Identification of Suppressors of top-2 Embryonic Lethality in Caenorhabditis elegans

Topoisomerase II is an enzyme with important roles in chromosome biology. This enzyme relieves supercoiling and DNA and RNA entanglements generated during mitosis. Recent studies have demonstrated that Topoisomerase II is also involved in the segregation of homologous chromosomes during the first meiotic division. However, the function and regulation of Topoisomerase II in meiosis has not been fully elucidated. Here, we conducted a genetic suppressor screen in Caenorhabditis elegans to identify putative genes that interact with topoisomerase II during meiosis. Using a temperature-sensitive allele of topoisomerase II, top-2(it7ts), we identified eleven suppressors of top-2-induced embryonic lethality. We used whole-genome sequencing and a combination of RNAi and CRISPR/Cas9 genome editing to identify and validate the responsible suppressor mutations. We found both recessive and dominant suppressing mutations that include one intragenic and 10 extragenic loci. The extragenic suppressors consist of a known Topoisomerase II-interacting protein and two novel interactors. We anticipate that further analysis of these suppressing mutations will provide new insights into the function of Topoisomerase II during meiosis.

Download Full-text

The Role of pkc-3 and Genetic Suppressors in Caenorhabditis elegans Epithelial Cell Junction Formation

Genetics ◽

10.1534/genetics.120.303085 ◽

2020 ◽

Vol 214 (4) ◽

pp. 941-959 ◽

Cited By ~ 4

Author(s):

José G. Montoyo-Rosario ◽

Stephen T. Armenti ◽

Yuliya Zilberman ◽

Jeremy Nance

Keyword(s):

Caenorhabditis Elegans ◽

Epithelial Cells ◽

Matrix Protein ◽

Extracellular Matrix Protein ◽

Cell Junction ◽

Loss Of Function ◽

Link Type ◽

Junction Formation ◽

Extragenic Suppressors

Epithelial cells form intercellular junctions to strengthen cell–cell adhesion and limit diffusion, allowing epithelia to function as dynamic tissues and barriers separating internal and external environments. Junctions form as epithelial cells differentiate; clusters of junction proteins first concentrate apically, then mature into continuous junctional belts that encircle and connect each cell. In mammals and Drosophila, atypical protein kinase C (aPKC) is required for junction maturation, although how it contributes to this process is poorly understood. A role for the Caenorhabditis elegans aPKC homolog PKC-3 in junction formation has not been described previously. Here, we show that PKC-3 is essential for junction maturation as epithelia first differentiate. Using a temperature-sensitive allele of pkc-3 that causes junction breaks in the spermatheca and leads to sterility, we identify intragenic and extragenic suppressors that render pkc-3 mutants fertile. Intragenic suppressors include an unanticipated stop-to-stop mutation in the pkc-3 gene, providing evidence for the importance of stop codon identity in gene activity. One extragenic pkc-3 suppressor is a loss-of-function allele of the lethal(2) giant larvae homolog lgl-1, which antagonizes aPKC within epithelia of Drosophila and mammals, but was not known previously to function in C. elegans epithelia. Finally, two extragenic suppressors are loss-of-function alleles of sups-1—a previously uncharacterized gene. We show that SUPS-1 is an apical extracellular matrix protein expressed in epidermal cells, suggesting that it nonautonomously regulates junction formation in the spermatheca. These findings establish a foundation for dissecting the role of PKC-3 and interacting genes in epithelial junction maturation.

Download Full-text

Caenorhabditis elegans mutants defective in the functioning of the motor neurons responsible for egg laying.

