Mutational accessibility of essential genes on chromosome I(left) in Caenorhabditis elegans

We have analyzed a region of approximately 5.4 million base pairs for mutations, which under standard laboratory conditions result in developmental arrest, sterility, or maternal-effect lethality in Caenorhabditis elegans. Lethal mutations were isolated, maintained, and genetically manipulated as homozygotes using sDp2– a duplication of the left half of chromosome I. All of the lethals and rearrangements used in this analysis were balanced by sDp2. Relatively low doses of mutagen, (approximately 15 mM ethylmethane sulfate; EMS), were used so as to limit the occurrence of second-site mutations, thus increasing the probability of recovering single nucleotide substitutions. Treatment of over 32,400 marked chromosomes resulted in 486 analyzed mutations. In this paper, we add 133 previously unidentified let genes, isolated in the EMS screens, and one let gene identified by a γ-ray induced mutation, to our collection of 103 essential genes. We also recovered lethal alleles of genes for which visible mutants already existed. In total, eight deficiencies and alleles of 237 essential genes were identified. Eighty-nine of the previously unidentified let genes are represented by more than one lethal allele. Statistical analysis indicates a minimum estimate of 400 essential genes in the region of chromosome I balanced by sDp2. This region occupies approximately half of chromosome I, and contains over 1135 protein-coding genes predicted from the genomic sequence data. Thus, approximately one-third of the predicted genes are estimated to be essential. Of these approximately 60% are represented by lethal alleles. Less than 2% of the lethal-bearing strains recovered in our analysis, including the eight genetically definable deficiencies, carried more than one lethal mutation. Several screens were used to recover mutations for this analysis. Because all the mutations were isolated using the same balancer, under similar screening conditions, it was possible to compare intervals within the sDp2 region with each other. The fraction of essential genes that present relatively large targets for EMS was highest within the central cluster (dpy-5 to unc-13). Received: 12 July 1999 / Accepted: 6 December 1999     

Genetic and molecular analysis of the dpy-14 region in Caenorhabditis elegans.

Summary Essential genes have been identified in the 1.5 map unit (m.u.)dpy-14-unc-29 region of chromosome I inCaenorhabditis elegans. Previous work defined nine genes with visible mutant phenotypes and nine genes with lethal mutant phenotypes. In this study, we have identified an additional 28 essential genes with 97 lethal mutations. The mutations were mapped using eleven duplication breakpoints, eight deficiencies and three-factor recombination experiments. Genes required for the early stages of development were common, with 24 of the 37 essential genes having mutant phenotypes arresting at an early larval stage. Most mutants of a gene have the same time of arrest; only four of the 20 essential genes with multiple alleles have alleles with different phenotypes. From the analysis of complementing alleles oflet-389, alleles with the same time-of-arrest phenotype were classified as either hypomorphic or amorphic. Mutants oflet-605, let-534 andunc-37 have both uncoordinated and lethal phenotypes, suggesting that these genes are required for the coordination of movement and for viability. The physical and genetic maps in thedpy-14 region were linked by positioning two N2/BO polymorphisms with respect to duplications in the region, and by localizing the right breakpoint of the deficiencyhDf8 on the physical map. Using cross-species hybridization toC. briggsae, ten regions of homology have been identified, eight of which are known to be coding regions, based on Northern analysis and/or the isolation of cDNA clones.     

Molecular analysis of Saccharomyces cerevisiae chromosome I. On the number of genes and the identification of essential genes using temperature-sensitive-lethal mutations.

