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Mutants remain a powerful means for dissecting gene function in model organisms such as Caenorhabditis elegans. Massively parallel sequencing has simplified the detection of variants after mutagenesis but determining precisely which change is responsible for phenotypic perturbation remains a key step. Genetic mapping paradigms in C. elegans rely on bulk segregant populations produced by crosses with the problematic Hawaiian wild isolate and an excess of redundant information from whole-genome sequencing (WGS). To increase the repertoire of available mutants and to simplify identification of the causal change, we performed WGS on 173 temperature-sensitive (TS) lethal mutants and devised a novel mapping method. The mapping method uses molecular inversion probes (MIP-MAP) in a targeted sequencing approach to genetic mapping, and replaces the Hawaiian strain with a Million Mutation Project strain with high genomic and phenotypic similarity to the laboratory wild-type strain N2. We validated MIP-MAP on a subset of the TS mutants using a competitive selection approach to produce TS candidate mapping intervals with a mean size < 3 Mb. MIP-MAP successfully uses a non-Hawaiian mapping strain and multiplexed libraries are sequenced at a fraction of the cost of WGS mapping approaches. Our mapping results suggest that the collection of TS mutants contains a diverse library of TS alleles for genes essential to development and reproduction. MIP-MAP is a robust method to genetically map mutations in both viable and essential genes and should be adaptable to other organisms. It may also simplify tracking of individual genotypes within population mixtures.

Temperature sensitive (TS) alleles are important tools for the genetic and functional analysis of essential genes in many model organisms. While isolating TS alleles is not difficult, determining the TS-conferring mutation can be problematic. Even with whole-genome sequencing (WGS) data there is a paucity of predictive methods for identifying TS alleles from DNA sequence alone. We assembled 173 TS lethal mutants ofCaenorhabditis elegansand used WGS to identify several hundred mutations per strain. We leveraged single molecule molecular inversion probes (MIPs) to sequence variant sites at high depth in the cross-progeny of TS mutants and a mapping strain with identified sequence variants but no apparent phenotypic differences from the reference N2 strain. By sampling for variants at ~1Mb intervals across the genome we genetically mapped mutant alleles at a resolution comparable to current standards in a process we call MIP-MAP. The MIP-MAP protocol, however, permits high-throughput sequencing of multiple TS mutation mapping libraries at less than 200K reads per library. Using MIP-MAP on a subset of TS mutants, via a competitive selection assay and standard recombinant mutant selection, we defined TS-associated intervals of 3Mb or less. Our results suggest this collection of strains contains a diverse library of TS alleles for genes involved in development and reproduction. MIP-MAP is a robust method to genetically map mutations in both viable and essential genes. The MIPs protocol should allow high-throughput tracking of genetic variants in any mixed population.

The adult Caenorhabditis elegans hermaphrodite gonad consists of two mirror-symmetric U-shaped arms, with germline nuclei located peripherally in the distal regions of each arm. The nuclei are housed within membrane cubicles that are open to the center, forming a syncytium with a shared cytoplasmic core called the rachis. As the distal germline nuclei progress through meiotic prophase, they move proximally and eventually cellularize as their compartments grow in size. The development and maintenance of this complex and dynamic germline membrane architecture are relatively unexplored, and we have used a forward genetic screen to identify 20 temperature-sensitive mutations in 19 essential genes that cause defects in the germline membrane architecture. Using a combined genome-wide SNP mapping and whole genome sequencing strategy, we have identified the causal mutations in 10 of these mutants. Four of the genes we have identified are conserved, with orthologs known to be involved in membrane biology, and are required for proper development or maintenance of the adult germline membrane architecture. This work provides a starting point for further investigation of the mechanisms that control the dynamics of syncytial membrane architecture during adult oogenesis.

