Mos1-Mediated Transgenesis to Probe Consequences of Single Gene Mutations in Variation-Rich Isolates of Caenorhabditis elegans

Caenorhabditis elegans, especially the N2 isolate, is an invaluable biological model system. Numerous additional natural C. elegans isolates have been shown to have unexpected genotypic and phenotypic variations which has encouraged researchers to use next generation sequencing methodology to develop a more complete picture of genotypic variations among the isolates. To understand the phenotypic effects of a genomic variation (GV) on a single gene, in a variation-rich genetic background, one should analyze that particular GV in a well understood genetic background. In C. elegans, the analysis is usually done in N2, which requires extensive crossing to bring in the GV. This can be a very time consuming procedure thus it is important to establish a fast and efficient approach to test the effect of GVs from different isolates in N2. Here we use a Mos1-mediated single-copy insertion (MosSCI) method for phenotypic assessments of GVs from the variation-rich Hawaiian strain CB4856 in N2. Specifically, we investigate effects of variations identified in the CB4856 strain on tac-1 which is an essential gene that is necessary for mitotic spindle elongation and pronuclear migration. We show the usefulness of the MosSCI method by using EU1004 tac-1(or402) as a control. or402 is a temperature sensitive lethal allele within a well-conserved TACC domain (transforming acidic coiled-coil) that results in a leucine to phenylalanine change at amino acid 229. CB4856 contains a variation that affects the second exon of tac-1 causing a cysteine to tryptophan change at amino acid 94 also within the TACC domain. Using the MosSCI method, we analyze tac-1 from CB4856 in the N2 background and demonstrate that the C94W change, albeit significant, does not cause any obvious decrease in viability. This MosSCI method has proven to be a rapid and efficient way to analyze GVs

Spindle assembly checkpoint genes reveal distinct as well as overlapping expression that implicates MDF-2/Mad2 in postembryonic seam cell proliferation in Caenorhabditis elegans

Background: The spindle assembly checkpoint (SAC) delays anaphase onset by inhibiting the activity of theanaphase promoting complex/cyclosome (APC/C) until all of the kinetochores have properly attached to thespindle. The importance of SAC genes for genome stability is well established; however, the roles these genes play,during postembryonic development of a multicellular organism, remain largely unexplored.Results: We have used GFP fusions of 5’ upstream intergenic regulatory sequences to assay spatiotemporalexpression patterns of eight conserved genes implicated in the spindle assembly checkpoint function inCaenorhabditis elegans. We have shown that regulatory sequences for all of the SAC genes drive ubiquitous GFPexpression during early embryonic development. However, postembryonic spatial analysis revealed distinct, tissuespecificexpression of SAC genes with striking co-expression in seam cells, as well as in the gut. Additionally, weshow that the absence of MDF-2/Mad2 (one of the checkpoint genes) leads to aberrant number and alignment ofseam cell nuclei, defects mainly attributed to abnormal postembryonic cell proliferation. Furthermore, we showthat these defects are completely rescued by fzy-1(h1983)/CDC20, suggesting that regulation of the APC/CCDC20 bythe SAC component MDF-2 is important for proper postembryonic cell proliferation.Conclusion: Our results indicate that SAC genes display different tissue-specific expression patterns duringpostembryonic development in C. elegans with significant co-expression in hypodermal seam cells and gut cells,suggesting that these genes have distinct as well as overlapping roles in postembryonic development that may ormay not be related to their established roles in mitosis. Furthermore, we provide evidence, by monitoring seamcell lineage, that one of the checkpoint genes is required for proper postembryonic cell proliferation. Importantly,our research provides the first evidence that postembryonic cell division is more sensitive to SAC loss, in particularMDF-2 loss, than embryonic cell division

Genome-wide variations in a natural isolate of the nematode Caenorhabditis elegans

Background Increasing genetic and phenotypic differences found among natural isolates of C. elegans have 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

