scholarly journals SUPPRESSION AND FUNCTION OF X-LINKED LETHAL AND STERILE MUTATIONS IN CAENORHABDITIS ELEGANS

Genetics ◽  
1981 ◽  
Vol 97 (1) ◽  
pp. 65-84
Author(s):  
Philip M Meneely ◽  
Robert K Herman
Keyword(s):  
Caenorhabditis Elegans ◽  
Linkage Map ◽  
X Chromosome ◽  
The Other ◽  
Null Alleles ◽  
Wild Type ◽  
Recessive Lethal ◽  
X Linkage ◽  
And Function ◽  
Null Mutants

ABSTRACT We have expanded our collection of recessive lethal and sterile mutants in the region of the X chromosome balanced by mnDp1(X;V), about 15% of the X linkage map, to a total of 54 mutants. The mutations have been mapped with respect to 20 overlapping deficiencies and five X duplications, and they have been assigned to 24 genes by complementation testing. Nine mutants are hermaphrodite-sterile: one of these is a sperm-defect mutant, two have abnormal gonadogeneses and six, in five genes, are maternally influenced mutants, producing inviable zygote progeny. One of the gonadogenesis mutants and two of the maternally influenced mutants are male fertile. All but one of the maternally influenced mutants give cross progeny when mated with wild-type males. Forty-three mutants were tested for suppression by homozygous sup-5(e1464), which is believed to be specific for null alleles. Ten mutants that were judged by independent criteria not to be null mutants are not suppressed. Nine of the other 33 mutants, in nine genes, are suppressed, five in both heterozygous and homozygous suppressor stocks and four only in homozygous suppressor stocks.

scholarly journals An efficient genome editing strategy to generate putative null mutants in Caenorhabditis elegans using CRISPR/Cas9

2018 ◽  
Author(s):  
Han Wang ◽  
Heenam Park ◽  
Jonathan Liu ◽  
Paul W. Sternberg
Keyword(s):  
Dna Repair ◽  
Caenorhabditis Elegans ◽  
Genome Editing ◽  
Gene Function ◽  
Unique Sequence ◽  
Target Site ◽  
Null Alleles ◽  
Wild Type ◽  
C Elegans ◽  
Null Mutants

AbstractNull mutants are essential for analyzing gene function. Here, we describe a simple and efficient method to generate Caenorhabditis elegans null mutants using CRISPR/Cas9 and short single stranded DNA oligo repair templates to insert a universal 43-nucleotide-long stop knock-in (STOP-IN) cassette into the early exons of target genes. This cassette has stop codons in all three reading frames and leads to frameshifts, which will generate putative null mutations regardless of the reading frame of the insertion position in exons. The STOP-IN cassette also contains an exogenous Cas9 target site that allows further genome editing and provides a unique sequence that simplifies the identification of successful insertion events via PCR. As a proof of concept, we inserted the STOP-IN cassette right at a Cas9 target site in aex-2 to generate new putative null alleles by injecting preassembled Cas9 ribonucleoprotein and a short synthetic single stranded DNA repair template containing the STOP-IN cassette and two 35-nucleotide-long homology arms identical to the sequences flanking the Cas9 cut site. We showed that these new aex-2 alleles phenocopied an existing loss-of-function allele of aex-2. We further showed that the new aex-2 null alleles could be reverted back to the wild-type sequence by targeting exogenous Cas9 cut site included in the STOP-IN cassette and providing a single stranded wild-type DNA repair oligo. We applied our STOP-IN method to generate new putative null mutants for additional 20 genes, including three pharyngeal muscle-specific genes (clik-1, clik-2, and clik-3), and reported a high insertion rate (46%) based on the animals we screened. We showed that null mutations of clik-2 cause recessive lethality with a severe pumping defect and clik-3 null mutants have a mild pumping defect, while clik-1 is dispensable for pumping. We expect that the knock-in method using the STOP-IN cassette will facilitate the generation of new null mutants to understand gene function in C. elegans and other genetic model organisms.SummaryWe report a simple and efficient CRISPR/Cas9 genome editing strategy to generate putative null C. elegans mutants by inserting a small universal stop knock-in (STOP-IN) cassette with stop codons in three frames and frameshifts. The strategy is cloning-free, with the mixture consisting of preassembled Cas9 ribonucleoprotein and single stranded repair DNA oligos directly injected into gonads of adult C. elegans. The universal STOP-IN cassette also contains a unique sequence that simplifies detection of successful knock-in events via PCR and an exogenous Cas9 target sequence that allows further genome editing.

scholarly journals The dpy-30 gene encodes an essential component of the Caenorhabditis elegans dosage compensation machinery.

