Page 90 - 72_04

ALMUDENA CROOKE Y COLS. AN. R. ACAD. NAC. FARM.

terminal region clearly differs from most eukaryotic and prokaryotic
G6PDs, and shows 6-phosphogluconolactonase activity; thus G6PD-
6PGL catalyses the first two stages of the pentose phosphate pathway
(21). The occurrence of large insertion sequences that differ with
respect to their homologous proteins in other species has been often
observed in many gene products of P. falciparum and other
Plasmodium species, but their structural functions and origins are
unknown (24, 25). We have been interested to know whether the low
complexity invasions of globular domains could have a role in
regulating protein turnover along the parasitic cycle, and therefore,
in the present paper, the unique bifunctional P. falciparum G6PD-
6PGL protein was explored as a model gene to study dynamics of its
protein processing along the intraerythrocytic cycle and under gene
silencing conditions.

EXPERIMENTAL PROCEDURES

Parasite cultures and electroporation

P. falciparum strain 3D7 was grown and double synchronised
using standard procedures (26,27). Parasites (ring stage 8-10%
parasitaemia) were transfected by electroporation with 40 µg of
dsRNA as described (8). The parasites transfected with dsRNA-G6PD
or dsRNA-Rab5a were kept for 24 h in 75 cm2 flasks. The growth and
development of each transfection was monitored by Giemsa staining
blood films.

dsRNA design

A 21 basepair dsRNA (sense: UACAUCAUGCACCAACGAAdTdT; antisense:
UUCGUUGGUGCAUGAUGUAdTdT) was designed for the target sequence
(UACAUCAUGCACCAACGAA) of the G6PD-6PGL gene, following Dharmacon
siDESIGN Center criteria (http://design.dharmacon.com/). In
addition, a dsRNA corresponding to the PfRab5a gene (GenBank™
accession number AE001399) (target sequence: UAUGCAAGUAUUGUCCCAC;
sense: UAUGCAAGUAUUGUCCCACdTdT; antisense: GUGGGACAAUACUUGCAUAdTdT)

632