scholarly journals A Naturally Occurring hPMS2 Mutation Can Confer a Dominant Negative Mutator Phenotype

1998 ◽  
Vol 18 (3) ◽  
pp. 1635-1641 ◽  
Author(s):  
Nicholas C. Nicolaides ◽  
Susan J. Littman ◽  
Paul Modrich ◽  
Kenneth W. Kinzler ◽  
Bert Vogelstein
Keyword(s):  
Mammalian Cells ◽  
Germ Line ◽  
Dominant Negative ◽  
Wild Type ◽  
Mutator Phenotype ◽  
Mmr Genes ◽  
Naturally Occurring ◽  
Hereditary Nonpolyposis ◽  
The Relationship ◽  
Present Experimental Evidence

ABSTRACT Defects in mismatch repair (MMR) genes result in a mutator phenotype by inducing microsatellite instability (MI), a characteristic of hereditary nonpolyposis colorectal cancers (HNPCC) and a subset of sporadic colon tumors. Present models describing the mechanism by which germ line mutations in MMR genes predispose kindreds to HNPCC suggest a “two-hit” inactivation of both alleles of a particular MMR gene. Here we present experimental evidence that a nonsense mutation at codon 134 of the hPMS2 gene is sufficient to reduce MMR and induce MI in cells containing a wild-type hPMS2 allele. These results have significant implications for understanding the relationship between mutagenesis and carcinogenesis and the ability to generate mammalian cells with mutator phenotypes.

scholarly journals The Naturally Occurring Variant of Estrogen Receptor (ER) ERΔE7 Suppresses Estrogen-Dependent Transcriptional Activation by Both Wild-Type ERα and ERβ

Endocrinology ◽  
2003 ◽  
Vol 144 (7) ◽  
pp. 2967-2976 ◽  
Author(s):  
Juana M. García Pedrero ◽  
Pedro Zuazua ◽  
Carlos Martínez-Campa ◽  
Pedro S. Lazo ◽  
Sofía Ramos
Keyword(s):  
Estrogen Receptor ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Activation Function ◽  
Inhibitory Effect ◽  
Dominant Negative ◽  
Wild Type ◽  
Independent Manner ◽  
Naturally Occurring ◽  
Estrogen Response

Abstract We have isolated and functionally characterized the exon 7-skipped variant (ERΔE7) of estrogen receptor (ER)α, which has emerged as the predominant variant expressed in multiple normal and tumoral tissues. However, to date no function has been established for this variant in mammalian cells. ERΔE7 exhibits a negligible ability to bind ligands, insensitivity to allosteric modulation by estrogen and antiestrogens, and loss of estrogen-dependent interaction with p160 coactivators such as SRC-1 and AIB1. ERΔE7 is able to form heterodimers with both ERα and ERβ in a ligand-independent manner. Transient expression experiments in HeLa cells show that increasing amounts of ERΔE7 result in a progressive inhibition of the estrogen-dependent transcriptional activation by both wild-type ERα and ERβ on estrogen response element-driven promoters. The inhibitory effect of ERΔE7 is due to the inhibition of binding of wild-type receptors to their responsive elements. Surprisingly, the activation function (AF)-1-dependent transactivation triggered by epithelial growth factor and phorbol-12-myristate-13-acetate is also abolished in ERΔE7 despite AF1 integrity, suggesting a cross-talk between AF1 and AF2 regions of the receptor. These results indicate that the naturally occurring variant ERΔE7 is a dominant negative receptor that, when expressed at high levels relative to wild-type ERs, might have profound effects on several estrogen-dependent functions.

scholarly journals RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (2) ◽  
pp. 758-766 ◽  
Author(s):  
R Ruggieri ◽  
A Bender ◽  
Y Matsui ◽  
S Powers ◽  
Y Takai ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Polarity ◽  
Copy Number ◽  
Mammalian Cells ◽  
Direct Interaction ◽  
Dominant Negative ◽  
Yeast Cells ◽  
Wild Type ◽  
Ras Gene ◽  
Ras Pathway

The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.

scholarly journals Primary Brain Calcification Causal PiT2 Transport-Knockout Variants can Exert Dominant Negative Effects on Wild-Type PiT2 Transport Function in Mammalian Cells

2016 ◽  
Vol 61 (2) ◽  
pp. 215-220 ◽  
Author(s):  
Frederik Tibert Larsen ◽  
Nina Jensen ◽  
Jacob Kwasi Autzen ◽  
Iben Boutrup Kongsfelt ◽  
Lene Pedersen
Keyword(s):  
Mammalian Cells ◽  
Dominant Negative ◽  
Transport Function ◽  
Wild Type ◽  
Negative Effects ◽  
Brain Calcification

scholarly journals Fluorescence Resonance Energy Transfer Microscopy of the Helicobacter pylori Vacuolating Cytotoxin within Mammalian Cells