Previous analyses of Saccharomyces cerevisiae chromosome I have suggested that the majority (greater than 75%) of single-copy essential genes on this chromosome are difficult or impossible to identify using temperature-sensitive (Ts-) lethal mutations. To investigate whether this situation reflects intrinsic difficulties in generating temperature-sensitive proteins or constraints on mutagenesis in yeast, we subjected three cloned essential genes from chromosome I to mutagenesis in an Escherichia coli mutator strain and screened for Ts- lethal mutations in yeast using the "plasmid-shuffle" technique. We failed to obtain Ts- lethal mutations in two of the genes (FUN12 and FUN20), while the third gene yielded such mutations, but only at a low frequency. DNA sequence analysis of these mutant alleles and of the corresponding wild-type region revealed that each mutation was a single substitution not in the previously identified gene FUN19, but in the adjacent, newly identified essential gene FUN53. FUN19 itself proved to be non-essential. These results suggest that many essential proteins encoded by genes on chromosome I cannot be rendered thermolabile by single mutations. However, the results obtained with FUN53 suggest that there may also be significant constraints on mutagenesis in yeast. The 5046 base-pair interval sequenced contains the complete FUN19, FUN53 and FUN20 coding regions, as well as a portion of the adjacent non-essential FUN21 coding region. In all, 68 to 75% of this interval is open reading frame. None of the four predicted products shows significant homologies to known proteins in the available databases.     

A Genetic Analysis of the Rose-Gespleten Region (68c8-69b5) of Drosophila Melanogaster

We describe a genetic analysis of the region 68C8-69B5 defined by Df(3L)vin-7. We have induced 35 new lethal mutations in this region, which together with 20 existing lethal mutations, visible mutations, genes identified by protein products and one gene deduced from complementation data fall into 37 complementation groups in this 35-band interval. Using existing and newly induced deficiencies we have assigned these to 11 intervals defined by deficiency breakpoints. Those mutations which fell in the same breakpoint interval as the Lsp-2 gene, which codes for the abundant larval serum protein 2, were the subject of detailed study. None was rescued by the active Lsp-2 gene transformed on to chromosome II and we conclude that, as yet, we have no lethal mutations of Lsp-2.     

Genetic analysis and complementation by germ-line transformation of lethal mutations in the unc-22 IV region of Caenorhabditis elegans

Summary The subject of this study is the organization of essential genes in the 2 map-unit unc-22 IV region of the Caenorhabditis elegans genome. With the goal of achieving mutational saturation of essential genes in this region, 6491 chromosomes mutagenized with ethyl methanesulfonate (EMS) were screened for the presence of lethal mutations in the unc-22 region. The genetic analysis of 21 lethal mutations in the unc-22 region resulted in the identification of 6 new essential genes, making a total of 36 characterized to date. A minimum of 49 essential genes are estimated to lie in this region. A set of seven formaldehyde-induced deficiencies of unc-22 and surrounding loci were isolated to facilitate the positioning of essential genes on the genetic and physical maps. In order to study essential genes at the molecular level, our approach was to rescue lethal mutations by the injection of genomic DNA in the form of cosmid clones into the germ-line of balanced heterozygotes carrying a lethal mutation. The cosmid clones containing let-56 and let-653 were identified by this method.     

Isolation of Mutations in the Drosophila Homologues of the Human Neurofibromatosis 2 and Yeast Cdc42 Genes Using a Simple and Efficient Reverse-Genetic Method

Reverse genetic analysis in Drosophila has been greatly aided by a growing collection of lethal P transposable element insertions that provide molecular tags for the identification of essential genetic loci. However, because the screens performed to date primarily have generated autosomal P-element insertions, this collection has not been as useful for performing reverse genetic analysis of X-linked genes. We have designed a reverse genetic screen that takes advantage of the hemizygosity of the X chromosome in males together with a cosmid-based transgene that serves as an autosomally linked duplication of a small region of the X chromosome. The efficacy and efficiency of this method is demonstrated by the isolation of mutations in Drosophila homologues of two well-studied genes, the human Neurofibromatosis 2 tumor suppressor and the yeast CDC42 gene. The method we describe should be of general utility for the isolation of mutations in other X-linked genes, and should also provide an efficient method for the isolation of new alleles of existing X-linked or autosomal mutations in Drosophila.     

