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‣ Selection of a Dominant Negative Retinoblastoma Protein (RB) Inhibiting Satellite Myoblast Differentiation Implies an Indirect Interaction between MyoD and RB

Li, Feng-Qian; Coonrod, Archie; Horwitz, Marshall
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/2000 Português
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Satellite myoblasts serve as stem cells in postnatal skeletal muscle, but the genes responsible for choosing between growth versus differentiation are largely undefined. We have used a novel genetic approach to identify genes encoding proteins whose dominant negative inhibition is capable of interrupting the in vitro differentiation of C2C12 murine satellite myoblasts. The screen is based on fusion of a library of cDNA fragments with the lysosomal protease cathepsin B (CB), such that the fusion protein intracellularly diverts interacting factors to the lysosome. Among other gene fragments selected in this screen, including those of known and novel sequence, is the retinoblastoma protein (RB) pocket domain. This unique dominant negative form of RB allows us to genetically determine if MyoD and RB associate in vivo. The dominant negative CB-RB fusion produces a cellular phenotype indistinguishable from recessive loss of function RB mutations. The fact that the dominant negative RB inhibits myogenic differentiation in the presence of nonlimiting concentrations of either RB or MyoD suggests that these two proteins do not directly interact. We further show that the dominant negative RB inhibits E2F1 but cannot inhibit a forced E2F1-RB dimer. Therefore...

‣ The Pol β-14 Dominant Negative Rat DNA Polymerase β Mutator Mutant Commits Errors during the Gap-Filling Step of Base Excision Repair in Saccharomyces cerevisiae†

Clairmont, Caroline A.; Sweasy, Joann B.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/1998 Português
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We demonstrated recently that dominant negative mutants of rat DNA polymerase β (Pol β) interfere with repair of alkylation damage in Saccharomyces cerevisiae. To identify the alkylation repair pathway that is disrupted by the Pol β dominant negative mutants, we studied the epistatic relationship of the dominant negative Pol β mutants to genes known to be involved in repair of DNA alkylation damage in S. cerevisiae. We demonstrate that the rat Pol β mutants interfere with the base excision repair pathway in S. cerevisiae. In addition, expression of one of the Pol β dominant negative mutants, Pol β-14, increases the spontaneous mutation rate of S. cerevisiae whereas expression of another Pol β dominant negative mutant, Pol β-TR, does not. Expression of the Pol β-14 mutant in cells lacking APN1 activity does not result in an increase in the spontaneous mutation rate. These results suggest that gaps are required for mutagenesis to occur in the presence of Pol β-14 but that it is not merely the presence of a gap that results in mutagenesis. Our results suggest that mutagenesis can occur during the gap-filling step of base excision repair in vivo.

‣ Dominant-Negative Inhibition of Prion Formation Diminished by Deletion Mutagenesis of the Prion Protein

Zulianello, Laurence; Kaneko, Kiyotoshi; Scott, Michael; Erpel, Susanne; Han, Dong; Cohen, Fred E.; Prusiner, Stanley B.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2000 Português
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Polymorphic basic residues near the C terminus of the prion protein (PrP) in humans and sheep appear to protect against prion disease. In heterozygotes, inhibition of prion formation appears to be dominant negative and has been simulated in cultured cells persistently infected with scrapie prions. The results of nuclear magnetic resonance and mutagenesis studies indicate that specific substitutions at the C-terminal residues 167, 171, 214, and 218 of PrPC act as dominant-negative, inhibitors of PrPSc formation (K. Kaneko et al., Proc. Natl. Acad. Sci. USA 94:10069–10074, 1997). Trafficking of substituted PrPC to caveaola-like domains or rafts by the glycolipid anchor was required for the dominant-negative phenotype; interestingly, amino acid replacements at multiple sites were less effective than single-residue substitutions. To elucidate which domains of PrPC are responsible for dominant-negative inhibition of PrPSc formation, we analyzed whether N-terminally truncated PrP(Q218K) molecules exhibited dominant-negative effects in the conversion of full-length PrPC to PrPSc. We found that the C-terminal domain of PrP is not sufficient to impede the conversion of the full-length PrPC molecule and that N-terminally truncated molecules (with residues 23 to 88 and 23 to 120 deleted) have reduced dominant-negative activity. Whether the N-terminal region of PrP acts by stabilizing the C-terminal domain of the molecule or by modulating the binding of PrPC to an auxiliary molecule that participates in PrPSc formation remains to be established.

