scholarly journals Distinct Morphological and Behavioural Alterations in ENU-Induced Heterozygous Trpc7K810Stop Mutant Mice

Genes ◽  
2021 ◽  
Vol 12 (11) ◽  
pp. 1732
Author(s):  
Birgit Rathkolb ◽  
Maike Howaldt ◽  
Stefan Krebs ◽  
Petra Prückl ◽  
Susanne Sauer ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Transient Receptor Potential ◽  
Stop Codon ◽  
Inbred Mice ◽  
Dominant Negative ◽  
Mutant Mice ◽  
Mutant Line ◽  
Dominant Negative Effect ◽  
Causative Mutation ◽  
Phenotypic Characterization

Trpc7 (transient receptor potential cation channel, subfamily C, member 7; 862 amino acids) knockout mice are described showing no clear phenotypic alterations, therefore, the functional relevance of the gene remains unclear. A complementary approach for the functional analysis of a given gene is the examination of individuals harbouring a mutant allele of the gene. In the phenotype-driven Munich ENU mouse mutagenesis project, a high number of phenotypic parameters was used for establishing novel mouse models on the genetic background of C3H inbred mice. The phenotypically dominant mutant line SMA002 was established and further examined. Analysis of the causative mutation as well as the phenotypic characterization of the mutant line were carried out. The causative mutation was detected in the gene Trpc7 which leads to the production of a truncated protein due to the novel stop codon at amino acid position 810 thereby affecting the highly conserved cytoplasmic C terminus of the protein. Trpc7 heterozygous mutant mice of both sexes were viable and fertile, but showed distinct morphological and behavioural alterations which is in contrast to the published phenotype of Trpc7 knockout mice. Thus, the Trpc7K810Stop mutation leads to a dominant negative effect of the mutant protein.

Regulation of TRPV1 by a novel renally expressed rat TRPV1 splice variant

2006 ◽  
Vol 290 (1) ◽  
pp. F117-F126 ◽  
Author(s):  
Wei Tian ◽  
Yi Fu ◽  
Donna H. Wang ◽  
David M. Cohen
Keyword(s):  
Splice Variant ◽  
Transient Receptor Potential ◽  
Calcium Influx ◽  
Splice Variants ◽  
Stop Codon ◽  
Rat Kidney ◽  
Dominant Negative ◽  
Epithelial Cell Line ◽  
Hek 293 ◽  
Donor And Acceptor

The capsaicin receptor and transient receptor potential channel TRPV1 senses heat, protons, and vanilloid agonists in peripheral sensory ganglia. Abundant data have suggested the presence of potentially novel splice variants in the kidney. We report a novel rat TRPV1 splice variant, TRPV1VAR, cloned from kidney papilla. TRPV1VAR cDNA was identified in multiple kidney tissues. Its sequence was fully compatible with potential splice donor and acceptor sites in the rat TRPV1 gene. TRPV1VAR is predicted to encode a truncated form of TRPV1 consisting of the NH2-terminal 248 residues of TRPV1 (all within the NH2-terminal intracellular domain) followed by five nonconsensus amino acids (Arg-Glu-Ala-Met-Trp) and a stop codon. The variant utilizes the same consensus Kozak sequence as canonical TRPV1. A band of the appropriate molecular mass was identified in rat kidney papillary (but not medullary) lysates immunoblotted with an antibody directed against the NH2 terminus of TRPV1, whereas an antibody recognizing the TRPV1 COOH terminus failed to detect it. Upon heterologous expression in HEK 293 cells, TRPV1VAR potentiated the ability of cotransfected TRPV1 to confer calcium influx in response to resiniferatoxin. TRPV1VAR did not influence expression or cell surface localization of cotransfected TRPV1. TRPV1VAR protein product associated with the NH2 terminus of canonical TRPV1. Interestingly, when expressed in the COS-7 epithelial cell line, TRPV1VAR functioned in a dominant-negative acting capacity, partially blocking TRPV1-dependent resiniferatoxin responsiveness. We conclude that TRPV1VAR is one of perhaps several TRPV1 splice variants expressed in rat kidney and that it may serve to modulate TRPV1 responsiveness in some tissues.

scholarly journals Essential Nonredundant Function of the Catalytic Activity of Histone Deacetylase 2 in Mouse Development

2015 ◽  
Vol 36 (3) ◽  
pp. 462-474 ◽  
Author(s):  
Astrid Hagelkruys ◽  
Katharina Mattes ◽  
Verena Moos ◽  
Magdalena Rennmayr ◽  
Manuela Ringbauer ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Regulation Of Gene Expression ◽  
Dominant Negative ◽  
Mutant Mice ◽  
Dominant Negative Effect ◽  
Mouse Development ◽  
Histone Deacetylase 2 ◽  
Histone Hyperacetylation ◽  
Negative Effect ◽  
The Impact

The class I histone deacetylases (HDACs) HDAC1 and HDAC2 play partially redundant roles in the regulation of gene expression and mouse development. As part of multisubunit corepressor complexes, these two deacetylases exhibit both enzymatic and nonenzymatic functions. To examine the impact of the catalytic activities of HDAC1 and HDAC2, we generated knock-in mice expressing catalytically inactive isoforms, which are still incorporated into the HDAC1/HDAC2 corepressor complexes. Surprisingly, heterozygous mice expressing catalytically inactive HDAC2 die within a few hours after birth, while heterozygous HDAC1 mutant mice are indistinguishable from wild-type littermates. Heterozygous HDAC2 mutant mice show an unaltered composition but reduced associated deacetylase activity of corepressor complexes and exhibit a more severe phenotype than HDAC2-null mice. They display changes in brain architecture accompanied by premature expression of the key regulator protein kinase C delta. Our study reveals a dominant negative effect of catalytically inactive HDAC2 on specific corepressor complexes resulting in histone hyperacetylation, transcriptional derepression, and, ultimately, perinatal lethality.

scholarly journals Dimerisation of APOBEC1 is dispensable for its RNA editing activity

2018 ◽  
Author(s):  
Martina Chieca ◽  
Marco Montini ◽  
Francesco Severi ◽  
Riccardo Pecori ◽  
Silvestro G. Conticello
Keyword(s):  
Rna Editing ◽  
Stop Codon ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Main Determinant ◽  
Apob Mrna ◽  
Dna And Rna ◽  
Negative Effect ◽  
Editing Activity ◽  
Nuclear Degradation

ABSTRACTAmong the AID/APOBECs -a family of DNA and RNA deaminases-APOBEC1 physiologically partakes into a complex that edits a CAA codon into UAA Stop codon in the transcript of Apolipoprotein B (ApoB), a protein crucial in the transport of lipids in the blood. Catalytically inactive mutants of APOBEC1 have a dominant negative effect on its activity, as they compete for the targeting to the ApoB mRNA. Here we show that catalytically inactive chimeras of APOBEC1 restricted to different compartments of the cell present different abilities to titrate APOBEC1-mediated RNA editing, and that the ability of APOBEC1 to interact with these mutants is the main determinant for its activity. Our results demonstrate that dimerisation, a feature common to other APOBECs targeting DNA, is not required for APOBEC1 activity on mRNA. Furthermore, APOBEC1-mediated RNA editing is a dynamic process where interplay among the components of the editing complex is regulated through the balance between availability of A1CF, one of APOBEC1 cofactors, and nuclear degradation of APOBEC1.

scholarly journals SNRNP200 Mutations Cause Autosomal Dominant Retinitis Pigmentosa

