scholarly journals ND9P, a Novel Protein With Armadillo-like Repeats Involved in Exocytosis: Physiological Studies Using Allelic Mutants in Paramecium

Genetics ◽  
2001 ◽  
Vol 157 (2) ◽  
pp. 611-620
Author(s):  
Marine Froissard ◽  
Anne-Marie Keller ◽  
Jean Cohen
Keyword(s):  
Membrane Fusion ◽  
Protein Interactions ◽  
Secretory Pathway ◽  
Point Mutations ◽  
Binding Activity ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Terminal Domain ◽  
Negative Effect ◽  
Regulated Secretory Pathway

Abstract In Paramecium, a number of mutants affected in the exocytotic membrane fusion step of the regulated secretory pathway have been obtained. Here, we report the isolation of one of the corresponding genes, ND9, previously suspected to encode a soluble protein interacting with both plasma and trichocyst membranes. Nd9p is a novel polypeptide that contains C-terminal Armadillo-like repeats. Point mutations were found in the first N-terminal quarter of the molecule and in the last putative Armadillo repeat, respectively, for the two thermosensitive mutants, nd9-1 and nd9-2. The different behaviors of these mutants in recovery experiments upon temperature shifts suggest that the N-terminal domain of the molecule may be involved in membrane binding activity, whereas the C-terminal domain is a candidate for protein-protein interactions. The nonsense nd9-3 mutation that produces a short N-terminal peptide has a dominant negative effect on the nd9-1 allele. We show here that, when overexpressed, the dominant negative effect can be produced even on the wild-type allele, suggesting competition for a common target. We suggest that Nd9p could act, like some SNARE proteins, at the membrane-cytosol interface to promote membrane fusion.

scholarly journals Novel Role for the C Terminus of Saccharomyces cerevisiae Rev1 in Mediating Protein-Protein Interactions

2006 ◽  
Vol 26 (21) ◽  
pp. 8173-8182 ◽  
Author(s):  
Sanjay D'Souza ◽  
Graham C. Walker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Protein Interactions ◽  
Sequence Similarity ◽  
Dna Lesions ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
Negative Effect

ABSTRACT The Saccharomyces cerevisiae REV3/7-encoded polymerase ζ and Rev1 are central to the replicative bypass of DNA lesions, a process called translesion synthesis (TLS). While yeast polymerase ζ extends from distorted DNA structures, Rev1 predominantly incorporates C residues from across a template G and a variety of DNA lesions. Intriguingly, Rev1 catalytic activity does not appear to be required for TLS. Instead, yeast Rev1 is thought to participate in TLS by facilitating protein-protein interactions via an N-terminal BRCT motif. In addition, higher eukaryotic homologs of Rev1 possess a C terminus that interacts with other TLS polymerases. Due to a lack of sequence similarity, the yeast Rev1 C-terminal region, located after the polymerase domain, had initially been thought not to play a role in TLS. Here, we report that elevated levels of the yeast Rev1 C terminus confer a strong dominant-negative effect on viability and induced mutagenesis after DNA damage, highlighting the crucial role that the C terminus plays in DNA damage tolerance. We show that this phenotype requires REV7 and, using immunoprecipitations from crude extracts, demonstrate that, in addition to the polymerase-associated domain, the extreme Rev1 C terminus and the BRCT region of Rev1 mediate interactions with Rev7.

scholarly journals A Knock-In Tristetraprolin (TTP) Zinc Finger Point Mutation in Mice: Comparison with Complete TTP Deficiency

2017 ◽  
Vol 38 (4) ◽  
Author(s):  
Wi S. Lai ◽  
Deborah J. Stumpo ◽  
Lianqun Qiu ◽  
Roberta Faccio ◽  
Perry J. Blackshear
Keyword(s):  
Zinc Finger ◽  
Mrna Decay ◽  
Rna Binding ◽  
Zinc Finger Protein ◽  
Binding Activity ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Negative Effect ◽  
A Minor ◽  
Inflammatory Syndrome