Genetics ◽

10.1093/genetics/121.4.703 ◽

1989 ◽

Vol 121 (4) ◽

pp. 703-721 ◽

Cited By ~ 8

Author(s):

C Desai ◽

H R Horvitz

Keyword(s):

Caenorhabditis Elegans ◽

Synaptic Transmission ◽

Motor Neurons ◽

Egg Laying ◽

The Other ◽

Single Type ◽

Sensitive Period ◽

Temperature Sensitive ◽

Sensitive Allele

Abstract We have isolated and characterized 45 Caenorhabditis elegans mutants presumed to be defective in the functioning of the hermaphrodite-specific neurons (HSNs). Like hermaphrodites that lack the HSN motor neurons, these mutants are egg-laying defective and do not lay eggs in response to exogenous imipramine but do lay eggs in response to exogenous serotonin. Twenty of the 45 mutations define 10 new egl genes; the other 25 mutations are alleles of five previously defined genes, four of which are known to affect the HSNs. Seven mutations in three genes cause the HSNs to die in hermaphrodites, as they normally do in males. These genes appear to be involved in the determination of the sexual phenotype of the HSNs, and one of them (egl-41) is a newly identified gene that may function generally in sex determination. Five of the 15 genes are defined only by mutations that have dominant effects on egg laying. One gene egl(n1108), is defined by a temperature-sensitive allele that has a temperature-sensitive period after HSN development is complete, suggesting that egl(n1108) may be involved in HSN synaptic transmission. Four of the genes are defined by single alleles, which suggests that other such genes remain to be discovered. Mutations in no more than 4 of the 15 genes specifically affect the HSNs, indicating that there are few genes with functions needed only in this single type of nerve cell.

Download Full-text

Null alleles of SAC7 suppress temperature-sensitive actin mutations in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.2308 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2308-2314 ◽

Cited By ~ 20

Author(s):

T M Dunn ◽

D Shortle

Keyword(s):

Saccharomyces Cerevisiae ◽

Normal Growth ◽

Null Alleles ◽

Growth Defect ◽

Temperature Sensitive ◽

Actin Assembly ◽

New Gene ◽

Cold Sensitive ◽

Sensitive Allele ◽

Extragenic Suppressors

Extragenic suppressors of a new temperature-sensitive mutation (act1-4) in the actin gene of Saccharomyces cerevisiae were isolated in an attempt to identify genes whose products interact directly with actin. One suppressor with a cold-sensitive growth phenotype defined the new gene, SAC7, which was mapped, cloned, sequenced, and disrupted. Genetic analysis of strains that are disrupted for SAC7 demonstrated that the protein is required for normal growth and actin assembly at low temperatures. Surprisingly, null mutations in SAC7 also suppressed the temperature-sensitive growth defect caused by the act1-1 and act1-4 mutations, whereas they were lethal in combination with the temperature-sensitive allele act1-2. These results support the notion that the SAC7 gene product is involved in the normal assembly or function or both of actin.

Download Full-text

Null alleles of SAC7 suppress temperature-sensitive actin mutations in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.2308-2314.1990 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2308-2314

Author(s):

T M Dunn ◽

D Shortle

Keyword(s):

Saccharomyces Cerevisiae ◽

Normal Growth ◽

Null Alleles ◽

Growth Defect ◽

Temperature Sensitive ◽

Actin Assembly ◽

New Gene ◽

Cold Sensitive ◽

Sensitive Allele ◽

Extragenic Suppressors

Download Full-text

Comprehensive Synthetic Genetic Array Analysis of Alleles That Interact with Mutation of the Saccharomyces cerevisiae RecQ Helicases Hrq1 and Sgs1

G3 Genes|Genome|Genetics ◽

10.1534/g3.120.401709 ◽

2020 ◽

Vol 10 (12) ◽

pp. 4359-4368

Author(s):

Elsbeth Sanders ◽

Phoebe A. Nguyen ◽

Cody M. Rogers ◽

Matthew L. Bochman

Keyword(s):