‣ Dominant-negative inhibition of prion replication in transgenic mice

Perrier, Véronique; Kaneko, Kiyotoshi; Safar, Jiri; Vergara, Julie; Tremblay, Patrick; DeArmond, Stephen J.; Cohen, Fred E.; Prusiner, Stanley B.; Wallace, Andrew C.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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Our discovery of dominant-negative inhibition of prion formation in cultured cells provided an explanation for the resistance of some sheep to scrapie and humans to Creutzfeldt–Jakob disease. To determine whether dominant-negative inhibition occurs in vivo, we produced transgenic (Tg) mice expressing prion protein (PrP) with either the Q167R or Q218K mutation alone or in combination with wild-type (wt) PrP. Tg(MoPrP,Q167R)Prnp0/0 mice expressing mutant PrP at levels equal to non-Tg mice remained healthy for >550 days, indicating that inoculation with prions did not cause disease. Immunoblots of brain homogenates and histologic analysis did not reveal abnormalities. Tg(MoPrP,Q167R)Prnp+/+ mice expressing both mutant and wt PrP did not exhibit neurologic dysfunction, but their brains revealed low levels of the PrP pathogenic isoform (PrPSc), and sections showed numerous vacuoles and severe astrocytic gliosis at 300 days after inoculation. Both Tg(MoPrP,Q218K)Prnp0/0 and Tg(MoPrP,Q218K)Prnp+/+ mice expressing high levels of the transgene product remained healthy for >300 days after inoculation. Neither PrPSc nor neuropathologic changes were found. Our studies demonstrate that although dominant-negative inhibition of wt PrPSc formation occurs...

‣ CLAVATA1 Dominant-Negative Alleles Reveal Functional Overlap between Multiple Receptor Kinases That Regulate Meristem and Organ Development

Diévart, Anne; Dalal, Monica; Tax, Frans E.; Lacey, Alexzandria D.; Huttly, Alison; Li, Jianming; Clark, Steven E.
Fonte: American Society of Plant Biologists Publicador: American Society of Plant Biologists
Tipo: Artigo de Revista Científica
Publicado em /05/2003 Português
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The CLAVATA1 (CLV1) receptor kinase controls stem cell number and differentiation at the Arabidopsis shoot and flower meristems. Other components of the CLV1 signaling pathway include the secreted putative ligand CLV3 and the receptor-like protein CLV2. We report evidence indicating that all intermediate and strong clv1 alleles are dominant negative and likely interfere with the activity of unknown receptor kinase(s) that have functional overlap with CLV1. clv1 dominant-negative alleles show major differences from dominant-negative alleles characterized to date in animal receptor kinase signaling systems, including the lack of a dominant-negative effect of kinase domain truncation and the ability of missense mutations in the extracellular domain to act in a dominant-negative manner. We analyzed chimeric receptor kinases by fusing CLV1 and BRASSINOSTEROID INSENSITIVE1 (BRI1) coding sequences and expressing these in clv1 null backgrounds. Constructs containing the CLV1 extracellular domain and the BRI1 kinase domain were strongly dominant negative in the regulation of meristem development. Furthermore, we show that CLV1 expressed within the pedicel can partially replace the function of the ERECTA receptor kinase. We propose the presence of multiple receptors that regulate meristem development in a functionally related manner whose interactions are driven by the extracellular domains and whose activation requires the kinase domain.

‣ Molecular Origins for the Dominant Negative Function of Human Glucocorticoid Receptor Beta

Yudt, Matthew R.; Jewell, Christine M.; Bienstock, Rachelle J.; Cidlowski, John A.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /06/2003 Português
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This study molecularly elucidates the basis for the dominant negative mechanism of the glucocorticoid receptor (GR) isoform hGRβ, whose overexpression is associated with human glucocorticoid resistance. Using a series of truncated hGRα mutants and sequential mutagenesis to generate a series of hGRα/β hybrids, we find that the absence of helix 12 is neither necessary nor sufficient for the GR dominant negative phenotype. Moreover, we have localized the dominant negative activity of hGRβ to two residues and found that nuclear localization, in addition to heterodimerization, is a critical feature of the dominant negative activity. Molecular modeling of wild-type and mutant hGRα and hGRβ provides structural insight and a potential physical explanation for the lack of hormone binding and the dominant negative actions of hGRβ.