ABSTRACTTristetraprolin (TTP) is a tandem CCCH zinc finger protein that can bind to AU-rich element-containing mRNAs and promote their decay. TTP knockout mice develop a severe inflammatory syndrome, largely due to excess tumor necrosis factor (TNF), whose mRNA is a direct target of TTP binding and destabilization. TTP's RNA binding activity and its ability to promote mRNA decay are lost when one of the zinc-coordinating residues of either zinc finger is mutated. To address several long-standing questions about TTP activity in intact animals, we developed a knock-in mouse with a cysteine-to-arginine mutation within the first zinc finger. Homozygous knock-in mice developed a severe inflammatory syndrome that was essentially identical to that of complete TTP deficiency, suggesting that TTP's critical anti-inflammatory role in mammalian physiology is secondary to its ability to bind RNA. In addition, there was no evidence for a “dominant-negative” effect of the mutant allele in heterozygotes, as suggested by previous experiments. Finally, mRNA decay experiments in mutant macrophages demonstrated that TTP can regulate the stability of its own mRNA, albeit to a minor extent. These studies suggest that RNA binding is an essential first step in the physiological activities of members of this protein family.

scholarly journals Mice Null for Sox18 Are Viable and Display a Mild Coat Defect

2000 ◽  
Vol 20 (24) ◽  
pp. 9331-9336 ◽  
Author(s):  
David Pennisi ◽  
Josephine Bowles ◽  
Andras Nagy ◽  
George Muscat ◽  
Peter Koopman
Keyword(s):  
Vascular Endothelium ◽  
Point Mutations ◽  
Hair Follicles ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Mild Phenotype ◽  
Negative Effect ◽  
Cardiovascular Defects ◽  
Sox Proteins ◽  
Hair Shafts

ABSTRACT We have previously shown that Sox18 is expressed in developing vascular endothelium and hair follicles during mouse embryogenesis and that point mutations in Sox18 are the underlying cause of cardiovascular and hair follicle defects inragged (Ra) mice. Here we describe the analysis of Sox18 −/− mice produced by gene targeting. Despite the profound defects seen in Ra mice,Sox18 −/− mice have no obvious cardiovascular defects and only a mild coat defect with a reduced proportion of zigzag hairs. A reduction in the amount of pheomelanin pigmentation in hair shafts was also observed; later-forming hair follicles showed a reduced subapical pheomelanin band, giving Sox18 −/−mice a slightly darker appearance than Sox18 +/+and Sox18 +/− siblings.Sox18 −/− mice are viable and fertile and show no difference in the ability to thrive relative to littermates. Because of the mild effect of the mutation on the phenotype ofSox18 −/− mice, we conclude that the semidominant nature of the Ra mutations is due to atrans-dominant negative effect mediated by the mutant SOX18 proteins rather than haploinsufficiency as has been observed for otherSOX genes. Due to the similarity of SOX18 to other subgroup F SOX proteins, SOX7 and −17, and the overlap in expression of these genes, functional redundancy amongst these SOX proteins could also account for the mild phenotype of Sox18 −/−mice.

scholarly journals Tumor-derived Mutations in the TRAIL Receptor DR5 Inhibit TRAIL Signaling through the DR4 Receptor by Competing for Ligand Binding

2007 ◽  
Vol 282 (38) ◽  
pp. 28189-28194 ◽  
Author(s):  
Lianghua Bin ◽  
Jacqueline Thorburn ◽  
Lance R. Thomas ◽  
Peter E. Clark ◽  
Robin Humphreys ◽  
...  
Keyword(s):  
Point Mutations ◽  
Inhibitory Effect ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Agonistic Antibody ◽  
Signaling Complex ◽  
Trail Receptors ◽  
Negative Effect ◽  
Mutant Receptors ◽  
Trail Signaling

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a cytokine that preferentially induces apoptosis in tumor cells compared with normal cells through two receptors (DR4 and DR5). Somatic mutations in these receptors have been found in different kinds of cancer; however, it is poorly understood how the mutations affect signaling. We found that point mutations (L334F, E326K, E338K, and K386N) that were identified in human tumors result in the DR5 receptor losing its ability to form a functional death-inducing signaling complex and induce apoptosis. The mutant receptors also have a “dominant negative” effect whereby they inhibit the ability of TRAIL to induce apoptosis through functional DR4 receptors. This dominant negative mechanism is achieved through competition for TRAIL binding as shown by experiments where the ability of the mutant DR5 receptor to bind with the ligand was abolished, thus restoring TRAIL signaling through DR4. The inhibitory effect on signaling through the wild-type DR4 protein can be overcome if the inhibitory mechanism is bypassed by using a DR4-agonistic antibody that is not subject to this competition. This study provides a molecular basis for the use of specific therapeutic agonists of TRAIL receptors in people whose tumors harbor somatic DR5 mutations.

