Expression of the Plasmodium berghei actin II gene is controlled by elements in a long genomic region.

Plasmodium parasites have two actin isoforms. Actin I is ubiquitously expressed, while the second actin isoform is expressed in the sexual stages and ookinetes. Reverse genetic analysis revealed two phenotypes in parasites lacking the protein: a block in male gametogenesis (exflagellation) and a second phenotype in oocyst development, dependent upon the expression of the gene in female gametocytes. Here, we report that the genetic complementation of two independent mutants lacking actin II does not fully restore wild-type function. Constructs were integrated in the c-rrna locus, previously used for expression of transgenes, in order to determine the dependence of expression on actin II flanking genomic regions. Partial restoration of male gametogenesis was achieved when the transgene contained, in addition to the coding region, 1.2 kb upstream of the actin II open reading frame. Another transgene, which comprised 2.7 kb of actin II 5' flanking regions and the cognate 3' downstream sequence, fully restored exflagellation. However, in both complemented strains, oocyst development was severely impaired compared to the WT. These data suggest that male gametocyte expression of actin II is dependent upon extensive flanking regions, while female expression requires even longer genomic sequences for correct expression of the gene.

Global expression profiling reveals shared and distinct transcript signatures in arrested act2(-) and CDPK4(-) Plasmodium berghei gametocytes.

Gametocytogenesis and gametogenesis in malaria parasites are complex processes of cell differentiation and development likely involving many gene products. Gametocytes develop in the blood of the vertebrate host but mature gametocytes are not activated until taken up by the mosquito vector. Several distinct mutants have been described that block gametogenesis but the detailed molecular causes for the mutant phenotypes are not understood. To investigate whether a block in gametogenesis also results in a changed transcriptional profile we studied two gene deletions mutants; act2(-) lacking stage-specific actin II and CDPK4(-) lacking calcium-dependent protein kinase 4. Whole genome microarray analysis was performed from RNA of mature gametocytes to compare the transcriptomes of the mutants with wild-type Plasmodium berghei. The microarray analysis identified ∼12% of all genes being differentially expressed in either or both mutants compared to normal gametocytes, as defined by at least two-fold change in transcript abundance. A large proportion of the differentially expressed genes overlapped in the two mutants, consistent with a related outcome of gametocyte arrest. Distinct profiles in each mutant were also observed. Among the down-regulated genes were thioredoxin 2 and members of the merozoite surface protein 7 family. Generation and characterization of a msp7(-)/mspr1(-)/mspr2(-) triple mutant and re-analysis of trx2(-) parasites revealed no impairment of life cycle progression. Together, our analysis provides a resource for molecular signatures of Plasmodium berghei gametogenesis and exemplifies the potential of expression profiling of distinct genetically arrested parasites.