‣ Probing the structure, function, and interactions of the Escherichia coli H-NS and StpA proteins by using dominant negative derivatives.

Williams, R M; Rimsky, S; Buc, H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1996 Português
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Twelve different dominant negative mutants of the Escherichia coli nucleoid-associated protein, H-NS, have been selected and characterized in vivo. The mutants are all severely defective in promoter repression activity in a strain lacking H-NS, and they all disrupt the repression normally exerted by H-NS at two of its target promoters. From the locations of the alterations in these mutants, which result in both large truncations and amino acid substitutions, we propose that H-NAS contains at least two distinct domains. The in vitro protein-protein cross-linking data presented in this report indicate that the proposed N-terminal domain of H-NS has a role in H-NS multimerization. StpA is a protein with known structural and functional homologies to H-NS. We have analyzed the extent of these homologies by constructing and studying StpA mutants predicted to be dominant negative. Our data indicate that the substitutions and deletions found in dominant negative H-NS have similar effects in the context of StpA. We conclude that the domain organizations and functions in StpA and H-NS are closely related. Furthermore, dominant negative H-NS can disrupt the activity of native StpA, and reciprocally, dominant negative StpA can disrupt the activity of native H-NS. We demonstrate that the N-terminal domain of H-NS can be chemically cross-linked to both full-length H-NS and StpA. We account for these observations by proposing that H-NS and StpA have the ability to form hybrid species.

‣ Efficient repression of endogenous major histocompatibility complex class II expression through dominant negative CIITA mutants isolated by a functional selection strategy.

Bontron, S; Ucla, C; Mach, B; Steimle, V
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1997 Português
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Major histocompatibility complex class II (MHC-II) molecules present peptide antigens to CD4-positive T cells and are of critical importance for the immune response. The MHC-II transactivator CIITA is essential for all aspects of MHC-II gene expression examined so far and thus constitutes a master regulator of MHC-II expression. In this study, we generated and analyzed mutant CIITA molecules which are able to suppress endogenous MHC-II expression in a dominant negative manner for both constitutive and inducible MHC-II expression. Dominant negative CIITA mutants were generated via specific restriction sites and by functional selection from a library of random N-terminal CIITA deletions. This functional selection strategy was very effective, leading to strong dominant negative CIITA mutants in which the N-terminal acidic and proline/serine/threonine-rich regions were completely deleted. Dominant negative activity is dependent on an intact C terminus. Efficient repression of endogenous MHC-II mRNA levels was quantified by RNase protection analysis. The quantitative effects of various dominant negative CIITA mutants on mRNA expression levels of the different MHC-II isotypes are very similar. The optimized dominant negative CIITA mutants isolated by functional selection should be useful for in vivo repression of MHC-II expression.

‣ Dominant negative mutant of ionotropic glutamate receptor subunit GluR3: implications for the role of a cysteine residue for its channel activity and pharmacological properties.

Watase, K; Sekiguchi, M; Matsui, T A; Tagawa, Y; Wada, K
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/03/1997 Português
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We reported that a 33-amino-acid deletion (from tyrosine-715 to glycine-747) in a putative extracellular loop of GluR3 produced a mutant that exhibited dominant negative effects upon the functional expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors [Sekiguchi et al. (1994) J. Biol. Chem. 269, 14559-14565]. In this study, we searched for a key residue in the dominant negative effects to explore the mechanism and examined the role of the residue in the function of the AMPA receptor. We prepared 20 GluR3 mutants with amino acid substitutions within the 33-amino-acid-region, and dominant negative effects were tested electrophysiologically in Xenopus oocytes co-expressing the mutant and normal subunits. Among the mutants, only a GluR3 mutant in which an original cysteine (Cys)-722 was replaced by alanine exhibited a dominant negative effect comparable with that of the original mutant in which the entire 33-amino-acid segment is deleted. The co-expression of the Cys-722 mutant did not inhibit the translation of normal subunits in oocytes. The Cys-722 mutant formed a functional homomeric receptor with significantly higher affinity for glutamate or kainate than a homomeric GluR3 receptor. The Cys-722 mutation greatly enhanced the sensitivity of GluR3 for aniracetam...

‣ Dominant negative mutants of the yeast splicing factor Prp2 map to a putative cleft region in the helicase domain of DExD/H-box proteins.

