Additional remarks phenotype | Mutant/mutation
In the 'promoter swap mutant': the promoter of smc4 has been replaced with the asexual blood stage promoter ama1 (PBANKA_0915000). This promoter is active in asexual blood stages but not in gametocytes.
Protein (function)
An essential component of chromosome dynamics is a family of structural maintenance of chromosomes proteins, originally described in budding yeast as stability of minichromosomes (SMC) proteins, which are implicated in chromosome segregation and condensation. Most eukaryotes have at least six genes encoding SMC proteins (each 110–170 kDa, with a central hinge region and N- and C-terminal globular domains with Walker A and Walker B motifs forming the ATPase head domain). The six SMCs can be classified as subunits of condensin (SMC2 and SMC4, required for chromosomal condensation), cohesin (SMC1 and SMC3, required for chromosomal segregation), and the SMC5-SMC6 complex (involved in DNA repair and homologous recombination. Higher eukaryotic organisms have two condensin complexes, condensin I and condensin II, whereas many single-celled organisms such as yeast have only one condensin complex.
SMC2 and SMC4 form the core structure for both condensin I and condensin II in higher eukaryotes and interact with three additional non-SMC components: one kleisin and two Heat protein subunits. Kleisin Ig (CAP-H), Heat IA (CAP-D2), and Heat IB (CAP-G) form the condensin I complex, whereas Kleisin IIb (CAP-H2), Heat IIA (CAP-D3), and Heat IIB (CAP-G2) form the condensin II complex.
Domain analysis of Plasmodium SMC2 and SMC4 revealed a conserved domain architecture for both SMC2 and SMC4 . A comparative sequence analysis revealed low
sequence similarity and identity (29%–34%), except for the SMC4 homolog in Arabidopsis thaliana (65%), although there was similarity in size and overall domain structure when compared with the proteins in the other studied organisms.
We found the P. berghei SMC4 N-terminal ATPase domain divided in two by a 44 amino acid insertion; a similar pattern has been observed in other Plasmodium species.
P. berghei SMC2 (PBANKA_1416900), SMC4 (PBANKA_1108700)
Phenotype
To investigate the function of SMC2 and SMC4 during cell division in male gametogenesis, we used a promoter trap double homologous recombination (PTD) approach to downregulate gene expression at this stage by placing each of the two genes under the control of the AMA1 promoter. AMA1 is known to be highly expressed in asexual blood stages, but not during sexual differentiation. This strategy resulted in the successful generation of two transgenic parasite lines: P(ama1)smc2 (SMC2PTD) and P(ama1)smc4 (SMC4PTD)
SMC2 is expressed both in asexual blood stages and (male) gametocytes. Knock-out of the smc2 gene is not possible since it has an essential role in asexual blood stages. In the promoter swap mutant SMC2 is expressed in asexual blood stages but is down-regulated in (male) gametocytes.
We were unable to detect any particular impaired phenotype in the SMC2PTD and SMC4PTD lines at the asexual blood stage (schizogony), and the parasite formed a similar number of schizonts and nuclei compared with the WTGFP line.
Fertilization and zygote formation leading to ookinete conversion was reduced to 10%–15% compared with the WTGFP line. The ookinete motility assay showed normal movement of SMC4PTD ookinetes compared with WT.
We observed an ~50% reduction in the number of exflagellation centers during male gametogenesis.
Fertilization and zygote formation leading to ookinete conversion was reduced to 10%–15% compared with the WTGFP line. The ookinete motility assay showed normal movement of SMC4PTD ookinetes compared with WT.
In the mosquito gut on 9, 14, and 21 days post-infection, we detected significantly fewer oocysts in the SMC2PTD and SMC4PTD lines. Furthermore, the oocysts were considerably smaller compared with those of WTGFP, with unequal distribution and clusters of DNA in some oocysts at 14 and 21 days post-infection. No sporogony or endomitosis was observed within oocysts.
We were also unable to detect sporozoites in the mosquito salivary glands; hence, no parasite transmission from infected mosquitoes to mice was observed for either SMC2PTD or SMC4PTD parasite lines in bite-back experiments
Additional information
Other mutants |