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Summary of Prioritized Research Needs |
 TOP Summary of Prioritized Research... 3.3.1 Overview3.3.2 Research Needs3.3.3 References |
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3.3.1 Overview |
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TOPSummary of Prioritized Research...3.3.1 Overview3.3.2 Research Needs3.3.3 References |
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Why genomics for LF? For more than 10 years (approximately 1982–1994), standard molecular cloning techniques were applied to the study of filarial parasites (particularly B. malayi and O. volvulus), but few genes were cloned and identified. By the end of 1994, only 60 Brugia genes had been submitted to the Genbank database. It was clear that a new approach for studying the filarial genome was needed to make rapid progress in understanding the biology and biochemistry of these parasites. The genome project approach represented a complete departure from the way parasite genes had been studied in the past. Genome projects are typically not directed at the identification of individual genes, but instead at the identification, cloning, and sequencing of all the organism’s genes.
At the first meeting of the Filarial Genome Project (1994), B. malayi was selected as the organism to be studied. This parasite was chosen over the other two medically important species of filarial parasites, W. bancrofti and O. volvulus, primarily because of the ready availability of all stages of the life cycle.4 In addition, molecular phylogenetic studies of filarial parasites had shown that all three of these species are closely related. Thus, much of the molecular data obtained on one species would be applicable to the other two.5
The genome of B. malayi6 is about the same size (100 million base pairs) as that of the free-living nematode Caenorhabditis elegans.7 The number of protein-coding genes in C. elegans is estimated to be approximately 19,000 and the number of genes in Brugia is expected to be about the same.
Filarial genomics: The 10 most important accomplishments to date. Filarial genomics now provide a basis for studying filarial parasites in ways that could not even be imagined only a few years ago. The 10 most important accomplishments of the first 10 years of filarial genomics include
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3.3.2 Research Needs |
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TOPSummary of Prioritized Research...3.3.1 Overview3.3.2 Research Needs3.3.3 References |
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For the future, a concerted effort must be made
2) Constructing cDNA and genomic libraries. Since fewer than 60 Brugia genes had been cloned by 1994, it was decided that the primary goal of the Filarial Genome Project would be the identification of at least 5,000 new Brugia genes.8 These new genes would help elucidate the biology of the organism and would aid in the identification of new vaccine candidates and drug targets. The plan was to identify these new genes by randomly selecting clones for DNA sequence analysis from new cDNA libraries that were to be constructed from all life cycle stages of B. malayi. The construction of many cDNA libraries from all of the life cycle stages would insure that a high proportion of the expressed genes of B. malayi would be represented. Such EST analysis is a rapid way to identify gene sequences, since no effort is made to completely sequence each cDNA clone.
Twelve Brugia cDNA libraries have been constructed in the bacteriophage lambda cloning vector ZAPII (Stratagene, La Jolla, CA) representing the following developmental stages: MF, L2, L3, molting L3 (L3M), L4, young adult (YA), adult male (AM), and adult female (AF). Unidirectional cloning was chosen to facilitate the use of these libraries in EST analyses. Seven of the Brugia cDNA libraries were constructed using conventional techniques,2 two were constructed using subtraction techniques,9 and three were constructed using PCR and the SL1 spliced leader sequence.10 The Brugia cDNA libraries (and cDNA libraries from O. volvulus and W. bancrofti) and all individual cDNA clones are available from the National Institute of Allergy and Infectious Diseases/National Institutes of Health (NIAID/NIH) Filariasis Research Reagent Repository Center (FRRRC) at Smith College (contact genome{at}smith.edu or www.filariasiscenter.org). For the future,
3) Sequencing cDNA clones (EST sequencing). By March 2004, there were already 26,215 ESTs containing more than 10 million basepairs of B. malayi sequence data submitted to the National Center for Biotechnology Information’s dbEST database (www.ncbi.nlm.nih.gov/dbEST/dbEST_summary.html). In addition, for O. volvulus, 14,974 ESTs have been submitted, while for W. bancrofti more than 6,000 ESTs are now available in the database. In total, this represents almost 20 million base pairs of expressed filarial parasite DNA sequences. All of these cDNA clones are available through the NIAID/NIH FRRRC at Smith College (contact genome{at}smith.edu or www.filariasiscenter.org). For the future,
4) Clustering the EST data and developing user-friendly bioinformatics tools. Since cDNA clones are selected randomly for sequencing and since duplication (especially of highly expressed genes) is unavoidable, the 26,000 Brugia EST represent approximately 8,945 Brugia clusters as determined by The Institute for Genome Research (TIGR) gene index (www.tigr.org/tdb/tgi/) and 8,392 as determined by the Nembase clustering11 (http://nema.cap.ed.ac.uk/fgp.html). The number of genes represented by the EST data set is determined using a clustering algorithm that groups ESTs derived from the same gene into clusters. If the total number of genes in B. malayi is similar to that predicted for C. elegans, this data set includes almost 50% of all Brugia genes. A unified nomenclature for naming filarial genes and gene clusters is in place12 (http://nema.cap.ed.ac.uk/fgn/funk/funkgenenames.html). Because these data are critical to gene identification,