Genetics ◽  
1994 ◽  
Vol 137 (4) ◽  
pp. 999-1018 ◽  
Author(s):  
D R Hsu ◽  
B J Meyer
Keyword(s):  
Caenorhabditis Elegans ◽  
Sex Determination ◽  
X Chromosome ◽  
Dosage Compensation ◽  
The Other ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Phenotypic Analysis ◽  
General Role ◽  
Essential Component

Abstract The need to regulate X chromosome expression in Caenorhabditis elegans arises as a consequence of the primary sex-determining signal, the X/A ratio (the ratio of X chromosomes to sets of autosomes), which directs 1X@A animals to develop as males and 2X/2A animals to develop as hermaphrodites. C. elegans possesses a dosage compensation mechanism that equalizes X chromosome expression between the two sexes despite their disparity in X chromosome dosage. Previous genetic analysis led to the identification of four autosomal genes, dpy-21, dpy-26, dpy-27 and dpy-28, whose products are essential in XX animals for proper dosage compensation, but not for sex determination. We report the identification and characterization of dpy-30, an essential component of the dosage compensation machinery. Putative null mutations in dpy-30 disrupt dosage compensation and cause a severe maternal-effect, XX-specific lethality. Rare survivors of the dpy-30 lethality are dumpy and express their X-linked genes at higher than wild-type levels. These dpy-30 mutant phenotypes superficially resemble those caused by mutations in dpy-26, dpy-27 and dpy-28; however, detailed phenotypic analysis reveals important differences that distinguish dpy-30 from these genes. In contrast to the XX-specific lethality caused by mutations in the other dpy genes, the XX-specific lethality caused by dpy-30 mutations is completely penetrant and temperature sensitive. In addition, unlike the other genes, dpy-30 is required for the normal development of XO animals. Although dpy-30 mutations do not significantly affect the viability of XO animals, they do cause them to be developmentally delayed and to possess numerous morphological and behavioral abnormalities. Finally, dpy-30 mutations can dramatically influence the choice of sexual fate in animals with an ambiguous sexual identity, despite having no apparent effect on the sexual phenotype of otherwise wild-type animals. Paradoxically, depending on the genetic background, dpy-30 mutations cause either masculinization or feminization, thus revealing the complex regulatory relationship between the sex determination and dosage compensation processes. The novel phenotypes caused by dpy-30 mutations suggest that in addition to acting in the dosage compensation process, dpy-30 may play a more general role in the development of both XX and XO animals.

scholarly journals A VISIBLE ALLELE OF THE MUSCLE GENE sup-10 X OF C. ELEGANS

Genetics ◽  
1986 ◽  
Vol 113 (1) ◽  
pp. 63-72
Author(s):  
Iva Greenwald ◽  
H Robert Horvitz
Keyword(s):  
Caenorhabditis Elegans ◽  
Protein Complex ◽  
Structure And Function ◽  
The Other ◽  
Wild Type ◽  
C Elegans ◽  
Muscle Structure ◽  
New Gene ◽  
Muscle Gene ◽  
And Function

ABSTRACT In this paper, we extend our previous analyses of a set of genes in Caenorhabditis elegans that are involved in muscle structure and function: unc-93 III, sup-9 II, sup-10 X and sup-11 I. We describe an unusual, visible allele of sup-10, examine how this allele interacts genetically with mutations in other genes of this set and propose that the wild-type products of the unc-93 and sup-10 loci may be components of a protein complex. We also describe a new gene of this set, sup-18 III, and the interaction of sup-18 alleles with mutations in the other genes.

scholarly journals Characterization of an Unstable Allele of the Arabidopsis HY4 Locus