Edwalds-Gilbert, G; Kim, D H; Kim, S H; Tseng, Y H; Yu, Y; Lin, R J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/2000 Português
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The Prp2 protein of Saccharomyces cerevisiae is an RNA-dependent ATPase required before the first transesterification reaction in pre-mRNA splicing. Prp2 binds to the spliceosome in the absence of ATP and is released following ATP hydrolysis. We determined what regions in Prp2 are essential for release from the spliceosome by analyzing dominant negative mutants in vivo and in vitro. We made mutations in conserved motif II (DExH) and motif VI (QRxGR) of the helicase (H) domain. Mutations that inactivated PRP2 had a dominant negative phenotype when overexpressed in vivo. To test whether mutations outside of the H domain could confer a dominant negative phenotype, we mutagenized a GAL1-PRP2 construct and screened for mutants unable to grow on galactose-containing media. Five dominant negative mutants were characterized; three mapped within the H domain and two mapped downstream of motif VI, indicating that an extended helicase domain is required for release of Prp2 from the spliceosome. Most mutants stalled in the spliceosome in vitro. However, not all mutants that were dominant negative in vivo were dominant negative in vitro, indicating that multiple mechanisms may cause a dominant negative phenotype. Structural modeling of the H domain of Prp2 suggests that mutants map to a cleft region found in helicases of known structure.

‣ Mutations in a dominant-negative isoform correlate with phenotype in inherited cardiac arrhythmias.

Mohammad-Panah, R; Demolombe, S; Neyroud, N; Guicheney, P; Kyndt, F; van den Hoff, M; Baró, I; Escande, D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1999 Português
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The long QT syndrome is characterized by prolonged cardiac repolarization and a high risk of sudden death. Mutations in the KCNQ1 gene, which encodes the cardiac KvLQT1 potassium ion (K+) channel, cause both the autosomal dominant Romano-Ward (RW) syndrome and the recessive Jervell and Lange-Nielsen (JLN) syndrome. JLN presents with cardiac arrhythmias and congenital deafness, and heterozygous carriers of JLN mutations exhibit a very mild cardiac phenotype. Despite the phenotypic differences between heterozygotes with RW and those with JLN mutations, both classes of variant protein fail to produce K+ currents in cultured cells. We have shown that an N-terminus-truncated KvLQT1 isoform endogenously expressed in the human heart exerts strong dominant-negative effects on the full-length KvLQT1 protein. Because RW and JLN mutations concern both truncated and full-length KvLQT1 isoforms, we investigated whether RW or JLN mutations would have different impacts on the dominant-negative properties of the truncated KvLQT1 splice variant. In a mammalian expression system, we found that JLN, but not RW, mutations suppress the dominant-negative effects of the truncated KvLQT1. Thus, in JLN heterozygous carriers, the full-length KvLQT1 protein encoded by the unaffected allele should not be subject to the negative influence of the mutated truncated isoform...

‣ Mapping of a Domain Required for Protein-Protein Interactions and Inhibitory Activity of a Helicobacter pylori Dominant-Negative VacA Mutant Protein

Torres, Victor J.; McClain, Mark S.; Cover, Timothy L.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/2006 Português
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The Helicobacter pylori VacA toxin is an 88-kDa secreted protein that causes multiple alterations in mammalian cells and is considered an important virulence factor in the pathogenesis of peptic ulcer disease and gastric cancer. We have shown previously that a VacA mutant protein lacking amino acids 6 to 27 (Δ6-27p88 VacA) is able to inhibit many activities of wild-type VacA in a dominant-negative manner. Analysis of a panel of C-terminally truncated Δ6-27p88 VacA proteins indicated that a fragment containing amino acids 1 to 478 (Δ6-27p48) exhibited a dominant-negative phenotype similar to that of the full-length Δ6-27p88 VacA protein. In contrast, a shorter VacA fragment lacking amino acids 6 to 27 (Δ6-27p33) did not exhibit detectable inhibitory activity. The Δ6-27p48 protein physically interacted with wild-type p88 VacA, whereas the Δ6-27p33 protein did not. Mutational analysis indicated that amino acids 351 to 360 are required for VacA protein-protein interactions and for dominant-negative inhibitory activity. The C-terminal portion (p55 domain) of wild-type p88 VacA could complement either Δ6-27p33 or Δ(6-27/351-360)p48, reconstituting dominant-negative inhibitory activity. Collectively, our data provide strong evidence that the inhibitory properties of dominant-negative VacA mutant proteins are dependent on interactions between the mutant VacA proteins and wild-type VacA...