The Caenorhabditis elegans genes dyf-6, daf-10, and osm-1 are among the set of genes that affect chemotaxis and the ability of certain sensory neurons to take up fluorescent dyes from the environment. Some genes in this category are known to be required for intraflagellar transport (IFT), which is the bidirectional movement of raft-like particles along the axonemes of cilia and flagella. The cloning of dyf-6, daf-10, and osm-1 are described here. The daf-10 and osm-1 gene products resemble each other and contain WD and WAA repeats. DYF-6, the product of a complex locus, lacks known motifs, but orthologs are present in flies and mammals. Phenotypic analysis of dyf-6 mutants expressing an OSM-6∷GFP reporter indicates that the cilia of the amphid and phasmid dendritic endings are foreshortened. Consistent with genetic mosaic analysis, which indicates that dyf-6 functions in neurons of the amphid sensilla, DYF-6∷GFP is expressed in amphid and phasmid neurons. Movement of DYF-6∷GFP within the ciliated endings of the neurons indicates that DYF-6 is involved in IFT. In addition, IFT can be observed in dauer larvae.

Homozygosity for a mutation in the idi-1 gene of Caenorhabditis elegans results in paralysis during the first larval stage, followed by an arrest of growth and development late in the first larval stage. Apoptotic corpses, which are apparently the result of normal programmed cell death, persist in the arrested larvae. In genetic mosaics, an additional defect becomes evident upon examination with Nomarski optics: cells that are genotypically mutant enlarge, and their cytoplasm becomes dimpled. Electron microscopy indicates that the dimpling reflects an accumulation of many enlarged lysosomes and autophagosomes. The mosaics demonstrate that the lethal mutation acts cell autonomously with respect to this vesicular abnormality and that there is a maternal effect with respect to the time of developmental arrest of mutant progeny. Cloning of the gene reveals that it is the only gene in C. elegans for isopentenyl-diphosphate isomerase, an enzyme that is important for the synthesis of lipophilic molecules, including farnesyl and geranyl diphosphates.

On the basis of synthetic lethality, five genes in Caenorhabditis elegans are known to be redundant with the mec-8 gene, which encodes a protein that contains two copies of an RNA recognition motif (RRM) and affects alternative RNA splicing. The molecular identities of two of the redundant genes, sym-1 and sym-5, were previously reported. The remaining three genes have now been cloned, and their synthetically lethal phenotypes with mec-8 are described in more detail. Animals homozygous for mec-8 and sym-2 loss-of-function mutations die during late embryogenesis. The SYM-2 predicted protein contains three RRMs; we propose that SYM-2 and MEC-8 can substitute for each other in promoting the maturation of the transcripts of a vital gene. Animals homozygous for mutations in mec-8 and in either sym-3 or sym-4 have the same striking defect: they arrest development just prior to or just after hatching with a pharynx that appears fully formed but is not properly attached to the body cuticle. sym-3 encodes a protein of unknown function with orthologs in Drosophila and mammals. sym-4 encodes a WD-repeat protein and may also have orthologs in Drosophila and mammals. We propose that SYM-3 and SYM-4 contribute to a common developmental pathway that is redundant with a MEC-8-dependent pathway.

For a nonessential diminutive organ comprised of only 22 nuclei, the Caenorhabditis elegans vulva has done very well for itself. The status of the vulva as an overachiever is in part due to its inherent structural simplicity as well as to the intricate regulation of its induction and development. Studies over the past twenty years have shown the vulva to be a microcosm for organogenesis and a model for the integration of complex signaling pathways. Furthermore, many of these signaling molecules are themselves associated with cancer in mammals. This review focuses on what is perhaps the most intriguing and complex story to emerge from these studies thus far, the role of the Synthetic Multivulval (SynMuv) genes in controlling vulval cell-fate adoption. Recent advances have led to a greater mechanistic understanding of how these genes function during vulval development and have also identified roles for these genes in diverse developmental processes.

The analysis of genetically mosaic worms, in which some cells carry a wild-type gene and others are homozygous mutant, can reveal where in the animal a gene acts to prevent the appearance of a mutant phenotype. In this primer article, we describe how Caenorhabditis elegans genetic mosaics are generated, identified and analyzed, and we discuss examples in which the analysis of mosaic worms has provided important information about the development of this organism.