Chaser (Csr), a New Gene Affecting Larval Foraging Behavior in Drosophila Melanogaster

Chaser (Csr) was uncovered in a gamma mutagenesis screen to identify genes that modify the larval foraging behavior of sitters to rovers. Rover larvae have significantly longer path lenghts than sitters while foraging on a yeast and water paste. This difference is influenced by one major gene, foraging (for), which has two naturally occurring alleles, for(R) (rover) and for(s) (sitter). In a mutagenesis screen for modifiers of for, we identified three lines with viable mutations on chromosome 3 that alter foraging behavior. Each of these mutations increased larval path lengths in for(s)/for(s) larvae in a dominant fashion, and were not separable by recombination. These mutations are therefore probably allelic and define a new gene that we have called Csr. Csr was genetically localized using the lethal-tagging technique. This technique resulted in seven lines with a significant decrease in larval path-length and recessive lethal mutations on chromosome 3. We refer to these as reverted Csr (Csr(rv)) lines. Deficiencies that uncovered cytologically visible chromosome rearrangements in three of the seven reverted lines were used in a complementation analysis. In this way we mapped the lethal mutations in the Csr(rv) lines to cytological region 95F7-96A1 on the right arm of chromosome 3.     

The Doublesex Locus of Drosophila Melanogaster and Its Flanking Regions: A Cytogenetic Analysis

The region of the third chromosome (84D-F) of Drosophila melanogaster that contains the doublesex (dsx) locus has been cytogenetically analyzed. Twenty nine newly induced, and 42 preexisting rearrangements broken in dsx and the regions flanking dsx have been cytologically and genetically characterized. These studies established that the dsx locus is in salivary chromosome band 84E1-2. In addition, these observations provide strong evidence that the dsx locus functions only to regulate sexual differentiation and does not encode a vital function. To obtain new alleles at the dsx locus and to begin to analyze the genes flanking dsx, 59 lethal and visible mutations in a region encompassing dsx were induced. These mutations together with preexisting mutations in the region were deficiency mapped and placed into complementation groups. Among the mutations we isolated, four new mutations affecting sexual differentiation were identified. All proved to be alleles of dsx, suggesting that dsx is the only gene in this region involved in regulating sexual differentiation. All but one of the new dsx alleles have equivalent effects in males and females. The exception, dsxEFH55, strongly affects female sexual differentiation, but only weakly affects male sexual differentiation. The interactions of dsxEFH55 with mutations in other genes affecting sexual differentiation are described. These results are discussed in terms of the recent molecular findings that the dsx locus encodes sex-specific proteins that share in common their amino termini but have different carboxyl termini. The 72 mutations in this region that do not affect sexual differentiation identify 25 complementation groups. A translocation, T(2;3)Es that is associated with a lethal allele in one of these complementation groups is also broken at the engrailed (en) locus on the second chromosome and has a dominant phenotype that may be due to the expression of en in the anterior portion of the abdominal tergites where en is not normally expressed. The essential genes found in the 84D-F region are not evenly distributed throughout this region; most strikingly the 84D1-11 region appears to be devoid of essential genes. It is suggested that the lack of essential genes in this region is due to the region (1) containing genes with nonessential functions and (2) being duplicated, possibly both internally and elsewhere in the genome.     

Genetic Analysis of Saccharomyces Cerevisiae Chromosome I: On the Role of Mutagen Specificity in Delimiting the Set of Genes Identifiable Using Temperature-Sensitive-Lethal Mutations

In a previous attempt to identify as many as possible of the essential genes on Saccharomyces cerevisiae chromosome I, temperature-sensitive (Ts(-)) lethal mutations that had been induced by ethyl methanesulfonate or nitrosoguanidine were analyzed. Thirty-two independently isolated mutations that mapped to chromosome I identified only three complementation groups, all of which had been known previously. In contrast, molecular analyses of segments of the chromosome have suggested the presence of numerous additional essential genes. In order to assess the degree to which problems of mutagen specificity had limited the set of genes detected using Ts(-) lethal mutations, we isolated a new set of such mutations after mutagenesis with UV or nitrogen mustard. Surprisingly, of 21 independently isolated mutations that mapped to chromosome I, 17 were again in the same three complementation groups as identified previously, and two of the remaining four mutations were apparently in a known gene involved in cysteine biosynthesis. Of the remaining two mutations, one was in one of the essential genes identified in the molecular analyses, and the other was too leaky to be mapped. These results suggest that only a minority of the essential genes in yeast can be identified using Ts(-) lethal mutations, regardless of the mutagen used, and thus emphasize the need to use multiple genetic strategies in the investigation of cellular processes.     