2021 ◽  
Vol 7 ◽  
Author(s):  
Tao Zhang ◽  
Jingshan Bai ◽  
Xinyi Zhang ◽  
Xiaowei Zheng ◽  
Nan Lu ◽  
...  
Keyword(s):  
Retinitis Pigmentosa ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Causative Mutation ◽  
Chinese Family ◽  
Zebrafish Embryos ◽  
Gene Recognition ◽  
Small Nuclear Ribonucleoprotein ◽  
Negative Effect ◽  
In The Wild

The small nuclear ribonucleoprotein 200 kDa (SNRNP200) gene plays a key role in the maturation of pre-message RNA (pre-mRNA) splicing with the indication for the etiology of retinitis pigmentosa (RP). Gene recognition can facilitate the diagnosis of these patients for better clinical management, treatment and counseling. This study aimed to outline the causative mutation in a Chinese family and the pathogenic mechanism of this SNRNP200 mutation in RP. Eighteen individuals from the affected family underwent a complete ophthalmic examination. Whole exome sequencing (WES) was conducted to identify the pathogenic variant in the proband, which was then confirmed by Sanger sequencing. Expression of the SNRNP200 transcript in zebrafish was identified via whole mount in situ hybridization. Morpholino oligonucleotide (MO) and SNRNP200 wild and mutant mRNA were injected into zebrafish embryos followed by analyses of the systemic changes and retinal phenotypes using immunofluorescence. Heterozygous SNRNP200c.C6088T (p.Arg2030Cys) mutation was ascertained in two members of this family: the proband and his father (II-2). Overexpression of SNRNP200Arg2030Cys, but not SNRNP200WT caused systemic deformities in the wild-type zebrafish embryos with the retina primarily injured, and significantly increased death rates in the morphant embryos, in which the orthologous zebrafish SNRNP200 gene was blocked. In conclusion, this study reports a novel heterozygous SNRNP200c.C6088T mutation, which is evidenced to cause RP via a dominant-negative effect.

scholarly journals The MADS-box gene FveSEP3 plays essential roles in flower organogenesis and fruit development in woodland strawberry

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Mengting Pi ◽  
Shaoqiang Hu ◽  
Laichao Cheng ◽  
Ruhan Zhong ◽  
Zhuoying Cai ◽  
...  
Keyword(s):  
Fruit Ripening ◽  
Fruit Development ◽  
Flower Development ◽  
Fruit Growth ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Causative Mutation ◽  
Parthenocarpic Fruit ◽  
Wild Type ◽  
Woodland Strawberry

AbstractFlower and fruit development are two key steps for plant reproduction. The ABCE model for flower development has been well established in model plant species; however, the functions of ABCE genes in fruit crops are less understood. In this work, we identified an EMS mutant named R27 in woodland strawberry (Fragaria vesca), showing the conversion of petals, stamens, and carpels to sepaloid organs in a semidominant inheritance fashion. Mapping by sequencing revealed that the class E gene homolog FveSEP3 (FvH4_4g23530) possessed the causative mutation in R27 due to a G to E amino acid change in the conserved MADS domain. Additional fvesep3CR mutants generated by CRISPR/Cas9 displayed similar phenotypes to fvesep3-R27. Overexpressing wild-type or mutated FveSEP3 in Arabidopsis suggested that the mutation in R27 might cause a dominant-negative effect. Further analyses indicated that FveSEP3 physically interacted with each of the ABCE proteins in strawberry. Moreover, both R27 and fvesep3CR mutants exhibited parthenocarpic fruit growth and delayed fruit ripening. Transcriptome analysis revealed that both common and specific differentially expressed genes were identified in young fruit at 6–7 days post anthesis (DPA) of fvesep3 and pollinated wild type when compared to unpollinated wild type, especially those in the auxin pathway, a key hormone regulating fruit set in strawberry. Together, we provided compelling evidence that FveSEP3 plays predominant E functions compared to other E gene homologs in flower development and that FveSEP3 represses fruit growth in the absence of pollination and promotes fruit ripening in strawberry.

scholarly journals Identification of Two DNMT3A Mutations Compromising Protein Stability and Methylation Capacity in Acute Myeloid Leukemia

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Samantha Bruno ◽  
Maria Teresa Bochicchio ◽  
Eugenia Franchini ◽  
Antonella Padella ◽  
Giovanni Marconi ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Protein Stability ◽  
Myeloid Leukemia ◽  
Stop Codon ◽  
Cpg Methylation ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Single Nucleotide ◽  
Negative Effect ◽  
Acute Myeloid

Somatic mutations of DNMT3A occur in about 20% of acute myeloid leukemia (AML) patients. They mostly consist in heterozygous missense mutations targeting a hotspot site at R882 codon, which exhibit a dominant negative effect and are associated with high myeloblast count, advanced age, and poor prognosis. Other types of mutations such as truncations, insertions, or single-nucleotide deletion also affect the DNMT3A gene, though with lower frequency. The present study aimed to characterize two DNMT3A gene mutations identified by next-generation sequencing (NGS), through analysis of protein stability and DNA methylation status at CpG islands. The first mutation was a single-nucleotide variant of DNMT3A at exon 20 causing a premature STOP codon (c.2385G > A; p.Trp795∗; NM_022552.4). The DNMT3A mutation load increased from 4.5% to 38.2% during guadecitabine treatment, with a dominant negative effect on CpG methylation and on protein expression. The second mutation was a novel insertion of 35 nucleotides in exon 22 of DNMT3A (NM_022552.4) that introduced a STOP codon too, after the amino acid Glu863 caused by a frameshift insertion (c.2586_2587insTCATGAATGAGAAAGAGGACATCTTATGGTGCACT; p. Thr862_Glu863fsins). The mutation, which was associated with reduced DNMT3A expression and CpG methylation, persisted at relapse with minor changes in the methylation profile and at protein level. Our data highlight the need to better understand the consequences of DNMT3A mutations other than R882 substitutions in the leukemogenic process in order to tailor patient treatments, thus avoiding therapeutic resistance and disease relapse.

scholarly journals NKCC1: Newly Found as a Human Disease-Causing Ion Transporter

Function ◽  
2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Rainelli Koumangoye ◽  
Lisa Bastarache ◽  
Eric Delpire
Keyword(s):  
De Novo ◽  
Stop Codon ◽  
Neurodevelopmental Disorder ◽  
Premature Stop Codon ◽  
Dominant Negative ◽  
De Novo Mutation ◽  
Dominant Negative Effect ◽  
Salt Wasting ◽  
Wide Range ◽  
Negative Effect

Abstract Among the electroneutral Na+-dependent chloride transporters, NKCC1 had until now evaded identification as a protein causing human diseases. The closely related SLC12A transporters, NKCC2 and NCC have been identified some 25 years ago as responsible for Bartter and Gitelman syndromes: two renal-dependent salt wasting disorders. Absence of disease was most surprising since the NKCC1 knockout mouse was shown in 1999 to be viable, albeit with a wide range of deleterious phenotypes. Here we summarize the work of the past 5 years that introduced us to clinical cases involving NKCC1. The most striking cases are of 3 children with inherited mutations, who have complete absence of NKCC1 expression. These cases establish that lack of NKCC1 causes deafness; CFTR-like secretory defects with mucus accumulation in lung and intestine; severe xerostomia, hypotonia, dysmorphic facial features, and severe neurodevelopmental disorder. Another intriguing case is of a patient with a dominant deleterious SLC12A2 allele. This de novo mutation introduced a premature stop codon leading to a truncated protein. This mutant transporter seems to exert dominant-negative effect on wild-type transporter only in epithelial cells. The patient who suffers from lung, bladder, intestine, pancreas, and multiple endocrine abnormalities has, however, normal hearing and cognition. Finally, new reports substantiate the haploinsufficiency prediction of the SLC12A2 gene. Cases with single allele mutations in SLC12A2 have been linked to hearing loss and neurodevelopmental disorders.

scholarly journals Transient Receptor Potential Melastatin 2 Enhances Vascular Reactivity During Development of Atherosclerosis in Mice

2021 ◽  
Vol 34 (1) ◽  
pp. 121-122
Author(s):  
Yi-quan Dai ◽  
Xiao-xiao Yan ◽  
Yi-chen Lin ◽  
Hong-yu Chen ◽  
Xiao-ru Liu
Keyword(s):  
Knockout Mice ◽  
Vascular Reactivity ◽  
Transient Receptor Potential ◽  
Gene Knockout ◽  
Receptor Potential ◽  
Blood Lipid ◽  
Normal Diet ◽  
Protein Levels ◽  
Transient Receptor Potential Melastatin ◽  
Transient Receptor

Abstract Background To investigate the function of transient receptor potential melastatin 2 (TRPM2) in vascular reactivity induced by 5-hydroxytryptamine (5-HT) in the aorta during development of atherosclerosis in mice. Methods Forty mice were randomly divided into 4 groups: C57BL/6J on normal diet (C57 + ND), C57BL/6J on high-fat diet (C57 + HFD), apolipoprotein E gene knockout mice (ApoE−/−) on ND (ApoE−/− + ND), and ApoE−/− on HFD (ApoE−/− + HFD). They were fed with a ND or HFD for 16 weeks. Aortic TRPM2 expression and isometric contractions were analyzed. Results In the ApoE−/− + HFD group, body weight, blood glucose, and blood lipid concentrations were increased, and aortic plaques were developed. Compared with the other 3 groups, aortic TRPM2 mRNA and protein levels were significantly increased in the ApoE−/− + HFD group (P < 0.01). Aortic reactivity to 5-HT was enhanced in ApoE−/− + HFD mice with lower EC50 values. The enhanced reactivity to 5-HT was significantly inhibited by TRPM2 inhibitors, N-p-amylcinnamoyl anthranilic acid (1 µmol/l) and 2-aminoethyl diphenylborinate (10 µmol/l). Conclusions Aortic TRPM2 expression is upregulated in ApoE knockout mice fed with a HFD. Upregulation of TRPM2 enhances 5-HT vascular reactivity during development of atherosclerosis.

scholarly journals A Dominant Negative Effect of eth-1r, a Mutant Allele of the Neurospora crassa S-Adenosylmethionine Synthetase-Encoding Gene Conferring Resistance to the Methionine Toxic Analogue Ethionine

Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1455-1462
Author(s):  
José L Barra ◽  
Mario R Mautino ◽  
Alberto L Rosa
Keyword(s):  
Neurospora Crassa ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Resistant Phenotype ◽  
Negative Effect ◽  
Encoding Gene ◽  
Adenosylmethionine Synthetase ◽  
Adomet Synthetase ◽  
Identical Gene

eth-1r a thermosensitive allele of the Neurospora crassa S-adenosylmethionine (AdoMet) synthetase gene that confers ethionine resistance, has been cloned and sequenced. Replacement of an aspartic amino acid residue (D48 → N48), perfectly conserved in prokaryotic, fungal and higher eukaryotic AdoMet synthetases, was found responsible for both thermosensitivity and ethionine resistance conferred by eth-1r. Gene fusion constructs, designed to overexpress eth-1r in vivo, render transformant cells resistant to ethionine. Dominance of ethionine resistance was further demonstrated in eth-1  +/eth-1r partial diploids carrying identical gene doses of both alleles. Heterozygous eth-1  +/eth-1r cells have, at the same time, both the thermotolerance conferred by eth-1  + and the ethionine-resistant phenotype conferred by eth-1r. AdoMet levels and AdoMet synthetase activities were dramatically decreased in heterozygous eth-1  +/eth-1r cells. We propose that this negative effect exerted by eth-1r results from the in vivo formation of heteromeric eth-1  +/eth-1r AdoMet synthetase molecules.

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