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1575-1585
Author(s):  
Edward P Bruggemann ◽  
Bernard Doan ◽  
Korie Handwerger ◽  
Gisela Storz
Keyword(s):  
Fast Neutron ◽  
Blue Light ◽  
Large Deletion ◽  
The Other ◽  
Epigenetic Mechanisms ◽  
Loss Of Function ◽  
Wild Type ◽  
Unusual Behavior ◽  
Recessive Lethal

Abstract The Arabidopsis HY4 gene encodes the nonessential blue light photoreceptor CRY1. Loss-of-function hy4 mutants have an elongated hypocotyl phenotype after germination under blue light. We previously analyzed 20 independent hy4 alleles produced by fast neutron mutagenesis. These alleles were grouped into two classes based on their genetic behavior and corresponding deletion size: (1) null hy4 alleles that were semidominant over wild type and contained small or moderate-sized deletions at HY4 and (2) null hy4 alleles that were recessive lethal and contained large HY4 deletions. Here we describe one additional fast neutron hy4 mutant, B144, that did not fall into either of these two classes. Mutant B144 was isolated as a heterozygote with an intermediate hy4 phenotype. One allele from this mutant, hy4-B144Δ, contains a large deletion at HY4 and is recessive lethal. The other allele from this mutant, HY4-B144*, appears to be intact and functional but is unstable and spontaneously converts to a nonfunctional hy4 allele. In addition, HY4-B144* is lethal in homozygotes and suppresses local recombination. We discuss genetic and epigenetic mechanisms that may account for the unusual behavior of the HY4-B144* allele.

CAENORHABDITIS ELEGANS DEFICIENCY MAPPING

Genetics ◽  
1984 ◽  
Vol 108 (2) ◽  
pp. 331-345
Author(s):  
D Christine Sigurdson ◽  
Gail J Spanier ◽  
Robert K Herman
Keyword(s):  
Caenorhabditis Elegans ◽  
Linkage Group ◽  
Ethyl Methanesulfonate ◽  
Single Site ◽  
Crossing Over ◽  
Wild Type ◽  
Nematode Caenorhabditis Elegans ◽  
Recessive Lethal ◽  
X Ray ◽  
New Mutations

ABSTRACT Six schemes were used to identify 80 independent recessive lethal deficiencies of linkage group (LG) II following X-ray treatment of the nematode Caenorhabditis elegans. Complementation tests between the deficiencies and ethyl methanesulfonate-induced recessive visible, lethal and sterile mutations and between different deficiencies were used to characterize the extents of the deficiencies. Deficiency endpoints thus helped to order 36 sites within a region representing about half of the loci on LG II and extending over about 5 map units. New mutations occurring in this region can be assigned to particular segments of the map by complementation tests against a small number of deficiencies; this facilitates the assignment of single-site mutations to particular genes, as we illustrate. Five sperm-defective and five oocyte-defective LG II sterile mutants were identified and mapped. Certain deficiency-by-deficiency complementation tests allowed us to suggest that the phenotypes of null mutations at two loci represented by visible alleles are wild type and that null mutations at a third locus confer a visible phenotype. A segment of LG II that is about 12 map units long and largely devoid of identified loci seems to be greatly favored for crossing over.

scholarly journals UNC-98 and UNC-96 Interact with Paramyosin to Promote Its Incorporation into Thick Filaments of Caenorhabditis elegans

2008 ◽  
Vol 19 (4) ◽  
pp. 1529-1539 ◽  
Author(s):  
Rachel K. Miller ◽  
Hiroshi Qadota ◽  
Kristina B. Mercer ◽  
Kim M. Gernert ◽  
Guy M. Benian
Keyword(s):  
Caenorhabditis Elegans ◽  
Light Microscopy ◽  
Polarized Light ◽  
Wild Type ◽  
Binding Region ◽  
Yeast Two Hybrid ◽  
Thick Filaments ◽  
Separate Region ◽  
Two Hybrid ◽  
Null Mutants

