scholarly journals HSP90 buffers newly induced mutations in massively mutated plant lines

2018 ◽  
Author(s):  
G. Alex Mason ◽  
Keisha D Carlson ◽  
Maximilian O Press ◽  
Kerry L Bubb ◽  
Christine Queitsch
Keyword(s):  
Genetic Variation ◽  
De Novo ◽  
Embryonic Lethality ◽  
Model Organisms ◽  
Dependent Manner ◽  
Wild Type ◽  
Induced Mutations ◽  
Emergent Feature ◽  
Phenotypic Robustness ◽  
Mutant Phenotypes

AbstractRobustness to both genetic and environmental change is an emergent feature of living systems. Loss of phenotypic robustness can be associated with increased penetrance of genetic variation. In model organisms and in humans, the phenotypic consequences of standing genetic variation can be buffered by the molecular chaperone HSP90. However, it has been argued that HSP90 has the opposite effect on newly introduced genetic variation. To test the buffering effect of HSP90 on new mutations, we introduced vast numbers of mutations into wild-type and HSP90-reduced plants and assessed embryonic lethality and early seedling phenotypes for thousands of offspring. Although the levels of newly introduced mutations were similar in the two backgrounds, the HSP90-reduced plants showed a significantly greater frequency of embryonic lethality and severe phenotypic abnormalities, consistent with higher penetrance and expressivity of newly introduced genetic variation. We further demonstrate that some mutant phenotypes were heritable in an HSP90-dependent manner, and we map candidate HSP90-dependent polymorphisms. Moreover, both sequence and phenotypic analyses of wild-type and HSP90-reduced plants suggest that the HSP90-dependent phenotypes are largely due the newly introduced mutations rather than to an increased mutation rate in HSP90-reduced plants. Taken together, our results support a model in which HSP90 buffers newly introduced mutations, and the phenotypic consequences of such mutations outweigh those of mutations arising de novo in response to HSP90 perturbation.

scholarly journals The Quorum-Sensing Molecule Autoinducer 2 Regulates Motility and Flagellar Morphogenesis in Helicobacter pylori

2007 ◽  
Vol 189 (17) ◽  
pp. 6109-6117 ◽  
Author(s):  
Bethany A. Rader ◽  
Shawn R. Campagna ◽  
Martin F. Semmelhack ◽  
Bonnie L. Bassler ◽  
Karen Guillemin
Keyword(s):  
Helicobacter Pylori ◽  
Quorum Sensing ◽  
Sigma Factor ◽  
Critical Role ◽  
Soft Agar ◽  
Dependent Manner ◽  
Flagellar Gene ◽  
Wild Type ◽  
Autoinducer 2 ◽  
Mutant Phenotypes

ABSTRACT The genome of the gastric pathogen Helicobacter pylori contains a homologue of the gene luxS, which has been shown to be responsible for production of the quorum-sensing signal autoinducer 2 (AI-2). We report here that deletion of the luxS gene in strain G27 resulted in decreased motility on soft agar plates, a defect that was complemented by a wild-type copy of the luxS gene and by the addition of cell-free supernatant containing AI-2. The flagella of the luxS mutant appeared normal; however, in genetic backgrounds lacking any of three flagellar regulators—the two-component sensor kinase flgS, the sigma factor σ28 (also called fliA), and the anti-sigma factor flgM—loss of luxS altered flagellar morphology. In all cases, the double mutant phenotypes were restored to the luxS + phenotype by the addition of synthetic 4,5-dihydroxy-2,3-pentanedione (DPD), which cyclizes to form AI-2. Furthermore, in all mutant backgrounds loss of luxS caused a decrease in transcript levels of the flagellar regulator flhA. Addition of DPD to luxS cells induced flhA transcription in a dose-dependent manner. Deletion of flhA in a wild-type or luxS mutant background resulted in identical loss of motility, flagella, and flagellar gene expression. These data demonstrate that AI-2 functions as a secreted signaling molecule upstream of FlhA and plays a critical role in global regulation of flagellar gene transcription in H. pylori.

scholarly journals How a Genetically Stable Extremophile Evolves: Modes of Genome Diversification in the Archaeon Sulfolobus acidocaldarius

2017 ◽  
Vol 199 (17) ◽  
Author(s):  
Dominic Mao ◽  
Dennis W. Grogan
Keyword(s):  
Dna Repair ◽  
Genetic Variation ◽  
Tandem Repeats ◽  
Local Population ◽  
Genome Structure ◽  
Sulfolobus Acidocaldarius ◽  
Hyperthermophilic Archaea ◽  
Wild Type ◽  
Induced Mutations ◽  
Content Type