Experiments with mammalian tissue culture cells have implicated the small GTPase Ras in the control of cellular proliferation. Evidence is presented here that this is not the case for a living animal, the nematode Caenorhabditis elegans: proliferation late in embryogenesis and throughout the four larval stages is not noticeably affected in animals lacking Ras in various parts of their cell lineages. Instead, genetic mosaic analysis of the let-60 gene suggests that Ras is required only, at least later in development (a maternal effect cannot be excluded), for establishment of a few temporally and spatially distinct cell fates. Only one of these, the duct cell fate, appears to be essential for viability.

A >23-kb gene that encodes a large integral membrane protein with a predicted structure similar to that of the low density lipoprotein (LDL) receptor-related protein (LRP) of mammals has been isolated and sequenced from the free-living nematode Caenorhabditis elegans. The 4753-amino acid predicted C. elegans product shares a nearly identical number and arrangement of amino acid sequence motifs with human LRP, and several exons of the C. elegans LRP gene correspond to exons of related parts of the human LDL receptor gene. The existence of an apparent homolog of LRP in C. elegans offers the possibility of genetic analysis of the in vivo roles of LRP and of the relationship between protein structure and function in a simple model organism.

The E. coli dnaK (groPC756) gene product is essential for bacteriophage lambda DNA replication. Bacterial DNA segments carrying this gene have been cloned onto a bacteriophage lambda vector. The product of the dnaK gene has been identified on SDS polyacrylamide gels after infection of UV-irradiated E. coli cells. The dnaK gene codes for a polypeptide with an apparent molecular weight of 93,000-Mr. Transducing phages carrying amber mutations in the dnaK gene fail to induce the synthesis of the 93,000-Mr polypeptide chain upon infection of sup+ bacteria, but do so upon infection of supF bacteria. E coli carrying the dnaK756 mutation are, in addition, temperature sensitive for growth at 43 degrees C. It is shown that the dnaK756 mutation results in an overproduction of the dnaK gene product at that temperature.

The isolation of a bacterial mutation in a gene, designated groPC, which affects the growth of phages lambda and P2 is described. Lambda replication is severely limited in the strain, and some lambda pi mutations, which map in (or near) the P gene, allow growth. The gro mutation, groPC259, is recessive to wild type and maps between threonine (thr) and diaminopimelate (dapB) on the E. coli chromosome. The possibility that the groPC gene is concerned with host DNA replication is discussed.

The lin-12 gene seems to control certain binary decisions during Caenorhabditis elegans development, from genetic and anatomical studies of lin-12 mutants that have either elevated or reduced levels of lin-12 activity1. We report here the complete DNA sequence of lin-12:13.5 kilobases (kb) derived from genomic clones and 4.5 kb from complementary DNA clones. It is of interest that the predicted product is a putative transmembrane protein, given that many of the decisions controlled by lin-12 activity require cell-cell interactions for the correct choice of cell fate2–5. In addition, the predicted lin-12 product may be classified into several regions, based on amino acid sequence similarities to other proteins. These include extensive overall sequence similarity to the Drosophila Notch protein6,7, which also is involved in cell-cell interactions that specify cell fate6–8; a repeated motif found9 in proteins encoded by the yeast cell-cycle control genes cdc10 (Schizosaccharomyces pombe)10 and SWI6 ( Saccharomyces cerevisiae)9; and a repeated motif exemplified by epidermal growth factor, found in many mammalian proteins11.

The FI gene of bacteriophage λ functions in head assembly, but its exact role is not well understood. FI mutants are leaky, producing between 0.1 and 0.5 viable particles per infected cell. In order to investigate the function of the FI product (gpFI) in vivo , mutants of λ were isolated that are able to grow in the absence of gpFI. These mutants, called fin (for FI independence) map in the region of gene Nul and the beginning of gene A . Proteins made in cells infected with the fin mutants were labelled with [ 35 S]methionine and analysed by polyacrylamide gel electrophoresis. In addition, the levels of activity of the A product were measured in the in vitro DNA packaging assay. As a result of these experiments, the fin mutants can be classified in two groups. Upon infection, fin mutants of one group selectively produce three to fivefold more gpA than do wild-type phage fin mutants of the second group do not overproduce any λ late gene product detectable by the autoradiographic technique. gpA overproducers can also be isolated by selecting for λ Aam Wam phages that can plate on a weak suII cell strain. The mutation responsible for this pseudoreversion is called A op and maps in the Nu1-A region. A op is also a fin mutation, since its presence in λ FI − enables it to plate on non-permissive hosts. Therefore, it seems that one condition sufficient for normal growth of FI − phage is the overproduction of gpA. The nature of the fin mutations that do not result in gpA overproduction is discussed.

