Colin Herd

A systematic genome-wide analysis of zebrafish protein-coding gene function

Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at a rapid rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in model vertebrate organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes, this number falls considerably short of the more than 22,000 mouse protein-coding genes. Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning, insertional mutagenesis, antisense morpholino oligonucleotides, targeted re-sequencing, and zinc finger and TAL endonucleases have made substantial contributions to our understanding of the biological activity of vertebrate genes, but again the number of genes studied falls well short of the more than 26,000 zebrafish protein-coding genes. Importantly, for both mice and zebrafish, none of these strategies are particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Here we describe an active project that aims to identify and phenotype the disruptive mutations in every zebrafish protein-coding gene, using a well-annotated zebrafish reference genome sequence, high-throughput sequencing and efficient chemical mutagenesis. So far we have identified potentially disruptive mutations in more than 38% of all known zebrafish protein-coding genes. We have developed a multi-allelic phenotyping scheme to efficiently assess the effects of each allele during embryogenesis and have analysed the phenotypic consequences of over 1,000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis.

Andersen-Tawil syndrome: new potassium channel mutations and possible phenotypic variation.

Objective: To evaluate clinical, genetic, and electrophysiologic features of patients with Andersen-Tawil syndrome (ATS) in the United Kingdom. Methods: Clinical and neurophysiologic evaluation was conducted of 11 families suspected to have ATS. Molecular genetic analysis of each proband was performed by direct DNA sequencing of the entire coding region of KCNJ2. Control samples were screened by direct DNA sequencing. The electrophysiologic consequences of several new mutations were studied in an oocyte expression system. Results: All 11 ATS families harbored pathogenic mutations in KCNJ2 with six mutations not previously reported. Some unusual clinical features including renal tubular defect, CNS involvement, and dental and phonation abnormalities were observed. Five mutations (T75M, D78G, R82Q, L217P, and G300D) were expressed, all of which resulted in nonfunctional channels when expressed alone, and co-expression with wild-type (WT) KCNJ2 demonstrated a dominant negative effect. Conclusion: Six new disease-causing mutations in KCNJ2 were identified, one of which was in a PIP2 binding site. Molecular expression studies indicated that five of the mutations exerted a dominant negative effect on the wild-type allele. KCNJ2 mutations are an important cause of ATS in the UK.

Functional profiling of a plasmodium genome reveals an abundance of essential genes

Summary The genomes of malaria parasites contain many genes of unknown function. To assist drug development through the identification of essential genes and pathways, we have measured competitive growth rates in mice of 2,578 barcoded Plasmodium berghei knockout mutants, representing >50% of the genome, and created a phenotype database. At a single stage of its complex life cycle, P. berghei requires two-thirds of genes for optimal growth, the highest proportion reported from any organism and a probable consequence of functional optimization necessitated by genomic reductions during the evolution of parasitism. In contrast, extreme functional redundancy has evolved among expanded gene families operating at the parasite-host interface. The level of genetic redundancy in a single-celled organism may thus reflect the degree of environmental variation it experiences. In the case of Plasmodium parasites, this helps rationalize both the relative successes of drugs and the greater difficulty of making an effective vaccine.

A Genome-Scale Vector Resource Enables High-Throughput Reverse Genetic Screening in a Malaria Parasite

Summary The genome-wide identification of gene functions in malaria parasites is hampered by a lack of reverse genetic screening methods. We present a large-scale resource of barcoded vectors with long homology arms for effective modification of the Plasmodium berghei genome. Cotransfecting dozens of vectors into the haploid blood stages creates complex pools of barcoded mutants, whose competitive fitness can be measured during infection of a single mouse using barcode sequencing (barseq). To validate the utility of this resource, we rescreen the P. berghei kinome, using published kinome screens for comparison. We find that several protein kinases function redundantly in asexual blood stages and confirm the targetability of kinases cdpk1, gsk3, tkl3, and PBANKA_082960 by genotyping cloned mutants. Thus, parallel phenotyping of barcoded mutants unlocks the power of reverse genetic screening for a malaria parasite and will enable the systematic identification of genes essential for in vivo parasite growth and transmission.