Polymorphic segmental duplication in the nematode Caenorhabditis elegans

<p>Abstract</p> <p>Background</p> <p>The nematode <it>Caenorhabditis elegans </it>was the first multicellular organism to have its genome fully sequenced. Over the last 10 years since the original publication in 1998, the <it>C. elegans </it>genome has been scrutinized and the last gaps were filled in November 2002, which present a unique opportunity for examining genome-wide segmental duplications.</p> <p>Results</p> <p>Here, we performed analysis of the <it>C. elegans </it>genome in search for segmental duplications using a new tool–OrthoCluster–we have recently developed. We detected 3,484 duplicated segments–duplicons–ranging in size from 234 bp to 108 Kb. The largest pair of duplicons, 108 kb in length located on the left arm of <it>Chromosome V</it>, was further characterized. They are nearly identical at the DNA level (99.7% identity) and each duplicon contains 26 putative protein coding genes. Genotyping of 76 wild-type strains obtained from different labs in the <it>C. elegans </it>community revealed that not all strains contain this duplication. In fact, only 29 strains carry this large segmental duplication, suggesting a very recent duplication event in the <it>C. elegans </it>genome.</p> <p>Conclusion</p> <p>This report represents the first demonstration that the <it>C. elegans </it>laboratory wild-type N2 strains has acquired large-scale differences.</p

mirTools: microRNA profiling and discovery based on high-throughput sequencing

miRNAs are small, non-coding RNA that negatively regulate gene expression at post-transcriptional level, which play crucial roles in various physiological and pathological processes, such as development and tumorigenesis. Although deep sequencing technologies have been applied to investigate various small RNA transcriptomes, their computational methods are far away from maturation as compared to microarray-based approaches. In this study, a comprehensive web server mirTools was developed to allow researchers to comprehensively characterize small RNA transcriptome. With the aid of mirTools, users can: (i) filter low-quality reads and 3/5′ adapters from raw sequenced data; (ii) align large-scale short reads to the reference genome and explore their length distribution; (iii) classify small RNA candidates into known categories, such as known miRNAs, non-coding RNA, genomic repeats and coding sequences; (iv) provide detailed annotation information for known miRNAs, such as miRNA/miRNA*, absolute/relative reads count and the most abundant tag; (v) predict novel miRNAs that have not been characterized before; and (vi) identify differentially expressed miRNAs between samples based on two different counting strategies: total read tag counts and the most abundant tag counts. We believe that the integration of multiple computational approaches in mirTools will greatly facilitate current microRNA researches in multiple ways. mirTools can be accessed at http://centre.bioinformatics.zj.cn/mirtools/ and http://59.79.168.90/mirtools

Clinical and molecular characterization of cystinuria in a French cohort: relevance of assessing large-scale rearrangements and splicing variants.

Cystinuria is an autosomal recessive disorder of dibasic amino acid transport in the kidney and the intestine leading to increased urinary cystine excretion and nephrolithiasis. Two genes, SLC3A1 and SLC7A9, coding respectively for rBAT and b0,+AT, account for the genetic basis of cystinuria. This study reports the clinical and molecular characterization of a French cohort including 112 cystinuria patients and 25 relatives from 99 families. Molecular screening was performed using sequencing and Quantitative Multiplex PCR of Short Fluorescent Fragments analyses. Functional minigene-based assays have been used to characterize splicing variants. Eighty-eight pathogenic nucleotide changes were identified in SLC3A1 (63) and SLC7A9 (25) genes, of which 42 were novel. Interestingly, 17% (15/88) and 11% (10/88) of the total number of variants correspond, respectively, to large-scale rearrangements and splicing mutations. Functional minigene-based assays were performed for six variants located outside the most conserved sequences of the splice sites; three variants affect splice sites, while three others modify exonic splicing regulatory elements (ESR), in good agreement with a new in silico prediction based on ΔtESRseq values. This report expands the spectrum of SLC3A1 and SLC7A9 variants and supports that digenic inheritance is unlikely. Furthermore, it highlights the relevance of assessing large-scale rearrangements and splicing mutations to fully characterize cystinuria patients at the molecular level

Comparative Genomics for the Elucidation of Multidrug Resistance in Candida lusitaniae

