Revisiting the Plasmodium falciparum RIFIN family: from comparative genomics to 3D-model prediction.

BACKGROUND: Subtelomeric RIFIN genes constitute the most abundant multigene family in Plasmodium falciparum. RIFIN products are targets for the human immune response and contribute to the antigenic variability of the parasite. They are transmembrane proteins grouped into two sub-families (RIF_A and RIF_B). Although recent data show that RIF_A and RIF_B have different sub-cellular localisations and possibly different functions, the same structural organisation has been proposed for members of the two sub-families. Despite recent advances, our knowledge of the regulation of RIFIN gene expression is still poor and the biological role of the protein products remain obscure. RESULTS: Comparative studies on RIFINs in three clones of P. falciparum (3D7, HB3 and Dd2) by Multidimensional scaling (MDS) showed that gene sequences evolve differently in the 5'upstream, coding, and 3'downstream regions, and suggested a possible role of highly conserved 3' downstream sequences. Despite the expected polymorphism, we found that the overall structure of RIFIN repertoires is conserved among clones suggesting a balance between genetic drift and homogenisation mechanisms which guarantees emergence of novel variants but preserves the functionality of genes. Protein sequences from a bona fide set of 3D7 RIFINs were submitted to predictors of secondary structure elements. In contrast with the previously proposed structural organisation, no signal peptide and only one transmembrane helix were predicted for the majority of RIF_As. Finally, we developed a strategy to obtain a reliable 3D-model for RIF_As. We generated 265 possible structures from 53 non-redundant sequences, from which clustering and quality assessments selected two models as the most representative for putative RIFIN protein structures. CONCLUSION: First, comparative analyses of RIFIN repertoires in different clones of P. falciparum provide insights on evolutionary mechanisms shaping the multigene family. Secondly, we found that members of the two sub-families RIF_As and RIF_Bs have different structural organization in accordance with recent experimental results. Finally, representative models for RIF_As have an "Armadillo-like" fold which is known to promote protein-protein interactions in diverse contexts.

A new parameter to study compositional properties of non-coding regions in eukaryotic genomes.

Genomes are characterized by global and local compositional properties that are interesting in an evolutionary perspective but also provide useful information for the identification of some functional elements. Following previous studies, in this work we investigated compositional properties of non-coding sequences in four eukaryotic genomes (C. elegans, D. melanogaster, M. musculus, H. sapiens). We developed a procedure based on Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) to identify pentamers that are over-represented in introns (intron vocabulary) and to define a new parameter (LD) that reflects oligonucleotide composition of a given sequence. We analyzed genomic sequences and we found that all non-coding parts of a genome are characterized by similar LD values. Furthermore, we used the new parameter to analyze potentially regulatory regions. We extracted non-redundant sets of promoter sequences for D. melanogaster and H. sapiens and we studied their compositional (G+C content and LD parameter) and conformational (bendability propensity) properties. We found that regions immediately surrounding transcription start sites are distinguishable because of their %G+C, LD and bendability values.

Genome-wide identification of genes upregulated at the onset of gametocytogenesis in Plasmodium falciparum.

A genome-wide expression analysis was undertaken to identify novel genes specifically activated from early stages of gametocytogenesis in Plasmodium falciparum. A comparative analysis was conducted on sexually induced cultures of reference parasite clone 3D7 and its gametocyteless derivative clone F12. Competitive hybridisations on long-oligomer microarrays representing 4488 P. falciparum genes identified a remarkably small number of transcripts differentially produced in the two clones. Upregulation of the mRNAs for the early gametocyte markers Pfs16 and Pfg27 was however readily detected in 3D7, and such genes were used as reference transcripts in a comparative time course analysis of 3D7 and F12 parasites between 30 and 40 h post-invasion in cultures induced to enter gametocytogenesis. One hundred and seventeen genes had expression profiles which correlated to those of pfs16 and pfg27, and Northern blot analysis and published proteomic data identified those whose expression was gametocyte-specific. Immunofluorescence analysis with antibodies against two of these gene products identified two novel parasite membrane associated, sexual stage-specific proteins. One was produced from stage I gametocytes and the second showed peak production in stage II gametocytes. The two proteins were named Pfpeg-3 and Pfpeg-4, for P. falciparum proteins of early gametocytes.

Functional genomics, new tools in malaria research.

The mosquito-transmitted unicellular parasite Plasmodium falciparum, the agent of malaria disease, still causes more than one million deaths every year in the tropical and subtropical areas of the world. New intervention strategies are needed to contrast the insurgence of resistance to effective drugs and insecticides. The complete annotated genomes of the human parasite P. falciparum and the rodent model P. yoelii is now available thus providing a prediction of their possible gene products. This makes feasible the application of functional genomics to malaria research with the final goal of providing a complete survey of Plasmodium life cycle. Genome-wide approaches to the study of transcriptome or proteome were successfully applied to malaria parasite with the promise for new drug and vaccine candidates in the next future.

Pentamer vocabularies characterizing introns and intron-like intergenic tracts from Caenorhabditis elegans and Drosophila melanogaster.

Overall compositional properties at the level of bases, dinucleotides and longer oligos characterize genomes of different species. In Caenorhabditis elegans, using recurrence analysis, we recognized the existence of a long-range correlation in the oligonucleotide usage of introns and intergenic regions. Through correlation analysis, this is confirmed here to be a genome-wide property of C. elegans non-coding portions. We then investigate the possibility of extracting a typical vocabulary through statistical analysis of experimentally confirmed introns of sufficient length (>1 kb), deprived of known splice signals, the focus being on distributed lexical features rather than on localized motifs. Lexical preferences typical of introns could be exposed using principal component analysis of pentanucleotide frequency distributions, both in C. elegans and in Drosophila melanogaster. In either species, the introns' pentamer preferences are largely shared by intergenic tracts. The pentamer vocabularies extracted for the two species exhibit interesting symmetry properties and overlap in part. A more extensive investigation of the interspecies relationship at the level of oligonucleotide preferences in non-coding regions, not related by sequence similarity, might form the basis of new approaches for the study of the evolutionary behaviour of these regions.