5) Completing the genomic sequencing of the Brugia genome. The complete genome sequence of B. malayi has recently been obtained through an NIH-funded project carried out at TIGR.13 The entire genome was sequenced using a whole genome shotgun strategy developed at TIGR. Approximately 1.3 million sequence reads have provided about 10x coverage (i.e., enough sequencing has been done to theoretically sequence the genome 10 times). This ensures that virtually all of the genome will be represented in the final sequence. Currently, all of the genomic data is being assembled and gaps in the sequence are being closed. The genome is also being annotated by the filarial research community; a process initiated at the "Brugia malayi Annotation Jamboree" held for two weeks at TIGR during January 2004. For the future,
6) Completing the sequencing of the Wolbachia genome from B. malayi and of the mitochondrial genomes from B. malayi and O. volvulus. Most filarial parasites contain an obligate intracellular bacterium most closely related to the Wolbachia intracellular bacteria found in mosquitoes and other arthropods.14 The genome was sequenced by a consortium of scientists led by New England Biolabs.15 Research over the past several years has shown that killing the Wolbachia bacteria results in reduced fecundity and often death of the filarial parasites.16–18 Since this intracellular bacterium contributes to the normal functioning of the filarial parasite, the bacterium itself is a target for novel chemotherapeutic and vaccination interventions. In addition, the complete sequence of the first mitochondrial genome from a filarial parasite (O. volvulus) was carried out at the University of Alabama Birmingham.19 The following year, the complete sequence of the B. malayi mitochondrial genome was completed at the University of Edinburgh. The mitochondrial genome provides additional targets for biological study and potential chemotherapy. What still remains are for
7) Developing a transgenic technique for Brugia. One of the principal barriers to rapid accumulation of knowledge on the biology and biochemistry of filarial parasites has been the inability of researchers to introduce DNA and RNA into the cells of these organisms. For example, the development of a transgenic technique (the ability to insert exogenous genes into the genome of the target organism) is important for the study of many organisms including bacteria, plants, and animals. Recently, a transgenic technique using a biolistic "gene gun" has been successfully applied to filarial parasites at the University of Alabama Birmingham.20 The development of this technique opens up the possibility of doing many gene function studies that were heretofore impossible in filarial parasites. Therefore,
8) Developing RNAi techniques for Brugia. Another important barrier to the rapid accumulation of knowledge on the biology and biochemistry of filarial parasites has been the inability of researchers to introduce RNA into the cells of these organisms. RNA interference has been used for the past several years in a wide variety of organisms as a means for silencing expression of specific genes. Such gene silencing is another valuable tool for studying gene function and for the identification of likely vaccine candidates and drug targets. This technique has been employed with great efficiency in C. elegans, a free-living nematode often used as a model system for filarial parasites. However, until recently, the technique had never been successfully applied to parasitic nematodes. However, in 2003 researchers at the University of Edinburgh successfully applied the RNAi technique in B. malayi adult female parasites,21 and in 2004 scientists at the New York Blood Center successfully applied RNAi to O. volvulus L3 larvae. Together, the development of a transgenic technique for introducing DNA and an RNAi technique for introducing RNA will dramatically revolutionize the way gene study can be undertaken in filarial parasites. Therefore,
9) Producing microarrays for filarial parasites. Another cutting-edge molecular biology technique now widely used in the study of genes in many organisms involves gene microarrays (gene chips). These arrays include thousands of genes on a single microscope slide or on a smaller silicon chip. These arrays enable the analysis of thousands of genes in a single experiment, instead of the more traditional analysis of one or a few genes at a time. Such technology has revolutionized the study of genes and gene expression. Brugia malayi microarrays have now been produced by a consortium of filarial researchers (led by scientists at Washington University and Smith College) at the Washington University Genome Center. These first microarrays for a filarial parasite are now available through the NIAID/NIH FRRRC at Smith College (genome{at}smith.edu or www.filariasiscenter.org). The arrays have been prepared using 65-mer oligonucleotides representing more than 3,000 genes of the parasite. It is still necessary for
10) Initiating comparative genomics studies among nematodes (including C. elegans). To take full advantage of the newly available, complete sequence of the genome of B. malayi this sequence must be compared to those of other nematodes. Fortunately, the complete genomic sequence of the free-living nematode, C. elegans, is also available. In addition, more than 400,000 EST sequences are now available from more than 30 species of nematodes, largely through the efforts of scientists at the Washington University Genome Center and the University of Edinburgh.22 Such genomic and EST sequence data is essential for gene identification, for studies of the conservation of gene order (synteny), and for developing generalizations concerning gene function, gene regulation, and parasite-specific pathways. Because such comparative data will be critical for identifying new genes useful in developing vaccines and new drugs for filarial parasites,
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3.3.3 References |
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TOPSummary of Prioritized Research...3.3.1 Overview3.3.2 Research Needs3.3.3 References |
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