Drosophila GABAergic systems II. Mutational analysis of chromosomal segment 64AB, a region containing the glutamic acid decarboxylase gene

The Drosophila melanogaster Gad gene maps to region 64A3-5 of chromosome 3L and encodes glutamic acid decarboxylase (GAD), the rate-limiting enzyme for the synthesis of the inhibitory neurotransmitter -aminobutyric acid (GABA). Because this neurotransmitter has been implicated in developmental functions, we have begun to study the role of GABA synthesis during Drosophila embryogenesis. We show that Gad mRNA is expressed in a widespread pattern within the embryonic nervous system. Similarly, GAD-immunoreactive protein is present during embryogenesis. These results prompted us to screen for embryonic lethal mutations that affect GAD activity. The chromosomal region to which Gad maps, however, has not been subjected to an extensive mutational analysis, even though it contains several genes encoding important neurobiological, developmental, or cellular functions. Therefore, we have initially generated both chromosomal rearangements and point mutations that map to the Drosophila 64AB interval. Altogether, a total of 33 rearrangements and putative point mutations were identified within region 64A3-5 to 64B12. Genetic complementation analysis suggests that this cytogenetic interval contains a minimum of 19 essential genes. Within our collection of lethal mutations are several chromosomal rearrangements, two of which are in the vicinity of the Gad locus. One of these rearrangements, Df(3L)C175, is a small deletion that removes the Gad locus and at least two essential genes; the second, T(2;3)F10, is a reciprocal translocation involving the second and third chromosomes with a break within region 64A3-5. Both of these rearrangements are associated with embryonic lethality and decreased GAD enzymatic activity.     

Drosophila GABAergic systems II. Mutational analysis of chromosomal segment 64AB,a region containing the glutamic acid decarboxylase gene

The Drosophila melanogaster Gad gene maps to region 64A3-5 of chromosome 3L and encodes glutamic acid decarboxylase (GAD), the rate-limiting enzyme for the synthesis of the inhibitory neurotransmitter γ-aminobutyric acid (GABA). Because this neurotransmitter has been implicated in developmental functions, we have begun to study the role of GABA synthesis during Drosophila embryogenesis. We show that Gad mRNA is expressed in a widespread pattern within the embryonic nervous system. Similarly, GAD-immunoreactive protein is present during embryogenesis. These results prompted us to screen for embryonic lethal mutations that affect GAD activity. The chromosomal region to which Gad maps, however, has not been subjected to an extensive mutational analysis, even though it contains several genes encoding important neurobiological, developmental, or cellular functions. Therefore, we have initially generated both chromosomal rearangements and point mutations that map to the Drosophila 64AB interval. Altogether, a total of 33 rearrangements and putative point mutations were identified within region 64A3-5 to 64B12. Genetic complementation analysis suggests that this cytogenetic interval contains a minimum of 19 essential genes. Within our collection of lethal mutations are several chromosomal rearrangements, two of which are in the vicinity of the Gad locus. One of these rearrangements, Df(3L)C175, is a small deletion that removes the Gad locus and at least two essential genes; the second, T(2;3)F10, is a reciprocal translocation involving the second and third chromosomes with a break within region 64A3-5. Both of these rearrangements are associated with embryonic lethality and decreased GAD enzymatic activity.     