ABSTRACT In order to analyze in molecular terms how Sulfolobus genomes diverge, damage-induced mutations and natural polymorphisms (PMs) were identified in laboratory constructs and wild-type isolates, respectively, of Sulfolobus acidocaldarius. Among wild-type isolates drawn from one local population, pairwise nucleotide divergence averaged 4 × 10−6, which is about 0.15% of the corresponding divergence reported for Sulfolobus islandicus. The most variable features of wild-type S. acidocaldarius genomes were homopolymer (mononucleotide) tracts and longer tandem repeats, consistent with the spontaneous mutations that occur under laboratory conditions. Natural isolates, however, also revealed large insertions/deletions and inversions, which did not occur in any of the laboratory-manipulated strains. Several of the large insertions/deletions could be attributed to the integration or excision of mobile genetic elements (MGEs), and each MGE represented a distinct system of site-specific recombination. The mode of recombination associated with one MGE, a provirus related to Sulfolobus turreted icosahedral virus, was also seen in certain chromosomal inversions. Artificially induced mutations, non-MGE insertions/deletions, and small PMs exhibited different distributions over the genome, suggesting that large-scale patterning of Sulfolobus genomes begins early in the divergence process. Unlike induced mutations, natural base pair substitutions occurred in clusters, and one cluster exhibited properties expected of nonreciprocal recombination (gene conversion) between dispersed imperfect repeats. Taken together, the results identify simple replication errors, slipped-strand events promoted by tandem repeats, homologous recombination, and rearrangements promoted by MGEs as the primary sources of genetic variation for this extremely acidophilic archaeon in its geothermal environment. IMPORTANCE The optimal growth temperatures of hyperthermophilic archaea accelerate DNA decomposition, which is expected to make DNA repair especially important for their genetic stability, yet these archaea lack certain broadly conserved types of DNA repair proteins. In this study, the genome of the extreme thermoacidophile Sulfolobus acidocaldarius was found to be remarkably stable, accumulating few mutations in many (though not all) laboratory manipulations and in natural populations. Furthermore, all the genetic processes that were inferred to diversify these genomes also operate in mesophilic bacteria and eukaryotes. This suggests that a common set of mechanisms produces most of the genetic variation in all microorganisms, despite the fundamental differences in physiology, DNA repair systems, and genome structure represented in the three domains of life.

scholarly journals How could fully scaled carps appear in natural waters in Madagascar?

2016 ◽  
Vol 283 (1837) ◽  
pp. 20160945 ◽  
Author(s):  
Jean-Noël Hubert ◽  
François Allal ◽  
Caroline Hervet ◽  
Monique Ravakarivelo ◽  
Zsigmond Jeney ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Natural Waters ◽  
Environmental Changes ◽  
Model Organisms ◽  
Homozygous Mutation ◽  
Loss Of Function ◽  
Wild Type ◽  
Scale Cover ◽  
Alternative Routes ◽  
Reduced Scale

The capacity of organisms to rapidly evolve in response to environmental changes is a key feature of evolution, and studying mutation compensation is a way to evaluate whether alternative routes of evolution are possible or not. Common carps ( Cyprinus carpio ) carrying a homozygous loss-of-function mutation for the scale cover gene fgfr1a1 , causing the ‘mirror’ reduced scale cover, were introduced in Madagascar a century ago. Here we show that carps in Malagasy natural waters are now predominantly covered with scales, though they still all carry the homozygous mutation. We also reveal that the number of scales in mutated carps is under strong polygenic genetic control, with a heritability of 0.49. As a whole, our results suggest that carps submitted to natural selection could evolve a wild-type-like scale cover in less than 40 generations from standing polygenic genetic variation, confirming similar findings mainly retrieved from model organisms.

scholarly journals Dnmt1 Overexpression Causes Genomic Hypermethylation, Loss of Imprinting, and Embryonic Lethality

2002 ◽  
Vol 22 (7) ◽  
pp. 2124-2135 ◽  
Author(s):  
Detlev Biniszkiewicz ◽  
Joost Gribnau ◽  
Bernard Ramsahoye ◽  
François Gaudet ◽  
Kevin Eggan ◽  
...  
Keyword(s):  
De Novo ◽  
Embryonic Stem ◽  
Artificial Chromosome ◽  
Embryonic Lethality ◽  
Imprinted Genes ◽  
Wild Type ◽  
Biallelic Expression ◽  
Loss Of Imprinting ◽  
Survival Factor ◽  
De Novo Methylation

