Targeted terminal deletions as a tool for functional genomics studies in Plasmodium.
We describe a transfection system that induces terminal deletions at specific chromosome ends in malaria parasites using a linear construct containing telomeric repeats at one end and plasmodial sequences able to drive homologous recombination at the other. A site-specific deletion was generated at one extremity of chromosome 5 of Plasmodium berghei, which was stably maintained in the parasite population selected after transfection. The telomeric repeat array introduced with the construct reached the average length observed in natural telomeres of Plasmodium, indicating that in vivo telomere addition occurred at the newly formed extremity. The expression of a mutant dhfr/ts gene conferring pyrimethamine resistance, used as a selectable marker, was not affected by the proximity to the telomeric sequences, either in the presence or absence of drug pressure. In addition, no transcriptional silencing was observed on insertion of the mutant dhfr/ts gene either in subtelomeric or internal positions that are transcriptionally silent in blood-stage parasites. This suggests that the activity of its promoter is not affected by the chromatin organization of the chromosomal context.
Pace
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Laboratorio di Biologia Cellulare, Istituto Superiore di Sanitá, 00161 Rome, Italy.
Scotti
R
R
Janse
C J
CJ
Waters
A P
AP
Birago
C
C
Ponzi
M
M
D000818:Animals; D018390:Gene Targeting; D017125:Genes, Protozoan / Q000235:genetics*; D005822:Genetic Vectors / Q000138:chemical synthesis; D018503:Genome, Protozoan; D016297:Mutagenesis, Site-Directed; D010962:Plasmodium berghei / Q000201:enzymology / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion / Q000235:genetics*; D016615:Telomere / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D013940:Thymidylate Synthase / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase