A double-mutant collection targeting MAP kinase related genes in Arabidopsis for studying genetic interactions

Alx4 and Cart1 are closely related members of the family of transcription factors that contain the paired-type homeodomain. In contrast to other types of homeodomains, the paired-type homeodomain has been shown to mediate high-affinity sequence-specific DNA binding to palindromic elements as either homodimers or as heterodimers with other family members. Alx4 and Cart1 are co-expressed at several sites during development, including the craniofacial mesenchyme, the mesenchymal derivatives of neural crest cells in the first branchial arch and the limb bud mesenchyme. Because of the molecular similarity and overlapping expression pattern, we have analyzed the functional and genetic relationships between Alx4 and Cart1. The two proteins have similar DNA-binding activity in vitro and can form DNA-binding heterodimers; furthermore, they activate transcription of reporter genes that contain high-affinity DNA-binding sites in cell culture in a similar manner. Therefore, at least by these criteria, the two proteins are functionally redundant. Analysis of double mutant animals reveals several genetic interactions. First, mutation of Cart1 exacerbates Alx4-dependent polydactyly in a manner that is dependent on gene dosage. Second, there are complex genetic interactions in the craniofacial region that reveal a role for both genes in the fusion of the nasal cartilages and proper patterning of the mandible, as well as other craniofacial structures. Third, double mutant mice show a split sternum that is not detected in mice with any other genotype. Interpreted in the context of the biochemical characterization, the genetic analysis suggests that Alx4 and Cart1 are indeed functionally redundant, and reveal both unique and redundant functions for these genes in development.

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Mapping DNA damage-dependent genetic interactions in yeast via party mating and barcode fusion genetics

10.1101/181750 ◽

2017 ◽

Author(s):

J. Javier Díaz-Mejía ◽

Albi Celaj ◽

Joseph C. Mellor ◽

Atina Coté ◽

Attila Balint ◽

...

Keyword(s):

Double Mutant ◽

Genetic Interaction ◽

Culture Conditions ◽

Genetic Interactions ◽

Specific Gene ◽

Functional Relationships ◽

Mutant Strains ◽

Genes Encoding ◽

Standard Culture ◽

Yeast Genetic

AbstractCondition-dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State-of-the-art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double mutant strains, does not scale readily to multi-condition studies. Here we describe Barcode Fusion Genetics to map Genetic Interactions (BFG-GI), by which double mutant strains generated via en masse ‘party’ mating can also be monitored en masse for growth and genetic interactions. By using site-specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG-GI enables multiplexed quantitative tracking of double mutants via next-generation sequencing. We applied BFG-GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4NQO), bleomycin, zeocin, and three other DNA-damaging environments. BFG-GI recapitulated known genetic interactions and yielded new condition-dependent genetic interactions. We validated and further explored a subnetwork of condition-dependent genetic interactions involving MAG1, SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to a decrease in the activation or activity of the checkpoint protein kinase Rad53.

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Genetic Analysis of the Caenorhabditis elegans MAP Kinase Gene mpk-1

Genetics ◽

10.1093/genetics/150.1.103 ◽

1998 ◽

Vol 150 (1) ◽

pp. 103-117 ◽

Cited By ~ 3

Author(s):

Mark R Lackner ◽

Stuart K Kim

Keyword(s):

Transcription Factor ◽

Caenorhabditis Elegans ◽

Map Kinase ◽

Double Mutant ◽

Cell Fates ◽

Gain Of Function ◽

Kinase Gene ◽

Epistasis Analysis ◽

Anchor Cell ◽

Ets Transcription Factor

Abstract The Caenorhabditis elegans mpk-1 gene encodes a MAP kinase protein that plays an important role in Ras-mediated induction of vulval cell fates. We show that mutations that eliminate mpk-1 activity result in a highly penetrant, vulvaless phenotype. A double mutant containing a gain-of-function mpk-1 mutation and a gain-of-function mek mutation (MEK phosphorylates and activates MPK-1) exhibits a multivulva phenotype. These results suggest that mpk-1 may transduce most or all of the anchor cell signal. Epistasis analysis suggests that mpk-1 acts downstream of mek-2 (encodes a MEK homolog) and upstream of lin-1 (encodes an Ets transcription factor) in the anchor cell signaling pathway. Finally, mpk-1 may act together with let-60 ras in multiple developmental processes, as mpk-1 mutants exhibit nearly the same range of developmental phenotypes as let-60 ras mutants.

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Seizure Suppression by shakB2, a Gap Junction Mutation in Drosophila

Journal of Neurophysiology ◽

10.1152/jn.01059.2004 ◽

2006 ◽

Vol 95 (2) ◽

pp. 627-635 ◽

Cited By ~ 23

Author(s):

Juan Song ◽

Mark A. Tanouye

Keyword(s):

Gap Junction ◽

Double Mutant ◽

Model System ◽

Genetic Interactions ◽

High Threshold ◽

Seizure Susceptibility ◽

Wild Type ◽

Giant Fiber ◽

Seizure Disorders ◽

Junction Proteins

Gap junction proteins mediate electrical synaptic transmission. In Drosophila, flies carrying null mutations in the shakB locus, such as shakB2, have behavioral and electrophysiological defects in the giant fiber (GF) system neurocircuit consistent with a loss of transmission at electrical synapses. The shakB2 mutation also affects seizure susceptibility. Mutant flies are especially seizure-resistant and have a high threshold to evoked seizures. In addition, in some double mutant combinations with “epilepsy” mutations, shakB2 appears to act as a seizure-suppressor mutation: shakB2 restores seizure susceptibility to the wild-type range in the double mutant. In double mutant combinations, shakB2 completely suppresses seizures caused by slamdance ( sda) , knockdown ( kdn), and jitterbug ( jbug) mutations. Seizures caused by easily shocked ( eas) and technical knockout ( tko) mutations are partially suppressed by shakB2. Seizures caused by bang-sensitive ( bas2) and bang- senseless ( bss1, bss2 alleles) mutations are not suppressed by shakB2. These results show the use of Drosophila as a model system for studying the kinds of genetic interactions responsible for seizure susceptibility, bringing us closer to unraveling the complexity of seizure disorders in humans.

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Identifying in vivo pathways using genome-wide genetic networks

Biochemical Society Transactions ◽

10.1042/bst0351538 ◽

2007 ◽

Vol 35 (6) ◽

pp. 1538-1541 ◽

Cited By ~ 3

Author(s):

J.V. Gray ◽

S.A. Krause

Keyword(s):

Double Mutant ◽

Biological Pathways ◽

Genetic Networks ◽

Genetic Interactions ◽

Yeast Genome ◽

Gene Products ◽

Global Networks ◽

Genome Wide ◽

Yeast Genes

Synthetic genetic interactions occur between two genes when the double mutant displays a phenotype much more severe than does either single mutant alone. Global networks of such interactions are now being systematically determined, spearheaded by the budding yeast genome. Genetic interactions reflect in vivo relationships between gene products. Extracting that functional information from such genetic networks is now possible by exploiting and modifying the key concept of congruence. Here, we focus on synthetic genetic interactions between pairs of null mutations in non-essential yeast genes. We summarize how to identify biological pathways from these emerging networks, using illustrative examples.

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Pigmentation and lysosome function in mice homozygous for both pale ear and Beige-J pigment genes

Genetics Research ◽

10.1017/s001667230001404x ◽

1980 ◽

Vol 35 (2) ◽

pp. 195-204 ◽

Cited By ~ 6

Author(s):

Edward K. Novak ◽

Richard T. Swank ◽

Miriam H. Meisler

Keyword(s):