ABSTRACT Biallelic expression of Igf2 is frequently seen in cancers because Igf2 functions as a survival factor. In many tumors the activation of Igf2 expression has been correlated with de novo methylation of the imprinted region. We have compared the intrinsic susceptibilities of the imprinted region of Igf2 and H19, other imprinted genes, bulk genomic DNA, and repetitive retroviral sequences to Dnmt1 overexpression. At low Dnmt1 methyltransferase levels repetitive retroviral elements were methylated and silenced. The nonmethylated imprinted region of Igf2 and H19 was resistant to methylation at low Dnmt1 levels but became fully methylated when Dnmt1 was overexpressed from a bacterial artificial chromosome transgene. Methylation caused the activation of the silent Igf2 allele in wild-type and Dnmt1 knockout cells, leading to biallelic Igf2 expression. In contrast, the imprinted genes Igf2r, Peg3, Snrpn, and Grf1 were completely resistant to de novo methylation, even when Dnmt1 was overexpressed. Therefore, the intrinsic difference between the imprinted region of Igf2 and H19 and of other imprinted genes to postzygotic de novo methylation may be the molecular basis for the frequently observed de novo methylation and upregulation of Igf2 in neoplastic cells and tumors. Injection of Dnmt1-overexpressing embryonic stem cells in diploid or tetraploid blastocysts resulted in lethality of the embryo, which resembled embryonic lethality caused by Dnmt1 deficiency.

scholarly journals BECN1 F121A mutation increases autophagic flux in aged mice and improves aging phenotypes in an organ-dependent manner

2022 ◽  
Author(s):  
Salwa Sebti ◽  
Zhongju Zou ◽  
Michael U Shiloh
Keyword(s):  
Skeletal Muscle ◽  
Model Organisms ◽  
Autophagic Flux ◽  
Dependent Manner ◽  
Multiple Model ◽  
Wild Type ◽  
Age Related ◽  
Organ Specific ◽  
Muscle Fiber Atrophy ◽  
Fiber Atrophy

Autophagy is necessary for lifespan extension in multiple model organisms and autophagy dysfunction impacts age-related phenotypes and diseases. Introduction of an F121A mutation into the essential autophagy protein BECN1 constitutively increases basal autophagy in young mice and reduces cardiac and renal age-related changes in longer-lived Becn1F121A mutant mice. However, both autophagic and lysosomal activity have been described to decline with age. Thus, whether autophagic flux is maintained during aging and whether it is enhanced in Becn1F121A mice is unknown. Here we demonstrate that old wild type mice maintained functional autophagic flux in heart, kidney and skeletal muscle but not liver, and old Becn1F121A mice had increased autophagic flux in those same organs compared to wild type. In parallel, Becn1F121A mice were not protected against age-associated hepatic phenotypes but demonstrated reduced skeletal muscle fiber atrophy. These findings identify an organ-specific role for the ability of autophagy to impact organ aging phenotypes.

scholarly journals Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro.

1995 ◽  
Vol 15 (8) ◽  
pp. 4125-4135 ◽  
Author(s):  
P Dent ◽  
D B Reardon ◽  
D K Morrison ◽  
T W Sturgill
Keyword(s):  
Protein Kinase ◽  
Plasma Membranes ◽  
De Novo ◽  
Serine Phosphorylation ◽  
Mitogen Activated Protein Kinase ◽  
Significant Activity ◽  
Dependent Manner ◽  
Wild Type ◽  
Independent Manner

The serine/threonine kinase Raf-1 functions downstream from Ras to activate mitogen-activated protein kinase kinase, but the mechanisms of Raf-1 activation are incompletely understood. To dissect these mechanisms, wild-type and mutant Raf-1 proteins were studied in an in vitro system with purified plasma membranes from v-Ras- and v-Src-transformed cells (transformed membranes). Wild-type (His)6- and FLAG-Raf-1 were activated in a Ras- and ATP-dependent manner by transformed membranes; however, Raf-1 proteins that are kinase defective (K375M), that lack an in vivo site(s) of regulatory tyrosine (YY340/341FF) or constitutive serine (S621A) phosphorylation, that do not bind Ras (R89L), or that lack an intact zinc finger (CC165/168SS) were not. Raf-1 proteins lacking putative regulatory sites for an unidentified kinase (S259A) or protein kinase C (S499A) were activated but with apparently reduced efficiency. The kinase(s) responsible for activation by Ras or Src may reside in the plasma membrane, since GTP loading of plasma membranes from quiescent NIH 3T3 cells (parental membranes) induced de novo capacity to activate Raf-1. Wild-type Raf-1, possessing only basal activity, was not activated by parental membranes in the absence of GTP loading. In contrast, Raf-1 Y340D, possessing significant activity, was, surprisingly, stimulated by parental membranes in a Ras-independent manner. The results suggest that activation of Raf-1 by phosphorylation may be permissive for further modulation by another membrane factor, such as a lipid. A factor(s) extracted with methanol-chloroform from transformed membranes or membranes from Sf9 cells coexpressing Ras and SrcY527F significantly enhanced the activity of Raf-1 Y340D or active Raf-1 but not that of inactive Raf-1. Our findings suggest a model for activation of Raf-1, wherein (i) Raf-1 associates with Ras-GTP, (ii) Raf-1 is activated by tyrosine and/or serine phosphorylation, and (iii) Raf-1 activity is further increased by a membrane cofactor.

