Identification of regulatory elements in the Plasmodium falciparum genome☆
Introduction
Malaria is a disease caused by infection with a protozoan parasite of the genus Plasmodium. The World Health Organization estimates that each year, 300–500 million individuals are infected with malaria, and 1–2 million succumb to the disease [1]. The majority of these deaths result from infection with P. falciparum, the most virulent of the Plasmodia that infect humans. One of the hallmark features of the malaria parasite is the complex life cycle that includes both mosquito and human hosts. This life cycle requires extensive control of gene expression since the parasite’s morphology and protein repertoire is markedly different at each life cycle stage [2], [3]. Microarray experiments have demonstrated that steady-state RNA levels for many mRNAs change throughout the parasitic life cycle, indicating control of gene expression at the level of RNA synthesis and/or stability [4], [5], [6], [7], [8], [9]. Indeed, nuclear run on experiments have directly demonstrated transcriptional regulation of several P. falciparum genes including genes required for pathogenesis (var) [10], sexual differentiation (pfg27/25) [11], DNA replication (DNA polymerase δ and topoisomerase I) [12], [13], and ribosomal RNA [14]. Thus, transcriptional regulation is an important control point for gene expression in this organism. However, there is also evidence for posttranscriptional control of gene expression in this parasite [12]. Therefore, expression of P. falciparum genes is likely controlled at multiple levels.
Nonetheless, the sequence elements necessary for control of gene expression have remained elusive. Transient transfection experiments have been useful in detecting large DNA fragments necessary to drive gene expression in Plasmodium sp. [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33]. However, these experiments have only been able to detect specific regulatory elements for a few genes [21], [22], [25], [27], [28], [30], [31], [32], and are too laborious for multi-gene analysis in this parasite. Furthermore, the few known sequences that regulate gene expression in P. falciparum may be unique to the parasite, making the search for regulatory elements even more difficult. As a result, regulatory elements have been identified for only a few of the predicted 5300 genes of P. falciparum. Thus, a faster and more efficient method for identifying regulatory elements in the genomes of Plasmodium sp. is needed, and will aid in the effort to better understand and potentially control malaria.
Therefore, we took advantage of the large amount of sequence information available for Plasmodium sp. [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], and devised a new bioinformatic strategy for regulatory element identification in these parasites. First, over-represented DNA elements upstream of the P. falciparum open reading frames were identified. Over-represented DNA sequence elements upstream of coordinately regulated genes or gene families are often regulatory elements [44], [45], and we predicted over-represented DNA sequences would also function as regulatory elements in P. falciparum. Since this is the case for mammalian and yeast hsp gene family members [46], [47], [48], [49], we chose the 18-member P. falciparum hsp gene family as a model system. Second, we evaluated whether predicted DNA elements are conserved between different Plasmodium species. Indeed, regulatory elements are often conserved in the genomes of related organisms, and have been the basis of a novel strategy for regulatory element identification in both prokaryotes [50] and eukaryotes [51], [52]. Thus, we also predicted that critical regulatory elements would be conserved between different Plasmodium sp., and this also became a criterion of regulatory element identification. Herein, the results of the analysis of P. falciparum hsp gene regulatory elements are presented, and many general features of Plasmodium sp. regulatory elements are described.
Section snippets
Parasite culture
Blood stage P. falciparum strains 3D7 and D10 were cultivated at 37 °C in RPMI–HEPES medium containing 0.2% sodium bicarbonate, 50 μg/ml hypoxanthine, 25 μg/ml gentamicin, 5% inactivated human O+ serum, 5% albumax II, and 5% human O+ blood using standard procedures [53].
Bioinformatics
Eighteen hsp genes in P. falciparum were identified by GO function using PlasmoDB release 4.0 (http://plasmodb.org) [54], [55], [56]. The DNA sequence 2 kb upstream from the predicted initiation codon of each P. falciparum hsp gene
Transient transfection analysis of hsp86 5′ and 3′ flanking regions
There is little information regarding the sequences necessary for gene expression in P. falciparum sp., even though the sequence of the entire genome is known. In order to elucidate the sequences necessary for gene expression in P. falciparum, the hsp genes were used as a model system. Hsp genes have been utilized as a model system to understand gene expression in many organisms including humans [47], [48], yeast [64], and Drosophila [65]. Eukaryotic hsp genes typically contain conserved
Discussion
Our long-term goal is to elucidate both regulatory sequences and regulatory mechanisms in P. falciparum. Thus, regulatory elements of the P. falciparum hsp gene family were elucidated using a combination of bioinformatic strategies and transient transfection experiments. Initially, over-represented motifs in the 5′ flanking regions of all 18 P. falciparum hsp genes were elucidated, as these sequences may regulate gene expression. The top-scoring motif amongst elements with five to seven
Acknowledgements
We thank Ali Sultan, Manoj Duraisingh, Sarah Volkman, Johanna Daily, Muhammad Zaman, Swati Pantakar, Connie Chow, Alissa Myrick, Susan Thomas, Anusha Munasinghe, and Heather Surkala for their helpful discussions and critical analysis of the manuscript. We also acknowledge Cathy Ndiaye and Gilberto Ramirez for excellent technical support. The work presented in this manuscript was supported by NIH Postdoctoral Fellowship AI050303-01 (K.T.M.), NIH grant GM61351-03 (D.F.W.), and Exxon-Mobil
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Supplementary data associated with this article can be found, in the online version, at doi: 10.1016/j.molbiopara.2003.11.004.