2002 ◽  
Vol 70 (7) ◽  
pp. 3824-3832 ◽  
Author(s):  
David C. Willhite ◽  
Dan Ye ◽  
Steven R. Blanke
Keyword(s):  
Helicobacter Pylori ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dominant Negative ◽  
Wild Type ◽  
Vacuolating Cytotoxin ◽  
Vacuolated Cells ◽  
Mutant Forms

ABSTRACT The Helicobacter pylori vacuolating cytotoxin (VacA) binds and enters mammalian cells to induce cellular vacuolation. To investigate the quaternary structure of VacA within the intracellular environment where toxin cytotoxicity is elaborated, we employed fluorescence resonance energy transfer (FRET) microscopy. HeLa cells coexpressing full-length and truncated forms of VacA fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) were analyzed for FRET to indicate direct associations. These studies revealed that VacA-CFP and VacA-YFP interact within vacuolated cells, supporting the belief that monomer associations at an intracellular site are important for the toxin's vacuolating activity. In addition, the two fragments of proteolytically nicked VacA, p37 and p58, interact when coexpressed within mammalian cells. Because p37 and p58 function in trans when expressed separately within mammalian cells, these data suggest that the mechanism by which these two fragments induce vacuolation requires direct association. FRET microscopy also demonstrated interactions between mutant forms of VacA, as well as wild-type VacA with mutant forms of the toxin within vacuolated cells. Finally, a dominant-negative form of the toxin directly associates with wild-type VacA in cells where vacuolation was not detectable, suggesting that the formation of complexes comprising wild-type and dominant-negative forms of toxin acts to block intracellular toxin function.

scholarly journals RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (2) ◽  
pp. 758-766
Author(s):  
R Ruggieri ◽  
A Bender ◽  
Y Matsui ◽  
S Powers ◽  
Y Takai ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Polarity ◽  
Copy Number ◽  
Mammalian Cells ◽  
Direct Interaction ◽  
Dominant Negative ◽  
Yeast Cells ◽  
Wild Type ◽  
Ras Gene ◽  
Ras Pathway

The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.

scholarly journals Different responses to cell crowding determine the clonal fitness of p53 and Notch inhibiting mutations in squamous epithelia

2020 ◽  
Author(s):  
Vasiliki Kostiou ◽  
Michael WJ Hall ◽  
Philip H Jones ◽  
Benjamin A Hall
Keyword(s):  
Areal Density ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Downstream Target ◽  
Normal Tissues ◽  
Fitness Advantage ◽  
Naturally Occurring ◽  
Type Population ◽  
Mutant Cells

AbstractThe growth and competition of cells in epithelial tissues plays an important role in both tissue homeostasis and the robustness of normal tissues to pre-cancer mutation. Whilst wild-type cells compete neutrally for dominance in the un-mutated tissue, naturally occurring mutations in individual cells may lend them a fitness advantage that can allow tissue colonisation. In mouse oesophageal epithelia, the growth of p53 mutants and a dominant negative mutant of the Notch downstream target Maml1 (DN_Maml1) have been shown to have different colonisation properties despite strong quantitative similarities in the growth of individual clones. Here we show that in order to recapitulate these behaviours whilst maintaining tissue turnover models need to take account of the response of cells to increased areal density in the tissue colonised by mutant cells. We demonstrate that p53 mutant clone growth approximates a logistic curve, but that without including limitations on mutation induced expansion the overall proliferation rate of the tissue drops due to space restrictions. In contrast, the ability of DN_Maml1 mutations to displace the wild-type population reflects a feedback that effects both mutant and wild-type cells equally. We go on to show how these distinct feedbacks are consistent with the distribution of mutations observed in human datasets.

scholarly journals Stat3 Activation Is Required for Cellular Transformation by v-src

1998 ◽  
Vol 18 (5) ◽  
pp. 2553-2558 ◽  
Author(s):  
Jacqueline F. Bromberg ◽  
Curt M. Horvath ◽  
Daniel Besser ◽  
Wyndham W. Lathem ◽  
James E. Darnell
Keyword(s):  
Cell Lines ◽  
Cellular Transformation ◽  
Human Tumors ◽  
Dominant Negative ◽  
Wild Type ◽  
Mutant Proteins ◽  
Transformed Cell ◽  
Transformed Cell Lines ◽  
Relationship Of ◽  
The Relationship