Saccharomyces Cerevisiae ◽

Single Gene ◽

Colony Growth ◽

Strand Break ◽

Telomere Maintenance ◽

Temperature Sensitive ◽

Recq Helicase ◽

Synthetic Genetic Array ◽

Link Type ◽

Sensitive Allele

Most eukaryotic genomes encode multiple RecQ family helicases, including five such enzymes in humans. For many years, the yeast Saccharomyces cerevisiae was considered unusual in that it only contained a single RecQ helicase, named Sgs1. However, it has recently been discovered that a second RecQ helicase, called Hrq1, resides in yeast. Both Hrq1 and Sgs1 are involved in genome integrity, functioning in processes such as DNA inter-strand crosslink repair, double-strand break repair, and telomere maintenance. However, it is unknown if these enzymes interact at a genetic, physical, or functional level as demonstrated for their human homologs. Thus, we performed synthetic genetic array (SGA) analyses of hrq1Δ and sgs1Δ mutants. As inactive alleles of helicases can demonstrate dominant phenotypes, we also performed SGA analyses on the hrq1-K318A and sgs1-K706A ATPase/helicase-null mutants, as well as all combinations of deletion and inactive double mutants. We crossed these eight query strains (hrq1Δ, sgs1Δ, hrq1-K318A, sgs1-K706A, hrq1Δ sgs1Δ, hrq1Δ sgs1-K706A, hrq1-K318A sgs1Δ, and hrq1-K318A sgs1-K706A) to the S. cerevisiae single gene deletion and temperature-sensitive allele collections to generate double and triple mutants and scored them for synthetic positive and negative genetic effects based on colony growth. These screens identified hundreds of synthetic interactions, supporting the known roles of Hrq1 and Sgs1 in DNA repair, as well as suggesting novel connections to rRNA processing, mitochondrial DNA maintenance, transcription, and lagging strand synthesis during DNA replication.

Download Full-text

gon-2, a Gene Required for Gonadogenesis in Caenorhabditis elegans

Genetics ◽

10.1093/genetics/147.3.1077 ◽

1997 ◽

Vol 147 (3) ◽

pp. 1077-1089 ◽

Cited By ~ 3

Author(s):

Andrew Y Sun ◽

Eric J Lambie

Keyword(s):

Caenorhabditis Elegans ◽

Larval Stage ◽

Cell Lineage ◽

Temperature Sensitive ◽

Loss Of Function ◽

Primary Defect ◽

Cell Fates ◽

Fourth Larval Stage ◽

Full Complement ◽

Sensitive Allele

The gonad of the Caenorhabditis elegans hermaphrodite is generated by the postembryonic divisions of two somatic precursors, Z1 and Z4, and two germline precursors, Z2 and Z3. These cells begin division midway through the first larval stage. By the end of the fourth larval stage, Z1 and Z4 produce 143 descendants, while Z2 and Z3 give rise to ∼1000 descendants. The divisions of Z2 and Z3 are dependent on signals produced by Z1 and Z4, but not vice versa. We have characterized the properties of five loss-of-function alleles of a newly described gene, which we call gon-2. In gon-2 mutants, gonadogenesis is severely impaired; in some animals, none of the gonad progenitors undergo any postembryonic divisions. Mutations in gon-2 have a partial maternal effect: either maternal or zygotic expression is sufficient to prevent the severe gonadogenesis defects. By cell lineage analysis, we found that the primary defect in gon-2 mutants is a delay (sometimes a complete block) in the onset and continuation of gonadal divisions. The results of upshift experiments using a temperature-sensitive allele suggest that zygotic expression of gon-2 begins early in embryogenesis, before the birth of Z1 and Z4. The results of downshift experiments suggest that Z1 and Z4 can generate the full complement of gonadal tissues even when gon-2 function is inhibited until the end of the second larval stage. Thus, gon-2 activity is probably not required for the specification of gonadal cell fates, but appears to be generally required for gonadal cell divisions.

Download Full-text

A Conserved NRDE-2/MTR-4 Complex Mediates Nuclear RNAi in Caenorhabditis elegans

Genetics ◽

10.1534/genetics.120.303631 ◽

2020 ◽

Vol 216 (4) ◽

pp. 1071-1085

Author(s):

Gang Wan ◽

Jenny Yan ◽

Yuhan Fei ◽

Daniel J. Pagano ◽

Scott Kennedy

Keyword(s):

