The RNA helicase Ddx52 functions as a growth switch in juvenile zebrafish

Vertebrate animals usually display robust growth trajectories during juvenile stages, and reversible suspension of this growth momentum by a single genetic determinant has not been reported. Here, we report a single genetic factor that is essential for juvenile growth in zebrafish. Using a forward genetic screen, we recovered a temperature-sensitive allele, pan (after Peter Pan), that suspends whole-organism growth at juvenile stages. Remarkably, even after growth is halted for a full 8-week period, pan mutants are able to resume a robust growth trajectory after release from the restrictive temperature, eventually growing into fertile adults without apparent adverse phenotypes. Positional cloning and complementation assays revealed that pan encodes a probable ATP-Dependent RNA Helicase (DEAD-Box Helicase 52; ddx52) that maintains the level of 47S precursor ribosomal RNA. Furthermore, genetic silencing of ddx52 and pharmacological inhibition of bulk RNA transcription similarly suspend the growth of flies, zebrafish and mice. Our findings reveal evidence that safe, reversible pauses of juvenile growth can be mediated by targeting the activity of a single gene, and that its pausing mechanism has high evolutionary conservation.

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The Mitochondrial Nucleoid Protein, Mgm101p, of Saccharomyces cerevisiae Is Involved in the Maintenance of ρ+ and ori/rep-Devoid Petite Genomes but Is Not Required for Hypersuppressive ρ− mtDNA

Genetics ◽

10.1093/genetics/160.4.1389 ◽

2002 ◽

Vol 160 (4) ◽

pp. 1389-1400

Author(s):

Xiao Ming Zuo ◽

G Desmond Clark-Walker ◽

Xin Jie Chen

Keyword(s):

Saccharomyces Cerevisiae ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Mitochondrial Rna ◽

Mtdna Copy Number ◽

Mitochondrial Rna Polymerase ◽

Mitochondrial Nucleoids ◽

Mtdna Replication ◽

Sensitive Allele ◽

Discriminatory Effect

Abstract The Saccharomyces cerevisiae MGM101 gene encodes a DNA-binding protein targeted to mitochondrial nucleoids. MGM101 is essential for maintenance of a functional ρ+ genome because meiotic segregants, with a disrupted mgm101 allele, cannot undergo more than 10 divisions on glycerol medium. Quantitative analysis of mtDNA copy number in a ρ+ strain carrying a temperature-sensitive allele, mgm101-1, revealed that the amount of mtDNA is halved each cell division upon a shift to the restrictive temperature. These data suggest that mtDNA replication is rapidly blocked in cells lacking MGM101. However, a small proportion of meiotic segregants, disrupted in MGM101, have ρ− genomes that are stably maintained. Interestingly, all surviving ρ− mtDNAs contain an ori/rep sequence. Disruption of MGM101 in hypersuppressive (HS) strains does not have a significant effect on the propagation of HS ρ− mtDNA. However, in petites lacking an ori/rep, disruption of MGM101 leads to either a complete loss or a dramatically decreased stability of mtDNA. This discriminatory effect of MGM101 suggests that replication of ρ+ and ori/rep-devoid ρ− mtDNAs is carried out by the same process. By contrast, the persistence of ori/rep-containing mtDNA in HS petites lacking MGM101 identifies a distinct replication pathway. The alternative mtDNA replication mechanism provided by ori/rep is independent of mitochondrial RNA polymerase encoded by RPO41 as a HS ρ− genome is stably maintained in a mgm101, rpo41 double mutant.

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Dad1p, Third Component of the Duo1p/Dam1p Complex Involved in Kinetochore Function and Mitotic Spindle Integrity

Molecular Biology of the Cell ◽

10.1091/mbc.12.9.2601 ◽

2001 ◽

Vol 12 (9) ◽

pp. 2601-2613 ◽

Cited By ~ 44

Author(s):

Maria Enquist-Newman ◽

Iain M. Cheeseman ◽

David Van Goor ◽

David G. Drubin ◽

Pamela B. Meluh ◽

...

