Background: The Amoebozoa constitute one of the primary divisions of eukaryotes, encompassing taxa of both biomedical and evolutionary importance, yet its genomic diversity remains largely unsampled. Here we present an analysis of a whole genome assembly of Acanthamoeba castellanii (Ac) the first representative from a solitary free-living amoebozoan. Results: Ac encodes 15,455 compact intron-rich genes, a significant number of which are predicted to have arisen through inter-kingdom lateral gene transfer (LGT). A majority of the LGT candidates have undergone a substantial degree of intronization and Ac appears to have incorporated them into established transcriptional programs. Ac manifests a complex signaling and cell communication repertoire, including a complete tyrosine kinase signaling toolkit and a comparable diversity of predicted extracellular receptors to that found in the facultatively multicellular dictyostelids. An important environmental host of a diverse range of bacteria and viruses, Ac utilizes a diverse repertoire of predicted pattern recognition receptors, many with predicted orthologous functions in the innate immune systems of higher organisms. Conclusions: Our analysis highlights the important role of LGT in the biology of Ac and in the diversification of microbial eukaryotes. The early evolution of a key signaling facility implicated in the evolution of metazoan multicellularity strongly argues for its emergence early in the Unikont lineage. Overall, the availability of an Ac genome should aid in deciphering the biology of the Amoebozoa and facilitate functional genomic studies in this important model organism and environmental host.

Background: Five regulatory factor X (RFX) transcription factors (TFs)–RFX1-5–have been previously characterized in the human genome, which have been demonstrated to be critical for development and are associated with an expanding list of serious human disease conditions including major histocompatibility (MHC) class II deficiency and ciliaophathies. Results: In this study, we have identified two additional RFX genes–RFX6 and RFX7–in the current human genome sequences. Both RFX6 and RFX7 are demonstrated to be winged-helix TFs and have well conserved RFX DNA binding domains (DBDs), which are also found in winged-helix TFs RFX1-5. Phylogenetic analysis suggests that the RFX family in the human genome has undergone at least three gene duplications in evolution and the seven human RFX genes can be clearly categorized into three subgroups: (1) RFX1-3, (2) RFX4 and RFX6, and (3) RFX5 and RFX7. Our functional genomics analysis suggests that RFX6 and RFX7 have distinct expression profiles. RFX6 is expressed almost exclusively in the pancreatic islets, while RFX7 has high ubiquitous expression in nearly all tissues examined, particularly in various brain tissues. Conclusion: The identification and further characterization of these two novel RFX genes hold promise for gaining critical insight into development and many disease conditions in mammals, potentially leading to identification of disease genes and biomarkers.

Background: The recent availability of genome sequences of multiple related Caenorhabditis species has made it possible to identify, using comparative genomics, similarly transcribed genes in Caenorhabditis elegans and its sister species. Taking this approach, we have identified numerous novel ciliary genes in C. elegans, some of which may be orthologs of unidentified human ciliopathy genes. Results: By screening for genes possessing canonical X-box sequences in promoters of three Caenorhabditis species, namely C. elegans, C. briggsae and C. remanei, we identified 93 genes (including known X-box regulated genes) that encode putative components of ciliated neurons in C. elegans and are subject to the same regulatory control. For many of these genes, restricted anatomical expression in ciliated cells was confirmed, and control of transcription by the ciliogenic DAF-19 RFX transcription factor was demonstrated by comparative transcriptional profiling of different tissue types and of daf-19(+) and daf-19(-) animals. Finally, we demonstrate that the dye-filling defect of dyf-5(mn400) animals, which is indicative of compromised exposure of cilia to the environment, is caused by a nonsense mutation in the serine/threonine protein kinase gene M04C9.5. Conclusion: Our comparative genomics-based predictions may be useful for identifying genes involved in human ciliopathies, including Bardet-Biedl Syndrome (BBS), since the C. elegans orthologs of known human BBS genes contain X-box motifs and are required for normal dye filling in C. elegans ciliated neurons.