Caenorhabditis Elegans ◽

Gene Silencing ◽

Small Interfering Rnas ◽

Interacting Protein ◽

C Elegans ◽

Link Type ◽

Evolutionarily Conserved ◽

Small Regulatory Rnas ◽

Regulatory Rnas ◽

Nuclear Rnai

Small regulatory RNAs, such as small interfering RNAs (siRNAs) and PIWI-interacting RNAs, regulate splicing, transcription, and genome integrity in many eukaryotes. In Caenorhabditis elegans, siRNAs bind nuclear Argonautes (AGOs), which interact with homologous premessenger RNAs to recruit downstream silencing effectors, such as NRDE-2, to direct cotranscriptional gene silencing [or nuclear RNA interference (RNAi)]. To further our understanding of the mechanism of nuclear RNAi, we conducted immunoprecipitation-mass spectrometry on C. elegansNRDE-2. The major NRDE-2 interacting protein identified was the RNA helicase MTR-4. Co-immunoprecipitation analyses confirmed a physical association between NRDE-2 and MTR-4. MTR-4 colocalizes with NRDE-2 within the nuclei of most/all C. elegans somatic and germline cells. MTR-4 is required for nuclear RNAi, and interestingly, MTR-4 is recruited to premessenger RNAs undergoing nuclear RNAi via a process requiring nuclear siRNAs, the nuclear AGO HRDE-1, and NRDE-2, indicating that MTR-4 is a component of the C. elegans nuclear RNAi machinery. Finally, we confirm previous reports showing that human (Hs)NRDE2 and HsMTR4 also physically interact. Our data show that the NRDE-2/MTR-4 interactions are evolutionarily conserved, and that, in C. elegans, the NRDE-2/MTR-4 complex contributes to siRNA-directed cotranscriptional gene silencing.

Download Full-text

Nicotinamide Supplementation Improves Oocyte Quality and Offspring Development by Modulating Mitochondrial Function in an Aged Caenorhabditis elegans Model

Antioxidants ◽

10.3390/antiox10040519 ◽

2021 ◽

Vol 10 (4) ◽

pp. 519

Author(s):

Hyemin Min ◽

Mijin Lee ◽

Kyoung Sang Cho ◽

Hyunjung Jade Lim ◽

Yhong-Hee Shim

Keyword(s):

Caenorhabditis Elegans ◽

Mitochondrial Function ◽

Stress Responses ◽

Oocyte Quality ◽

Embryonic Lethality ◽

Reproductive Aging ◽

Control Female ◽

Offspring Development ◽

Developmental Growth ◽

Ros Accumulation

Aging is associated with a decline in the quality of biological functions. Among the aging processes, reproductive aging is a critical process because of its intergenerational effects. However, the mechanisms underlying reproductive aging remain largely unknown. Female reproductive aging is the primary reason for limited fertility in mammals. Therefore, we attempted to investigate a modulator that can control female reproductive aging using a Caenorhabditis elegans model. In the present study, we examined the role of nicotinamide (NAM) in oocyte quality and offspring development. The levels of reactive oxygen species (ROS) and oxidative stress responses in aged oocytes, embryonic lethality, and developmental growth of the offspring were examined with maternal NAM supplementation. Supplementation with NAM improved oocyte quality, decreased embryonic lethality, and promoted germ cell apoptosis. Furthermore, NAM supplementation in aged mothers reduced ROS accumulation and improved mitochondrial function in oocytes. Consequently, the developmental growth and motility of offspring were improved. These findings suggest that NAM supplementation improves the health of the offspring produced by aged mothers through improved mitochondrial function. Taken together, our results imply that NAM supplementation in the aged mother improves oocyte quality and protects offspring by modulating mitochondrial function.