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

The Novel ◽

Yeast Saccharomyces Cerevisiae ◽

Sensitive Allele ◽

Two Hybrid ◽

Kinetochore Function

We showed recently that a complex between Duo1p and Dam1p is required for both spindle integrity and kinetochore function in the budding yeast Saccharomyces cerevisiae. To extend our understanding of the functions and interactions of the Duo1p/Dam1p complex, we analyzed the novel gene product Dad1p (for Duo1 and Dam1 interacting). Dad1p physically associates with Duo1p by two-hybrid analysis, coimmunoprecipitates with Duo1p and Dam1p out of yeast protein extracts, and shows interdependent localization with Duo1p and Dam1p to the mitotic spindle. These results indicate that Dad1p functions as a component of the Duo1p/Dam1p complex. Like Duo1p and Dam1p, Dad1p also localizes to kinetochore regions in chromosomes spreads. Here, we also demonstrate by chromatin immunoprecipitation that Duo1p, Dam1p, and Dad1p associate specifically with centromeric DNA in a manner that is dependent upon Ndc10 and partially dependent upon the presence of microtubules. To explore the functions of Dad1p in vivo, we generated a temperature-sensitive allele, dad1-1. This allele shows spindle defects and a mitotic arrest phenotype that is dependent upon the spindle assembly checkpoint. In addition, dad1-1 mutants undergo chromosome mis-segregation at the restrictive temperature, resulting in a dramatic decrease in viability.

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Characterization of an essential Saccharomyces cerevisiae gene related to RNA processing: cloning of RNA1 and generation of a new allele with a novel phenotype.

Molecular and Cellular Biology ◽

10.1128/mcb.5.5.907 ◽

1985 ◽

Vol 5 (5) ◽

pp. 907-915 ◽

Cited By ~ 17

Author(s):

N S Atkinson ◽

R W Dunst ◽

A K Hopper

Keyword(s):

Rna Processing ◽

Elevated Temperatures ◽

Growth Defect ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Multicopy Plasmid ◽

Recessive Lethal ◽

Recessive Lethal Mutation ◽

Sensitive Allele

The RNA1 gene product is believed to be involved in RNA metabolism due to the phenotype of a single conditionally lethal, temperature-sensitive allele, rna1-1. We cloned the RNA1 gene and determined that it produces a 1,400-nucleotide polyadenylated transcript. On a multicopy plasmid, the mutant rna1-1 allele partially complements the rna1-1 temperature-sensitive growth defect. This suggests that the temperature-sensitive nature of the rna1-1 allele results from the synthesis of a product with lowered activity or stability at elevated temperatures or from a decrease in synthesis of the rna1-1 product at the restrictive temperature. A chromosomal disruption of RNA1 behaves as a recessive lethal mutation. Haploids bearing the disruption were isolated by sporulating a diploid heterozygous for the disrupted allele and the rna1-1 allele and possessing an episomal copy of the RNA1 gene. Analysis of the rescued haploids bearing the chromosomal disruption indicated that the recessive lethal phenotype of the RNA1 disruption is not merely due to a block in spore germination. Unexpectedly, diploids heterozygous for the disruption and the rna1-1 alleles become aneuploid for chromosome XIII at a frequency of 2 to 5%. It appears that the disrupted RNA1 allele on a multicopy plasmid also promotes aneuploidy for chromosome XIII. Promotion of aneuploidy seems to be a phenotype of this particular allele of RNA1.

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Comprehensive synthetic genetic array analysis of alleles that interact with mutation of the Saccharomyces cerevisiae RecQ helicases Hrq1 and Sgs1

10.1101/2020.08.28.272666 ◽

2020 ◽

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 ◽

Sensitive Allele ◽

Dna Maintenance

ABSTRACTMost 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 sgsl-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.

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Characterization of an essential Saccharomyces cerevisiae gene related to RNA processing: cloning of RNA1 and generation of a new allele with a novel phenotype

Molecular and Cellular Biology ◽

10.1128/mcb.5.5.907-915.1985 ◽

1985 ◽

Vol 5 (5) ◽

pp. 907-915

Author(s):

N S Atkinson ◽

R W Dunst ◽

A K Hopper

Keyword(s):

Rna Processing ◽

Elevated Temperatures ◽

Growth Defect ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Multicopy Plasmid ◽

Recessive Lethal ◽

Recessive Lethal Mutation ◽

Sensitive Allele

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Mutations in polycombeotic, a Drosophila polycomb-group gene, cause a wide range of maternal and zygotic phenotypes.