We show that a collection of 93 E. coli mutations which map between thr and leu and which block phage lambda DNA replication define two closely linked cistrons. Work published in the accompanying paper shows that these mutations also affect host DNA replication, so we designate them dnaJ and dnaK; the gene order is thr--dnaK--dnaJ--leu. Demonstration of two cistrons was possible with the isolation of lambda transducing phages carrying one or the other or both of the dna genes. These phages were employed in phage vs bacterial complementation studies which unambiguously show that dnaK and dnaJ are different cistrons.

The function of the cell division cycle gene, CDC4, is required in Saccharomyces cerevisiae for progression beyond the G1 phase of the cell cycle. The wild-type gene was isolated from a plasmid library by selection for complementation of a recessive, temperature-sensitive allele. Hybridization of genomic sequences with the cloned gene revealed the presence of a duplicated sequence. Both CDC4 and the duplicated sequence were subjected to DNA sequence analysis. These analyses revealed (1) that CDC4 contains a large open reading frame encoding a protein of 779 amino acids, and (2) that the duplicated sequence bears strong homology with the carboxy-terminal segment of this open reading frame. Presence of a nonsense codon within the duplicated sequence suggested that it does not encode a functional product. Disruption of the duplicated sequence within the yeast genome provided a more critical test for function. The absence of any detectable phenotype for this disruption confirms that the sequence should be considered a pseudogene. The marker inserted to disrupt the sequence also served to map the duplication and to establish that it is not genetically linked to CDC4. The structural features determined suggest evolutionary relationships between these genes as well as between the CDC4 product and other proteins.

Analysis of Escherichia coli mutants unable to propagate bacteriophage λ has revealed the existence of the dnaj and dnaK bacterial genes. These two genes have been shown to be closely linked and to map at 0.3 on the E. coli genetic map, between the thr and leu loci. DnaJ and dnaK bacterial mutants were found to block λ propagation at the level of λ DNA replication by specifically interfering with the function of the A gene P product. In addition, these bacterial mutants were shown to be temperature sensitive for bacterial growth at high temperature. The dnaj and dnaK genes have been cloned onto bacteriophage λ vectors and amber mutations in these genes have been isolated. The dnaJ and dnaK gene products have been identified as polypeptides with apparent molecular weights of 37,000 and 93,000 respectively. It has been shown that the dnaK756 mutation results in the overproduction of the dnaK756 product at high temperature.

We report here significant primary sequence homology among the predicted translational products of three genes: CDC4 , CDC36 and ets. CDC4 and CDC36 are Saccharomyces cerevisiae cell division cycle genes, while ets is a transformation-specific sequence of avian erythroblastosis virus E26. The deduced primary structures of the three gene products were compared by computer to a large data base of known and predicted protein sequences. The search revealed 22.0-25.5% identity over regions of 140-206 codons, respectively between the different pairwise combinations. For these particular sequences, these identity scores fall 3.4-4.0 standard deviations above the empirically-determined mean values of fortuitous similarity. S. cerevisiae calls require CDC36 and CDC4 in order to complete two early events in the cell cycle: execution of start ( CDC36 ) and spindle pole body separation ( CDC4 ). In virus E26, the ets sequence is linked in frame with delta gag and mybE in the tripartite structure 5'-delta gag- mybE -ets-3', comprising the E26 transforming oncogene. The homologies described here suggest that the biochemical functions or regulation of the CDC4 , CDC36 and ets products may be related.