The Let-60 Locus Controls the Switch between Vulval and Nonvulval Cell Fates in Caenorhabditis Elegans

During induction of the Caenorhabditis elegans hermaphrodite vulva by the anchor cell of the gonad, six multipotent vulval precursor cells (VPCs) have two distinct fates: three VPCs generate the vulva and the other three VPCs generate nonspecialized hypodermis. Genes that control the fates of the VPCs in response to the anchor cell signal are defined by mutations that cause all six VPCs to generate vulval tissue (Multivulva or Muv) or that cause all six VPCs to generate hypodermis (Vulvaless or Vul). Seven dominant Vul mutations were isolated as dominant suppressors of a lin-15 Muv mutation. These mutations are dominant alleles of the gene let-60, previously identified only by recessive lethal mutations. Our genetic studies of these dominant Vul recessive lethal mutations, recessive lethal mutations, intragenic revertants of the dominant Vul mutations, and the closely mapping semi-dominant multivulva lin-34 mutations suggest that: (1) loss-of-function mutations of let-60 are recessive lethal at a larval stage, but they also cause a Vul phenotype if the lethality is rescued maternally by a lin-34 gain-of-function mutation. (2) The dominant Vul alleles of let-60 are dominant negative mutations whose gene products compete with wild-type activity. (3) lin-34 semidominant Muv alleles are either gain-of-function mutations of let-60 or gain-of-function mutations of an intimately related gene that elevates let-60 activity. We propose that let-60 activity controls VPC fates. In a wild-type animal, reception by a VPC of inductive signal activates let-60, and it generates into a vulval cell type; in absence of inductive signal, let-60 activity is low and the VPC generates hypodermal cells. Our genetic interaction studies suggest that let-60 acts downstream of let-23 and lin-15 and upstream of lin-1 and lin-12 in the genetic pathway specifying the switch between vulval and nonvulval cell types.     

Phenotype of the tomato high pigment-2 mutant is caused by a mutation in the tomato homolog of DEETIOLATED1.

Tomato high pigment (hp) mutants are characterized by their exaggerated photoresponsiveness. Light-grown hp mutants display elevated levels of anthocyanins, are shorter and darker than wild-type plants, and have dark green immature fruits due to the overproduction of chlorophyll pigments. It has been proposed that HP genes encode negative regulators of phytochrome signal transduction. We have cloned the HP-2 gene and found that it encodes the tomato homolog of the nuclear protein DEETIOLATED1 (DET1) from Arabidopsis. Mutations in DET1 are known to result in constitutive deetiolation in darkness. In contrast to det1 mutants, tomato hp-2 mutants do not display any visible phenotypes in the dark but only very weak phenotypes, such as partial chloroplast development. Furthermore, whereas det1 mutations are epistatic to mutations in phytochrome genes, analysis of similar double mutants in tomato showed that manifestation of the phenotype of the hp-2 mutant is strictly dependent upon the presence of active phytochrome. Because only one DET1 gene is likely to be present in each of the two species, our data suggest that the phytochrome signaling pathways in which the corresponding proteins function are regulated differently in Arabidopsis and tomato.     

Isolation and Genetic Characterization of Cell-Lineage Mutants of the Nematode CAENORHABDITIS ELEGANS

Twenty-four mutants that alter the normally invariant post-embryonic cell lineages of the nematode Caenorhabditis elegans have been isolated and genetically characterized. In some of these mutants, cell divisions fail that occur in wild-type animals; in other mutants, cells divide that do not normally do so. The mutants differ in the specificities of their defects, so that it is possible to identify mutations that affect some cell lineages but not others. These mutants define 14 complementation groups, which have been mapped. The abnormal phenotype of most of the cell-lineage mutants results from a single recessive mutation; however, the excessive cell divisions characteristic of one strain, CB1322, require the presence of two unlinked recessive mutations. All 24 cell-lineage mutants display incomplete penetrance and/or variable expressivity. Three of the mutants are suppressed by pleiotropic suppressors believed to be specific for null alleles, suggesting that their phenotypes result from the complete absence of gene activity.     

