High-throughput capturing and characterization of mutations in essential genes of Caenorhabditis elegans

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

Functional categorization of essential genes identified in this study using GO terms. The Y-axis indicates the GO term categories. The X-axis represents the number of genes in each category. Random sampling of 1000 iterations was done by selecting equal number of genes from either all sDp2 genes or the set of all essential genes identified by RNAi. Error bars represent standard error.

Figure 2

This figure represents the normalized transcript level (read number per coding length per million reads) for each gene across the developmental stages including 23 embryo stages separated by 30 minute interval, four larval stages (L1-L4), pre-gravid young adult, and gravid young adult. For comparing germline expression, we’ve included the transcript level from JK1107 carrying a mutation in glp-1, which is essential for mitotic germ cell proliferation [71]. The heatmap represents normalized transcript level from high (yellow) to low (blue). Seven distinct clusters that are based on their expression pattern are shown by colored branches. Purple: early-embryonic; Brown: early- and mid-embryonic; Red: mid-embryonic; Blue: late-embryonic; Green: mid- and late-embryonic; Orange: gastrulation; Black: larval.