Lysosomal Enzyme ◽

Double Mutant ◽

Gene Dosage ◽

Cumulative Effect ◽

Enzyme Concentration ◽

Genetic Interactions ◽

Gene Dosage Effect ◽

Pigment Genes ◽

Independent Effects ◽

Pigmentation Phenotype

SUMMARYWe have examined mice doubly homozygous for both pale ear (ep/ep) and beige (bgJ/bgJ) mutations in order to detect genetic interactions between these 2 loci affecting pigmentation and lysosome physiology. The doubly homozygous mouse has a new pigmentation phenotype consistent with independent effects ofepandbg. The beige (Brandt, Elliott & Swank, 1975) and pale ear (Novak & Swank, 1979)genes have abnormal kidney lysosomal enzyme accumulation caused by defective secretion into urine. No cumulative effect on these functions was observed in the new double mutant phenotype. The new phenotype has giant lysosomes typical of the beige mutation. Unexpectedly, the beige gene corrects the effect of the pale ear on serum lysosomal enzyme concentration. There is also a gene dosage effect of the beige gene on this serum lysosomal enzyme phenotype. The results suggest that the beige and pale ear genes affect the same pathway(s) of lysosome biosynthesis and/or processing. The action of the beige gene may precede that of the pale ear gene in lysosome physiology.

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Saccharomyces cerevisiae PAC2 Functions With CIN1, 2 and 4 in a Pathway Leading to Normal Microtubule Stability

Genetics ◽

10.1093/genetics/146.3.849 ◽

1997 ◽

Vol 146 (3) ◽

pp. 849-857 ◽

Cited By ~ 7

Author(s):

M Andrew Hoyt ◽

Jennifer P Macke ◽

B Tibor Roberts ◽

John R Geiser

Keyword(s):

Saccharomyces Cerevisiae ◽

Double Mutant ◽

Genetic Interactions ◽

Deletion Mutants ◽

Guanine Nucleotide ◽

Microtubule Stability ◽

Cold Sensitive ◽

Guanine Nucleotide Binding ◽

Functional Pathway ◽

Microtubule Function

The products of the Saccharomyces cerevisiae CIN1, CIN2 and CIN4 genes participate in a nonessential pathway required for normal microtubule function. In this article, we demonstrate that the product of PAC2 also functions in this pathway. PAC2 deletion mutants displayed phenotypes and genetic interactions similar to those caused by cin1Δ, cin2Δ and cin4Δ. These include cold-sensitive microtubule structures and sensitivity to the microtubule depolymerizing agent benomyl. Involvement in a common functional pathway is indicated by the observation that all double mutant combinations are viable and no more affected than any single mutant. In addition, extra copies of CIN1 were found to suppress the benomyl sensitivity of pac2Δ, cin2Δ and cin4Δ, but not that caused by other mutations that affect microtubule function. Cin1p and Pac2p were found to be related in sequence to mammalian proteins that aid in the folding of β-tubulin into an assembly-competent state. Alleles of CIN1 were identified that could suppress the benomyl sensitivity of cin4-4 in a highly specific fashion. Our findings suggest that the guanine nucleotide-binding Cin4p interacts with Cin1p and regulates its tubulin folding activity.

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PIK3CAH1047R-induced paradoxical ERK activation results in resistance to BRAFV600E specific inhibitors in BRAFV600E PIK3CAH1047R double mutant thyroid tumors

10.1101/164533 ◽

2017 ◽

Author(s):

Matthias A. Roelli ◽

Dorothée Ruffieux-Daidié ◽

Amandine Stooss ◽

Oussama ElMokh ◽

Wayne A. Phillips ◽

...

Keyword(s):

Thyroid Cancer ◽

Mouse Model ◽

Map Kinase ◽

Double Mutant ◽

Great Success ◽

Thyroid Carcinomas ◽

Map Kinase Pathway ◽

Phosphoinositide 3 Kinase ◽

Kinase Pathway ◽

Specific Inhibitors

AbstractThyroid carcinomas are the most prevalent endocrine cancers. The BRAFV600E mutation is found in 40% of the papillary type and 25% of the anaplastic type. BRAFV600E inhibitors have shown great success in melanoma but, they have been, to date, less successful in thyroid cancer. About 50% of anaplastic thyroid carcinomas present mutations/amplification of the phosphatidylinositol 3’ kinase. Here we propose to investigate if the hyper activation of that pathway could influence the response to BRAFV600E specific inhibitors.To test this, we used two mouse models of thyroid cancer. Single mutant (BRAFV600E) mice responded to BRAFV600E-specific inhibition (PLX-4720), while double mutant mice (BRAFV600E; PIK3CAH1047R) showed resistance and even signs of aggravation. This resistance was abrogated by combination with a phosphoinositide 3-kinase inhibitor. At the molecular level, we could show that this resistance was concomitant to a paradoxical activation of the MAP-Kinase pathway, which could be overturned by phosphoinositide 3-kinase inhibition in vivo in our mouse model and in vitro in human double mutant cell lines.In conclusion, we reveal a phosphoinositide 3-kinase driven, paradoxical MAP-Kinase pathway activation as mechanism for resistance to BRAFV600E specific inhibitors in a clinically relevant mouse model of thyroid cancer.