Genetic modifiers of the Drosophila NSF mutant, comatose, include a temperature-sensitive paralytic allele of the calcium channel alpha1-subunit gene, cacophony

The N-ethylmaleimide-sensitive fusion protein (NSF) has been implicated in vesicle trafficking in perhaps all eukaryotic cells. The Drosophila comatose (comt) gene encodes an NSF homolog, dNSF1. Our previous work with temperature-sensitive (TS) paralytic alleles of comt has revealed a function for dNSF1 at synapses, where it appears to prime synaptic vesicles for neurotransmitter release. To further examine the molecular basis of dNSF1 function and to broaden our analysis of synaptic transmission to other gene products, we have performed a genetic screen for mutations that interact with comt. Here we report the isolation and analysis of four mutations that modify TS paralysis in comt, including two intragenic modifiers (one enhancer and one suppressor) and two extragenic modifiers (both enhancers). The intragenic mutations will contribute to structure-function analysis of dNSF1 and the extragenic mutations identify gene products with related functions in synaptic transmission. Both extragenic enhancers result in TS behavioral phenotypes when separated from comt, and both map to loci not previously identified in screens for TS mutants. One of these mutations is a TS paralytic allele of the calcium channel alpha1-subunit gene, cacophony (cac). Analysis of synaptic function in these mutants alone and in combination will further define the in vivo functions and interactions of specific gene products in synaptic transmission.     

Mutations with Dominant Effects on the Behavior and Morphology of the Nematode CAENORHABDITIS ELEGANS

We have analyzed 31 mutations that have dominant effects on the behavior or morphology of the nematode Caenorhabditis elegans. These mutations appear to define 15 genes. We have studied ten of these genes in some detail and have been led to two notable conclusions. First, loss of gene function for four of these ten genes results in a wild-type phenotype; if these genes represent a random sample from the genome, then we would estimate that null mutations in about half of the genes in C. elegans would result in a nonmutant phenotype. Second, the dominant effects of mutations in nine of these ten genes are caused by novel gene functions, and in all nine cases the novel function is antagonized by the wild-type function.     

Coliphage P1 morphogenesis: analysis of mutants by electron microscopy.

We used electron microscopy and serum blocking power tests to determine the phenotypes of 47 phage P1 amber mutants that have defects in particle morphogenesis. Eleven mutants showed head defects, 30 showed tail defects, and 6 had a defect in particle maturation (which could be either in the head or in the tail). Consideration of previous complementation test results, genetic and physical positions of the mutations, and phenotypes of the mutants allowed assignment of most of the 47 mutations to genes. Thus, a minimum of 12 tail genes, 4 head genes, and 1 particle maturation gene are now known for P1. Of the 12 tail genes, 1 (gene 19, located within the invertible C loop) codes for tail fibers, 6 (genes 3, 5, 16, 20, 21, and 26) code for baseplate components (although one of these genes could code for the tail tube), 1 (gene 22) codes for the sheath, 1 (gene 6) affects tail length, 2 (genes 7 and 25) are involved in tail stability, and 1 (gene 24) either codes for a baseplate component or is involved in tail stability. Of the four head genes, gene 9 codes for a protein required for DNA packaging. The function of head gene 4 is unclear. Head gene 8 probably codes for a minor head protein, whereas head gene 23 could code for either a minor head protein or the major head protein. Excluding the particle maturation gene (gene 1), the 12 tail genes are clustered in three regions of the P1 physical genome. The four head genes are at four separate locations. However, some P1 head genes have not yet been detected and could be located in two regions (for which there are no known genes) adjacent to genes 4 and 8. The P1 morphogenetic gene clusters are interrupted by many genes that are expressed in the prophage.     

Five SWI genes are required for expression of the HO gene in yeast

High-frequency mating type interconversion in yeast requires the HO gene, which encodes a site-specific endonuclease that initiates the switching process. We have isolated and analyzed switching-defective mutants. These mutants define five complementation and linkage groups, SWI 1 to SWI 5. We have shown by two assays, Northern hybridization and beta-galactosidase activity in strains containing an HO-lacZ fusion, that mutants defective any SWI gene fail to express the HO gene. In addition, all of the swi mutants exhibit other phenotypes, the most notable being the inviability of double mutants defective in SWI 4 and in either SWI 1, SWI 2 or SWI 3. These results indicate that the SWI genes function in some way as positive regulators of HO expression and have additional cellular roles.     

Identification of novel Drosophila meiotic genes recovered in a P-element screen.

