Pfnek-1, a NIMA-related kinase from the human malaria parasite Plasmodium falciparum Biochemical properties and possible involvement in MAPK regulation.

We have cloned Pfnek-1, a gene encoding a novel protein kinase from the human malaria parasite Plasmodium falciparum. This enzyme displays maximal homology to the never-in-mitosis/Aspergillus (NIMA)/NIMA-like kinase (Nek) family of protein kinases, whose members are involved in eukaryotic cell division processes. Similar to other P. falciparum protein kinases and many enzymes of the NIMA/Nek family, Pfnek-1 possesses a large C-terminal extension in addition to the catalytic domain. Bacterially expressed recombinant Pfnek-1 protein is able to autophosphorylate and phosphorylate a panel of protein substrates with a specificity that is similar to that displayed by other members of the NIMA/Nek family. However, the FXXT motif usually found in NIMA/Nek protein kinases is substituted in Pfnek-1 by a SMAHS motif, which is reminiscent of a MAP/ERK kinase (MEK) activation site. Mutational analysis indicates that only one of the serine residues in this motif is essential for Pfnek-1 kinase activity in vitro. We show (a) that recombinant Pfnek-1 is able to specifically phosphorylate Pfmap-2, an atypical P. falciparum MAPK homologue, in vitro, and (b) that coincubation of Pfnek-1 and Pfmap-2 results in a synergistic increase in exogenous substrate labelling. This suggests that Pfnek-1 may be involved in the modulation of MAPK pathway output in malaria parasites. Finally, we demonstrate that recombinant Pfnek-1 can be used in inhibition assays to monitor the effect of kinase inhibitors, which opens the way to the screening of chemical libraries aimed at identifying potential new antimalarials.

Repetitive sequences upstream of the pfg27/25 gene determine polymorphism in laboratory and natural lines of Plasmodium falciparum.

The structure of the genomic region located upstream of the gametocyte-specific gene pfg27/25 of Plasmodium falciparum was analysed in laboratory lines and field isolates of the parasite. The gene is located in a subtelomeric region of chromosome 13 in parasite clones 3D7 and HB3. Analysis of laboratory lines and field isolates of P. falciparum indicated that polymorphism upstream of pfg27/25 is mainly due to the structure of a repetitive DNA region located at about half a kilobase from the pfg27/25 coding sequence. Different types of repetitive sequences are present in this region, whose copy number is variable in different parasite lines. In addition a GC-rich sequence element contained in this region, which is proposed to be the startpoint of pfg27/25 mRNA, presents either a direct or a reverse orientation in different parasite lines. Genomic deletions upstream of the pfg27/25 gene are also described in two laboratory lines of the parasite, which eliminate two newly identified malaria genes. orf P and orf Gap, from the genome of these parasites. One of them, orf Gap, deleted from the reference parasite clone 3D7, is abundantly expressed as mature mRNA in asexual parasites. PCR analysis on 64 field isolates of P. falciparum indicated that orf P and orf Gap sequences are present in all tested samples of naturally propagating parasites.

Population structure of Aedes albopictus (Skuse): the mosquito which is colonizing Mediterranean countries.

Multilocus electrophoresis analysis has been used to study the genetic structure of 18 populations of Aedes albopictus newly introduced to Italy, in comparison with two populations in the United States, four in Japan, and four in Indonesia. Allozyme analysis revealed that 15 out of the 18 studied loci were polymorphic among the 28 populations. No significant deviations from Hardy-Weinberg equilibrium were found at polymorphic loci. High genetic affinity was observed between the Italian populations and those from the United States and Japan. The analysis of variance in allele frequencies showed that variance among subpopulations accounted for most of the total variance, suggesting that isolation of the Italian populations is not related to distance. Analysis of linkage disequilibrium using Ohta's method shows that the variance in the frequency of allele combinations could be explained by the action of the genetic drift which accompanies the establishment of new populations. The colonization process of Ae. albopictus in Italy is following a trend similar to that previously observed in the U.S. A., probably because both infestations derive from several successive introductions, each with large numbers of individuals.

Chromosome mapping in Cryptosporidium parvum and establishment of a long-range restriction map for chromosome VI.

We used contour-clamped homogeneous electric field (CHEF) gel electrophoresis and Southern blot hybridization to analyze the molecular karyotype of Cryptosporidium parvum and establish the chromosomal location of 12 single copy genes. In agreement with previous studies, the molecular karyotype of C. parvum was found to consist of partially co-migrating chromosomes ranging in size from 0.97 to 1.55 Mb and segregating into five distinct electrophoretic bands. Hybridization results allowed the definition of a linkage group comprised of five distinct loci located on chromosome VI. Southern hybridization and restriction analysis of total C. parvum chromosomes or isolated chromosome VI using gene-specific probes and an oligonucleotide specific for C. parvum telomeres allowed the development of a long-range restriction map of chromosome VI.

Genome plasticity and sexual differentiation in Plasmodium.

Spontaneous subtelomeric deletions of Plasmodium chromosomes have been observed both in natural infections and in laboratory maintained parasites. In the latter case, functions dispensable for asexual parasite multiplication and encoded at the extremities of the chromosomes are easily lost. In particular, spontaneous subtelomeric deletions have been characterised which affect gametocytogenesis both in Plasmodium berghei maintained in laboratory animals and in Plasmodium falciparum propagated in in vitro cultures. In order to identify these genetic determinants, and, potentially, other genes located subtelomerically, we designed a transfection system able to induce and select for controlled, site-specific subtelomeric deletions.

Multilocus genotypic analysis of Cryptosporidium parvum isolates from different hosts and geographical origins.

The genetic analysis of oocysts recovered from the stools of humans and animals infected with Cryptosporidium parvum has consistently shown the existence of two distinct genotypes. One of the genotypes is found exclusively in some human infections, whereas the other genotype is found in human as well as in animal infections. On the basis of these observations and the results of published epidemiological studies with single polymorphic markers, the existence of two separate transmission cycles has been postulated, one exclusively anthroponotic and the other involving both animals and humans. To test this hypothesis, C. parvum isolates of different geographic and host origins were analyzed by using unlinked genetic polymorphisms. A total of 28 isolates originating from Europe, North and South America, and Australia were examined. Isolates clustered into two groups, one comprising both human and animal isolates and the other comprising isolates only of human origin. The absence of recombinant genotypes is consistent with two reproductively isolated populations within the species C. parvum.