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1.
Cell Microbiol ; 13(12): 1956-74, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21899698

ABSTRACT

Human FACT (facilitates chromatin transcription) consists of the proteins SPT16 and SSRP1 and acts as a histone chaperone in the (dis)assembly of nucleosome (and thereby chromatin) structure during transcription and DNA replication. We identified a Plasmodium berghei protein, termed FACT-L, with homology to the SPT16 subunit of FACT. Epitope tagging of FACT-L showed nuclear localization with high expression in the nuclei of (activated) male gametocytes. The gene encoding FACT-L could not be deleted indicating an essential role during blood-stage development. Using a 'promoter-swap' approach whereby the fact-l promoter was replaced by an 'asexual blood stage-specific' promoter that is silent in gametocytes, transcription of fact-l in promoter-swap mutant gametocytes was downregulated compared with wild-type gametocytes. These mutant male gametocytes showed delayed DNA replication and gamete formation. Male gamete fertility was strongly reduced while female gamete fertility was unaffected; residual ookinetes generated oocysts that arrested early in development and failed to enter sporogony. Therefore FACT is critically involved in the formation of fertile male gametes and parasite transmission. 'Promoter swapping' is a powerful approach for the functional analysis of proteins in gametocytes (and beyond) that are essential during asexual blood-stage development.


Subject(s)
Germ Cells/physiology , Histone Chaperones/metabolism , Plasmodium berghei/physiology , Protozoan Proteins/metabolism , Animals , Anopheles/parasitology , Cell Nucleus/metabolism , DNA Replication , DNA, Protozoan/genetics , DNA, Protozoan/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Down-Regulation , Epitope Mapping , Female , Fertility , Flagella/metabolism , Gene Expression Regulation , Gene Knockdown Techniques , Genetic Vectors/genetics , Genetic Vectors/metabolism , Germ Cells/metabolism , Histone Chaperones/genetics , Mice , Oocysts/metabolism , Oocysts/physiology , Plasmodium berghei/genetics , Plasmodium berghei/metabolism , Promoter Regions, Genetic , Protein Biosynthesis , Protozoan Proteins/genetics , Transcription, Genetic
2.
Cell Microbiol ; 11(8): 1272-88, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19438517

ABSTRACT

Malaria parasites invade erythrocytes of their host both for asexual multiplication and for differentiation to male and female gametocytes - the precursor cells of Plasmodium gametes. For further development the parasite is dependent on efficient release of the asexual daughter cells and of the gametes from the host erythrocyte. How malarial parasites exit their host cells remains largely unknown. We here report the characterization of a Plasmodium berghei protein that is involved in egress of both male and female gametes from the host erythrocyte. Protein MDV-1/PEG3, like its Plasmodium falciparum orthologue, is present in gametocytes of both sexes, but more abundant in the female, where it is associated with dense granular organelles, the osmiophilic bodies. Deltamdv-1/peg3 parasites in which MDV-1/PEG3 production was abolished by gene disruption had a strongly reduced capacity to form zygotes resulting from a reduced capability of both the male and female gametes to disrupt the surrounding parasitophorous vacuole and to egress from the host erythrocyte. These data demonstrate that emergence from the host cell of male and female gametes relies on a common, MDV-1/PEG3-dependent mechanism that is distinct from mechanisms used by asexual parasites.


Subject(s)
Erythrocytes/metabolism , Germ Cells/physiology , Plasmodium berghei/physiology , Protozoan Proteins/metabolism , Animals , Anopheles , Female , Fertilization , Genes, Protozoan , Host-Pathogen Interactions , Malaria/metabolism , Malaria/parasitology , Male , Mice , Microscopy, Electron, Transmission , Plasmodium berghei/ultrastructure , Protozoan Proteins/chemistry , Sequence Analysis, Protein , Sex Factors
3.
Int J Parasitol ; 37(7): 735-42, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17362967

ABSTRACT

The use of transposable elements as a gene-trapping strategy is a powerful tool for gene discovery. Herein we describe the development of a transposable system, based on the bacterial Tn5 transposon, which has been used successfully in Leishmania braziliensis. The transposon carries the neomycin phosphotransferase gene, which is expressed only when inserted in-frame with a Leishmania gene present in the target DNA. Four cosmid clones from a L. braziliensis genomic library were used as targets in transposition reactions and four insertional libraries were constructed and transfected in L. braziliensis. Clones resistant to G418 were selected and analysed by immunofluorescence in order to identify the subcellular localisation of the protein coded by the trapped gene. A definitive subcellular localisation for neomycin phosphotransferase/targeted protein fusion was not obtained in any of the four Leishmania clones investigated. However, the constructed transposable element is highly efficient considering the frequency of insertion in large targets and is therefore a useful tool for functional genetic studies in Leishmania. Our data confirm the utility of the Tn5 transposon system for insertion of sequencing priming sites into target DNA. Furthermore, the high frequency of insertion and even distribution are important in studying genomic regions bearing long and polymorphic repetitive sequences.


Subject(s)
DNA Transposable Elements/genetics , Leishmania braziliensis/genetics , Amino Acid Sequence , Animals , Base Sequence , Gene Library , Genomics/methods , Microscopy, Confocal , Molecular Sequence Data , Mutagenesis, Insertional , Transfection
4.
Mol Biochem Parasitol ; 137(1): 81-6, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15279954

ABSTRACT

We have carried out a survey of the genome of Leishmania (Viannia) braziliensis by shotgun sequencing. Approximately 15% of the haploid genome of the parasite (5.15 Mb of genomic sequence) was obtained. A large number of known and putative genes, predicted to be involved in several cellular processes, were identified. Some genomic features were investigated, such as the general G + C content, which was found to be lower than L. major (57% versus 63%). BlastN searches revealed that 60.2% of the clusterized GSS sequences displayed similarity to L. major genomic sequences, while a BlastX search showed that 45.3% of the thus obtained predicted protein sequences showed similarity to annotated proteins of L. major. Further comparison of the degree of conservation between L. major and L. braziliensis revealed that coding regions are much more conserved than non-coding ones. The shotgun sequence analysis of Leishmania braziliensis appears to be an efficient and suitable strategy contributing to the search for vaccines and novel drug targets. The sequence data described in this paper have been submitted to the dbGSS database under the following accession numbers (BX530413 to BX530454; BX530456 to BX530718; BX538354 to BX539305; BX539350 to BX540325; BX541002 to BX544869; BX544893 to BX545685; BX897701 to BX897710; BX905184 to BX907797; BX907798 to BX908381; BX908403 to BX908718). All data including sequences are also available at (www.ebi.ac.uk/embl/).


Subject(s)
Genome, Protozoan , Leishmania braziliensis/genetics , Sequence Analysis, DNA , Animals , Base Composition , Conserved Sequence , DNA, Protozoan/chemistry , DNA, Protozoan/isolation & purification , Genes, Protozoan , Leishmania major/genetics , Molecular Sequence Data , Multigene Family , Open Reading Frames , Sequence Homology
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