The segregation of homologous chromosomes from one another is the essence of meiosis. In many organisms, accurate segregation is ensured by the formation of chiasmata resulting from crossing over. Drosophila melanogaster females use this type of recombination-based system, but they also have mechanisms for segregating achiasmate chromosomes with high fidelity. We describe a P-element mutagenesis and screen in a sensitized genetic background to detect mutations that impair meiotic chromosome pairing, recombination, or segregation. Our screen identified two new recombination-deficient mutations: mei-P22, which fully eliminates meiotic recombination, and mei-P26, which decreases meiotic exchange by 70% in a polar fashion. We also recovered an unusual allele of the ncd gene, whose wild-type product is required for proper structure and function of the meiotic spindle. However, the screen yielded primarily mutants specifically defective in the segregation of achiasmate chromosomes. Although most of these are alleles of previously undescribed genes, five were in the known genes alphaTubulin67C, CycE, push, and Trl. The five mutations in known genes produce novel phenotypes for those genes.     

Genetic Analysis of X-Chromosome Dosage Compensation in Caenorhabditis elegans

We have shown that the phenotypes resulting from hypomorphic mutations (causing reduction but not complete loss of function) in two X-linked genes can be used as a genetic assay for X-chromosome dosage compensation in Caenorhabditis elegans between males (XO) and hermaphrodites (XX). In addition we show that recessive mutations in two autosomal genes, dpy-21 V and dpy-26 IV, suppress the phenotypes resulting from the X-linked hypomorphic mutations, but not the phenotypes resulting from comparable autosomal hypomorphic mutations. This result strongly suggests that the dpy-21 and dpy-26 mutations cause increased X expression, implying that the normal function of these genes may be to lower the expression of X-linked genes. Recessive mutations in two other dpy genes, dpy-22 X and dpy-23 X, increase the severity of phenotypes resulting from some X-linked hypomorphic mutations, although dpy-23 may affect the phenotypes resulting from the autosomal hypomorphs as well. The mutations in all four of the dpy genes show their effects in both XO and XX animals, although to different degrees. Mutations in 18 other dpy genes do not show these effects.     

BIN2, a new brassinosteroid-insensitive locus in Arabidopsis

Brassinosteroids (BRs) play important roles throughout plant development. Although many genes have been identified that are involved in BR biosynthesis, genetic approaches in Arabidopsis have led to the identification of only one gene, BRI1, that encodes a membrane receptor for BRs. To expand our knowledge of the molecular mechanism(s) of plant steroid signaling, we analyzed many dwarf and semidwarf mutants collected from our previous genetic screens and identified a semidwarf mutant that showed little response to exogenous BR treatments. Genetic analysis of the bin2 (BR-INSENSITIVE 2) mutant indicated that the BR-insensitive dwarf phenotype was due to a semidominant mutation in the BIN2 gene that mapped to the middle of chromosome IV between the markers CH42 and AG. A direct screening for similar semidwarf mutants resulted in the identification of a second allele of the BIN2 gene. Despite some novel phenotypes observed with the bin2/+ mutants, the homozygous bin2 mutants were almost identical to the well-characterized bri1 mutants that are defective in BR perception. In addition to the BR-insensitive dwarf phenotype, bin2 mutants exhibited BR insensitivity when assayed for root growth inhibition and feedback inhibition of CPD gene expression. Furthermore, bin2 mutants displayed an abscisic acid-hypersensitive phenotype that is shared by the bri1 and BR-deficient mutants. A gene dosage experiment using triploid plants suggested that the bin2 phenotypes were likely caused by either neomorphic or hypermorphic gain-of-function mutations in the BIN2 gene. Thus, the two bin2 mutations define a novel genetic locus whose gene product might play a role in BR signaling.     

The Genetics of Levamisole Resistance in the Nematode CAENORHABDITIS ELEGANS

We have characterized a small group of genes (13 loci) in the nematode Caenorhabditis elegans that, when mutated, confer resistance to the potent anthelmintic levamisole. Mutants at the 7 loci conferring the most extreme resistance generally possess almost identical visible and pharmacological phenotypes: uncoordinated motor behavior, most severe in early larval life, extreme resistance to cholinergic agonists and sensitivity to hypo-osmotic shock. Mutants with exceptional phenotypes suggest possible functions for several of the resistance loci. The most extreme mutants can readily be selected by their drug resistance (211 mutants, as many as 74 alleles of one gene). The more common resistance loci are likely to be unessential genes, while loci identified by only a few alleles may be essential genes or genes conferring resistance only when mutated in a special way. We propose that these mutants represent a favorable system for understanding how a small group of related genes function in a simple animal. The extreme drug resistance of these mutants makes them useful tools for the genetic manipulation of C. elegans. And, as the most resistant class of mutants might lack pharmacologically functional acetylcholine receptors (Lewis et al. 1980), these mutants may also be of some neurobiological significance.     