scholarly journals Functional Characterization of the N Terminus of Sir3p

1998 ◽  
Vol 18 (10) ◽  
pp. 6110-6120 ◽  
Author(s):  
Monica Gotta ◽  
Francesca Palladino ◽  
Susan M. Gasser
Keyword(s):  
Functional Characterization ◽  
Dominant Negative ◽  
Full Length ◽  
Dominant Negative Effect ◽  
Terminal Domain ◽  
Allosteric Effector ◽  
Elevated Expression ◽  
Negative Effect ◽  
Punctate Pattern

ABSTRACT Silent information regulator 3 is an essential component of theSaccharomyces cerevisiae silencing complex that functions at telomeres and the silent mating-type loci, HMR andHML. We show that expression of the N- and C-terminal-encoding halves of SIR3 in transpartially complements the mating defect of the sir3 null allele, suggesting that the two domains have distinct functions. We present here a functional characterization of these domains. The N-terminal domain (Sir3N) increases both the frequency and extent of telomere-proximal silencing when expressed ectopically inSIR + yeast strains, although we are unable to detect interaction between this domain and any known components of the silencing machinery. In contrast to its effect at telomeres, Sir3N overexpression derepresses transcription of reporter genes inserted in the ribosomal DNA (rDNA) array. Immunolocalization of Sir3N-GFP and Sir2p suggests that Sir3N directly antagonizes nucleolar Sir2p, releasing an rDNA-bound population of Sir2p so that it can enhance repression at telomeres. Overexpression of the C-terminal domain of either Sir3p or Sir4p has a dominant-negative effect on telomeric silencing. In strains overexpressing the C-terminal domain of Sir4p, elevated expression of either full-length Sir3p or Sir3N restores repression and the punctate pattern of Sir3p and Rap1p immunostaining. The similarity of Sir3N and Sir3p overexpression phenotypes suggests that Sir3N acts as an allosteric effector of Sir3p, either enhancing its interactions with other silencing components or liberating the full-length protein from nonfunctional complexes.

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.

scholarly journals Restoration of Silencing in Saccharomyces cerevisiae by Tethering of a Novel Sir2-Interacting Protein, Esc8

Genetics ◽  
2002 ◽  
Vol 162 (2) ◽  
pp. 633-645 ◽  
Author(s):  
Guido Cuperus ◽  
David Shore
Keyword(s):  
Protein A ◽  
Dominant Negative ◽  
Class I ◽  
Dominant Negative Effect ◽  
Rna Pol Ii ◽  
Interacting Protein ◽  
Nucleosome Remodeling ◽  
Negative Effect ◽  
Close Homolog

Abstract We previously described two classes of SIR2 mutations specifically defective in either telomeric/HM silencing (class I) or rDNA silencing (class II) in S. cerevisiae. Here we report the identification of genes whose protein products, when either overexpressed or directly tethered to the locus in question, can establish silencing in SIR2 class I mutants. Elevated dosage of SCS2, previously implicated as a regulator of both inositol biosynthesis and telomeric silencing, suppressed the dominant-negative effect of a SIR2-143 mutation. In a genetic screen for proteins that restore silencing when tethered to a telomere, we isolated ESC2 and an uncharacterized gene, (YOL017w), which we call ESC8. Both Esc2p and Esc8p interact with Sir2p in two-hybrid assays, and the Esc8p-Sir2 interaction is detected in vitro. Interestingly, Esc8p has a single close homolog in yeast, the ISW1-complex factor Ioc3p, and has also been copurified with Isw1p, raising the possibility that Esc8p is a component of an Isw1p-containing nucleosome remodeling complex. Whereas esc2 and esc8 deletion mutants alone have only marginal silencing defects, cells lacking Isw1p show a strong silencing defect at HMR but not at telomeres. Finally, we show that Esc8p interacts with the Gal11 protein, a component of the RNA pol II mediator complex.

scholarly journals Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil Differentiation

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4154-4166 ◽  
Author(s):  
Robert L. Ilaria ◽  
Robert G. Hawley ◽  
Richard A. Van Etten
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cytokine Signaling ◽  
Transactivation Domain ◽  
Negative Effects ◽  
Murine Bone Marrow ◽  
Negative Effect

Abstract STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR→AAA and R290QQ→AAA), two in the DNA-binding domain (E437E→AA and V466VV→AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/▵53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, ▵53C. Stable expression of either the WKR or ▵53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3–dependent proliferation and granulocyte colony-stimulating factor (G-CSF)–dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF–dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.

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