Download Full-text

The Novel Adaptor Protein, Mti1p, and Vrp1p, a Homolog of Wiskott-Aldrich Syndrome Protein-Interacting Protein (WIP), May Antagonistically Regulate Type I Myosins in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/160.3.923 ◽

2002 ◽

Vol 160 (3) ◽

pp. 923-934

Author(s):

Junko Mochida ◽

Takaharu Yamamoto ◽

Konomi Fujimura-Kamada ◽

Kazuma Tanaka

Keyword(s):

Adaptor Protein ◽

Actin Binding ◽

Temperature Sensitive ◽

Null Mutation ◽

Type I ◽

Cortical Actin ◽

Tail Region ◽

Interacting Protein ◽

Wiskott Aldrich Syndrome ◽

Syndrome Protein

Abstract Type I myosins in yeast, Myo3p and Myo5p (Myo3/5p), are involved in the reorganization of the actin cytoskeleton. The SH3 domain of Myo5p regulates the polymerization of actin through interactions with both Las17p, a homolog of mammalian Wiskott-Aldrich syndrome protein (WASP), and Vrp1p, a homolog of WASP-interacting protein (WIP). Vrp1p is required for both the localization of Myo5p to cortical patch-like structures and the ATP-independent interaction between the Myo5p tail region and actin filaments. We have identified and characterized a new adaptor protein, Mti1p (Myosin tail region-interacting protein), which interacts with the SH3 domains of Myo3/5p. Mti1p co-immunoprecipitated with Myo5p and Mti1p-GFP co-localized with cortical actin patches. A null mutation of MTI1 exhibited synthetic lethal phenotypes with mutations in SAC6 and SLA2, which encode actin-bundling and cortical actin-binding proteins, respectively. Although the mti1 null mutation alone did not display any obvious phenotype, it suppressed vrp1 mutation phenotypes, including temperature-sensitive growth, abnormally large cell morphology, defects in endocytosis and salt-sensitive growth. These results suggest that Mti1p and Vrp1p antagonistically regulate type I myosin functions.

Download Full-text

Two Pleiotropic Classes of daf-2 Mutation Affect Larval Arrest, Adult Behavior, Reproduction and Longevity in Caenorhabditis elegans

Genetics ◽

10.1093/genetics/150.1.129 ◽

1998 ◽

Vol 150 (1) ◽

pp. 129-155 ◽

Cited By ~ 23

Author(s):

David Gems ◽

Amy J Sutton ◽

Mark L Sundermeyer ◽

Patrice S Albert ◽

Kevin V King ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Adult Longevity ◽

Temperature Sensitive ◽

Adult Behavior ◽

Dauer Larva ◽

Class 1 ◽

Low Levels ◽

Dauer Larvae ◽

Overlapping Classes ◽

Receptor Family

Abstract The nematode Caenorhabditis elegans responds to overcrowding and scarcity of food by arresting development as a dauer larva, a nonfeeding, long-lived, stress-resistant, alternative third-larval stage. Previous work has shown that mutations in the genes daf-2 (encoding a member of the insulin receptor family) and age-1 (encoding a PI 3-kinase) result in constitutive formation of dauer larvae (Daf-c), increased adult longevity (Age), and increased intrinsic thermotolerance (Itt). Some daf-2 mutants have additional developmental, behavioral, and reproductive defects. We have characterized in detail 15 temperature-sensitive and 1 nonconditional daf-2 allele to investigate the extent of daf-2 mutant defects and to examine whether specific mutant traits correlate with each other. The greatest longevity seen in daf-2 mutant adults was approximately three times that of wild type. The temperature-sensitive daf-2 mutants fell into two overlapping classes, including eight class 1 mutants, which are Daf-c, Age, and Itt, and exhibit low levels of L1 arrest at 25.5°. Seven class 2 mutants also exhibit the class 1 defects as well as some or all of the following: reduced adult motility, abnormal adult body and gonad morphology, high levels of embryonic and L1 arrest, production of progeny late in life, and reduced brood size. The strengths of the Daf-c, Age, and Itt phenotypes largely correlated with each other but not with the strength of class 2-specific defects. This suggests that the DAF-2 receptor is bifunctional. Examination of the null phenotype revealed a maternally rescued egg, L1 lethal component, and a nonconditional Daf-c component. With respect to the Daf-c phenotype, the dauer-defective (Daf-d) mutation daf-12(m20) was epistatic to daf-2 class 1 alleles but not the severe class 2 alleles tested. All daf-2 mutant defects were suppressed by the daf-d mutation daf-16(m26). Our findings suggest a new model for daf-2, age-1, daf-12, and daf-16 interactions.

Download Full-text