Genetics of Extracellular Protease Production in SACCHAROMYCOPSIS LIPOLYTICA

Mutants of Saccharomycopsis lipolytica with reduced ability to produce zones of clearing on skim-milk agar plates were isolated and their properties studied. For 18 mutants it was possible to score unambiguously segregants of crosses between these mutants and wild type for extracellular protease production. These mutants all produce reduced levels of extracellular protease in liquid culture. The mutations are recessive and are in nuclear genes. The 18 mutations define 10 or 11 complementation groups, no two of which are closely linked. Mutants in four of the complementation groups also produced reduced levels of extracellular RNAse, and the reduced levels of extracellular protease and RNAse production segregate together. Five of the mutants exhibited reduced mating frequency, and one mutant was osmotic remedial for extracellular protease production. These results demonstrate that many genes can affect extracellular protease production. Besides mutations in the structural gene and in regulatory genes, mutations are likely to be in genes involved in steps common to the production of several extracellular enzymes or in genes coding for cell wall or membrane components necessary for extracellular enzyme production.     

Mating type regulation of cellular tolerance to DNA damage is specific to the DNA post-replication repair and mutagenesis pathway

In order to help further define DNA post-replication repair (PRR), a conditional synthetic lethal screen was employed to identify new genes involved in the PRR pathway. A synthetic lethal screen with the mms2 mutation resulted in the recovery of two suppressor mutations responsible for regulating PRR. The recovered suppressors are the mating type genes and SIR3. Indeed, controlled expression of both mating type genes or deletion of SIR3 rescued the conditional synthetic lethal mutant phenotypes. Furthermore, comprehensive analyses suggest that mating type heterozygosity confers tolerance to a broad range of DNA damage, and that this effect is limited to all PRR pathway mutations, but does not apply to base excision repair, nucleotide excision repair or recombination repair mutants. In addition, the tolerance conferred to PRR mutants as a result of mating type heterozygosity is dependent on a functional homologous recombination but not the non-homologous end-joining pathway. Thus, mating type status appears to be responsible for signalling DNA content and possibly cell cycle stage, allowing the cell to select the most efficient means to repair the DNA damage.     

Functional redundancy and new roles for genes of the autonomous floral-promotion pathway

The early-flowering habit of rapid-cycling accessions of Arabidopsis (Arabidopsis thaliana) is, in part, due to the genes of the autonomous floral-promotion pathway (AP). The AP promotes flowering by repressing expression of the floral inhibitor FLOWERING LOCUS C (FLC). AP mutants are therefore late flowering due to elevated levels of FLC, and this late-flowering phenotype is eliminated by loss-of-function mutations in FLC. To further investigate the role of the AP, we created a series of double mutants. In contrast to the phenotypes of single mutants, which are largely limited to delayed flowering, a subset of AP double mutants show a range of defects in growth and development. These phenotypes include reduced size, chlorophyll content, growth rate, and fertility. Unlike the effects of the AP on flowering time, these phenotypes are FLC independent. Recent work has also shown that two AP genes, FCA and FPA, are required for the repression and, in some cases, proper DNA methylation of two transposons. We show that similar effects are seen for all AP genes tested. Microarray analysis of gene expression in AP single and double mutants, however, suggests that the AP is not likely to play a broad role in the repression of gene expression through DNA methylation: very few of the genes that have been reported to be up-regulated in DNA methylation mutants are misexpressed in AP mutants. Together, these data indicate that the genes of the AP play important and sometimes functionally redundant roles in aspects of development in addition to flowering time.     