scholarly journals Radiation-induced neoantigens broaden the immunotherapeutic window of cancers with low mutational loads

2021 ◽  
Vol 118 (24) ◽  
pp. e2102611118
Author(s):  
Danielle M. Lussier ◽  
Elise Alspach ◽  
Jeffrey P. Ward ◽  
Alexander P. Miceli ◽  
Daniele Runci ◽  
...  
Keyword(s):  
T Cell ◽  
De Novo ◽  
Clinical Findings ◽  
External Beam Radiation ◽  
Dependent Manner ◽  
Induced Mutations ◽  
Beam Radiation ◽  
Radiation Induced

Immunotherapies are a promising advance in cancer treatment. However, because only a subset of cancer patients benefits from these treatments it is important to find mechanisms that will broaden the responding patient population. Generally, tumors with high mutational burdens have the potential to express greater numbers of mutant neoantigens. As neoantigens can be targets of protective adaptive immunity, highly mutated tumors are more responsive to immunotherapy. Given that external beam radiation 1) is a standard-of-care cancer therapy, 2) induces expression of mutant proteins and potentially mutant neoantigens in treated cells, and 3) has been shown to synergize clinically with immune checkpoint therapy (ICT), we hypothesized that at least one mechanism of this synergy was the generation of de novo mutant neoantigen targets in irradiated cells. Herein, we use KrasG12D x p53−/− sarcoma cell lines (KP sarcomas) that we and others have shown to be nearly devoid of mutations, are poorly antigenic, are not controlled by ICT, and do not induce a protective antitumor memory response. However, following one in vitro dose of 4- or 9-Gy irradiation, KP sarcoma cells acquire mutational neoantigens and become sensitive to ICT in vivo in a T cell-dependent manner. We further demonstrate that some of the radiation-induced mutations generate cytotoxic CD8+ T cell responses, are protective in a vaccine model, and are sufficient to make the parental KP sarcoma line susceptible to ICT. These results provide a proof of concept that induction of new antigenic targets in irradiated tumor cells represents an additional mechanism explaining the clinical findings of the synergy between radiation and immunotherapy.

scholarly journals CaMK (CMK-1) and O-GlcNAc transferase (OGT-1) modulate mechanosensory responding and habituation in an interstimulus interval-dependent manner in Caenorhabditis elegans

2017 ◽  
Author(s):  
Evan L. Ardiel ◽  
Troy A. McDiarmid ◽  
Tiffany A. Timbers ◽  
Kirsten C. Y. Lee ◽  
Javad Safaei ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Interstimulus Interval ◽  
Behavioral Plasticity ◽  
Neural Circuit ◽  
Model Organisms ◽  
Dependent Manner ◽  
Mechanical Stimuli ◽  
C Elegans ◽  
Mutant Phenotypes ◽  
Glcnac Transferase

AbstractThe ability to learn is an evolutionarily conserved adaptation that remains incompletely understood. Genetically tractable model organisms facilitate mechanistic explanations of learning that span genetic, neural circuit, and behavioural levels. Many aspects of neural physiology, including processes that underlie learning (e.g. neurotransmitter release and long-lasting changes in synaptic strength), are regulated by brief and local changes in [μm] levels of free intracellular Ca2+. On this scale, changes in [Ca2+] activate many Ca2+-sensors, including the Ca2+/calmodulin-dependent kinases (CaMKs). Here we reveal that the Caenorhabditis elegans ortholog of CaMK1/4, CMK-1, functions in primary sensory neurons to regulate responses to mechanical stimuli and behavioral plasticity, specifically habituation, a conserved form of non-associative learning. The habituation phenotypes of cmk-1 mutants were dependent on interstimulus interval (ISI), such that CMK-1 slows habituation at short ISIs, but promotes it at long ISIs. We predicted potential CaMK phosphorylation targets from catalytic site analysis of the human and C. elegans CaMKs and mutant analysis of these candidates implicated O-linked N-acetylglucosamine (O-GlcNAc) transferase, OGT-1, in mechanosensitivity and learning. Cell specific rescue and knockdown experiments showed that both CMK-1 and OGT-1 function cell autonomously in mechanosensory neurons to modulate learning. Interestingly, despite their similar mutant phenotypes, detailed behavioral analysis of double mutants demonstrated that CMK-1 and OGT-1 act in parallel genetic pathways. Our research identifies CMK-1 and OGT-1 as co-expressed yet independent regulators of mechanosensitivity and learning.