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Differential Requirement of SAGA Subunits for Mot1p and Taf1p Recruitment in Gene Activation

Molecular and Cellular Biology ◽

10.1128/mcb.25.12.4863-4872.2005 ◽

2005 ◽

Vol 25 (12) ◽

pp. 4863-4872 ◽

Cited By ~ 16

Author(s):

Chris J. C. van Oevelen ◽

Hetty A. A. M. van Teeffelen ◽

H. T. Marc Timmers

Keyword(s):

Saccharomyces Cerevisiae ◽

Double Mutant ◽

Gene Activation ◽

Transcription Activation ◽

Tata Binding Protein ◽

Genetic Interactions ◽

Hexose Transporter ◽

Phenotypic Analysis ◽

Transcription Factor Complexes ◽

Mutant Strains

ABSTRACT Transcription activation in yeast (Saccharomyces cerevisiae) involves ordered recruitment of transcription factor complexes, such as TFIID, SAGA, and Mot1p. Previously, we showed that both Mot1p and Taf1p are recruited to the HXT2 and HXT4 genes, which encode hexose transporter proteins. Here, we show that SAGA also binds to the HXT2 and HXT4 promoters and plays a pivotal role in the recruitment of Mot1p and Taf1p. The deletion of either SPT3 or SPT8 reduces Mot1p binding to HXT2 and HXT4. Surprisingly, the deletion of GCN5 reduces Taf1p binding to both promoters. When GCN5 is deleted in spt3Δ or spt8Δ strains, neither Mot1p nor Taf1p binds, and this results in a diminished recruitment of TATA binding protein and polymerase II to the HXT4 but not the HXT2 promoter. This is reflected by the SAGA-dependent expression of HXT4. In contrast, SAGA-independent induction of HXT2 suggests a functional redundancy with other factors. A functional interplay of different SAGA subunits with Mot1p and Taf1p was supported by phenotypic analysis of MOT1 SAGA or TAF1/SAGA double mutant strains, which revealed novel genetic interactions between MOT1 and SPT8 and between TAF1 and GCN5. In conclusion, our data demonstrate functional links between SAGA, Mot1p, and TFIID in HXT gene regulation.

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SHORT INTEGUMENTS 2 Promotes Growth During Arabidopsis Reproductive Development

Genetics ◽

10.1093/genetics/155.2.899 ◽

2000 ◽

Vol 155 (2) ◽

pp. 899-907

Author(s):

Jean Broadhvest ◽

Shawn C Baker ◽

Charles S Gasser

Keyword(s):

Cell Division ◽

Cell Expansion ◽

Double Mutant ◽

Reproductive Development ◽

Flower Morphology ◽

Genetic Interactions ◽

Pleiotropic Effects ◽

Evolutionary Origin ◽

Morphological Development ◽

Primary Role

Abstract The short integuments 2 (sin2) mutation arrests cell division during integument development of the Arabidopsis ovule and also has subtle pleiotropic effects on both sepal and pistil morphology. Genetic interactions between sin2 and other ovule mutations show that cell division, directionality of growth, and cell expansion represent at least partially independent processes during integument development. Double-mutant analyses also reveal that SIN2 shares functional redundancy with HUELLENLOS in ovule primordium outgrowth and proximal-distal patterning and with TSO1 in promotion of normal morphological development of the four whorls of primary floral organs. All of these observations are consistent with SIN2 being a promoter of growth and cell division during reproductive development, with a primary role in these processes during integument development. On the basis of the floral pleiotropic effects observed in a majority of ovule mutants, including sin2, we postulate a relationship between ovule genes and the evolutionary origin of some processes regulating flower morphology.

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