Genetic Analysis of Chromosomal Region 67a-D of Drosophila Melanogaster

In an effort to (1) characterize the 67 interval of chromosome 3 of Drosophila melanogaster genetically and (2) isolate mutations of the 67B1 small heat shock protein (hsp) gene cluster specifically, we undertook a mutational analysis of the 67A-D subinterval. Using a deficiency of the 67A2 to 67D11-13 region, Df(3L)AC1, we screened 8700 diepoxybutane-treated chromosomes and 7800 ethyl methanesulfonate-treated chromosomes for visible and lethal mutations throughout this interval and recovered 74 independent recessive lethal mutations, but no visible mutations. One of the lethal mutations, d29A6, was identified as an overlapping deficiency extending from 66F3 to 67B1. An additional 6000 diepoxybutane-treated chromosomes were screened for lethality over d29A6, yielding another four lethal mutations within the 67A2-B1 subinterval. These 78 lethal mutations, along with two others isolated in other laboratories, define 23 essential loci--6 within the 67A2-B1 subinterval and 17 within the 67A2 to D11-13 subinterval. Many of these loci appear to be required for imaginal development only, exhibiting late larval to pharate adult lethal phases. Examination of the 67A2-B1 lethal complementation groups for (1) earlier onset of lethality following a heat shock, (2) missing or altered small hsps on two-dimensional protein gels, and (3) restoration of viability by transformed wild-type copies of the small hsp genes indicates that none of these mutations affect the small hsps. On the basis of this analysis and the known homology of the genes, we conclude that the small hsps are functionally equivalent.     

Genetic and bioinformatic analysis of 41C and the 2R heterochromatin of Drosophila melanogaster: a window on the heterochromatin-euchromatin junction

Genomic sequences provide powerful new tools in genetic analysis, making it possible to combine classical genetics with genomics to characterize the genes in a particular chromosome region. These approaches have been applied successfully to the euchromatin, but analysis of the heterochromatin has lagged somewhat behind. We describe a combined genetic and bioinformatics approach to the base of the right arm of the Drosophila melanogaster second chromosome, at the boundary between pericentric heterochromatin and euchromatin. We used resources provided by the genome project to derive a physical map of the region, examine gene density, and estimate the number of potential genes. We also carried out a large-scale genetic screen for lethal mutations in the region. We identified new alleles of the known essential genes and also identified mutations in 21 novel loci. Fourteen complementation groups map proximal to the assembled sequence. We used PCR to map the endpoints of several deficiencies and used the same set of deficiencies to order the essential genes, correlating the genetic and physical map. This allowed us to assign two of the complementation groups to particular "computed/curated genes" (CGs), one of which is Nipped-A, which our evidence suggests encodes Drosophila Tra1/TRRAP.     

Alignment of the genetic and physical maps in the dpy-5 bli-4 (I) region of C. elegans by the serial cosmid rescue of lethal mutations

Lethal mutations in the 0.5 map unit region between dpy-5 and bli-4 on chromosome I in Caenorhabditis elegans were serially rescued using cosmid-containing transgenic strains. All the lethal mutations analyzed came from a set of 495 EMS-induced, sDp2-rescued lethals described previously. Germline transformation with cosmid DNA was used to create 25 transgenic strains bearing heritable extrachromosomal arrays. These arrays were used as small duplications for the fine-scale mapping of essential genes, via the rescue of lethal mutations. Lethal mutations in 13 essential genes have been phenotypically rescued, allowing the alignment of the genetic and physical maps in this region. Extrachromosomal arrays were found to be transmitted 2- to 7-fold less frequently in oocytes than in hermaphrodite sperm for 12 of the 16 arrays that were examined. Three of these strains showed a subsequent 4- to 13-fold increase in array stability in oocytes. This phenomenon may be influenced by cosmid sequences. Early mitotic loss of the arrays was observed in all 17 transgenic strains examined, suggesting that loss of the array can occur at any time during development when cell divisions are occurring. As a result of this work, 13 of the essential loci positioned between dpy-5 and bli-4 are anchored to the physical map, thereby providing links between the physical and genetic maps on average every 85 kb.     

