var genes PFD0995/PFD1000c are highlighted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254424&req=5

Figure 2: var promoter profile of different adhesive parasites phenotypes. The pie charts present relative levels of transcription of each var promoter type from parasites selected on – upsA, upsB/upsBsh, upsC, upsD and upsE. In hatched dark grey, the fraction of transcripts from upsC var genes PFD0995/PFD1000c are highlighted.
Mentions: The herein used stationary assay of cytoadherence is believed to select for the strongest binding parasite phenotype. In order to elucidate from which promoter type the most abundant transcripts were generated, the relative var transcript copy numbers of each transcript were grouped corresponding to their promoter type upsA, B, C, D and E. As shown in Figure 2, most of the transcribed var genes were from upsC promoters, even when omitting the signal from the PFD0995/PFD1000c locus.

Bottom Line: Each cytoadherence selected parasite line also adhered to untransfected CHO-745 cells and upregulation of the var gene PFD995/PFD1000c was consistently associated with cytoadherence to all but one CHO cell line.In addition, parasites panned on different CHO cell lines revealed candidate var genes which reproducibly associated to the respective cytoadherent phenotype.The transcription profile obtained by RT-PCR/cloning/sequencing differed significantly from that of RT-quantitative PCR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, Avenida Prof, Lineu Prestes 1374, São Paulo - SP, Brazil. uta.goelnitz@gmx.de

ABSTRACT

Background: Cytoadherence of Plasmodium falciparum-infected red blood cells is mediated by var gene-encoded P. falciparum erythrocyte membrane protein-1 and host receptor preference depends in most cases on which of the 50-60 var genes per genome is expressed. Enrichment of phenotypically homogenous parasites by panning on receptor expressing cells is fundamental for the identification of the corresponding var transcript.

Methods: P. falciparum 3D7 parasites were panned on several transfected CHO-cell lines and their var transcripts analysed by i) reverse transcription/PCR/cloning/sequencing using a universal DBLalpha specific oligonucleotide pair and ii) by reverse transcription followed by quantitative PCR using 57 different oligonucleotide pairs.

Results: Each cytoadherence selected parasite line also adhered to untransfected CHO-745 cells and upregulation of the var gene PFD995/PFD1000c was consistently associated with cytoadherence to all but one CHO cell line. In addition, parasites panned on different CHO cell lines revealed candidate var genes which reproducibly associated to the respective cytoadherent phenotype. The transcription profile obtained by RT-PCR/cloning/sequencing differed significantly from that of RT-quantitative PCR.

Conclusion: Transfected CHO cell lines are of limited use for the creation of monophenotypic cytoadherent parasite lines. Nevertheless, 3D7 parasites can be reproducibly selected for the transcription of different determined var genes without genetic manipulation. Most importantly, var transcription analysis by RT-PCR/cloning/sequencing may lead to erroneous interpretation of var transcription profiles.

Show MeSH