Genetics ◽

10.1093/genetics/125.1.91 ◽

1990 ◽

Vol 125 (1) ◽

pp. 91-101 ◽

Cited By ~ 8

Author(s):

M D Phillips ◽

A Shearn

Keyword(s):

Extreme Temperature ◽

Polycomb Group ◽

Null Alleles ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Polycomb Group Genes ◽

Wide Range ◽

Small Disc ◽

Sensitive Allele ◽

Anterior Segments

Abstract The polycomb-group genes, a set of genes characterized by mutations that cause similar phenotypes and dosage-dependent interactions, are required for the normal expression of segment-specific homeotic loci. Here we report that polycombeotic (formerly 1(3)1902), originally identified by a lethal mutation that causes a small-disc phenotype, is also a member of this group of essential genes. Adults homozygous for temperature-sensitive pco alleles that were exposed to the restrictive temperature during larval life display the second and third leg to first leg transformation characteristic of polycomb-group mutants. Adult females homozygous for temperature-sensitive alleles exposed to the restrictive temperature during oogenesis produce embryos that show anterior segments with structures normally unique to the eighth abdominal segment, another transformation characteristic of polycomb-group mutants. Mutations in the polycombeotic gene also cause defects not reported for mutations in other polycomb-group genes. Females homozygous for the most extreme temperature-sensitive allele are sterile, and larvae homozygous for null alleles have small imaginal discs and reduced frequencies of mitotic figures in the brain. Dominant mutations originally identified as enhancers or suppressors of zeste are gain-of-function alleles of polycombeotic. The type and variety of defects displayed by different mutations in this gene indicate that the product might be involved in chromosome structure and/or function.

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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.

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MUTATIONS AFFECTING CELL DIVISION IN TETRAHYMENA PYRIFORMIS. I. SELECTION AND GENETIC ANALYSIS

Genetics ◽

10.1093/genetics/83.3.489 ◽

1976 ◽

Vol 83 (3) ◽

pp. 489-506

Author(s):

Joseph Frankel ◽

Leslie M Jenkins ◽

F Paul Doerder ◽

E Marlo Nelsen

Keyword(s):

Cell Division ◽

Genetic Analysis ◽

Single Gene ◽

Specific Effect ◽

Tetrahymena Pyriformis ◽

Recessive Mutation ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Induced Mutations ◽

Mendelian Segregation

ABSTRACT Fourteen nitrosoguanidine-induced mutations that bring about temperature-sensitive morphological abnormalities resulting from a specific effect on cell division have been isolated as heterozygous phenotypic assortants in Tetrahymena pyriformis syngen 1. Genetic analysis revealed all to be single-gene recessives. Detailed analysis of the kinetics of assortment for one of the mutated alleles revealed a rate (0.0104 pure lines per fission) consistent with that previously observed at other loci in this organism. The mutations fall into six complementation groups (mo1, mo2, mo3, mo6, mo8, and mo12). Homozygotes of mo2 are unconditionally expressed, while all alleles of mo1, mo6, mo8, and mo12 are heat sensitive for division arrest. At the mo3 locus two alleles are heat sensitive, one is primarily cold sensitive, while two are sensitive to both heat and cold. Two out of three combinations of different mo3alleles show conventional Mendelian segregation of conditions of expression. Different alleles of mo1, mo3, mo8, and mo12 also manifest differences in penetrance at the restrictive temperature. Despite these differences involving expression, the abnormal phenotypes themselves are locus-specific and distinctive; in the one case (mo1a and mo1b) in which two alleles manifest somewhat different phenotypes, the F1 between them is intermediate. One additional recessive mutation (fat1) brings about a nonconditional lengthening of the cell cycle, with some arrest of cell division at the restrictive temperature. These findings demonstrate that selection of heterozygotes undergoing phenotypic assortment can be an effective method for obtaining substantial numbers of a desired class of temperature-sensitive mutations in T. pyriformis.

