ABSTRACT
We present a total of approximately 15 kb of DNA sequences, encompassing four chorion genes Ccs18, Ccs15, Ccs19, Cc16 and their flanking DNA in the medfly C. capitata. Comparison of coding regions, introns and intergenic sequences in five Dipteran species, D. melanogaster, D. subobscura, D. virilis, D. grimshawi and C. capitata documented an extensive divergence in introns and coding regions, but few well conserved elements in the proximal 5' flanking regions in all species. These elements are related to conserved regulatory features of three of the genes, including tissue- and temporal regulation. In the fourth, gene s15, significant alterations in the 5' flanking region may be responsible for its changed temporal regulation in C. capitata. One long intergenic sequence, located in the distal 5' flanking region of gene s18, is homologous to ACE3, a major amplification control element and contains an 80-bp A/T-rich sequence, known to stimulate strong binding of the origin recognition complex (ORC) in D. melanogaster. Analysis of the nucleotide composition of all chorion genes in C. capitata and D. melanogaster showed that C. capitata exhibit less biased representation of synonymous codons than does D. melanogaster.
Subject(s)
Diptera/genetics , Egg Proteins/genetics , Animals , Base Sequence , DNA/chemistry , DNA/genetics , Evolution, Molecular , Gene Order , Molecular Sequence Data , Multigene Family/genetics , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Nucleic AcidABSTRACT
Laminin is a major constituent of the basal lamina surrounding the midgut of the malaria vectors that has been implicated in the development of the Plasmodium oocyst. In this report we describe the cloning of the Anopheles gambiae gene encoding the laminin gamma 1 polypeptide and follow its expression during mosquito development. To further investigate the putative role of laminin in the transmission of the malaria parasite we studied the potential binding of the P25 surface protein of Plasmodium berghei using a yeast two-hybrid system. Heterodimer formation was observed and does not require any additional protein factors since purified fusion proteins can also bind each other in vitro. Laminin gamma 1 also interacts with the paralogue of P25, namely P28, albeit more weakly, possibly explaining why the two parasite proteins can substitute for each other in deletion mutants. This represents the first direct evidence for molecular interactions between a surface protein of the Plasmodium parasite with an Anopheles protein; the strong interplay between laminin gamma 1 and P25 suggests that this pair of proteins may function as a receptor/ligand complex regulating parasite development in the mosquito vector.
Subject(s)
Anopheles/metabolism , Insect Proteins/metabolism , Laminin/metabolism , Plasmodium berghei/metabolism , Protozoan Proteins/metabolism , Amino Acid Sequence , Animals , Anopheles/chemistry , Anopheles/genetics , Anopheles/parasitology , Dimerization , Gene Expression Regulation, Developmental , Insect Proteins/chemistry , Insect Proteins/genetics , Laminin/chemistry , Laminin/genetics , Malaria/parasitology , Molecular Sequence Data , Molecular Weight , Plasmodium berghei/chemistry , Plasmodium berghei/genetics , Protein Binding , Protozoan Proteins/chemistry , Two-Hybrid System TechniquesABSTRACT
We have developed an in vitro culture system for early sporogonic stages of Plasmodium berghei, which can be used to study developmental events normally taking place in the midgut of an infected mosquito. These include penetration of insect cells by the mature ookinete, transformation into oocysts and the early development of the latter, sustained through several rounds of nuclear division. The system, based upon co-culture of enriched ookinetes with several established insect cell lines, was used to study the development of mutant ookinetes lacking both the Pbs21 and Pbs25 surface proteins. Motility and entry of double knockout and Pbs21 single knockout ookinetes into the insect cells are normal, but the number of ookinetes successfully transforming into oocysts expressing the CSP protein are substantially reduced. Finally, using the yeast two-hybrid system we also show that Pbs25 has the capacity to homodimerise as well as to form heterodimers with Pbs21.
Subject(s)
Plasmodium berghei/growth & development , Plasmodium berghei/genetics , Protozoan Proteins/metabolism , Aedes , Animals , Anopheles , Cell Line , Coculture Techniques , Culture Media, Conditioned , Drosophila melanogaster , Locomotion , Mice , Microscopy, Fluorescence , Microscopy, Phase-Contrast , Mutation , Protozoan Proteins/genetics , Rats , Recombinant Fusion Proteins , Two-Hybrid System TechniquesSubject(s)
Myosins/metabolism , Plasmodium berghei/growth & development , Plasmodium berghei/metabolism , Amino Acid Sequence , Animals , Life Cycle Stages , Molecular Sequence Data , Myosins/chemistry , Myosins/genetics , Plasmodium berghei/genetics , Plasmodium berghei/pathogenicity , Plasmodium falciparum/genetics , Plasmodium falciparum/metabolism , Sequence Analysis, DNAABSTRACT
We report the isolation, full sequence characterization, amplification and expression properties of medfly chorion genes corresponding to the autosomal chorion locus of Drosophila. These genes are found adjacent to the paramyosin gene and are organized in the same order and tandem orientation as their Drosophila homologues, although they are spaced further apart. They show substantial sequence divergence from their Drosophila homologues, including novel peptide repeats and a new spacing of the tyrosines, which are known to be cross-linked in Dipteran chorion. The genes are amplified and expressed during oogenesis, as in Drosophila. Three of them are expressed in the same relative temporal order as in Drosophila but the fourth gene, the homologue of s15, shows a clear shift to an earlier expression period. This is the first known instance of changed temporal regulation in dipteran chorion genes.