Background: Increasing genetic and phenotypic differences found among natural isolates ofC. eleganshave encouraged researchers to explore the natural variation of this nematode species.Results: Here we report on the identification of genomic differences between the reference strain N2 and the Hawaiian strain CB4856, one of the most genetically distant strains from N2. To identify both small- and large-scale genomic variations (GVs), we have sequenced the CB4856 genome using both Roche 454 (~400 bps single reads) and Illumina GA DNA sequencing methods (101 bps paired-end reads). Compared to previously described variants (available in WormBase), our effort uncovered twice as many single nucleotide variants (SNVs) and increased the number of small InDels almost 20-fold. Moreover, we identified and validated large insertions, most of which range from 150 bps to 1.2 kb in length in the CB4856 strain. Identified GVs had a widespread impact on protein-coding sequences, including 585 single-copy genes that have associated severe phenotypes of reduced viability in RNAi and genetics studies. Sixty of these genes are homologs of human genes associated with diseases. Furthermore, our work confirms previously identified GVs associated with differences in behavioural and biological traits between the N2 and CB4856 strains.Conclusions: The identified GVs provide a rich resource for future studies that aim to explain the genetic basis for other trait differences between the N2 and CB4856 strains.

Essential genes are critical for the development of all organisms and are associated with many human diseases. These genes have been a difficult category to study prior to the availability of balanced lethal strains. Despite the power of targeted mutagenesis, there are limitations in identifying mutations in essential genes. In this paper, we describe the identification of coding regions for essential genes mutated using forward genetic screens in Caenorhabditis elegans. The lethal mutations described here were isolated and maintained by a wild-type allele on a rescuing duplication. We applied whole genome sequencing to identify the causative molecular lesion resulting in lethality in existing C. elegans mutant strains. These strains are balanced and can be easily maintained for subsequent characterization. Our method can be effectively used to analyze mutations in a large number of essential genes. We describe here the identification of 64 essential genes in a region of chromosome I covered by the duplication sDp2. Of these, 42 are nonsense mutations, six are splice signal mutations, one deletion, and 15 are non-synonymous mutations. Many of the essential genes in this region function in cell cycle, transcriptional regulation, and RNA processing. The essential genes identified here are represented by mutant strains, many of which have more than one mutant allele. The genetic resource can be utilized to further our understanding of essential gene function and will be applicable to the study of C. elegans development, conserved cellular function, and ultimately lead to improved human health.

Essential genes are required for an organism’s viability and their functions can vary greatly, spreading across many pathways. Due to the importance of essential genes, large scale efforts have been undertaken to identify the complete set of essential genes and to understand their function. Studies of genome architecture and organization have found that genes are not randomly disturbed in the genome. Using combined genetic mapping, Illumina sequencing, and bioinformatics analyses, we successfully identified 44 essential genes with 130 lethal mutations in genomic regions of C. elegans of around 7.3 Mb from Chromosome I (left). Of the 44 essential genes, six of which were genes not characterized previously by mutant alleles, let-633/let-638 (B0261.1), let-128 (C53H9.2), let-511 (W09C3.4), let-162 (Y47G6A.18), let-510 (Y47G6A.19), and let-131 (Y71G12B.6). Examine essential genes with Hi-C data shows that essential genes tend to cluster within TAD units rather near TAD boundaries. We have also shown that essential genes in the left half of chromosome I in C. elegans function in enzyme and nucleic acid binding activities during fundamental processes, such as DNA replication, transcription, and translation. From protein-protein interaction networks, essential genes exhibit more protein connectivity than non-essential genes in the genome. Also, many of the essential genes show strong expression in embryos or early larvae stages, indicating that they are important to early development. Our results confirmed that this work provided a more comprehensive picture of the essential gene and their functional characterization. These genetic resources will offer important tools for further heath and disease research.