Additional remarks phenotype | Mutant/mutation
The mutant expresses GFP under control of the uis4 promoter
Protein (function)
Phenotype
The upregulated in infectious sporozoites 4 (uis4) promoter (of rodent malaria parasites) has been shown to have very low activity in oocyst sporozoites but produces one of the most abundantly transcribed mRNAs in salivary gland sporozoites. Expression of uis4 of rodent malaria parasites continues through early and mid-liver stage, where the UIS4 protein serves an essential role. Moreover, the uis4 mRNA is known to be translationally repressed until transmission to the mammalian host. However, the cis element that provides for translational repression activity is known to not be provided by the promoter/5′UTR, and thus this promoter is useful for exceptionally high expression of transgenes in salivary gland sporozoites/early-mid liver stage of rodent malaria parasites. In agreement with previous work, robust expression in salivary gland sporozoites through mid-liver stage was observed. Additionally, a few asexual blood stage schizonts, male and female gametocytes, and oocysts also had robust expression.
Additional information
From the literature, promoters that have been described to be strong and constitutive or stage specific as observed in other Plasmodium species were selected for study. To produce as diverse of a promoter collection as possible, individual promoters with expression profiles that span the complete life cycle of Plasmodium parasites were chosen. The clag-a promoter (PY17X_1402200) has been defined as restricting transcription to the asexual blood stage of the related rodent malaria parasite P. berghei. The dynein heavy chain delta (“dd”, male, PY17X_0418900) and lap40 (also called ccp2, female, PY17X_1323300) promoters have been commonly used in the “820” male/female fluorescent protein reporter line (820cl1m1cl1). The trap promoter (PY17X_1354800) is known to be active throughout sporozoite development and is critical for salivary gland invasion, gliding motility, and infectivity, whereas the uis4 promoter (PY17X_0502200) flips from being weakly active in oocyst sporozoites to becoming one of the strongest promoters in salivary gland sporozoites. Finally, the lisp2 promoter (PY17X_1004400) becomes active in midliver stage and remains so throughout liver stage development [34]. Finally, the bip promoter (PY17X_0822200) was also selected, as it is known to be a strong and constitutive promoter in eukaryotes due to the integral role that the BiP protein plays in the translocation of nascent, unfolded proteins into the ER. In previous work, a 1.5 kb portion of sequence upstream of the bip coding sequence was used to serve as a promoter for CRISPRbased gene editing. Here, two truncated variants were created in an attempt to produce a strong, minimal constitutive promoter. Promoters tested here typically included 1.6–1.8 kb of sequence upstream of (and including) the start codon, and were designed to place a unique restriction site 3′ of the start codon to avoid effects upon translation that may occur if placed upstream of it.
Other mutants |