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Requirement for Three Novel Protein Complexes in the Absence of the Sgs1 DNA Helicase in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/157.1.103 ◽

2001 ◽

Vol 157 (1) ◽

pp. 103-118 ◽

Cited By ~ 16

Author(s):

Janet R Mullen ◽

Vivek Kaliraman ◽

Samer S Ibrahim ◽

Steven J Brill

Keyword(s):

Saccharomyces Cerevisiae ◽

Genome Stability ◽

Protein Complexes ◽

Ring Finger ◽

Dna Helicase ◽

Open Reading Frames ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Dna Helicases ◽

Cell Extracts

Abstract The Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ family of DNA helicases and is required for genome stability, but not cell viability. To identify proteins that function in the absence of Sgs1, a synthetic-lethal screen was performed. We obtained mutations in six complementation groups that we refer to as SLX genes. Most of the SLX genes encode uncharacterized open reading frames that are conserved in other species. None of these genes is required for viability and all SLX null mutations are synthetically lethal with mutations in TOP3, encoding the SGS1-interacting DNA topoisomerase. Analysis of the null mutants identified a pair of genes in each of three phenotypic classes. Mutations in MMS4 (SLX2) and SLX3 generate identical phenotypes, including weak UV and strong MMS hypersensitivity, complete loss of sporulation, and synthetic growth defects with mutations in TOP1. Mms4 and Slx3 proteins coimmunoprecipitate from cell extracts, suggesting that they function in a complex. Mutations in SLX5 and SLX8 generate hydroxyurea sensitivity, reduced sporulation efficiency, and a slow-growth phenotype characterized by heterogeneous colony morphology. The Slx5 and Slx8 proteins contain RING finger domains and coimmunoprecipitate from cell extracts. The SLX1 and SLX4 genes are required for viability in the presence of an sgs1 temperature-sensitive allele at the restrictive temperature and Slx1 and Slx4 proteins are similarly associated in cell extracts. We propose that the MMS4/SLX3, SLX5/8, and SLX1/4 gene pairs encode heterodimeric complexes and speculate that these complexes are required to resolve recombination intermediates that arise in response to DNA damage, during meiosis, and in the absence of SGS1/TOP3.

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A genetic screen for temperature-sensitive morphogenesis-defectiveCaenorhabditis elegansmutants

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab026 ◽

2021 ◽

Vol 11 (4) ◽

Author(s):

Molly C Jud ◽

Josh Lowry ◽

Thalia Padilla ◽

Erin Clifford ◽

Yuqi Yang ◽

...

Keyword(s):

Cell Shape ◽

The Body ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Fundamental Biological Process ◽

Embryonic Morphogenesis ◽

Cell Shape Changes ◽

Development Temperature ◽

Multicellular Organisms ◽

Shape Changes

AbstractMorphogenesis involves coordinated cell migrations and cell shape changes that generate tissues and organs, and organize the body plan. Cell adhesion and the cytoskeleton are important for executing morphogenesis, but their regulation remains poorly understood. As genes required for embryonic morphogenesis may have earlier roles in development, temperature-sensitive embryonic-lethal mutations are useful tools for investigating this process. From a collection of ∼200 such Caenorhabditis elegans mutants, we have identified 17 that have highly penetrant embryonic morphogenesis defects after upshifts from the permissive to the restrictive temperature, just prior to the cell shape changes that mediate elongation of the ovoid embryo into a vermiform larva. Using whole genome sequencing, we identified the causal mutations in seven affected genes. These include three genes that have roles in producing the extracellular matrix, which is known to affect the morphogenesis of epithelial tissues in multicellular organisms: the rib-1 and rib-2 genes encode glycosyltransferases, and the emb-9 gene encodes a collagen subunit. We also used live imaging to characterize epidermal cell shape dynamics in one mutant, or1219ts, and observed cell elongation defects during dorsal intercalation and ventral enclosure that may be responsible for the body elongation defects. These results indicate that our screen has identified factors that influence morphogenesis and provides a platform for advancing our understanding of this fundamental biological process.

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