‣ The dominant-negative effect of the Q218K variant of the prion protein does not require protein X

Lee, Cheng I.; Yang, Qingyuan; Perrier, Veronique; Baskakov, Ilia V.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /10/2007 Português
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Previous studies identified several single-point mutants of the prion protein that displayed dominant-negative effects on prion replication. The dominant-negative effect was assumed to be mediated by protein X, an as-yet-unknown cellular cofactor that is believed to be essential for prion replication. To gain insight into the mechanism that underlies the dominant-negative phenomena, we evaluated the effect of the Q218K variant of full-length recombinant prion protein (Q218K rPrP), one of the dominant-negative mutants, on cell-free polymerization of wild-type rPrP into amyloid fibrils. We found that both Q218K and wild-type (WT) rPrPs were incorporated into fibrils when incubated as a mixture; however, the yield of polymerization was substantially decreased in the presence of Q218K rPrP. Furthermore, in contrast to fibrils produced from WT rPrP, the fibrils generated in the mixture of WT and Q218K rPrPs did not acquire the proteinase K-resistant core of 16 kDa that was shown previously to encompass residues 97–230 and was similar to that of PrPSc. Our studies demonstrate that the Q218K variant exhibits the dominant-negative effect in cell-free conversion in the absence of protein X, and that this effect is, presumably, mediated by physical interaction between Q218K and WT rPrP during the polymerization process.

‣ Dominant-Negative Mutants Identify a Role for Girk Channels in D3 Dopamine Receptor-Mediated Regulation of Spontaneous Secretory Activity

Kuzhikandathil, Eldo V.; Oxford, Gerry S.
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/06/2000 Português
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The human D3 dopamine receptor can activate G-protein–coupled inward rectifier potassium channels (GIRKs), inhibit P/Q-type calcium channels, and inhibit spontaneous secretory activity in AtT-20 neuroendocrine cells (Kuzhikandathil, E.V., W. Yu, and G.S. Oxford. 1998. Mol. Cell. Neurosci. 12:390–402; Kuzhikandathil, E.V., and G.S. Oxford. 1999. J. Neurosci. 19:1698–1707). In this study, we evaluate the role of GIRKs in the D3 receptor-mediated inhibition of secretory activity in AtT-20 cells. The absence of selective blockers for GIRKs has precluded a direct test of the hypothesis that they play an important role in inhibiting secretory activity. However, the tetrameric structure of these channels provides a means of disrupting endogenous GIRK function using a dominant negative approach. To develop a dominant-negative GIRK mutant, the K+ selectivity amino acid sequence -GYG- in the putative pore domain of the human GIRK2 channels was mutated to -AAA-, -GLG-, or -GFG-. While the mutation of -GYG- to -GFG- did not affect channel function, both the -AAA- and -GLG- GIRK2 mutants were nonfunctional. This suggests that the aromatic ring of the tyrosine residue rather than its hydroxyl group is involved in maintaining the pore architecture of human GIRK2 channels. When expressed in AtT-20 cells...

‣ Characterisation of a dominant negative androgen receptor in prostate cancer cells.

Centenera, Margaret Mary
Fonte: Universidade de Adelaide Publicador: Universidade de Adelaide
Tipo: Tese de Doutorado
Publicado em //2008 Português
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Prostate cancer is the second leading cause of death from cancer in Australian men. As prostate cancer cells are reliant on androgens for growth and survival, the standard therapy for metastatic disease is androgen ablation therapy (AAT). AAT inhibits androgen signalling by altering androgen synthesis or prevent binding of androgens to their intracellular mediator, the androgen receptor (AR). Although initially effective, virtually all patients relapse, beyond which there are limited treatment options. The failure of AAT is not necessarily due to a decreased requirement for androgen signalling, but rather the AR is able to maintain signalling and tumour growth in an androgen-depleted environment. Therefore novel strategies that directly target the AR may provide a more effective therapeutic approach. We have endeavoured to suppress AR activity in prostate cancer cells by utilising a dominant negative AR. The most effective dominant negative construct developed, ARi41O, lacks amino acids 39-410 in the AR amino terminal transactivation domain. In studies of transcriptional activity, ARi410 has no intrinsic activity but inhibits the activity of wild type AR (wtAR) and also clinically relevant AR variants, by up to 95%. The objective of this thesis was to characterise the mechanisms of action of ARi410 and assess the functional effects of introducing this dominant negative receptor into prostate cancer cells. To investigate the mechanism by which ARi410 suppresses AR activity...