PlasmoGEM, a database supporting a community resource for large-scale experimental genetics in malaria parasites

The Plasmodium Genetic Modification (PlasmoGEM) database (http://plasmogem.sanger.ac.uk) provides access to a resource of modular, versatile and adaptable vectors for genome modification of Plasmodium spp. parasites. PlasmoGEM currently consists of >2000 plasmids designed to modify the genome of Plasmodium berghei, a malaria parasite of rodents, which can be requested by non-profit research organisations free of charge. PlasmoGEM vectors are designed with long homology arms for efficient genome integration and carry gene specific barcodes to identify individual mutants. They can be used for a wide array of applications, including protein localisation, gene interaction studies and high-throughput genetic screens. The vector production pipeline is supported by a custom software suite that automates both the vector design process and quality control by full-length sequencing of the finished vectors. The PlasmoGEM web interface allows users to search a database of finished knock-out and gene tagging vectors, view details of their designs, download vector sequence in different formats and view available quality control data as well as suggested genotyping strategies. We also make gDNA library clones and intermediate vectors available for researchers to produce vectors for themselves.

Complete avian malaria parasite genomes reveal features associated with lineage-specific evolution in birds and mammals.

Avian malaria parasites are prevalent around the world and infect a wide diversity of bird species. Here, we report the sequencing and analysis of high-quality draft genome sequences for two avian malaria species, Plasmodium relictum and Plasmodium gallinaceum We identify 50 genes that are specific to avian malaria, located in an otherwise conserved core of the genome that shares gene synteny with all other sequenced malaria genomes. Phylogenetic analysis suggests that the avian malaria species form an outgroup to the mammalian Plasmodium species, and using amino acid divergence between species, we estimate the avian- and mammalian-infective lineages diverged in the order of 10 million years ago. Consistent with their phylogenetic position, we identify orthologs of genes that had previously appeared to be restricted to the clades of parasites containing Plasmodium falciparum and Plasmodium vivax, the species with the greatest impact on human health. From these orthologs, we explore differential diversifying selection across the genus and show that the avian lineage is remarkable in the extent to which invasion-related genes are evolving. The subtelomeres of the P. relictum and P. gallinaceum genomes contain several novel gene families, including an expanded surf multigene family. We also identify an expansion of reticulocyte binding protein homologs in P. relictum, and within these proteins, we detect distinct regions that are specific to nonhuman primate, humans, rodent, and avian hosts. For the first time in the Plasmodium lineage, we find evidence of transposable elements, including several hundred fragments of LTR-retrotransposons in both species and an apparently complete LTR-retrotransposon in the genome of P. gallinaceum.

Complete avian malaria parasite genomes reveal host-specific parasite evolution in birds and mammals

Avian malaria parasites are prevalent around the world, and infect a wide diversity of bird species. Here we report the sequencing and analysis of high quality draft genome sequences for two avian malaria species, Plasmodium relictum and Plasmodium gallinaceum. We identify 50 genes that are specific to avian malaria, located in an otherwise conserved core of the genome that shares gene synteny with all other sequenced malaria genomes. Phylogenetic analysis suggests that the avian malaria species form an outgroup to the mammalian Plasmodium species. Consistent with their phylogenetic position, we identify orthologs of genes that had previously appeared to be restricted to the clades of parasites containing P. falciparum and P. vivax - the species with the greatest impact on human health. The subtelomeres of P. relictum and P. gallinaceum contain several novel gene families, including an expanded surf multigene family. We also identify an expansion of reticulocyte binding protein homologs in P. relictum and within these proteins proteins, we detect distinct regions that are specific to non-human primate, humans, rodent and avian hosts. For the first time in the Plasmodium lineage we find evidence of transposable elements, including several hundred fragments of LTR-retrotransposons in both species and an apparently complete LTR-retrotransposon in the genome of P. gallinaceum.

Erratum: The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma (Nature (2012) 486 (266-270) DOI: 10.1038/nature11114)

This corrects the article DOI: 10.1038/nature11114

Erratum: Corrigendum: The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma

This corrects the article DOI: 10.1038/nature11114

Corrigendum: The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma

This corrects the article DOI: 10.1038/nature11114