Egg-laying defective mutants of the nematode Caenorhabditis elegans

We have isolated 145 fertile mutants of C. elegans that are defective in egg laying and have characterized 59 of them genetically, behaviorally and pharmacologically. These 59 mutants define 40 new genes called egl. for egg-laying abnormal. Most of the other mutants are defective in previously identified genes. The egl mutants differ with respect to the severity of their egg-laying defects and the presence of behavioral or morphological pleiotropies. We have defined four distinct categories of mutants based on their responses to the pharmacological agents serotonin and imipramine, which stimulate egg laying by wild-type hermaphrodites. These drugs test the functioning of the vulva, the vulval and uterine muscles and the hermaphrodite-specific neurons (HSNs), which innervate the vulval muscles. Mutants representing 14 egl genes fail to respond to serotonin and to imipramine and are likely to be defective in the functioning of the vulva or the vulval and uterine muscles. Four mutants (representing four different genes) lay eggs in response to serotonin but not to imipramine and appear to be egg-laying defective because of defects in the HSNs; three of these four were selected specifically for these drug responses. Mutants representing seven egl genes lay eggs in response to serotonin and to imipramine. One egl mutant responds to imipramine but not to serotonin. The remaining egl mutants show variable or intermediate responses to the drugs. Two of the HSN-defective mutants, egl-1 and her-1(n695), lack HSN cell bodies and are likely to be expressing the normally male-specific program of HSN cell death. Whereas egl-1 animals appear to be defective specifically in HSN development, her-1(n695) animals exhibit multiple morphological pleiotropies, displaying partial transformation of the sexual phenotype of many cells and tissues. At least two of the egl mutants appear to be defective in the processing of environmental signals that modulate egg laying and may define new components of the neural circuitry that control egg laying.     

Control of vulval competence and centering in the nematode Oscheius sp. 1 CEW1

To compare vulva development mechanisms in the nematode Oscheius sp. 1 to those known in Caenorhabditis elegans, we performed a genetic screen for vulva mutants in Oscheius sp. 1 CEW1. Here we present one large category of mutations that we call cov, which affect the specification of the Pn.p ventral epidermal cells along the antero-posterior axis. The Pn.p cells are numbered from 1 to 12 from anterior to posterior. In wild-type Oscheius sp. 1 CEW1, the P(4-8).p cells are competent to form the vulva and the progeny of P(5-7).p actually form the vulva, with the descendants of P6.p adopting a central vulval fate. Among the 17 mutations (defining 13 genes) that we characterize here, group 1 mutations completely or partially abolish P(4-8).p competence, and this correlates with early fusion of the Pn.p cells to the epidermal syncytium. In this group, we found a putative null mutation in the lin-39 HOM-C homolog, the associated phenotype of which could be weakly mimicked by injection of a morpholino against Osp1-lin-39 in the mother's germ line. Using cell ablation in a partially penetrant competence mutant, we show that vulval competence is partially controlled by a gonadal signal. Most other mutants found in the screen display phenotypes unknown in C. elegans. Group 2 mutants show a partial penetrance of Pn.p competence loss and an abnormal centering of the vulva on P5.p, suggesting that these two processes are coregulated by the same pathway in Oscheius sp. 1. Group 3 mutants display an enlarged competence group that includes P3.p, thus demonstrating the existence of a specific mechanism inhibiting P3.p competence. Group 4 mutants display an abnormal centering of the vulval pattern on P7.p and suggest that a specific mechanism centers the vulval pattern on a single Pn.p cell.     

The Multivulva Phenotype of Certain Caenorhabditis Elegans Mutants Results from Defects in Two Functionally Redundant Pathways

We previously identified Caenorhabditis elegans mutants in which certain of the six vulval precursor cells adopt fates normally expressed by other vulval precursor cells. These mutants define genes that appear to function in the response to an intercellular signal that induces vulval development. The multivulva (Muv) phenotype of one such mutant, CB1322, results from an interaction between two unlinked mutations, lin-8(n111) II and lin-9(n112) III. In this paper, we identify 18 new mutations, which are alleles of eight genes, that interact with either lin-8(n111) or lin-9(n112) to generate a Muv phenotype. None of these 20 mutations alone causes any vulval cell lineage defects. The "silent Muv" mutations fall into two classes; hermaphrodites carrying a mutation of each class are Muv, while hermaphrodites carrying two mutations of the same class have a wild-type vulval phenotype. Our results indicate that the Muv phenotype of these mutants results from defects in two functionally-redundant pathways, thereby demonstrating that redundancy can occur at the level of gene pathways as well as at the level of gene families.