Multidrug resistance (MDR) has emerged in hospitals due to the use of several agents administered in combination or sequentially to the same individual. We reported earlier MDR in Candida lusitaniae during therapy with amphotericin B (AmB), azoles, and candins. Here, we used comparative genomic approaches between the initial susceptible isolate and 4 other isolates with different MDR profiles. From a total of 18 nonsynonymous single nucleotide polymorphisms (NSS) in genome comparisons with the initial isolate, six could be associated with MDR. One of the single nucleotide polymorphisms (SNPs) occurred in a putative transcriptional activator (MRR1) resulting in a V668G substitution in isolates resistant to azoles and 5-fluorocytosine (5-FC). We demonstrated by genome editing that MRR1 acted by upregulation of MFS7 (a multidrug transporter) in the presence of the V668G substitution. MFS7 itself mediated not only azole resistance but also 5-FC resistance, which represents a novel resistance mechanism for this drug class. Three other distinct NSS occurred in FKS1 (a glucan synthase gene that is targeted by candins) in three candin-resistant isolates. Last, two other NSS in ERG3 and ERG4 (ergosterol biosynthesis) resulting in nonsense mutations were revealed in AmB-resistant isolates, one of which accumulated the two ERG NSS. AmB-resistant isolates lacked ergosterol and exhibited sterol profiles, consistent with ERG3 and ERG4 defects. In conclusion, this genome analysis combined with genetics and metabolomics helped decipher the resistance profiles identified in this clinical case. MDR isolates accumulated six different mutations conferring resistance to all antifungal agents used in medicine. This case study illustrates the capacity of C. lusitaniae to rapidly adapt under drug pressure within the host.IMPORTANCE Antifungal resistance is an inevitable phenomenon when fungal pathogens are exposed to antifungal drugs. These drugs can be grouped in four distinct classes (azoles, candins, polyenes, and pyrimidine analogs) and are used in different clinical settings. Failures in therapy implicate the sequential or combined use of these different drug classes, which can result in some cases in the development of multidrug resistance (MDR). MDR is particularly challenging in the clinic since it drastically reduces possible treatment alternatives. In this study, we report the rapid development of MDR in Candida lusitaniae in a patient, which became resistant to all known antifungal agents used until now in medicine. To understand how MDR developed in C. lusitaniae, whole-genome sequencing followed by comparative genome analysis was undertaken in sequential MDR isolates. This helped to detect all specific mutations linked to drug resistance and explained the different MDR patterns exhibited by the clinical isolates

Analysis of exome data for 4293 trios suggests GPI-anchor biogenesis defects are a rare cause of developmental disorders.

Over 150 different proteins attach to the plasma membrane using glycosylphosphatidylinositol (GPI) anchors. Mutations in 18 genes that encode components of GPI-anchor biogenesis result in a phenotypic spectrum that includes learning disability, epilepsy, microcephaly, congenital malformations and mild dysmorphic features. To determine the incidence of GPI-anchor defects, we analysed the exome data from 4293 parent-child trios recruited to the Deciphering Developmental Disorders (DDD) study. All probands recruited had a neurodevelopmental disorder. We searched for variants in 31 genes linked to GPI-anchor biogenesis and detected rare biallelic variants in PGAP3, PIGN, PIGT (n=2), PIGO and PIGL, providing a likely diagnosis for six families. In five families, the variants were in a compound heterozygous configuration while in a consanguineous Afghani kindred, a homozygous c.709G>C; p.(E237Q) variant in PIGT was identified within 10-12 Mb of autozygosity. Validation and segregation analysis was performed using Sanger sequencing. Across the six families, five siblings were available for testing and in all cases variants co-segregated consistent with them being causative. In four families, abnormal alkaline phosphatase results were observed in the direction expected. FACS analysis of knockout HEK293 cells that had been transfected with wild-type or mutant cDNA constructs demonstrated that the variants in PIGN, PIGT and PIGO all led to reduced activity. Splicing assays, performed using leucocyte RNA, showed that a c.336-2A>G variant in PIGL resulted in exon skipping and p.D113fs*2. Our results strengthen recently reported disease associations, suggest that defective GPI-anchor biogenesis may explain ~0.15% of individuals with developmental disorders and highlight the benefits of data sharing

Draft genome of an iconic Red Sea reef fish, the blacktail butterflyfish (Chaetodon austriacus): Current status and its characteristics

Butterflyfish are among the most iconic of the coral reef fishes and represent a model system to study general questions of biogeography, evolution and population genetics. We assembled and annotated the genome sequence of the blacktail butterflyfish (Chaetodon austriacus), an Arabian region endemic species that is reliant on coral reefs for food and shelter. Using available bony fish (superclass Osteichthyes) genomes as a reference, a total of 28 926 high-quality protein-coding genes were predicted from 13 967 assembled scaffolds. The quality and completeness of the draft genome of C. austriacus suggest that it has the potential to serve as a resource for studies on the co-evolution of reef fish adaptations to the unique Red Sea environment, as well as a comparison of gene sequences between closely related congeneric species of butterflyfish distributed more broadly across the tropical Indo-Pacific