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Plasmodial chromatin - structure and function

Objective

* to identify P. falciparum centromere sequences
* to analyze spatial and temporal chromatin changes and their effect on transcriptional activity in P. falciparum
* to develop an in vitro plasmodial telomerase assay
* to identify and clone the P. falciparum telomerase including its RNA subunit
* to characterize subtelomeric domains in P. vivax
* to identify and clone non-histone nuclear proteins
* to identify origins of replication
Expected Outcome

* The study aims at characterizing those structural aspects of plasmodial chromatin that are relevant to the multiple functions in which chromatin is involved, such as transcription, replication, segregation, chromosomal stability versus (possibly programmed) rearrangements.

* The study will aid in identifying new targets for rational drug design.
* Development of an in vitro P. falciparum telomerase assay. An in vitro assay for plasmodial telomerase activity will be established. Different oligonucleotides will be tested for function as templates for the plasmodial telomerase. The effect of inhibitors on in vitro telomerase activity and in vivo parasite viability will be tested.

* Cloning of the plasmodial telomerase. The gene encoding the protein subunit of the plasmodial telomerase will be cloned a) using a structural approach, based on conserved telomerase sequences, b) by screening a P. falciparum expression library with antibodies to the Tetrahymena telomerase. The RNA subunit of the plasmodial telomerase will be cloned by screening a genomic P. falciparum libraries with oligonucleotide complementary to plasmodial telomeres.

* Studies on plasmodial replication. Electron-microscopic studies will be conducted using decondensed chromatin to determine areas where replication had initiated. In addition, two-dimensional gel electrophoresis as described by Brewer and Fangman will be used to visualize replication intermediates.

* Identification of functional centromere sequences. Centromeric sequences will be identified using a functional, transfection based approach. A wild-type P. falciparum strain will be transfected with a P. falciparum library cloned in a vector, which contains a selectable marker. Repeated rounds of selection and plasmid recovery, followed by re-transformation, will be employed to enrich for sequences containing a plasmodial centromere. Centromeres will be further dissected by deletion analysis. Protein binding motifs will be tested in gel retardation assays, using nuclear proteins.

* Spatial and temporal changes of plasmodial chromatin. The chromatin structure of telomeric repeat sequences, subtelomeric regions, internal chromosome domains and the transition between these different domains will be investigated. The possible effect of chromatin changes on the transcriptional activity of developmentally expressed genes will be investigated.

* Characterizing protein components of plasmodial chromatin. Monoclonal antibodies against NAP (Nucleosome Assembly Protein) will be used in immunofluorescence studies to examine the association of this protein with the nuclear material. In vitro reconstitution experiments are planned to test the activity of the NAP protein in chromatin assembly.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Bayerische Julius-Maximilians-Universität Würzburg
Address
11,Röntgenring 11
97070 Würzburg
Germany

Participants (5)

INSTITUT PASTEUR
France
Address
Rue Du Docteur Roux 25
75724 Paris
Instituto Nacional de Salud
Colombia
Address
50,Avenida El Dorado 50
10 Bogota
Istituto Superiore di Sanità
Italy
Address
Viale Regina Elena 299
00161 Roma
UNIVERSITAETSKLINIKUM HEIDELBERG
Germany
Address
Im Neuenheimer Feld 324
69120 Heidelberg
Universidade de São Paulo
Brazil
Address
1374,Avenida Prof. Lineu Pretes 1374
5508 São Paulo