Mutations in unc-96 or -98 cause reduced motility and a characteristic defect in muscle structure: by polarized light microscopy birefringent needles are found at the ends of muscle cells. Anti-paramyosin stains the needles in unc-96 and -98 mutant muscle. However there is no difference in the overall level of paramyosin in wild-type, unc-96, and -98 animals. Anti-UNC-98 and anti-paramyosin colocalize in the paramyosin accumulations of missense alleles of unc-15 (encodes paramyosin). Anti-UNC-96 and anti-UNC-98 have diffuse localization within muscles of unc-15 null mutants. By immunoblot, in the absence of paramyosin, UNC-98 is diminished, whereas in paramyosin missense mutants, UNC-98 is increased. unc-98 and -15 or unc-96 and -15 interact genetically either as double heterozygotes or as double homozygotes. By yeast two-hybrid assay and ELISAs using purified proteins, UNC-98 interacts with paramyosin residues 31-693, whereas UNC-96 interacts with a separate region of paramyosin, residues 699-798. The importance of surface charge of this 99 residue region for UNC-96 binding was shown. Paramyosin lacking the C-terminal UNC-96 binding region fails to localize throughout A-bands. We propose a model in which UNC-98 and -96 may act as chaperones to promote the incorporation of paramyosin into thick filaments.

scholarly journals Lethal and amanitin-resistance mutations in the Caenorhabditis elegans ama-1 and ama-2 genes.

Genetics ◽  
1988 ◽  
Vol 120 (2) ◽  
pp. 409-422
Author(s):  
T M Rogalski ◽  
A M Bullerjahn ◽  
D L Riddle
Keyword(s):  
Caenorhabditis Elegans ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Resistance Mutations ◽  
Recessive Lethal ◽  
New Gene ◽  
Alpha Amanitin

Abstract Mutants of Caenorhabditis elegans resistant to alpha-amanitin have been isolated at a frequency of about 1.6 x 10(-6) after EMS mutagenesis of the wild-type strain, N2. Four new dominant resistance mutations have been studied genetically. Three are alleles of a previously identified gene, ama-1 IV, encoding the largest subunit of RNA polymerase II. The fourth mutation defines a new gene, ama-2 V. Unlike the ama-1 alleles, the ama-2 mutation exhibits a recessive-lethal phenotype. Growth and reproduction of N2 was inhibited at a concentration of 10 micrograms/ml amanitin, whereas ama-2/+ animals were inhibited at 100 micrograms/ml, and 800 micrograms/ml was required to inhibit growth of ama-1/+ larvae. We have also determined that two reference strains used for genetic mapping, dpy-11(e224)V and sma-1(e30)V, are at least four-fold more sensitive to amanitin that the wild-type strain. Using an amanitin-resistant ama-1(m118) or ama-1(m322) strain as a parent, we have isolated amanitin-sensitive mutants that carry recessive-lethal ama-1 alleles. The frequency of EMS-induced lethal ama-1 mutations is approximately 1.7 x 10(-3), 1000-fold higher than the frequency of amanitin-resistance alleles. Nine of the lethal alleles are apparent null mutations, and they exhibit L1-lethal phenotypes at both 20 degrees and 25 degrees. Six alleles result in partial loss of RNA polymerase II function as determined by their sterile phenotypes at 20 degrees. All but one of these latter mutations exhibit a more severe phenotype at 25 degrees C. We have also selected seven EMS-induced revertants of three different ama-1 lethals. These revertants restore dominant resistance to amanitin. The selection for revertants also produced eight new dominant amanitin resistance alleles on the balancer chromosome, nT1.

scholarly journals A SECOND INFORMATIONAL SUPPRESSOR, SUP-7 X, IN CAENORHABDITIS ELEGANS

Genetics ◽  
1981 ◽  
Vol 97 (2) ◽  
pp. 307-325
Author(s):  
Robert H Waterston
Keyword(s):  
Suppressor Gene ◽  
Null Alleles ◽  
Chain Gene ◽  
Wild Type ◽  
Heavy Chain Gene ◽  
Myosin Heavy Chain Gene ◽  
C Elegans ◽  
X Linkage ◽  
Suppressor Mutations ◽  
Dose Dependent