Abstract 5495: Role of SIRT1 in Smooth Muscle Cell Function and Vascular Remodeling

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Daniel Sedding ◽  
Sabina Vogel ◽  
Harald Tillmanns
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Cell Function ◽  
Model Organisms ◽  
Dependent Manner ◽  
Wild Type ◽  
Vascular Homeostasis

Background: The class III histone deacetylase SIRT1 has been identified as a key regulator of ageing and longevity in model organisms such as S. cerevisiae and C. elegans, which regulates cellular functions such as differentiation, senescence and metabolism. However, the role of SIRT1 for Smooth muscle cell (VSMC) function and vascular homeostasis or during vascular remodelling remains unknown. Methods and Results: Here, we show that SIRT1 is highly expressed in intact blood vessels in vivo as well as in cultured VSMC. Stimulation of SIRT1 activity by either treatment with the SIRT1 activator resveratrol or adenoviral overexpression of wild type SIRT1 but not with an inactive SIRT1 mutant attenuated serum-induced VSMC proliferation in a dose dependent manner in vitro. In contrast, treatment of VSMC with the small molecule weight inhibitors of SIRT1, nicotinamide and sirtinol, augmented the proliferative and migratory activity of VSMC. Consistent with these data, MEF cells isolated from SIRT −/− mice showed an augmented proliferative response to serum stimulation but were also more resistant to starving-induced apoptosis compared to WT-MEF cells. Silencing of endogenous SIRT1 using siRNA resulted in an increased proliferation, migration and apoptosis of VSMC. In vivo, following arterial injury of the mouse femoral artery, SIRT1 was downregulated in the developing neointima. Adenoviral-mediated reconstitution of wild type SIRT1 but not of the inactive SIRT1 mutant prevented neointima formation in vivo. Conclusion: Thus, these data identify SIRT1 as a key regulator of vascular proliferative disease processes and indicate that SIRT1 plays an essential role in proliferative migratory and apoptotic processes which regulate vascular homeostasis and remodeling.

scholarly journals RhlR, but not RhlI, allowsP. aeruginosabacteria to evadeDrosophilaTep4-mediated opsonization

2017 ◽  
Author(s):  
Samantha Haller ◽  
Adrien Franchet ◽  
Abdul Hakkim ◽  
Jing Chen ◽  
Eliana Drenkard ◽  
...  
Keyword(s):  
Intestinal Barrier ◽  
Infection Model ◽  
Dependent Manner ◽  
Intestinal Infection ◽  
Wild Type ◽  
Independent Function ◽  
C Elegans ◽  
Injury Model ◽  
Mutant Phenotypes ◽  
Cellular Immune

ABSTRACTWhenDrosophilaflies feed onPseudomonas aeruginosastrain PA14, some bacteria cross the intestinal barrier and start proliferating inside the hemocoel. This process is limited by hemocytes through phagocytosis. We have previously shown that the PA14 quorum-sensing regulator RhlR is required for these bacteria to elude the cellular immune response. RhlI synthesizes the auto-inducer signal that activates RhlR. Here, we compare the null mutant phenotypes ofrhlRandrhlIin a variety of infection assays inDrosophilaand in the nematodeCaenorhabditis elegans. Surprisingly, inDrosophila, unlikeΔrhlRmutants,ΔrhlImutants are only modestly attenuated for virulence and are poorly phagocytosed and opsonized in a Thioester-containing Protein4-dependent manner. Likewise, ΔrhlIbut not ΔrhlRmutants colonize the digestive tract ofC. elegansand kill it as efficiently as wild-type PA14. Thus, RhlR has an RhlI-independent function in eluding detection or counter-acting the action of the immune system. In contrast to the intestinal infection model,Tep4mutant flies are more resistant to PA14 in a septic injury model, which also depends onrhlR. Thus, the Tep4 putative opsonin can either be protective or detrimental to host defense depending on the infection route.

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