|Successful modification||The parasite was generated by the genetic modification |
|The mutant contains the following genetic modification(s)||
|Reference (PubMed-PMID number)||
Reference 1 (PMID number) : 16888139|
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|Parent parasite used to introduce the genetic modification|
|Rodent Malaria Parasite||P. berghei|
|Parent strain/line||P. berghei ANKA|
|Name parent line/clone
P. berghei ANKA cl15cy1
|Other information parent line||A reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).|
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|The mutant parasite was generated by|
|Name PI/Researcher||G.R. Mair, C.J. Janse, A.P. Waters|
|Name Group/Department||Leiden Malaria Research Group|
|Name Institute||Leiden University Medical Center (LUMC)|
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|Name of the mutant parasite|
|Principal name||560cl1; 611cl1|
|Is the mutant parasite cloned after genetic modification||Yes|
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|Asexual blood stage||Not different from wild type|
|Gametocyte/Gamete||Normal production of gametocytes and gametes. A complete block of development of fertilized female gametes (zygotes) into mature ookinetes. Northern analysis of mRNA of gametocytes showed a nearly complete loss of transcripts (mRNA) of genes that are translationally repressed (for example genes encoding the ookinete surface proteins P25, P28). Microarray analysis of mRNA of gametocytes showed a significant reduction (> 2x) of transcripts of 370 genes; including seven of nine genes previously shown to be translationally repressed.|
|Fertilization and ookinete||Normal production of gametocytes and gametes (see also phenotype 'Gametocyte/gamete'). A complete block of development of fertilized female gametes (zygotes) into mature ookinetes. Normal development into ookinetes requires meiotic DNA replication in the zygote 2-3 hours after fertilization of female gametes. In the mutant, all zygotes aborted development before meiosis.|
|Liver stage||Not tested|
|Additional remarks phenotype|
The mutant lacks expression of DOZI (protein development of zygote inhibited; ATP-dependent RNA helicase, putative).
DOZI is an RNA helicase that is highly up-regulated in female gametocytes and shows high sequence homology to the DDX6 family of RNA helicases. It contains domains involved in RNA-binding and RNA-unwinding activity. In other eukaryotes the DDX6 family of DEAD-box RNA helicases is tightly linked both to storage of mRNAs encoding proteins associated with progression through meiosis into translationally silent mRNPs and with the transport of mRNA to degradation centers in the cell (P-bodies). These helicases are found in organisms as diverse as yeast (e.g., Dhh1p) and humans (e.g., RCK/p54).
The phenotype analyses indicate an essential role of DOZI in the development of fertilized female gametes (zygotes).
The phenotype analyses indicate that DOZI has a central role in the silencing and maintenance of steady-state levels of a population of gametocyte-specific transcripts. The loss of DOZI severely affected the capacity of the parasite to store and stabilize a discrete subset of mRNAs in the female gametocyte, resulting in a failure to synthesize specific proteins and to complete normal zygote development. Translational repression of transcripts encoding these proteins in Plasmodium may function to specifically regulate gene expression during meiosis in the zygote.
DOZI is also produced in male gametocytes, although in much lower abundance than in females. Male and female gametes of pbdozi– have been crossed with gametes of mutant parasite lines that are defective in male or in female gamete production. These cross-fertilization assays revealed that male pbdozi– gametes were able to fertilize wild-type DOZI-expressing female gametes, resulting in the development of ookinetes. By contrast, development of zygotes from pbdozi– females fertilized by wild-type males was incomplete and similar to development of zygotes from the pbdozi– line. These crosses demonstrate that the block in zygote/ookinete development is essentially due to a lack of DOZI of female gametocyte origin.
RMgm-133: A mutant expressing a (c-terminal) GFP tagged DOZI
RMgm-361: A mutant which lacks expression of DOZI and expresses GFP under control of a male and RFP under control of a female gametocyte specific promoter.