ABSTRACT More than 30 independent suppressor mutations have been obtained in the nematode C. elegans through reversion analysis of two unc-13 mutants. Many of the new isolates map to the region of the previously identified informational suppressor, sup-5 III (Waterston and Brenner 1978). Several of the other suppressor mutations map to the left half of the X-linkage group and define a second suppressor gene, sup-7 X. In tests against 40 mutations in six genes, the sup-7(st5) allele was found to suppress to a greater extent the same alleles acted on by sup-5(e1464). Like sup-5(e1464), sup-7(st5) acts on null alleles of the myosin heavy-chain gene unc-54 I (MacLeod et al. 1977; MacLeod, Waterston and Brenner 1977) and the putative paramyosin gene unc-15 I (Waterston et al. 1977). Chemical analysis of unc-15(e1214); sup-7(st5) animals show that paramyosin is restored to more than 30% of the wild-type level. —As was observed for sup-5(e1464), suppression by sup-7(st5) is dose dependent and is greater in animals grown at 15° than at 25°. However, associated with this increased suppression is a decreased viability of sup-7(st5) homozygotes. Reversion of the lethality has resulted in the isolation of deficiency mutations that complement st5 lethality, but lack suppressor function. These properties of sup-7(st5) suggest that it, like sup-5(e1464), is an informational suppressor of null alleles, and its reversion via deficiencies further narrows the possible explanations of its action.

scholarly journals CROSSOVER SUPPRESSORS AND BALANCED RECESSIVE LETHALS IN CAENORHABDITIS ELEGANS

Genetics ◽  
1978 ◽  
Vol 88 (1) ◽  
pp. 49-65
Author(s):  
Robert K Herman
Keyword(s):  
Caenorhabditis Elegans ◽  
Linkage Group ◽  
The Other ◽  
Crossing Over ◽  
Linkage Groups ◽  
Recessive Lethal ◽  
X Ray ◽  
C Elegans ◽  
Complementation Groups ◽  
The Right

ABSTRACT Two dominant suppressors of crossing over have been identified following X-ray treatment of the small nematode C. elegans. They suppress crossing over in linkage group II (LGII) about 100-fold and 50-fold and are both tightly linked to LGII markers. One, called C1, segregates independently of all other linkage groups and is homozygous fertile. The other is a translocation involving LGII and X. The translocation also suppresses rrossing over along the right half of X and is homozygous lethal. CI has been used as a balancer of LGII recessive lethal and sterile mutations induced by EMS. The frequencies of occurrence of lethals and steriles were approximately equal. Fourteen mutations were assigned to complementation groups and mapped. They tended to map in the same region where LGII visibles are clustered.

GENETIC STUDIES OF UNUSUAL LOCI THAT AFFECT BODY SHAPE OF THE NEMATODE CAENORHABDITIS ELEGANS AND MAY CODE FOR CUTICLE STRUCTURAL PROTEINS

Genetics ◽  
1986 ◽  
Vol 113 (3) ◽  
pp. 621-639
Author(s):  
Meredith Kusch ◽  
R S Edgar
Keyword(s):  
Caenorhabditis Elegans ◽  
Body Shape ◽  
Structural Proteins ◽  
Null Alleles ◽  
Wild Type ◽  
Nematode Caenorhabditis Elegans ◽  
Genetic Studies ◽  
Wild Type Phenotype ◽  
Mutant Phenotypes ◽  
Collagen Genes

ABSTRACT In Caenorhabditis elegans, four loci (sqt-1, sqt-2, sqt-3 and rol-8) in which mutations affect body shape and cuticle morphology have unusual genetic properties. (1) Mutant alleles of sqt-1 can interact to produce animals with a variety of mutant phenotypes: left roller, right roller, dumpy and long. At least three mutant phenotypes are specified by mutations in the sqt-3 locus. (2) Most alleles at these loci are either dominant or cryptic dominant (i.e., are dominant only in certain genetic backgrounds). (3) Most alleles of these loci exhibit codominance. (4) Two putative null alleles of the sqt-1 locus produce a wild-type phenotype. (5) Many alleles of these genes demonstrate unusual intergenic interactions that are not the result of simple epistasis: animals doubly heterozygous for mutations at two loci often display unexpected and unpredictable phenotypes. We suggest that these genetic properties might be expected of genes, such as the collagen genes, the products of which interact to form the animal's cuticle, and which are member genes of a gene family.

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