Vital Genes That Flank Sex-Lethal, an X-Linked Sex-Determining Gene of DROSOPHILA MELANOGASTER

The X-chromosome:autosome balance in D. melanogaster appears to control both sex determination and dosage compensation through effects on a maternally influenced sex-linked gene called Sex-lethal (Sxl; 1-19.2). To facilitate molecular and genetic analysis of Sxl, we attempted to determine the locations of all ethyl methanesulfonate (EMS)-mutable genes vital to both sexes in the region between 6E1 and 7B1. This area includes approximately 1 cM of the genetic map on each side of Sxl and was reported by C. B. Bridges to contain 26 salivary gland polytene chromosome bands. The region appears rather sparsely populated with genes vital to both sexes, since the 122 recessive lethal mutations we recovered fell into only nine complementation groups. From one to 38 alleles of each gene were recovered. There was a preponderance of embryonic lethals in this area, although the lethal periods of loss-of-function mutations included larval, pupal and adult stages as well. Since the screen required that mutations be recessive and lethal to males, our failure to recover new Sxl alleles was the result expected for a gene with a female-specific function. An attempt was made to identify recessive male-specific lethals in this region, but none were found. Precise map positions were determined for eight of the nine vital genes. An interesting feature of the map is the location of Sxl in the middle of a 0.6- to 0.7-cM interval that appears to be devoid of genes vital to both sexes. The genetic location was determined of breakpoints near Sxl for all available chromosome rearrangements. Sxl is most likely located just to the left of band 7A1. We determined the relationship of our EMS-induced mutations in these nine genes to alleles induced by others. From this we conclude that the various genes appear to differ significantly from each other in their relative sensitivity to mutation by EMS vs. X rays.     

Lethal mutations defining 112 complementation groups in a 4.5 Mb sequenced region of Caenorhabditis elegans chromosome III

The central gene cluster of chromosome III was one of the first regions to be sequenced by the Caenorhabditis elegans genome project. We have performed an essential gene analysis on the left part of this cluster, in the region around dpy-17III balanced by the duplication sDp3. We isolated 151 essential gene mutations and characterized them with regard to their arrest stages. To facilitate positioning of these mutations, we generated six new deficiencies that, together with preexisting chromosomal rearrangements, subdivide the region into 14 zones. The 151 mutations were mapped into these zones. They define 112 genes, of which 110 were previously unidentified. Thirteen of the zones have been anchored to the physical sequence by polymerase chain reaction deficiency mapping. Of the 112 essential genes mapped, 105 are within these 13 zones. They span 4.2?Mb of nucleotide sequence. From the nucleotide sequence data, 920 genes are predicted. From a Poisson distribution of our mutations, we predict that 234 of the genes will be essential genes. Thus, the 105 genes constitute 45% of the estimated number of essential genes in the physically defined zones and between 2 and 5% of all essential genes in C. elegans.     

Lethal mutations defining 112 complementation groups in a 4.5?Mb sequenced region of Caenorhabditis elegans chromosome III

The central gene cluster of chromosome III was one of the first regions to be sequenced by the Caenorhabditis elegans genome project. We have performed an essential gene analysis on the left part of this cluster, in the region around dpy-17III balanced by the duplication sDp3. We isolated 151 essential gene mutations and characterized them with regard to their arrest stages. To facilitate positioning of these mutations, we generated six new deficiencies that, together with preexisting chromosomal rearrangements, subdivide the region into 14 zones. The 151 mutations were mapped into these zones. They define 112 genes, of which 110 were previously unidentified. Thirteen of the zones have been anchored to the physical sequence by polymerase chain reaction deficiency mapping. Of the 112 essential genes mapped, 105 are within these 13 zones. They span 4.2 Mb of nucleotide sequence. From the nucleotide sequence data, 920 genes are predicted. From a Poisson distribution of our mutations, we predict that 234 of the genes will be essential genes. Thus, the 105 genes constitute 45% of the estimated number of essential genes in the physically defined zones and between 2 and 5% of all essential genes in C. elegans. Received: 23 April 1998 / Accepted: 18 August 1998