‣ Structural Analysis of Human Growth Hormone with Respect to the Dominant Expression of GH Mutations in Isolated Growth Hormone Deficiency Type II; Structural Analysis of Human Growth Hormone with Respect to the Dominant Expression of GH Mutations in Isolated Growth Hormone Deficiency Type II; Strukturanalyse des humanen Wachstumshormons zur Erforschung der dominanten Expression von GH-1 Genmutationen beim isolierten Wachstumshormonmangel Typ II

Iliev, Daniel
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
Português
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Human GH protein consists of four alpha-helices and contains two disulfide bridges. Isolated growth hormone deficiency type II (IGHD II) is mainly caused by heterozygous splice site mutations of GH-1 leading to the disruption of one disulfide bridge (Cys53-Cys165) and to the loss of amino acids (aa) 32-71 which comprise the complete loop between alpha-helices 1 and 2. The mutant GH protein exerts a dominant-negative effect on wild-type (wt) GH secretion by unclear mechanisms. For the study of the structure-function relationship of GH mutants concerning the dominant-negative effect, expression vectors harbouring mutated GH cDNAs (the mutations affecting either the linker region between alpha-helices 1 and 2 or the disulfide bridge Cys182-Cys189) were transiently cotransfected with a vector encoding wtGH (pwtGH) into GH4C1 cells. Plasmids encoding either â-galactosidase, luciferase or IGFBP-2 were cotransfected with pwtGH and either of the GH mutants. For the study of a potential dominant-negative effect due to disturbed Zn2+-binding, expression vectors harbouring mutated GH cDNAs were constructed in which triplets encoding histidine and glutamine residues were mutated to triplets encoding alanine residues. These plasmids were transiently cotransfected with a vector encoding wtGH (pwtGH) into GH4C1 cells. Compared to the control transfection with pwtGH...

‣ A spontaneous dominant-negative mutation within a 35S::AtMYB90 transgene inhibits flower pigment production in tobacco

Velten, Jeff; Cakir, Cahid; Cazzonelli, Christopher I.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica Formato: 12 pages
Português
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BACKGROUND In part due to the ease of visual detection of phenotypic changes, anthocyanin pigment production has long been the target of genetic and molecular research in plants. Specific members of the large family of plant myb transcription factors have been found to play critical roles in regulating expression of anthocyanin biosynthetic genes and these genes continue to serve as important tools in dissecting the molecular mechanisms of plant gene regulation. FINDINGS A spontaneous mutation within the coding region of an Arabidopsis 35S::AtMYB90 transgene converted the activator of plant-wide anthocyanin production to a dominant-negative allele (PG-1) that inhibits normal pigment production within tobacco petals. Sequence analysis identified a single base change that created a premature nonsense codon, truncating the encoded myb protein. The resulting mutant protein lacks 78 amino acids from the wild type C-terminus and was confirmed as the source of the white-flower phenotype. A putative tobacco homolog of AtMYB90 (NtAN2) was isolated and found to be expressed in flower petals but not leaves of all tobacco plants tested. Using transgenic tobacco constitutively expressing the NtAN2 gene confirmed the NtAN2 protein as the likely target of PG-1-based inhibition of tobacco pigment production. CONCLUSIONS Messenger RNA and anthocyanin analysis of PG-1Sh transgenic lines (and PG-1Sh x purple 35S::NtAN2 seedlings) support a model in which the mutant myb transgene product acts as a competitive inhibitor of the native tobacco NtAN2 protein. This finding is important to researchers in the field of plant transcription factor analysis...