ABSTRACT Stat3 activation has been associated with cytokine-induced proliferation, anti-apoptosis, and transformation. Constitutively activated Stat3 has been found in many human tumors as well as v-abl- and v-src-transformed cell lines. Because of these correlations, we examined directly the relationship of activated Stat3 to cellular transformation and found that wild-type Stat3 enhances the transforming potential of v-src while three dominant negative Stat3 mutants inhibit v-srctransformation. Stat3 wild-type or mutant proteins did not affect v-ras transformation. We conclude that Stat3 has a necessary role in v-src transformation.

scholarly journals Short Stature is Progressive in Patients with Heterozygous NPR2 Mutations

2020 ◽  
Vol 105 (10) ◽  
pp. 3190-3202 ◽  
Author(s):  
Patrick C Hanley ◽  
Harsh S Kanwar ◽  
Corine Martineau ◽  
Michael A Levine
Keyword(s):  
Short Stature ◽  
Mammalian Cells ◽  
Extended Family ◽  
Medical Center ◽  
Academic Medical Center ◽  
Dominant Negative ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Wild Type ◽  
Z Scores

Abstract Background NPR2 encodes atrial natriuretic peptide receptor B (ANPRB), a regulator of skeletal growth. Biallelic loss-of-function mutations in NPR2 result in acromesomelic dysplasia Maroteaux type (AMDM; OMIM 602875), while heterozygous mutations may account for 2% to 6% of idiopathic short stature (ISS). Objective Describe the physical proportions and growth characteristics of an extended family with novel NPR2 mutations including members with AMDM, ISS, or normal stature. Design and Participants We performed whole exome sequencing in 2 healthy parents and 2 children with AMDM. Detailed genotyping and phenotyping were performed on members of a multigenerational family in an academic medical center. We expressed mutant proteins in mammalian cells and characterized expression and function. Results The sisters with AMDM were compound heterozygotes for missense mutations in the NPR2 gene, a novel p.P93S (maternal) and the previously reported p.R989L (paternal). Both mutant ANPRB proteins were normally expressed in HEK293T cells and exhibited dominant negative effects on wild-type ANPRB catalytic activity. Heterozygous relatives had proportionate short stature (height z-scores −2.06 ± 0.97, median ± SD) compared with their wild-type siblings (−1.37 ± 0.59). Height z-scores progressively and significantly decreased as NPR2-heterozygous children matured, while remaining constant in their wild-type siblings. Conclusions Biallelic NPR2 mutations cause severe skeletal dysplasia (AMDM), whereas heterozygous mutations lead to a subtler phenotype characterized by progressive short stature with by increasing loss of height potential with age.

scholarly journals The T/G mutation in exon 8 of HMSH2 gene in the sporadic colon cancer patients

2006 ◽  
Vol 53 (2) ◽  
pp. 57-60
Author(s):  
Eva Langner ◽  
Karolina Przybyzowska ◽  
Galbfach Przemyszaw ◽  
Janusz Kunierz ◽  
Beata Smolarz ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Colon Cancer ◽  
Histological Grade ◽  
Clinical Parameters ◽  
Dna Mismatch ◽  
Mmr Genes ◽  
Significant Difference ◽  
Hereditary Nonpolyposis ◽  
Sporadic Colon Cancer ◽  
The Relationship

The DNA mismatch repair (MMR) system guards against genomic instability, therefore the mutations in the human MMR genes cause the majority of the hereditary nonpolyposis colorectal cancer (HNPCC) and a small percentage of the sporadic colon cancer. hMSH2 is one of MMR genes involved in the correction of mispairing during replication and its mutations are associated with both - microsatellite instability and the hereditary and sporadic colon tumor genesis. The aim of this study was to analyze the T/G mutation (codon 458) in exon 8 of hMSH2 gene in the sporadic colon cancer cells. We also examined the relationship between the T/G mutation of hMSH2 gene, and the selected prognostic factors such as Dukes? stage, histological grade and lymph node metastasis. We analyzed samples of tumor from 75 patients with sporadic colorectal cancers. The mutation in the hMSH2 gene ware determined by the RFLP-PCR. We found T/G mutation in exon 8 of hMSH2 gene in 5 patients (6,7%). There was no statistically significant difference between this mutation and selected clinical parameters. The results of our studies revealed that mutations of hMSH2 gene may lead to development of colorectal cancer. No dependence between the mutation of hMSH2 gene and clinical parameters, suggests that the mutation of hMSH2 gene may have a critical significance for the first steps of carcinogenesis in colon epithelial.

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