‣ Identification of Dominant Negative Human Immunodeficiency Virus Type 1 Vif Mutants That Interfere with the Functional Inactivation of APOBEC3G by Virus-Encoded Vif ▿

Walker, Robert C.; Khan, Mohammad A.; Kao, Sandra; Goila-Gaur, Ritu; Miyagi, Eri; Strebel, Klaus
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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APOBEC3G (A3G) is a host cytidine deaminase that serves as a potent intrinsic inhibitor of retroviral replication. A3G is packaged into human immunodeficiency virus type 1 virions and deaminates deoxycytidine to deoxyuridine on nascent minus-strand retroviral cDNA, leading to hyper-deoxyguanine-to-deoxyadenine mutations on positive-strand cDNA and inhibition of viral replication. The antiviral activity of A3G is suppressed by Vif, a lentiviral accessory protein that prevents encapsidation of A3G. In this study, we identified dominant negative mutants of Vif that interfered with the ability of wild-type Vif to inhibit the encapsidation and antiviral activity of A3G. These mutants were nonfunctional due to mutations in the highly conserved HCCH and/or SOCS box motifs, which are required for assembly of a functional Cul5-E3 ubiquitin ligase complex. Similarly, mutation or deletion of a PPLP motif, which was previously reported to be important for Vif dimerization, induced a dominant negative phenotype. Expression of dominant negative Vif counteracted the Vif-induced reduction of intracellular A3G levels, presumably by preventing Vif-induced A3G degradation. Consequently, dominant negative Vif interfered with wild-type Vif's ability to exclude A3G from viral particles and reduced viral infectivity despite the presence of wild-type Vif. The identification of dominant negative mutants of Vif presents exciting possibilities for the design of novel antiviral strategies.

‣ A dominant negative mutation in the conserved RNA helicase motif 'SAT' causes splicing factor PRP2 to stall in spliceosomes.

Plumpton, M; McGarvey, M; Beggs, J D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/02/1994 Português
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To characterize sequences in the RNA helicase-like PRP2 protein of Saccharomyces cerevisiae that are essential for its function in pre-mRNA splicing, a pool of random PRP2 mutants was generated. A dominant negative allele was isolated which, when overexpressed in a wild-type yeast strain, inhibited cell growth by causing a defect in pre-mRNA splicing. This defect was partially alleviated by simultaneous co-overexpression of wild-type PRP2. The dominant negative PRP2 protein inhibited splicing in vitro and caused the accumulation of stalled splicing complexes. Immunoprecipitation with anti-PRP2 antibodies confirmed that dominant negative PRP2 protein competed with its wild-type counterpart for interaction with spliceosomes, with which the mutant protein remained associated. The PRP2-dn1 mutation led to a single amino acid change within the conserved SAT motif that in the prototype helicase eIF-4A is required for RNA unwinding. Purified dominant negative PRP2 protein had approximately 40% of the wild-type level of RNA-stimulated ATPase activity. As ATPase activity was reduced only slightly, but splicing activity was abolished, we propose that the dominant negative phenotype is due primarily to a defect in the putative RNA helicase activity of PRP2 protein.

‣ Dominant-negative interference with defence signalling by truncation mutations of the tomato Cf-9 disease resistance genes

Barker, Claire; Baillie, Brett; Hammond-Kosack, Kim E; Jones, Jonathan D G; Jones, David
Fonte: Blackwell Publishing Ltd Publicador: Blackwell Publishing Ltd
Tipo: Artigo de Revista Científica
Português
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The tomato Cf-9 gene confers resistance to races of the leaf mould fungus Cladosporium fulvum that carry the Avr9 avirulence gene. Cf-9 was isolated by transposon tagging using a modified maize Dissociation (Ds) element. This generated an allelic series of Ds-induced mutations of Cf-9, of which two were found to confer novel phenotypes in a screen for mutants affecting wild-type Cf-9 function in trans. Genetic and molecular analysis of these mutants suggested semidominant, Avr9-dependent, negative-interfering mutations involving Ds insertions in a defined subregion of Cf-9. Interference was associated with expression of the 5′-end of Cf-9 upstream of the Ds insertions in these mutants, suggesting that truncated Cf-9 proteins were the likely cause of interference. Transgenic tomato lines harbouring Cf-9 constructs with premature stop codons in positions similar to the Ds insertions also showed interference, indicating that the presence of Ds was not required for interference to occur. Interestingly, interference in these transgenic lines was completely dominant and was associated with a pronounced developmental phenotype that was dependent on co-expression of Cf-9, Avr9 and a truncated Cf-9 transgene. However, interference with a weakly autoactive Hcr9 gene was Avr9-independent and did not cause a developmental phenotype...